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[ [ "Reformation of soft soil and system therefor", "A system and a method for draining soft soil A bordered with soil B are disclosed.", "The system includes airtight sheet means 10 which covers the soft soil A to assist in vacuuming the soft soil A.", "The system further includes water gathering pipes 13 and water drain tank means 16 to receive water from the water gathering pipes 13.", "The water gathering pipes 13 and the water drain tank means 16 provide water passages and air passages separately so as to expedite the drainage operation." ], [ "1.A method for reforming target soft soil by vacuuming and draining the target soft soil covered with airtight sheet means, comprising separating water expelling routes and air expelling routes.", "2.A method of claim 1, further comprising installing water drain tank means under water gathering pipes which are in fluid connection with horizontal drains and vertical drains so as to receive underground water from the water gathering pipes.", "3.A method of claim 2, further comprising providing separator means which separates underground water coming from said water gathering pipes into water and air and sends the separated water into said water drain tank means.", "4.A method of claim 2, further comprising providing water drain pump means to expel the water in said water drain tank means out of the target soft soil.", "5.A method of claim 3, further comprising providing water drain pump means to expel the water in said water drain tank means out of the target soft soil.", "6.A method of claim 1, further comprising providing first water drain tank means in connection with water gathering pipes, providing second water drain tank means in connection with said first water drain tank means such that water is received into said second water drain tank means from said first water drain tank means via connection pipes, providing said second water drain tank means with water drain pump means, and expelling water from said second water drain tank means with said water drain pump means.", "7.A method of claim 2, wherein said horizontal drains and said water gathering pipes each have a water passage and an air passage separately.", "8.A drain system for reforming target soft soil by vacuuming and draining the target soft soil, comprising separately provided water expelling routes and air expelling routes.", "9.A drain system of claim 8, wherein the system comprises vertical drains installed in said target soft soil, horizontal drains laid on said target soft soil in contact with top portions of said vertical drains, water gathering pipes in contact with said horizontal drains, and water drain tank means installed below and in connection with said water gathering pipes, said water gathering pipes and said water drain tank means each providing a water passage and an air passage separately.", "10.A drain system of claim 9, wherein said water gathering pipes and said water drain tank means are connected via separator means.", "11.A drain system of claim 9, wherein said water drain tank means is provided with water drain pump means.", "12.A drain system of claim 10, wherein said water drain tank means is provided with water drain pump means.", "13.A drain system of claim 8, further comprising watertight type vacuum pump means having cooling water circulation tank means.", "14.A drain system of claim 8, further comprising vertical pipes installed in the vicinity of the target soft soil to evaporate underground water therefrom.", "15.A drain system of claim 14, wherein said vertical pipes are drain pipes.", "16.A drain system of claim 15, wherein said drain pipes are provided with air blower means.", "17.A drain system of claim 9, further comprising first water drain tank means in connection with said water gathering pipes and second water drain tank means in connection with said first water drain tank means such that water is received into said second water drain tank means from said first water drain tank means via connection pipes, wherein said second water drain tank means is provided with water drain pump means which expels water from said second water drain tank means.", "18.A drain system of claim 9, wherein said horizontal drains and said water gathering pipes each have a water passage and an air passage separately." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention This invention generally relates to a method and a system for reforming soft soil such as muddy soil or swampy soil by draining underground water therefrom.", "More particularly, this invention relates to a method and a system for efficiently hardening soft soil by adequately separating air passages and water passages in the drainage routes.", "2.Background Art JP Patent Application Laid-Open No.11-131465 teaches use of vertical drains which are laid vertically in target soft soil to vacuum the soil and drain underground water.", "FIG.", "11 shows a conventional drain system comprised of vertical drains 1 , horizontal drains 2 laid in contact with the vertical drains 1 , water gathering pipes 3 laid in contact with the horizontal drains 2 , and airtight sheet means 6 which covers target soft soil A after the vertical drains 1 , the horizontal drains 2 and the water gathering pipes 3 have been installed in place.", "The drain system is further comprised of vacuum tank means 4 placed in connection with the water gathering pipes 3 and vacuum pump means 5 placed in connection with the vacuum tank means 4 .", "In use of the conventional drain system shown in FIG.", "11 , the vacuum pump means 5 vacuums the vacuum tank means 4 .", "When a check valve (not shown) provided on the vacuum tank means 4 is opened, the water gathering pipes 3 are vacuumed.", "The vacuuming effect propagates to the horizontal drains 2 and the vertical drains 1 which are in connection with the horizontal drains 2 and reduces their respective inner pressures to below 0.4 atm.", "The target soft soil A is gradually evacuated from around the vertical drains 1 where air is drawn into the vertical drains 1 .", "The evacuated regions gradually spread throughout the soft soil A.", "Spread of the evacuated regions in the soft soil A directs underground water and underground air towards the vertical drains 1 and the water and air drawn into the vertical drains 1 travel up through the vertical drains 1 .", "The water and air are sucked into the horizontal drains 2 and then into the water gathering pipes 3 .", "The continued drainage of the target soft soil A further spreads the evacuated regions.", "The whole of the target soft soil A will eventually be vacuumed to around 0.4 atm, and underground water and air are eventually drained out of the soft soil A, leading to compaction of the soft soil A to a harder and stabler soil state.", "It is to be noted that in the conventional system the vacuuming routes and water drain routes are common.", "Therefore, sucked air and water flow together all through the common routes comprised of the vertical drains 1 , the horizontal drains 2 , the water gathering pipes 3 , the vacuum tank means 4 and the vacuum pump means 5 .", "Initially, underground water and air flow into the water gathering pipes 3 in large quantities from the horizontal drains 2 , which stuffs the water gathering pipes 3 and therefore impedes subsequent vacuuming of the target soft soil A as will be readily understood by those skilled in the art.", "In addition, as the compaction of the soft soil A progresses and the soft soil A sinks, the vertical distance from the underground water level to the vacuum pump means 5 widens, and efficiency of drainage degrades as will be readily understood by those with ordinary skills." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 is a schematic diagram showing a drain system of the present invention; FIG.", "2 is a schematic diagram showing a vacuum tank of the present invention; FIG.", "3 is a schematic diagram partially showing an assistant system of the present invention; FIG.", "4 is a schematic diagram partially showing another assistant system of the present invention; FIG.", "5 is a schematic diagram showing another drain system of the present invention; FIG.", "6 is a schematic diagram showing a first drain tank of the drain system of FIG.", "5 ; FIG.", "7 is a schematic diagram showing a second drain tank of the drain system of FIG.", "5 ; FIG.", "8 is a schematic diagram partially showing a connection of a vertical drain and a horizontal drain of the present invention; FIG.", "9 is a schematic diagram showing a sectional view of a water gathering pipe of the present invention; FIG.", "10 is a schematic diagram showing a sectional view of another water gathering pipe of the present invention; and FIG.", "11 is a schematic diagram showing a conventional drain system.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention This invention generally relates to a method and a system for reforming soft soil such as muddy soil or swampy soil by draining underground water therefrom.", "More particularly, this invention relates to a method and a system for efficiently hardening soft soil by adequately separating air passages and water passages in the drainage routes.", "2.Background Art JP Patent Application Laid-Open No.11-131465 teaches use of vertical drains which are laid vertically in target soft soil to vacuum the soil and drain underground water.", "FIG.", "11 shows a conventional drain system comprised of vertical drains 1, horizontal drains 2 laid in contact with the vertical drains 1, water gathering pipes 3 laid in contact with the horizontal drains 2, and airtight sheet means 6 which covers target soft soil A after the vertical drains 1, the horizontal drains 2 and the water gathering pipes 3 have been installed in place.", "The drain system is further comprised of vacuum tank means 4 placed in connection with the water gathering pipes 3 and vacuum pump means 5 placed in connection with the vacuum tank means 4.In use of the conventional drain system shown in FIG.", "11, the vacuum pump means 5 vacuums the vacuum tank means 4.When a check valve (not shown) provided on the vacuum tank means 4 is opened, the water gathering pipes 3 are vacuumed.", "The vacuuming effect propagates to the horizontal drains 2 and the vertical drains 1 which are in connection with the horizontal drains 2 and reduces their respective inner pressures to below 0.4 atm.", "The target soft soil A is gradually evacuated from around the vertical drains 1 where air is drawn into the vertical drains 1.The evacuated regions gradually spread throughout the soft soil A.", "Spread of the evacuated regions in the soft soil A directs underground water and underground air towards the vertical drains 1 and the water and air drawn into the vertical drains 1 travel up through the vertical drains 1.The water and air are sucked into the horizontal drains 2 and then into the water gathering pipes 3.The continued drainage of the target soft soil A further spreads the evacuated regions.", "The whole of the target soft soil A will eventually be vacuumed to around 0.4 atm, and underground water and air are eventually drained out of the soft soil A, leading to compaction of the soft soil A to a harder and stabler soil state.", "It is to be noted that in the conventional system the vacuuming routes and water drain routes are common.", "Therefore, sucked air and water flow together all through the common routes comprised of the vertical drains 1, the horizontal drains 2, the water gathering pipes 3, the vacuum tank means 4 and the vacuum pump means 5.Initially, underground water and air flow into the water gathering pipes 3 in large quantities from the horizontal drains 2, which stuffs the water gathering pipes 3 and therefore impedes subsequent vacuuming of the target soft soil A as will be readily understood by those skilled in the art.", "In addition, as the compaction of the soft soil A progresses and the soft soil A sinks, the vertical distance from the underground water level to the vacuum pump means 5 widens, and efficiency of drainage degrades as will be readily understood by those with ordinary skills.", "SUMMERY OF THE INVENTION Therefore, it is an object of the present invention to provide an improved drainage method and an improved drain system for reforming or hardening soft ground or soil, wherein the drain system provides separated water passages and air passages.", "The drainage method of the present invention which uses a drain system of the invention provides an improved efficiency in draining underground water from soft soil and hardening the soft soil in a relatively short period of time by adequately separating the water passages and the air passages of the system.", "The drain system of the present invention provides an improved efficiency in draining underground water from soft soil and hardening the soft soil in a relatively short period of time by adequately separating its water passages and air passages.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a schematic diagram showing a drain system of the present invention; FIG.", "2 is a schematic diagram showing a vacuum tank of the present invention; FIG.", "3 is a schematic diagram partially showing an assistant system of the present invention; FIG.", "4 is a schematic diagram partially showing another assistant system of the present invention; FIG.", "5 is a schematic diagram showing another drain system of the present invention; FIG.", "6 is a schematic diagram showing a first drain tank of the drain system of FIG.", "5; FIG.", "7 is a schematic diagram showing a second drain tank of the drain system of FIG.", "5; FIG.", "8 is a schematic diagram partially showing a connection of a vertical drain and a horizontal drain of the present invention; FIG.", "9 is a schematic diagram showing a sectional view of a water gathering pipe of the present invention; FIG.", "10 is a schematic diagram showing a sectional view of another water gathering pipe of the present invention; and FIG.", "11 is a schematic diagram showing a conventional drain system.", "BEST MODE OF THE INVENTION The present invention is described hereunder using the drawings which accompany the specification.", "In FIG.", "1 is shown a drain system according to an embodiment of the present invention which efficiently drains and hardens target soft soil A surrounded by soil B.", "The vacuuming and drainage routes include vertical drains 11, horizontal drains 12 which are laid in contact with the vertical drains 11, water gathering pipes 13 which are laid in contact with the horizontal drains 12, vacuum tank means 14 placed in connection with the horizontal drains 12, and vacuum pump means 15 placed in connection with the vacuum tank means 14.The vertical drain 11 comprises an elongated net body made of a synthetic resin material and a substantially equally elongated nonwoven fabric body which is folded in half along its longitudinal center line and longitudinally sandwiches the net body in a sheath-blade relationship.", "The horizontal drain 12 also comprises an elongated net body made of a synthetic resin material and a substantially equally elongated nonwoven fabric body which is folded in half along its center line and longitudinally sandwiches the net body in a sheath-blade relationship.", "The vertical drain 11 and the horizontal drain 12 may be of any elongated materials as long as they can provide both water and air passages without clogging even when bent or deformed with ground pressure.", "The vertical drains 11 are vertically installed at predetermined intervals, length and breadth, in the target soft soil A with their top portions bent and laid on the surface of the soft soil A.", "The horizontal drains 12 are laid on the surface over the target soft soil A in contact with the bent top portions of the vertical drains 11.Air and water together enter the vertical drain 11 and the horizontal drain 12 through the nonwoven fabric bodies and flow through the net bodies and the nonwoven fabric bodies.", "A plurality of water gathering pipes 13 are placed horizontally in fluid association with the horizontal drains 12.The water gathering pipes 13 have many through holes in the cylindrical walls to draw in air and water from the horizontal drains 12.The water gathering pipes 13 are connected to the vacuum tank means 14 which is connected to the vacuum pump means 15 installed outside the target soft soil A or in the soil B.", "Any type of vacuum pump means 15 may be used.", "Watertight type pump means 15 may be advantageously used.", "The vertical drains 11, the horizontal drains 12 and the water gathering pipes 13 are covered with airtight sheet means 10 to provide effectuate evacuation of the soft soil A.", "Any type of airtight sheet means 10 may be used.", "A synthetic film laminated fibrous sheet may be advantageously used.", "The drain system of FIG.", "1 includes water drain tank means 16 installed in connection with the water gathering pipes 13 via water/air separator means 17.The separator means 17 gravitationally separates water from air and lets only water drop into the drain tank means 16.The accumulated water in the drain tank means 16 is pumped out with water drain pump means 18 and drained out of the target soil A through connection pipes 19 and water drain pipes 20.Any type and size of drain tank means 16 may be used.", "Also, any type of drain pump means 18 may be used.", "Water meter means may be additionally provided in the drain tank means 16 to control the water levels.", "The water drain tank means 16 may alternatively be provided with automatic drain control means to control the drain pump means 18 so as to provide controlled drainage.", "The connection pipes 19 and the water drain pipes 20 should be installed below the water gathering pipes 13 to utilize the work of gravity.", "The sizes of the connection pipes 19 or the water drain pipes 20 may be decided according to requirements.", "In this embodiment, the connection pipes 19 and the water drain pipes 20 are provided with check valves 21 to prevent counter water flows.", "The drain system of this embodiment provides separate air and water expelling routes or passages.", "Water is accumulated in the water drain tank means 16 and expelled through the connection pipes 19 and the water drain pipes 20.Air and the water which has not entered the water drain tank means 16 are together sent into the vacuum tank means 14.The air and the water in the vacuum tank means 14 are expelled out of the vacuum tank means 14 with the vacuum pump means 15.FIG.", "2 shows a watertight type vacuum pump 30 used as part of the vacuum pump means 14 which includes a water circulation tank 30.The water tight vacuum pump 30 requires cooling water.", "According to the present invention, the underground water supplied into the vacuum pump means 14 is limited.", "As a compensation, the water circulation tank 30 supplies water to the vacuum pump means 14 through a circulation pipe 32.The circulation water may be cooled with cooling means 31.The water drain pipe 20 is connected to the vacuum tank means 14.If underground water supplied through the water drain pipe 20 is cool enough, the cooling means 31 is not required.", "FIG.", "3 shows an assistant system of the present invention, which sends air continuously or periodically into the soft soil A and/or the soil B to promote drainage operation after the soft soil A has been sufficiently vacuumed and drained to a considerable extent.", "This system includes a plurality of vertical drain pipes 40 which are connected with water gathering pipes 43, air blower means 41 and air compression control means 42.In an embodiment, this system blows air into the soft soil A in a controlled manner so that the pressure in the soft soil A does not exceed a desired pressure level, e.g.", "0.4 atm.", "It was found that the air blown into the target soft soil A helps press down the underground water level and promotes plasticity and unsaturation of the soft soil A, effectively improving water drainage from the soft soil A. Alternatively, the vertical pipes 40 may be used only for air extraction from the soft soil A.", "FIG.", "4 introduces another assistant system of the present invention.", "In an embodiment using the system shown in FIG.", "4, a plurality of vertical drain pipes 41 are vertically installed in the peripheral soil B (preferably within a peripheral belt zone of several meters wide around the soft soil A) at 0.3 m-1 m intervals in fluid association with water gathering pipes 43.The underground water in the peripheral soil B is drawn into the vertical drain pipes 41 together with air, and the soil B gradually dries, which helps in hardening the surface soil layers of the soil B, promoting separation of the peripheral soil B from the soil A, which by turn expedites sinking of the soft soil A independently from the soil B.", "FIG.", "5 introduces another drain system of the present invention.", "The system shown in FIG.", "5 includes first water drain tank means 54 which is connected with vertical drains 51 and horizontal drains 52 via water gathering pipes 53 (see also FIG.", "6).", "The water gathering pipes 53 are laid above the first drain tank means 54 and send the water received from the horizontal drains 52 to the first drain tank means 54.The vertical drains 51, the horizontal drains 52 and the water gathering pipes 53 are covered with airtight sheet means 50.This system functions similarly with the system introduced with reference to FIG.", "1.This system, however, additionally includes second water drain tank means 55 (see FIG.", "7) to expel water out of the soft soil A, which is connected with the first tank means 54 via pipe means 56.The underground water drained into the first drain tank 54 is sent to the second drain tank 55 and expelled therefrom through a drain pipe 58 with a drain pump 57 provided within the second drain tank 55 as shown in FIGS.", "6 and 7.The drain pipe 58 is provided with a check valve 59 to prevent counter water flow.", "The air drawn into the second drain tank 55 together with water from the water gathering pipe 13 is expelled through an exhaust pipe 60 which is connected to the vacuum tank 14.FIG.", "8 shows a contact portion between the vertical drain 71 and the horizontal drain 72.FIG.", "9 shows a water gathering pipe 73.FIG.", "10 shows a modification to the water gathering pipe 73 of FIG.", "9.The horizontal drains 72 and water gathering pipes 73 provide air passages and water passages separately.", "The horizontal drain 72 which is comprised of two elongated net bodies which are sandwiched by an elongated nonwoven fabric body sandwiches a bent top portion of the vertical drain 71 between the upper portion 72b and the lower portion 72a of the horizontal drain 72 as shown in FIG.", "8.The upper portion 72b provides the air passage and the lower portion 72a provides the water passage as the “heavier” underground water coming from the vertical drain 71 runs through the lower portion 72a and the “lighter” air coming from the vertical drain 71 runs through the upper portion 72b.", "The water gathering pipe 73 also provides an air passage and a water passage separately.", "The water gathering pipe 73 shown in FIG.", "9 is provided with many through holes in the cylindrical wall.", "An elongated inner partition 73a having a slit or slits axially divides the inside of the water gathering pipe 73 to provide the air passage above and the water passage below.", "In the modified water gathering pipe 73 shown in FIG.", "10, the slit in the partition 73a of the water gathering pipe 73 is provided with check valve means 73b to prevent the water running in the water passage below from entering the air passage above.", "The horizontal drain 72 and the water gathering pipe 73 of the present invention each having a water passage and a air passage separately provide efficient and quick drainage of the target soft soil A, an improvement over conventional systems.", "In the following, a drainage method of the present invention is described using the drawings that accompany this specification.", "First, a method using the drain system of FIG.", "1 is described.", "A plurality of vertical drains 11 are driven into a target soft soil A with mandrel means (not shown) at appropriate intervals, length and breadth.", "A preferred interval is about 1 m. The mandrel means are removed from the soft soil A after the vertical drains 11 have been installed in the soft soil A.", "The vertical drains 11 provide water drainage and air suction routes.", "Next, a plurality of horizontal drains 12 are laid on the ground surface of the target soft soil A in contact with the upper end portions 11a of the vertical drains 11.A plurality of water gathering pipes 13 are then laid in contact with the horizontal drains 12.The water gathering pipes 13 are provided with through holes in their cylindrical walls to suck up water and air therethrough.", "The water gathering pipes 13 are connected to vacuum tank means 14 installed outside the target soil A, which is connected to vacuum pump means 15 installed outside the target soft soil A.", "The vacuum pump means 15 evacuates the vacuum tank means 14, the water gathering pipes 13, the horizontal drains 12 and the vertical drains 11 in this order to below 0.4 atm or so.", "After having installed the vertical drains 11, the horizontal drains 12 and the water gathering pipes 13, airtight sheet means 10 is spread over the vertical drains 11, the horizontal drains 12 and the water gathering pipes 13 to promote vacuuming of the soft soil A.", "Underground water and air are attracted into the vertical drains 11.The drawn water and air go up the vertical drains 11 and run into the horizontal drains 12 and then into the water gathering pipes 13.As shown in FIG.", "1, water drain tank means 16 is provided below the water gathering pipes 13.The water in the water gathering pipes 13 is drawn into the drain tank means 16 through separator means 17 which separates air from water.", "The soft soil A is gradually compacted and sinks, widening the vertical distance between the vacuum pump means 14 and the water drain tank means 16.When the distance reaches about 10 meters, the pump means 14 loses its pumping function.", "In order to pump up underground water continuously to the ground surface, the drain tank means 16 is internally provided with drain pump means 18.The drain pump means 18 pumps the water in the drain tank means 16 out of the soft soil A through connection pipes 19 and drain pipes 20.The drain pump means 18 is thus capable of further hardening the soft soil A when continuously used after the vacuum pump means 14 cannot provide vacuuming effect any longer.", "It is to be noted that the drain tank means 16 may not require assistance of the drain pump means 14 depending on the target soil condition.", "In the method using the devices shown in FIGS.", "5-7, vertical drains 51, horizontal drains 52, water gathering pipes 53, first water drain tank means 54 and second water drain tank means 55 are used.", "The first water drain tank means 54 and the second water drain tank means 55 are connected by connection pipes 56.The water gathered in the water gathering pipes 13 is drained into the first water drain tank means 54, then into the second water drain tank means 55.The water gathered in the second water drain tank means 55 is pumped with drain pump means 57 out of the target soil A.", "In the method using the devices shown in FIGS.", "8 to 10, both the horizontal drains 72 and the water gathering pipes 73 have separate air passages and water passages, capable of efficiently draining underground water.", "Vacuuming processes may be controlled in accordance with requirements and given conditions of target soil A.", "INDUSTRIAL UTILIZATION OF THE INVENTION The present invention is capable of efficiently draining and hardening soft soil by using a drain system having separated water routes and air routes.", "The efficiency is improved by use of water drain tank means having water drain pump means, which provides continuous drainage and hardening of soft soil after vacuum pump means can no longer provides its expected function." ] ]	Patent_10451309
[ [ "Method and apparatus for background segmentation based on motion localization", "A system (1000) and method of detecting static background on a video sequence of images with moving foreground objects is described.", "The method includes localizing moving objects in each frame and training a background model using the rest of the image.", "The system is also capable of handling occasional background changes and camera movements." ], [ "1.A method of extracting a background image, comprising: localizing a moving object in a video sequence based on a change in the moving object over a plurality of frames of the video sequence, the moving object occupying frame areas of changing color; and training a background model for the plurality of frames outside of the frame areas of changing color.", "2.The method of claim 1, wherein localizing comprises localizing the moving object using a change detection mask.", "3.The method of claim 1, wherein localizing comprises: determining a boundary for the moving object that is of homogenous color; and constructing a hull around the moving object using the boundary.", "4.The method of claim 3, wherein determining a boundary comprises: determining a maximum contour of a plurality of contours of the moving object, the maximum contour having the largest area of the plurality of contours; determining other contours of the moving object; and joining the maximum contour with the other contours.", "5.The method of claim 4, further comprising: eliminating the smallest contour from joining with the maximum contour.", "6.The method of claim 4, wherein joining comprises joining one of the other contours with the maximum contour if the distance between the maximum contour and the one of the other contours is less than a predetermined distance.", "7.The method of claim 6, wherein the frames comprise a plurality of pixels and wherein the predetermined distance is based on a probability that a pixel of the plurality of pixels is considered moving given that it corresponds to the moving object.", "8.The method of claim 7, wherein the predetermined distance is based on a probability that the pixel is considered moving given that it is static.", "9.The method of claim 3, wherein the frames comprise a plurality of pixels and wherein the hull is constructed to contain only pixels of changing colors over consecutive frames.", "10.The method of claim 3, wherein constructing the hull comprises: determining all connected components in the boundary, wherein each of the components has a contour having an area; filtering out a smallest area contour; selecting a maximum area contour; and joining the maximum area contour with other contours of the connected components.", "11.The method of claim 1, wherein the frames comprise a plurality of pixels and wherein training comprises characterizing a pixel color at a given time with a value based on a state, each pixel corresponding to a state of a plurality of states.", "12.The method of claim 11, wherein the plurality of states includes an untrained background state.", "13.The method of claim 11, wherein the plurality of states includes a trained background state.", "14.The method of claim 11, wherein the plurality of states includes a foreground state.", "15.The method of claim 11, wherein the plurality of states includes an unknown background state.", "16.The method of claim 11, wherein training comprises: training the background model for the pixel in a foreground; and changing the state of the pixel to an untrained background if the pixel represents a static behavior for a certain period of time.", "17.The method of claim 16, further comprising changing the state to a trained background after a predetermined number of two frames.", "18.The method of claim 1, wherein the video sequence is recorded with a video camera and wherein the method further comprises: detecting a motion of the video camera; and compensating for the motion of the video camera.", "19.The method of claim 18, detecting the motion comprises: selecting a frame feature; and tracking the frame features over the plurality of frames.", "20.The method of claim 19, wherein compensating comprises resetting the background model when the motion has stopped.", "21.A machine readable medium having stored thereon instructions/which when executed by a processor, cause the processor to perform the following: localizing a moving object in a video sequence based on a change in the moving object over a plurality of frames of the video sequence/the moving object occupying frame areas of changing color; and training a background model for the plurality of frames outside of the frame areas of changing color.", "22.The machine readable medium of claim 21, wherein localizing comprises localizing the moving object using a change detection mask.", "23.The machine readable medium of claim 21, wherein localizing comprises: determining a boundary for the moving object that is of homogenous color; and constructing a hull around the moving object using the boundary.", "24.The machine readable medium of claim 23, wherein determining a boundary comprises: determining a maximum contour of a plurality of contours of the moving object, the maximum contour having the largest area of the plurality of contours; determining other contours of the moving object; and joining the maximum contour with the other contours.", "25.The machine readable medium of claim 24, wherein the processor further performs: determining a smallest contour of the plurality of contours; and eliminating the smallest contour from joining with the maximum contour.", "26.The machine readable medium of claim 24, wherein joining comprises joining one of the other contours with the maximum contour if the distance between the maximum contour and the one of the other contours is less than a predetermined distance.", "27.The machine readable medium of claim 23, wherein the processor performing constructing the hull comprises the processor performing: determining all connected components in the boundary, wherein each of the components has a contour having an area; filtering out a smallest area contour; selecting a maximum area contour; and joining the maximum area contour with other contours of the connected components.", "28.The machine readable medium of claim 21, wherein the frames comprise a plurality of pixels and wherein the processor performing training, comprises the processor performing characterizing a pixel color at a given time with a value based on a state, each pixel corresponding to a state of a plurality of states.", "29.The machine readable medium of claim 28, wherein the processor performing training comprises the processor performing: training the background model for the pixel in a foreground; and changing the state of the pixel to an untrained background if the pixel represents a static behavior for a certain period of time.", "30.The machine readable medium of claim 21, wherein the video sequence is recorded with a video camera and wherein the processor further performs: detecting a motion of the video camera; and compensating for the motion of the video camera.", "31.The machine readable medium of claim 30, wherein the processor performing detecting the motion comprises the processor performing the following: selecting a frame feature; and tracking the frame features over the plurality of frames.", "32.The machine readable medium of claim 30, wherein the processor performing compensating comprises the processor performing the following: resetting the background model when the motion has stopped.", "33.An apparatus for extracting a background image, comprising: means for localizing a moving object in a video sequence based on a change in the moving object over a plurality of frames of the video sequence, the moving object occupying frame areas of changing color; and means for training a background model for the plurality of frames outside of the frame areas of changing color.", "34.The apparatus of claim 33, wherein the means for localizing comprises: means for determining a boundary for the moving object that is of homogenous color; and means for constructing a hull around the moving object using the boundary.", "35.The apparatus of claim 33, wherein the video sequence is recorded with a video camera and wherein the apparatus further comprises: means for detecting a motion of the video camera; and means for compensating for the motion of the video camera.", "36.An apparatus for extracting a background image, comprising: a processor to execute one or more routines to localize a moving object in a video sequence based on a change in the moving object over a plurality of frames of the video sequence, the moving object occupying frame areas of changing color, and to train a background model for the plurality of frames outside of the frame areas of changing color; and a storage device coupled with the processor, the storage device having stored therein the one or more routines to localize the moving object and train the background model.", "37.The apparatus of claim 36, wherein the processor executes one or more routines to localize the moving object using a change detection mask.", "38.The apparatus of claim 36, wherein the processor executes one or more routines to determine a boundary for the moving object that is of homogenous color and to construct a hull around the moving object using the boundary.", "39.The apparatus of claim 36, further comprising a display coupled with the processor to display the plurality of frames of the video sequence.", "40.The apparatus of claim 36, further comprising a camera coupled with the processor to record the plurality of frames of the video sequence.", "41.The apparatus of claim 40, wherein the processor executes one or more routines to detect a motion of the video camera to compensate for the motion of the video camera." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Video conferencing and automatic video surveillance has been growing area driven by the increasing availability of lower priced systems and improvements in motion detection technology.", "Video display technology provides for the display of sequences of images through a display image rendering device such as a computer display.", "The sequence of images is time varying such that it can adequately represent motion in a scene.", "A frame is a single image in the sequence of images that is sent to the monitor.", "Each frame is composed of picture elements (pels or pixels) that are the basic unit of programming color in an image or frame.", "A pixel is the smallest area of a monitor's screen that can be turned on or off to help create the image with the physical size of a pixel depending on the resolution of the computer display.", "Pixels may be formed into rows and columns of a computer display in order to render a frame.", "If the frame contains a color image, each pixel may be turned on with a particular color in order to render the image.", "The specific color that a pixel describes is some blend of components of the color spectrum such as red, green, and blue.", "Video sequences may contain both stationary objects and moving objects.", "Stationary objects are those that remain stationary from one frame to another.", "As such, the pixels used to render a stationary object's colors remain substantially the same over consecutive frames.", "Frame regions containing objects with stationary color are referred to as background.", "Moving objects are those that change position in a frame with respect to a previous position within an earlier frame in the image sequence.", "If an object changes its position in a subsequent frame with respect to its position in a preceding frame, the pixels used to render the object's image will also change color over the consecutive frames.", "Such frame regions are referred to as foreground.", "Applications such as video display technology often rely on the detection of motion of objects in video sequences.", "In many systems, such detection of motion relies on the technique of background subtraction.", "Background subtraction is a simple and powerful method of identifying objects and events of interest in a video sequence.", "An essential stage of background subtraction is training a background model to learn the particular environment.", "Most often this implies acquiring a set of images of a background for subsequent comparison with test images where foreground objects might be present.", "However this approach experiences problems in applications where the background is not available or changes rapidly.", "Some prior art methods that deal with these problems are often referred to as background segmentation.", "The approaches to the task of background segmentation can be roughly classified into two stages: motion segmentation and background training.", "Motion segmentation is used to find regions in each frame of an image sequence that correspond to moving objects.", "Motion segmentation starts from a motion field obtained from optical flow calculated on two consecutive frames.", "The motion field is divided into two clusters using k-means.", "The largest group is considered a background.", "Background training trains background models on the rest of the image.", "Model-based background extraction extracts background from “museum-like” color images based on assumptions about image properties.", "This includes small numbers of objects on a background that is relatively smooth with spatial color variations and slight textures.", "The problem with these prior background segmentation solutions is that they propose pixel-based approaches to motion segmentation.", "A pixel-based approach analyses each pixel to make a decision whether it belongs to background or not.", "Hence, the time T of processing each pixel (T) is the sum of motion detection time (T 1 ) and background training time (T 2 ).", "If a frame consists of N pixels then the time of processing a single frame is TN. Such an approach may be robust but it is very time-consuming." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>The present invention is illustrated by way of example and not intended to be limited by the figures of the accompanying drawings.", "FIG.", "1 illustrates one embodiment of a method for extracting a background image from a video sequence.", "FIG.", "2A illustrates an exemplary frame from a video sequence.", "FIG.", "2B illustrates another exemplary frame from the video sequence subsequent to the frame of FIG.", "2A .", "FIG.", "2C illustrates an exemplary embodiment of a change detection image.", "FIG.", "2D illustrates an exemplary embodiment of the border contours of the change detection image of FIG.", "2C .", "FIG.", "2E illustrates an exemplary embodiment of hull construction.", "FIG.", "3 illustrates one embodiment of an iterative construction of a hull.", "FIG.", "4 illustrates one embodiment of a background training scheme.", "FIG.", "5 illustrates an exemplary embodiment of the relative dispersion of running averages depending on a.", "FIG.", "6 illustrates exemplary features to track on an exemplary frame background.", "FIG.", "7 illustrates one embodiment of camera motion detection and compensation.", "FIG.", "8 is an exemplary illustration of the percent of moving pixels segmented by a motion localization algorithm.", "FIG.", "9 is an exemplary illustration of the percent of background pixels segmented as foreground obtained with a motion localization algorithm.", "FIG.", "10 illustrates one embodiment of a computer system with a camera.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION This invention relates to the field of motion detection and, in particular, to background segmentation based on motion localization.", "BACKGROUND OF THE INVENTION Video conferencing and automatic video surveillance has been growing area driven by the increasing availability of lower priced systems and improvements in motion detection technology.", "Video display technology provides for the display of sequences of images through a display image rendering device such as a computer display.", "The sequence of images is time varying such that it can adequately represent motion in a scene.", "A frame is a single image in the sequence of images that is sent to the monitor.", "Each frame is composed of picture elements (pels or pixels) that are the basic unit of programming color in an image or frame.", "A pixel is the smallest area of a monitor's screen that can be turned on or off to help create the image with the physical size of a pixel depending on the resolution of the computer display.", "Pixels may be formed into rows and columns of a computer display in order to render a frame.", "If the frame contains a color image, each pixel may be turned on with a particular color in order to render the image.", "The specific color that a pixel describes is some blend of components of the color spectrum such as red, green, and blue.", "Video sequences may contain both stationary objects and moving objects.", "Stationary objects are those that remain stationary from one frame to another.", "As such, the pixels used to render a stationary object's colors remain substantially the same over consecutive frames.", "Frame regions containing objects with stationary color are referred to as background.", "Moving objects are those that change position in a frame with respect to a previous position within an earlier frame in the image sequence.", "If an object changes its position in a subsequent frame with respect to its position in a preceding frame, the pixels used to render the object's image will also change color over the consecutive frames.", "Such frame regions are referred to as foreground.", "Applications such as video display technology often rely on the detection of motion of objects in video sequences.", "In many systems, such detection of motion relies on the technique of background subtraction.", "Background subtraction is a simple and powerful method of identifying objects and events of interest in a video sequence.", "An essential stage of background subtraction is training a background model to learn the particular environment.", "Most often this implies acquiring a set of images of a background for subsequent comparison with test images where foreground objects might be present.", "However this approach experiences problems in applications where the background is not available or changes rapidly.", "Some prior art methods that deal with these problems are often referred to as background segmentation.", "The approaches to the task of background segmentation can be roughly classified into two stages: motion segmentation and background training.", "Motion segmentation is used to find regions in each frame of an image sequence that correspond to moving objects.", "Motion segmentation starts from a motion field obtained from optical flow calculated on two consecutive frames.", "The motion field is divided into two clusters using k-means.", "The largest group is considered a background.", "Background training trains background models on the rest of the image.", "Model-based background extraction extracts background from “museum-like” color images based on assumptions about image properties.", "This includes small numbers of objects on a background that is relatively smooth with spatial color variations and slight textures.", "The problem with these prior background segmentation solutions is that they propose pixel-based approaches to motion segmentation.", "A pixel-based approach analyses each pixel to make a decision whether it belongs to background or not.", "Hence, the time T of processing each pixel (T) is the sum of motion detection time (T1) and background training time (T2).", "If a frame consists of N pixels then the time of processing a single frame is TN. Such an approach may be robust but it is very time-consuming.", "BRIEF DESCRIPTION OF THE DRAWINGS The present invention is illustrated by way of example and not intended to be limited by the figures of the accompanying drawings.", "FIG.", "1 illustrates one embodiment of a method for extracting a background image from a video sequence.", "FIG.", "2A illustrates an exemplary frame from a video sequence.", "FIG.", "2B illustrates another exemplary frame from the video sequence subsequent to the frame of FIG.", "2A.", "FIG.", "2C illustrates an exemplary embodiment of a change detection image.", "FIG.", "2D illustrates an exemplary embodiment of the border contours of the change detection image of FIG.", "2C.", "FIG.", "2E illustrates an exemplary embodiment of hull construction.", "FIG.", "3 illustrates one embodiment of an iterative construction of a hull.", "FIG.", "4 illustrates one embodiment of a background training scheme.", "FIG.", "5 illustrates an exemplary embodiment of the relative dispersion of running averages depending on a.", "FIG.", "6 illustrates exemplary features to track on an exemplary frame background.", "FIG.", "7 illustrates one embodiment of camera motion detection and compensation.", "FIG.", "8 is an exemplary illustration of the percent of moving pixels segmented by a motion localization algorithm.", "FIG.", "9 is an exemplary illustration of the percent of background pixels segmented as foreground obtained with a motion localization algorithm.", "FIG.", "10 illustrates one embodiment of a computer system with a camera.", "DETAILED DESCRIPTION In the following description, numerous specific details are set forth such as examples of specific systems, techniques, components, etc.", "in order to provide a thorough understanding of the present invention.", "It will be apparent, however, to one skilled in the art that these specific details need not be employed to practice the present invention.", "In other instances, well known components or methods have not been described in detail in order to avoid unnecessarily obscuring the present invention.", "The present invention includes various steps, which will be described below.", "The steps of the present invention may be performed by hardware components or may be embodied in machine-executable instructions, which may be used to cause a general-purpose or special-purpose processor programmed with the instructions to perform the steps.", "Alternatively, the steps may be performed by a combination of hardware and software.", "The present invention may be provided as a computer program product, or software, that may include a machine-readable medium having stored thereon instructions, which may be used to program a computer system (or other electronic devices) to perform a process according to the present invention.", "A machine readable medium includes any mechanism for storing or transmitting information in a form (e.g., software, processing application) readable by a machine (e.g., a computer).", "The machine-readable medium may includes, but is not limited to, magnetic storage medium (e.g., floppy diskette); optical storage medium (e.g., CD-ROM); magneto-optical storage medium; read only memory (ROM); random access memory (RAM); erasable programmable memory (e.g., EPROM and EEPROM); flash memory; electrical, optical, acoustical or other form of propagated signal (e.g., carrier waves, infrared signals, digital signals, etc.", "); or other type of medium suitable for storing electronic instructions.", "The present invention may also be practiced in distributed computing environments where the machine readable medium is stored on and/or executed by more than one computer system.", "In addition, the information transferred between computer systems may either be pulled or pushed across the communication medium connecting the computer systems.", "Some portions of the description that follow are presented in terms of algorithms and symbolic representations of operations on data bits that may be stored within a memory and operated on by a processor.", "These algorithmic descriptions and representations are the means used by those skilled in the art to effectively convey their work.", "An algorithm is generally conceived to be a self-consistent sequence of acts leading to a desired result.", "The acts are those requiring manipulation of quantities.", "Usually, though not necessarily, these quantities take the form of electrical or magnetic signals capable of being stored, transferred, combined, compared, and otherwise manipulated.", "It has proven convenient at times, principally for reasons of common usage, to refer to these signals as bits, values, elements, symbols, characters, terms, numbers, parameters, or the like.", "A method and system for extracting a background image from a video sequence with foreground objects is described.", "Background regions in a frame that are not occluded by foreground objects during a video sequence may be captured by processing individual frames of the video sequence.", "FIG.", "1 illustrates one embodiment of a method for extracting a background image from a video sequence.", "In one embodiment, the method may include localization of moving objects in an image using a change detection mask, step 110, and training a background model of the remaining regions of the image, step 120.In localizing moving objects, step 110, the boundaries of moving objects that are of homogenous color for at least two consecutive frames are marked by constructing one or several hulls that enclose regions corresponding to the moving objects.", "The rest of the image is regarded as background and is used for training a background model in step 120.In one embodiment, the background may also be used to detect and compensate for camera motion, step 130.FIGS.", "2A and 2B shows two consecutive frames from the same video sequence.", "As an example of step 110 of FIG.", "1, suppose that the images in the video sequence represents only one moving object 205 (e.g., parts of a walking person) that is color homogenous.", "On frame 255, parts of the walking person 205 may have changed position relative to their position in frame 250.The difference of these two image frames 250 and 255 is the object, or parts thereof, that has moved and is shown as the change detection image 209 illustrated in FIG.", "2C.", "For example, the person's left foot 261 is almost invisible in the image 209 because the person is taking a step with the right leg 264 while keeping the left foot 262 substantially immovable on the floor.", "As such, the person's left foot 262 does not appear in change detection image 209.In contrast, the heel 263 of the person's right foot 264 has risen from frame 250 to frame 255 and, therefore, appears in change detection image 209.The application of a change detection mask 219 marks only the border contours 210, 211, and 212 of color homogenous moving regions 209, not the entire regions themselves, as illustrated in FIG.", "2D.", "For example: contour 210 corresponds to the border around the torso, arms, and outer legs of object 205; contour 211 corresponds to the border around the inner legs of moving object 205; and contour 212 corresponds to the head and neck of moving object 205.As a result, the change detection mask 219 contains a much fewer number of pixels than the entire number of pixels in a frame.", "The use of a change detection algorithm for a high resolution image with subsequent processing of the change detection mask for motion localization takes much less time than the application of a complicated raster technique like optical flow.", "All moving objects are localized by applying a fast connected components analysis to the change detection mask 219 that constructs a hull 239 around the contour of each moving region, as illustrated in FIG.", "2E.", "For example, hull 220 is constructed around contour 210, hull 221 is constructed around contour 211, and hull 222 is constructed around contour 212.Let It be the image at time t, mt ⊂ It—the set of pixels that correspond to actually moving objects and Mt ⊂ It—the set of pixels that belong to one of the hulls.", "Localization means that Mt should enclose mt.", "In practice, if a pixel p belongs to St=It−Mt then it corresponds to a static object with a high degree of confidence.", "In order to find moving objects, a change detection algorithm is applied to the video sequence frames (e.g., frames 250 and 255).", "In one embodiment, for example, a change detection algorithm as discussed in “Introductory Techniques for 3-D Computer Vision” by Emaluel Trucco and Alessandro Verri, Prentice Hall, 1998, may be used.", "Alternatively, other change detection algorithms may be used.", "Moreover, a change detection algorithm may be selected based on a particular application need.", "If for any n  X t ( n ) - X t - 1 ( n )  < β CD ( n ) then the pixel is considered moving, where βCD(n) is the maximum change in successive running average values such that the background model for the pixel is considered trained.", "The threshold βCD(n) is chosen as a multiplication of σ(n) calculated from a sequence of images of a static scene, where is a standard deviation of a Normal distribution of a pixel color in case of one or several color channels.", "In one embodiment, the change detection mask marks noise and illumination change regions in addition to boundaries of color homogenous moving regions.", "As previously mentioned, to localize the moving object, a hull of these regions is constructed so that it contains moving pixels and does not occupy static pixels as far as possible.", "The moving object is the accumulation of the change detection regions at the current time moment t. For the sake of simplicity, an assumption may be made that there is only one moving object.", "All connected components in the change detection mask and their contours are found.", "In one embodiment, in order to get rid of noise contours (e.g., contour 231 of FIG.", "2D), regions with small areas are filtered out.", "Then, the contour Cmax with the biggest area (which corresponds to the object or its boundary) is selected, for example, contour 220 of FIG.", "2D.", "An iterative construction of the hull H is started by jointing Cmax with other contour areas (e.g., contours 221 and 222).", "These other contour areas represent other moving regions of the moving object 205.FIG.", "3 illustrates one embodiment of an iterative construction of a hull.", "In step 310, for all contours Cb their convex hulls are constructed.", "A convex hull is the smallest convex polygon that contains one or several moving region components.", "A convex hull of a contour Ci is denoted by Hi and the convex hull of Cmax is denoted by Hmax.", "In step 320 the index k is found such that the euclidean distance between Hk and Hmax is the minimum one: k=arg min(dist(Hi, Hmax)) and dk=min dist (Hi, Hmax).", "In step 340, determine if a convex hull is within the minimum distance Dmax of the convex hull of Cmax (dk is less than a threshold Dmax).", "If so, then a convex hull Ĥmax is constructed around the set of hulls Hk and Hmax, step 350.If not, then repeat step 340 for the next contour, step 345.In step 360, denote Hmax=Ĥmax and, in step 370 determine all contours have been considered.", "Then, repeat from step 320 unless all Ci have already been considered.", "Otherwise go to step 380.In step 380, set the moving region equal to the latest maximum contour (Mt=Hmax).", "The above steps may be generalized for the case of several moving objects.", "The quality of the above algorithm can be estimated using two values.", "The first is the conditional probability that the pixel is considered moving given that it really corresponds to a moving object: P1=P(pεMt\|pεmt).", "The second is the conditional probability that the pixel is considered moving given that it is static: P2 =P(pεMt\|pεIt−mt).", "where It is the image at time t, mt is the set of pixels of It that corresponds to moving objects, and Mt is the set of pixels of It that have experience considerable change in color over the last one or few frames.", "P1 needs to be as big as possible while P2 should be small.", "If P1 is not big enough then a corrupt background may be trained while having P2 not sufficiently small will increase the training time.", "P1 and P2 should evidently grow with increase of Dmax This defines Dmax to be minimum value providing P1 higher than a certain level of confidence.", "The selection of Dmax is discussed below in relation to FIG.", "8.As previously discussed, the change detection mask marks only boundaries of homogenous moving regions.", "Moreover, it may not mark regions that move sufficiently slow.", "Hence, some slowly moving objects may constantly go to background and some moving objects may occasionally be considered to belong to background.", "One solution to the first problem is to perform change detection several times with different reference frames, for example, one frame before the current frame, two frames before the current frame, etc.", "One solution to the second problem is to perform background training taking into account that some background frames might be corrupted.", "At this point two characteristics of the motion localization algorithm are of interest: the probability P(m) that a moving pixel is misclassified m times in a row and the index m* such that P(m) is below a level of confidence, m may be used as a parameter for the background training algorithm.", "Referring again to FIG.", "1, when all the moving regions in a current frame are localized, step 110, a background model with given static pixels of the current frame is trained, step 120.A pixel color may be characterized at a give time moment with three values {X(n)}n=1 .", ".", ".", "3, which in case of a static pixel may be reasonably modeled by Normal distributions N (μ(n), σ(n)) with unknown means μ(n) and standard deviations σ(n).", "The training is multistage in order to remove out-liers produced by mis-prediction during step 110.Occasional background changes may be handled in a similar manner.", "If a foreground pixel represents a Normal distribution with small deviation for a long time, it is considered to be a change in the background and the background model is immediately updated.", "The background subtraction, for example, as discussed in “Non-Parametric Model for Background Subtraction,” Ahmed Elgammal, David Harwood, Larry Davis, Proc.", "ECCV, Vol.", "2, pp.", "751-767, 2000, may be used to segment background on every image.", "In an alternative embodiment, other background subtraction techniques may be used.", "During training process, a calculation of the values of μ(n) is performed using a running average update: μti(n)=(1−α)μti-1(n)+αXti(n), (1) where ti mark the frames where the pixel was classified as static.", "When the sequence converges, that is the difference between μti and μti-1 is swall:  μ t i ( n ) - μ t i - 1 ( n )  < β ( n ) , ( 2 ) the background model is considered trained in this pixel and μ(n)=μti(n).", "Therefore each pixel can correspond to one of four states, as illustrated in FIG.", "4: unknown background state 410 (that corresponds to pixels that have never been in St), untrained background state 420 (when statistics are being collected and inequality (2) is not satisfied), trained background state 430 (inequality (2) is satisfied), and foreground state 440 (when the background is trained and foreground is detected on the current image with background subtraction).", "The possible transitions are shown in FIG.", "4.Transition A 471 takes place when pixel appears in St for the first time.", "Transition B 472 occurs when the pixel's model is considered to be sufficiently trained.", "Transition C 473 occurs when the foreground is static for a long time period.", "For the sake of simplicity, a pixel at the given time moment t may be characterized with only one value Xt.", "Equation (1) and inequality (2) contain unknown parameters a and β which define the training process.", "The appropriate choice of these parameters gives a fast and at the same time statistically optimal background training.", "Assuming that X1=I+Δt where I is a constant color value of a background pixel and Δ is a zero-mean Gaussian noise in the color of a pixel at time t with standard deviation σΔ, then for δt=μt−I we will have the following equation δt=(1−α)δti-1+αΔti, where δt is the difference of the running average and constant background color.", "δt will be normally distributed with mean <δt> and deviation σt <δti>=(1−α)iδto, where a is the running average constant α t l 2 = δ Δ 2 ⁡ ( α 2 - α ⁢ ( 1 - ( 1 - α ) 2 ⁢ i ) + ( 1 - α ) 2 ⁢ i ) ( 3 ) In order to have a robust background, the background should be trained long enough to make sure that it is not trained by a moving object.", "In other words, if the pixel value changes significantly, the training should endure for at least m* frames.", "Hence, the following inequality should be fulfilled: β≦α(1−α)m-1δto, (4) where δto is equal to σ Δ and m is the minimum number of successive frames such that the probability P(m) is below the level of confidence; in other words, an assumption may be made that no pixel is misclassified through all m successive frames.", "In one embodiment, there may be no reason to make β smaller than the value defined in inequality 4 since it will dramatically increase the background training time.", "At the same time, the standard deviation of δm* should be as small as possible.", "It can be proved that ζ=σti2/σΔ2 as a function of αε[0,1] has one minimum a=ai where α i = arg ⁢ ⁢ min α ∈ [ 0 , 1 ] ⁢ { ( α 2 - α ⁢ ( 1 - ( 1 - α ) 2 ⁢ i ) + ( 1 - α ) 2 ⁢ i ) ) } ( 5 ) Examples of ζ (α) for different frame numbers are shown in FIG.", "5.FIG.", "5 illustrates an exemplary embodiment of the relative dispersion of the running average depending on α.", "In one embodiment, solid line 510 corresponds to a 5th frame, dashed line 520 corresponds to a 10th frame, and dash-dotted line 530 corresponds to a 20th frame.", "Choosing either too low or too high value of a would result in a big statistical uncertainty of δ and of running average μ. α=αm*.", "may be chosen so that with a static background pixel, the running average μtm accepted as a background pixel value would have a minimum possible standard deviation.", "Given m, inequality 4 and equation 5 define the optimal values of β and α.", "In one embodiment, background changes may be considered in training the background model.", "Suppose that the camera is not moving but the background has changed significantly, though remaining static afterwards.", "For example, one of static objects has been moved to a different position.", "The system marks the previous and current places of the object as foreground.", "Such pixels are not usual foreground pixels but, rather, they are static foreground.", "This property enables the tracking of such background changes and the adaptation of the background model.", "The model is trained for each pixel in the foreground and, if it represents a static behavior for a long period of time, its state is changed to an untrained background.", "After a predetermined number of frames (e.g., three frames) it will become a trained background.", "Referring again to FIG.", "1, in one embodiment, the background may also be used to detect and compensate for camera motion, step 130.The methods described herein may be generalized to the case of a moving camera by incorporation of fast global motion detection.", "When part of the image becomes a trained background state 430 of FIG.", "4, background subtraction 450 may be applied to every frame and a global motion estimation algorithm run on the found background mask.", "FIG.", "7 illustrates one embodiment of camera motion detection and compensation.", "In one embodiment, frame features are selected to track on a background, step 710, for example, corners 681-693 as illustrated in FIG.", "6.Optical flow may be used to track a few strong features in background to determine the camera motion, step 720.In one embodiment, feature selection techniques such as those discussed in “Good Features To Track,” Jianbo Shi, Carlo Tomasi, Proc.", "CVPR, pp.", "593-600, 1994, may be used to select features.", "In one embodiment, feature tracking techniques such as those discussed in “Introductory Techniques for 3-D Computer Vision” by Emaluel Trucco and Alessandro Verri, Prentice Hall, 1998, may be used to track features.", "Alternatively, other features and feature selection and tracking methods may be used.", "Once global motion is detected in the background indicating camera motion, step 730 then the background model is reset, step 740, by setting all pixels to unknown background state (e.g., state 410 of FIG.", "4).", "Feature tracking provides a good global motion estimation with points being tracked in a stable manner for a long time.", "If the background pixels are all lost, then the percent of moving pixels from change detection algorithm may be tracked.", "If a false end of motion is detected (a low change detection rate might take place during camera movement, for example, because of a homogenous background), the motion localization and training steps 110 and 120 of FIG.", "1 will filter out incorrect pixel values.", "When the camera stops moving, step 760, then the background model starts training again for each pixel value (step 120 of FIG.", "1).", "Some experimental results using the motion localization and background training methods are presented below.", "It should be noted that the experimental results are provided only to help describe the present invention and are not meant to limit the present invention.", "In one embodiment, the scheme discussed herein was implemented using Intel® Image Processing Library (IPL) and Intel® Open Source Computer Vision Library (OpenCV), with the system capable of processing 320×240 images for 15 milliseconds (ms).", "The testing was performed on a large number of video sequences taken with a raw USB video camera.", "The motion localization threshold, Dmax, may be selected, in one embodiment, according to FIG.", "8.FIG.", "8 illustrates exemplary results of testing the algorithm on a video sequence and comparing these results with foreground segmentation based on background subtraction.", "The value of P1 represents the percent of pixels from the foreground that were classified as moving pixels.", "In alternative embodiments, Dmax may be selected based on other empirical data or by other means, for examples, simulations, models, and assumptions.", "FIG.", "9 illustrates the percent of background pixels segmented as foreground obtained with the same methods.", "P1 and P2 discussed above may be varied by using the parameter Dmax.", "For Dmax=15, the number n(m) of foreground pixels that are mis-classified m times in a row are calculated.", "The results are presented in the following table: m 1 2 3 4 5 6 n 542 320 238 128 3 0 Taking m=5 gives α˜0.25 and β˜0.71 for inequality (4) and equation (5) presented above.", "FIG.", "10 illustrates one embodiment of a computer system (e.g., a client or a server) in the form of a digital processing system representing an exemplary server, workstation, personal computer, laptop computer, handheld computer, personal digital assistant (PDA), wireless phone, television set-top box, etc., in which features of the present invention may be implemented.", "Digital processing system 1000 may be used in applications such as video surveillance, video conferencing, robot vision, etc.", "Digital processing system 1000 includes one or more buses or other means for transferring data among components of digital processing system 1000.Digital processing system 1000 also includes processing means such as processor 1002 coupled with a system bus for processing information.", "Processor 1002 may represent one or more general purpose processors (e.g., a Motorola PowerPC processor and an Intel Pentium processor) or special purpose processor such as a digital signal processor (DSP)(e.g., a Texas Instrument DSP).", "Processor 1002 may be configured to execute the instructions for performing the operations and steps discussed herein.", "For example, processor 1002 may be configured to process algorithms to localize a moving object in frames of a video sequence.", "Digital processing system 1000 further includes system memory 1004 that may include a random access memory (RAM), or other dynamic storage device, coupled to memory controller 1065 for storing information and instructions to be executed by processor 1002.Memory controller 1065 controls operations between processor 1002 and memory devices such as memory 1004.Memory 1004 also may be used for storing temporary variables or other intermediate information during execution of instructions by processor 1002.Memory 1004 represents one or more memory devices, for example, memory 1004 may also include a read only memory (ROM) and/or other static storage device for storing static information and instructions for processor 1002.Digital processing system 1000 may also include an I/O controller 1070 to control operations between processor 1002 and one or more input/output (I/O) devices 1075, for examples, a keyboard and a mouse.", "I/O controller 1070 may also control operations between processor 1002 and peripheral devices, for example, a storage device 1007.Storage device 1007 represents one or more storage devices (e.g., a magnetic disk drive or optical disc drive) coupled to I/O controller 1070 for storing information and instructions.", "Storage device 1007 may be used to store instructions for performing the steps discussed herein.", "I/O controller 1070 may also be coupled to BIOS 1050 to boot digital processing system 1000.Digital processing system also includes a video camera 1071 for recording and/or playing video sequences.", "Camera 1071 may be coupled to I/O controller 1070 using, for example, a universal serial bus (USB) 1073.Alternatively, other types of buses may be used to connect camera 1071 to I/O controller 1070, for example, a fire wire bus.", "Display device 1021, such as a cathode ray tube (CRT) or Liquid Crystal Display (LCD), may also be coupled to I/O controller 1070 for displaying video sequences to a user.", "A communications device 1026 (e.g., a modem or a network interface card) may also be coupled to I/O controller 1070.For example, the communications device 1026 may be an Ethernet card, token ring card, or other types of interfaces for providing a communication link to a network for which digital processing system 1000 is establishing a connection.", "For example, communication device 1026 may be used to receive data relating to video sequences from another camera and/or computer system over a network.", "It should be noted that the architecture illustrated in FIG.", "10 is only exemplary.", "In alternative embodiments, other architectures may be used for digital processing system 1000.For examples, memory controller 1065 and the I/O controller 1070 may be integrated into a single component and/or the various components may be coupled together in other configurations (e.g., directly to one another) and with other types of buses.", "A novel and fast method of background extraction from a sequence of images with moving foreground objects has been presented.", "The method employs image and contour processing operations and is capable of robust extraction of background for a small number of frames.", "For example, the methods may operate for about 30 frames on a typical videoconferencing image sequence with a static background and a person in the foreground.", "This is a significant advantage in the context real-time video applications such as surveillance and robotic vision over prior art systems that rely on computationally expensive operations.", "The methods of the present invention may be applied to a wide range of problems that deal with stationary background and objects of interest in foreground.", "In addition, the versatility of the system allows for the selection of a change detection algorithm to a particular application need.", "Such methods may also be used in conjunction with video compression taking advantage of the knowledge of static regions in a sequence.", "In the foregoing specification, the invention has been described with reference to specific exemplary embodiments thereof.", "It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention as set forth in the appended claims.", "The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense." ] ]	Patent_10451517
[ [ "Gasket for a filtration element and module integrating a filtration element fitted with such a gasket", "The invention provides a sealing gasket for mounting in a passage (5) of a support plate (4) to surround the end of a tubular-shaped filter element (3) provided with at least one fluid flow channel (31), the channel lying within a flow section Sc, the gasket being constituted in the form of a sleeve: possessing a height h not less than the height hps of the passage (5) in the support plate (4); and presenting an overlap bore (15) for the filter element (3) defined between one end (16) of the sleeve and a shoulder (17) which co-operates with the other end of the sleeve to define a channel bore (19) for the fluid, the shoulder (17) possessing a surface for acting as an abutment for the terminal portion of the filter element and presenting dimensions that are adapted to extend outside the flow section Sc so as to avoid impeding fluid circulation, the overlap bore (15) being provided with a groove (28) adjacent to the shoulder (17) in order to allow the gasket to creep." ], [ "1.A sealing gasket for mounting in a passage (5) of a support plate (4) to surround the end of a tubular-shaped filter element (3) provided with at least one fluid flow channel (31), the channel lying within a flow section Sc, the gasket being characterized in that it is made in the form of a sleeve: Possessing a height h not less than the height hps of the passage (5) in the support plate (4); and Presenting an overlap bore (15) for the filter element (3) defined between one end (16) of the sleeve and a shoulder (17) which co-operates with the other end of the sleeve to define a channel bore (19) for the fluid, the shoulder (17) possessing a surface for acting as an abutment for the terminal portion of the filter element and presenting dimensions that are adapted to extend outside the flow section Sc so as to avoid impeding fluid circulation, the overlap bore (15) being provided with a groove (28) adjacent to the shoulder (17) in order to allow the gasket to creep.", "2.A sealing gasket according to claim 1, characterized in that the groove (28) presents a diameter dg and a height hg such that the ratio dg/dm lies in the range 1 to 1.5 and the ratio hg/hci lies in the range 0.2 to 1, with dm being the diameter of the filter element (3), and with hci being the height of the channel bore (19).", "3.A sealing gasket according to claim 1, characterized in that the overlap bore (15) possesses a height hp and a diameter dp such that the ratio dp/dm lies in the range 0.6 to 1, and the ratio hp/dm lies in the range 0.2 to 1.5, where dm is the diameter of the filter element (3).", "4.A sealing gasket according to claim 1, characterized in that the channel bore (19) possesses an inside diameter dci and an inside height hci lying in the range dci/2 to dci/24 and such that the ratio dci/dm lies in the range 0.77 to 0.9.5.A sealing gasket according to claim 1, characterized in that the sleeve possesses on the outside, starting from the end (18) into which the channel bore (19) opens out, a diameter dcc extending over a height hce, so as to form an outside collar (21), the ratio dce/dm lying in the range 1.1 to 2 and the ratio hce/hl lying in the range 1.5 to 10, where hl is the height of a gasket-receiving countersink (23) formed in the backplate (11) for fixing on the support plate (4).", "6.A sealing gasket according to claim 5, characterized in that the outside of the sleeve, starting from the collar (21), possesses a first tapering portion (25) and a second tapering portion (26) extending to its end (16) into which the overlap bore (15) opens out.", "7.A sealing gasket according to claim 6, characterized in that: The first tapering portion (25) possesses a maximum outside diameter dpc1ma, a minimum outside diameter dpc1mi and an outside height hpc1 lying in the range dm/5 to dm/20, and such that the ratio dpc1ma/dce lies in the range 0.77 to 1, and the ratio dpc1mi/dpc1ma lies in the range 0.83 to 1; and The second tapering portion (26) possesses a maximum outside diameter dpc2ma equal to the minimum outside diameter dpc1mi of the first tapering portion (25) and a minimum outside diameter dpc2mi and an outside height hpc2 such that the ratio dp/dpc2mi lies in the range 0.8 to 1, and the ratio hpc2/dm lies in the range 0.2 to 1.5.8.A multiple sealing gasket characterized in that it comprises a series of gaskets (6) in accordance with claim 1 interconnected by connection zones (30).", "9.A multiple sealing gasket according to claim 8, characterized in that the connection zones (30) are of a height hmp such that the ratio hmp/hce lies in the range 1 to 0.2.10.A filter module of the type comprising at least one filter element (3) supported at each of its terminal portions by a support plate (4) provided with a passage (5) and having a backplate (11) fixed thereon, which backplate is provided with a countersink (23) for each filter element (3), the module being characterized in that for each passage (5) of the support plates (4), it includes a gasket (6) in accordance with claim 1.11.A filter module of the type comprising at least one filter element (3) supported at each of its terminal portions by a support plate (4) provided with a passage (5) and having a backplate (11) fixed thereon, which backplate is provided with a countersink (23) for each filter element (3), the module being characterized in that it includes a gasket (6) in accordance with claim 1, fitted to each support plate (4).", "12.A filter module of the type comprising at least one filter element (3) supported at each of its terminal portions by a support plate (4) provided with a passage (5) and having a backplate (11) fixed thereon, which backplate is provided with a countersink (23) for each filter element (3), the module being characterized in that it includes a multiple gasket in accordance with claim 8 fitted to each support plate (4)." ], [ "The present invention relates to the technical field of molecular or particulate separation using filter or separation elements generally referred to as membranes, adapted to separate molecules or particles contained in a fluid medium for processing.", "The present invention relates more particularly to technical means adapted to provide sealing for such filter or separation elements.", "The subject matter of the invention finds a particularly advantageous application in the field of filtering, in the broad sense, a fluid medium for processing, and in particular nanofiltering, ultrafiltering, microfiltering, etc.", "In the state of the art, it is known to use a filter module constituted by a metal case fitted at each end with a support plate arranged to present one or more packages for allowing filter elements of tubular shape to be positioned relative to one another.", "The filter elements thus extend inside the case parallel to one another and they are mounted in leaktight manner at each end to a support plate.", "Each filter element has at least one flow channel for the fluid to be processed, the channel extending from one terminal portion to the other terminal portion of the element.", "Filter elements perform cross-flow filtering of the fluid in order to obtain, at the peripheral surface of the filter elements, an outlet filtrate that is designed to be recovered in a collection volume situated between the support plates and the case.", "In order to ensure sealing between the terminal portions of the filter elements and the support plates, sealing gaskets are positioned and clamped by means of a metal backplate fixed on each support plate.", "The sealing gaskets are made of deformable material such as an elastomer or a rubber so that on being tightened they provide sealing between the support plates and the filter elements.", "Unfortunately, it sometimes happens that filter elements are broken at their terminal portions positioned in the support plates.", "The Applicant has found that the main cause of filter elements breaking at the support plates comes from friction between the filter element and the support plate and/or the clamping back plate.", "On the basis of that observation, the Applicant has developed a novel sealing gasket for a filter element that is designed to avoid the elements becoming eroded by contact with the support plates and/or the clamping backplates, without disturbing fluid flow.", "The object of the invention is thus to propose a sealing gasket for mounting in a passage of a support plate to surround the end of a filter element of tubular shape that is provided with at least one flow channel for a fluid, said channel lying within a flow section.", "According to the invention, the sealing gasket is made in the form of a sleeve: possessing a height not less than the height of the passage in the support plate; and presenting an overlap bore for the filter element defined between one end of the sleeve and a shoulder which co-operates with the other end of the sleeve to define a channel bore for the fluid, the shoulder possessing a surface for acting as an abutment for the terminal portion of the filter element and presenting dimensions that are adapted to extend outside the flow section so as to avoid impeding fluid circulation, the overlap bore being provided with a groove adjacent to the shoulder in order to allow the gasket to creep.", "The sealing gasket of the invention makes it possible to avoid any contact between the filter elements and the metal portions constituted by the clamping backplates and by the support plates.", "By implementing an abutment for the filter elements, the filter elements are prevented from moving under the effect of the pressure difference that arises between their upstream and downstream terminal portions, such that a filter element can no longer move, and consequently can no longer come into contact with the metal backplate.", "Various other characteristics appear from the description given below with reference to the accompanying drawings which, as non-limiting examples, show embodiments and implementations of the invention.", "FIG.", "1 is an elevation section view of an embodiment of a filter module using a sealing gasket in accordance with the invention.", "FIG.", "2 is a transverse view looking substantially along lines II-II of FIG.", "1.FIG.", "3 shows a filter element fitted on each of its terminal portions with a sealing gasket of the invention.", "FIG.", "4 is a transverse view on a larger scale seen looking substantially along liens IV-IV of FIG.", "3 and showing a characteristic detail of the invention.", "FIG.", "5 is a view on a larger scale showing various characteristics of an embodiment of a sealing gasket of the invention.", "FIG.", "6 shows a multiple sealing gasket in accordance with the invention.", "As can be seen more clearly in FIGS.", "1 and 2, the subject matter of the invention is implemented for a device or module 1 performing cross-flow filtering for a fluid to be treated that can be of any kind.", "In a case or cylinder 2, the module 1 comprises one, and more generally a series of tubular-shaped filter elements 3 extending parallel to one another and represented in FIG.", "1 solely by their axes.", "As can be seen more clearly from FIGS.", "3 and 4, each filter element 3 possesses a right cross-section of outside shape that is hexagonal, for example, or circular as in the example shown.", "Each filter element 3 comprises at least one, and in the example shown, three channels 31 extending parallel to the longitudinal axis of the filter element 3 so as to open out in each of its terminal portions 3a and 3b.", "The surface of each channel 31 is covered in at least one separating layer (not shown) that is designed to come into contact with the fluid medium for processing that flows inside the channel.", "The nature of the or each separating layer is selected as a function of the separation or filtering power that is to be obtained.", "At each of their terminal portions 3a, 3b, the filter elements 3 are mounted on a support plate 4 which is fixed in leaktight manner to each end of the case 2.In conventional manner, each support plate 4 has a number of passages 5 equal to the number of filter elements 3 that are mounted inside the case 2.Each passage 5 is preferably of tapering shape with its smaller end opening to the inside of the enclosure defined by the case 2.Each passage 5 enables a terminal portion of a tubular filter element 3 to be positioned.", "Each passage 5 is designed to be fitted with a sealing gasket 6 in accordance with the invention so as to ensure that the filter elements 3 are mounted in leaktight manner on the support plates 4.Between themselves and the case 2, the support plates 4 define a collecting enclosure 8 which communicates through at least one outlet 9 for delivering the filtrate, i.e.", "the fluid medium that has passed through the filter elements 3.In conventional manner, each support plate 4 is designed to have mounted thereon by any appropriate means a clamping backplate 11 that serves to deform the sealing gasket 6 in order to obtain good sealing.", "As can be seen more precisely in FIGS.", "3 to 5, each sealing gasket 6 is in the form of a sleeve of axial height h that is not less than the height hps of the support plate 4.Thus, the sealing gasket 6 is capable of covering the filter element 3 over a length that is not less than the height hps of the support plate 4.Under such conditions, the filter element 3 cannot come into contact with the support plate 4 so the filter element 3 is not subjected to friction or to erosion that might lead to breakage thereof.", "According to another characteristic of the invention, the sealing gasket 6 has an overlap bore 15 for the filter element 3 which extends between one end 16 of the sleeve and a shoulder 17 which co-operates with the other end 18 of the sleeve to define a fluid-channeling bore 19.As can be seen more clearly in FIG.", "3, the shoulder 17 serves as an abutment for the general portion of the filter element 3.This shoulder 17 presents a surface of dimensions that are adapted to extend outside the free flow section Sc for the fluid.", "As can be seen more precisely in FIG.", "4, each filter element 3 comprises at least one, and in the example shown, three flow channels 31 lying within the free flow section Sc.", "In other words, this flow section Sc corresponds to the envelope containing all of the flow channels 31, such that a peripheral surface exists around said section extending to the periphery of the filter element.", "Thus, the shoulder 17 presents dimensions that are adapted to extend outside the flow section Sc of the filter element so as to avoid impeding the flow of fluid flowing out from the channels 31.According to a characteristic of the invention, the overlap bore 15 possesses a determined height hp and a determined diameter dp.", "Considering that the diameter of the filter element 3 is equal to dm, the ratio of the diameter dp of the overlap bore over the diameter dm of the filter element lies in the range 0.6 to 1, and the ratio between the height hp of the overlap bore 15 and the diameter dm of the filter element 3 lies in the range 0.2 to 1.5.According to another characteristic of the invention, the channel bore 19 has a determined inside diameter dci and a determined inside height hci.", "The inside height hci of the channel bore 19 lies between the inside diameter dci of the channel bore divided by 2 and the inside diameter dci of the channel bore divided by 24.Furthermore, the ratio of the inside diameter dci of the channel bore 19 divided by the diameter dm of the filter element 3 lies in the range 0.77 to 0.9.According to a preferred embodiment characteristic, each sealing gasket 6 possesses a determined outside diameter dce starting from the end 18 into which the channel bore 19 opens out, this determined diameter dce extending over a determined height hce so as to form an outside collar 21.Over its entire height, this collar 21 thus presents a diameter that is constant.", "The ratio of the diameter dce of the collar 21 over the diameter dm of the filter element lies in the range 1.1 to 2.According to a preferred embodiment characteristic, facing each passage 5, each backplate 11 has a countersink 23 formed in that one of the main faces of the backplate 11 that faces the adjacent support plate 4.Each countersink 23 is in communication with a through hole 24 formed in the backplate 11 and is adapted to receive a sealing gasket 6.More precisely, each countersink 23 is designed to receive the gasket 6 starting from its end 18 so as to receive at least part of the collar 21 of the sleeve.", "According to an embodiment characteristic, the height hce of the outer collar 21 is such that the ratio of said height hce over the height h1 of the countersink 23 lies in the range 1.5 to 10.According to another characteristic of the invention, each sealing gasket 6 possesses on its outside, starting from the collar 21, a first portion 25 of tapering shape and a second portion 26 of tapering shape extending to the end 16 into which the overlap bore 15 opens out.", "According to an embodiment characteristic, the first tapering portion 25 possesses a maximum outside diameter dpc1ma, a minimum outside diameter dpc1mi and an outside height hpc1.The outside height hpc1 of the first tapering portion 25 lies between the diameter dm of the filter element divided by 5 and the diameter dm of the filter element divided by 20.According to another preferred embodiment characteristic, the ratio between the maximum outside diameter dpc1ma of the first tapering portion 25 over the diameter dce of the collar lies in the range 0.77 to 1, while the ratio of the minimum outside diameter dpc1mi of the first tapering portion 25 over the maximum outside diameter dpc1ma of the first tapering portion lies in the range 0.83 to 1.According to another preferred embodiment characteristic, the second tapering portion 26 possesses a maximum outside diameter dpc2ma equal to the minimum outside diameter dpc1mi of the first tapering portion 25.This second tapering portion 26 also possesses a determined minimum outside diameter dpc2mi and a determined outside height hpc2.The ratio of the diameter dp of the overlap bore 15 over the minimum outside diameter dpc2mi of the second tapering portion 26 lies in the range 0.8 to 1, while the ratio of the diameter hpc2 of the second tapering portion 26 over the diameter dm of the filter element 3 lies in the range 0.2 to 1.5.According to another characteristic of the invention, the overlap bore 15 has a groove 28 adjacent to the shoulder 17 in order to allow the constituent material of the sealing gasket 6 to creep.", "Thus, when the backplate 11 is clamped on the support plate 4, a portion of the gasket material can creep into the inside of the groove 28 while not obstructing the flow section Sc of the filter element.", "According to a preferred embodiment characteristic, the groove 28 presents a determined diameter dg and a determined height hg such that the ratio of the groove diameter dg over the diameter dm of the filter element lies in the range 1 to 1.5, while the ratio of the groove height hg over the inside diameter hci of the channel bore 19 lies in the range 0.2 to 1.In the above example, each support plate 4 has a series of distinct individual gaskets 6 each mounted in a respective passage 5.According to another embodiment characteristic shown more particularly in FIG.", "6, a series of sealing gaskets 6 for mounting on a support plate 4 can be interconnected via connection zones 30 in such a manner as to constitute a single part.", "Each connection zone 30 possesses a determined height hmp such that the ratio hmp over the height hce of the collar 21 lies in the range 1 to 0.2.The invention is not limited to the examples described and shown, since various modifications can be applied thereto without going beyond the ambit of the invention." ] ]	Patent_10451532
[ [ "PACKAGED INTEGRATED CIRCUITS AND METHODS OF PRODUCING THEREOF", "A packaged integrated circuit and method for producing thereof, including an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, a package enclosing the integrated circuit substrate and defining first and second planar surfaces generally parallel to the substrate plane and a plurality of electrical contacts, each connected to the electrical circuitry at the substrate plane, at least some of the plurality of electrical contacts extending onto the first planar surface and at least some of the plurality of electrical contacts extending onto the second planar surface." ], [ "1.A packaged integrated circuit comprising: an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon; a package enclosing said integrated circuit substrate and defining first and second planar surfaces generally parallel to said substrate plane; and a plurality of electrical contacts, each connected to said electrical circuitry at said substrate plane, at least some of said plurality of electrical contacts extending onto said first planar surface and at least some of said plurality of electrical contacts extending onto said second planar surface.", "2.A packaged integrated circuit according to claim 1 and wherein said package is a chip-scale package.", "3.A packaged integrated circuit according to claim 1 and wherein said package includes at least one portion which is at least partially transparent to visible radiation.", "4.A packaged integrated circuit according to claim 1 and wherein said package includes at least one portion which is at least partially transparent to infra-red radiation.", "5.A packaged integrated circuit according to claim 2 and wherein said package includes at least one portion which is at least partially transparent to visible radiation.", "6.A packaged integrated circuit according to claim 2 and wherein said package includes at least one portion which is at least partially transparent to infra-red radiation.", "7.A packaged integrated circuit assembly comprising: a packaged integrated circuit including an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, a package enclosing said integrated circuit substrate and defining first and second planar surfaces generally parallel to said substrate plane and a plurality of electrical contacts, each connected to said electrical circuitry at least some of said plurality of electrical contacts extending onto said first planar surface and at least some of said plurality of electrical contacts extending onto said second planar surface; and at least one additional electrical circuit element mounted onto and supported by said second planar surface and electrically coupled to at least one of said plurality of electrical contacts extending therealong.", "8.A packaged integrated circuit assembly according to claim 7 and wherein said at least one additional electrical circuit element comprises an electrical component selected from the group consisting of: passive electrical elements, light generating elements, heat generating elements, light detecting elements, integrated circuits, hybrid circuits, environmental sensors, radiation sensors, micromechanical sensors, mechanical actuators and force sensors.", "9.A packaged integrated circuit according to claim 7 and wherein said package is a chip-scale package.", "10.A packaged integrated circuit according to claim 7 and wherein said package includes at least one portion which is at least partially transparent to visible radiation.", "11.A packaged integrated circuit according to claim 7 and wherein said package includes at least one portion which is at least partially transparent to infra-red radiation.", "12.A packaged integrated circuit according to claim 9 and wherein said package includes at least one portion which is at least partially transparent to visible radiation.", "13.A packaged integrated circuit according to claim 9 and wherein said package includes at least one portion which is at least partially transparent to infra-red radiation.", "14.A method for producing packaged integrated circuits comprising: producing, on a wafer scale, an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon; providing wafer scale packaging enclosing said integrated circuit substrate and defining first and second planar surfaces generally parallel to said substrate plane; forming on said wafer scale packaging a plurality of electrical contacts, each connected to said electrical circuitry at said substrate plane, at least some of said plurality of electrical contacts extending onto said first planar surface and at least some of said plurality of electrical contacts extending onto said second planar surface; and separating said integrated circuit substrate in said wafer scale packaging into a plurality of individual chip packages.", "15.A method for producing packaged integrated circuits according to claim 14 and wherein said plurality of individual chip packages are chip scale packages.", "16.A method according to claim 14 and wherein said package includes at least one portion which is at least partially transparent to visible radiation.", "17.A method according to claim 14 and wherein said package includes at least one portion which is at least partially transparent to infra-red radiation.", "18.A method according to claim 15 and wherein said package includes at least one portion which is at least partially transparent to visible radiation.", "19.A method according to claim 15 and wherein said package includes at least one portion which is at least partially transparent to infra-red radiation.", "20.A method for producing packaged integrated circuit assemblies, the method comprising: producing, on a wafer scale, an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon; providing wafer scale packaging enclosing said integrated circuit substrate and defining first and second planar surfaces generally parallel to said substrate plane; forming on said wafer scale packaging a plurality of electrical contacts, each connected to said electrical circuitry, at least some of said plurality of electrical contacts extending onto said first planar surface and at least some of said plurality of electrical contacts extending onto said second planar surface; separating said integrated circuit substrate in said wafer scale packaging into a plurality of individual chip packages; and mounting onto said at second planar surface of at least one of said plurality of individual chip packages, at least one additional electrical circuit element, said at least one additional electrical circuit element being supported by said second planar surface and electrically coupled to at least one of said plurality of electrical contacts extending therealong.", "21.A method of forming a packaged integrated circuit assembly according to claim 20 and wherein said at least one additional electrical circuit element comprises an electrical component selected from the group consisting of: passive electrical elements, light generating elements, heat generating elements, light detecting elements, integrated circuits, hybrid circuits, environmental sensors, radiation sensors, micromechanical sensors, mechanical actuators and force sensors.", "22.A method for producing packaged integrated circuits according to claim 20 and wherein said plurality of individual chip packages are chip scale packages.", "23.A packaged integrated circuit according to claim 20 and wherein said package includes at least one portion which is at least partially transparent to visible radiation.", "24.A packaged integrated circuit according to claim 20 and wherein said package includes at least one portion which is at least partially transparent to infra-red radiation.", "25.A packaged integrated circuit according to claim 22 and wherein said package includes at least one portion which is at least partially transparent to visible radiation.", "26.A packaged integrated circuit according to claim 22 and wherein said package includes at least one portion which is at least partially transparent to infra-red radiation.", "27.A method for producing packaged integrated circuit assemblies, the method comprising: producing, on a wafer scale, an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon; providing wafer scale packaging enclosing said integrated circuit substrate and defining first and second planar surfaces generally parallel to said substrate plane; forming on said wafer scale packaging a plurality of electrical contacts, each connected to said electrical circuitry, at least some of said plurality of electrical contacts extending onto said first planar surface and at least some of said plurality of electrical contacts extending onto said second planar surface; mounting onto said at second planar surface of said wafer scale packaging, at least one additional electrical circuit element, said at least one additional electrical circuit element being supported by said second planar surface and electrically coupled to at least one of said plurality of electrical contacts extending therealong; and separating said integrated circuit substrate in said wafer scale packaging into a plurality of individual chip packages.", "28.A method of forming a packaged integrated circuit assembly according to claim 27 and wherein said at least one additional electrical circuit element comprises an electrical component selected from the group consisting of: passive electrical elements, light generating elements, heat generating elements, light detecting elements, integrated circuits, hybrid circuits, environmental sensors, radiation sensors, micromechanical sensors, mechanical actuators and force sensors.", "29.A method for producing packaged integrated circuits according to claim 27 and wherein said plurality of individual chip packages are chip scale packages.", "30.A packaged integrated circuit according to claim 27 and wherein said package includes at least one portion which is at least partially transparent to visible radiation.", "31.A packaged integrated circuit according to claim 27 and wherein said package includes at least one portion which is at least partially transparent to infra-red radiation.", "32.A packaged integrated circuit according to claim 29 and wherein said package includes at least one portion which is at least partially transparent to visible radiation.", "33.A packaged integrated circuit according to claim 29 and wherein said package includes at least one portion which is at least partially transparent to infra-red radiation." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Various types of packaged integrated circuits are known in the prior art.", "The following patents and published patent applications of the present inventor and the references cited therein are believed to represent the state of the art: U.S. Pat.", "Nos.", "4,551,629; 4,764,846; 4,794,092; 4,862,249; 4,984,358; 5,104,820; 5,126,286; 5,266,833; 5,546,654; 5,567,657; 5,612,570; 5,657,206; 5,661,087; 5,675,180; 5,703,400; 5,837,566; 5,849,623; 5,857,858; 5,859,475; 5,869,353; 5,888,884; 5,891,761; 5,900,674; 5,938,45; 5,985,695; 6,002,163; 6,046,410; 6,080,596; 6,092,280; 6,098,278; 6,124,637; 6,134,118.EP 490739 A1; JP 63-166710 WO 85/02283; WO 89/04113; WO 95/19645 The disclosures in the following publications: “Three Dimensional Hybrid Wafer Scale Integration Using the GE High Density Interconnect Technology” by R. J. Wojnarowski, R. A. Filliion, B. Gorowitz and R. Sala of General Electric Company, Corporate Research & Development, P.O.", "Box 8, Schenectady, N.Y. 12301, USA, International Conference on Wafer Scale Integration, 1993.“M-DENSUS”, Dense-Pac Microsystems, Inc., Semiconductor International, December 1997, p. 50; “Introduction to Cubic Memory, Inc.” Cubic Memory Incorporated, 27 Janis Way, Scotts Valley, Calif. 95066, USA; “A Highly Integrated Memory Subsystem for the Smaller Wireless Devices” Intel(r) Stacked-CSP, Intel Corporation, January 2000; “Product Construction Analysis (Stack CSP)”, Sung-Fei Wang, ASE, R & D Group, Taiwan, 1999; “Four Semiconductor Manufacturers Agree to Unified Specifications for Stacked Chip Scale Packages”, Mitsubishi Semiconductors, Mitsubishi Electronics America, Inc., 1050 East Arques Avenue, Sunnyvale, Calif. 94086, USA; “Assembly & Packaging, John Baliga, Technology News, Semiconductor International, December 1999; “<6 mils Wafer Thickness Solution (DBG Technology)”, Sung-Fei Wang, ASE, R & D Group, Taiwan, 1999; “Memory Modules Increase Density”, DensePac Micro Systems, Garden Grove, Calif., USA, Electronics Packaging and Production, p. 24, Nov. 1994; “First Three-Chip Staked CSP Developed”, Semiconductor International, January 2000, p. 22; “High-Density Packaging: The Next Interconnect Challenge”, Semiconductor International, February 2000, pp.", "91-100; “3-D IC Packaging”, Semiconductor International, p. 20, May 1998; “High Density Pixel Detector Module Using Flip Chip and Thin Film Technology” J. Wolf, P. Gerlach, E. Beyne, M. Topper, L. Dietrich, K. H. Becks, N. Wermes, O. Ehrmann and H. Reichl, International System Packaging Symposium, January 1999, San Diego; “Copper Wafer Bonding”, A.", "Fan, A. Rahman and R. Rief, Electrochemical and Solid State Letters, 2(10), pp.", "534-536, 1999; “Front-End 3-D Packaging”, J. Baliga, Semiconductor International, December 1999, p 52, are also believed to represent the state of the art." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention seeks to provide improved packaged integrated circuits and methods for producing same.", "There is thus provided in accordance with a preferred embodiment of the present invention a packaged integrated circuit including an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, a package enclosing the integrated circuit substrate and defining first and second planar surfaces generally parallel to the substrate plane and a plurality of electrical contacts, each connected to the electrical circuitry at the substrate plane, at least some of the plurality of electrical contacts extending onto the first planar surface and at least some of the plurality of electrical contacts extending onto the second planar surface.", "Further in accordance with a preferred embodiment of the present invention the package is a chip-scale package.", "Additionally in accordance with a preferred embodiment of the present invention the package includes at least one portion which is at least partially transparent to visible radiation.", "Alternatively the package includes at least one portion which is partially transparent to infra-red radiation.", "There is also provided in accordance with another preferred embodiment of the present invention a packaged integrated circuit assembly including a packaged integrated circuit including an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, a package enclosing the integrated circuit substrate and defining first and second planar surfaces generally parallel to the substrate plane and a plurality of electrical contacts, each connected to the electrical circuitry at least some of the plurality of electrical contacts extending onto the first planar surface and at least some of the plurality of electrical contacts extending onto the second planar surface and at least one additional electrical circuit element mounted onto and supported by the second planar surface and electrically coupled to at least one of the plurality of electrical contacts extending therealong.", "Further in accordance with a preferred embodiment of the present invention the additional electrical circuit element includes an electrical component selected from the group consisting of: passive electrical elements, light generating elements, heat generating elements, light detecting elements, integrated circuits, hybrid circuits, environmental sensors, radiation sensors, micromechanical sensors, mechanical actuators and force sensors.", "Additionally in accordance with a preferred embodiment of the present invention the package includes at least one portion which is at least partially transparent to visible radiation.", "Alternatively the package includes at least one portion which is at least partially transparent to infra-red radiation.", "Still further in accordance with a preferred embodiment of the present invention the package is a chip-scale package.", "There is further provided in accordance with a preferred embodiment of the present invention a method for producing packaged integrated circuits.", "The method includes producing, on a wafer scale, an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, providing wafer scale packaging enclosing the integrated circuit substrate and defining first and second planar surfaces generally parallel to the substrate plane, forming on the wafer scale packaging a plurality of electrical contacts, each connected to the electrical circuitry at the substrate plane, at least some of the plurality of electrical contacts extending onto the first planar surface and at least some of the plurality of electrical contacts extending onto the second planar surface and separating the integrated circuit substrate in the wafer scale packaging into a plurality of individual chip packages.", "Further in accordance with a preferred embodiment of the present invention the plurality of individual chip packages are chip scale packages.", "Additionally in accordance with a preferred embodiment of the present invention the package includes at least one portion which is at least partially transparent to visible radiation.", "Alternatively the package includes at least one portion which is at least partially transparent to infra-red radiation.", "There is also provided in accordance with yet another preferred embodiment of the present invention a method for producing packaged integrated circuit assemblies.", "The method includes producing, on a wafer scale, an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, providing wafer scale packaging enclosing the integrated circuit substrate and defining first and second planar surfaces generally parallel to the substrate plane, forming on the wafer scale packaging a plurality of electrical contacts, each connected to the electrical circuitry, at least some of the plurality of electrical contacts extending onto the first planar surface and at least some of the plurality of electrical contacts extending onto the second planar surface, separating the integrated circuit substrate in the wafer scale packaging into a plurality of individual chip packages and mounting onto the at second planar surface of at least one of the plurality of individual chip packages, at least one additional electrical circuit element, the at least one additional electrical circuit element being supported by the second planar surface and electrically coupled to at least one of the plurality of electrical contacts extending therealong.", "Further in accordance with a preferred embodiment of the present invention the additional electrical circuit element includes an electrical component selected from the group consisting of: passive electrical elements, light generating elements, heat generating elements, light detecting elements, integrated circuits, hybrid circuits, environmental sensors, radiation sensors, micromechanical sensors, mechanical actuators and force sensors.", "Additionally in accordance with a preferred embodiment of the present invention the package includes at least one portion which is at least partially transparent to visible radiation.", "Alternatively the package includes at least one portion which is at least partially transparent to infra-red radiation.", "There is further provided in accordance with yet another preferred embodiment of the present invention a method for producing packaged integrated circuit assemblies.", "The method includes producing, on a wafer scale, an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, providing wafer scale packaging enclosing the integrated circuit substrate and defining first and second planar surfaces generally parallel to the substrate plane, forming on the wafer scale packaging a plurality of electrical contacts, each connected to the electrical circuitry, at least some of the plurality of electrical contacts extending onto the first planar surface and at least some of the plurality of electrical contacts extending onto the second planar surface, mounting onto the at second planar surface of the wafer scale packaging, at least one additional electrical circuit element, the at least one additional electrical circuit element being supported by the second planar surface and electrically coupled to at least one of the plurality of electrical contacts extending therealong and separating the integrated circuit substrate in the wafer scale packaging into a plurality of individual chip packages.", "Further in accordance with a preferred embodiment of the present invention the additional electrical circuit element includes an electrical component selected from the group consisting of: passive electrical elements, light generating elements, heat generating elements, light detecting elements, integrated circuits, hybrid circuits, environmental sensors, radiation sensors, micromechanical sensors, mechanical actuators and force sensors.", "Additionally in accordance with a preferred embodiment of the present invention the package includes at least one portion which is at least partially transparent to visible radiation.", "Alternatively the package includes at least one portion which is at least partially transparent to infra-red radiation." ], [ "FIELD OF THE INVENTION The present invention relates to integrated packaging, packaged integrated circuits and methods of producing packaged integrated circuits.", "REFERENCE TO CO-PENDING APPLICATIONS Applicants hereby claim priority of Israel Patent Application No.", "140,482, filed Dec. 21, 2001, entitled “Packaged Integrated Circuits and Methods of Producing Thereof”, and U.S. patent application Ser.", "No.", "09/758,906 filed Jan. 11, 2001, entitled “Packaged Integrated Circuits and Methods of Producing Thereof”.", "BACKGROUND OF THE INVENTION Various types of packaged integrated circuits are known in the prior art.", "The following patents and published patent applications of the present inventor and the references cited therein are believed to represent the state of the art: U.S. Pat.", "Nos.", "4,551,629; 4,764,846; 4,794,092; 4,862,249; 4,984,358; 5,104,820; 5,126,286; 5,266,833; 5,546,654; 5,567,657; 5,612,570; 5,657,206; 5,661,087; 5,675,180; 5,703,400; 5,837,566; 5,849,623; 5,857,858; 5,859,475; 5,869,353; 5,888,884; 5,891,761; 5,900,674; 5,938,45; 5,985,695; 6,002,163; 6,046,410; 6,080,596; 6,092,280; 6,098,278; 6,124,637; 6,134,118.EP 490739 A1; JP 63-166710 WO 85/02283; WO 89/04113; WO 95/19645 The disclosures in the following publications: “Three Dimensional Hybrid Wafer Scale Integration Using the GE High Density Interconnect Technology” by R. J. Wojnarowski, R. A. Filliion, B. Gorowitz and R. Sala of General Electric Company, Corporate Research & Development, P.O.", "Box 8, Schenectady, N.Y. 12301, USA, International Conference on Wafer Scale Integration, 1993.“M-DENSUS”, Dense-Pac Microsystems, Inc., Semiconductor International, December 1997, p. 50; “Introduction to Cubic Memory, Inc.” Cubic Memory Incorporated, 27 Janis Way, Scotts Valley, Calif. 95066, USA; “A Highly Integrated Memory Subsystem for the Smaller Wireless Devices” Intel(r) Stacked-CSP, Intel Corporation, January 2000; “Product Construction Analysis (Stack CSP)”, Sung-Fei Wang, ASE, R & D Group, Taiwan, 1999; “Four Semiconductor Manufacturers Agree to Unified Specifications for Stacked Chip Scale Packages”, Mitsubishi Semiconductors, Mitsubishi Electronics America, Inc., 1050 East Arques Avenue, Sunnyvale, Calif. 94086, USA; “Assembly & Packaging, John Baliga, Technology News, Semiconductor International, December 1999; “<6 mils Wafer Thickness Solution (DBG Technology)”, Sung-Fei Wang, ASE, R & D Group, Taiwan, 1999; “Memory Modules Increase Density”, DensePac Micro Systems, Garden Grove, Calif., USA, Electronics Packaging and Production, p. 24, Nov. 1994; “First Three-Chip Staked CSP Developed”, Semiconductor International, January 2000, p. 22; “High-Density Packaging: The Next Interconnect Challenge”, Semiconductor International, February 2000, pp.", "91-100; “3-D IC Packaging”, Semiconductor International, p. 20, May 1998; “High Density Pixel Detector Module Using Flip Chip and Thin Film Technology” J. Wolf, P. Gerlach, E. Beyne, M. Topper, L. Dietrich, K. H. Becks, N. Wermes, O. Ehrmann and H. Reichl, International System Packaging Symposium, January 1999, San Diego; “Copper Wafer Bonding”, A.", "Fan, A. Rahman and R. Rief, Electrochemical and Solid State Letters, 2(10), pp.", "534-536, 1999; “Front-End 3-D Packaging”, J. Baliga, Semiconductor International, December 1999, p 52, are also believed to represent the state of the art.", "SUMMARY OF THE INVENTION The present invention seeks to provide improved packaged integrated circuits and methods for producing same.", "There is thus provided in accordance with a preferred embodiment of the present invention a packaged integrated circuit including an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, a package enclosing the integrated circuit substrate and defining first and second planar surfaces generally parallel to the substrate plane and a plurality of electrical contacts, each connected to the electrical circuitry at the substrate plane, at least some of the plurality of electrical contacts extending onto the first planar surface and at least some of the plurality of electrical contacts extending onto the second planar surface.", "Further in accordance with a preferred embodiment of the present invention the package is a chip-scale package.", "Additionally in accordance with a preferred embodiment of the present invention the package includes at least one portion which is at least partially transparent to visible radiation.", "Alternatively the package includes at least one portion which is partially transparent to infra-red radiation.", "There is also provided in accordance with another preferred embodiment of the present invention a packaged integrated circuit assembly including a packaged integrated circuit including an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, a package enclosing the integrated circuit substrate and defining first and second planar surfaces generally parallel to the substrate plane and a plurality of electrical contacts, each connected to the electrical circuitry at least some of the plurality of electrical contacts extending onto the first planar surface and at least some of the plurality of electrical contacts extending onto the second planar surface and at least one additional electrical circuit element mounted onto and supported by the second planar surface and electrically coupled to at least one of the plurality of electrical contacts extending therealong.", "Further in accordance with a preferred embodiment of the present invention the additional electrical circuit element includes an electrical component selected from the group consisting of: passive electrical elements, light generating elements, heat generating elements, light detecting elements, integrated circuits, hybrid circuits, environmental sensors, radiation sensors, micromechanical sensors, mechanical actuators and force sensors.", "Additionally in accordance with a preferred embodiment of the present invention the package includes at least one portion which is at least partially transparent to visible radiation.", "Alternatively the package includes at least one portion which is at least partially transparent to infra-red radiation.", "Still further in accordance with a preferred embodiment of the present invention the package is a chip-scale package.", "There is further provided in accordance with a preferred embodiment of the present invention a method for producing packaged integrated circuits.", "The method includes producing, on a wafer scale, an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, providing wafer scale packaging enclosing the integrated circuit substrate and defining first and second planar surfaces generally parallel to the substrate plane, forming on the wafer scale packaging a plurality of electrical contacts, each connected to the electrical circuitry at the substrate plane, at least some of the plurality of electrical contacts extending onto the first planar surface and at least some of the plurality of electrical contacts extending onto the second planar surface and separating the integrated circuit substrate in the wafer scale packaging into a plurality of individual chip packages.", "Further in accordance with a preferred embodiment of the present invention the plurality of individual chip packages are chip scale packages.", "Additionally in accordance with a preferred embodiment of the present invention the package includes at least one portion which is at least partially transparent to visible radiation.", "Alternatively the package includes at least one portion which is at least partially transparent to infra-red radiation.", "There is also provided in accordance with yet another preferred embodiment of the present invention a method for producing packaged integrated circuit assemblies.", "The method includes producing, on a wafer scale, an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, providing wafer scale packaging enclosing the integrated circuit substrate and defining first and second planar surfaces generally parallel to the substrate plane, forming on the wafer scale packaging a plurality of electrical contacts, each connected to the electrical circuitry, at least some of the plurality of electrical contacts extending onto the first planar surface and at least some of the plurality of electrical contacts extending onto the second planar surface, separating the integrated circuit substrate in the wafer scale packaging into a plurality of individual chip packages and mounting onto the at second planar surface of at least one of the plurality of individual chip packages, at least one additional electrical circuit element, the at least one additional electrical circuit element being supported by the second planar surface and electrically coupled to at least one of the plurality of electrical contacts extending therealong.", "Further in accordance with a preferred embodiment of the present invention the additional electrical circuit element includes an electrical component selected from the group consisting of: passive electrical elements, light generating elements, heat generating elements, light detecting elements, integrated circuits, hybrid circuits, environmental sensors, radiation sensors, micromechanical sensors, mechanical actuators and force sensors.", "Additionally in accordance with a preferred embodiment of the present invention the package includes at least one portion which is at least partially transparent to visible radiation.", "Alternatively the package includes at least one portion which is at least partially transparent to infra-red radiation.", "There is further provided in accordance with yet another preferred embodiment of the present invention a method for producing packaged integrated circuit assemblies.", "The method includes producing, on a wafer scale, an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, providing wafer scale packaging enclosing the integrated circuit substrate and defining first and second planar surfaces generally parallel to the substrate plane, forming on the wafer scale packaging a plurality of electrical contacts, each connected to the electrical circuitry, at least some of the plurality of electrical contacts extending onto the first planar surface and at least some of the plurality of electrical contacts extending onto the second planar surface, mounting onto the at second planar surface of the wafer scale packaging, at least one additional electrical circuit element, the at least one additional electrical circuit element being supported by the second planar surface and electrically coupled to at least one of the plurality of electrical contacts extending therealong and separating the integrated circuit substrate in the wafer scale packaging into a plurality of individual chip packages.", "Further in accordance with a preferred embodiment of the present invention the additional electrical circuit element includes an electrical component selected from the group consisting of: passive electrical elements, light generating elements, heat generating elements, light detecting elements, integrated circuits, hybrid circuits, environmental sensors, radiation sensors, micromechanical sensors, mechanical actuators and force sensors.", "Additionally in accordance with a preferred embodiment of the present invention the package includes at least one portion which is at least partially transparent to visible radiation.", "Alternatively the package includes at least one portion which is at least partially transparent to infra-red radiation.", "BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be understood and appreciated more fully from the following detailed description, taken in conjunction with the drawings in which: FIG.", "1 is a simplified pictorial illustration of a chip-scale packaged integrated circuit constructed and operative in accordance with a preferred embodiment of the present invention; FIGS.", "2A, 2B and 2C are simplified pictorial illustrations of three examples of packaged integrated circuit assemblies constructed and operative in accordance with a preferred embodiment of the present invention; FIGS.", "3A and 3B are simplified illustrations of a first series of stages in the production of chip-scale packaged integrated circuits in accordance with a preferred embodiment of the present invention; FIGS.", "3C, 3D, 3E and 3F, are simplified sectional illustrations of a first series of stages in the production of chip-scale packaged integrated circuits in accordance with a preferred embodiment of the present invention; FIG.", "4A is a simplified pictorial illustration of an in-production packaged wafer following the stage illustrated in FIG.", "3F and following a first grooving stage; FIG.", "4B is a simplified pictorial illustration of an in-production packaged wafer following the stages illustrated in FIGS.", "3F and 4A and following a second grooving stage; FIGS.", "5A, 5B, 5C, 5D and 5E are simplified sectional illustrations taken along lines VI-VI in FIG.", "4A of a second series of stages in the production of chip-scale packaged integrated circuits in accordance with a preferred embodiment of the present invention; FIGS.", "6A, 6B, 6C, 6D and 6E are simplified sectional illustrations taken along lines V-V in FIG.", "4B of the second series of stages in the production of chip-scale packaged integrated circuits in accordance with a preferred embodiment of the present invention; and FIGS.", "7A and 7B taken together illustrate apparatus and methodologies for producing integrated circuit devices in accordance with a preferred embodiment of the present invention.", "DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Reference is now made to FIG.", "1, which is a simplified pictorial illustration of a chip-scale packaged integrated circuit constructed and operative in accordance with a preferred embodiment of the present invention.", "FIG.", "1 illustrates a preferred embodiment of integrated circuit device constructed and operative in accordance with a preferred embodiment of the present invention and includes a relatively thin and compact, environmentally protected and mechanically strengthened packaged integrated circuit 10, having a multiplicity of electrical contacts plated along edge surfaces and planar surfaces thereof.", "In contrast with prior art devices, such as those described in applicant's published PCT application WO 95/19645, the packaged integrated circuit shown in FIG.", "1 is characterized in that it has electrical contacts 12 extending along a first planar surface 14 thereof and also has electrical contacts 16 extending along an oppositely facing second planar surface 18 thereof This arrangement enables the packaged integrated circuit to be conveniently mounted in a stacked arrangement.", "As seen in FIG.", "1, the packaged integrated circuit 10 includes a plurality of generally planar edge surfaces which extend non-perpendicularly with respect to planar surfaces 14 and 18.These edge surfaces include first and second edge surfaces 20 and 22, each of which intersects the plane of a silicon substrate 24 on which is formed an integrated circuit 26 and extends from a location slightly beyond that plane to planar surface 14.There are also provided third and fourth edge surfaces 30 and 32, each of which intersects the plane of silicon substrate 24 and extends from a location slightly beyond that plane to planar surface 18.There are also provided fifth and sixth edge surfaces 40 and 42, neither of which intersects the plane of silicon substrate 24.Each of edge surfaces 40 and 42 intersects a respective one of surfaces 30 and 32 and extends therefrom to planar surface 14.There are additionally provided seventh and eighth edge surfaces 50 and 52, neither of which intersects the plane of silicon substrate 24.Each of edge surfaces 50 and 52 intersects a respective one of surfaces 20 and 22 and extends therefrom to planar surface 18.It is seen that contacts 12 extend along respective edge surfaces 20 and 22 and onto planar surface 14 and are in electrical contact with edges of pads 60 extending from silicon substrate 24 in the plane thereof It is also seen that contacts 16 extend along respective edge surfaces 30 and 32 and onto planar surface 18 and are in electrical contact with edges of pads 62 extending from silicon substrate 24 in the plane thereof.", "Reference is now made to FIGS.", "2A, 2B and 2C, which are simplified pictorial illustrations of three examples of packaged integrated circuit assemblies constructed and operative in accordance with a preferred embodiment of the present invention.", "FIG.", "2A illustrates a packaged integrated circuit 70 having mounted onto a planar surface 72 thereof, a plurality of other electrical devices, such as integrated circuits 78 and 74.It is seen that, for example, integrated circuit 74 electrically engages a pair of contacts 76 formed on planar surface 72, while integrated circuit 78 electrically engages six contacts 76 formed on planar surface 72.FIG.", "2B illustrates a packaged integrated circuit 80 having mounted onto a planar surface 82 thereof, a plurality of other electrical devices, such as four integrated circuits 84.It is seen that, for example, integrated circuits 84 each electrically engage a pair of contacts 86 formed on planar surface 82.FIG.", "2C illustrates a pair of packaged integrated circuits 90 and 92 mounted in a stacked arrangement, wherein contacts 94 of integrated circuit 92 are in electrical contact with corresponding contacts 96 of integrated circuit 90.It is appreciated that stacks having more than two integrated circuits of this type may be provided and that the integrated circuits need not be stacked in registration with each other, thus providing branched stacks.", "Reference is now made to FIGS.", "3A, 3B, 3C, 3D, 3E and 3F, which are simplified pictorial and sectional illustrations of a first series of stages in the production of chip-scale packaged integrated circuits in accordance with a preferred embodiment of the present invention.", "In accordance with a preferred embodiment of the present invention, and as illustrated in FIGS.", "3A, 3B and 3C a complete silicon wafer 120 having a plurality of finished dies 122 formed thereon by conventional techniques, is bonded at its active surface 124 to a protective insulating cover plate 126 via a layer 128 of epoxy.", "The insulating cover plate 126 typically comprises glass, quartz, sapphire or any other suitable insulative substrate.", "FIG.", "3A illustrates the initial mutual arrangement of cover plate 126 and wafer 120, FIG.", "3B illustrates the final placement and FIG.", "3C shows the bonding in a sectional illustration.", "The cover plate 126 may be opaque or transparent or may be colored or tinted in order to operate as a spectral filter.", "Alternatively, a dichroic or colored spectral filter may be formed on at least one surface of the cover plate 126.It is appreciated that certain steps in the conventional fabrication of silicon wafer 120 may be eliminated when the wafer is used in accordance with the present invention.", "These steps include the provision of via openings above pads, wafer back grinding and wafer back metal coating.", "The complete silicon wafer 120 may be formed with an integral color filter array by conventional lithography techniques at any suitable location therein.", "Prior to the bonding step of FIGS.", "3A, 3B & 3C, a filter may be formed and configured by conventional techniques over the cover plate 126, such that the filter plane lies between cover plate 126 and the epoxy layer 128.Following the bonding step described hereinabove, the silicon wafer 120 is preferably ground down to a decreased thickness, typically 100 microns, as shown in FIG.", "3D.", "This reduction in wafer thickness is enabled by the additional mechanical strength provided by the bonding thereof of the insulating cover plate 126.Following the reduction in thickness of the wafer, which is optional, the wafer is etched, using a photolithography process, along its back surface along predetermined dice lines which separate the individual dies.", "Etched channels 130 are thus produced, which extend entirely through the thickness of the silicon substrate, typically 100 microns thick.", "The etched wafer is shown in FIG.", "3E.", "The aforementioned etching typically takes place in conventional silicon etching solution, such as a combination of 2.5% hydrofluoric acid, 50% nitric acid, 10% acetic acid and 37.5% water, so as to etch the silicon down to the field oxide layer, as shown in FIG.", "3E.", "The result of the silicon etching is a plurality of separated dies 140, each of which includes silicon of thickness of about 100 microns.", "As seen in FIG.", "3F, following the silicon etching, a second insulating packaging layer 142 is bonded over the dies 140 on the side thereof opposite to insulating packaging layer 126.A layer 144 of epoxy lies between the dies 140 and the layer 142 and epoxy also fills the interstices defined by etched channels 130 between dies 140.In certain applications, the packaging layer 142 and the epoxy layer 144 are both transparent in the relevant spectral wavebands, such as, the visible waveband or the infrared waveband.", "The sandwich of the etched wafer 120 and the first and second insulating packaging layers 126 and 142 is then partially cut along lines 150, lying along the interstices between adjacent dies 140 to define notches along the outlines of a plurality of pre-packaged integrated circuits.", "It is noted that lines 150 are selected such that the edges of the dies along the notches are distanced from the outer extent of the silicon 140 by at least a distance d, as shown in FIG.", "3F.", "It is noted that partial cutting of the sandwich of FIG.", "3F along lines 150 exposes edges of a multiplicity of pads on the silicon wafer 120, which pad edges, when so exposed, define contact surfaces on dies 140.These contact surfaces are in electrical contact with the contacts, such as contacts 12 or 16 shown in FIG.", "1 and are designated in FIG.", "1 by reference numerals 60 or 62 respectively.", "It is a particular feature of the present invention that notches are formed in the sandwich of FIG.", "3F in a grid pattern, wherein notches in a first direction are formed inwardly from a first planar surface of the sandwich and cut through the plane of the active surface of silicon substrate 120 and notches in a second direction, orthogonal to the first direction are formed inwardly from a second planar surface of the sandwich, parallel to the first planar surface and opposite thereto, and also cut through the plane of the active surface of silicon substrate 120.FIG.", "4A illustrates notching of the sandwich of FIG.", "3F, producing notches 180 which extend typically inwardly from substrate 142 and engaging a plane 160 of the active surface of silicon substrate 120.FIG.", "4B illustrates notching of the sandwich of FIG.", "4A, producing notches 181 inwardly from substrate 126.It is seen that the notches 181 of FIG.", "4B extend perpendicularly to notches 180 of FIGS.", "4A & 4B and that both notches 180 and 181 pass through plane 160.Reference is now made to FIGS.", "5A, 5B, 5C, 5D & 5E which are simplified sectional illustrations taken along lines V-V in FIG.", "4A and lines VI-VI in FIG.", "4B of a second series of stages in the production of chip-scale packaged integrated circuits in accordance with a preferred embodiment of the present invention.", "FIG.", "5A is a sectional illustration of the sandwich of FIG.", "3F, which illustrates more clearly than in FIG.", "3F, the dies 140 and pads 172 extending outwardly thereof in the plane 160 (FIGS.", "4A and 4B).", "The remaining structural elements shown in FIG.", "3F are identified by the same reference numerals in FIG.", "5A.", "FIG.", "5B shows the notches 180 illustrated in FIG.", "4A.", "FIG.", "5C illustrates a preferred cross sectional configuration of a notch 180 produced by partially cutting as described hereinabove in connection with FIG.", "4A.", "Vertical lines 182 indicate the intersection of the notch 180 with the pads 172, defining exposed sectional pad surfaces 62 (FIG.", "1).", "Vertical lines 184 indicate the location of a subsequent final cut which separates the dies into individual integrated circuits at a later stage.", "FIG.", "5D illustrates the formation of metal contacts 16 (FIG.", "1) along the edges 30 and 32 and part of the surface 18 (FIG.", "1).", "These contacts, which may be formed by any suitable metal deposition technique, are seen to extend inside notch 180, thus establishing electrical contact with surfaces 62 of pads 172.It is noted that metal contacts are formed onto the dies in electrical contact with surfaces 62 of pads 172 without first separating the dies into individual chips.", "FIG.", "5E illustrates subsequent dicing of the individual dies on the wafer, along the lines 184, subsequent to metal contact formation thereon, into individual pre-packaged integrated circuit devices.", "Reference is now made to FIGS.", "6A, 6B, 6C, 6D and 6E, which are simplified sectional illustrations taken along lines V-V in FIG.", "4B of the second series of stages in the production of chip-scale packaged integrated circuits in accordance with a preferred embodiment of the present invention.", "FIG.", "6A is a sectional illustration of the sandwich of FIG.", "3F, which illustrates more clearly than in FIG.", "3F, the dies 140 and the pads 272 extending outwardly thereof in the plane 160 (FIGS.", "4A and 4B) in directions perpendicular to the directions along which extend pads 172.The remaining structural elements shown in FIG.", "3F are identified by the same reference numerals in FIG.", "6A.", "FIG.", "6B shows the notches 181 illustrated in FIG.", "4B.", "FIG.", "6C illustrates a preferred cross sectional configuration of a notch 181 produced by partially cutting as described hereinabove in connection with FIG.", "4B.", "Vertical lines 282 indicate the intersection of the notch 181 with pads 272, defining exposed sectional pad surfaces 60 (FIG.", "1).", "Vertical lines 284 indicate the location of a subsequent final cut which separates the dies into individual integrated circuits at a later stage.", "FIG.", "6D illustrates the formation of metal contacts 12 (FIG.", "1) along the edges 20 and 22 and part of the surface 14 (FIG.", "1).", "These contacts, which may be formed by any suitable metal deposition technique, are seen to extend inside notch 181, thus establishing electrical contact with surfaces 60 of pads 272.It is noted that metal contacts are formed onto the dies in electrical contact with surfaces 60 of pads 272 without first separating the dies into individual chips.", "FIG.", "6E illustrates subsequent dicing of the individual dies on the wafer, subsequent to metal contact formation thereon, into individual pre-packaged integrated circuit devices.", "Reference is now made to FIGS.", "7A and 7B, which together illustrate apparatus and methodologies for producing integrated circuit devices in accordance with a preferred embodiment of the present invention.", "A conventional wafer fabrication facility 380 provides complete wafers 120 (FIG.", "3A).", "Individual wafers 120 are bonded on their active surfaces to protective layers, such as glass layers 126 (FIG.", "3A), using epoxy 128 (FIG.", "3C), by bonding apparatus 382, preferably having facilities for rotation of the wafer 120, the layer 126 and the epoxy 128 so as to obtain even distribution of the epoxy.", "The bonded wafer 121 (FIG.", "3C) is thinned (FIG.", "3D) at its non-active surface as by grinding apparatus 384, such as Model 32BTGW using 12.5A abrasive, which is commercially available from Speedfam Machines Co. Ltd. of England.", "The wafer 121 is then etched at its non-active surface, preferably by photolithography, such as by using conventional spin-coated photoresist, which is commercially available from Hoechst, under the brand designation AZ 4562.The photoresist is preferably mask exposed by a suitable UV exposure system 385, such as a Karl Suss Model KSMA6, through a lithography mask 386 to define etched channels 130 (FIG.", "3E).", "The photoresist is then developed in a development bath (not shown), baked and then etched in a silicon etch solution 388 located in a temperature controlled bath 390.Commercially available equipment for this purpose include a Chemkleen bath and an WHRV circulator both of which are manufactured by Wafab Inc. of the U.S.A.. A suitable conventional silicon etching solution is Isoform Silicon etch, which is commercially available from Micro-Image Technology Ltd. of England.", "The wafer is conventionally rinsed after etching.", "The resulting etched wafer is shown in FIG.", "3E.", "Alternatively, the foregoing wet chemical etching step may be replaced by dry plasma etching.", "The etched wafer is bonded on the non-active side to another protective layer.", "142 by bonding apparatus 392, which may be essentially the same as apparatus 382, to produce a doubly bonded wafer sandwich 393 as shown in FIG.", "3F.", "Notching apparatus 394 initially partially cuts the bonded wafer sandwich 393 of FIG.", "3F inwardly from layer 142 to a configuration shown in FIG.", "4A including notches 180 (FIG.", "4A).", "Notching apparatus 394 thereafter partially cuts the bonded wafer sandwich 393 of FIG.", "3F inwardly from layer 126 to a configuration shown in FIG.", "4B including notches 180 and 181 (FIG.", "4B) and cuts the bonded wafer sandwich 393 of FIG.", "3F inwardly from layer 142 a configuration shown in FIG.", "4B including notches 180 and 181 (FIG.", "4B), extending mutually non-collinear and normally mutually perpendicular to each other.", "The notched wafer 393 is then subjected to anti-corrosion treatment in a bath 396, containing a chromating solution 398, such as described in any of the following U.S. Pat.", "Nos.", "2,507,956; 2,851,385 and 2,796,370, the disclosure of which is hereby incorporated by reference.", "Conductive layer deposition apparatus 400, which operates by vacuum deposition techniques, such as a Model 903M sputtering machine manufactured by Material Research Corporation of the U.S.A., is employed to produce a conductive layer initially on surfaces 30, 32 and 18 of each die of the wafer as shown in FIG.", "1 and thereafter on surfaces 20, 22 and 14 of each die of the wafer as shown in FIG.", "1.Configuration of contact strips 12 and 16 as shown in FIG.", "1, is carried out preferably by using conventional electro-deposited photoresist, which is commercially available from DuPont under the brand name Primecoat or from Shipley, under the brand name Eagle.", "The photoresist is applied to the wafers in a photoresist bath assembly 402 which is commercially available from DuPont or Shipley.", "The photoresist is preferably light configured by a UV exposure system 404, which may be identical to system 385, using masks 405 and 406 to define suitable etching patterns.", "The photoresist is then developed in a development bath 407, and then etched in a metal etch solution 408 located in an etching bath 410, thus providing a conductor configuration such as that shown in FIG.", "1.The exposed conductive strips 12 and 16 shown in FIG.", "1 are then plated, preferably by electroless plating apparatus 412, which is commercially available from Okuno of Japan.", "The wafer is then diced into individual pre-packaged integrated circuit devices.", "Preferably the dicing blade 414 is a diamond resinoid blade of thickness 4-12 mils.", "The resulting dies appear as illustrated generally in FIG.", "1.It will be appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described hereinabove.", "Rather the scope of the present invention includes both combinations and subcombinations of the various features described hereinabove as well as variations and modifications which would occur to persons skilled in the art upon reading the specification and which are not in the prior art." ] ]	Patent_10451564
[ [ "Conveyor lift", "A conveyor lift having a device for engaging a product to transfer the product from a conveyor at one level to a conveyor at another level wherein the lift is driven by at least one drive comprising a motor, and a coupling to couple the motor to the lift wherein the coupling comprises a clutch comprising a pair of drive members which are relatively rotatable about, and axially movable along, an axis, a relievable biasing device to bias the drive members axially towards each other so as to be disengageably connected in torque transmitting relationship and a relieving element operable to release the biasing device to interrupt transmission of said torque and the coupling also incorporating a torque indicating device comprising first and second torque transmitting parts, torque transmitting elements connecting said first and second parts, the device being rotatable about the axis and having torque sensing apparatus on at least one of said torque transmitting elements and responsive to torque transmitted by said torque transmitting apparatus to provide a signal dependent upon said transmitted torque." ], [ "1.A conveyor lift having a device for engaging a product to transfer the product from a conveyor at one level to a conveyor at another level wherein the lift is driven by at least one drive comprising a motor, and a coupling to couple the motor to the motor wherein the coupling comprises a clutch comprising a pair of drive members which are relatively rotatable about, and axially movable along, an axis; a relievable biasing device to bias the drive members axially towards each other so as to be disengageably connected in torque transmitting relationship and a relieving apparatus operable to release the biasing device to interrupt transmission of said torque and the coupling also incorporating a torque indicating device comprising first and second torque transmitting parts, torque transmitting elements connecting said first and second parts, the device being rotatable about the axis and having torque sensing apparatus on at least one of said torque transmitting elements and responsive to torque transmitted by said torque transmitting element to provide a signal dependent upon said transmitted torque.", "2.A conveyor lift according to claim 1 where said signal is transmitted to a receiving apparatus by means of a slip ring between the torque indicating device and a fixed element.", "3.A conveyor lift according to claim 1 wherein the torque transmitting device is provided with a communicating apparatus including a transmitter of electromagnetic radiation receiving said signal produced by said torque sensing apparatus and communicating an output signal dependent upon said torque to receiving apparatus which provides an output responsive to said transmitted torque.", "4.A conveyor lift according to any of the preceding claims wherein the first part of the torque indicating device comprises a radially inner part, relative to said axis, and the second part comprises a radially outer part, relative to said axis and the torque transmitting elements comprise at least one radially and circumferentially extending torque transmitting element extending between said parts to transmit torque therebetween.", "5.A device according to claim 1, claim 3 or claim 4 when dependant on claim 3 wherein the electromagnetic radiation is of radio frequency and is transmitted from an aerial to the receiving means.", "6.A device according to any one of the preceding claims wherein the drive members are disengageably connectable in torque transmitting relationship by a plurality of torque transmitting elements interposed between the drive members and which are biased into torque transmitting relationship with torque transmitting abutments by the biasing means when the clutch is engaged and which are moveable out of torque transmitting relationship with a torque transmitting abutments.", "7.A device according to claim 6 wherein the torque transmitting elements of the clutch are rotatable and are guided by a cage which is free to rotate relative to both drive members during disengagement and there are provided: a) means to maintain the cage space further from one of said drive members when the clutch is disengaged than when the clutch is engaged, and b) a guide member biasing means to remove the guide members apart when the clutch is disengaged to provide a separation between the parts of the drive members which are engaged by the torque transmitting elements which is greater than the diameter of the torque transmitting elements to maintain the torque transmitting elements out of engagement with the torque transmitting abutment of said one drive member when the clutch is disengaged.", "8.A clutch according to any one of the preceding claims wherein the clutch is a torque limiting clutch and said movement of the torque transmitting elements out of out torque transmitting relationship occurs when the torque exceeds a predetermined value and is as a result of movement of the drive members axially apart by the torque transmitting elements and the relieving means relieves the biasing means when the torque exceeds the predetermined value.", "9.A conveyor lift according to any one of claims 1 to 8 having control means for managing the relief of said biasing device or devices based on transmitted torque.", "10.A conveyor lift according to any one of the preceding claims wherein the lift is provided with a main drive and an auxiliary drive each of which comprises a motor and a coupling to couple the motor to the lift.", "11.A conveyor lift according to claim 10 wherein the main drive and the auxiliary drive are each in accordance with any one of claims 1 to 9.12.A conveyor lift according to claim 10 or claim 11 wherein the relieving means of one of said clutches is actuated to cause the clutch not to transmit torque when the torque indicating device of the coupling comprising said one clutch senses a predetermined torque and the relieving means of the other of said clutches is de-activated to cause said other clutch to transmit torque.", "13.A conveyor lift according to claim 10 or claim 11 wherein a control apparatus is provided alternately to activate one of said drives to cause it not to transmit torque to the lift and de-activate the other of said drives to cause it to transmit torque to the lift.", "14.A conveyor lift according to claim 13 wherein the control apparatus is manually operable.", "15.A conveyor lift according to claim 13 wherein the control apparatus is automatically operated on the basis of the time for which each of said drives has been activated.", "16.A conveyor lift according to any one of the preceding claims wherein the biasing means comprises air pressure means and the relieving means comprises means to relieve said air pressure.", "17.A conveyor lift according to any one of claims 1 to 7, or 8 to 15 when dependent on any one of claims 1 to 7, wherein the biasing means comprises a spring means and the relieving means comprises air pressure means.", "18.A conveyor lift substantially as hereinbefore described with reference to the accompanying drawings.", "19.A method of operating a conveyor lift according to claim 1 comprising performing a step selected from the group comprising: a) wherein the clutch is a torque limiting clutch and relative movement of said drive members when the transmitted torque exceeds a predetermined value causes operation of the said relieving apparatus, b) wherein the conveyor lift is provided with a main drive and an auxiliary drive each of which comprises a motor and a coupling to couple the motor to the lift wherein a control device is provided wherein the relieving apparatus of one of said clutches is actuated to cause the clutch not to transmit torque when the torque indicating device of the coupling comprising said one clutch senses a predetermined torque and the relieving apparatus of the other of said clutches is de-activated to cause said other clutch to transmit torque, c) wherein the lift is provided with a main drive and an auxiliary drive each of which comprises a motor and a coupling to couple the motor to a lift and said control device is operated alternately to de-activate one of said relieving apparatus to cause it to transmit torque to the lift and to de-activate the other of said relieving apparatus to cause it not to transmit torque to the lift, d) the control device referred in c) is a manually operable device or is operable depending upon the time for which each drive has been operating or may be operable depending on the time for which each drive has been operating.", "e) a predetermined torque may be indicated by the torque indicating apparatus, f) where the lift is provided with a main drive an auxiliary drive each of which comprises a motor and a coupling to couple the motor to the drive the coupling may be arranged to changeover the drive from the main drive motor to the auxiliary drive motor or vice versa at a predetermined torque, g) the torque indicating device may provide a signal in accordance with a predetermined torque so that a maintenance operative may investigate the drive showing said predetermined torque and may cause a changeover of the drive from said one drive to the other drive.", "h) apparatus may be provided to monitor the condition of the or each drive and/or of the lift by sensing a variation in torque.", "i) apparatus may be provided to record the variation in torque of the or each drive in any desired manner using the output of the torque indicating device.", "20.A method substantially as hereinbefore described with reference to the accompanying drawings.", "21.Any novel feature or novel combination of features described herein and/or shown in the accompanying drawings." ], [ "DESCRIPTION OF INVENTION This invention relates to a conveyor lift having a device for engaging a product to transfer the product from a conveyor at one level to a conveyor at another level.", "Plants, for example motor vehicle assembly plants, may have a conveyor at one level and a conveyor at another level and such a conveyor lift for transferring a car body panel or other product between the conveyors.", "The conveyor lift may be driven by a main drive which has a motor, usually a gearbox, and a coupling to couple the motor to the lift.", "The motor, gearbox when provided and coupling may be duplicated to provide an auxiliary drive if desired, to give a reserve or alternative drive for the conveyor lift, for example, in the case of a failure of the main drive.", "Downtime of the conveyor lift leads to costly loss of production and even a changeover from the main drive to the auxiliary drive takes a relatively long time typically approximately 1 hour and such changeover requires the conveyor lift and the associated production line to be at a standstill during the changeover time.", "An object of the invention is to provide a new and improved conveyor lift whereby the above mentioned problems are overcome or are reduced.", "According to a first aspect of the present invention we provide a conveyor lift having a device for engaging a product to transfer the product from a conveyor at one level to a conveyor at another level wherein the lift is driven by at least one drive comprising a motor, and a coupling to couple the motor to the lift wherein the coupling comprises a clutch comprising a pair of drive members which are relatively rotatable about, and axially movable along, an axis, a relievable biasing device to bias the drive members axially towards each other so as to be disengageably connected in torque transmitting relationship and a relieving apparatus operable to release the biasing device to interrupt transmission of said torque and the coupling also incorporating a torque indicating device comprising first and second torque transmitting parts, torque transmitting elements connecting said first and second parts, the device being rotatable about the axis and having torque sensing apparatus on at least one of said torque transmitting elements and responsive to torque transmitted by said torque transmitting element to provide a signal dependent upon said transmitted torque.", "Said signal may be transmitted to a receiving apparatus by means of a slip ring between the torque indicating device and a fixed element.", "Alternatively, the torque transmitting device may be provided with a communicating apparatus including a transmitter of electromagnetic radiation receiving said signal produced by said torque sensing apparatus and communicating an output signal dependent upon said torque to receiving apparatus which provides an output responsive to said transmitted torque.", "The first part of the torque indicating device may comprise a radially inner part, relative to said axis, and the second part comprises a radially outer part, relative to said axis and the torque transmitting elements comprise at least one radially and circumferentially extending torque transmitting element extending between said parts to transmit torque therebetween.", "The electromagnetic radiation may be of radio frequency and be transmitted from an aerial to the receiving means.", "The drive members may be disengageably connectable in torque transmitting relationship by a plurality of torque transmitting elements interposed between the drive members and which are biased into torque transmitting relationship with torque transmitting abutments by the biasing means when the clutch is engaged and which are moveable out of torque transmitting relationship with a torque transmitting abutments.", "The torque transmitting elements of the clutch may be rotatable and are guided by a cage which is free to rotate relative to both drive members during disengagement and there are provided: a) means to maintain the cage space further from one of said drive members when the clutch is disengaged than when the clutch is engaged, and b) a guide member biasing means to remove the guide members apart when the clutch is disengaged to provide a separation between the parts of the drive members which are engaged by the torque transmitting elements which is greater than the diameter of the torque transmitting elements to maintain the torque transmitting elements out of engagement with the torque transmitting abutment of said one drive member when the clutch is disengaged.", "The clutch may be a torque limiting clutch and said movement of the torque transmitting elements out of out torque transmitting relationship may occur when the torque exceeds a predetermined value and is as a result of movement of the drive members axially apart by the torque transmitting elements and the relieving means relieves the biasing means when the torque exceeds the predetermined value.", "The conveyor lift may have a control means for managing the relief of said biasing devices or devices based on transmitted torque.", "The lift may be provided with a main drive and an auxiliary drive each of which comprises a motor and a coupling to couple the motor to the lift.", "The main drive and the auxiliary drive may each be in accordance with any one of the preceding statements of invention.", "The relieving means of one of said clutches may be actuated to cause the clutch not to transmit torque when the torque indicating device of the coupling comprising said one clutch senses a predetermined torque and the relieving means of the other of said clutches may be de-activated to cause said other clutch to transmit torque.", "A control apparatus may be provided alternately to activate one of said drives to cause it not to transmit torque to the lift and de-activate the other of said drives to cause it to transmit torque to the lift.", "The control apparatus may be manually operable.", "The control apparatus may be automatically operated on the basis of the time for which each of said drives has been activated.", "The biasing means may comprise air pressure means and the relieving means comprises means to relieve said air pressure.", "Alternatively, the biasing means may comprise a spring means and the relieving means comprise air pressure means.", "According to another aspect of the invention we provide a method of operating a conveyor lift according to the first aspect of the invention comprising performing a step selected from the group comprising: a) wherein the clutch is a torque limiting clutch and relative movement of said drive members when the transmitted torque exceeds a predetermined value causes operation of the said relieving apparatus, b) wherein the conveyor lift is provided with a main drive and an auxiliary drive each of which comprises a motor and a coupling to couple the motor to the lift wherein a control device is provided wherein the relieving apparatus of one of said clutches is actuated to cause the clutch not to transmit torque when the torque indicating device of the coupling comprising said one clutch senses a predetermined torque and the relieving apparatus of the other of said clutches is de-activated to cause said other clutch to transmit torque, c) wherein the lift is provided with a main drive and an auxiliary drive each of which comprises a motor and a coupling to couple the motor to a lift and said control device is operated alternately to de-activate one of said relieving apparatus to cause it to transmit torque to the lift and to deactivate the other of said relieving apparatus to cause it not to transmit torque to the lift, d) the control device referred in c) may be a manually operable device or may be operable depending upon the time for which each drive has been operating, e) a predetermined torque may be indicated by the torque indicating apparatus, f) where the lift is provided with a main drive an auxiliary drive each of which comprises a motor and a coupling to couple the motor to the drive the coupling may be arranged to changeover the drive from the main drive motor to the auxiliary drive motor or vice versa at a predetermined torque, g) the torque indicating device may provide a signal in accordance with a predetermined torque so that a maintenance operative may investigate the drive showing said predetermined torque and may cause a changeover of the drive from said one drive to the other drive.", "h) apparatus may be provided to monitor the condition of the or each drive and/or of the lift by sensing a variation in torque.", "i) apparatus may be provided to record the variation in torque of the or each drive in any desired manner using the output of the torque indicating device.", "A conveyor lift in accordance with the present invention enables the drive to be manually controlled so as to either drive or not drive as desired and therefore a change from the main drive motor to the auxiliary drive motor and vice versa can be made simply by actuating the relieving apparatus of the coupling where drive is not required to be transmitted and de-activating the relieving apparatus of the coupling where drive is to be transmitted.", "Any abnormal change in torque is indicated by the torque sensing apparatus and then if desired, where an alternative drive is provided, the coupling may be arranged to changeover the drive from the main drive motor to the auxiliary drive motor or vice versa at a predetermined torque value.", "In addition or alternatively a signal may be provided so that a maintenance operative may investigate the drive showing an abnormal torque before failure thereof and/or if desired before changeover to the other motor.", "The couplings may be arranged so that the drive motors are operated alternatively without losing any time in the changeover.", "If desired the condition of the drive and/or of the lift may be monitored by sensing a variation in, or a predetermined, torque using the torque sensing apparatus.", "A user may record any variation in torque in any desired manner by using the output from the torque sensing apparatus.", "If a predetermined torque level occurs then the drive is protected against this by the provision of the above mentioned coupling as a torque limiting coupling.", "Embodiments of the invention will now be described by way of example with reference to the accompanying drawings wherein:— FIG.", "1 is a side view of a lower part of a conveyor lift embodying the invention, FIG.", "1a is a diagrammatic view of a conveyor lift embodying the invention, FIG.", "2 is a plan view of the arrangement shown in FIG.", "1, FIG.", "3 is an end view on the line 3-3 of FIG.", "1, FIG.", "4 is a view on the line 4-4 of FIG.", "1, FIG.", "5 is a vertical cross-sectional view of a coupling used in the lift shown in FIG.", "1-4, taken on the longitudinal axis thereof and on the line 5-5 of FIG.", "6, FIG.", "6 is an axial view of the torque indicating device of FIGS.", "1-5, FIG.", "7 is a section on the line 7-7 of FIG.", "6, FIG.", "8 is a block circuit diagram of a torque communicating device of FIGS.", "1 to 7, FIG.", "9 is a circuit diagram in respect of the torque communicating device of FIG.", "1 to 7, FIG.", "9a is a circuit diagram showing part B of FIG.", "9 in more detail, FIG.", "10 is a flow chart which illustrates the cycle of the torque communicating device of FIGS.", "1 to 9a.", "FIG.", "10a is a circuit diagram in respect of a modification of the circuit diagram shown in FIG.", "9, FIGS.", "11 and 12 are longitudinal sectional views through alternative arrangements of coupling for use in the conveyor lift described with reference to FIGS.", "1 to 10, FIG.", "13 is a longitudinal sectional view of a further alternative coupling for use in the conveyor lift of FIGS.", "1 to 4.FIG.", "14 is a longitudinal sectional view of a still further coupling for use in the conveyor lift of FIGS.", "1 to 4.Referring to the figures, a vertical drop lift is shown for transporting a motor vehicle body from a conveyor disposed at a one, lower, level to a conveyor disposed at another, higher level.", "As best shown in FIGS.", "1 and 2 a lower sub-assembly comprises a frame 10 from which a pair of upright plates 11 extend upwards and support an upper plate 12.Four trapezoidal plates 13 are provided to brace the frame 10, plates 11 and plate 12.Bolted to the underside of the plate 12 are a pair of pillow blocks 14 by which a drive shaft 15 is rotatably supported.", "The drive shaft 15 has fixed to rotate therewith a pair of sprockets 16 around which are entrained roller link chains 17 which emerge in a vertical direction through generally oval-shaped openings 18 provided in the plate 12 and which are entrained, at their upper end, about a further pair of sprockets 19 carried by an upper shaft 20 rotatably mounted on pillow blocks 21 of an upper sub-assembly U.", "The roller link chains 17 are provided with at least one device D for engaging a product such as a car body, to be lifted.", "The device D is shown only diagrammatically and in use will be shaped as necessary to engage the product concerned.", "The shaft 15 has fixed thereto a brake disc 30 with which a pair of callipers of a spring actuated calliper brake 31 are engaged, the brake pads of the calliper being indicated at 34.An air supply 35 is supplied to the brake 31 so as to hold the pads 34 out of braking engagement with the disc 30 and which is interrupted to allow spring application of the brake and thus rapid halting of the lift and in particular to prevent reversal of the lift and downward movement of a car body or other product carried thereby.", "The shaft 15 is connected by a coupling device 40, hereinafter described in more detail, to an output shaft 41 of a gearbox 42 driven by a main drive electric motor 43.Shaft 15 is also connected by a further coupling device 40a to an output shaft 41a of a gearbox 42a of an auxiliary drive electric motor 43a.", "Because of the distance between the pillow block 14 disposed between the brake and the sprockets 16 and the gearbox 42 an additional pillow block 44 is provided for the shaft 15 carried on a bracket 45 mounted on a block 46 whereby the motor 42 is supported above a base level 47.Referring now to FIG.", "5 each coupling 40, 40a is identical and thus only the coupling 40a will be described and illustrated in detail in FIG.", "5.The coupling 40a comprises a torque limiting clutch, indicated generally at 50 to connect the output shaft 41a of the auxiliary gearbox 42a with the shaft 15.The clutch 50 comprises a driving member 51 and a driven member 52.The driving member 51 is connected by a splined connection to the output shaft 41a whilst the driven member 52 is connected by suitable bolts 52a to an element coupling 53 which is splined to a flange 53a fastened to an end part of the shaft 15.The driving member 51 has a driving clutch plate 54 splined thereto as shown at S for axial sliding and non-rotatable movement relative to the driving member 51.The driving clutch plate 54 is connected in torque transmitting relationship with a driven clutch plate 55.Interposed therebetween is a cage plate 56 which has a plurality of apertures 57 therein for torque transmitting elements in the form of balls 58.The apertures 57 are of generally cylindrical configuration but are provided with smaller ends at the end thereof adjacent the driven clutch plate 55.The thickness of the cage plate 56 relative to the diameter of the balls 58 is such that the smaller diameter end of the apertures 57 nearly engages the balls 58 as shown when the clutch is engaged i.e.", "on a chord approximately midway between the centre of the ball and its surface.", "The driving and driven clutch plates 54, 55 are provided with torque transmitting abutments 60, 61 respectively which are in the form of generally frusto-conical recesses having an included angle of approximately 70°.", "If desired other angles may be used, for example 90°, but a smaller angle such a 70° is preferred because this permits transmittal of a greater torque between the clutch plates for a given force biasing the plates together by a relievable resilient biasing means 62.If desired the abutments may be of other shape such as generally prism shaped and again the abutment surfaces would be similarly inclined.", "That is the included angle could be 70° or 90° and hence would be inclined at 55° or 45° respective to a plane normal to the axis of rotation of X-X of the clutch C. The relievable resilient biasing means 62 in the present example, comprises a ball bearing 63 the inner race 64 of which is engaged by the driving clutch plate 54 and the outer race 65 of which is engaged by a piston 66 slidable on inner cylindrical surfaces 67, 68 of a member 69 which is carried on the outer race 70 of a second ball bearing 71 the inner race 32 of which is engaged with the driving clutch member 51.An ‘O’ ring seal R is provided between the surfaces 67, 68 and the piston 66.Alternatively, if desired, a plurality of pistons and associated cylinders may be provided circumferentially spaced around the clutch in equi-angular positions, for example four pistons and cylinders or eight pistons and cylinders.", "Air is fed from a source of air under pressure (P) via a passageway 69a through the member 69 into a volume V so that when air is supplied under pressure the piston 66 is biased to the right in FIG.", "5 to bias the driving clutch plate 54 towards the driven clutch plate 55 and thus to force the balls 58 into torque transmitting relationship with the abutments 60,61 on the plates 54, 55 respectively i.e.", "when the relieving means is de-activated.", "If desired any other form of relievable resilient biasing means may be provided.", "A combined cage and driving member biasing assembly comprises a plurality of coil compression springs 73 provided at equi-angularly disposed dispositions around the axis X-X.", "If desired the springs 73 may not be equi-angular.", "At their right hand ends the springs 73 are housed in a blind bore 74 of a ring member 75 which engages a shoulder 76 of the member 51.At their left hand end the springs 73 are received in a blind bore 77 of a further ring member 78 which is slidably mounted on the member 51 and has an outwardly extending flange 79.The flange 79 is adapted to engage the cage plate 56 whilst an end surface 81 of the ring member 78 is adapted to engage the driving plate 54.As a result, the cage plate 56 and the driving plate 54 are biased to the left in FIG.", "5 by the springs 73.In the present example 12 springs 73 are provided circumferentially around the axis X-X and similarly a plurality, in the present example, 4 pins 85 are provided between through bores in the rings 75 and 78 for guidance purposes.", "The output member 52 is rotatably mounted relative to the input member 51 by a pair of ball bearings 86.As best shown in FIGS.", "5 to 7 the drive plate 55 is connected in torque transmitting relationship with a torque indicating device 101.The torque indicating device 101 comprises an outer part 122 which is ring shaped and an inner part 124 which is also ring shaped.", "The outer part 122 has three axially extending apertures 125 within which three bolts 121 are received to connect the outer part 122 to the output member 52.The inner part 124 is provided with six apertures 126 in which are received six bolts 100 to connect the inner part 124 to the driven plate 55.The outer part 122 and the inner part 124 are interconnected by a pair of diametrically opposed torque transmitting element 128 which, as best shown in FIGS.", "5 and 7, are relatively thin in the axial direction of the device, i.e.", "in a direction parallel to a central axis X-X of the device and are relatively wide in a circumferential direction, as shown in FIG.", "6.The inner and outer parts are also connected together by eight radially and longitudinally extending webs 129 which are relatively thin in the circumferential direction, as shown in FIG.", "6, but are relatively wide in the axial direction, as shown in FIG.", "7, and thus the webs 129 provide axial stability for the connection between the inner and outer parts 124, 122.The torque transmitting elements 128 and the webs 129 are connected to the outer and inner parts 122, 124 by virtue of being integral therewith and so are connected thereto in an “encastré” structural mode.", "As shown in FIG.", "6, these parts being omitted from FIGS.", "5 and 7 for clarity, an apparatus 130 for determining strain is applied to each of the torque transmitting elements 128.In the present example the apparatus 130 comprises a conventional strain gauge bridge with the strain gauges being configured so that their resistance varies in accordance with strain of the elements 128 as a result of the torque being transmitted.", "The output of the apparatus 130 for determining strain is connected to a microprocessor system 131 which is connected to a transmitting aerial 133 which extends circumferentially around the outer circumference of the outer part 122 and is insulated therefrom by an insulating ring 134.The aerial 133 comprises two generally semi-circular parts 133a, 133b which are connected to a negative terminal and a positive terminal respectively of a battery B and provide a current path into the microprocessor system 131.The battery B is housed in a blind recess 132, of circular cross-section and extending radially relative to the axis X-X provided in the inner part 124 and in a corresponding radially extending cylindrical aperture 135 provided in the outer part 122.The battery is retained in position by the insulating ring 134 and aerial part 133a which are held in place by screws 136, 136a-b which are received in threaded inserts 137a-b which are insulated from the outer part 122.The threaded insert 137b receives a further screw 138a which anchors a terminal tag 139a in electrically conducting relationship with the insert 137b and hence with the aerial part 133a and the tag 139a is connected by a wire 140a to a positive terminal of the microprocessor system 131.Similarly, the insert 137b receives a further screw 138b which anchors a tag 139b connected by a wire 140b to a negative terminal of the microprocessor system.", "A frusto-conical spring 141 is received in the recess 132 and engages the negative terminal of the battery B.", "The spring 141 is connected by a further wire 140c to a terminal tag 139c which is anchored by a further screw 138c to the insert 137b.", "The aerial part 133a has a further screw 142 threadedly engaged therewith, the inner end of which engages the positive terminal of the battery B.", "The aerial parts 133a and 133b thus provide a current path from the positive and negative terminals respectively of the battery B to the positive and negative terminals of the microprocessor system 131.Referring now to FIG.", "8, there is illustrated diagrammatically the electric circuit of the torque transmitting device.", "The strain gauge means 130 comprises a conventional strain gauge bridge arrangement 144, the output of which is fed to an amplifier 45 and power is supplied on lines 146 and 147.The line 146 is connected to the positive battery terminal and provides positive supply to the amplifier 245, a single to bi-polar converter 148 and a low power radio frequency oscillator 149 which provides a transmitter which acts as a communicating means to communicate an output signal dependant upon the torque sensed by the strain gauge means.", "If desired a transmitter of electromagnetic radiation of other than radio frequency may be provided in association with a suitable receiver, such as a transmitter of electromagnetic radiation in the visible light or infra-red band.", "For example, by virtue of liquid crystal display of digits, or a suitable bar code or the like.", "The output from the amplifier 145 is fed to the single to bi-polar converter 148 the output of which is fed on lines 150 and 151 to an eight bit microprocessor 152, the line 151 carrying a polarity detection or sign bit.", "The microprocessor 152 is connected to a negative terminal of the battery on line 154 and is also supplied with a voltage reference signal on the line 153.The microprocessor provides a serial output on line 155 to the oscillator 149 and the oscillator 149 provides a signal on line 156 which is supplied to the aerial 133, for example on lines 140a, 140b and transmitted therefrom as a radio frequency signal.", "The microprocessor 152 is also connected on line 157 to the oscillator 149.The microprocessor system comprises a central processor unit (CPU) connected to an address bus, a data bus and a control bus.", "The address bus is connected to a random access memory (RAM) serving as a working store, a programmable read-only memory (PROM) serving as a store for the operating program of the system and input and output buses to which the lines 150, 151, 153 and 154 are connected.", "In addition, the microprocessor provides a switched output to line 147 or line 157.Referring now to FIGS.", "9 and 9a, FIG.", "9a is a circuit diagram in respect of the amplifier 145, the single to bi-polar converter 148, the oscillator.", "149 and the microprocessor 152.The strain gauge bridge 144 is connected as shown in FIG.", "9a to the terminal P1, P2, P5, P6, of the differential amplifier provided by the components within the block identified in dashed line in FIG.", "9a at 145, whilst a gain setting resistor, in the form of a potentiometer, is connected to terminals P3 and P4.The differential amplifier 145 includes a reference voltage circuit comprising integrated circuit U4 and which also supplies the single to bi-polar converter which is provided by the components within the dashed line 148 of FIG.", "9a.", "The converter 148 performs an absolute value and sign detection on the output from the amplifier 145 and also conditions a signal so that it covers the full input range of the analogue to digital converter of the microprocessor provided by the components in the dashed line box 62 of FIG.", "9a.", "The microprocessor 152 performs the analogue to digital conversion and produces a serial bit stream on line 155 to modulate the RF oscillator provided by the components in the dashed line box 149 of FIG.", "9a.", "The absolute value signal is supplied from the converter 148 on line 150 whilst the polarity detector is supplied on line 151 in FIG.", "9a.", "FIG.", "9 also illustrates how a battery assembly B is connected in circuit.", "The RF oscillator 149 comprises a single transistor stage using a crystal element operating in fundamental mode.", "Since the whole circuit must be operated at very low power levels so as to give a long battery life, for example six months, and since there is no requirement for range, no power amplification stage is necessary or desirable.", "Only sufficient radio frequency power is developed in order to fully drive a receiver at a distance of, for example, 25 mm or less away from the aerial.", "The aerial 133 is directly loosely coupled into the oscillator collector circuit by a transformer coupling comprising, for example, four turns of wire 133a around an inductor, the other end being connected to the metalwork of the sensor assembly which is grounded or forms a counterpoise through the bulk of the apparatus with which the device is used.", "This increases the radiated radio frequency power considerably.", "Narrow band frequency modulation is achieved by changing the DC bias point and hence the parasitic capacitances in the base/emitter and collector/base circuits.", "The frequency change is approximately ±100 hertz from a nominal mean centre frequency, which is itself correctly adjusted with a small variable capacitor across the crystal.", "If desired, the frequency change may of be other value such as ±500 hz from said nominal mean centre frequency.", "The receiver R is of any suitable kind, for example, a simple “data sheet”, single conversion superhet design using, for example, an MC3361 chip.", "A fundamental crystal is used to give 455 kilohertz IF.", "The output from the FM modulator is AC coupled with a comparator which drives the output at TTL levels.", "The squelch circuit operates at high HF noise to clamp the input to the comparator and the desired information can be presented as described hereinafter.", "The microprocessor controls the operation according to the program in PROM.", "Referring to the flow chart of FIG.", "10, the operation is as follows:— The clock of the microprocessor initially switches power on to lines a and b.", "The microprocessor then receives signals of the absolute value of the torque on line 150 in analogue form from the single to bi-polar converter 148 and the power on line 147 is then switched off.", "The microprocessor performs an analogue to digital conversion and eight bit serialises the torque value and ascribes a sign in accordance with the polarity detector signal supplied on line 151.The microprocessor then digitises the input from the voltage reference and eight bit serialises this information.", "The microprocessor compares the torque value with full scale output of the A/D stage and if the torque is less than 5% of this the microprocessor switches the power off on line b and the system remains in this condition until the microprocessor clock reaches the end of a pre-programmed sleep mode which lies in the range 50 to 1000 milliseconds, in the present example 5 hertz.", "Thereafter the microprocessor switches power on at lines 147 and 157 and the cycle is repeated as previously described.", "The bridge is provided with a scaling resistor 158 which can be adjusted to give a full scale output of the A/D converter stage for a predetermined torque.", "Alternatively, if the torque comparison indicates that the torque is not less than 5%, the power on line 157 is maintained and after a short delay, which typically lies in the range 150 milliseconds, and in the present example is 100 hertz, the cycle is repeated by the switching of power on to both lines 147 and 157.The circuit is powered, in the present example, by the battery B which comprises a lithium thionyl chloride battery of 1500 mAh capacity.", "The battery provides a d.c. current at a voltage lying in the range 3.3-3.7 volts.", "This voltage is determined by the reciprocal of the digitised VREF, since all A/D conversions are ratiometric.", "When the torque is less than a predetermined value then, as explained above, the circuit is arranged to sample the torque at a relatively low frequency.", "In the present example 5 hertz.", "However, when, as a result of such a sampling, the torque is detected to be greater than the predetermined value a higher sample and transmission rate is signalled to occur, in the present example 100 hertz.", "As a result, because the circuit is not sampling and transmitting a signal continuously, the power consumption of the device is relatively small compared with consumption which would be the case if the sampling were performed continuously.", "In the present example the signal is a frequency modulated signal at a nominal frequency of 27.145 MHz.", "The radiated signal is detected by a receiver R located adjacent the aerial 133, in the present example 5 mm apart but which may lie preferably in the range 2 mm to 10 mm, for example, up to 25 mm apart.", "The receiver R is supplied with power on line R1 and is arranged to display the detected torque and, if desired, power and/or battery state, in any desired manner.", "If desired the receiver may provide a signal to a printer to provide a permanent record and/or to a computer system or other device.", "If desired the circuit illustrated and described hereinbefore may be modified as illustrated in FIG.", "10a in which power is supplied to a regulator R which supplies an output of 0 and +5 volts.", "This output from the regulator R is then supplied to the various stages of the circuit as illustrated in FIG.", "10a in which the same reference numerals are used to refer to corresponding parts as were used in the previous figures, except that the single to bi-polar converter 48 is not used.", "If desired the component 48 may be provided where it is desirable for reasons of resolution.", "If desired, the A/D and the microcompressor may be both of 8 bit type or alternatively the microcompressor may be of 8 bit type and the A/D of 10 bit type and the 10 bit A/D may be provided off-board or on-board as desired.", "As can be seen from FIG.", "10A, if desired, an interface I to a slip-ring assembly may be provided instead of a low power oscillator 49.Such an arrangement is utilised in the slip-ring version of the device illustrated in FIG.", "12.FIG.", "11 shows an alternative configuration of coupling to that shown in FIG.", "5 in which the same reference numerals are used to refer to corresponding parts as are used in FIG.", "5.The electric circuitry and components as well as the manner of operation are the same as hereinbefore described with reference to FIG.", "5 modified as necessary mutatis mutandis.", "If desired, instead of an electromagnetic communication apparatus as described hereinbefore the signal dependent upon said transmitted torque, may be transmitted to a receiving apparatus by providing a slip ring between the torque transmitting device and a fixed element or ground.", "FIG.", "12 shows a coupling embodying the present invention in which a slip ring is shown at 200 and a pick-up at 201.The torque sensing apparatus is provided on torque transmitting element 202 and either the analogue signal resulting from the torque sensing apparatuses may be fed via the slip ring to a stationary position at which the analogue signal is operated upon by electronics functioning in the same manner as described hereinbefore or by conventional signal processing equipment (for example an analogue meter) or, if desired, the analogue signals from the torque sensing apparatuses may be processed in a similar manner to that described hereinbefore and the processed signal which in the example described hereinbefore is fed to an electromagnetic transmitter is, in the embodiment shown in FIG.", "12, fed to the slip ring 200 and the processed signal is picked up by the pick up 201.Alternatively or in addition, the sensitivity of the torque transmitting means may be adapted by altering the configuration of the torque transmitting means.", "In use, when the driving member 51 is rotated through the shaft 41a by the motor 43a then so long as torque is to be transmitted to the driven clutch member 55 and thence to the torque indicating device 101 the relieving apparatus is de-activated so that air under pressure is supplied to the volume V to bias the plates 54, 55 towards each other and so long as the torque to be transmitted is lower than a predetermined torque then the torque is transmitted between the clutch members 54, 55 by the balls 58 and torque transmitting abutments 60, 61.If the torque to be transmitted increases above a predetermined torque the balls 58 begin to roll out of their associated abutments 60, 61 as the force applied by the piston 66 is less than the resultant force tending to bias the driving plate 54 to the left in FIG.", "5.As the driving plate 54 is forced to the left away from the driven plate 55 the piston 66 is pushed to the left in FIG.", "5 and this causes a relieving means actuating disc 86 to engage a sensor of a relieving apparatus shown at 87, such as a micro-switch, to open an electrically operated valve in the supply of air to the passage 69a so that the air supply is cut off and the pressure within the volume V is released thereby relieving the resilient bias of the driving plate 54 to the right in FIG.", "5.In this condition the relieving apparatus is activated.", "In addition, as the balls 58 ride out of their associated abutments they rotate and cause the cage plate 56 to rotate at half the relative speed between the driving and driven clutch plates relative to each clutch plate.", "Because of the above described configuration of the apertures 57 and their relationship with the size of the balls 58 the cage plate 56 is also moved to the left of the figure away from the driven clutch plate 56.When the pressure in the volume V is relieved as described hereinbefore the coil springs 73 bias the driving clutch plate 54 and the cage plate 56 to the left in FIG.", "5.As the driving and driven members continue to rotate relative to one another the balls 58 continue to rotate until they become circumferentially aligned with the next adjacent torque transmitting abutments.", "When the balls are thus aligned springs 73 urge the cage plate 56 to the left which movement is permitted by the alignment of the balls 58 with the torque transmitting abutment 60 in the driving clutch plate 54 and thus the cage plate guides the balls 58 into the abutment and maintains the balls therein whilst the driving clutch plate 54 continues to rotate relative to the driven clutch plate 55.The cage plate 56 thus maintains the balls 58 out of position for engagement with the abutments 61 of the driven clutch plate 55 so that relative rotation between the driven and driving clutch plates can continue without any risk of the balls chattering as the result of engagement with the abutments 61.Reengagement is achieved simply by applying air pressure to the piston 66 so that the abutments 61 in the driven clutch plate 55 are moved into torque transmitting engagement with the balls 58 when they become suitably aligned therewith as a result of the relative rotation between the driving and driven clutch members.", "The predetermined torque at which the clutch disengages can be easily adjusted by varying the pressure of the air acting on the piston 66.The relieving means may be arranged to relieve the relievable resilient biasing means when the driven clutch plate 20 has moved through an axial distance somewhat less than that required completely to disengage the balls 58 from the recesses 60, 61 but complete disengagement is achieved by the hereinbefore described springs 73 when the biasing means is relieved.", "Although the clutch described hereinbefore has been described in its function as a torque limiting clutch, if desired it may be used as simple clutch either under the control of an operator or under the control of automatic operating means in dependence on the torque indicated by the torque indicating device 101 described above.", "In such a case drive is transmitted when air under pressure is supplied to the relevant volume V and drive is disconnected when the air supply to the relevant volume V is relieved by an appropriate mechanism in the air supply means.", "In the previously described couplings 40/40a air under pressure is applied to the volume V to bias the plates 54 and 55 towards each other and so enable torque to be transmitted between the plates by the balls 58, or other rolling elements, and the recesses 60 and 61.Referring now to FIG.", "13, a further alternative coupling 40/40a to that shown in FIG.", "5 is illustrated in which the same reference numerals are used for corresponding parts as are used in FIG.", "5.In this embodiment a clutch plate 354 is axially slidably and non-rotatably mounted on a drive member 51.The plate 354 has a plurality of circumferentially disposed pins 356 which are held in torque transmitting relationship with detents 361 provided in a driven clutch plate 355 by a plurality of coil compression springs 300.In this embodiment the pins 356 and the detents 361 have mutually engaging surfaces of frusto-conical configuration having a cone angle such that the clutch will disengage when a predetermined torque is exceeded so that in this the clutch will act as a torque-limiting clutch.", "If desired, in a modification, an alternative angle may be used such that the clutch will stay in engagement with any positive spring pressure or applied torque.", "In either case rolling elements such as balls and recesses may be used instead of pins and detents as in the previously described embodiments.", "The angle of the recesses are chosen in relation to the rolling element diameter to provide either a torque-limiting clutch or not.", "In this embodiment, or the modification thereof described above, a pneumatic system is provided to overcome the load applied by the springs 300 to the clutch plate 354.This comprises a sleeve 301 which is axially immovably mounted on the clutch plate 355 by means of a ball bearing 302 and is prevented from rotation by a suitable anti-rotation device attached to the sleeve 301 at the point shown at 311 and as desired at other corresponding points.", "Axially slidably mounted on the sleeve 301 is a flange member 303 which is made an air-tight fit by means of O-ring seals 304a, 304b.", "Thus a sealed volume 305 is formed between the flange part 303 and a transversely extending limb 306 of, or attached to, the sleeve part 301.Air under pressure may be introduced into the volume 305 via an air port 307.When air under pressure is introduced into the volume 305 an axial load is applied to a flange part 308 of the flange member 303 to cause it to apply an axial load to a plate 309 through a bearing 310 which allows the clutch to rotate relative to the parts 303 and 301.The plate 309 is connected to the clutch plate 354 by, for example, a split ring 311.If the thus applied axial load exceeds the load applied by the springs 300, the plate 309 moves the clutch plate 354 to disengage the clutch by disengaging the pins 356 from the detents 361.In a modification of this embodiment, the coupling 40a may act as a holding brake.", "In FIG.", "13, when the clutch plate 54 is in its disengaged position, the left hand face 354a of the clutch plate 354 is spaced from the limb 306.If desired, for example by removing an abutment part 354b, the clutch plate 354 may abut the limb 306 in its disengaged position to prevent co-rotation thereof and such that the coupling 40a acts as a holding brake.", "Metal-to-metal contact between the surface 354a and the limb 306 may provide sufficient frictional contact, or alternatively a suitable surface may be provided on one or both of the surface 354a and limb 306 as desired.", "In other respects the embodiment described with reference to FIG.", "13 is similar to the other embodiments and so further discussion is not required.", "If it is desired to operate the clutch automatically in accordance with the torque sensed by the device, the apparatus, which in the present example is the apparatus described with reference to FIG.", "13 is provided with additional components as shown in FIG.", "14.The device illustrated in FIG.", "14 the same as that illustrated in FIG.", "13; as is its manner of operation and the same reference numerals have been used in FIG.", "14 as were used in FIG.", "13.The torque sensing device has a receiver 400 which receives a signal dependent upon torque transmitted by the device through the aerial 33.The receiver 400 provides a signal on a line 401 which is proportional to the torque sensed and this signal is fed to an output device 402.The output device 402 provides a relay output when the torque sensed by the device achieves a predetermined torque level set in the output device 402.The relay output is fed on line 403 to a plant controller 404 which provides an output signal on a line 405 to switch a solenoid or other valve 406.The solenoid valve 406, is of conventional type and can either connect the inlet 307 to an air supply under pressure P as indicated by the line 407 or to exhaust by the line 408.In use, when the torque exceeds the predetermined level set in the output device 402 a relay out put is fed on lines 403 and 405 to the air solenoid valve 406 to cause air under pressure to be fed to the inlet 307 to cause the clutch to be disengaged as described hereinbefore in connection with FIG.", "13.It is desired to reengage the clutch a signal is fed to the air solenoid valve 406 to cause it to connect the inlet 307 to atmosphere instead of to supply air under pressure P on line 407.Of course similar arrangements may be provided as desired with other embodiments such as the embodiment shown in FIG.", "5 where in this case a solenoid valve is arranged to connect the inlet 69a to exhaust when the torque exceeds a predetermined value.", "In all the embodiments, to enable display of power, a reflective target T is provided at one position, or at a plurality of circumferentially spaced positions on the outer parts 122, or on any other convenient rotating component of the device.", "Passage of the target T past a photo electric cell P is detected and the time taken for a complete revolution is thus measured to provide the speed of rotation of the device.", "The torque transmitted is multiplied by the thus determined speed to enable power transmitted by the device to be displayed.", "The conveyor lift described hereinbefore therefore is capable of operation in a plurality of different modes.", "In the illustrated example the conveyor lift is provided with a main drive motor and an auxiliary drive motor with associated drives but, if desired, the conveyor lift may be provided with only a single drive motor and associated drive.", "Further, whether a single drive or a pair of drives is provided, the clutch described hereinbefore may function as a torque limiting clutch or clutches or the pressure of air supplied to the volume V, in association with the other element parameters of the clutch, may be such that the or each clutch may not function as a torque limiting clutch.", "Whether the or each clutch operates as a torque limiting clutch or not, the or each clutch is provided with relieving means which can be de-actuated so as to enable torque to be transmitted by the clutch or actuated so as interrupt transmission of torque.", "A conveyor lift in accordance with the present invention enables the drive to be manually controlled so as to either drive or not drive as desired and therefore a change from the main drive motor to the auxiliary drive motor and vice versa can be made simply by actuating the relieving apparatus of the coupling where drive is required to be transmitted and de-activating the relieving apparatus of the coupling where drive is not required.", "This may be done manually or automatically, for example, on the basis of the time for which a drive has been operating.", "Alternatively, any attainment of a predetermined torque, for example, the occurrence of an abnormal torque level, is indicated by the torque sensing apparatus and then if desired, where an alternative drive is provided, the coupling may be arranged to changeover the drive from the main drive motor to the auxiliary drive motor or vice versa at a predetermined torque value this may be done manually or automatically.", "In addition or alternatively a signal may be provided so that a maintenance operative may investigate the drive showing an abnormal torque before failure thereof and/or if desired before changeover to the other motor.", "The couplings may be arranged so that the drive motors are operated alternatively without losing any time in the changeover.", "If desired the condition of the drive and/or of the lift may be monitored by sensing a predetermined torque or a predetermined variation in torque using the torque sensing apparatus.", "A user, such as a maintenance operative, may record a predetermined torque or a predetermined variation in torque in any desired manner by using the output from the torque sensing apparatus.", "If an undesirable increase in torque occurs, then the drive is protected against this by the provision of the above mentioned coupling as a torque limiting coupling.", "If desired the torque indicating device may be disposed at a different location in the drive part from the motor to the sprockets 16 to that illustrated in FIG.", "4.A more detailed description and of various modifications of the torque limiting clutch and of the torque indicating apparatus described hereinbefore are contained in our European patent EP 0310020 B1 and British patent GB 2286055 B respectively the contents of which are incorporated herein by reference.", "If desired, the clutch part of the coupling described hereinbefore may comprise any other suitable form of clutch and for example it may be a clutch which is engaged by springs and disengaged by the application of air or the clutch may be a pin clutch in which pins are axially moveable into inter engagement with sockets to transmit torque or may be a spring clutch or an electromagnetic clutch.", "In the present specification “comprise” means “includes or consists of” and “comprising” means “including or consisting of”.", "The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof." ] ]	Patent_10451571
[ [ "Phage diplay libraries of human vh fragments", "Phage display libraries are taught in which the recombinant phage population displays a plurality of potential binding fragments having preferred characteristics of solubility and/or intermolecular interaction.", "Also taught are methods of biasing display libraries to produce variants which more closely approximate the preferred characteristics of the parental binding fragment." ], [ "1.A library for expression of immunoglobulin heavy chain domains, said library comprising a repertoire of nucleic acid sequences each encoding a polypeptide comprising a VH, said VH comprising a CDR and any one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q, said repertoire comprising a diversity of sequences which differ from one another at least in a subsequence coding for part of the CDR so as to provide nucleic acid encoding a repertoire of polypeptides comprising VHS differing at least in said CDR and comprising at least one amino acid of said group.", "2.A library according to claim 1, wherein said group further comprises 74-A, 83-K, 84 P and wherein said repertoire of nucleic acid sequences each encode a polypeptide comprising a VH which comprises any one, two, three, four, five, six, seven or eight amino acids of said group and include at least one amino acid of the group as defined in claim 1.3.A library according to claim 1, wherein said CDR is a CDR3.4.", "(canceled) 5.A library according to claim 1, wherein said CDRs comprise random sequences.", "6.A library according to claim 1, wherein said nucleic acid encoding a repertoire of immunoglobulin heavy chain variable domains further comprises a subsequence encoding one or more constant domains for expression of Ig-type chains.", "7.", "(canceled) 8.A library which expresses VHS, said library comprising a set of framework regions carrying a diversity of CDR sequences, said library having a diversity of binding activities, said frame work regions comprising one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q.", "9.", "(canceled) 10.A library according to claim 8, which expresses VHS having CDR diversity in only the CDR3 sequences.", "11.A library produced by the method of claim 7.12.", "(canceled) 13.", "(canceled) 14.", "(canceled) 15.", "(canceled) 16.", "(canceled) 17.", "(canceled) 18.A polypeptide comprising a VH, said polypeptide derived from a library according to claim 1.19.A combinatorial library comprising variants of a parental VH ligand binding molecule, wherein said parental ligand binding molecule comprises an immunoglobulin VH fragment comprising at least in substantial part, at least the FR regions of the immunoglobulin VH domain depicted in FIG.", "2 and wherein said variants comprise, at least in substantial part, at least the FR regions of the immunoglobulin VH domain depicted in FIG.", "2 including at least one of amino acid residues 6,23,82a, 93 and 108 depicted in FIG.", "2, and differ from said parental ligand binding molecule in amino acid residues constituting part of at least one of the CDRs of said parental ligand binding molecule.", "20.A library according to claim 19, wherein said variants comprise each of amino acid residues 6,23,82a, 93 and 108 depicted in FIG.", "2 subject only to conservative substitutions which on the whole do not adversely affect the solubility properties of such variants.", "21.", "(canceled) 22.", "(canceled) 23.", "(canceled) 24.", "(canceled) 25.", "(canceled) 26.", "(canceled) 27.", "(canceled) 28.", "(canceled) 29.", "(canceled) 30.", "(canceled) 31.", "(canceled) 32.A library according to claim 19, wherein said parental ligand binding molecule comprises at least in substantial part the FR2 region of the immunoglobulin VH domain depicted in FIG.", "2, including residues 44,45 and 47 depicted in FIG.", "2, and wherein the FR2 regions is at least partially randomized to generate variants having one or more hydrophilic amino acids at VH-VL interface.", "33.", "(canceled) 34.", "(canceled) 35.A library according to claim 20, wherein said variants vary from said parental ligand binding molecule in an amino acids 100i to 100n identified FIG.", "2.36.", "(canceled) 37.A method of identifying a polypeptide comprising a VH which binds to a target ligand, comprising the steps of: (a) screening polypeptide members of the library of claim 1 for their ability to preferentially bind to the target ligand; and (b) identifying at least one polypeptide member which binds to the target ligand.", "38.", "(canceled) 39.An immunoglobulin domain comprising a polypeptide comprising a VH, said VH comprising a CDR and any one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q.", "40.An immunoglobulin domain according to claim 38, wherein said group further comprises 74-A, 83-K, 84-P.", "41.", "(canceled) 42.", "(canceled) 43.", "(canceled) 44.", "(canceled) 45.An immunoglobulin domain comprising a variant of a parental VH ligand binding molecule, wherein said parental ligand binding molecule comprises an immunoglobulin VH fragment comprising at least in substantial part, at least the FR regions of the immunoglobulin VH domain depicted in FIG.", "2 and wherein said variants comprise, at least in substantial part, at least the FR regions of the immunoglobulin VH domain depicted in FIG.", "2 including at least one of amino acid residues 6,23,82a, 93 and 108 depicted in FIG.", "2, and differ from said parental ligand binding molecule in amino acid residues constituting part of at least one of the CDRs of said parental ligand binding molecule.", "46.An immunoglobulin domain according to claim 45, wherein said variant comprises each of amino acid residues 6,23,82a, 93 and 108 depicted in FIG.", "2 subject only to conservative substitutions which on the whole do not adversely affect the solubility properties of said variant.", "47.", "(canceled) 48.", "(canceled) 49.", "(canceled) 50.", "(canceled) 51.", "(canceled) 52.", "(canceled) 53.", "(canceled) 54.", "(canceled) 55.", "(canceled) 56.An immunoglobulin domain according to claim 45, wherein said parental ligand binding molecule comprises at least in substantial part the FR2 region of the immunoglobulin VH domain depicted in FIG.", "2, including residues 44,45 and 47 depicted in FIG.", "2, and wherein the FR2 regions is at least partially randomized to generate variants having one or more hydrophilic amino acids at VH-VL interface.", "57.", "(canceled) 58.", "(canceled)" ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Developments in antibody engineering and recombinant DNA technology have made it possible to generate forms of recombinant antibody fragments which, in many ways, are functional substitutes of larger intact immunoglobulin molecules.", "Single heavy domain antibody fragments (“dAb”) have been the subject of several reports in the patent and scientific literature.", "The literature reports efforts to generate phage display libraries of such fragments for biopanning against a target ligand.", "U.S. Pat.", "No.", "5,702,892 ('892) discloses a phage display library constructed in an M13 derived expression vector, in which recombinant phage of the library contain a polynucleotide encoding a fusion protein which comprises a phage coat protein and an immunoglobulin heavy chain binding-fragment.", "The heavy-chain binding-fragment spans from a position upstream of CDR1 to a position downstream of CDR3.", "'892 describes that the DNA sequence encoding the CDR3 region and/or the CDR1 region may be randomly varied so that the population of phage expresses a series of potential heavy chain binding domains for panning against the target ligand.", "U.S. Pat.", "No.", "5,759,808 discloses a phage display library comprising a population of phage based on random variation of a cDNA sequence obtained from lymphocytes of camelids previously immunized with target antigens.", "Camelid heavy chain antibodies occur naturally, in a composition of about 45%, as heavy chain dimers.", "Heavy chain antibodies specific for a target antigen may be generated by immunizing a member of the camelid species with the target antigen (see Lauwereys et al.", "(1998) The EMBO J.", "17, 3512-3520).", "Hamers-Casterman et al.", "(1993) Nature 363, 446-448 report that camelid heavy chain antibodies are naturally more hydrophilic at amino acid residues at locations 44, 45 and 47 (Kabat numbering system), in FR2, which corresponds to the surface where they normally contact the VL domain Another salient feature of a camelid V H is that it generally has a comparatively longer CDR3 with a high incidence of cysteines and thus may form, via paired cysteines in CDR1 and CDR3, exposed loops, which are more amenable to binding into cavities such as the active site of enzymes and antibodies (Desmyter et al.", "(1996) Nat.", "Struct.", "Biol.", "Vol.", "3, No.", "9, p. 803).", "However, it has been questioned whether single domain antibodies with desired affinities can be generated with such configurations in the absence of prior immunization, i.e.", "with a na ve library (Lauwereys et al.", "(1998) supra).", "The present invention discloses advances in the technology related to creating libraries containing immunoglobulin-like proteins that specifically bind target ligands eg.", "antigens." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>According to one aspect, the invention is directed to a library for expression of immunoglobulin heavy chain domains, said library comprising a repertoire of nucleic acid sequences each encoding a polypeptide comprising a V H , said V H comprising a CDR and any one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q, said repertoire comprising a diversity of sequences which differ from one another at least in a subsequence coding for part of the CDR so as to provide nucleic acid encoding a repertoire of polypeptides comprising V differing at least in said CDR and comprising at least one amino acid of said group.", "In addition to a library of nucleic acid molecules, the term expression library is understood to specifically include a phage, viral, bacterial or other cell surface display library, a ribosome display library or any other functional nucleic acid expression system which permits the expression products to be screened.", "According to another aspect, the invention is directed to a method for generating a V H expression library having a diversity of CDR sequences, said method comprising: providing expression vectors, said vectors comprising a nucleic acid sequence which encodes a polypeptide comprising a V H , said V H comprising a CDR and any one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q; introducing by mutagenesis a diversity of CDR sequences into said V H nucleic acid sequence; and recovering an expression library having a diversity of binding activities.", "According to another aspect, the invention is directed to an expression library which expresses polypeptides comprising Vs, said library comprising a set of framework regions carrying a diversity of CDR sequences, said library having a diversity of binding activities, said frame work regions comprising one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q.", "The invention is also directed to a method of identifying a polypeptide comprising a V H , which binds to a target ligand, the method comprising the steps of screening polypeptide members of this immediately aforementioned expression library for their respective ability to bind to the target ligand; and identifying at least one polypeptide member which binds to the target ligand.", "According to another aspect, the invention is directed to a polypeptide comprising a V H , said polypeptide derived from a library according to any of the preceding paragraphs.", "The invention is directed to a population of variants of at least one parental V H ligand-binding molecule (dAb), wherein said parental V H ligand-binding molecule comprises an immunoglobulin V H binding fragment comprising, at least in substantial part, at least the framework (FR) regions of the immunoglobulin V H fragment depicted FIG.", "2 , wherein said variants comprise at least in substantial part, the FR regions of the immunoglobulin V H fragment depicted FIG.", "2 , including at least one (and preferably all) of amino acid residues, 6, 23, 82a, 93, and 108, and differ from said parental ligand-binding molecule in amino acid residues constituting at least part of at least one of the CDRs of said parental ligand-binding molecule: Preferably said population of variants is constituted by one or more combinatorial libraries of such variants, for example, protein arrays, phage display libraries, ribosome display libraries etc.", "It is to be understood that the variants may (though not necessarily) form part of another structure or molecule, for example in the case of phage display, part of the coat protein of the phage.", "Accordingly, the term variant is used broadly to refer to variants of the essential molecule (a ligand-binding molecule) when forming part of another structure or molecule (eg.", "as in phage display or ribosome display) or when independent of any such combination, eg.", "in the case of protein arrays whose members may not be associated with individual supporting structures/molecules.", "In a particularly preferred embodiment said parental V H ligand binding molecule and said variants comprise each of amino acid residues 6, 23, 82a, 93 and 108, depicted in FIG.", "2 and more preferably each of amino acid residues 6, 23, 74, 82a, 83, 84, 93, and 108 depicted in FIG.", "3 .", "More preferably, said parental V H ligand binding molecules and said variants include the entirety of the FR regions the parental V H ligand molecule depicted in FIG.", "2 or 3 , optionally subject to one or more additions, deletions and/or substitutions, (preferably conservative amino acid substitutions) which, if they do not improve, preferably at least do not adversely affect the solubility properties of said variants.", "Optionally, said parental V H ligand binding molecules and variants additionally include, the entirety of the CDR1 and CDR2 depicted in FIG.", "2 or 3 , optionally subject to one or more additions, deletions and/or substitutions (preferably conservative amino acid substitutions) which, if they do not improve, preferably at least do not adversely affect the solubility properties of said variants.", "In a particularly preferred embodiment, said parental V H ligand molecule and said variants, additionally comprise, the entirety of CDR3 of the parental V H ligand binding molecule depicted in FIG.", "2 with the exception of amino acids 100i to 100n, which are randomized to create said population of variants subject only to one or more additions, deletions, and/or substitutions, (preferably conservative amino acid substitutions) which, if they do not improve, preferably at least do not adversely affect the solubility properties of said variants.", "In another embodiment, said parental V H ligand binding molecule and said variants comprise one or more amino substitutions relative to FIG.", "2 , including any combinations thereof.", "In a further preferred embodiment nucleotides corresponding to amino acid residues 3-16 are optimized to remove a putative recombination site.", "For purposes herein, it is to be understood that an adverse affect on the solubility properties is to be assessed in terms of the percentage of dimer and higher aggregate forms, relative to monomer, as determined by size exclusion chromatography (i.e.", "the respective areas under the peaks representing monomer, dimer and higher aggregate forms, as illustrated in FIG.", "5 ).", "In another aspect, the invention is directed to a ligand-binding molecule or variant which has been identified as binding to a target ligand by screening a combinatorial library of the invention for one or more ligand-binding molecules which specifically recognize said target ligand.", "The invention is also directed more generally to any specific such ligand-binding molecule which is derived from such combinatorial library of the invention.", "It is understood herein that such specific ligand-binding molecule may be directly obtained from such a library or may be indirectly derived, for example, through the course of further antibody engineering or other modification steps (eg.", "creating fragments, derivatives, a secondary library, etc) using a ligand-binding molecule directly or indirectly obtained from such library.", "It also understood that the invention excludes known ligand-binding molecules.", "In one embodiment of this aspect of the invention the target ligand is a cancer antigen.", "FIGS.", "2, 3 and 4 depict variations, more fully described below, on the preferred immunoglobulin V H ligand binding fragment and/or nucleic acid construct depicted in FIG.", "1 .", "FIG.", "1 describes a wild-type parental immunoglobulin V H binding fragment derived from human monoclonal antibody BT32/A6 (hereinafter referred to as “A6”) partially described in U.S. Pat.", "No.", "5,639,863 (hereinafter referred to as the '863 patent).", "As described in our co-pending application PCT/CA00/01027, the contents of which are hereby incorporated by reference, A6 has preferred solubility and other characteristics which lend themselves well to the creation of libraries, including naive libraries, of various types of human immunoglobulin fragments including dAbs as more fully described below.", "Accordingly, A6, and in particular, as more fully described below, at least a substantial part of the framework (FR) regions of the A6 V H fragment depicted in FIG.", "2 , alone or in combination with features of its CDR3, provides a useful departure point, in the form of a parental ligand-binding molecule, for the randomization or partial randomization of amino acid residues which tend to play a predominant role in ligand-binding, namely the CDR regions of the heavy chain and particularly the CDR3 of the heavy chain.", "As more fully described below, the nucleic acid changes (removal of the recombination site) relative to A6 wild-type FIG.", "1 ) reflected in FIG.", "4 may be incorporated into FIGS.", "2 and 3 respectively.", "The combinatorial library of the invention may be generated by phage display.", "In a preferred embodiment of one aspect of the invention, the invention is directed to a phage display library displaying a plurality of different variants of a parental V H ligand-binding molecule, wherein said parental ligand-binding molecule comprises an immunoglobulin V H binding fragment comprising, at least in substantial part, at least the FR regions of the immunoglobulin V H fragment depicted in FIG.", "3 including at least one of amino acid residues 6, 23, 74, 82a, 83, 84, 93 and 108, and wherein said variants are encoded by nucleic acid sequences which vary from the nucleic acid sequence encoding said parental ligand-binding molecule in a subsequence (at least one) encoding at least part of one of the CDRs of said parental ligand-binding molecule, preferably the CDR3, whereby said plurality of variants comprise at least, in substantial part, the FR regions of the immunoglobulin V H fragment depicted in FIG.", "3 , including at least one of said amino acid residues and are differentiated, at least in part, by amino acid variations encoded by variations in said subsequence.", "In a preferred embodiment, in addition to substantial preservation and optional improvement of the FR regions of A6, the A6-based parental ligand molecule comprises (and therefore preserves within members of the library), in substantial part (subject to at least partial randomization of selected regions of one of the CDRs (preferably the CDR3), to create binding diversity within the library, one or more of the CDR regions of the A6 V H fragment.", "In a further preferred embodiment, at least the length of the wild-type V H CDR3 (23 amino acids) and preferably also elements of its amino acid composition, is preserved or at least partially preserved (approximately 16-23 amino acids and more particularly 18 to 23 amino acids).", "Optionally the CDR3 may also be lengthened by approximately 1 to 10 residues.", "The library may optionally have representation of binding molecules having CDR3s of varying lengths.", "It is known that a dAb molecule, due to the removal of its light chain partner, tends to, in most, if not all cases, aggregate, in varying degrees due to the “sticky” nature of the VL interface.", "This stickiness is attributable, at least in part, to the hydrophobic nature of the V H residues at this interface.", "This stickiness results in substantial dimer and/or multimer formation which may reduce, on the whole, the solubility characteristics of members of the library.", "Accordingly, in a further preferred aspect of the invention A6 V H amino acid residues at the VL interface are substituted by residues which tend to minimize aggregate formation, for example, hydrophilic amino acids, including one or more of the highlighted substitutions reflected in FIG.", "8 , relative to FIG.", "1 .", "Alternatively, in yet a further preferred embodiment of the invention, more fully described below, such substitutions are not fixed within the entire population of the library, but are introduced by randomizing or partially randomizing various A6 V H amino acid residues, particularly including FR residues, among the residues at the interface.", "(see for example, Padlan et al “Anatomy of the Antibody Molecule” Molecular Immunology Vol.", "31, p 169-217, Table 25 for itemization and related discussion of these residues).", "Particularly in the case of larger size libraries, for example those generated by ribosome display, it is possible to introduce diversity in one or more FR regions containing amino acid residues identified herein as important for improving solubility properties, in addition to one or more regions affecting the specificity of the dAb, eg.", "the V H CDR3 and CDR1.Alternatively, in yet a further preferred embodiment of the invention, FR regions, other than, or in addition to, modifications to the VL interface (FR2) may be modified by at least partial randomization, for example, one or both of FR1 (one or more of residues 4 to 21) and FR4 (one or more of residues 100o to 113) to improve, on the whole, the solubility characteristics of members of the library (for example, biasing at least some and preferably all of one or both of these sets of residues [at least 70% or more], preferably 90% in favour of the parental amino acid constitution to achieve 10% randomization).", "In the case of A6 dAb fragments, it has been found that recombination events within the nucleic acid sequence encoding the V H binding fragment tend to result in deletions yielding shorter molecules, with possible compromise in binding characteristics.", "Thus, in a further preferred aspect of the invention nucleic acid sequences which promote such recombination events (at putative recombination sites) are substituted, to oppose this tendency, preferably in a manner that does not result in an amino acid change.", "These changes may be incorporated, for example, into the nucleotide sequences encoding the parental V H ligand-binding molecule depicted in FIG.", "2 or 3 .", "In particularly preferred aspects, the present invention provides a heterogeneous population of genetic packages (eg.", "phage) having a genetically determined outer surface protein, wherein the genetic packages collectively display a plurality of different, preferably human (i.e.", "having substantial identity, preferably at least 80% homology to human framework and other conserved regions) V H ligand-binding fragments, each genetic package including a nucleic acid construct coding for a fusion protein which comprises at least a portion of the outer surface protein and a variant of at least one soluble parental ligand-binding fragment preferably derived from or having a substantial part of the FR regions of the amino acid sequence identified in one of FIG.", "2 or 3 (or a sequence at least 80%, preferably 85 to 100%, more preferably 90-100%, homologous (% identity) thereto), including at least one and preferably all of amino acid residues 6, 23, 82a, 93 and 108 depicted in FIG.", "2 or 3 , wherein the variant V H ligand-binding fragments preferably span from a position upstream of an immunoglobulin heavy chain CDR1 to a position downstream of CDR3 (preferably including substantially all of FR1 and/or FR4), and wherein at least part of a CDR, preferably the CDR3, is a randomly generated variant of a CDR of said parental V H ligand binding-fragment and wherein the fusion protein is preferably expressed in the absence of an immunoglobulin light chain whereby the variant V H ligand-binding fragments are, on the whole, better adapted to be or better capable of being expressed as soluble proteins.", "Various combinations of 2, 3 and 4 substitutions relative to wild-type A6 at amino acid residues 6, 23, 82a, 93 and 108 are also contemplated by the invention.", "In yet another embodiment of the invention, by biasing the amino acid constitution, preferably on an individual amino acid by amino acid basis, in favor of the wild-type or parental amino acid constitution, even portions of the parental ligand-binding molecule that are randomized in favor of generating variability in the variant binding fragments can be engineered to maintain favorable solubility characteristics of the parental binding domain.", "In one embodiment of the invention, a portion of the construct encoding at least part of the CDR3 is biased or partially biased in favor of the parental amino acid constitution.", "In a further preferred embodiment, the parental V H binding-fragment naturally has a long CDR3 that is amenable to forming exposed loops for binding into cavities.", "In a most preferred embodiment, the parental V H ligand-binding fragment is built on a human framework or is adapted from or adaptable to a human framework.", "In another preferred embodiment, the preferred binding region of the variants (corresponding to the randomized or partially randomized part of the CDR3) is located in carboxy terminal region of the CDR3.In summary, according to a preferred embodiment of the invention, a substantial part of the amino acid sequence identified in FIG.", "2 , preferably including at least part of the CDR3, supplies the preferred amino acid constitution of the various preferred parental ligand-binding molecules, such that a population of variant heavy chain ligand-binding molecules built on this framework of amino acids are on the whole better adapted to be or better capable of being expressed as soluble proteins.", "Generally, the importance of amino acid residues 6, 23, 82a, 93, and 108, shown in FIG.", "2 , particularly when all five are combined, is that they can be used to significantly augment the solubility properties of a parental V H ligand binding fragment, preferably one, like A6, that has useful solubility properties to begin with, to produce a library of dAb variants for paining against a target ligand, said variants on the whole being better adopted to be or better capable of being expressed as soluble proteins." ], [ "FIELD OF THE INVENTION The present invention relates to combinatorial libraries including phage display libraries which display single domain heavy chain binding fragments having preferred characteristics of solubility.", "BACKGROUND OF THE INVENTION Developments in antibody engineering and recombinant DNA technology have made it possible to generate forms of recombinant antibody fragments which, in many ways, are functional substitutes of larger intact immunoglobulin molecules.", "Single heavy domain antibody fragments (“dAb”) have been the subject of several reports in the patent and scientific literature.", "The literature reports efforts to generate phage display libraries of such fragments for biopanning against a target ligand.", "U.S. Pat.", "No.", "5,702,892 ('892) discloses a phage display library constructed in an M13 derived expression vector, in which recombinant phage of the library contain a polynucleotide encoding a fusion protein which comprises a phage coat protein and an immunoglobulin heavy chain binding-fragment.", "The heavy-chain binding-fragment spans from a position upstream of CDR1 to a position downstream of CDR3.", "'892 describes that the DNA sequence encoding the CDR3 region and/or the CDR1 region may be randomly varied so that the population of phage expresses a series of potential heavy chain binding domains for panning against the target ligand.", "U.S. Pat.", "No.", "5,759,808 discloses a phage display library comprising a population of phage based on random variation of a cDNA sequence obtained from lymphocytes of camelids previously immunized with target antigens.", "Camelid heavy chain antibodies occur naturally, in a composition of about 45%, as heavy chain dimers.", "Heavy chain antibodies specific for a target antigen may be generated by immunizing a member of the camelid species with the target antigen (see Lauwereys et al.", "(1998) The EMBO J.", "17, 3512-3520).", "Hamers-Casterman et al.", "(1993) Nature 363, 446-448 report that camelid heavy chain antibodies are naturally more hydrophilic at amino acid residues at locations 44, 45 and 47 (Kabat numbering system), in FR2, which corresponds to the surface where they normally contact the VL domain Another salient feature of a camelid VH is that it generally has a comparatively longer CDR3 with a high incidence of cysteines and thus may form, via paired cysteines in CDR1 and CDR3, exposed loops, which are more amenable to binding into cavities such as the active site of enzymes and antibodies (Desmyter et al.", "(1996) Nat.", "Struct.", "Biol.", "Vol.", "3, No.", "9, p. 803).", "However, it has been questioned whether single domain antibodies with desired affinities can be generated with such configurations in the absence of prior immunization, i.e.", "with a nave library (Lauwereys et al.", "(1998) supra).", "The present invention discloses advances in the technology related to creating libraries containing immunoglobulin-like proteins that specifically bind target ligands eg.", "antigens.", "SUMMARY OF THE INVENTION According to one aspect, the invention is directed to a library for expression of immunoglobulin heavy chain domains, said library comprising a repertoire of nucleic acid sequences each encoding a polypeptide comprising a VH, said VH comprising a CDR and any one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q, said repertoire comprising a diversity of sequences which differ from one another at least in a subsequence coding for part of the CDR so as to provide nucleic acid encoding a repertoire of polypeptides comprising V differing at least in said CDR and comprising at least one amino acid of said group.", "In addition to a library of nucleic acid molecules, the term expression library is understood to specifically include a phage, viral, bacterial or other cell surface display library, a ribosome display library or any other functional nucleic acid expression system which permits the expression products to be screened.", "According to another aspect, the invention is directed to a method for generating a VH expression library having a diversity of CDR sequences, said method comprising: providing expression vectors, said vectors comprising a nucleic acid sequence which encodes a polypeptide comprising a VH, said VH comprising a CDR and any one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q; introducing by mutagenesis a diversity of CDR sequences into said VH nucleic acid sequence; and recovering an expression library having a diversity of binding activities.", "According to another aspect, the invention is directed to an expression library which expresses polypeptides comprising Vs, said library comprising a set of framework regions carrying a diversity of CDR sequences, said library having a diversity of binding activities, said frame work regions comprising one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q.", "The invention is also directed to a method of identifying a polypeptide comprising a VH, which binds to a target ligand, the method comprising the steps of screening polypeptide members of this immediately aforementioned expression library for their respective ability to bind to the target ligand; and identifying at least one polypeptide member which binds to the target ligand.", "According to another aspect, the invention is directed to a polypeptide comprising a VH, said polypeptide derived from a library according to any of the preceding paragraphs.", "The invention is directed to a population of variants of at least one parental VH ligand-binding molecule (dAb), wherein said parental VH ligand-binding molecule comprises an immunoglobulin VH binding fragment comprising, at least in substantial part, at least the framework (FR) regions of the immunoglobulin VH fragment depicted FIG.", "2, wherein said variants comprise at least in substantial part, the FR regions of the immunoglobulin VH fragment depicted FIG.", "2, including at least one (and preferably all) of amino acid residues, 6, 23, 82a, 93, and 108, and differ from said parental ligand-binding molecule in amino acid residues constituting at least part of at least one of the CDRs of said parental ligand-binding molecule: Preferably said population of variants is constituted by one or more combinatorial libraries of such variants, for example, protein arrays, phage display libraries, ribosome display libraries etc.", "It is to be understood that the variants may (though not necessarily) form part of another structure or molecule, for example in the case of phage display, part of the coat protein of the phage.", "Accordingly, the term variant is used broadly to refer to variants of the essential molecule (a ligand-binding molecule) when forming part of another structure or molecule (eg.", "as in phage display or ribosome display) or when independent of any such combination, eg.", "in the case of protein arrays whose members may not be associated with individual supporting structures/molecules.", "In a particularly preferred embodiment said parental VH ligand binding molecule and said variants comprise each of amino acid residues 6, 23, 82a, 93 and 108, depicted in FIG.", "2 and more preferably each of amino acid residues 6, 23, 74, 82a, 83, 84, 93, and 108 depicted in FIG.", "3.More preferably, said parental VH ligand binding molecules and said variants include the entirety of the FR regions the parental VH ligand molecule depicted in FIG.", "2 or 3, optionally subject to one or more additions, deletions and/or substitutions, (preferably conservative amino acid substitutions) which, if they do not improve, preferably at least do not adversely affect the solubility properties of said variants.", "Optionally, said parental VH ligand binding molecules and variants additionally include, the entirety of the CDR1 and CDR2 depicted in FIG.", "2 or 3, optionally subject to one or more additions, deletions and/or substitutions (preferably conservative amino acid substitutions) which, if they do not improve, preferably at least do not adversely affect the solubility properties of said variants.", "In a particularly preferred embodiment, said parental VH ligand molecule and said variants, additionally comprise, the entirety of CDR3 of the parental VH ligand binding molecule depicted in FIG.", "2 with the exception of amino acids 100i to 100n, which are randomized to create said population of variants subject only to one or more additions, deletions, and/or substitutions, (preferably conservative amino acid substitutions) which, if they do not improve, preferably at least do not adversely affect the solubility properties of said variants.", "In another embodiment, said parental VH ligand binding molecule and said variants comprise one or more amino substitutions relative to FIG.", "2, including any combinations thereof.", "In a further preferred embodiment nucleotides corresponding to amino acid residues 3-16 are optimized to remove a putative recombination site.", "For purposes herein, it is to be understood that an adverse affect on the solubility properties is to be assessed in terms of the percentage of dimer and higher aggregate forms, relative to monomer, as determined by size exclusion chromatography (i.e.", "the respective areas under the peaks representing monomer, dimer and higher aggregate forms, as illustrated in FIG.", "5).", "In another aspect, the invention is directed to a ligand-binding molecule or variant which has been identified as binding to a target ligand by screening a combinatorial library of the invention for one or more ligand-binding molecules which specifically recognize said target ligand.", "The invention is also directed more generally to any specific such ligand-binding molecule which is derived from such combinatorial library of the invention.", "It is understood herein that such specific ligand-binding molecule may be directly obtained from such a library or may be indirectly derived, for example, through the course of further antibody engineering or other modification steps (eg.", "creating fragments, derivatives, a secondary library, etc) using a ligand-binding molecule directly or indirectly obtained from such library.", "It also understood that the invention excludes known ligand-binding molecules.", "In one embodiment of this aspect of the invention the target ligand is a cancer antigen.", "FIGS.", "2, 3 and 4 depict variations, more fully described below, on the preferred immunoglobulin VH ligand binding fragment and/or nucleic acid construct depicted in FIG.", "1.FIG.", "1 describes a wild-type parental immunoglobulin VH binding fragment derived from human monoclonal antibody BT32/A6 (hereinafter referred to as “A6”) partially described in U.S. Pat.", "No.", "5,639,863 (hereinafter referred to as the '863 patent).", "As described in our co-pending application PCT/CA00/01027, the contents of which are hereby incorporated by reference, A6 has preferred solubility and other characteristics which lend themselves well to the creation of libraries, including naive libraries, of various types of human immunoglobulin fragments including dAbs as more fully described below.", "Accordingly, A6, and in particular, as more fully described below, at least a substantial part of the framework (FR) regions of the A6 VH fragment depicted in FIG.", "2, alone or in combination with features of its CDR3, provides a useful departure point, in the form of a parental ligand-binding molecule, for the randomization or partial randomization of amino acid residues which tend to play a predominant role in ligand-binding, namely the CDR regions of the heavy chain and particularly the CDR3 of the heavy chain.", "As more fully described below, the nucleic acid changes (removal of the recombination site) relative to A6 wild-type FIG.", "1) reflected in FIG.", "4 may be incorporated into FIGS.", "2 and 3 respectively.", "The combinatorial library of the invention may be generated by phage display.", "In a preferred embodiment of one aspect of the invention, the invention is directed to a phage display library displaying a plurality of different variants of a parental VH ligand-binding molecule, wherein said parental ligand-binding molecule comprises an immunoglobulin VH binding fragment comprising, at least in substantial part, at least the FR regions of the immunoglobulin VH fragment depicted in FIG.", "3 including at least one of amino acid residues 6, 23, 74, 82a, 83, 84, 93 and 108, and wherein said variants are encoded by nucleic acid sequences which vary from the nucleic acid sequence encoding said parental ligand-binding molecule in a subsequence (at least one) encoding at least part of one of the CDRs of said parental ligand-binding molecule, preferably the CDR3, whereby said plurality of variants comprise at least, in substantial part, the FR regions of the immunoglobulin VH fragment depicted in FIG.", "3, including at least one of said amino acid residues and are differentiated, at least in part, by amino acid variations encoded by variations in said subsequence.", "In a preferred embodiment, in addition to substantial preservation and optional improvement of the FR regions of A6, the A6-based parental ligand molecule comprises (and therefore preserves within members of the library), in substantial part (subject to at least partial randomization of selected regions of one of the CDRs (preferably the CDR3), to create binding diversity within the library, one or more of the CDR regions of the A6 VH fragment.", "In a further preferred embodiment, at least the length of the wild-type VH CDR3 (23 amino acids) and preferably also elements of its amino acid composition, is preserved or at least partially preserved (approximately 16-23 amino acids and more particularly 18 to 23 amino acids).", "Optionally the CDR3 may also be lengthened by approximately 1 to 10 residues.", "The library may optionally have representation of binding molecules having CDR3s of varying lengths.", "It is known that a dAb molecule, due to the removal of its light chain partner, tends to, in most, if not all cases, aggregate, in varying degrees due to the “sticky” nature of the VL interface.", "This stickiness is attributable, at least in part, to the hydrophobic nature of the VH residues at this interface.", "This stickiness results in substantial dimer and/or multimer formation which may reduce, on the whole, the solubility characteristics of members of the library.", "Accordingly, in a further preferred aspect of the invention A6 VH amino acid residues at the VL interface are substituted by residues which tend to minimize aggregate formation, for example, hydrophilic amino acids, including one or more of the highlighted substitutions reflected in FIG.", "8, relative to FIG.", "1.Alternatively, in yet a further preferred embodiment of the invention, more fully described below, such substitutions are not fixed within the entire population of the library, but are introduced by randomizing or partially randomizing various A6 VH amino acid residues, particularly including FR residues, among the residues at the interface.", "(see for example, Padlan et al “Anatomy of the Antibody Molecule” Molecular Immunology Vol.", "31, p 169-217, Table 25 for itemization and related discussion of these residues).", "Particularly in the case of larger size libraries, for example those generated by ribosome display, it is possible to introduce diversity in one or more FR regions containing amino acid residues identified herein as important for improving solubility properties, in addition to one or more regions affecting the specificity of the dAb, eg.", "the VH CDR3 and CDR1.Alternatively, in yet a further preferred embodiment of the invention, FR regions, other than, or in addition to, modifications to the VL interface (FR2) may be modified by at least partial randomization, for example, one or both of FR1 (one or more of residues 4 to 21) and FR4 (one or more of residues 100o to 113) to improve, on the whole, the solubility characteristics of members of the library (for example, biasing at least some and preferably all of one or both of these sets of residues [at least 70% or more], preferably 90% in favour of the parental amino acid constitution to achieve 10% randomization).", "In the case of A6 dAb fragments, it has been found that recombination events within the nucleic acid sequence encoding the VH binding fragment tend to result in deletions yielding shorter molecules, with possible compromise in binding characteristics.", "Thus, in a further preferred aspect of the invention nucleic acid sequences which promote such recombination events (at putative recombination sites) are substituted, to oppose this tendency, preferably in a manner that does not result in an amino acid change.", "These changes may be incorporated, for example, into the nucleotide sequences encoding the parental VH ligand-binding molecule depicted in FIG.", "2 or 3.In particularly preferred aspects, the present invention provides a heterogeneous population of genetic packages (eg.", "phage) having a genetically determined outer surface protein, wherein the genetic packages collectively display a plurality of different, preferably human (i.e.", "having substantial identity, preferably at least 80% homology to human framework and other conserved regions) VH ligand-binding fragments, each genetic package including a nucleic acid construct coding for a fusion protein which comprises at least a portion of the outer surface protein and a variant of at least one soluble parental ligand-binding fragment preferably derived from or having a substantial part of the FR regions of the amino acid sequence identified in one of FIG.", "2 or 3 (or a sequence at least 80%, preferably 85 to 100%, more preferably 90-100%, homologous (% identity) thereto), including at least one and preferably all of amino acid residues 6, 23, 82a, 93 and 108 depicted in FIG.", "2 or 3, wherein the variant VH ligand-binding fragments preferably span from a position upstream of an immunoglobulin heavy chain CDR1 to a position downstream of CDR3 (preferably including substantially all of FR1 and/or FR4), and wherein at least part of a CDR, preferably the CDR3, is a randomly generated variant of a CDR of said parental VH ligand binding-fragment and wherein the fusion protein is preferably expressed in the absence of an immunoglobulin light chain whereby the variant VH ligand-binding fragments are, on the whole, better adapted to be or better capable of being expressed as soluble proteins.", "Various combinations of 2, 3 and 4 substitutions relative to wild-type A6 at amino acid residues 6, 23, 82a, 93 and 108 are also contemplated by the invention.", "In yet another embodiment of the invention, by biasing the amino acid constitution, preferably on an individual amino acid by amino acid basis, in favor of the wild-type or parental amino acid constitution, even portions of the parental ligand-binding molecule that are randomized in favor of generating variability in the variant binding fragments can be engineered to maintain favorable solubility characteristics of the parental binding domain.", "In one embodiment of the invention, a portion of the construct encoding at least part of the CDR3 is biased or partially biased in favor of the parental amino acid constitution.", "In a further preferred embodiment, the parental VH binding-fragment naturally has a long CDR3 that is amenable to forming exposed loops for binding into cavities.", "In a most preferred embodiment, the parental VH ligand-binding fragment is built on a human framework or is adapted from or adaptable to a human framework.", "In another preferred embodiment, the preferred binding region of the variants (corresponding to the randomized or partially randomized part of the CDR3) is located in carboxy terminal region of the CDR3.In summary, according to a preferred embodiment of the invention, a substantial part of the amino acid sequence identified in FIG.", "2, preferably including at least part of the CDR3, supplies the preferred amino acid constitution of the various preferred parental ligand-binding molecules, such that a population of variant heavy chain ligand-binding molecules built on this framework of amino acids are on the whole better adapted to be or better capable of being expressed as soluble proteins.", "Generally, the importance of amino acid residues 6, 23, 82a, 93, and 108, shown in FIG.", "2, particularly when all five are combined, is that they can be used to significantly augment the solubility properties of a parental VH ligand binding fragment, preferably one, like A6, that has useful solubility properties to begin with, to produce a library of dAb variants for paining against a target ligand, said variants on the whole being better adopted to be or better capable of being expressed as soluble proteins.", "BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described with reference to the drawings, wherein: FIG.", "1 is a sequence diagram showing the A6 VH ligand-binding molecule from which we construct parental VH ligand binding molecules according to the invention.", "FIG.", "2 is a sequence diagram showing a preferred parental VH ligand-binding molecule (A6VH-L1) according to the invention, incorporating amino acid substitutions E6A, S23A, S82aN, 93A and T108Q.", "FIG.", "3 is a sequence diagram showing the A6VH-L2 ligand-binding molecule according to the invention, incorporating 3 additional amino acid substitutions at positions 74, 83 and 84.FIG.", "4 is a sequence diagram showing the A6VH ligand-binding molecule (encoded by A6 chi (−)) according to the invention, in which nucleic acids corresponding to amino acids 3 to 16 of A6 have been modified to remove a putative recombination site, leaving the amino acid constitution of A6 unchanged.", "FIG.", "5 is a size exclusion chromatogram of A6VH-L1 dAb following IMAC purification comparing A6VH-L1 (plot A) to wildtype A6VH (plot B) and showing a much greater proportion of monomer (represented by the largest peak).", "Plot C represents a camelized version of A6 with amino acid substitutions of positions 44, 45, 47 and 94 as described herein and in the literature.", "FIG.", "6 is a size exclusion chromatogram of Yst9.1-L3-9, which was used for binding against Yst9.1 scFv, depicted in FIG.", "7.FIG.", "7 is a sensorgram showing the binding of 10 (M Yst9.1-L3-9 binding to immobilized Yst9.1 scFv.", "The control surface (BSA) data has been subtracted from the active surface (Yst9.1) data in this sensorgram.", "The rate constants were calculated by separate fitting of the association (fit shown) and dissociation data to a 1:1 interaction model.", "FIG.", "8 is a sequence diagram showing amino acid substitutions for optional camelization according to one embodiment of the invention.", "FIG.", "9 is a diagrammatic representation of vector, SJFI used to create the vector into which the library may be cloned.", "FIG.", "10 is a sequence diagram depicting the library construction strategy for library A6VH-L1.FIG.", "11 is a sequence diagram depicting the library construction strategy for library A6VH-L1a.", "DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS In a preferred embodiment, the invention is directed to a population of genetic packages having a genetically determined outer surface protein including genetic packages which collectively display a plurality of different ligand-binding molecules in association with the outer surface protein, each package including a nucleic acid construct coding for a fusion protein which is at least a portion of the outer surface protein and a variant of at least one soluble parental ligand-binding molecule derived from or having the amino acid sequence identified in FIG.", "2 (or a sequence preferably at least 80% homologous to the framework and conserved regions thereof), which includes at least one and preferably all of amino acids 6, 23 82a, 94 and 108 shown in FIG.", "2 (or conservative substitutions of those residues which, if they do not improve, at least do not adversely affect the solubility properties of said parental VH ligand binding molecule), wherein at least part of the construct, preferably including at least part of the CDR3 identified in FIG.", "2, encodes or is biased in favor of encoding, the amino acid constitution of the parental ligand binding fragment such that the plurality of different ligand-binding domains are on the whole better adapted to be or better capable of being expressed as soluble proteins.", "The variant VH ligand-binding molecules are preferably characterized by a CDR3 having 16 to 33 amino acids.", "Preferably, the replicable genetic package is a recombinant phage and the heterogeneous population of replicable genetic packages collectively constitute a phage display library.", "In a further embodiment, the VL interface is engineered to avoid hydrophobic amino acids.", "In another embodiment, the VL interface is engineered for amino acids, which form weak interactions.", "In another embodiment the parental ligand binding molecule has a camelid type VL interface.", "In another embodiment, at least one of the VL interface amino acids are randomized or partially randomized in the construction of the library.", "Preferably the potential VH binding fragments include the entire FR1 through to FR4 regions, although it is to be understood that partial deletions, for example, within CDR2, are contemplated to be within the scope of the invention.", "Preferably, CDR3s of a variety of different lengths from 16 to 33 amino acids are predominantly represented among the potential VH binding fragments.", "Preferably CDR3s of a variety of different lengths, from 18 to 28 amino acids, or from 20 to 25, or from 18 to 23, amino acids are predominantly represented in the library.", "In a preferred embodiment of the invention, the parental VH ligand-binding fragment is built on a human framework and preferably is the parental VH ligand-binding fragment identified in FIG.", "2 which has a CDR3 of 23 amino acids in length.", "More importantly, the invention encompasses a phage display library which is constructed using a parental VH ligand-binding molecule derived from native A6VH (FIG.", "1), or is built on any framework which is at least 80% (preferably 85%, more preferably 90 to 95%) homologous to the framework and other conserved regions of a fully human VH chain.", "The invention also contemplates that the parental VH binding-fragment, though not human, is adapted (egg.", "humanized) or adaptable (egg.", "to be adapted after selection of preferred binders) to a human framework.", "In another embodiment, the invention also contemplates the random, biased or fixed occurrence of features disclosed in the camelid literature, for example pairable cysteines in CDR1 and CDR3 (optional) and/or the substitution of hydrophilic amino acids at least one of positions 44, 45, and 47 and preferably also positions 93 and 94 (Kabat numbering system).", "In a most preferred embodiment of the invention, the parental ligand-binding molecule is a VH fragment derived from a human IgM heavy chain, and preferably comprises FR1 through FR4 of the VH chain.", "A partial sequence of the preferred antibody BT32/A6 (A6) is disclosed in U.S. Pat.", "No.", "5,639,863, incorporated herein by reference.", "The entire sequence is supplied now in FIG.", "1.In FIG.", "1, the CDR regions are demarcated.", "The amino acid residue numbers in FIG.", "1 and throughout the disclosure refer to the Kabat numbering system (Kabat et al.", "1991, Sequences of Proteins of Immunological Interest, publication No.", "91-3242, U.S. Public Health Services, NIH, Bethesda Md.)", "except in the sequence listings and where explicitly stated or otherwise implied.", "FIG.", "1 corresponds to SEQ.", "ID.", "NO.", "1 (nucleic acid) and SEQ.", "ID.", "NO.", "2 (amino acid).", "FIG.", "1 demarcates and labels regions CDR1 (corresponding to SEQ.", "ID.", "NO.", "3), CDR2 (SEQ.", "ID.", "NO.", "4) and CDR3 (SEQ.", "ID.", "NO.", "5).", "The A6 framework provides preferred solubility characteristics for creating dAb libraries.", "The term preferred solubility characteristics, as used herein, refers to at least one of the several, often correlated, characteristics including good yield, expression as a soluble product (as opposed to inclusion bodies) within the periplasm of the host organism, eg.", "Escherichia Coli, and a reduced tendency to dimerize and form other aggregates.", "The term “VH” is used to refer to the variable heavy chain domain and variants thereof made by routine skill in the art, including known forms, truncated and fusion protein forms thereof, which variants retain its essential binding characteristics.", "This term is used interchangeably with the terms such as VH binding fragment, VH ligand binding fragment, dAb and immunoglobulin VH fragment, to which the same meaning is ascribed.", "The term “subsequence” is used to refer to a subset of nucleotides within a referenced nucleic acid sequence and includes a single nucleotide and consecutive and non-consecutive nucleotides within a sequence of greater than two consecutive nucleotides.", "The terms “polypeptide”, “peptide” and “protein”, unless the context implies otherwise, are used interchangeably herein, to refer to polymers of amino acid residues of any length.", "The term “combinatorial library” is used herein to refer to a set of molecules, typically belonging to a defined (narrowly or broadly) class comprising a substantial number of potentially useful variants, wherein the variations in the molecule represent a complete or partial set of permutations or combinations of at least some constituent elements of a reference molecule, which is typically a template or “parental” molecule, or simply the class itself.", "For clarity, in the case of polypeptides and nucleic acids, the constituent elements are amino acids and nucleic acid bases, respectively.", "As used herein, the phrase “in substantial part” refers to variations relative to a referenced molecule which do not significantly impair the “functionality” of that molecule.", "In the case of the parental ligand-binding molecule and variants thereof functionality refers primarily to the solubility and binding characteristics of the molecule.", "Such variations (i.e.", "the referenced molecule in substantial part) can be tested systematically to assess their impact.", "In the case of framework regions, in contrast to CDR regions, due to the substantial conservation of the framework amino acid residues, a substantial part of the framework would preferably refer to at least 80% identity of the amino acid residues and more preferably an 85 to 100% identity, and even more preferably at least a 90% identity of the amino acid residues.", "However, it is understood that each of the previous percentages could be relaxed to discount instances where the absence of identity in a given residue, is due to a well recognized conservative amino acid substitution, or where a particular class of functionality is noted, e.g.", "hydrophilic, if the substitution is with a residue of the same class.", "In the case of CDR residues, these numbers could be considerably even more relaxed, and includes 50 to 100% homology, and all incremental percentages and all ranges of percentages therebetween.", "The term “in substantial part”, in reference to portions of framework and CDR regions, also contemplates the possibility of additions and deletions in those regions which preferably do not impact the solubility and binding characteristics of the ligand-binding molecule in question.", "The term ligand-binding fragment is used broadly to define the whole or any part of an antibody that is capable of specifically binding to any ligand, in the broadest sense of the term ligand.", "An A6-based human heavy domain ligand-binding-fragment is well suited for the development of a combinatorial library (optionally a phage display library) that is used to generate soluble binding fragments that are useful for human diagnosis and therapy (due to limited HAMA response).", "These phage display libraries are used to selectively generate molecular probes that specifically interact with a ligand, including without limitation, natural and synthetic molecules and macromolecules and can be used in vitro (i.e., a diagnostic) and in vivo (i.e., a diagnostic and/or therapeutic) as indicators, inhibitors and immunological agents.", "The types of natural and synthetic molecules and macromolecules include but are not limited to: antibodies and fragments thereof; enzymes; cell receptors; proteins, polypeptides, peptides; polynucleotides, oligonucleotides; carbohydrates such as polysaccharides, oligosaccharides, saccharides; lipids; organic-based and inorganic-based molecules such as antibiotics, steroids, hormones, pesticides, herbicides, dyes, polymers.", "Conventional antibodies such as those found in human or murine species are composed of two identical light chains and two identical heavy chains.", "The combining sites of these antibodies are formed by association of the variable domains of both chains.", "This association is mediated through hydrophobic interactions at the interface.", "Structural and biochemical studies have shown that the heavy chain variable domain (VH) provides most of the antigen-contacting residues (Padlan, 1994) (Chothia & Lesk, 1987) (Chothia, Novotny, et al., 1985).", "This finding has formed the basis for the development of single heavy domain antibodies (dAbs)—recombinant antigen binding fragments consisting of only the VH (Ward, Gussow, et al., 1989) (Cai & Garen, 1996).", "However, in the absence of their VL partners, VHs have been found to be insoluble, presumably because of the exposed hydrophobic VL interface (Ward, Gussow, et al., 1989).", "Heavy chain antibodies, found in camelids (Hamers, Atarhouch, et al., 1993) (Sheriff & Constantine,), lack light chains and as a result have variable domains that reflect the absence of a VL partner.", "Single domain antibodies derived from these antibodies are highly soluble and the structural basis of solubility has been partially elucidated.", "First, conserved human/murine interface residues such as Val37, Gly44, Leu45 and Trp47 are generally replaced in heavy chain antibodies by tyrosine or phenylalanine, glutamate, arginine or cysteine, and glycine, respectively.", "These mutations increase the hydrophilicity of the VL interface either by non-polar to polar substitutions or, in a more subtle way, by inducing local conformational changes (Desmyter, Transue, et al., 1996) (Spinelli, Frenken, et al.,).", "This explanation is supported by experiments in which an insoluble human VH was made soluble by introducing the aforementioned mutations at positions 44, 45 and 47 (Davies & Riechmann, 1994).", "Second, in the solved structures of two camel dabs, the CDR3s fold back on the VH surface, masking a significant surface area of the VL interface (Desmyter, Transue, et al., 1996)(Decanniere, Desmyter, et al., 1999).", "Several other features of VHHs are noteworthy.", "One is the frequent occurrence of the cysteine residues in CDR1 and CDR3 (Muyldermans, Atarhouch, et al., 1994) (Lauwereys, Arbabi, et al., 1998 (Vu, Ghahroudi, et al., 1997).", "While the location of the CDR1 cysteine is typically fixed at position 33, that of the CDR3 cysteine varies.", "These two residues form a disulfide linkage between CDR1 and CDR3 (Desmyter, Transue, et al., 1996) (Davies & Riechmann, 1996).", "In the crystal structure of a dAb-lysozyme complex, the disulfide linkage imparts rigidity on the CDR3 loop which extends out of the combining site and penetrates deep into the active site of lysozyme (Desmyter, Transue, et al., 1996).", "A second feature is the longer average length of the VHH CDR3, relative to human or murine VHs (Muyldermans, Atarhouch, et al., 1994).", "A longer CDR3, which is a feature of A6, increases the antigen binding surface and, to some extent, compensates for the absence of the antigen binding surface provided by the VL in conventional antibodies (Desmyter, Transue, et al., 1996).", "A third feature is the absence of the CDR3 salt linkage that is typically present in conventional antibodies and formed by arginine or lysine residues at position 94 and aspartate at position 101 (Desmyter, Transue, et al., 1996) (Muyldermans, Atarhouch, et al., 1994) (Spinelli, Frenken, et al., 1996) (Davies & Riechmann, 1996) (Chothia & Lesk, 1987) (Morea, Tramontano, et al., 1998).", "As antigen binding fragments, dAbs are an attractive alternative to scFvs because of their much smaller size and the fact that they demonstrate affinities comparable to those demonstrated by scFvs (Ward, Gussow, et al., 1989) (Spinelli, Frenken, et al., 1996) (Lauwereys, Arbabi, et al., 1998) (Davies & Riechmann, 1995) (Arbabi, Desmyter, et al., 1997) (Reiter, Schuck, et al., 1999).", "Smaller size is an advantage in applications requiring tissue penetration and rapid blood clearance.", "Smaller molecules also offer a tremendous advantage in terms of structural studies (Davies & Riechmann, 1994) (Constantine, Goldfarb, et al., 1992 (Constantine, Goldfarb, et al., 1993).", "Phage antibody library construction is much simpler and more efficient if single domain antibodies (dabs) are used instead of Fabs or single chain Fvs.", "Randomization can be introduced at a much higher percentage of CDR positions without exceeding practical library size.", "The problem of shuffling original VL-VH pairings is also avoided.", "Camelid phage dAb libraries constructed from the VHH repertoire of camels immunized with target antigens have performed well (Arbabi, Desmyter, et al., 1997) (Lauwereys, Arbabi, et al., 1998) (Decanniere, Desmyter, et al., 1999).", "However, construction of libraries from immunized camels presents obvious problems.", "In addition, the non-human nature of products from these libraries limits their usefulness.", "Synthetic dAb libraries (Davies & Riechmann, 1995) (Reiter, Schuck, et al., 1999), particularly those based on a human VH framework, alleviate these problems.", "As described in detail in Example 1, a dAb phage display library, according to the invention was constructed by randomizing positions 100i-100n of the parental VH ligand binding fragment identified in FIG.", "2 (A6VH-L1).", "The actual library size was 2.4×108 with 6.4×107 possible sequences.", "The sequences of 27 clones randomly picked from the unpanned library confirmed the integrity of the library (Table 1).", "TABLE 1 Sequences of the randomized region (100i-n) of randomly picked clones from the A6VH-L1a library.", "1.DQFTHS 15.CVRGAE 2.SSMYGN 16.SPSLAA 3.IKMQQN 17.HASGRS 4.SVDARD 18.GYMCSL 5.VSRFGA 19.HNKDLA 6.GLGSPK 20.LADLYM 7.IDAKWA 21.WRRAHE 8.VSRFGA 22.SDLFAR 9.HCLPDG 23.VSRFGA 10.RWR?VP 24.RYRHST 11.VSRFGA 25.ARLAGP 12.LECEGC 26.SYRPYL 13.RNVGAL 27.VVLGNS 14.RRSDYL As shown in FIG.", "5, this potential VH ligand binding fragment shown has a substantially improved percentage of monomer relative to A6 wild-type.", "The A6VH-L1 library was panned against the anti-FLAG IgG M-2, H11 scFv and Brucella scFv.", "M-2 Results Three rounds of panning were performed.", "Twenty one clones from rounds 2 and 3 were sequenced (Table 2).", "The motif recognized by M-2 is XYKXXD.", "As we observed previously with the camelized A6VH library, the epitope was preferentially positioned at the C-terminus of the randomized region with the D residue of non-randomized FDI sequence forming part of the epitope.", "As shown in Table, the sequences of the M-2 binders identified by phage ELISA reasonably reproduce the motif recognized by M-2.TABLE 2 Sequences of M-2 binders identified by phage ELISA.", "Sequence # of clones (A) deleted constructs EVQLQAS----- 6 SGYYEDDYRLFDIWGQGTQVTVSS EVQLQASGGGLVQP----- 1 GYYDSSGYYKDLDTRFDIWGQGTQVTVSS EVQLQASGGGLVQPGGSLRLS----- RLKVEYYDSSYYGDHYKWFDIWGQ GTQVTVSS 1 (B) Full length constructs (CDR3 only) DRLKVEYYDSSGYYRNEYKEFDI 1 DRLKVEYYDSSGYYVDEYKSFDI 1 DRLKVEYYDSSGYYAGRYKDFDI 1 DRLKVEYYDSSGYYRSDYKRFDI 1 DRLKVEYYDSSGYYASHYKDFDI 1 DRLKVEYYDSSGYYVDGYKDFDI 1 DRLKVEYYDSSGYYTADYKMFDI 1 DRLKVEYYDSSGYYDMDYKTFDI 1 DRLKVEYYDSSGYYKS?YKSFDI 1 DRLKVEYYDSSGYYDYKSQDFDI 1 DRLKVEYYDSSGYYKNWDSTFDI 1 DRLKVEYYDSSGYYKDWDSSFDI 1 DRLKVEYYDSSGYYKDGDSFFDI 1 H11 Results Three rounds of panning were performed in each instance.", "No deleted sequences were observed for either antigen.", "Twenty-one H11 binders were sequenced.", "The sequence FSSP is present in 13 of the 21 H11 binders (Table 3).", "TABLE 3 CDR3 sequences H11 binders identified by phage ELISA.", "The randomized region is shown in bold.", "CDR3 # of clones (A)H11 scFv DRLKVEYYDSSG--YSFSSPFDI 7 DRLKVEYYDSSGYYYDFSSPFDI 3 DRLKVEYYDSSGYYNLFSSPFDI 1 DRLKVEYYDSSGYYSEFSSPFDI 1 DRLKVEYYDSSGYYSDFSSPFDI 1 DRLKVEYYDSSGYYTDMSWEFDI 1 DRLKVEYYDSSGYYDLGSWEFDI 1 DRLKVEYYDSSGYYDYVSWEFDI 1 DRLKVEYYDSSGYYDWGSWEFDI 1 DRLKVEYYDSSGYYDG?TWDFDI 1 DRLKVEYYDSSGYYWEGSGLFDI 1 DRLKVEYYDSSGYYIWYSGLFDI 1 DRLKVEYYDSSGYYSSWASAFDI 1 Brucella scFv (Yst9.1) Clones obtained by panning against Yst9.1 scFv were selected for expression.", "Two clones had VSRFGA sequence.", "The VSRFGA dAb was designated Yst9.1-L3-9.The VSRFGA clone was expressed in good yield (approximately 18 mg/liter of bacterial culture) and was characterized by BIACORE.", "Yst9.1-L3-9 dAb Binding to Immobilized Yst9.1 scFv Following IMAC, the dAb was purified by Superdex 75 size exclusion chromatography prior to BIACORE analysis.", "It gave an elution profile that was similar to the parent A6VH-L1 molecule (FIG.", "6).", "For the determination of binding kinetics and affinities, the purified dAb was injected over active (1800 RU of Yst9.1) and reference surfaces (9400 RU of BSA) to generate the sensorgram and constants presented in FIG.", "7.The Yst9.1 surface was regenerated with 10 mM HCl (6 sec contact).", "All proteins were immobilized on CM5 chips by amine coupling.", "A 2000 RU H11 scFv surface was also used as a reference.", "The dAb did not bind to BSA.", "The subtracted data, using the BSA surface as a reference, for dAb binding to Yst9.1 scFv fit quite well to a 1:1 interaction model.", "A KD of 117 nM was calculated.", "According to another embodiment of the invention, the parental ligand-binding fragment has additional amino acid substitutions at VL interface which reduce the tendency to aggregation attributable to the “stickiness” of the VH dAb at this interface.", "For example, substitutions at positions 44, 45 and 47 relative to the wild-type A6VH (FIG.", "1) are illustrated in FIG.", "8.According to another preferred embodiment of the invention, the parental ligand-binding fragment has a long CDR3.In a particularly preferred embodiment, the library is constructed using the A6VH-L1 parental VH ligand binding molecule as a template and preserves the entire length of this CDR3 and, additionally, at least one of positions 44, 45, 47 and 94 (and preferably all) is altered, (preferably including 44 or 45) to camelid type residues.", "Davies and Riechmann (1995) constructed a camelized dAb library by randomizing CDR3 amino acid residues but the library was ten times larger and yielded anti-hapten dAbs with dissociation constants in the range of 100400 nM.", "However, the isolated anti-protein dAbs had weak affinity (Davies & Riechmann, 1995) (Davies & Riechmann, 1996).", "Therefore, a smaller library such as the one constructed here may therefore contain only weak anti-protein dAbs.", "The isolation of such dAbs would be difficult with monovalent display (Lowman, Bass, et al., 1991).", "In a phage vector format the dAb are displayed 3-5 copies and therefore there is potential for avidity which increases the likelihood of isolating weak binders (Nissim, Hoogenboom, et al., 1994).", "Additional embodiments of the randomization strategy for the libraries of the invention, are described below.", "The present inventors have also found a method of enhancing the probability that the binding fragments displayed in the library have characteristics which approximate the desired solubility characteristics found in the wild type binding fragment.", "During construction of the library, nucleotides of the variable region are added in a step-wise addition and by selecting a nucleotide ratio which is biased in favor of producing amino acids which reflect the DNA of the parental or wild type species.", "Thus, a method for biasing a library in favor of obtaining selected percentages of wild type amino acid residues is achieved by creating residue substitutions by using different spiking levels of the various dNTPs as described below.", "When creating a phage library, the randomization of amino acids is often achieved by DNA synthesis.", "A primer is annealed next to DNA encoding for the variable region, and nucleotides are randomly added to synthesize randomized variable regions.", "Normally, at the step of synthesizing the DNA used to produce the variable region of the phage library, one uses a nucleotide ratio of 1:1:1:1, which generates a totally random variable region.", "By the present method, during synthesis of the variable region, the likelihood of achieving affinity or other desirable traits found in the wild type as follows.", "At each step of adding a nucleotide to the DNA variable region, one selects a dNTP ratio which is biased in favor of producing amino acids which reflect the DNA of the parental (wild type) species.", "Table 4 charts particular amino acid residues or sequences of residues and preferred types of amino acid substitutions according to various examples of the invention to be defined hereafter.", "The selection of amino acids for randomization or partial randomization is based on adopting one or more of a variety of approaches including one of more of the following: 1.universal recognition of wild-type amino acids through a broad-based biasing in favour of the wild-type amino acids in one or more regions of interest (approximately 10%-90% biasing) in order to maintain the characteristics of the parental VH ligand-binding molecule; 2.selective recognition of amino acids that are important to maintain as wild-type through biasing (approximately 10-100%) in order maintain conserved or strategic regions of amino acid residues of the parental VH ligand-binding molecule; and 3.recognition of selected amino acids as important for intermolecular interaction and biasing those amino acids to wild-type and amino acids of the same type.", "TABLE 4 Amino Acid Residue #s Description of Various Preferred Amino Acid Constitutions a.", "At least one of 100a- Randomize; 100h, preferably at each At least approximately 10% biasing in favor of wild-type amino position of 100a-100h acids; At least approximately 50% biasing in favor of wild-type amino acids; At least approximately 90% biasing in favor of wild-type amino acids; Randomize, but bias 100f to wild-type (approximately 10-100%) b.", "At least one amino Randomize; acid of: 100a-100b and Randomize with bias to wild-type (approximately 10-100%), 100g-100h preferably at preferably at least approximately 50% wild-type, alternatively at least each position of 100a- approximately 90% wild-type amino acids; 100b and 100g-100h Randomize with bias to one of the amino acids selected from the group consisting of tyrosine, histidine, glutamine, asparagine, lysine, aspartic acid and glutamic acid (approximately 10-100%) c. At least one of 100b- Randomize; 100g, preferably at each Delete; position of 100b-100g d. 100a-100h Random additions of up to 10 amino acids; Random deletions of up to 7 amino acids; e. 95-100o Randomize; Random additions of up to 10 amino acids; Random deletions of up to 7 amino acids; f. At least one of 95- Randomize; 100, preferably at each Randomize with bias to wild-type (approximately 10-100%), position of 95-100 preferably at least approximately 50% wild-type, or preferably at least approximately 90% wild-type amino acids; Invariant (primer spans this region) g. 101-102 Invariant (primer spans this region) conserved amino acids N/A i.", "At least one amino Randomize with bias to wild-type (approximately 10-100%), acid of 100a-100b, preferably at least approximately 50% wild-type, more preferably at 100g-100h and 100l- least approximately 90% wild-type amino acids; 100o, preferably at each Randomize with bias to one of the amino acids selected from the position of 100a-100b, group consisting of tyrosine, histidine, glutamine, asparagine, lysine, 100g-100h and 100l- aspartic acid and glutamic acid (approximately 10-100%); 100o Bias to aromatic amino acids (10-100%) j.", "95-100h Randomize but maintain any 5-10 consecutive amino acids as wild- type k. 100a-100o Randomize but maintain any 5-10 consecutive amino acids as wild- type Unless otherwise necessarily implied as a result of logistical considerations, it is to be understood that the various embodiments which relate to choice of amino acids for random, biased or fixed substitution (specified in column 1) as well as the various embodiments related to types of substitutions (column 2) are not mutually exclusive.", "Moreover the various permutations and combinations of such substitutions are hereby contemplated as embodiments of the invention.", "For example, substitutions referred to in row a.", "(any one or more amino acids and preferably all amino acids of residues 100a-100h) #3 (at least approximately 50% wild-type amino acids) may combined with row b.", "(any one or more and preferably all of amino acids residues 100a, 100b, 100g and 100h) #2 (for instance, at least approximately 90% wild-type amino acids) so that, for instance, any 3 of the amino acids in 100a-100h are biased in favor of wild-type in approximately 50% of the variant VH ligand-binding fragments and 100a and 100b are biased in favor of wild-type in 90% of potential binding fragments.", "By necessary implication the three amino acids that are biased in favor of wild-type are not residues 100a and 100b, but they may be any other three residues.", "Accordingly, the broadest possible interpretation is to be given to the disclosure of the various combinations and permutations of the embodiments disclosed herein.", "Furthermore, it is to be understood that each of the various embodiments described herein are disclosed, except insofar as logistically impossible, in reference to each of the various aspects and definitions of the invention.", "Moreover, it is to be understood that phrases such as at least approximately 10%, or approximately 10-100% are intended to specify a preference for each of the unit percentages between about 7 and 100% that are practically achievable by oligonucleotide primer design and PCR amplification described herein below, as well as other well known PCR techniques and techniques of Controlled Mutation described in the art, and routine variations of such techniques.", "By the same token, phrases such as at least 80% are intended to specify a preference for each of the unit percentages between 80% and 100%.", "It is to be understood that biasing of a percentage less than 100% implies unless otherwise implied or stated that the remaining percentage is fully randomized.", "Furthermore, it is to be understood, for example, that 90% biasing in favor of wild-type amino acids at a given amino acid position is to be approximated by controlling the percentage amounts of each of the three relevant nucleotides (so that, for example, the product of the probabilities of occurrence of the three desired nucleotides in sequence in the growing chain is 90%) so as to supply 90% of correct coding triplet(s) and a total of 10% of random coding triplets, having regard to the degeneracy of the genetic code (for example if two different coding triplets result in a given amino acid, then the sum of the probabilities of achieving those two triplets will have to equal 90%).", "This is preferably accomplished on an amino acid by amino acid basis so that, for example the probability of achieving two and three wild-type amino acids in sequence, in the case of 90% biasing is 0.81 and 0.73, respectively, etc.", "It is to be understood that this high level of biasing may be suitable only for part of the coding sequence into which variability is introduced and that higher levels of biasing are acceptable, when for example substantially all of the amino acids of a long CDR3 are biased, as disclosed in one of embodiments herein.", "Accordingly there is a balance to be struck between a large diverse library and biasing for multifactorial characteristics such as solubility.", "Nevertheless it is contemplated that the library produced may be a pooled library in which several libraries each having varying degrees of biasing to wild-type, for example, 60%, 50%, 40% and 30%, are pooled together to obtain the both desired variability and similarity.", "The preferred parental binding-fragment may be engineered to maximize the desired characteristic (e.g.", "solubility, intermolecular interaction) and then made the subject of libraries with varying degrees of biasing.", "In this connection, the library could be biased to be rich in amino acids, which are highly soluble.", "It is to be understood that both arms (halves) of the preferred longer loop forming CDR3s may be biased to amino acids that are favored for intermolecular interaction, preferably charged amino acids, so as to provide a method of generating, in addition to loop size, varying loop structures.", "This bias may be systematically introduced or systematically reduced by randomization, in cooperating pooled libraries having varying degrees of biasing.", "With respect to the application of these methods to parental VH, preferably, CDR3s of a variety of different lengths from 16 to 33 amino acids are predominantly represented among the variant VH ligand-binding fragments.", "Preferably CDR3s of a variety of different lengths, from 18 to 25 amino acids, or, from 18 to 23 amino acids are predominantly represented in the library.", "Although the term “predominant” ordinarily implies a majority representation of the specified long CDR3 variant VH ligand-binding fragments, the invention also contemplates an even less substantial representation, especially within a reasonably large size library (>107).", "Preferably, the specified long CDR3 variant VH ligand-binding fragments have a majority representation within the library and more preferably an even greater or exclusive representation.", "Optionally, the parental VH ligand-binding molecule is reduced in size and the parental VH ligand-binding molecule is optionally modified by deleting a portion of the CDR2.In another embodiment, CDR3s of the same length as that of the parental VH ligand-binding molecule are predominantly or exclusively represented in the variant VH ligand-binding fragments.", "In another aspect, the CDR3 region is specifically retained along with human sequence elements of other regions that confer favorable characteristics solubility, to create a phage display library having favorable characteristics of solubility, preferably when compared with variant VH ligand-binding fragments that have fully randomized hypervariable regions particularly CDR3).", "In particular, the present inventors have found that favorable solubility characteristics of a parental VH ligand-binding molecule can be maintained in the population of variant VH ligand-binding fragments in the course of randomizing the hypervariable regions by biasing all or selected amino acids residues to wild-type and/or biasing in favor of amino acids residues that favor certain or a variety of types of intermolecular interaction.", "This is respectively accomplished by increasing the percentage amounts of nucleotide bases that represent wild-type amino acids and/or amino acids that provide favorable intermolecular interactions during the randomization procedure e.g.", "site directed PCR mutagenesis.", "Thus, variant VH ligand-binding fragments having relatively long CDR3s of varying lengths are produced by randomly or partially randomly inserting varying numbers of nucleotide triplets in any part of a randomized portion of the parental VH framework.", "Primers of the desired length and nucleotide composition are synthesized followed by PCR amplification.", "Desired randomization can be achieved by biasing nucleotide composition of the primer.", "The production of displays of long CDR3 variant binders may also be accomplished by pooling several libraries of variant VH ligand-binding fragments having randomized or partially randomized CDR3s of different respective uniform lengths.", "These strategies are not mutually exclusive.", "The additional following terms are used herein as follows, unless the context logically implies otherwise: “Biasing”, “biased in favor of” and related forms of these terms are generally intended to refer to weighting in the course of introducing variation in the parental ligand-binding molecule.", "“Homologous” or “homology” as used herein refers to “identity” or “similarity” as used in the art, meaning relationships between two or more polynucleotide or amino acid sequences, as determined by comparing the sequences.", "In the art, identity also means the degree of sequence relatedness between polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.", "Both identity and similarity can be readily calculated (Lesk, A. M., ed., Computational Molecular Biology, Oxford University Press, New York, 1988; Smith, D. W., ed., Biocomputing: Informatics and Genome Projects, Academic Press, New York, 1993; Griffin, A. M., and Griffin, H. G., eds., Computer Analysis of Sequence Data, Part I, Humana Press, New Jersey, 1994; von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, 1987; and Gribskov, M. and Devereux, J., eds., Sequence Analysis Primer, M Stockton Press, New York, 1991).", "While there exist a number of methods to measure identity and similarity between two polynucleotide sequences, both terms are well known to skilled artisans (von Heinje, G., 1987; Gribskov, M. and Devereux, J., 1991; and Carillo, H., and Lipman, D., 1988).", "Methods commonly employed to determine identity or similarity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D. (1988, SIAM J.", "Applied Math., 48: 1073).", "Methods to determine identity and similarity are codified in computer programs.", "Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al.", "(1984), Nucleic Acids Research 12(1): 387), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al.", "(1990), J. Molec.", "Biol.", "215: 403).", "“Percent homology” or “/homologous” or related terms include both of the following interpretations/methods of calculation: 1) an approximate percentage of the sequence referenced in terms of the number of common residues (e.g.", "80% of 11 is understood to be an approximation insofar as application of the percentage does not yield a unit number of residues, in which case both the immediately higher number and immediately lower unit numbers, 9 and 8 respectively, are deemed to be covered); 2) the percentage of binding fragments theoretically achievable that have the full wild-type sequence, which is calculated as a product of the probabilities that the wild-type amino acid will occur at a given amino acid position.", "“Conserved” regions refer to those which are commonly found in at least other antibodies of the same type or in at least the same species of mammal.", "“Wild-type” refers to the parental binding-fragment, which may be a variant of the natural or to the native A6 VH parental ligand-binding fragment, depending on the context.", "“Step-wise” refers to the addition of, for example, nucleic acids, in a manner such that the quantity of nucleic acids added at each step is rigorously control, usually one nucleic acid at a time.", "“Spanning” does not preclude deletions or additions within the parental VH binding-fragment that are not inimical to the operation of the invention.", "“Camelid type” refers specifically to one or more features of the camelid VL interface.", "“Soluble” includes the generally ascribed meaning in the art and without limitation includes (based on solubility correlated phenomena) the relative amounts of naturally-folded recombinant protein released from the cell.", "“Percent biasing” or “% of binding fragments” (or “biasing 10-100%”, etc.)", "refers to biasing on an individual amino acid basis (though other techniques to accomplish the same effect might apparent to those skilled in the art).", "Similarly, the specification that wild-type amino acids occur at a specified position or series of positions in, for example, at least approximately 50% of potential binding fragments is intended to mean both that 50% biasing is sought at a given such position or that a total of 50% of the correct nucleotide triplets are represented.", "“Approximately” in reference to percentages is intended to accommodate attrition of various desired variant VH ligand-binding fragments, the assumption that the probabilistic outcomes will not be achieved in practice and that certain variation in methods to accomplish the specified results is deemed to be suitable.", "The term 50% in reference to an uneven number of amino acids residues means that either one more or one less than half of the amino acids is referred to.", "The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.", "Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology” (D. M. Wei & C. C. Blackwell, eds.", "); “Gene Transfer Vectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds., 1994); “Current Protocols in Immunology” (J. E. Coligan et al., eds., 1991).", "These references are incorporated herein by reference.", "These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention.", "Particularly useful techniques for particular embodiments will be discussed in the sections that follow.", "Recombinant genetic techniques have allowed cloning and expression of antibodies, functional fragments thereof and the antigens recognized.", "These engineered antibodies provide novel methods of production and treatment modalities.", "For instance, functional immunoglobulin fragments have been expressed in bacteria and transgenic tobacco seeds and plants.", "Skerra (1993) Curr.", "Opin.", "Immunol.", "5: 256: 262; Fiedler and Conrad (1995) Bio/Technology 13: 1090-1093; Zhang et al.", "(1993) Cancer Res.", "55: 3384-3591; Ma et al.", "(1995) Science 268: 916; and, for a review of synthetic antibodies, see Barbas (1995) Nature Med.", "1: 836-839.These and more current references describing these techniques, which these references, particularly those well known to persons practicing in the relevant arts, are hereby incorporated herein by reference.", "Nucleotide sequences can be isolated, amplified, and processed by standard recombinant techniques.", "Standard techniques in the art include digestion with restriction nucleases, and amplification by polymerase chain reaction (PCR), or a suitable combination thereof PCR technology is described in U.S. Pat.", "Nos.", "4,683,195; 4,800,159; 4,754,065; and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al., eds., Birkauswer Press, Boston (1994).", "In addition to the specific PCR methods of biasing to wild-type A6 amino acid residues detailed below, it is possible to produce multiple different oligonucleotide primers consisting of specified amino acid residues (one or more) of the wild-type A6 molecule (e.g.", "CDR3 residues), mixing these in appropriate concentrations with a completely randomized (e.g.", "CDR3) oligonucleotide primer and subjecting the mixture of oligonucleotide primers to PCR.", "This will result in a biased phage library population of one's choosing (i.e.", "the amounts of the selectively randomized and totally randomized primers in the mixture will determine the percent of each CDR3 representation in the library).", "Polynucleotides comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification.", "Polynucleotides can be introduced into host cells by any means known in the art.", "Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, f-mating or electroporation.", "Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell by standard methods.", "See, e.g., Sambrook et al.", "(1989).", "RNA can also be obtained from transformed host cell, or it can be obtained directly from the DNA by using a DNA-dependent RNA polymerase.", "Suitable cloning and expression vectors include any known in the art, e.g., those for use in bacterial, mammalian, yeast and insect expression systems.", "Specific vectors and suitable host cells are known in the art and are not described in detail herein.", "See e.g.", "Gacesa and Ramji, Vectors, John Wiley & Sons (1994).", "Phage display techniques are generally described or referenced in some of the preceding general references, as well as in U.S. Pat.", "Nos.", "4,593,002; 5,403,484; 5,837,500; 5,571,698; 5,750,373; 5,821,047; 5,223,409 and 5,702,892.“Phage Display of Peptides and Proteins”, (Kay, Brian K. et al., 1996); “Methods in Enzymology”, Vol.", "267 (Abelson, John N., 1996); “Immunology Methods Manual”, (Lefkovits, Ivan, 1997); “Antibody phage display technology and its applications”, (Hoogenboom, Hennie R. et al., 1998).", "Immunotechnology 4 p. 1-20; Cesareni G et al.", "Phage displayed peptide libraries.", "Comb Chem High Throughput Screen.", "1999 February; 2(1): 1-17; Yip, Y L et al.", "Epitope discovery using monoclonal antibodies and phage peptide libraries.", "Comb Chem High Throughput Screen.", "1999 June; 2(3): 125-38; Rodi D J et al.", "Phage-display technology—finding a needle in a vast molecular haystack.", "Curr Opin Biotechnol.", "1999 February; 10(1): 87-93.Generally, DNA encoding millions of variants of a parental binding-fragment can be batch-cloned into the phage genome as a fusion to the gene encoding one of the phage coat proteins (pIII, pVI or pVIII).", "Upon expression, the coat protein fusion will be incorporated into new phage particles that are assembled in the bacterium.", "Expression of the fusion product and its subsequent incorporation into the mature phage coat results in the ligand being presented on the phage surface, while its genetic material resides within the phage particle.", "This connection between ligand genotype and phenotype allows the enrichment of specific phage, e.g.", "using selection on immobilized target.", "Phage that display a relevant ligand will be retained, while non-adherent phage will be washed away.", "Bound phage can be recovered from the surface, reinfected into bacteria and re-grown for further enrichment, and eventually for analysis of binding.", "The success of ligand phage display hinges on the combination of this display and enrichment method, with the synthesis of large combinatorial repertoires on phage.", "While the use of phage is described as an embodiment for the production of libraries for displaying, and selecting particular binding fragments, it is to be understood that and suitable genetic package may be used for the production of libraries of the invention.", "Such suitable genetic packages include cells, spores and viruses (see U.S. Pat.", "No.", "5,571,698), or any other suitable replicable genetic packages.", "With respect to cell based approaches, another popular method of presenting a library is the two-hybrid system (Feilds and Sternglanz, 1994, Trends in Genetics 10: 286-292).", "Those skilled in the art will appreciate that in vitro systems (non-cell based) may be equally applicable to the methods of the present invention, for example ribosome display (Hanes et al., 1998) or RNA-peptide fusion (Mattheakis et al., 1994, Proc Natl Acad Sci USA 91: 9022-26; Hanes et al., 1999, Curr Top Microbiol Immunol 243: 107-22).", "Ribosome display is a well documented technique that may be useful for generating libraries.", "This-entirely in vitro method allows for libraries with a diversity of >1012.In this method, a peptide is displayed on the surface of a ribosome that is translating it.", "Briefly, a library of mRNA molecules (we could start with A6) is translated in vitro translation system to the 3′ end, such that the ribosome does not fall off.", "The protein emerges from the ribosome in such a way that it can fold, but does not fall off.", "In some instances, there is an additional folding step in an oxiding environment (important for proteins with disulfide bonds).", "The whole complex of folded protein, ribosome and mRNA, which is stable for several days, can then be panned against a ligand that is recognized by the translated protein.", "(For example, the translated protein could be an antibody and the ligand is its antigen).", "The mRNA can then be amplified by reverse transcription and PCR.", "This technique has been used to successfully generate scFv antibody fragments with high affinity for their target.", "Reference is made to Hanes, J., Jermutus, L., Weber-Bomhauser, S., Bosshard, H. R. & Pluckthun, A. Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries.", "Proc.", "Natl.", "Acad.", "Sci.", "USA 95, 14130-14135 (1998); Schaffitzel, C., Hanes, J., Jermutus, L. & Pluckthun, A Ribosome display: an in vitro method for selection and evolution of antibodies from libraries.", "Journal of Immunological Methods 231, 119-135 (1999); He, M. et al.", "Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display.", "Journal of Immunological Methods 231, 105-117 (1999); Roberts, R. W. Totally in vitro protein selection using mRNA-protein fusions and ribosome display.", "Current Opinion in Chemical Biology 3, 268-273 (1999); Williams, C. Biotechnology match making: screening orphan ligands and receptors.", "Current Opinion in Biotechnology 11, 42-46 (2000); Mattheakis, L. C., Bhatt, R. R. & Dower, W. J.", "An in vitro polysome display system for identifying ligands from very large peptide libraries.", "Proc.", "Natl.", "Acad.", "Sci.", "USA 91, 9022-9026 (1994).", "Construction of A6VH-L1 Library EXAMPLE 1 A dAb phage display library was constructed employing the VH portion of A6 as a starting template and amino acid substitutions at positions 6, 23, 82a, 93 and 108, as shown in Table 5 below.", "In addition, silent mutations were introduced in codons for amino acids 3-16 (FR1) to remove a putative recombination site.", "All mutations were introduced by splice overlap extension—PCR(SOE).", "The modified VH was used as a scaffold for complete randomization at positions 100i-100n located at the carboxy terminal of the CDR3 of A6.TABLE 5 A6VH-L1 Template Position and Amino Acid Identity Position 6 23 82a 93 108 Wild Type A6VH E S S V T Modified A6VH A A N A Q Removal of Putative Recombination Site The codons for amino acids 3-16, which surround the recombination site, were changed.", "Using the Chi.R/FP and Chi.F/RP primer pairs (Table 6) and pSJF-A6VH plasmid as template, 5′ and 3′ fragments were synthesized by PCR in a total volume of 50 μl containing 10 pmol of each primer, 2 mM of each of the four dNTPs, 1× buffer and 2.5 units of AmpliTaq™ DNA polymerase (Perkin Elmer).", "The PCR protocol consisted of an initial denaturation step at 94° C. for 3 min followed by 30 cycles at 94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 1 min and a final extension step at 72° C. for 10 min.", "The two fragments were gel purified using the Q1Aquick Gel Extraction™ kit (QIAGEN) and a larger construct was assembled from the 5′ and 3′ fragments by performing splice overlap extension (SOE) PCR using RP and FP.", "Briefly, the reaction vial containing both 5′ and 3′ fragments, 200 μM each of the four dNTPs, 5 μl 10× buffer (NEB), and 2 units of Vent DNA polymerase (NEB) were subjected to 7 cycles of 1 min at 94° C. and 2.5 min at 72° C. To amplify the assembled construct, RP and FP primers were added at a final concentration of 1 pmol/μl and the mixture was subjected to 30 cycles of 1 min at 94° C., 30 sec at 55° C., and 1 min at 72° C. The amplified product was purified (Q1Aquick PCR Purification™ kit) and used as template for the mutagenesis steps.", "Sequencing revealed that the putative recombination sequences had been removed.", "Modification of Positions 6, 23, 82a, 93 and 108 Employing SOE, positions 23, 82a, 93 were mutated initially (Table 5).", "Using the Chi template, three fragments were synthesised by PCR using the A6VH.S23A.R/A6VH.S82aN/V93A.F (fragment 1), A6VH.S23A.F/RP (fragment 2) and A6VH.V93A.R/FP (fragment 3) primer pairs Crable 6).", "The first two fragments were assembled by SOE using RP/A6VH.S82aNN93A and the resultant fragment was spliced to the third fragment using RP/FP primers.", "Sequencing revealed the desired mutations at positions 23, 82a and 93.This product in turn was used as template for the final round of SOE experiments to alter positions 6 and 108.First, two fragments were synthesized using the A6VH.E6A.R/A6VH.T108Q.F and A6VH.E6A.F/RP primer pairs followed by SOE using RP/A6VH.T108Q.F primers.", "The final construct was purified, digested with BamHI and EcoRI, purified again and ligated to BamHI/EcoRI-restricted expression plasmid.", "The colonies were then screened to identify clones containing the mutated A6VH.", "TABLE 6 PRIMERS USED CHI SITE REMOVAL AND MUTAGENSIS OF A6VH- Chi.R CAATTACAAGAAAGTGGTGGCGGACTGGTGCAACCAGGAGGTTCCCTGAGACTC Chi.F ACTTTCTTGTAATTGGACCTCGGCCTGCGC A6VH.S23A.R CTCTCCTGTGCTGCCTCTGGA A6VH.S23A.F TCCAGAGGCAGCACAGGAGAG A6VH.S82aN/V93A.F CGCACAGTAATACACAGCCGTGTCCTCAGCTCTCAGACTGTTCATTTGAAGATA A6VH.V93A.R GTGTATTACTGTGCGAAAGACAGG A6VH.E6A.R CAATTACAAGCTAGTGGTGGC A6VH.E6A.F GCCACCACTAGCTTGTAATTG A6VH.T108Q.F TATGGATCCTGAGGAGACGGTGACCTGTGTCCCTTGGCC A6VH.ApaII.R CATGACCACAGTGCACAGGAGGTCCAATTACAAGCTAGA A6VH.NOT.T108Q CGATTCTGCGGCCGCTGAGGAGACGGTGACCTGTGTCCCTTGGCCCCAGATATC RP GCGGATAACAATTTCACACAGGAA FP CGCCAGGGTTTTCCCAGTCACGAC Cloning, Expression and Evaluation of Dimer/Multimer Formation The modified A6VH, designated A6VH-L1 was cloned into a vector for expression in E. coli using EcoR1 and BamH1.Thirty ml of LB containing 100 ug/ml ampicillin was inoculated with a single colony harboring pSJF2-dAb and the culture was shaken at 240 rpm at 37° C. overnight.", "In the morning the entire overnight culture was used to inoculate 1 liter of M9 medium supplemented with 5 μg/ml vitamin B1, 0.4% casamino acids and 100 μg/ml ampicillin.", "The culture was shaken at room temperature for 30 hr at 160 rpm and subsequently supplemented with 100 ml of 10× induction medium and 100 ul of 1M isopropylthio-β-D-galactoside.", "The culture was shaken for another 60 hr, the periplasmic fraction was extracted by osmotic shock method (Anand et al., 1991), and the presence of dAb in the extract was detected by Western blotting (MacKenzie 1994).", "The periplasmic fraction was dialyzed extensively in 10 mM HEPES (N-[2-hydroxyethyl]piperazine-N′ [2-ethanesulfonic acid]) buffer pH 7.0, 500 mM NaCl.", "The presence of the dAb C-terminal His5 tag allowed a one step protein purification by immobilized metal affinity chromatography using HiTrap Chelating™ column (Phamacia).", "The 5-ml column was charged with Ni2+ by applying 30 ml of a 5 mg/ml NiCl2.6H2O solution and subsequently washed with 15 ml deionized water.", "Purification was carried out as described (MacKenzie, 1994) except that the starting buffer was 10 mM HEPES buffer, 10 mM imidazole, 500 mM NaCl, pH 7.0, and the bound protein was eluted with a 10-500 mM imidazole gradient.", "The purityof the protein was determined by SDS-PAGE (Laemmli).", "To detect the presence of dimer/multimer dAb in the protein preparation, gel filtration chromatography was performed using Superdex75 (Pharmacia) as described (Deng et al., 1995).", "Library Construction A PCR product was generated using A6VH-L1 as template and primers RP and A6VH.Rndm100i-n.F, CCTTGGCCCCAGATATCAAA6[(A/C)NN]GTAATAACCACTACTATC.", "The latter primer was degenerate in the region encoding residues 100i-n. A second PCR employed 180 pmol of the above product and 100 pmol of each of the two primers A6VH.Apal.R, CATGACCACAGTGCACAGGAGGTCCAATTACAAGCTAG, and A6VH.NotT108Q.F, CGATTCTGCGGCCGCTGAGGAGACGGTGACCTGTGTCCCTTGGCCCCAGATATC.", "The second set of primers are complimentary to the 5′ and 3′ ends of the dAb genes and incorporate ApalI and NotI restriction sites (underlined sequences) at the end of the amplified genes.", "The amplified products were purified, cut sequentially with ApalI and NotI restriction endonucleases, purified again, and ligated to the ApalI/NotI-treated fd-tet phage vector.", "Following this, 1.5 μg of the desalted ligated product was mixed with 40 μl of competent E. coli strain TG1 and the cells were transformed by electroporation.", "Transformation, library phage amplification and purification and library size determination were performed as described below.", "The randomization strategy is depicted in FIG.", "11.Library Size Determination.", "To determine the size of the library, immediately following the transformation and after the addition of the SOC medium an small aliquot of the electroporated cells were serially diluted in exponentially growing TG1 cells.", "Two hundred μl of the diluted cells was mixed with 3 ml of 50° C. agarose top and immediately poured onto 2×YT plates pre-warmed to 37° C. Plates were incubated overnight at 37° C. and the number of plaques were used to determine the size of the library.", "Panning Panning was performed using the Nunc-Immuno MaxiSorp™ 8-well strips (Nunc).", "Briefly, the wells were coated overnight by adding 150 μl of 100 μg/ml antigen in PBS.", "In the morning, they were rinsed three times with PBS and subsequently blocked with 400 μl PBS-2% (w/v) skim milk (2% MPBS) at 37° C. for 2 hr.", "The wells were rinsed as above and 1012 transducing units phage in 2% MPBS were added.", "The mixture was incubated at room temperature for 1.5 hr after which the unbound phage in the supernatant was removed.", "The wells were rinsed 10 times with PBS-0.1% (v/v) Tween 20 and then 10 times with PBS to remove the detergent.", "The bound phage was eluted by adding freshly prepared 200 μl 100 mM triethylamine, pipetting the content of the well up and down several times and incubating the mixture at room temperature for 10 min.", "The eluted phage was transfered to a tube containing 100 μl 1 M Tris-HCl, pH 7.4 and vortexed to neutralize triethylamine.", "Following this, 10 ml exponentially growing TG1 culture was infected with 150 μl eluted phage by incubating the mixture at 37° C. for 30 min.", "Serial dilutions of the infected cells were used to determine the titer of the eluted phage as described in the previous section.", "The remaining of the infected cells were spun down and then resuspend in 900 μl 2xYT.", "The cells were mixed in 300 μl aliquots with 3 ml agarose top and the phage propagated on the plates overnight at 37° C. In the morning the phage was purified, the titer was determined, and a total of 1011 transducing units phage were used for further rounds of selection.", "EXAMPLE 2 A library (A6VH-L1a) in which residues 100i and 100n were deleted and residues 95-100h were randomized with 50% biasing in favour of the parental amino acid constitution, was also constructed.", "The procedure for A6VH-L1 a construction was identical to that for A6VH-L1 except that the randomization primer used in the first PCR step was A6VH.95-100hRndm CCCTTGGCCCCAGATATCAAA14[(A/C)NN]TTTCGCACAGTAATACAC.", "The sequence of the A6VH for this library is shown in FIG.", "12.EXAMPLE 3 A6VH-L2, a parental VH ligand binding molecule in which each of positions 6, 23, 74, 82a, 83, 84, 93 and 108 were modified as shown in FIG.", "3 relative to the native A6VH(FIG.", "1), was also constructed.", "Mutations at positions 74, 83 and 84 were introduced with a single mutagenic primer, A6VH.S74A.F, (5′CATTTGAAG-ATACAGAGTGTTCTTGGCATTGTCTCT3′), which when used together with RP (see Example 1 above) generated an N-terminal fragment.", "A second primer, A6VH.R83K/A84P.R., (5′TATCTTCAAATGAACAGTCTGAAAC-CAGAGGACACGGCT3′), together with FP (see Example 1 above) generated a C-terminal fragment.", "The two fragments were joined by SOE, using RP and FP as primers, to give the gene encoding A6VH-L2.This molecule showed slightly better levels of monomer production when expressed and assessed by size exclusion chromatography when compared with A6VH-L1.TABLE 7 Amino acid identity at positions 6, 23, 74, 82a, 83, 84, 93 and 108 for A6VH and the mutants of A6VH.3 Amino acid Identity Kabat # 6 23 74 82a 83 84 93 108 A6VH E S S S R A V T A6VH-L1 A A S N R A A Q A6VH-L2 A A A N K P A Q EXAMPLE 4 Surface Plasmon Resonance Binding studies were performed using BIACORE Upgrade (Biacore Inc., Piscataway, N.J.) as described (Jönsson et al., 1991).", "Approximately 14, 000 RU of anti-FLAG M2 IgG or control IgG were immobilized on CM5 sensor chips by amine coupling.", "Single-domain antibodies were passed over the sensor chips surfaces in 10 mM HEPES buffer, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% P-20 (Biacore Inc.) at 25° C. and at a flow rate of 5 μl/min.", "Sensorgram data were analyzed using the BIAevaluation 3.0 software package (Biacore Inc.).", "EXAMPLE 5 Enzyme-Linked Immunosorbent Assay (ELISA) Nunc-Immuno MaxiSorp™ plates (Nunc) were coated overnight at 4° C. with 150 μl of 10 μg/ml of 3B1 scFv or BSA in PBS.", "The contents were removed and the plates were tapped on a paper towel to remove any liquid remaining in the wells.", "The wells were blocked by adding 300 μl of 2% MPBS and incubating for 2 hr at 37° C. The contents of the wells were emptied as before, 100 μl of purified dAb phage in 2% MPBS was added, and the wells were incubated at room temperature for 1.5 hr.", "The contents were emptied again and the wells were washed 5 times with PBS-0.05% (v/v) Tween 20 and subsequently blotted on a paper towel to remove any remaining wash buffer.", "One Hundred μl of recommended dilution of RP/Anti-M13 monoclonal antibody conjugate (Amersham Pharmacia Biotech) in 2% MPBS was added and the wells were incubated at room temperature for 1 hr.", "The wells were washed six times as before and the binding of dAb to the antigen was detected colorimetrically by adding 100 μl of equal mixtures of TMB Peroxidase Substrate and H2O2 (Kirkegaard and Perry Laboratories, Gaithersberg, Md., USA) at room temperature for several minutes.", "The reaction was stopped by adding 100 μl of 1 M H3PO4 and the A450 was measured by DYNATECH MR5000 plate reader (Dynatech Laboratories, Chantilly, Va., USA).", "EXAMPLE 6 Introducing Genetic Variation into the Sequence Corresponding to the A6 Heavy Chain CDR3 Region—Randomized Residues Oligonucleotides comprising randomly mutated CDR3 regions were prepared on an Applied Biosystems 394 DNA synthesizer as described above.", "1.Production of 23 Randomized Residues (CDR3 1-23): The anti-codon formula [(A/C)NN] is used resulting in a reduction in possible codon usage from 64 to 32 and reduces the number of possible stop codons.", "Position one, therefore, comprises only A and C in the synthetic reaction mixture.", "For complete randomization of the second and third positions of the codons the dNTP mixture comprise 25% each of A,G,C and T. The 3′ oligonucleotide randomizing primer was designed such that the last 15 nucleotides of framework 3 and the first 17 nucleotides of framework 4 were kept constant for hybridization.", "The nucleotides encoding the intervening amino acids, namely amino acids 1-23 of the CDR3 region were randomized using the following primer: 5′ (GTTGTCCCTTGGCCCCA n[(A/C)NN]TTTCACACAGTAATA] 3′ (Where n=23, antisense strand).", "Using a 50% A and 50% C for the first nucleotide position for each anti-codon triplet and 25% each of A, C, G, and T for the second and third nucleotide positions for n=23, complete randomization of the 23 amino acids of the A6 CDR3 is achieved.", "2.Synthesis of CDRs Comprising 15-23 Residues The primers are adapted by reducing n to 15-23 in the above primer formulae whilst keeping the flanking nucleotides constant.", "3.For Synthesis of CDR3s Comprising 24-33 Residues The primers would be adapted by increasing n to 24-33 in the above primer formula while keeping the flanking nucleotides constant.", "EXAMPLE 7 Selective Randomization Biasing for 50% Homology to Parental Tyrosine To achieve approximately 50% homology to wild type at any one position in the A6 dAb CDR3 region during antisense synthesis using the DNA synthesizer, the following example would be used.", "In the case of tyrosine, which is encoded by TAC or TAT (antisense strand GTA or ATA) the nucleotides would be spiked as follows for the antisense strand.", "First anticodon nucleotide position: 80% of A and 20% of C is added to the dNTP solution, and G and T are not added to reduce codon degeneracy.", "Second anticodon nucleotide position: 80% T and approximately 6.67% of C, 6.67 of A and 6.67% of G. Third anticodon nucleotide position the mixture: 80% of A and approximately 6.67% of T and 6.67% of G and 6.7% C. The calculated probability of tyrosine would thus be 0.8×0.8×0.8×100%=51.2%.", "Thus approximately 51% of the chains of the library will contain a wild-type A6 tyrosine in that specified position.", "EXAMPLE 8 Selective Randomization Biasing for 50% Homology to Parental Serine Using the same strategy in order to achieve approximately 50% homology to wild type serine at one or more positions, the following example is useful.", "Using only A and/or C in the first anticodon position the amino acid serine could have two codons these are AGT, TCT and TCG (antisense ACT, AGA and CGA, respectively).", "The nucleotide spiking levels would be as follows: First anticodon nucleotide position: 50% A and 50% C. Second anticodon nucleotide position: 35.35% C, 35.35% G, 14.65% A and 14.65% T Third anticodon nucleotide position: 35.35% A, 35.35% T, 14.65% C and 14.65% G. The probability of producing serine for any given fragment, using this strategy is (1×[0.3535+0.3535]×[0.3535+0.3535]×100%=50%.", "Thus, approximately 50% of the chains will have a serine in the selected position.", "EXAMPLE 9 Selective Randomization Biasing for 50% Homology to Parental Serine To achieve approximately 10% homology to wild type at any one position in the A6 dAb CDR3 region during antisense synthesis using the DNA synthesizer, the following example can be used.", "In the case of tyrosine which is encoded by TAC or TAT (antisense strand GTA or ATA) the nucleotides would be spiked as follows for the anti sense strand.", "First anticodon nucleotide position: 47% of A and 53% of C is added; G and T are not added to reduce codon degeneracy.", "Second anticodon nucleotide position: 47% T and approximately 17.67% of C, 17.67 of A and 17.67% of G. Third anticodon nucleotide position: 47% of A and approximately 17.67% of T and 17.67% of G and 17.67% C. The calculated probability of tyrosine is thus 0.47×0.47×0.47×100%=10.4%.", "Thus approximately 10% of the chains of the library will contain a wild-type A6 tyrosine in that specified position.", "EXAMPLE 10 Selective Randomization Biasing for 50% Homology to Parental Serine To achieve approximately 90% homology to wild-type amino acids at any positions in the A6 dAb CDR3 region during antisense synthesis using the DNA synthesizer, the following example would be used.", "In the case of tyrosine which is encoded by TAC or TAT (antisense strand GTA or ATA) the nucleotides would be spiked as follows: First anticodon nucleotide position: 97% of A and 3% of C is added, G and T are not added to reduce codon degeneracy.", "For this reason, only A and C are used in the first anticodon position for all 20 naturally occurring amino acids.", "Second antcodon nucleotide position: 97% T and approximately 1% of C, 1% of A and 1% of G. Third anticodon nucleotide position: 97% of A and approximately 1% of T and 1% of G and 1% C. The calculated probability of tyrosine would be 0.97×0.97×0.97×100%=91.3%.", "Thus approximately 90% of the chains of the library will contain a wild-type A6 tyrosine in that specified position.", "Using the approaches in the examples above, approximately 10% to approximately 90% of wild type amino acid representation at one or more specified amino acid residues in the A6 CDR3 can be calculated and applied to the DNA synthesizer.", "The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof.", "Certain adaptations and modifications of the invention will be obvious to those skilled in the art.", "Therefore, the presently discussed embodiments are considered to be illustrative and not restrictive.", "It is understood that the claims may refer to aspects or embodiments of the invention that are only inferentially referred to in the disclosure.", "All references disclosed in this application are hereby incorporated by reference.", "Additional References Anand, N. N., Dubuc, G., Phipps, J., MacKenzie, C. R., Sadowska, J., Young, N. M., Bundle, D. R., and Narang, S. A.", "(1991).", "Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen.", "Gene 100: 39-44.Arbabi, G. M., Desmyter, A., Wyns, L., Hamers, R., and Muyldermans, S. (1997).", "Selection and identification of single domain antibody fragments from camel heavy-chain antibodies.", "FEBS Lett 414: 521-526.Bodenhausen, G. and Ruben, D. J., Chem.", "Phys.", "Lett., 69, 185-188, 1980.Cai, X. and Garen, A.", "(1996).", "A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient.", "Proc Natl Acad Sci USA 93: 6280-6285.Chothia, C and Lesk, A. M. (1987).", "Canonical structures for the hypervariable regions of immunoglobulins.", "J Mol Biol 196: 901-917.Chothia, C., Novotny, J., Bruccoleri, R., and Karplus, M. (1985).", "Domain association in immunoglobulin molecules.", "The packing of variable domains.", "J Mol Biol 186: 651-663.Clackson, T., Hoogenboom, H. R., Griffiths, A. D., and Winter, G. (1991).", "Making antibody fragments using phage display libraries.", "Nature 352: 624-628.Constantine, K. L., Goldfarb, V., Wittekind, M., Anthony, J., Ng, S. C., and Mueller, L. (1992).", "Sequential 1H and 15N NMR assignments and secondary stricture of a recombinant anti-digoxin antibody VL domain.", "Biochemistry 31: 5033-5043.Constantine, K. L., Goldfarb, V., Wittekind, M., Friedrichs, M. S., Anthony, J., Ng, S. C., and Mueller, L. (1993).", "Aliphatic 1H and 13C resonance assignments for the 26-10 antibody VL domain derived from heteronuclear multidimensional NMR spectroscopy.", "J Biomol NMR 3: 41-54.Dan, M., Earley, E. M., Griffin, M. C., Maiti, P. K., Prashar, A. K., Yuan, X. Y., Friesen, A. D., and Kaplan, H. 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[ [ "Solder foil semiconductor device and electronic device", "A solder foil formed from a material comprising particles of Cu, etc.", "as metal particles and Sn particles as solder particles by rolling is suitable for solder bonding at a high temperature side in temperature-hierarchical bonding, and semiconductor devices and electronic devices produced by use of such solder bonding have distinguished reliability of mechanical characteristics, etc." ], [ "1.A solder foil, characterized by being formed from a material comprising metal particles and solder particles by rolling.", "2.A solder foil, characterized by being formed from a material comprising Cu particles and Sn particles by rolling.", "3.A solder foil characterized by being formed from a solder comprising Cu and Sn by pressing, where Cu is in a particulate state and Sn is in a state of filling in spaces between the Cu particles.", "4.A solder foil according to claim 2, characterized in that when the solder foil is subjected to reflowing, at least one portion of the Cu particle surface is covered by Cu6Sn5.5.A solder foil according to claim 2, characterized in that when the solder foil is subjected to reflowing, the Cu particles and Sn after plastic deformation are bonded to each other by a compound comprising Cu6Sn5.6.A solder foil according to claim 2, characterized in that the Cu particles have particle sizes of 10-40 μm.", "7.A solder foil according to claim 2, characterized in that the Cu particles have particle sizes of 3-10 μm.", "8.A solder particles according to claim 2, characterized in that the Cu particles have a Ni plating or Ni/Au plating layer on the surfaces.", "9.A solder foil according to claim 2, characterized in that at least Cu-exposed portion of the foil are Sn-plated.", "10.A solder foil according to claim 1, characterized in that the solder foil has a thickness of 80 μm-150 μm.", "11.A solder foil according to claim 1, characterized in that the solder foil has a thickness of 150 μm-250 μm.", "12.A solder foil according to claim 1, characterized in that plastic particles are further contained.", "13.A solder foil according to claim 2, characterized in that other particles having a lower coefficient of thermal expansion than that of Cu is further contained.", "14.A solder foil according to claim 2, characterized in that other particles having a lower coefficient of thermal expansion than that of Cu are particles of Invar alloy series, silica, alumina, AlN or SiC.", "15.A solder foil according to claim 2, characterized in that In particles are further contained.", "16.A solder foil according to claim 2, characterized in that the Cu particles and the Sn particles are mixed together in vacuum, in a reductive atmosphere or in an inert gas atmosphere, and then formed into a foil by pressing.", "17.A solder foil according to claim 2, characterized in that a draft percentage is 15%-20%.", "18.A solder foil, characterized by being formed from a material comprising metal fibers and solder particles by rolling.", "19.A solder foil, characterized by being formed from a solder comprising Cu metal fibers and Sn particles by rolling.", "20.A solder foil according to claim 19, characterized in that the Cu metal fibers of the solder material are in an oblong shape.", "21.A solder foil, characterized by being formed from a solder material comprising particles of Al, An or Ag, and Sn particles by rolling.", "22.A solder foil, characterized by being formed from a solder material comprising particles of Zn—Al system alloy or Au—Sn system alloy, and Sn particles by rolling.", "23.An electronic device, which comprises a first electronic device, a second electronic device, and a third electronic device, characterized in that the first electronic device and the second electronic device are bonded to each other by a first solder foil according to claim 1, and the second electronic device and the third electronic device are bonded to each other by a second solder having a lower melting point than that of the first solder foil.", "24.An electronic device, characterized in that a first electronic device and a second electronic device are bonded to each other by a solder foil formed from a material comprising Cu particles and Sn particles by rolling, and the second electronic device and a third electronic device are bonded to each other by a solder having a lower melting point than that of the solder foil.", "25.A semiconductor device, which comprises a semiconductor chip, a tab on which the semiconductor chip is to be disposed, and a lead serving as a connector terminal to the outside, an electrode on the semiconductor chip being bonded to the lead by wire bonding, characterized in that the semiconductor chip and the tab are bonded to each other by a solder foil formed from a material comprising a mixture of metal particles and solder particles.", "26.A semiconductor device according to claim 25, characterized in that the solder foil is a solder foil formed from a material comprising a mixture of metal particles and solder particles by rolling.", "27.A semiconductor device according to claim 25, characterized in that the solder foil is a solder foil formed from a material comprising Cu particles and Sn particles by rolling.", "28.An electronic device, which comprises a circuit board and a semiconductor chip, an electrode on the circuit board and an electrode on the semiconductor chip being bonded to each other by wire bonding, characterized in that the circuit board and the semiconductor chip are bonded to each other by a solder foil comprising a mixture of metal particles and solder particles.", "29.An electronic device, which comprises a circuit board, and a semiconductor chip, an electrode on the circuit board and an electrode on the semiconductor chip being bonded to each other by wire bonding, characterized in that the circuit board and the semiconductor chip are bonded to each other by a solder foil formed from a solder material comprising Cu particles and Sn particles by rolling." ], [ "<SOH> BACKGROUND ART <EOH>In the case of solders of Sn—Pb system, it has been possible to carry out temperature-hierarchical bonding, which comprises soldering lead (Pb)-rich Pb-5Sn (melting point: 314-310° C.), Pb-10Sn (melting point: 302-275° C.), etc.", "as high temperature series solders at temperatures of approximately 330° C., and then bonding a Sn-37Pb eutectic mixture (melting point: 183° C.) as low temperature series solders without melting the first soldered portions.", "These solders have been capable of bonding Si chips, etc., which are flexible and high in deformability and thus is easy to break, to a substrate having a different coefficient of thermal expansion.", "Such a temperature-hierarchical bonding has been applied to semiconductor devices of chip die-bonding type, semiconductor devices of chip flip-chip-bonding type such as BGA, CSP, etc.", "That is, this means that a solder for use within the semiconductor device and a solder for bonding a semiconductor device itself to a substrate undergo temperature-hierarchical bonding.", "In every field, there is now a growing tendency to make the solder lead-free.", "Sn—Ag eutectic series (melting point: 221° C.), Sn—Ag—Cu eutectic series (melting point: 221°-217° C.) and Sn—Cu eutectic series (melting point: 227° C.) are now the mainstreams of Pb-free solders.", "A lower soldering temperature is desirable for the surface mounting in view of the heat resistance of parts, but actually it is approximately 235°-245° C. in the case of Sn—Ag—Cu eutectic series having a possibly lowest bonding temperature in view of the necessity for assuring a wettability to attain the reliability and also in view of considerable temperature fluctuations throughout a substrate in spite of using a furnace with distinguished temperature uniformity control.", "Thus, solders for the hierarchical bonding, which can withstand such a soldering temperature, must have a melting point of at least 250° C., but actually any Pb-free solders for use on the high temperature side of temperature-hierarchical bonding, which can be used in combination thereof, is not yet available.", "Sn-5Sb (melting point: 240°-232° C.) is a most possible Pb-free solder composition, but is not available for the temperature-hierarchical bonding because of melting thereof.", "Au-20Sn (melting point: 280° C.) is known as a solder of higher temperature series, but it is so hard and expensive that its use is restricted to a narrow range.", "Particularly in bonding Si chips to a material having a different coefficient of thermal expansion and also in bonding of large chips, the Au-20Sn solder is not available, because it is so hard as to break Si chips with high possibility." ], [ "<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.", "1 is views showing manufacturing steps of a composite metal from composite balls.", "FIG.", "2 is cross-sectional views of a model before and after rolling of elastomer plastic balls in a dispersion state, respectively.", "FIG.", "3 is a cross-sectional view of a model according to one embodiment of die bonding process.", "FIG.", "4 is a cross-sectional view of model showing a die bonded joint by using a solder foil comprising Cu and Sn in combination.", "FIG.", "5 is cross-sectional views of a model of bonding LSI and cap to a substrate.", "FIG.", "6 is a cross-sectional view of power module.", "FIG.", "7 is cross-sectional views of a model of mounting modules onto a printed circuit board.", "FIG.", "8 is cross-sectional views of a model of mounting RF module.", "FIG.", "9 is process flow charts each showing a process for mounting an RF module.", "FIG.", "10 is a plan view of a high power output resin package and cross-sectional views of models thereof.", "FIG.", "11 is a flow chart showing a process for making a high power output resin package.", "FIG.", "12 is a cross-sectional view of a model of plastic package.", "FIG.", "13 is a perspective view and a cross-sectional view of a model of a combination with metal fibers.", "FIG.", "14 is a perspective view of a model with crossed metal fibers.", "FIG.", "15 is cross-sectional views of a model with network fibers.", "FIG.", "16 is a plan view and a cross-sectional view showing long and slender fibers disposed at random, followed by flattening.", "FIG.", "17 is a cross-sectional view of a model with oblong metal fibers and non-metal fibers.", "detailed-description description=\"Detailed Description\" end=\"lead\"?", "In the drawings, reference numerals denote the following members: 1 .", ".", ".", "carbon jig, 2 .", ".", ".", "Cu ball, 3 .", ".", ".", "Sn ball, 4 .", ".", ".", "Sn, 5 .", ".", ".", "roll, 6 .", ".", ".", "plastic ball, 7 .", ".", ".", "resistance heater tool, 8 .", ".", ".", "Si chip, 9 .", ".", ".", "vacuum suction hole, 10 .", ".", ".", "nitrogen, 11 .", ".", ".", "solder foil, 12 .", ".", ".", "silicone gel, 13 .", ".", ".", "Al 2 O 3 substrate, 14 .", ".", ".", "W (sintered)-Cu plated electrode, 15 .", ".", ".", "heater for preheating, 16 .", ".", ".", "thermocouple for temperature measurement, 17 .", ".", ".", "Cu—Sn combination foil, 18 .", ".", ".", "bump, 19 .", ".", ".", "soft resin, 20 .", ".", ".", "lead, 21 .", ".", ".", "solder ball bump, 22 .", ".", ".", "printed circuit board, 23 .", ".", ".", "Al fin, 24 .", ".", ".", "bonded portion with fin, 25 .", ".", ".", "bonded portion with lead, 26 .", ".", ".", "lead, 27 .", ".", ".", "solder foil, 28 .", ".", ".", "terminal of substrate, 29 .", ".", ".", "module substrate, 30 .", ".", ".", "terminal, 31 .", ".", ".", "Cu, 32 .", ".", ".", "organic substrate, 33 .", ".", ".", "Cu throughhole conductor, 34 .", ".", ".", "Ag—Pd conductor, 35 .", ".", ".", "wire bond, 36 .", ".", ".", "AlN interposer substrate, 37 .", ".", ".", "connection terminal, 38 .", ".", ".", "Cr—Cu—Au, 39 .", ".", ".", "die bonding, 40 .", ".", ".", "solder foil, 41 .", ".", ".", "press, 42 .", ".", ".", "Ni—Au-plated metallization layer, 43 .", ".", ".", "interposer substrate, 44 .", ".", ".", "Cr—Ni—Au metallization layer, 45 .", ".", ".", "electroless Ni plating, 46 .", ".", ".", "electrical Ni plating, 47 .", ".", ".", "solder, 48 .", ".", ".", "Cu disc, 49 .", ".", ".", "Cu base, 50 .", ".", ".", "Al 2 O 3 insulation substrate, 51 .", ".", ".", "Cu lead, 52 .", ".", ".", "chip part, 53 .", ".", ".", "Cu pad, 54 .", ".", ".", "TQFP-LSI, 55 .", ".", ".", "Sn—Ag—Cu system solder, 56 .", ".", ".", "lead, 57 .", ".", ".", "dam cut portion, 58 .", ".", ".", "resin, 59 .", ".", ".", "throughhole, 60 .", ".", ".", "W—Ni—Au thick film electrode, 61 .", ".", ".", "W—Ni (or Ag—Pd, Ag) thick film conductor, 62 .", ".", ".", "Au-plated electrode, 63 .", ".", ".", "caulked portion, 64 .", ".", ".", "heat diffusion plate (header), 65 .", ".", ".", "lead frame, 66 .", ".", ".", "tab, 67 .", ".", ".", "electroconductive paste, 68 .", ".", ".", "solder, 69 .", ".", ".", "fiber, 70 .", ".", ".", "Cu netting (lateral cross-section), 71 .", ".", ".", "Cu netting (longitudinal cross-section), 72 .", ".", ".", "solder (sea), 73 .", ".", ".", "long and slender fiber, and 74 .", ".", ".", "oblong fiber" ], [ "TECHNICAL FIELD The present invention relates to a solder foil formed from a material comprising metal particles such as copper (Cu) particles or balls and solder particles such as tin (Sn) particles or balls by rolling, and a semiconductor device and an electronic device using the solder foil.", "BACKGROUND ART In the case of solders of Sn—Pb system, it has been possible to carry out temperature-hierarchical bonding, which comprises soldering lead (Pb)-rich Pb-5Sn (melting point: 314-310° C.), Pb-10Sn (melting point: 302-275° C.), etc.", "as high temperature series solders at temperatures of approximately 330° C., and then bonding a Sn-37Pb eutectic mixture (melting point: 183° C.) as low temperature series solders without melting the first soldered portions.", "These solders have been capable of bonding Si chips, etc., which are flexible and high in deformability and thus is easy to break, to a substrate having a different coefficient of thermal expansion.", "Such a temperature-hierarchical bonding has been applied to semiconductor devices of chip die-bonding type, semiconductor devices of chip flip-chip-bonding type such as BGA, CSP, etc.", "That is, this means that a solder for use within the semiconductor device and a solder for bonding a semiconductor device itself to a substrate undergo temperature-hierarchical bonding.", "In every field, there is now a growing tendency to make the solder lead-free.", "Sn—Ag eutectic series (melting point: 221° C.), Sn—Ag—Cu eutectic series (melting point: 221°-217° C.) and Sn—Cu eutectic series (melting point: 227° C.) are now the mainstreams of Pb-free solders.", "A lower soldering temperature is desirable for the surface mounting in view of the heat resistance of parts, but actually it is approximately 235°-245° C. in the case of Sn—Ag—Cu eutectic series having a possibly lowest bonding temperature in view of the necessity for assuring a wettability to attain the reliability and also in view of considerable temperature fluctuations throughout a substrate in spite of using a furnace with distinguished temperature uniformity control.", "Thus, solders for the hierarchical bonding, which can withstand such a soldering temperature, must have a melting point of at least 250° C., but actually any Pb-free solders for use on the high temperature side of temperature-hierarchical bonding, which can be used in combination thereof, is not yet available.", "Sn-5Sb (melting point: 240°-232° C.) is a most possible Pb-free solder composition, but is not available for the temperature-hierarchical bonding because of melting thereof.", "Au-20Sn (melting point: 280° C.) is known as a solder of higher temperature series, but it is so hard and expensive that its use is restricted to a narrow range.", "Particularly in bonding Si chips to a material having a different coefficient of thermal expansion and also in bonding of large chips, the Au-20Sn solder is not available, because it is so hard as to break Si chips with high possibility.", "DISCLOSURE OF INVENTION An object of the present invention is to provide a semiconductor device and an electronic device by quite a novel solder bonding, and particularly to provide solder bonding on the high temperature side in the temperature hierarchical bonding.", "Another object of the present invention is to provide quite a novel solder foil.", "The present invention provides a solder foil formed from a material comprising metal particles such as Cu particles or balls and solder particles such as Sn particles or balls by rolling.", "The present invention also provides an electronic device, which comprises a first electronic device, a second electronic device, and a third electronic device, characterized in that the first electronic device and the second electronic device are bonded to each other by a first solder foil as described above, and the second electronic device and the third electronic device are bonded, to each other by a second solder having a lower melting point than that of the first solder.", "The present invention further provides a semiconductor device, which comprises a semiconductor chip, a tab on which the semiconductor chip is to be disposed, and a lead serving as a connection terminal to the outside, an electrode on the semiconductor chip being bonded to the lead by wire bonding, characterized in that the semiconductor chip and the tab are bonded to each other by a solder foil formed from a material comprising a mixture of metal particles and solder particles as described above.", "BRIEF DESCRIPTION OF DRAWINGS FIG.", "1 is views showing manufacturing steps of a composite metal from composite balls.", "FIG.", "2 is cross-sectional views of a model before and after rolling of elastomer plastic balls in a dispersion state, respectively.", "FIG.", "3 is a cross-sectional view of a model according to one embodiment of die bonding process.", "FIG.", "4 is a cross-sectional view of model showing a die bonded joint by using a solder foil comprising Cu and Sn in combination.", "FIG.", "5 is cross-sectional views of a model of bonding LSI and cap to a substrate.", "FIG.", "6 is a cross-sectional view of power module.", "FIG.", "7 is cross-sectional views of a model of mounting modules onto a printed circuit board.", "FIG.", "8 is cross-sectional views of a model of mounting RF module.", "FIG.", "9 is process flow charts each showing a process for mounting an RF module.", "FIG.", "10 is a plan view of a high power output resin package and cross-sectional views of models thereof.", "FIG.", "11 is a flow chart showing a process for making a high power output resin package.", "FIG.", "12 is a cross-sectional view of a model of plastic package.", "FIG.", "13 is a perspective view and a cross-sectional view of a model of a combination with metal fibers.", "FIG.", "14 is a perspective view of a model with crossed metal fibers.", "FIG.", "15 is cross-sectional views of a model with network fibers.", "FIG.", "16 is a plan view and a cross-sectional view showing long and slender fibers disposed at random, followed by flattening.", "FIG.", "17 is a cross-sectional view of a model with oblong metal fibers and non-metal fibers.", "In the drawings, reference numerals denote the following members: 1 .", ".", ".", "carbon jig, 2 .", ".", ".", "Cu ball, 3 .", ".", ".", "Sn ball, 4 .", ".", ".", "Sn, 5 .", ".", ".", "roll, 6 .", ".", ".", "plastic ball, 7 .", ".", ".", "resistance heater tool, 8 .", ".", ".", "Si chip, 9 .", ".", ".", "vacuum suction hole, 10 .", ".", ".", "nitrogen, 11 .", ".", ".", "solder foil, 12 .", ".", ".", "silicone gel, 13 .", ".", ".", "Al2O3 substrate, 14 .", ".", ".", "W (sintered)-Cu plated electrode, 15 .", ".", ".", "heater for preheating, 16 .", ".", ".", "thermocouple for temperature measurement, 17 .", ".", ".", "Cu—Sn combination foil, 18 .", ".", ".", "bump, 19 .", ".", ".", "soft resin, 20 .", ".", ".", "lead, 21 .", ".", ".", "solder ball bump, 22 .", ".", ".", "printed circuit board, 23 .", ".", ".", "Al fin, 24 .", ".", ".", "bonded portion with fin, 25 .", ".", ".", "bonded portion with lead, 26 .", ".", ".", "lead, 27 .", ".", ".", "solder foil, 28 .", ".", ".", "terminal of substrate, 29 .", ".", ".", "module substrate, 30 .", ".", ".", "terminal, 31 .", ".", ".", "Cu, 32 .", ".", ".", "organic substrate, 33 .", ".", ".", "Cu throughhole conductor, 34 .", ".", ".", "Ag—Pd conductor, 35 .", ".", ".", "wire bond, 36 .", ".", ".", "AlN interposer substrate, 37 .", ".", ".", "connection terminal, 38 .", ".", ".", "Cr—Cu—Au, 39 .", ".", ".", "die bonding, 40 .", ".", ".", "solder foil, 41 .", ".", ".", "press, 42 .", ".", ".", "Ni—Au-plated metallization layer, 43 .", ".", ".", "interposer substrate, 44 .", ".", ".", "Cr—Ni—Au metallization layer, 45 .", ".", ".", "electroless Ni plating, 46 .", ".", ".", "electrical Ni plating, 47 .", ".", ".", "solder, 48 .", ".", ".", "Cu disc, 49 .", ".", ".", "Cu base, 50 .", ".", ".", "Al2O3 insulation substrate, 51 .", ".", ".", "Cu lead, 52 .", ".", ".", "chip part, 53 .", ".", ".", "Cu pad, 54 .", ".", ".", "TQFP-LSI, 55 .", ".", ".", "Sn—Ag—Cu system solder, 56 .", ".", ".", "lead, 57 .", ".", ".", "dam cut portion, 58 .", ".", ".", "resin, 59 .", ".", ".", "throughhole, 60 .", ".", ".", "W—Ni—Au thick film electrode, 61 .", ".", ".", "W—Ni (or Ag—Pd, Ag) thick film conductor, 62 .", ".", ".", "Au-plated electrode, 63 .", ".", ".", "caulked portion, 64 .", ".", ".", "heat diffusion plate (header), 65 .", ".", ".", "lead frame, 66 .", ".", ".", "tab, 67 .", ".", ".", "electroconductive paste, 68 .", ".", ".", "solder, 69 .", ".", ".", "fiber, 70 .", ".", ".", "Cu netting (lateral cross-section), 71 .", ".", ".", "Cu netting (longitudinal cross-section), 72 .", ".", ".", "solder (sea), 73 .", ".", ".", "long and slender fiber, and 74 .", ".", ".", "oblong fiber BEST MODES FOR CARRYING OUT THE INVENTION Among the inventions disclosed herein to attain the aforementioned objects, typical ones are briefly outlined below: (1) A solder foil formed from a material comprising metal particles and solder particles by rolling.", "(2) A solder foil formed from a solder material comprising Cu particles and Sn particles by rolling.", "(3) A solder foil formed from a solder comprising Cu and Sn by pressing, where Cu is in a particulate state and Sn is in a state of filling interspaces between the Cu particles.", "(4) A solder foil as described in said item (2) or (3), wherein when the solder foil is subjected to reflowing at least one portion of the Cu particle surfaces is covered by Cu6Sn5.", "(5) A solder foil as described in said item (2) or (3), wherein when the solder foil is subjected to reflowing the Cu particles and the Sn after plastic deformation are bonded to each other by a compound comprising Cu6Sn5.", "(6) A solder foil as described in said items (2) to (5), wherein the Cu particles have particle sizes of 10-40 μm.", "(7) A solder foil as described in said items (2) to (5), wherein the Cu particles have particle sizes of 3-10 μm.", "(8) A solder foil as described in said items (2) to (7), wherein the Cu particles have a Ni plating or Ni/Au plating layer on the surfaces.", "(9) A solder foil as described in said items (2) to (8), wherein at least Cu-exposed portions of the foil are Sn-plated.", "(10) A solder foil as described in said items (1) to (9), wherein the solder foil has a thickness of 80 μm-150 μm.", "(11) A solder foil as described in said items (1) to (9), wherein the solder foil has a thickness of 150 μm-250 μm.", "(12) A solder foil as described in said items (1) to (11), wherein plastic particles are further contained.", "(13) A solder foil as described in said item (2) or (3), wherein other particles having a lower coefficient of thermal expansion than that of Cu are further contained.", "(14) A solder foil as described in said item (2) or (3), wherein other particles having a lower coefficient of thermal expansion than that of Cu are particles of Invar alloy series, silica, alumina, AlN (aluminum nitride), or SiC.", "(15) A solder foil as described in said item (2) or (3), wherein In particles are further contained.", "(16) A solder foil as described in said items (2) or (3), wherein the Cu particles and the Sn particles are mixed together in vacuum, in a reductive atmosphere or in an inert gas atmosphere, and then formed into a foil by pressing.", "(17) A solder foil as described in said item (2) or (3), wherein a draft percentage is 15%-20%.", "(18) A solder foil formed from a material comprising metal fibers and solder particles by rolling.", "(19) A solder foil formed from a solder material comprising Cu metal fibers and Sn particles by rolling.", "(20) A solder foil as described in said item (19), wherein the Cu metal fibers of the solder material are in an oblong shape.", "(21) A solder foil formed from a solder material comprising particles of Al, Au or Ag, and Sn particles by rolling.", "(22) A solder foil formed from a solder material comprising particles of Zn—Al system alloy or Au—Sn system alloys, and Sn particles by rolling.", "(23) An electronic device, which comprises a first electronic device, a second electronic device, and a third electronic device, wherein the first electronic device and the second electronic device are bonded to each other by a first solder foil as described in said items (1) to (22), and the second electronic device and the third electronic device are bonded to each other by a second solder having a lower melting point than that of the first solder foil.", "(24) An electronic device, wherein a first electronic device and a second electronic device are bonded to each other by a solder foil formed from a material comprising Cu particles and Sn particles by rolling, and the second electronic device and a third electronic device are bonded to each other by a solder having a lower melting point than that of the solder foil.", "(25) A semiconductor device, which comprises a semiconductor chip, a tab on which the semiconductor chip is to be disposed, and a lead serving as a connector terminal to the outside, an electrode on the semiconductor chip being bonded to the lead by wire bonding, wherein the semiconductor chip and the tab are bonded to each other by a solder foil formed from a material comprising a mixture of metal particles and solder particles.", "(26) A semiconductor device as described in said item (25), wherein the solder foil is a solder foil formed from a material comprising a mixture of metal particles and solder particles by rolling.", "(27) A semiconductor device as described in said item (25), wherein the solder foil is a solder foil formed from a material comprising Cu particles and Sn particles by rolling.", "(28) An electronic device, which comprises a circuit board and a semiconductor chip, an electrode on the circuit board and an electrode on the semiconductor chip being bonded to each other by wire bonding, wherein the circuit board and the semiconductor chip are bonded to each other by a solder foil comprising a mixture of metal particles and solder particles.", "(29) An electronic device, which comprises a circuit board and a semiconductor chip, an electrode on the circuit substrate and an electrode on the semiconductor chip being bonded to each other by wire bonding, wherein the circuit board and the semiconductor chip are bonded to each other by a solder foil formed from a solder material comprising Cu particles and Sn particles by rolling.", "Or, a solder foil characterized by being formed by mixing metal balls, which comprise solder-wettable simple metal, alloy, compound or their mixture, with solder balls, which comprise at least one of Sn and In, followed by pressing to fill gaps therebetween and successive rolling; Or a solder foil formed by mixing metal balls, which comprise solder-wettable simple metal, alloy, compound or their mixture, with solder balls, which comprise at least one of Sn and In, placing the resulting mixture in advance into an easy-to-roll mold to which a uniform pressure is to be applied, and uniformly pressing the mixture to fill gaps therebetween, thereby eliminating the gaps, followed by rolling the resulting combination solder material; Or, the above-mentioned solder foil, wherein the solder balls further contain at least one of Ag, Bi, Cu, Zn, Ni, Pd, Au, Sb, etc.", "in addition to Sn and In; Or, the afore-mentioned solder foil, wherein the metal balls comprises Cu, Cu alloy, Cu6Sn5 compound, Ag, Ag—Sn compound, Au, Au—Sn compound, Al, Al—Ag compound, Al—Au compound, Zn—Al system solder or their mixtures; Or, the afore-mentioned solder foil, wherein the rolled foil or the combination solder material is plated with a plating material comprising Sn and at least one of Bi, In, Ag, Au, Cu, Ni and Pd; Or, the afore-mentioned solder foil, wherein the metal balls comprising the simple metal, alloy, compound or their mixture, when not wettable, are provided with a solder-wettable metallization layer such as a plating of Ni, Ni—Au, Cu, Ag, Sn, Au, or the like, or their combination plating, or a further plating of Sn system in addition thereto on the surfaces of the metal balls; Or, the afore-mentioned solder foil, wherein the solder foil has a particle size distribution corresponding to the closest packing of the metal balls, which comprises the simple metal, alloy, compound or their mixture; Or, the afore-mentioned solder foil, wherein the combination solder material further contains plastic balls having a solder-wettable metallization layer on the surfaces in a dispersed state to reduce the rigidity of the combination solder material; Or, the above-mentioned solder foil, wherein the combination solder material further contains particles having a lower coefficient of thermal expansion than that of the metal balls comprising the simple metal, alloy, compound or their mixture and having a solder-wettable metallization layer on the surfaces or further a solder plating of Sn, In, etc.", "thereon in a dispersion state to reduce the coefficient of thermal expansion of the combination solder material; Or, the afore-mentioned solder foil, wherein the particles having a lower coefficient of thermal expansion includes Invar alloy system (Fe-36Ni alloy), silica, alumina, AlN, SiC, etc.", "; Or, the afore-mentioned solder foil, wherein materials for the plastic balls include polyimide-based resin, heat-resistant epoxy resin, silicone-based resin, various polymer beads, or their modified products or their mixtures; Or, the afore-mentioned solder foil in the form of band, wire, ball, or block; Or, the afore-mentioned solder foil, wherein metal fibers or copper plated fibers of carbon, glass, ceramics, etc.", "or a dispersion mixture of the metal balls in the metal fibers are used in place of the metal balls; Or, the afore-mentioned solder foil, wherein cross-wise disposed assembly of metal fibers or copper-plated fibers of carbon, glass, ceramics, etc.", "or a dispersion mixture of the metal balls with the cross-wise disposed assembly of the fibers is used in place of the metal balls; Or, the afore-mentioned solder foil, wherein network of metal fibers or copper-plated fibers of carbon, glass, ceramics, etc.", "or dispersion of the metal balls in the netting is used in place of the metal balls; Or, the afore-mentioned solder foil, wherein the fibers have a diameter of 1-20 μm, preferably 3-15 μm; Or, the afore-mentioned solder foil, wherein metal short fibers or copper-plated short fibers of carbon, glass, ceramics, etc.", "or dispersion of the metal balls in the short fibers are used in place of the metal balls; Or, the afore-mentioned solder foil, wherein the short fibers have a diameter of 1-10 μm, preferably 1-5 μm, and an aspect ratio (length/diameter) of 2-5.When metal balls of Cu, etc.", "and Sn system solder balls are mixed in a proportion of about 50% each and rolled, the Cu particles are brought into contact with each other, while Sn enters into gaps therebetween to form a combination solder.", "When the resulting foil is inserted between a chip and a substrate, followed by pressing and reflowing, the Cu balls themselves are linked to one another by a Cu—Sn compound at the combination solder portions, while a compound of the Cu balls with the chip electrode or a compound of the Cu balls with the substrate terminal is formed between the combination solder portions and the chip and between the combination solder portions and the substrate to produce a lead-free temperature-hierarchical bonding structure assuring a good bonding strength even at such high temperatures as 280° C. That is, a temperature-hierarchical bonding method can be provided in the lead-free solder field.", "In the temperature-hierarchical bonding, the already bonded solder of high temperature side, even if partly melted but so far as the remaining portions are not melted, can keep a sufficient strength enough to withstand the successive solder bonding process.", "We are now making study of solder materials of dispersion mixtures of metal balls (Cu, Ag, Au, surface-treated Al, Zn—Al system solders, etc.)", "with solder balls.", "Once the bonding is established with such solder materials, even by passage through a reflow furnace (250° C. maximum) for Sn—Ag—Cu system solder as successive solder bonding the established precedent bonding can be maintained even at the set reflow furnace temperature (250° C. maximum) because of the bonding by an intermetallic compound of higher melting point (Cu6Sn5) between the Cu balls themselves, between the Cu balls and the chips and between the Cu balls and the substrate, though the Sn in the bonded portions can melt, thereby assuring the sufficient bonding strength.", "That is, temperature-hierarchical bonding can be attained for the Sn—Ag—Cu system solder.", "Effect of the intermetallic compound formation is not limited to Cu—Sn, but the same effect can be obtained with compounds of Ni—Sn (Ni5Sn4), Ag—Sn (Ag3Sn), etc., and also with Au—Sn.", "Solders comprising In in place of Sn have the same effect.", "Diffusion-formed alloy layer has a high melting point, though there is a difference in growth rate of alloy layer, and never melts at 280° C., once formed.", "Bonding by such solder materials remains in an incompletely restricted state of Cu balls themselves and there is some degree of freedom in the vertical and horizontal directions in case of, for example, die bonded joints.", "Thus, mechanical characteristics of intermediate level between Cu balls and solder balls can be expected and also thermal fatigue resistance by Sn and high reliability by prevention of crack development by Cu particles (balls) can be expected even in temperature cycle tests.", "However, it has been clarified through our studies that the combination paste comprising a mixture of Cu balls and solder balls has a high probability of void formation, because the Sn system solder per se has a property of less wetting extendability on the Cu balls, though the Cu balls have many wettable portions, all the Cu balls are not completely wetted and furthermore the Cu balls and the solder balls are initially restricted by cross-linking therebetween, and when the solder balls melt, the spaces remain as voids in the melted solder ball positions.", "Thus, the combination paste tends to produce a large number of voids inevitably and thus may be converted to unsuitable solder materials, though dependent on desired bonding purpose.", "It is preferable to eliminate voids when electronic parts are to be mounted, but in the case of face-to-face bonding as in, for example, die bonding of Si chips, bonding of power module, etc.", "voids are structurally hard to eliminate.", "The remaining voids will give rise to such problems as crack development, obstruction to necessary heat diffusion, etc.", "due to the presence of voids.", "Thus, we tried to use a solder foil prepared by placing the solder material into an easy-to-roll mold in advance, uniformly pressing, the entirety in vacuum, in a reductive atmosphere or in an inert gas atmosphere, thereby subjecting Sn system solder balls to plastic flow through between metal balls to obtain a composite molding in which gaps are filled with the solder (Sn system solder after plastic deformation), and rolling the combination molding.", "It was confirmed that, for example, in the case of preparing a solder foil for die bonding Si chips from the combination molding, match balls of Cu—Cu, etc.", "were brought into contact with one another by pressing and an intermetallic compound was easily formed between the metal balls by die bonding to make organic linkage by the metal of high melting point entirely and assure a satisfactory strength even at 280° C. As a matter of course, the voids were compressed in vacuum and collapsed at the bonded portions, thereby enabling bonding with less voids.", "It was also confirmed that in the case of using a low temperature hot press in a nitrogen gas atmosphere and using Cu balls and Sn system solder balls having a larger ball diameter (about 40 μm), the Sn system solder showed a void-filling ratio of 97% or more.", "Furthermore, application of Sn plating of appropriate layer thickness to the foil surface could prevent materials susceptible to considerable oxidation from oxidation.", "It was also confirmed that when a lap joint prepared by bonding Cu foil leads to each other by this solder was subjected to a shearing tensile test at 270° C. and a pulling speed of 50 mm/min., a test result of about 0.3 kgf/mm2 showing a satisfactory strength at a high temperature was obtained.", "The combination molding is to fill spaces in the material with metal balls in advance, and thus it can be expected that there are correspondingly less voids in a void ratio on the same level as or less than that of the conventional solder foil (because of the inherent structure hardly capable of forming larger voids).", "Thus, it has been an important task for the soldering according to the combination molding method to eliminate voids because of the inherent larger soldering area.", "For example, suitable lead-free materials can be provided thereby for Si die bonding, power module bonding, etc.", "(where “lead-free” is in the sense that any lead should not be contained intensively).", "That is, high temperature, lead-free materials with high reliability of temperature-hierarchical bonding, etc.", "can be provided thereby.", "In the paste solder method, on the other hand, it has been difficult to make the solder fluxless due to the easy oxidation, but the combination molding method can solve the fluxless problem.", "That is, in the field where flux residues should be avoided, it has been necessary to wash the remaining, flux out after bonding by the paste solder method, and the washing has be omitted by the fluxless combination molding method.", "Even in the case of hard and highly rigid solder having a desirable melting point, for example, Au-20Sn, Au-(50-55)Sn (melting point: 309°-370° C,), Au-12Ge (melting point: 356° C.), etc., they are used as metal balls and mixed with more soft, elastic rubber particles together with soft solder balls of Sn, In, etc.", "to make a dispersion in the metal balls, whereby the solidus temperature of the solder containing the metal balls can be kept at about 280° C. or higher, so that good bonding strength at high temperatures can be obtained, while any deformation can be lessened by soft Sn or In and rubber existing between the metal balls.", "Thus, a novel effect of making up for disadvantages of these hard and highly rigid solders can be expected.", "Modes for carrying out the present invention will be described below.", "FIG.", "1 outlines step of preparing a composite metal from a combination of balls (metal balls and solder balls), where (a) shows a state of Cu balls 2 as metal balls and Sn balls 3 as solder balls provided in carbon jig 1 in a vacuum hot press, (b) shows a cross-sectional profile model of a block of a combination of the balls after plastic flow of solder by vacuum hot pressing, where Sn and Cu are deformed into a “sea-and-island structure”, and (c) shows a model of rolling the block of a combination of balls by rolls 5 to form a solder foil.", "In FIG.", "1, Cu balls, 10-40 μm in diameter are mixed together in such a volume ratio as to make 50-60% of Cu balls.", "Contact area between the Cu balls can be increased by adding microfine Cu particles to the Cu balls in a closest packing ratio (refer to e.g.", "Shigeo Miwa: Outline of Powder Technology, page 39, published by Nikkan Kogyo Shinbun K. K. in Feb. 5, 1981).", "The closest packing can make a theoretical volume ratio of Cu about 74% and that of the solder 26%.", "So can do the microfine particles, not more than 10 μm in diameter, resulting in formation of finer network of alloy layer.", "This is suitable for high density fine bonding.", "In the case of, e.g.", "Cu balls, 3-8 μm in diameter and Sn balls, 10-40 μm in diameter, or in the case of Cu balls, 3-10 μm in diameter and Sn balls, 10-40 μm in diameter, or in the case of Cu balls, 5-15 μm in diameter and Sn balls, 10-40 μm in diameter, the solder packing density of foils will be decreased, but a good bonding result will be obtained.", "As to the diameters (sizes) of Cu balls, Sn balls, etc., all the particles are not always included in the disclosed sizes, but it is needless to say that larger or smaller balls than the disclosed sizes may be included within such a range as not to affect the effect of the invention.", "These balls are mixed together in a nitrogen gas atmosphere and placed in a pressure vessel comprising a carbon jig, as shown in FIG.", "1(a).", "After evacuation, only Sn undergoes plastic deformation to fill gaps between the Cu balls by applying pressure uniformly and circumferentially to the mixture with time.", "Sn has a melting point of 232° C., but can be made to flow even at room temperature with prolonged time.", "When Sn fails to flow to every corners at room temperature, the flowing can be easily made by elevating the temperature to some degrees (100°-150° C.).", "So far as Cu and Sn fail to react with each other in this step, the restriction is not so high on the interface that the degree of freedom becomes higher and Sn can be more easily deformed (made to flow).", "The block of a combination of the balls formed by the vacuum hot press is further rolled through rolls 5 to form a solder foil.", "The gaps between the Cu balls are much more eliminated by rolling, so that the solder foil with much less voids can be obtained.", "In that case the block of a combination of the balls is directed to preparation of a solder foil having a thickness of 150 μm (±10 μm), and it is desirable to form the block of shape similar to foil shape in advance because the draft percentage can be reduced.", "Increase in the draft percentage will increase a contact area of Cu ball themselves and thus will intensity restriction by the increased contact area.", "In view of flexibility to deformation by temperature cycle, etc., it is desirable to reduce the contact area.", "Ultimate draft percentage is preferably not more than 20%, more preferably 15-20%.", "When Cu, etc.", "are exposed from the solder foil so formed, it is preferable to prevent the exposed Cu, etc.", "from oxidation by further plating Sn to a thickness of 0.5-2 μm.", "From the viewpoints of easy preparation, easy uniform dispersion at the mixing, easy handling, etc., it is preferable that the metal balls such as Cu balls and the solder balls are spherical, though the spherical shape is not always necessary.", "Cu balls with considerably rugged surfaces, Cu balls of bar shapes, needle shapes, fiber shapes, angular shapes or dendritic shapes may be recommended, or their combinations will be also recommended, so far as Cu members can be entangled with one another after bonding.", "However, when the Cu members themselves are so restricted by the compression as to lower the degree of freedom, the cushionability will be lost at the stage of soldering and bonding failure will be easy to occur.", "In that case, Cu balls with considerably rugged surfaces, or Cu balls of bar shapes, needle shapes, fiber shapes, angular shapes or dendritic shapes or their combinations are preferable rather than true ball shape.", "As shown in FIG.", "2, metallized by electroless Ni plating-Au plating or electroless Ni plating-solder plating) plastic balls (rubber) 6 as a heat-resistant, soft elastomer can be further dispersed therein besides Cu balls 2 and Sn balls 3 to lower a Young's modulus, thereby assuring the cushionability.", "FIG.", "2(a) shows a state before rolling and FIG.", "2(b) shows that after rolling.", "The resin balls have a diameter of ideally not more than 10 μm, preferably 1 μm level, for example, desirably 0.5-5 μm.", "A mixing proportion even of a few % by volume of the resin balls is effective.", "Two terms “particles” and “balls” are herein used as to “metal” and “solder”, but these two terms are used herein substantially in the same meaning, as is clear from the foregoing description.", "In further distinction therebetween, the term “particles” has a rather broad sense including the term “balls”.", "The case of using Al as another example of metal balls will be described below.", "Generally, metals of high melting points are hard, but a low cost, soft metal is pure Al.", "Pure Al (99.99%) is soft (Hv 17), but is usually less wettable.", "Thus, it is preferable to apply Ni—Au plating or Ni—Sn plating or the like to pure Al.", "Coating of Al surface with Au by sputtering, etc.", "can be recommended.", "It is difficult to prepare microfine particles of pure Al due to a safety problem of explosion, etc., but the safety can be assured by preparing the microfine particles in an inert gas atmosphere and successively applying Ni—Au plating to the surfaces, thereby preventing Al from contact with the atmosphere.", "An oxide film, even if formed on the Al particles to some extent, can be removed by a plating treatment without any problem.", "Furthermore, the oxide film on Al can be easily broken in the rolling step to expose fresh Al surfaces, so that bonding is not so affected thereby.", "The metallization layer on the Al surface is not restricted thereto, but it is necessary that after the preparation of a solder foil the solder can wet Cu, Ni, etc.", "to assure a sufficient bonding strength at high temperatures.", "Thus, it is necessary that bonding can be made between the Al particles and the Ni-plated Cu plate and between the Al particles and Ni plating on the Si chips by formation of an Sn compound with the metallization layer and Ni.", "In the preparation of a block of a combination of balls, Al is liable to disperse in vacuum and particularly at high temperatures, and thus Ag-containing Sn solder is used to form a compound with Al.", "Besides Ag, a very small amount of Zn, Cu, Ni, Sb, etc.", "is added to Sn to facilitate reaction with Al, thereby making a solder for Al bonding.", "When a very small amount of Ag, Zn, Cu, Ni, Sb, etc.", "is added to Sn, it is not necessary to provide a metallization layer on the Al surface with the result of a larger merit of cost.", "The Al surface can be wetted entirely or spotwise, depending on relation to the existing state of a metallization layer, that is, on whether the metallization layer is to be formed entirely or spotwise.", "In the case of spotwise wetting, restriction can be reduced at the stage of stress deformation, resulting in easy deformation, while the unwetted portions can absorb energy as a friction loss.", "Thus, solder materials with a good deformability as well as an assured bonding strength can be obtained thereby.", "In place of formation of Al into a ball shape, it is possible to use other shapes, prepared by applying a plating of Sn, Ni—Sn, Au or the like to an Al wire, approximately 20-40 μm in diameter, and cutting the plated Al wire into a particulate shape or a stick shape.", "Al particles in a ball shape can be mass-produced at a low cost by atomization in a nitrogen gas atmosphere.", "Description of Au balls will be made below.", "In the preparation of a block of a combination of balls, Au balls can be easily wetted by a Sn system solder, and in the case of bonding for a short time any metallization layer is not required.", "In the case of prolonged soldering time, Sn diffuses so considerably that a fear of formation of a brittle Au—Sn compound arises.", "Thus, In plating, etc.", "of less Au diffusibility are useful for making a soft structure, or Ni, N—Au, etc.", "can be used as a barrier.", "Au balls can be easily deformed by making the barrier layer as thin as possible.", "Any other metallized structure can be used, so far as it can suppress growth of an alloy layer with Au.", "Diffusion can be suppressed up to the rolling step by controlling the temperature.", "In the case of short-time bonding by die bonding, the effect of Au flexibility can be expected without any provision of a barrier, because the alloy layer formed on the grain boundaries is very thin.", "A combination of Au balls and In solder balls is also available.", "Description of Ag balls will be made below.", "The same things as mentioned above as to Cu balls can be applied also to Ag balls, but mechanical properties of Ag3Sn compound are quite satisfactory, so that Ag particles can be bonded to one another by the compound according to the ordinary process.", "Ag balls can be used in mixture with Cu, etc.", "Cases of using alloy materials as metal balls will be described below.", "Typical examples of alloy series include Zn—Al system, Au—Sn system, etc.", "The Zn—Al system solders have a melting point ranging mostly from 330° to 370° C., which is in a suitable temperature range for temperature hierarchical bonding with Sn—Ag—Cu system, Sn—Ag system and Sn—Cu system solders.", "These alloys can be used as metal balls.", "Typical examples of Zn—Al system include Zn—Al—Mg, Zn—Al—Mg—Ga, Zn—Al—Ge and Zn—Al—Mg—Ge, which can further contain at least one of Sn, In, Ag, Cu, Au, Ni, etc.", "However, it is pointed out that in the case of Si bonding there is a fear of Si chip cracking because Zn—Al system undergoes vigorous oxidation and the rigidity of the solder is very high, etc.", "(Shimizu et al: Zn—Al—Mg—Ga alloy for Pb-free solders directed to die attaching, Mate 99, 1999-2).", "In the case of using it as metal balls in the block of a combination of balls, these problems must be solved.", "From the necessity for solving these problems, Ni-solder plated or Au-plated, heat-resistant plastic balls are uniformly mixed with and dispersed through between Sn balls and Zn—Al system balls to lower the rigidity of solder and reduce the Young's modulus.", "When Sn balls are mixed in a proportion of 10-50% on the basis of the entire mixture, molten Sn balls enter into the spaces between the Zn—Al system solder balls.", "In that case, Zn—Al balls themselves are bonded to one another to some extent, but deposited low-temperature, soft Sn—Zn phases or undissolved Sn remain in the most cases.", "Such Sn, Sn—Zn phases and rubber of plastic balls take part in the deformation.", "In the case of actual bonding using the solder foil, the Sn can absorb deformation by a portion of Sn layer made to remain after, for example, die bonding.", "It can be further expected that the rigidity can be lowered by a double action of the plastic balls and the Sn layer.", "In that case, the solidus temperature of the Zn—Al system solder is kept at 280° C. or higher, and thus there is no problem of strength at high temperatures.", "It is desirable that the plastic balls have a smaller diameter than that of Zn—Al system balls and are uniformly dispersed.", "When soft and elastic plastic balls of 1 μm level in diameter are deformed at the stage of deformation, remarkable effects on thermal shock damping and mechanical shock damping can be obtained.", "Heat-resistant plastic balls are commercially available.", "Since the plastic balls are substantially uniformly dispersed through between the Zn—Al system solder balls, such dispersion is mostly not broken by short-time melting at the stage of bonding.", "The heat-resistant resin has a thermal decomposition temperature of approximately 300° C., and thus more heat-resistant materials are desirable to use, but there is no problem in the case of short-time die bonding.", "As described before, in the case of molding by the vacuum hot press, uniform pressing at a temperature incapable of melting Sn on the Sn-plated plastic balls (melting point of Sn: 232° C.) gives rise to plastic flowing, where the Zn—Al balls are seldom deformed.", "Spaces are uniformly filled with the plastic balls, Sn, etc.", "by uniform pressing, followed by rolling to a thickness of approximately 150 μm to form a solder foil.", "For use in die bonding, the solder foil can be coiled around a roll and supplied to a continuous process.", "Zn—Al is easily oxidized, and thus in view of its storage it is desirable to apply Cu-substituted Sn plating to the surface.", "The Sn and Cu are dissolved into Zn—Al system solder at the stage of, for example, die bonding.", "The presence of Sn on the surface can facilitate bonding to Ni—Au plating on the Cu electrode.", "Si chip sides can be easily bonded to, for example, Ti—Ni—Au metallization layer.", "Since the growth rate of alloy layer of Ni and Sn (Ni3Sn4) is higher than that of Cu—Sn at temperatures of not lower than 200° C., there is no bonding failure due to insufficient formation of the compounds.", "In some cases, formation of a block of a combination of Zn—Al system solder balls and plastic balls can be recommended.", "Temperature-hierarchical bonding is enabled by adding to the Zn—Al system solder larger amounts of Sn and In to such a level as to assure the 280° C. level of the solidus temperature.", "Addition of increased amounts of Sn and In gives rise to partial formation of low strength phase of Zn—Sn eutectic alloy, etc., but the bonding strength depends on the solid phase of Zn—Al system as a skeleton, and thus there is no problem of strength at high temperatures.", "By applying Cu-substituted Sn plating to the Zn—Al system solder, and elevating the temperature to the liquidus temperature of Zn—Al system solder or higher, Sn can easily extend the wetting area and dissolved into the Zn—Al system solder, while bringing thin Cu layer into solid solution.", "When Sn is too much (for example, about 7%), Sn fails to undergo solid solution into Zn—Al, while depositing a low-temperature Sn—Zn phase on the grain boundries.", "By intensive dispersion and deposition of a large number of Sn phases, the Sn—Zn phase can be allocated to deformation, whereas the Zn—Al system solid phase can be allocated to bonding strength.", "Thus, by applying Sn plating to the Zn—Al system solder balls and intensively allowing the Sn phase incapable of undergoing solid solution into the balls to remain, deformation can be absorbed by the Sn layer to lower the rigidity of Zn—Al.", "That is, the rigidity of the solder at the bonded portions can be lowered and the bonding failure can be made less.", "FIG.", "3 shows one embodiment of die bonding Si chip 8 to W—Cu plating metallization layer 14 (Ni plating will do in place of the W—Cu plating) on Al2O3 substrate 13, using the aforementioned solder foil 11.A typical example of solder foil 11 is a combination of Cu as metal balls and Sn as a solder.", "Cu is relatively soft and very reactive with Sn, and the mechanical properties of the intermetallic compound (Cu6Sn5) are so distinguished that brittleness is hard to come out even if the compound grows thickly.", "Even if the growth of the compound is considerable to show an adverse effect, it is possible to retard the growth rate of the alloy layer by adding a very small amount of Cu or the like to Sn.", "It is also possible to suppress the growth of the alloy layer by applying a thin Ni plating of Ni, Ni—Au or the like to Cu, where it is important that the Cu balls are surely linked to one another by the intermetallic compound at the stage of short-time soldering, and also it is desirable to actively conduct the reaction.", "Thus, excess growth is no problem.", "It is rather important in the bonding between Sn and the chip and between Sn and the substrate to improve the wettability and wetting extendability of Sn.", "Thus, effects of flowability improvement by addition of a very small amount of Cu and Bi to Sn and wettability improvement by reduction in the surface tension can be expected.", "Furthermore, an effect by addition of a very small amount of Ni, Ag, Zn etc.", "can be also expected due to the improvement of the strength on the boundaries.", "Replacement of Sn with Sn—Sb (5-10%) can contribute to an improvement of the melting point of Sn, and Sb concentration of the solder can be increased by formation of Cu—Sb compound and Ni—Sn compound, whereby the melting point of the solder can be increased to 246° C. Pure Al balls further softer than Cu as another typical example has a distinguished temperature cycle deformability.", "Reaction between Al balls and a chip or a metallization layer on the substrate is a problem.", "By applying Ni plating or Ni—Au flash plating to the Al surface, good bonding strength by Sn can be likewise assured between the Al balls themselves, between the Al balls and Ni-plated chip or Ni-plated substrate.", "Intermetallic compound between Ni and Sn is usually Ni3Sn4, which has a higher growth rate than that of Cu—Sn at 200° C. or higher, and thus there is no fear of insufficient reaction.", "Where Cu and Ni are present together, an alloy layer containing (NiCu)3Sn4 can be partly formed sometimes.", "To allow the solder to react directly with Al balls, a very small amount of Ag, Ni, Zn, Ti, etc.", "is added to Sn, whereby bonding between the Al balls themselves can be also obtained, depending on bonding conditions.", "The same steps can be also taken toward Au balls.", "Au is soft and can easily form a compound with Sn, and thus can provide a useful composition apart from the cost side.", "However, compound of Sn-rich system have low melting points.", "To obtain a melting point of 280° C. or higher, it is necessary to make compounds of AuSn and AuSn2 in a Sn composition ratio of not more than 55% Sn.", "Thus, it is necessary to make the bonded portion Sn-poor by elevating the soldering temperature.", "Formation of Au—Sn and AuSn can be easily made by providing, for example, Cr—Ni—Sn metallization layer on the Si chip side.", "In view of cost reduction, etc., it is possible to mix Au balls with Cu balls, Al balls, Ag balls or the like.", "Ag balls are likewise a strong candidate, and can provide unmeltable linked bonding even at 280° C. by formation of an Ag3Sn compound having a high melting point.", "An application example to Zn—Al system hard balls having a low melting point will be given below.", "Zn—Al system is generally settled down to an Al range of 3-5% from the viewpoint of melting point and brittleness, and furthermore Mg, Ge, Ga or the like is added thereto to lower the melting point and furthermore Sn or In is added thereto to lower the solidus temperature.", "Sometimes, Cu, Ag, Ni or the like is added thereto to secure better wettability and strength.", "Their melting points are on a level of 280°-360° C. For example, in the case of Zn-4Al-2Mg-1Ag-10Sn, when Sn balls are mixed therewith as solder balls, a portion of Sn will undergo solid solution into the Zn—Al system balls even if these two are melted, while the most portions remain as Sn.", "In that case, excess Sn, In or the like, which fails to underdo solid solution in the solder can be dispersed into the solder in an isolated state by thorough dispersion in a particulate state, and thus similar effect can be expected.", "Application of thick Sn plating to the Zn—Al system balls is one of solutions for dispersing Sn in an isolated state.", "In the case of Zn—Al system balls, the entirety is melted at the stage of soldering, and thus the surface shape made by the action of surface tension can characteristically take a natural shape.", "Zn—Al system also undergoes vigorous surface oxidation, and thus it is necessary to take a step for preventing oxidation, the step including preheating.", "When used as a foil, oxidation can be effectively prevented by applying Cu (0-0.2μm thick)-Sn (1 μm thick) plating to the surface.", "By making Sn present between the Zn—Al system balls themselves, Sn can play a roll of a buffering agent against temperature cycle deformation, but in an unsatisfactory case, microfine Sn plated plastic balls (rubber) are mixed therewith to make their dispersion, whereby deformability and shock resistance can be further improved and also the Young's modulus can be lowered together with an improvement of thermal fatigue resistance.", "An—Sn system, etc.", "are likewise available as hard alloy series of low melting point, for which the same steps can be taken.", "An electrode with W (sintered)-Cu plating (3 μm thick) (or W—Ni plating) 14 is formed on Al2O3 substrate 13 to be used.", "Besides, mullite, glass ceramic, AlN, etc.", "are also available as a ceramic substrate.", "When a flux is used at the stage of bonding, simple Cu electrode can be used as such, if an inert gas atmosphere or a reductive atmosphere is available at the initial preheating stage and thereafter.", "Si chip 8 to be used has a size of 5 mm □, and solder foil 11 has a size of 4 mm □×0.15 mm thick, but there is no restriction to the chip size.", "That is, a larger-size chip can be used.", "Against secondary reflowing at the post step, the compound layer can assure good strength at high temperatures, and Sn system solder can mostly act on the successively occurring thermal fatigue, while partially elastically bonded portions can exert a maximum effect on stresswise severe portions (though unbearable portions will be broken).", "Thus, the life can be improved, as compared with the case without any elastic bonding.", "Thus, the compound must be partly formed into a network in the solder, far from such an image as strongly constrained by the compound layer.", "By forming the compound at the bonding boundaries on the chip periphery subject to large strains and stresses, breakage is hard to take place due to strong bonding.", "On the other hand, when the solder foil in the same peripheral position has less network bonds in the center level position, the stresses and strains are shifted from the outermost peripheral position onto the center level position of the solder foil, thereby lessening the stresses on the upside and downside boundary positions.", "At first, Al2O3 substrate 13 is fixed onto a frame base by vacuum suction, while Si chip 8 is also held on resistance heater tool 7 serving also as a mounting jig by vacuum suction 9.Si chip 8 is brought into contact with Al2O3 substrate 13 through solder foil 11 by descending resistance heater tool 7 and kept to heating (maximum 380° C.) and pressing (initially 2 kgf) for 5 seconds.", "Thermocouple 16 for temperature measurement is embedded in the tool at a position near the contact between the tool and the chip to conduct temperature control.", "When the temperature of solder foil 11 reaches the melting point, Sn, etc.", "of the solder foil melt immediately, and pressing force is applied to the bonding between the metal balls themselves to start melting.", "To prevent crushing of bonding between the metal balls, resistance heating tool 7 is made to move, upon reaching a set temperature, from the position of pressing solder foil 11 as a starting point to a position of not more than about 10% (maximum 20%) of the solder foil thickness, thereby controlling an amount of the solder extruded from the chip.", "The thickness of the solder foil affects thermal fatigue life, and thus usually it is 80-150 μm.", "The solder foil thickness and solder foil dimension relative to the chip dimension serve to control a crushing rate of solder foil.", "In the present system, inclusion of Cu to a half and also its network linkage can ensure distinguished thermal conduction, and thus even a solder foil thickness of 200-250 μm is thermally distinguished, as compared with the conventional system.", "Preheating 15 of Al2O3 substrate is set to about 100° C. Abrupt temperature increase or decrease gives a large stress on the joints, and thus preheating is important also in the sense of damping a thermal shock.", "In the case of die bonding by a resistance heater, a mechanism of locally injecting nitrogen 10 from circumferential positions is used to prevent oxidation of solder foil 11 at the stage of bonding.", "Nitrogen 10 is injected also to the circumference of resistance heater tool 7 onto which Si chip 8 is attached by suction, where maintenance of the bonding portions at an oxygen purity on a level of 50-100 ppm can be recommended.", "Such a solder foil can fulfil die bonding of Si chip etc.", "or also bonding of power module, etc.", "at about 270° C. maximum in a hydrogen furnace or a furnace in an inert gas atmosphere such as nitrogen gas, etc.", "In the case of using the furnace, bonding can be carried out at a maximum temperature of 260°-350° C. for Sn, but conditions must be selected in view of the compound formation state.", "FIG.", "4 shows a cross-sectional view of a model of typical bonded portion by die bonding with a resistance heater or in a hydrogen furnace or a furnace in an inert gas atmosphere such as nitrogen gas, etc.", "Upside surface of the chip so die bonded is linked to the substrate terminal by wire bonding, etc., followed by cap sealing the chip or resin sealing, and further by bonding small-sized parts, etc.", "to the substrate circumference (in this case a solder foil matched to the thermal and provisionally fixed to electrodes, etc.", "on chip parts in advance can be bonded to the substrate, or the foil thermally compression bonded thereto can be bonded to the substrate in a reflow furnace), and external connection terminals are provided (usually by bonding with a solder of Sn-3Ag-0.5Cu, etc.)", "on the backside, etc.", "of the substrate to complete a module.", "Alloy layers are firmly formed between Cu balls 2 themselves, between Cu balls and metallization layer 44 on the chip side (for example, Cr—Ni—Au:Au is very thin, and thus substantially an alloy layer is formed between Cu—Sn—Ni), and between Cu balls and metallization layer 12 on the substrate side (for example, Ni plating on Ag—Pd conductor; formation of an alloy layer between Cu—Sn—Ni), respectively to maintain a firmly linked state.", "Combinations of the metallization layer on the chip side are various, and those which react with Sn of the solder are mostly Cu or Ni.", "Sometimes, Au is used in the surface layer mostly to prevent oxidation, but Au undergoes solid solution into Sn on a level of not more than 0.1 μm and thus takes no part in the alloy layer formation.", "The substrate side surface, on the other hand, is also various, and a reactive layer with Sn consists of Ni or Cu as in the case of chips.", "In special cases, thick film conductors of Ag, Ag—Pt, Ag—Pd, Au—Pd, etc.", "are available.", "In the bonding of power module, the presence of voids considerably affects thermal conduction characteristics, and thus it is most important to eliminate voids.", "In the case of a solder paste, a large amount of gas is generated by reaction with a flux, evaporation of solvents, etc., and thus the solder paste is applied to die bonding of joint structures capable of easily discharging gases, such as long and slender terminals, small-sized Si chips, etc.", "That is, in the die bonding of medium or large-sized Si chips, die bonding by a resistance heater using a solder foil in an inert gas atmosphere without any flux or die bonding in a hydrogen furnace or in a furnace in an inert gas atmosphere such as nitrogen, etc.", "is commonly used.", "Voids involved in the solder foil prepared according to the present invention has such a tendency that voids will increase with decreasing Cu particle size, but the voids will be dispersed structurally more finely than the particle size and are far from the image of large voids so far observed.", "It can be expected that voids have less influence on the characteristics.", "In the case of using Cu particles and Sn particles, both having particles of 3-8 μm, the solder foil has a solder filling ratio of about 80% (void ratio 20%).", "When the foil was sandwiched between Sn-plated Cu plates and subjected to die bonding in a nitrogen gas atmosphere by a die bonder, an intermetallic compound of Cu6Sn5 was formed firmly between the Cu balls and the Cu plates, and excess Sn was absorbed into microspaces (voids) in the solder.", "It was found that sufficiently bonded portions could be obtained.", "From the results of cross-sectional investigation it was confirmed that the solder filing ratio in the foil was improved after bonding, as compared with the ratio before bonding.", "It was found therefrom that the conventional void problem was substantially negligible in the present system.", "When Cu particle size is reduced to a 3 μm level or less, bonding must be carried out at high soldering temperatures, such as 300° C. or higher, or when the retention time at high temperatures is prolonged, the Cu particle shape will be collapsed due to the vigorous reaction with Sn, and Cu—Sn compound may serve to conduct linkage, while such characteristics as high temperature strength, etc.", "per se undergo no change thereby.", "Particularly to suppress the reaction, chemical Ni/Au plating, etc.", "can be applied (where the compound is hard to form thickly even at high temperatures) or Ag particles, etc.", "can be also used.", "When Cu particles are coarse such as 30 μm level, the void ratio is not more than 3% in a dispersed state of voids, which seem to have no influence on the characteristics.", "The solder foil prepared in the steps described in the foregoing embodiments can be coiled around a reel and can be supplied continuously to the successive process steps including a cutting step.", "In the case of using it in bonding of the sealing portions and terminal connection portions of necessary parts for the temperature-hierarchical bonding, the solder-foil matched to the desired shape by punching, laser processing, etc.", "can be used.", "The sealing portions and terminal connection portions of the parts are heated and pressed in a nitrogen gas atmosphere by a pulse type, pressing heating tool to conduct bonding without any flux.", "To prevent oxidation at the stage of preheating and assure good wettability, Sn-plated solder foil is desirable.", "In the case of bonding parts of a few terminals provided at a coarse pitch, etc., bonding can be easily carried out due to easy disposition of solder foil, easy positioning of terminals of the parts, and easy compression bonding by resistance heating electrode with a pulse current.", "FIG.", "5(a) shows a cross-sectional view of BGA or CSP type chip carrier where solder foil 39 as shown in FIG.", "5(c) and described above is placed between chip 8 and interposer substrate 36 and subjected to die bonding in a nitrogen gas atmosphere by a resistance heater by pulse heating without any flux, and terminals on the chip is linked to terminals on interposer substrate 36 by wire bonding 35 of Au wire, while a solder foil is placed between cap 23 of Ni-plated Al, etc.", "and interposer substrate 36 and subjected to sealing therebetween in a nitrogen gas atmosphere by a resistance heater without any flux.", "The solder foil can be subjected to bonding upon provisional fixing to the member to be bonded.", "Interposer substrate 36 is brought into a vertical electrical connection through throughholes not shown in the drawing, that is, electrical connection of chip 8 to external connection terminals.", "This structure is a typical example of the ordinary module structure, and chip parts such as resistors, condensers, etc.", "can be mounted on interposer substrate 3, though not shown in the drawing.", "In the case of chips of high power output, an AlN interposer substrate of distinguished thermal conductivity is preferably used from the viewpoint of heat radiation efficiency.", "Solder composition for the external connection terminals of the module is Sn-3Ag-0.5Cu, and can be supplied in the form of balls in the case of broad pitches, but formed into a paste in the case of narrow pitches.", "Then, the module is mounted onto a printed circuit board and subjected to reflow bonding together with other parts at the same time at 240° C. maximum by Sn-3Ag-0.5Cu solder (melting point: 217°-221° C.).", "As described before, bonding of the solder foil itself can be maintained at the reflow temperature, and thus the bonding to the printed circuit board can be made with high reliability.", "That is, temperature-hierarchical bonding can be obtained in the bonding in the case of module packaging and also in the bonding onto the printed circuit board.", "Though the form of external connection terminals is various, temperature-hierarchical bonding can be anyway attained in the bonding of external connection terminals to the printed circuit board by using a solder foil.", "Needless to say, this structure is also applicable to the so called BGA type semiconductor device, in which a semiconductor chip is bonded to a substrate by die bonding with a solder foil, terminals on the semiconductor chip are bonded to terminals on the substrate by wire bonding, and solder balls acting as external connection terminals are formed on the backside of the substrate.", "In that case, resin molding is applied to the chip-to-be-mounted side.", "To improve the wettability of the outer circumferences of the bonded portions, the chip carrier is subjected to reflowing in a nitrogen gas furnace, a hydrogen furnace or the like after the bonding by a resistance heater by pulse heating, whereby good joints can be formed.", "FIG.", "5(b) shows an embodiment of sealing in the structure shown in FIG.", "5(a) where Ni-plated Al fin 23 is sealed in a nitrogen gas atmosphere to interposer substrate 43 with a foil placed thereon by a resistance heater without any flux.", "Left view of FIG.", "5(b) shows punched solder foil 40 comprising Cu balls and Sn balls, and right view of FIG.", "5(b) shows a cross-sectional view of a model of sealing solder foil 40 (cross-section along line B-B′ of the left view) and Ni-plated Al fin 23 to terminals (Ni—Au flash 42) on the interposer substrate by heating in a nitrogen gas atmosphere by resistance heater press 41 by pulse heating.", "After bonding in the state shown in the right view of FIG.", "5(b), the shape of bonded portion 24 in FIG.", "5(a) can be obtained.", "The solder foil used is as shown in FIG.", "5(c) and as so far described above.", "Reflow bonding without any flux in a furnace in a reductive atmosphere using a hydrogen gas, etc.", "is available.", "In the case of using rosin-based flux assuring a long-term insulation, there is no corrosion problem, and thus washing-free reflow bonding is also available, depending on products.", "In the case of using metal balls having a high melting point, the key to the reflowing is to bring out a state of solder foil in contact with the side to be bonded so as to facilitate diffusion bonding on both sides of the solder foil, and thus compression contacting is preferable.", "That is, a process including a provisional fixing step or a pressing step is preferably used.", "For example, it is preferable to supply a solder foil fixed to electrode portions of leads and parts by compression bonding in advance.", "All the Zn—Al system are of melting type and thus have no such fear.", "FIG.", "6 shows an embodiment of application to power module bonding.", "In most cases, Si chip 8 has a dimension of 10 mm □ level, and thus soft, Pb-rich, high temperature system have been so far used.", "On the other hand, Pb-free solders include, for example, Sn-3.5Ag (221° C.), Sn-0.7Cu (227° C.) and Sn-5Sb (235° C.).", "In view of the fact that Sb has an environmental load problem, only Sn-3.5Ag and Sn-0.7Cu are available in the current situation.", "Zn—Al system are hard and have a high possibility to cause Si chip cracking as each.", "In that case, Pb-5Sn system solders have been so far used, because even the conventional solders such as Sn-5Sb system, etc.", "have failed to assure the reliability due to high temperature heat generation, far from the original function of high temperature solder for temperature-hierarchical bonding.", "No Pb-free, soft solders are available yet as a substitute for the high Pb content solder, and thus the present solder foil can be used for this purpose as a substitute.", "The 230° C. level state, which is rarely encountered in vehicles, is specified for vehicle solders as requirements, and requirements for withstanding reflowing at 260° C. is specified for the vehicle solders.", "In the case of the composite solders, Sn melts at the stage of reflowing at 260° C., whereas network linkage is maintained by the intermetallic compound to assure good strength at high temperatures.", "In vehicles, etc.", "having chances of exposing to high temperature on a 220° C. level, Sn-(5-7)% Sb solder (melting point: 236-243° C.) balls are used as Sn system solders to prevent instantaneous partial melting, whereby the Sb concentration reaches 10% or more by reaction between the Sn balls and Cu balls and reaction between Sn and substrate terminals (Cu and Ni) to elevate the lower limit temperature to a 245° C. level, which is higher than the melting point of Sn (232° C.).", "That is, a fear of partial melting at 220° C. can be eliminated.", "Shearing strength at 280° C. of the present bonding system can be kept at 1N/mm2 or more.", "Different from Sn—Pb eutectic mixture, it is said that Sn—Ag—Cu system, on the other hand, has a high strength, a high rigidity and a poor deformability, and thus has an adverse effect on devices, parts, etc.", "Soft solders of Sn—In system, Sn—Cu—In system, Sn—(0-1)Ag—Cu, Sn-(0-1)Ag—Cu—In system, etc.", "themselves can correspond to deformation, though melting points of the solders are somewhat decreased to a 200° C. level, and thus it can be expected that they can be used as temperature-hierarchical solders for packaging of shock resistance-requiring portable appliances.", "As a matter of course, the necessary strength for the stage of second soldering can be obtained as a high temperature strength by linkage of compounds with network Cu, and particularly at the outermost circumference of chips, parts, etc.", "subject to maximum stress and strain, formation of such a network as to prevent breakage near the boundaries but break within the solder by formation of compounds with Cu balls is desirable.", "For this purpose, a solder foil comprising Cu balls and Sn balls is used.", "That is, soft Cu balls, 10-30 μm in diameter, and Sn balls, 10-30 μm in diameter, are mixed together in a ratio of approximately 1:1 by weight, and Sn is made to undergo plastic flow between the Cu balls in vacuum or in a reductive atmosphere, followed by rolling to prepare a solder foil.", "Alternatively, soft Cu balls, 3-8 μm in diameter, are mixed together in a ratio of approximately 1:1 and Sn is made to undergo plastic flow between Cu balls in vacuum or in a reductive atmosphere, followed by rolling to prepare a solder foil.", "The solder foil is cut to the necessary dimensions and the cut solder foils are inserted between Ni-plated Cu lead 51 and Si chip 8, between Si chip 8 and Cu disk plate (or Mo disk plate) 48 with Ni plating 46, between Cu disk plate 48 and alumina insulation substrate 50 with Ni plating 49 on W metallization layer, and between said alumina insulation substrate 50 and Cu base plate 49 with Ni electrolplating 46, respectively, and subjected all together to reflow bonding in a hydrogen furnace at 280° C., whereby bonding can be made by intermetallic compounds of Cu or Ni between Cu balls, between Cu balls and Cu lead, between Cu balls and chips, between Cu balls and Ni-plated Cu plates, between Cu balls and Ni-plated alumina insulation substrate, between Cu balls and Ni-plated Cu base, etc.", "The resulting joints are linked by high temperature-resistant intermetallic compounds (Cu6Sn5 in the case of Cu and Ni3Sn4 in the case of Ni), and thus good strength can be maintained at 260° C. (satisfactory even at 260°-280° C.) without any reflow problem in the successive steps.", "It has been confirmed by temperature cycle tests and power cycle tests that the resulting joints have an equivalent duration to that of the conventional Pb-rich solder.", "Furthermore, dispersion of rubber of Sn-plated plastic balls can lower the Young's modulus, thereby much more improving the thermal shock resistance, and enabling bonding of much large-sized Si chips.", "Packaging can be made by compression bonding at 350° C. for 5 seconds (5-10 seconds are satisfactory) while injecting nitrogen through a pulse-heating type die bonder.", "Wetting at the outer circumference of chips can be maintained and bonding on the bonded boundaries can be assured by provisional fixing by pulse heating to make sure of contacting at the boundaries and then by reflowing in a hydrogen furnace in bulk.", "It is desirable to form smooth fillets at the chip circumference, and thus a layer of only Sn can be provided at the outer circumference of solder foil.", "When a rolled solder foil comprising a mixture in dispersion of balls of Sn, In, or the like in place of the Cu balls, and solder balls of Zn—Al system (Zn—Al—Mg, Zn—Al—Ge, Zn—Al—Mg—Ge, Zn—Al—Mg—Ga, etc.", "), and further rubber of Sn-plated plastic balls is used, temperature cycle resistance and shock resistance can be likewise lessened and high relaibility can be obtained.", "Single Zn—Al system solder is hard (Hv approximately 120-160) and has a high rigidity, and thus large-sized Si chips have a fear of easy breakage.", "Thus, by providing a soft, low-temperature resistant layer of Sn or In partly at the peripheries of the balls or dispersing the rubber around the balls, a deformability can be effectively brought about to lower the rigidity and increase the relaibility.", "By adding Ni-plated or Ni—Au-plated particles of low thermal expansion filler (SiO2, AlN, Invar alloy, etc.)", "thereto, the coefficient of thermal expansion can be made to approach that of Si, etc., whereby the working stress can be made lower and prolonged duration can be expected.", "FIG.", "7 shows embodiments of mounting an RF (radio frequency) module for high frequency for use in signal processing of portable telephones, etc.", "Generally, a method of die bonding the backside of a device to an interposer substrate having a distinguished thermal conduction, followed by wire bonding to the terminal portions of the interposer substrate is suitable for such a type of mode.", "In many cases, chip parts such as R, C, etc.", "are provided on several chips and their surrounding, followed by MCM (multichip module) formation.", "Conventional HIC (hybrid IC), power MOSIC, etc.", "are typical cases.", "Module substrates include, for example, Si thin film substrate, AlN substrates with a low coefficient of thermal expansion and high thermal conduction, glass ceramic substrates with a low coefficient of thermal expansion, Al2O3 substrates with a coefficient of thermal expansion near that of GaAs, metallic core polymer substrates of Invar alloy, etc.", "with a high thermal resistance and improved thermal conduction, etc.", "FIG.", "7(a) shows an embodiment of mounting Si chips 8 on Si module substrate 29.R, C, etc.", "can be formed as thin films on Si module substrate 29, thereby enabling much higher density mounting, and only Si chips 8 can be mounted mostly as flip chips.", "Mounting on printed circuit board 22 can be carried out through QFP-LSI type, soft Cu-based leads 20.Bonding between leads 20 and Si module substrate 29 can be carried out by compression heating, using cut solder foils 17 of the present invention.", "Then, final protection and reinforcement are carried out with soft resin 19 such as silicone, etc.", "Solder bumps 18 for Si chips are made of Sn-3Ag (melting point: 221° C.) and bonded to interposer substrate 29.Bonding to printed circuit board 22 is carried out with Sn—Ag—Cu system, Pb-free solder 21.Solder bumps 18, even if remelted at the stage of reflowing of Sn—Ag—Cu system, Pb-free solder 21, hardly undergo any change by the weight of Si chips 8 at the stage of mounting onto printed circuit substrate 22 and also have no stress burden by virtue of Si—Si bonds without any reliability problem.", "After the mounting on printed circuit substrate 22, Si chips 8 can be coated with silicone gel 12, etc.", "for protection purpose.", "Alternatively, when Au ball bumps are used in place of solder bumps 18 for Si chips 8 and Sn plating is applied to terminals formed on interposer substrate 29, Au—Sn bond can be obtained by compression heat bonding without melting at a reflow temperature of 250° C. at the stage of mounting on printed circuit substrate 22.Thus, temperature-hierarchical bonding can be obtained and bonding can fully withstand reflowing.", "In the bonding with solder foil 17, good bonding by the intermetallic compounds formed between metal balls of Cu, etc.", "are maintained, as already described before, and good strength can be assured even at a reflow temperature of 250° C. at the stage of mounting on printed circuit board 22, whereby temperature-hierarchical lead-free bonding, which has been so far an important task, can be obtained.", "When such thick film substrates as AlN substrates, glass ceramic substrates, Al2O3 substrates, etc.", "are used in place of Si substrates, mounting of chip parts such as R, C, etc.", "are required for making functional devices.", "On the other hands, R and C can be formed by laser trimming of thick film paste.", "In the case of forming R and C from the thick film paste, the same mounting method as for the Si substrates can be used.", "FIG.", "7(b) shows an embodiment of insulation sealing a module using GaAs chips 8 and Al2O3 module substrate 29 with distinguished thermal conduction and mechanical characteristics with a case of Al fin 23.Since GaAs and Al2O3 have nearly equal coefficients of thermal expansion, flip chip mounting has no reliability problem.", "Bonding of these chip parts to the terminal is carried out by provisionally fixing a solder foil with a thickness t: 0.05-0.10 onto devices and chip parts with a small number of terminals, if the terminal area is 0.6 mm □ or more, or by provisionally fixing it to the terminals on the substrate, followed by individually subjecting to compression bonding in a nitrogen gas atmosphere by a resistance heater or reflowing in a reductive atmosphere or in an inert gas atmopshere.", "A solder foil with a thickness t: 0.15-0.25 can be also used.", "To correspond to high power output, generally the solder foil of the present invention is used for chip mounting (chip backside 8) and subjected to die bonding, whereas terminals are subjected to wire bonding.", "In the case of Al fin bonding, a solder foil in such a shape as to surround the circumference of the fin is used and subjected to compression bonding in a nitrogen gas atmosphere by a resistance heater.", "Left side of FIG.", "7(c) is an example of terminal bonding, whereas right side thereof is an example of bonding of Al fin 23, where solder foil 27 is inserted between terminal 28 of module substrate and lead or terminal of the fin bonding portion, followed by bonding in both examples.", "In that case, it is preferable that the solder foil is provisionally fixed either to the substrate side or to the fin side.", "In the case of Al, Ni plating, etc.", "are applied to the terminal portion.", "FIG.", "7(d) shows a step model of mounting C such as Invar alloy, etc.", "onto organic substrate 32.When a polymer substrate of metal core polyimide resin with low thermal expansion and distinguished heat resistance, etc., a build-up substrate corresponding to high density mounting, etc.", "are used for heat-generating chips GaAs chips can be directly mounted thereon.", "In the case of highly heat-generating chips, dummy terminals can be provided to conduct heat directly to metal.", "RF modules have been described above as an application embodiment to the device of the present invention, but application to SAW (elastic surface wave) device structures used as a band pass filter for various mobile telecommunication equipment, PA (high frequency power amplifier) modules, other modules and devices can be likewise made.", "Product field covers not only portable telephones, note-type personal computers, etc.", "but also module-mounted appliances applicable to new household electrical appliances, etc.", "in the degitalization age.", "FIG.", "8 shows further application embodiments to RF module mounting, where FIG.", "8(a) is a cross-sectional view of the module and FIG.", "8(b) shows a plan view of the upper side thereof upon taking member 23 away therefrom.", "In the actual structure, several MOSFET devices of chip 8, about 2 mm □, capable of generating radio wave are mounted by bonding in a face-up manner to correspond to conversion to the multiband, and furthermore a high frequency circuit capable of efficiently generating radio wave is formed in the surrounding position by R or C chip part 52, etc.", "Chip parts are down-sized, and 1005, etc.", "can be used, so that the longitudinal and lateral dimensions of the module is approximately 7×14 with high density mounting.", "In view of only the functional side of solder, an example of a model with only one device and one chip part as mounted is typically shown.", "As will be given later, chip 8 and chip part 52 are solder bonded onto Al2O3 substrate 13.Terminal of chip 8 is connected to an electrode on Al2O3 substrate 13 by wire bonding, and furthermore electrically connected to thick film electrode 60 serving as an outer connector on the backside of the substrate through throughhole 59 and thick film conductor 61.Chip part 52 is solder bonded to an electrode on the substrate 13 and furthermore electrically connected to the thick film electrode 60 serving as an external connector on the backside of the substrate through throughhole 59 and wiring 61.Though not shown in the drawings, electrode 62 on the substrate and throughhole 59, which are connected to the chip or chip part, are electrically connected to each other by wiring.", "Member (Al fin) 23 covering the entire modules and Al2O3 substrate 13 are joined together by caulking, etc.", "The present module can be packaged by solder bonding to thick film electrode 60 serving as an external connector to a printed circuit board, etc., where temperature-hierarchical bonding is required.", "FIG.", "9 is flow charts showing 4 processes on the basis of die bonding of Si (or GaAs) chip, using a solder foil in the structure shown in FIG.", "8.Processes (1) and (2) are systems of selecting the conventional Ag paste for small-sized R or C chip parts such as 1005, etc.", "from the viewpoint of workability, where process (1) is a system of die bonding with a solder foil in a nitrogen gas atmosphere for a short time without any flux in a clean substrate surface state, followed by wire bonding and bonding of chip parts by an Ag paste, whereas process (2) is a system of bonding chip parts by an Ag paste of first, where a furnace, when used for resin hardening, will foul the substrate surface, leading to a fear of adverse effect on wire bonding in the successive steps, and thus in that case washing must be carried out before wire bonding.", "Process (3) is a system of supplying a mixed paste of metal balls and solder balls with distinguished workability to small-sized chip parts to assure likewise temperature-hierarchical bonding on the high temperature side, though the principle of joining is the same as in the case of solder foil, where supply of the mixed paste can be made by printing or by a dispenser.", "Washing must be carried out after reflowing, and since extreme void elimination is required for high power output Si chips, die bonding of chips is carried out with a solder foil suitable for the void elimination, followed by the final wire bonding.", "When die bonding and wire bonding are carried out at first in process (3), step of flux washing can be omitted.", "Process (4) is a system of die bonding and wire bonding at first, which involves two plans for the poststep.", "One plan is to bond chip parts one by one in a nitrogen gas atmosphere without any flux in the poststep, but this plan takes a long time as a disadvantage.", "The other plan is to bond chip parts with a flux on a provisional fixing level, followed by reflow bonding in bulk.", "More particularly, after die bonding and wire bonding, it is preferable that a composite solder foil comprising, for example, Zu balls and Sn balls, with approximately 1 μm-thick Sn plating on the surface (in almost all cases, chip parts are Ni-plated, and in these cases Sn plating is unnecessary) is cut to substantially the electrode dimension and a provisionally fixed to the electrode portion of the parts by compression heating (where a flux may be used), and the solder-foil provisionally fixed parts are provisionally fixed to the W—Ni—Au plated electrode portion on the Al2O3 substrate by heat compression bonding to such a degree as to undergo plastic deformation.", "When the individual parts are pressed one by one at 300°-350° C. for 5 seconds in a nitrogen atmosphere by a pulse-type resistance heater, intermetallic compounds are surely formed to serve to attain linkage.", "Needless to say, satisfactory strength even at high temperatures of 260° C. or higher can be maintained.", "By passing through a reflow furnace (270°-320° C. maximum), the compression bonded portions, Cu and Ni together can undergo linkage by the alloy layer.", "Complete linkage is not necessary.", "Partial linkage has no problem at high temperatures even though the strength is low.", "Small-sized chip parts are not so elevated to high temperatures as the devices are, but in the case that deterioration of Ag paste is a problem when used for a long time, high reliability can be assured by using a solder as the essential component of the present composition.", "Only a problem is a time-consuming one in surely fixing small-sized chip parts by heat pressure bonding one by one.", "FIG.", "8(c) shows one embodiment of solder bonding of the aforementioned module to printed circuit board 22, where besides the module, electronic parts 52 and BAG type semiconductor devices are also solder bonded.", "Semiconductors are provided by bonding semiconductor chip 8 onto interposer substrate 43 in a face-up manner by the aforementioned solder foil, and bonding the terminal of semiconductor chip 8 to the terminal on interposer substrate 43 by wire bonding 35, while the surroundings are resin sealed with resin 58.Solder ball bumps 21 are formed on the underside of interposer substrate 43.For solder ball bumps 21, a solder of, for example, Sn-2.5Ag-0.5Cu is used.", "Sn-(1-2.5)Ag-0.5Cu is desirable for solder ball 30.For example, Sn-1.0Ag-0.5Cu can be used.", "Electronic parts are also solder bonded to the backside of the interposer substrate as an example of so called “both side mounting”.", "According to one mode of mounting, for example, a Sn-3Ag-0.5Cu solder (melting point: 2170°-221° C.) paste is printed onto the electrode portion on a printed circuit board at first.", "Then, to conduct solder bonding from the mounting side of electronic part 54, electronic part 50 is mounted thereon and solder bonding can be attained by reflow bonding at 240° C. maximum.", "Then, electronic part, module and semiconductor device are mounted and reflow bonded at 240° C. maximum to attain both-side mounting.", "In this manner, heat-resistant, light parts are reflow bonded at first, and then less heat resistant, heavy parts are bonded as a general rule.", "In the case of reflow bonding afterwards, it is desirable not to remelt the solder on the initially bonded side.", "As already described before, bonding of solder foil itself used is bonding within the module can be maintained at a reflowing temperature at the stage of mounting onto the printed circuit board in that case, and thus modules and semiconductor devices can be bonded to the printed circuit board with high reliability.", "That is, temperature-hierarchical bonding can be attained between the bonding of semiconductor devices or bonding within the modules and the bonding onto the printed circuit board.", "In the foregoing embodiment, bonding onto both sides of the printed circuit board is made by the same solder, but in the case of weight-negligible, small-sized parts such as 1005, etc.", "as electronic parts 54, even if the solder melts at the stage of reflow bonding of electronic parts, modules and semiconductor devices, the small-sized parts never fall off because the small-sized parts themselves are of high weight, and the action of surface tension is superior to the gravity.", "Thus, even bonding only by Sn without any formation of intermetallic compounds with the terminals on the board, as in the worst case, will be no problem.", "In view of the productivity, a solder paste comprising a mixture of Cu and Sn is preferred for small-sized parts mounted within the module to the provisional fixing of a solder foil comprising a mixture of Cu and Sn.", "An application embodiment to resin package of high power output chips such as motor drive IC, etc.", "will be given below.", "FIG.", "10(a) is a plan view showing joining of lead frame 65 to heat diffusion plate 64 face-to-face by caulking, where caulking positions 63 are two.", "FIG.", "10(b) is a cross-sectional view of a package and FIG.", "10(c) is a partially enlarged view thereof.", "Heat from heat-generating chip 8 on a 3 W level is conducted to heat diffusion plate (Cu-based composite material of low thermal expansion) as a leader through solder 47.Lead material is composed of, for example, 42 alloy series materials.", "FIG.", "11 shows a flow diagram of packaging process.", "At first, a lead frame and a heat diffusion plate (heat sink) are joined together by caulking.", "Then, semiconductor chip 8 is die bonded onto heat diffusion plate 64 joined by caulking through solder and dam 57.Die-bonded semiconductor chip 8 is wire bonded to lead 56 by gold wire 35, etc., as shown in the drawing, followed by resin molding, dam cutting and application of Sn-based solder.", "Then, the lead is shaped by cutting and the heat diffusion plate is cut to finish the outer profile.", "The electrode on the backside of Si chip 8 can be a commonly used metallization layer of Cr—Ni—Au, Cr—Cu—Au, Ti—Pt—Au, Ti—Ni—Au, etc.", "In the case that Au is rich, formation of an Au-rich compound of Au—Sn having a higher melting point can be recommended.", "Die bonding of the chip was carried out at 350° C. under an initial compression of 2 kgf for 5 seconds by a pulse type, resistance heater, while injecting a nitrogen gas thereto, where control of solder film thickness was carried out by setting a position by 10 μm downward from the initial compression position (film thickness: 70 μm) according to a system of mechanismwise maintaining the film thickness to improve the thermal fatigue resistance.", "Besides the above chip die bonding conditions, the chip die bonding was also carried out at 350° C. under an initial compression of 1 kgf for 5-10 seconds, where, even if control of the solder film thickness was set to a position by 10 μm downward from the initial compression position (film thickness: 150 μm), the same results was obtained.", "Reduction in void ratio was important because of the high power output chip, and not more than 5% as a target was attained.", "The solder contains Cu balls in a uniformly dispersed state, and thus larger voids are structurally hard to generate.", "Against severe thermal fatigue, Sn and Sn-based solder themselves have a distinguished thermal fatigue resistance and also have a distinguished deformability.", "Furthermore, intermetallic compounds are formed networkwise between the Cu particles and between the Cu particles and the electrode, and thus good strength can be maintained even at high temperatures of 260° C. or higher.", "When the bonds between the Cu particles, etc.", "are too strong (alloy layers are much more formed on boundaries between the Cu particles, etc.", "), the Cu balls, etc.", "are restricted to lose the degree of freedom, resulting in strong elastic bonds.", "Thus, such bonds are not good for devices, etc.", "There must be appropriate bonds.", "Particularly, in the chip peripheral region, the conventional solder is broken in a position near the stress-concentrated bonding boundaries, but is hardly broken within the solder.", "In the case of present solder, breakage is hard to take place at the boundaries due to reaction between the bonding boundaries and the Cu balls, whereas breakable network is formed within the solder.", "After die bonding and wire bonding, resin molding is conducted and the dam is cut.", "Then, Pb-free solder plating of Sn—Bi, Sn—Ag, or Sn—Cu system is applied to the lead to a thickness of 2-8 μm.", "Then, the lead is shaped by cutting, and unwanted parts are cut off from the heat diffusion plate to finish the outer profile.", "FIG.", "12 shows an application embodiment to an ordinary plastic package.", "Backside of Si chip is bonded to tab 66 of 42 Alloy by electroconductive paste 67.Device is linked to lead 56 by wire bonding 35 and molded by resin 58.Then, Sn—Bi system plating, which can meet the Pb-free requirements, is applied to the lead.", "Heretofore, Sn-37Pb eutectic solder having a melting point of 183° C. has been used for mounting onto a printed circuit board, and thus reflow bonding at 220° C. maximum has been available.", "To meet the Pb-free requirements, reflow bonding must be carried out with Sn-3Ag-0.5Cu (melting point: 217°-221° C.), and thus the maximum temperature is 240° C., which is by about 20° C. higher than the conventional maximum temperature.", "Thus, when the conventional heat-resistant electroconductive paste or adhesive is used for bonding between Si chip 8 and tab 66 of 42 Alloy, the bonding strength is lowered at high temperatures and adverse effects on the subsequent reliability are highly expectable.", "Use of the present solder foil in place of the electroconductive paste can assure good strength at high temperatures of 270°-350° C. maximum, and thus the temperature-hierarchical bonding by a Pb-free solder can be attained.", "The application to a plastic package can be applied to all the plastic package structures of bonding Si chips to tabs.", "Gull-Wing type, Flap type, J-Lead type, Butt-Lead type and Leadless type are structurally available.", "FIG.", "13 shows one embodiment of model structure at the stage before formation of a composite solder foil.", "Sn-plated metal fibers 69 of Cu, etc.", "in diameters on a 3-15 μm level (surface treatment with Ni/Au, etc.", "may be carried out to suppress reaction between Cu and Sn in the case of shaping and molding at high temperatures) are arranged in parallel with one another, and a mixture of solder balls of Sn, etc.", "and metal balls of Sn-plated Cu, etc.", "in an appropriate proportion (about 50%) is placed thereon, followed by shaping and rolling to form a foil with a thickness on a 150-250 μm level.", "To lower the Young's modulus, Sn-plated, heat-resistant plastic balls, or Cu/Sn-plated silica of low thermal expansion, Invar alloy, etc.", "as a substitute for some portions of the metal balls may be added thereto.", "By shaping and rolling, the soft solder balls enters into gaps between the metal balls and metal fibers to form the sea in the “sea-and-island structure”.", "The metal fibers are not limited to said diameter of 3-15 μm, and serve as nuclei on the center level of the foil, whereas the metal balls play an important roll on the bonding boundaries with members to be bonded.", "In the continuous rolling, the metal fibers are oriented in the rolling direction to facilitate the rolling work.", "In place of the metal fibers, Cu (or Cu/solder)-plated carbon fibers capable of conversion to finer filaments and lower thermal expansion, Ni/Au, Ni/solder or Cu (or Cu/solder)-plated fibers of ceramics, glass, Invar alloy, etc.", "are available.", "FIG.", "13 shows an embodiment of parallel arrangement of metal fibers serving as nuclei of the solder foil, whereas FIG.", "14 shows a stable structure of crossed arrangement of metal fibers (where crossing angle is as desired).", "A mixture of solder balls of Sn, etc.", "and metal balls of Sn-plated Cu, etc.", "in an appropriate proportion (about 50%) enters into gaps between the crossed metal fibers.", "Similar modifications to those as in FIG.", "13 are available.", "FIG.", "15 are cross-sectional views of foils where network metal fibers 71 are used.", "Cross-sections of network metal fibers extended backwards are shown by mark “x” 70 in the drawing.", "FIG.", "15(a) shows a solder foil composed of network metal fibers and a solder.", "Reduction in network mesh sizes is limited, and the minimum mesh size of the currently commercially available network metal fibers is 325, particle size through which particles can pass is as large as 44 μm, and also the sizes of metal fibers, which form the network, are large, so that the contact area at the bonding boundaries is small (area for compound formation).", "Thus, maintenance of good strength at high temperatures is a problem.", "Thus, a mixture of solder balls of Sn, etc.", "and metal balls 2 of Sn-plated Cu, etc.", "in an appropriate proportion (about 50%) is filled in gaps between the networks 70 and 71 to form a solder foil, whose cross-sectional view is shown in FIG.", "15(b).", "Solder 72 is in such a structure as filled in the gaps.", "When maintenance of good strength at high temperature is required, the Cu balls are mixed in a somewhat larger proportion to concentrate on compound formation at the bonding boundaries with member to be bonded.", "In the case of making much of thermal fatigue of joints, the solder is mixed in a somewhat larger proportion to enable control of concentrating on thermal fatigue resistance of the solder.", "The metal balls to be filled are not limited to ball form, but fiber form, etc.", "as will be mentioned below, are also available.", "Mixing proportion of metal balls to solder depends on form, contact state, etc.", "of metal, and is much variable.", "FIG.", "16 shows a model of such a state, where long and slender metal fibers 73 are flattened at random to make a frame, as in making paper, and a mixture of solder balls 68 of Sn, etc.", "and metal balls 2 of Sn-plated Cu, etc.", "in an appropriate proportion (about 50%) is spread on both sides of the frame to fill the gaps in the frame.", "FIG.", "16(a) is a plan view thereof, and FIG.", "16(b) is a cross-sectional view thereof.", "FIG.", "17 shows use of oblong metal fibers or Cu (or Cu/solder)-plated oblong carbon fibers capable of conversion to low thermal expansion, or Ni/Au, Ni/solder or Cu (or Cu/solder)-plated oblong fibers of ceramics, glass, Invar alloy, etc.", "in place of the metal balls.", "Use of oblong fibers can considerably increase the mixing proportion of solder.", "Metal balls can be filled into the gaps between the oblong fibers to intensity networks by compound formation.", "The structure is restricted only by metal balls and becomes rigid, but by dispersion of oblong fibers in this manner a structure with distinguished deformability and elasticity can be expected, and it seems that good performance at the stage of die bonding or against thermal fatigue can be obtained.", "The length of oblong fibers is desirably 1/10 or less, so far as the foil thickness is 200 μm.", "For example, such a ranges as diameter level: 1-5 μm and length level: 5-15 μm are desirable.", "INDUSTRIAL APPLICABILITY The present invention provides an electronic device and a semiconductor device based on quite novel solder bonding.", "Particularly, solder bonding on the high temperature side in the temperature-hierarchical bonding can be obtained.", "Furthermore, the present invention provides a novel solder foil with a highly industrial utility." ] ]	Patent_10451610
[ [ "Surface-functionalised carrier material, method for the production thereof and solid phase synthesis method", "The invention concerns a novel surface-functionalized carrier material with a polymeric surface and at least one linker compound according to the general formula (I), which is covalently bound to the surface.", "In the formula, P indicates the polymeric surface; R2 has the meaning OR4 or NR4R5 and R1, R4 and R5, independently of one another, indicate H, an alkyl group or an aryl group; R3 indicates H, an alkyl, an aryl, an acyl, an alkoxycarbonyl or an aryloxycarbonyl group; and the alkyl, aryl, acyl, alkoxycarbonyl and/or aryloxycarbonyl group of the radicals R1, R3, R4 and R5, independently of one another, are substituted or unsubstituted.", "The material according to the invention can be very easily produced by photochemical coupling and serves for the solid-phase synthesis of amino acids, peptides, proteins or molecules with at least one peptidic structural unit." ], [ "1.A surface-functionalized carrier material with a polymeric surface and at least one linker compound according to general formula (I), which is covalently bound to the latter: in which P indicates the polymeric surface; R2 has the meaning OR4 or NR4R5 and R1, R4 and R5, independently of one another, indicate H, an alkyl group or an aryl group; R3 indicates H, an alkyl, an aryl, an acyl, an alkoxycarbonyl or an aryloxycarbonyl group; and the alkyl, aryl, acyl, alkoxycarbonyl and/or aryloxycarbonyl group of the residues R1, R3, R4 and R5, independently of one another, are substituted or unsubstituted.", "2.The surface-functionalized carrier material according to claim 1, further characterized in that the polymeric surface (P) and/or the carrier material is an organic polymer.", "3.The surface-functionalized carrier material according to claim 2, further characterized in that the organic polymer is polypropylene, polyethylene, polysulfone, polyether sulfone, polystyrene, polyvinyl chloride, polyacrylonitrile, cellulose, amylose, agarose, polyamide, polyimide, polytetrafluoroethylene, polyvinylidene difluoride, polyester, polycarbonate, polyacrylate, polyacrylamide or a derivative of these or a copolymer or a blend thereof.", "4.The surface-functionalized carrier material according to claim 1, further characterized in that the carrier material is an inorganic and/or mineral material.", "5.The surface-functionalized carrier material according to claim 4, further characterized in that the carrier material is a glass, a silicate, a ceramic material or a metal.", "6.The surface-functionalized carrier material according to one of claims 2 to 5, further characterized in that the carrier material is a composite of at least one inorganic and/or mineral material and at least one organic polymer.", "7.The surface-functionalized carrier material according to one of the preceding claims, further characterized in that the carrier material is present in the form of a membrane, a film, a plate, a microtiter plate, a test tube, a glass slide, a fiber, a hollow fiber, a nonwoven material, a woven fabric, a powder, a granulate, or of particles, and may be porous or nonporous.", "8.The surface-functionalized carrier material according to claim 7, further characterized in that the carrier material is present in the form of a membrane with a symmetric or asymmetric pore structure.", "9.The surface-functionalized carrier material according to claim 7 or 8, further characterized in that a pore size amounts to 1 nm to 10 μm.", "10.The surface-functionalized carrier material according to one of the preceding claims, further characterized in that the alkyl groups of the radicals R1, R3, R4 and R5 and the acyl and the alkoxycarbonyl groups of the radical R3 are C1 to C20 alkyl units.", "11.The surface-functionalized carrier material according to one of the preceding claims, further characterized in that the aryl groups of the radicals R1, R3, R4 and R5 and the aryloxycarbonyl group of the radical R3 is a phenyl group.", "12.A method for the production of a surface-functionalized carrier material according to formula (I): wherein P indicates the polymeric surface of the carrier material; R2 has the meaning OR4 or NR4R5 and R1, R4 and R5, independently of one another, indicate H, an alkyl group or an aryl group; R3 indicates H, an alkyl, an aryl, an acyl, an alkoxycarbonyl or an aryloxycarbonyl group; and the alkyl, aryl, acyl, alkoxycarbonyl and/or aryloxycarbonyl group of the radicals R1, R3, R4 and R5, independently of one another, are substituted or unsubstituted, with the steps: a) Introducing a linker compound according to general formula (II): in which R1, R2 and R3 have the above meaning, onto a polymeric surface (P) of a carrier material, b) Irradiating the surface with light of the UV-vis spectral region, whereby a covalent bond forms between the linker compound according to formula (II) and the polymeric surface (P) with the formation of the surface-functionalized carrier material according to formula (I).", "13.The method according to claim 12, further characterized in that the irradiation is conducted with light of a wavelength region of 250 to 500 nm.", "14.The method according to claim 13, further characterized in that lasers, UV tubes or mercury vapor lamps, optionally with the use of suitable filters, are utilized as light sources.", "15.The method according to one of claims 12 to 14, further characterized in that the irradiation of the surface is conducted in the presence or in the absence of a sensitizer.", "16.The method according to one of claims 12 to 15, further characterized in that after the irradiation, unreacted substances are removed by washing with a washing fluid.", "17.The method according to claim 16, further characterized in that water, an organic solvent or a solvent mixture is used as the washing fluid.", "18.The method according to one of claims 12 to 17, further characterized in that a material according to one of claims 2 to 9 is used as the polymeric surface (P) and/or carrier material.", "19.Use of a surface-functionalized carrier material according to general formula (I) for the covalent immobilization of biomolecules, particularly of amino acids, peptides or proteins or molecules with amino and/or carboxyl groups.", "20.A method for the synthesis of amino acids, peptides, proteins or molecules with at least one peptide structural unit at solid phases, wherein the first amino acid to be utilized for the synthesis is covalently bound to a surface-functionalized carrier material and the chain extension is conducted by successive coupling of additional amino acids and/or chemical modification is produced, is hereby characterized in that a surface-functionalized carrier material according to general formula (I) is used according to one of claims 1 to 11 as the solid phase.", "21.The method according to claim 20, further characterized in that the first amino acid utilized for the synthesis is bound to the carrier material by a peptide bond, selectively, either between the amino group of the amino acid and the C1 position of the linker compound or between the carboxyl group of the amino acid and the amino group of the linker compound.", "22.The method according to claim 21, further characterized in that the N or C-terminal binding of the amino acid to the linker compound is controlled by blocking the amino group or the carboxyl group of the amino acid and/or the C1 position or the amino group of the linker compound with chemical protective groups." ], [ "The invention concerns a surface-functionalized carrier material comprising a carrier material and at least one linker compound covalently bound to a polymeric surface of the latter, a method for the production thereof as well as a method for the solid-phase synthesis of amino acids, peptides or molecules with at least one peptide structural unit.", "Conducting the synthesis of peptides or more complex molecules with peptide structural units in the form of so-called solid-phase syntheses is known.", "For this purpose, an amino acid which represents virtually the first molecular member of the peptide sequence to be produced is covalently bound to a carrier material that is not soluble in water and whose surface bears suitable functional groups.", "Further extension of the chain is produced by successively binding additional amino acids, which correspond to the sequence to be constructed, to the first amino acid or to the free end of the forming peptide chain.", "In addition to pure chain extension, the conducting of chemical modifications at the immobilized amino acid or the immobilized peptide is also known.", "Polystyrene is primarily used as the basic material for the solid phase (the carrier material) (refer to: F. Z. Dörwald, Organic Synthesis on Solid Phases, Wiley Publishing Co. Chemistry, Weinheim 2000, pp.", "414 ff).", "Two strategies are distinguished with respect to the direction of synthesis of the peptide to be produced.", "In the Merrifield strategy, which is also denoted as type A extension, a surface functionalization of the polystyrene is conducted by derivatizing with chloromethyl, hydroxymethyl or acrylamide groups (R. B. Merrifield, J.", "Am.", "Chem.", "Soc.", "1963, 85, pp.", "2149-2154; R. Arshady et al., J. Chem.", "Soc.", "Perkin Trans.", "I 1981, pp.", "529-537).", "The covalent coupling of the first amino acid to these groups is conducted via the carboxyl group of the amino acid, i.e., the C terminal.", "The further construction of the chain includes a condensation of the subsequent amino acid to the amino group (the N terminal) of the already immobilized amino acid, or—in further synthesis—the immobilized peptide.", "According to the Merrifield strategy, synthesis is produced accordingly from the C terminal to the N terminal of the peptide.", "Also, in the Boc strategy derived from the Merrifield concept (R. Arshady et al., J. Chem.", "Soc.", "Perkin Trans I 1981, 529-537) or the Fmoc strategy (L. A. Carpino, G. Y. Han, J. Org.", "Chem.", "1972, 37, 3404-3409), in which the amino group of the respective amino acids to be coupled is protected by specific protective groups, the peptide is finally bound to the solid phase via the carbonyl function of the first amino acid.", "In contrast, in the inverse strategy (type B extension), chloroformic acid ester units immobilized on the surface of polystyrene are used as the starting point for the peptide synthesis (R. L. Letsinger, M. J. Kornet, J.", "Am.", "Chem.", "Soc.", "1963, 85, 2149-2154; R. Matsueda et al., J.", "Am.", "Chem.", "Soc.", "1975, 97, 2573-2575).", "The synthesis is conducted here by N-terminal coupling of the amino acids protected as tert-butyl esters in the sequence given in advance by the target sequence in the direction of the C terminal of the peptide.", "It is a disadvantage in this prior art that, independent of the desired synthesis strategy, carrier materials with different functionalizations must be utilized, since the chemical properties of the respective surface function of the carrier material permit exclusively an N terminal or a C terminal coupling of the amino acid.", "The object of the present invention is thus to provide a surface-functionalized carrier material, which permits a user selectively a C terminal or an N terminal binding of an amino acid or another molecule with appropriate functional groups.", "A method for the production of surface-functionalized carrier material as well as a method for solid-phase synthesis will also be provided.", "The first aspect of this object is solved by a surface-functionalized carrier material with a polymeric surface and at least one linker compound according to general formula (I), which is covalently bound to the surface: in which P indicates the polymeric surface; R2 has the meaning OR4 or NR4R5 and R1, R4 and R5, independently of one another, indicate H, an alkyl group or an aryl group; R3 indicates H, an alkyl, an aryl, an acyl, an alkoxycarbonyl or an aryloxycarbonyl group; and the alkyl, aryl, acyl, alkoxycarbonyl and/or aryloxycarbonyl group of the radicals R1, R3, R4 and R5, independently of one another, are substituted or unsubstituted.", "The linker compound covalently bound to the carrier material can be based on the amino acid glycine, whose amino and/or carboxyl group is optionally derivatized and which is covalently bound at its Cα position via a hydroxyethyl unit to the polymeric surface of the carrier material.", "The hydroxyethyl unit at the C position bearing the hydroxy group may also be substituted.", "The material according to the invention is accordingly characterized in that it bears two free functions, i.e., the amino and the carboxyl groups of the glycine, at which a biomolecule, particularly an amino acid, can be condensed.", "The material according to the invention thus involves a doubly surface-functionalized material.", "The alkyl groups of the radicals R1, R3, R4 and R5 as well as the alkyl residues of the acyl or the alkoxycarbonyl group of the radical R3 are preferably branched or unbranched, saturated or unsaturated C1 to C20 units.", "Advantageously, phenyl groups can be utilized as the aryl groups of the radicals R1, R3, R4 and R5 as well as the aryl residue of the aryloxycarbonyl group of the radical R3.The polymeric surface and/or the carrier material itself is an organic polymer according to an advantageous embodiment, particularly polypropylene, polyethylene, polysulfone, polyether sulfone, polystyrene, polyvinyl chloride, polyacrylonitrile, cellulose, amylose, agarose, polyamide, polyimide, polytetrafluoroethylene, polyvinylidene difluoride, polyester, polycarbonate, polyacrylate, polyacrylamide or a derivative of these or a copolymer or a blend thereof.", "In addition, however, inorganic and/or mineral materials can also be utilized as carrier materials, particularly glasses, silicates, ceramic materials or metals.", "It is also conceivable to use composites of one or more inorganic and/or mineral materials and one or more organic polymers.", "In the case of pure inorganic and/or mineral carrier materials, a coating with one of the named organic polymer materials may be necessary in order to make possible a binding of the linker compound.", "With respect to its external configuration, the carrier material can be present in the form of a membrane, a film, a plate, a microtiter plate, a test tube, a glass slide, a fiber, a hollow fiber, a nonwoven material, a woven fabric, a powder, a granulate or in the form of particles.", "Thus, the carrier material may have a porous or nonporous structure each time.", "It is most preferably provided that the carrier material is present in the form of a membrane with a symmetrical or asymmetrical pore structure, whereby a pore size can lie in the range of 1 nm to 10 μm.", "The surface-bifunctionalized polymer material is produced preferably by: a) Introducing a linker compound according to general formula II in which R1, R2 and R3 have the above meaning, onto a polymeric surface (P) of a carrier material, and b) Irradiating the surface with light of the UV-vis spectral region, whereby a covalent bond forms between the compound according to formula (II) and the polymeric surface (P) with the formation of the surface-functionalized carrier material according to formula (I).", "The glycine derivative according to formula (II) is introduced onto the carrier material or its polymeric surface, which is selected from the above-described materials, by impregnating, moistening or coating, depending on the external shape of the carrier material.", "The surface can be exposed—although this is not absolutely necessary—particularly advantageously in the presence of a sensitizer.", "The yield of the photoreaction can be increased in this way.", "Irradiation is preferably conducted with light of the wavelength region of 250 to 500 nm.", "The selection of the wavelength or the wavelength region used primarily depends on the radical R1, the polymeric surface and the presence of and the type of sensitizer.", "Suitable light sources are, for example, lasers, UV tubes or mercury vapor lamps, whereby the wavelength region can be limited optionally by the use of suitable filters.", "The light-induced coupling of molecules to polymeric surfaces is known, for example, from WO 91/16425 or EP 0 562,373 A2, and will not be explained in further detail here.", "After the photochemical conversion has been produced, unreacted linker compound, by-products and, optionally, the sensitizer can be removed by washing with water, an organic solvent or a solvent mixture, and the functionalized carrier material is dried.", "The dried product has a very good stability and can be stored at room temperature for weeks and months.", "The surface-functionalized carrier material according to the invention can be used for the covalent immobilization of biomolecules, particularly of amino acids, peptides or proteins or other molecules with amino and/or carboxyl groups.", "It can also be utilized particularly advantageously for the solid-phase synthesis of amino acids, peptides, proteins or molecules with at least one peptide structural unit.", "Thus, a first amino acid utilized for the synthesis can be bound to the carrier material by a peptide bond, selectively, either between the amino group of the amino acid and the C1 position of the linker compound or between the carboxyl group of the amino acid and the amino group of the linker compound.", "If a selective C- or N-terminal bond is desired, the coupling can be controlled in the known way by use of chemical protective groups for blocking the N terminal or the C terminal of the amino acid and/or the C1 position or the amino group of the linker compound.", "Thus, the terminal of the amino acid which is not to be bound or the function of the linker compound that is not to be connected is blocked by the protective group.", "Relative to the linker compound, the coupling of the biomolecule can be controlled by suitable selection of the radicals R2 or R3.The region-specific immobilization of an amino acid is shown in FIG.", "3, wherein the radicals R1 and R3 of the surface-immobilized linker compound (a) are each hydrogens and the radical R2 is a hydroxyl group.", "According to the upper branch of Figure (b), the first amino acid, whose amino function is protected with a tert-butoxycarbonyl protective group (Boc), is condensed by its free carboxyl function at the amino group of the carrier material, with the formation of an amide bond.", "The carboxyl group can be activated beforehand by reacting the amino acid with dicyclohexylcarbodiimide (DCCl).", "This measure is also known to the person skilled in the art.", "The peptide chain of the covalently coupled amino acid can now be extended in such a way that after acidic hydrolytic removal of the Boc protective group, additional amino acids that are protected at the N terminal can be condensed at the now free N terminal of the first amino acid (type A extension).", "The individual method steps of coupling and cleavage of the protective groups are repeated until the desired peptide sequence is completely constructed.", "The finished peptide is cleaved by addition of dilute hydrofluoric acid.", "The inverse strategy (type B extension) proceeds according to the lower branch (C) of FIG.", "3.Here, the carboxyl group of the amino acid to be immobilized is protected by a tert-butyl ester.", "Consequently, the amino acid can be condensed with the surface-functionalized carrier material only via the amino group of the amino acid to the carboxyl group of the carrier material.", "For further peptide construction, the tert-butyl group is first removed by saponification, and then other amino acids, which are appropriately protected, are added by condensation.", "The invention will be explained in further detail below in examples of embodiment.", "EXAMPLE 1 A polypropylene membrane (diameter 30 mm) was immersed for 30 minutes in a 0.1 M solution of N-Boc-L-β-benzoylalanine, i.e., an amino acid of the general formula (II) (compare FIG.", "2) with R1=phenyl, R2=OR4, R3=t-butoxycarbonyl (Boc) and R4=H, in dichloromethane, and then dried in high vacuum at 3·10−5 Torr for 30 minutes.", "The membrane was irradiated for 30 minutes with the light of an HBO (maximum-pressure light) 500 at a distance of 20 cm with the use of a cutoff filter, which filters out light below 290 nm.", "The membrane was then washed five times with a total of 150 ml of dichloromethane and dried for 30 minutes in high vacuum at 3·10−5 Torr.", "The the surface functionalization according to formula (I) (compare FIG.", "1), in which R1 to R4 have the above-named meanings, was detected by comparison of the FT-IR spectra for the polypropylene membrane before and after treatment.", "EXAMPLE 2 A polypropylene membrane (diameter 30 mm) was immersed for 30 minutes in a 0.1 M solution of N-Boc-L-P -benzoylalanine methyl ester, i.e., an amino acid of the general formula (II) with R1=phenyl, R2=OR4, R3=t-butoxycarbonyl (Boc) and R4=methyl, in dichloromethane, and then dried in high vacuum at 3·10−5 Torr for 30 minutes.", "The membrane was irradiated for 30 minutes with the light of an HBO 500 at a distance of 20 cm with the use of a cutoff filter, which filters out light below 290 nm.", "The membrane was then washed five times with a total of 150 ml of dichloromethane and dried for 30 minutes in high vacuum at 3·10−5 Torr.", "The surface functionalization according to formula (I), in which R1 to R4 have the above-named meanings, was detected by comparison of the FT-IR spectra for the polypropylene membrane before and after treatment.", "EXAMPLE 3 A polypropylene membrane (diameter 30 mm) was irradiated in a glass dish, filled with a 0.1 M solution of N-Boc-L-β-benzoylalanine, i.e., an amino acid of the general formula (II) with R1=phenyl, R2=OR4, R3=t-butoxycarbonyl (Boc) and R4=H, in benzene for 60 minutes with the light of an HBO 500 at a distance of 20 cm with the use of a tilted mirror and a cutoff filter, which filters out light below 290 nm.", "The membrane was then washed once with 50 ml of benzene and twice with 50 ml of dichloromethane and dried for 30 minutes in high vacuum at 3·10−5 Torr.", "The surface functionalization according to formula (I), in which R1 to R4 have the above-named meanings, was detected by comparison of the FT-IR spectra for the polypropylene membrane before and after treatment." ] ]	Patent_10451654
[ [ "Interconnecting module for the base of electronic equipment casing", "This interconnection module for the base of an electronic device casing carries out the transfer, at the faces of the printed circuit boards (4, 5) supporting the components of the device housed in the casing, of a field of connection points with the external environment of the casing embodied by the rear ends of the pins of a semi-connector 3 fixed to the back of the casing while still retaining the compactness of the casing despite a very large number of connection points with the external environment of the casing (several hundreds of them).", "This module takes the form of a three-panel structure with a central panel 7 fixed to the rear ends of the pins of the semi-connector (3) mounted on the back of the casing and two side panels (8, 9) folded and placed flat against each other, joined to the longitudinal edges of the central panel (7) by flexible printed circuit elements (10, 11) and supporting the field of connection points transferred to the level of the faces of the boards (4,5) of the electronic device.", "Electronic connections link the two fields of connection points in going through the joining parts (10, 11)." ], [ "1.Interconnecting module for the base of an electronic device casing including two groups of printed circuit boards bearing electronic device components, each group having one or more stacked printed circuit boards, and that has a back equipped with a field of contacts constituting electrical connection points accessible from the exterior of the casing, said interconnecting module providing for the transfer, at the level of the two groups of printed circuit boards of the connection points of the casing accessible from the exterior, comprising a field of electrical connection points transferred to the interior of the casing so as to be facing the two groups of printed circuit boards and tracks setting up the electrical links between this transferred field of connection points and the field of contacts with which the back of the casing is equipped, comprising: a central panel in printed circuit form, fixed to the back of the field of contacts of the connection points of the casing with the external environment, parallel to the back of the casing, and supporting the electrical connection tracks, on a portion of their paths starting from the contacts of this field of connection points with the external environment, said tracks being divided, on this portion of their paths, into at least two groups going towards opposite edges of the central panel, called longitudinal edges, and two side panels in printed circuit form, folded and placed flat against each other between the two groups of printed circuit boards, attached on either side of the central panel, each by an edge of one of the longitudinal edges of the central panel, by means of a linking part in the form of a flexible printed circuit having a size sufficient to enable them to be folded and placed flat against each other, sharing the transferred field of connection points and the linking tracks that lead thereto from the field of contacts of the connection points of the casing with the external environment, the transferred field of connection points being equipped with through-contacts, the connection points of the part of the transferred field borne by a side panel being shifted laterally with respect to the connection points of the matching part of the transferred field borne by the other side panel, the side panels supporting the connection tracks on the portion of their paths that leads to their part of the transferred field of connection points and being provided with at least one aperture leaving space for the through-contacts of the other side panel, the connecting tracks that lead to the part of the transferred field of connection points of a side panel passing, in order to meet contacts of the field of connection points accessible from the exterior of the casing, through the linking part attaching one of the edges of the side panel considered with one of the longitudinal edges of the central panel.", "2.The module according to claim 1, wherein the central and side panels are rigid panels.", "3.The module according to claim 1, adapted to a casing comprising a metal shielding partition wall between the two groups of printed circuit boards characterized in that said metal shielding partition wall interposed between the two side panels that are folded and placed flat against each other.", "4.The module according to claim 1, wherein the two side panels are folded and placed flat against each other in parallel to a plane perpendicular to the central panel.", "5.The module according to claim 1, wherein the two side panels are folded and placed flat against each other, in parallel and on either side of a plane perpendicular to the central panel, parallel to the longitudinal edges of this central panel and passing through its middle.", "6.The module according to claim 1, wherein the ends of the through-contacts of the transferred field of connection points form pins for semi-connectors mounted back-to-back, on each side of the block constituted by the two side panels folded and placed flat against each other, designed to co-operate by fitting together with semi-connectors having matching shapes mounted on the printed circuit board of each of the two groups of printed circuit boards that directly face the two side panels.", "7.The module according to claim 1, wherein the side panels are provided with an internal field of electrical connection points having no link with the contacts of the field of connection points with the environment outside the casing, borne by the central panel, said internal field of connection points comprising through-contacts forming pins of semi-connectors on the two opposite faces of the block constituted by the two side panels, folded and placed flat against each other, these pins of semi-connectors being designed to co-operate by fitting together with semi-connectors of matching shapes mounted on the printed circuit board of each of the two groups of printed circuit boards that directly face the two side panels.", "8.The module according to claim 7, wherein the semi-connectors mounted on each side of the block constituted by the two side panels folded and placed flat against each other comprise, without distinction, pins constituted by through-contacts belonging to the transferred field of connection points and to the internal field of connection points.", "9.The module according to claim 1, wherein the part of the transferred field of connection points borne by a side panel is shifted laterally with respect to the matching part of the transferred field of connection points borne by the other side panel, each side panel comprising an aperture allowing passage to the part of the transferred field of connection points that is borne by the other side panel.", "10.The module according to claim 9, wherein the semi-connectors equipping the block formed by the side panels folded and placed flat against each other have different thicknesses as a function of the side panel that supports them so that all are flush with the same level on each face of the block although the levels at which they are affixed depend on the side panel that supports them.", "11.The module according to claim 10, wherein the differences in thickness imposed on the semi-connectors equipping the block constituted by the side panels folded and placed flat against each other, so that they are flush at the same level, are obtained by means of thickness shims.", "12.The module according to claim 9, wherein the side panels have identical dissymmetrical contours and are placed so as to be folded and positioned flat against each other so as to have non-coinciding contours.", "13.The module according to claim 1, wherein the semi-connectors equipping the block constituted by the side panels folded and placed flat against each other co-operate with the semi-connectors of matching shapes mounted on the printed circuit boards of each of the two groups of printed circuit boards coming into a position that directly faces the two side panels whose pins are constituted by through-contacts forming, at their other end, other pins for the semi-connectors mounted on an opposite face reproducing the semi-connectors of the two side panels and making the fields of connection points of the side panels accessible to the next printed circuit board belonging to the same group.", "14.The module according to claim 13, wherein the printed circuit boards of a group comprising through-contacts aligned with those of the semi-connectors borne by the side panels, forming, on one face of the board, pins of nestable semi-connectors with shapes matching the nestable semi-connectors of the side panels and, on the other face, nestable semi-connectors having the same shapes as the semi-connectors of the side panels so that, in fitting together, they make it possible, with the printed circuit boards of a group, to form a stack of printed circuit boards having access to all the connection points of the fields of connection points of the side panels." ], [ "The present invention relates to interconnecting modules used in electronic device casings in order to electrically connect their printed circuit boards with one another and with the external environment.", "It relates more particularly to the interconnecting modules used as backplanes in detachable electronic device casings which are mounted detachably on supporting frames such as cradles, seats, cubicles or shelves etc.", "and equipped at the back with one or more semi-connectors designed to co-operate by being fitted together with semi-connectors of matching shape mounted on the supporting frames to electrically connect the electronic device, housed in the casings, with the external environment in which this device is designed to function.", "The utility of detachable casings for electronic device lies in the fact that it facilitates maintenance and trouble-shooting for an electronic system, whose elements are distributed among several casings, by greatly simplifying replacement operations through the standard exchange of the casings.", "There are many sorts of known detachable casings.", "The trend is toward standardization, namely toward the utmost possible elimination of the specializing of casings as a function of the specific characteristics of connection of the printed circuit boards that they are designed to receive.", "This trend toward the standardization of the casings implies that the interconnecting modules used as backplanes enable all the printed circuit boards housed in a casing, without distinction, whatever the specific characteristics of the electronic device whose components they bear, to have a possibility of accessing all the points of connection of the casing with the external environment.", "This constraint relating to the possibility of access of all the printed circuit boards housed in the casing to all the connection points of the casing with the external environment raises a problem of the space requirement of the interconnecting modules used on the backplane to set up an interface between the printed circuit boards housed in the casing and the semi-connectors mounted on the back of the casing once the number of connection points exceeds about a hundred.", "This is especially true as compactness is sought for the casings, which is often the case for devices designed to the incorporated into mobiles.", "The present invention is aimed at obtaining an interconnecting module for a base of an electronic device casing, mounted on printed circuit boards, that is compact while at the same time allowing the casing to have a large number of electrical connection points with the outer environment accessible to all its printed circuit boards.", "An object of the invention is an interconnecting module for a base of an electronic device casing that contains two groups of printed circuit boards bearing electronic device components, each group being constituted by one or more stacked printed circuit boards, and that has a back equipped with at least one field of contacts constituting electrical connection points accessible from the exterior of the casing.", "This interconnecting module provides for the transfer, at the level of the two groups of printed circuit boards, of the connection points of the casing accessible from the exterior.", "It comprises a field of electrical connection points transferred to the interior of the casing so as to be facing the two groups of printed circuit boards and tracks setting up the electrical links between this transferred field of connection points and the field of contacts with which the back of the casing is equipped.", "The module comprises a flat support in the form of a three-panel structure with: a central panel, in printed circuit form, fixed to the back of the field of contacts of the connection points of the casing with the external environment, parallel to the back of the casing, and supporting the electrical connection tracks, on a portion of their paths starting from the contacts of this field of connection points with the external environment, said tracks being divided, on this portion of their paths, into at least two groups going towards opposite edges of the central panel, called longitudinal edges, and two side panels in printed circuit form, folded and placed flat against each other between the two groups of printed circuit boards, attached on either side of the central panel, each by an edge of one of the longitudinal edges of the central panel, by means of a linking part in the form of a flexible printed circuit having a size sufficient to enable them to be folded and placed flat against each other, sharing the transferred field of connection points and the linking tracks that lead thereto from the field of contacts of the connection points of the casing with the external environment, the transferred field of connection points being equipped with through-contacts, the connection points of the part of the transferred field borne by a side panel being shifted laterally with respect to the connection points of the matching part of the transferred field borne by the other side panel, the side panels supporting the connection tracks on the portion of their paths that leads to their part of the transferred field of connection points and being provided with at least one aperture leaving space for the through-contacts of the other side panel, the connecting tracks that lead to the part of the transferred field of connection points of a side panel passing, in order to meet contacts of the field of connection points accessible from the exterior of the casing, through the linking part attaching one of the edges of the side panel considered with one of the longitudinal edges of the central panel.", "Advantageously, the central and side panels are rigid panels.", "Advantageously, a metal shielding partition wall is interposed between the two side panels that are folded and placed flat against each other.", "Advantageously, the two side panels are folded and placed flat against one another in parallel to a plane perpendicular to the central panel.", "Advantageously, the two side panels are folded and placed flat against each other, in parallel and on either side of a plane perpendicular to the central panel, parallel to its longitudinal edges and passing through the middle of the central panel.", "Advantageously, the ends of the through-contacts of the transferred field of connection points form pins for semi-connectors mounted back-to-back, on each side of the block constituted by the two side panels folded and placed flat against each other, designed to co-operate by fitting together with semi-connectors having matching shapes mounted on the printed circuit board of each of the two groups of printed circuit boards that directly face the two side panels.", "Advantageously, the side panels are provided with an internal field of electrical connection points having no link with the contacts of the field of connection points with the environment outside the casing, borne by the central panel, the internal field of connection points comprising through-contacts forming pins of semi-connectors on the two opposite faces of the block constituted by the two side panels, folded and placed flat against each other, the pins of semi-connectors being designed to co-operate by fitting together with semi-connectors of matching shapes mounted on the printed circuit boards of each of the two groups of printed circuit boards that directly face the two side panels.", "Advantageously, the semi-connectors mounted on each side of the block constituted by the two side panels folded and placed flat against each other comprise, without distinction, pins constituted by through-contacts belonging to the transferred field of connection points and to the internal field of connection points.", "Advantageously, the part of the transferred field of connection points borne by a side panel is shifted laterally with respect to the matching part of the transferred field of connection points borne by the other side panel, each side panel comprising an aperture allowing passage to the part of the transferred field of connection points that is borne by the other side panel.", "Advantageously, the semi-connectors equipping the block formed by the side panels folded and placed flat against each other have different thicknesses as a function of the side panel that supports them so that all are flush with the same level on each face of the block although the levels at which they are affixed depend on the side panel that supports them.", "Advantageously, the side panels have dissymmetrical identical contours and are placed so as to be folded and positioned flat against each other so as to have non-coinciding contours.", "Advantageously, the semi-connectors with matching shapes mounted on the printed circuit boards of each of the two groups of printed circuit boards that directly face the two side panels have pins consisting of through-contacts forming other pins, at their other end, for the semi-connectors mounted on the opposite face, reproducing the semi-connectors of the two side panels and making the fields of connection points of the side panels accessible to the next board of the printed circuit boards belonging to the same group.", "Advantageously, the printed circuit boards of a group comprise through-contacts aligned with those of the fields of connection points borne by the side panels forming, on one face of the boards, pins of nestable semi-connectors having shapes that match the nestable semi-connectors of the side panels and, on the other face, nestable semi-connectors having the same shapes as the semi-connectors of the side panels so that, when they fit together, they make it possible, with the printed circuit boards of a group, to form a stack of printed circuit boards having access to all the connection points of the fields of connection points of the side panels.", "Other features and advantages of the invention shall appear from the following description of an embodiment given by way of an example.", "This description is made with reference to the appended drawing, wherein: FIG.", "1 is a drawing illustrating the architecture of an interconnecting module according to the invention and one of its possible arrangements within a casing, FIG.", "2 is a view in perspective of an interconnecting module according to the invention mounted on the back of a semi-connector designed to equip the back of a casing to provide for the connections with the external environment, FIG.", "3 is a view in section of a casing equipped with the interconnecting module illustrated in FIG.", "2, and FIG.", "4 is a view in perspective of another interconnecting module according to the invention.", "FIG.", "1 shows a sectional view of the rear part of an electronic device casing having the usual parallelepiped shape of a large book, with a casing 1 having an aperture 2 on the back of the casing, revealing a multiple-pin semi-connector 3 designed to fit into a semi-connector with a matching shape equipping a cradle designed to receive the casing.", "The casing contains one or more electronic devices whose components are mounted on two printed circuit boards 4 and 5 positioned in parallel to the biggest faces of the casing and separated from each other by a metal shielding plate 6.It is assumed that the electronic device or devices whose components are mounted on the printed circuit wafers 4, 5 exchange electrical signals with other electronic devices housed in other casings and therefore need to be connected with them through the environment external to the casing.", "The multiple-pin, semi-connector 3 fixed to the back of the casing and accessible from the exterior fulfils this function of electric connection with the environment external to the casing, at the transition between the casing and its cradle.", "However, inside the casing, the connections are still to be made between the rear ends of the pins of the semi-connector 3 and the tracks of the printed circuit boards 4, 5 bearing the components of the device or devices housed in the casing.", "The connections are generally made inside a casing, by means of a backplane board consisting of a single rigid printed circuit.", "This single rigid printed circuit is then fixed directly to the rear ends of the pins of the semi-connector 3 which are pointed to the inside of the casing, parallel to the back wall of the casing.", "Facing the edges of the printed circuit board supporting the components of the devices housed in the casing, this single rigid printed circuit is equipped with rows of nestable semi-connectors designed to get coupled with semi-connectors of matching shapes fixed to the edges of these boards and provided with a network of tracks setting up the necessary electrical links between the pins of the different semi-connectors.", "Once the number of connecting points to be made becomes great, this manner of making the connections inside the casing, at the interface between the printed circuit boards of the electronic devices and the nestable semi-connector or semi-connectors fixed to the back of the casing and giving access to the external environment has a drawback.", "This drawback is that substantial space becomes necessary within the casing, in order to house the network of connectors setting up the electrical links at the edges of the printed circuit boards.", "Thus, it is not infrequent that it becomes necessary to provide for several hundreds of points of connection with the external environment, for example 400 to 600 connection points, on an electronic device casing.", "Thus, the casing must be designed to be far bulkier than would be the case for the simple housing of the printed circuit boards bearing the components of the electronic device placed in the casing.", "The result of this is a lack of compactness of the casings which often entails penalties for devices designed to be fitted into mobiles where space is very often costly.", "A known way of restricting the space requirement of the backplane board of an electronic device casing consists achieving the utmost possible reduction in the number of internal connections that the board sets up, ensuring only indispensable connections.", "This amounts to customizing the casings according to the specific connection requirements of the types of printed circuit boards that these casings are effectively designed to contain.", "This runs counter to the present trend toward the standardization of the casings where it is sought, as far as possible, to avoid customizing casings according to the type of printed circuit board that they are designed to contain, since it is very rare for two types of boards to have the same connection requirements.", "Here, an interconnecting module playing the role of the backplane board is proposed.", "This module enables all the printed circuit boards of a casing to have access to all the connection points of the casing with the external environment while at the same time being very compact.", "In this interconnecting module, the compactness is obtained by transferring the connections with the printed circuit boards 4, 5 that support the components of the electronic device housed in the casing from the edge of these components toward their flanks.", "As shown in FIG.", "1, the interconnecting module takes the form of a three-panel panel with a central panel 7 fixed to the back of the pins of the semi-connector 3 mounted on the real wall of the casing and two side panels 8, 9 joined by one side to the lateral edges of the central panel 7, by means of two flexible printed circuit 10, 11 folded and placed back-to-back against each other and fixed to the shielding partition wall 6.More specifically, the central panel 7 is formed by a rigid printed circuit that is fixed, inside the casing, to the rear ends of the pins of the semi-connector 3 and bears electric linking tracks starting from the pins of the semi-connector 3 and separating into two groups toward its two opposite edges attached to the side panels 8, 9 by the flexible printed circuits 10, 11 The side panels 8, 9 are formed by two rigid printed circuits folded and placed against each other back-to-back, perpendicularly to the central panel 7, on either side of the shielding partition wall 6, the flexible printed circuit 10, 11 having widths sufficient to enable this arrangement.", "They share, on the one hand, a transferred field of connection points which, facing the flanks of the printed circuit boards 4,5 supporting the components of the electronic device housed in the casing, reproduce the field of connection points with the external environment corresponding to the pins of the semi-connector 3 fixed to the back of the casing.", "On the other hand, they share the extensions of the electrical connection tracks coming from the central panel by means of the joining flexible printed circuits 10, 11.The side panels 8, 9, as well as the shielding partition wall 6 at the side panels 8, 9 are drilled with networks of holes that correspond with each other and enable the passage of through-contacts 12 materializing the transferred field of connection points.", "Out of three aligned holes crossed by one and the same through-contact 12, only one made in one of the side panels is used as a destination for an electrical linking track with a contact pin of the semi-connector 3 fixed to the back of the casing, the other two holes, one made in the shielding plate 6 and the other in the other side panel being simple holes, either large enough to prevent any contact that could set up electrical transmission with the through-contact 12 or made with an insulating wall.", "The two ends of the through-contacts 12 constitute pins for the nestable semi-connectors whose bases 13, 14 are fixed to the opposite flanks of the block constituted by the side panels 8, 9 folded and placed flat against each other back-to-back.", "These ends are directed toward semi-connectors 15, 16 having matching shapes, fixed to the facing faces of the printed circuit boards 4,5 bearing the components of the electronic device housed in the casing.", "The electrical linking tracks which, on the side panels, end in the networks of holes provided with through-contacts 12 are distributed more or less equitably, on the two side panels 8, 9 and are divided into two groups with different paths passing through the two longitudinal edges of the central panel 7.This distribution facilitates the routing of the different electrical linking tracks since it doubles the possibility of designing tracks.", "This reduces the number of layers and hence the complexity of the printed circuits used for the central panels 7 and side panels 8, 9.The making of the interconnecting module in the form of three rigid printed circuit panels joined by flexible printed circuits shall not be described in detail because it is known to those skilled in the art.", "In the context of multiple-layer printed circuits, the three-panel structure may be obtained at each layer, before the lay-up process and before the layers are fixedly joined together by bonding.", "The use of a transferred field of connection points, electrically linked with the field of connection points of the casing with the external environment, reoriented from the edge to the flanks of the printed circuit boards bearing the components of the electronic device housed in the casing by means of an interconnecting module formed by folding printed circuits, makes it possible to take the space needed for the nestable connectors for connection with these printed circuit circuits.", "This space is taken not from the spacing between boards but on the surface of the boards, thus preserving the compactness of the casing.", "Indeed, the spacing between the printed circuit boards bearing the components of the electronic device housed in the casing is always small because of the small height of the components.", "The arrangement of the connectors on the edges of the boards necessarily dictates an increase in this pacing once the number of connection points to be made becomes great.", "This increase implies the poor use of the volume between boards.", "However, the arrangement of the semi-connectors on the surface of the boards results in only a small increase in the depth of the casing.", "The transferred field of connection points may comprise a greater number of elements than the field of connection points with the external environment corresponding to the pins of the semi-connector 3 mounted on the back of the casing, the supernumerary elements being also equipped with through-contacts and constituting an internal field of connection points between the two printed circuit boards 4, 5 without any electrical links with the pins of the semi-connector 3 mounted on the back of the back.", "It may be that the printed circuit boards 4, 5 are not isolated but form part of two stacks of boards.", "They are then also provided with through-contacts.", "The ends of these through-contacts that face the side walls 8, 9 constitute pins for the semi-connectors 15, 16 designed to fit together with the semi-connectors 13, 14 of the side panels 8, 9.The opposite ends of these through-contacts having their back facing the side walls 8, 9 constitute pins for semi-connectors (not shown) that reproduce the semi-connectors 13, 14 of the side walls 8, 9 for the next boards of the stack.", "FIG.", "2 gives a view in perspective of an exemplary embodiment of the interconnecting module shown schematically in FIG.", "1.This module is mounted on the rear ends of the pins of a nestable semi-connector designed to be fixed to the back of the casing to provide for connections with the external environment of the casing.", "The body 20 of the nesting connector designed to be fixed to the back of the casing can be seen from the rear.", "The rear ends of the pins of this nesting connector fixed to the back of the casing are masked by the interconnecting module 30.The three-panel structure of this interconnecting module can be distinguished with its rigid central panel 31 coming above the body 20 of the connector, its two rigid side panels 32,33 folded and placed flat against each other in a plane perpendicular to the central panel 31 and the flexible printed circuits 34,35 joining each of the side panels 32,33 to a distinct longitudinal edge of the central panel 31.The elongated, rectangular central panel 31 is positioned in parallel to the back of the casing, fixed to the rear ends of the pins of the semi-connector whose body 20 can be glimpsed.", "The two side panels 32, 33, which have a same elongated rectangular contour here, are folded and placed flat against each other on either side of a plane perpendicular to the back of the casing passing through the longitudinal median line of the central panel 31 which is also that of the semi-connector fixed to the back of the casing, the flexible printed circuits 34, 35 joining them by a longitudinal edge to the longitudinal edges of the central panel 31 having a width sufficient to enable this arrangement.", "At a position shifted so as to be before the faces of the printed circuit boards housed in the casing, each side panel 32 and 33 reproduces the field of connection points with the external environment of the casing, embodied at the central panel 31 by the rear ends of the pins of the semi-connector fixed to the back of the casing.", "At the two side panels 31, 32, this transferred field takes the form of two networks of holes, one on each side panel 32 or 33.These two networks of holes get superimposed when the two side panels 32, 33 are folded and placed flat against each other.", "Through-contacts are threaded through these networks of holes.", "On each face of the block constituted by the two side panels 32, 33 that are folded and placed flat against each other, these through-contacts serve as pins for nestable semi-connectors whose guide slots 36,37,38,39 can be seen.", "These pins are designed, as can be seen more clearly in FIG.", "3, to co-operate with semi-connectors 15, 16 of matching shapes that equip the two printed circuit boards 40, 41 housed in the casing.", "As indicated here above, the network of pairs of aligned holes materializing the transferred field of connection points within the block constituted by the two side panels 32, 33 folded and placed flat against each other is connected by electrical linking tracks to the field of connection points of the casing with the external environment, formed on the central panel 31 by the rear ends of the connection pins of the semi-connector fixed to the back of the casing.", "These tracks which cannot be seen in FIG.", "2, are distributed more or less equitably between the two side panels 32 and 33, and the flexible printed circuits 34, 35 for joining the side panels 32, 33 to the central panel 31.On the side panel 32, 33 side, they lead to one of the holes of a pair occupied by a through-contact, the hole made on the side panels 32 or 33 bearing the track while the other hole belonging to the other side panel remains without any assigned track.", "FIG.", "3 is a cross-section view of a casing along its thickness.", "This casing repeats the main characteristics of the architecture described with reference to FIG.", "1.It is parallelepiped-shaped with a rectangular-sectioned wall 1.It contains one or more electronic devices whose components are mounted on two printed circuit boards 4,5 placed flat on either side of a metal shielding partition wall 6′ placed at the center of the casing.", "It is equipped with a multiple-pin nestable semi-connector 3 mounted in an aperture 2 of its back wall providing the electrical connections of the devices housed in the casing with the environment external to the casing and an interconnecting module forming the interface between this semi-connector 3 and the printed circuit boards 4, 5.The interconnecting module is the one shown in FIG.", "2, with its three-panel conformation.", "Its rigid central panel 31 is fixed to the rear ends of the pins of the semi-connector 3, parallel to the back wall of the casing.", "Its side panels 32 and 33, attached to the longitudinal edges of the central panel 31 by two flexible printed circuits 34, 35, are folded, placed flat against each other, fixed through an aperture of the shielding partition wall 6′.", "The block that they constitute supports nestable semi-connectors 36, 37 designed to co-operate with semi-connectors 16, 15 having matching shapes, mounted on the mutually facing sides of the printed circuit boards 4, 5 supporting the components of the electronic device housed in the casing.", "The main difference with the interconnecting module shown schematically in FIG.", "1 lies at the level of the block constituted by the two side panels 36, 37, folded and placed directly flat against each other and not on either side of the shielding plate.", "This difference in the mounting of the side panels 36, 37 facilitates the making of the casing since its shielding plate no longer has to present a network of individual holes for the through-contacts but only one general aperture.", "Above all, it facilitates the mounting of the through-contacts through the side panels 36, 37 since the block that they constitute no longer include the shielding plate.", "Another difference consists of the presence, through an aperture of the shielding plate 6′, of an insulating plate 40 supporting a field of through-contacts used as pins for nestable semi-connectors 41, 42 mounted on the opposite faces of the plate 40 and co-operating with semi-connectors 43, 44 having matching shapes fixed to the mutually facing sides of the printed circuit boards 4, 5 supporting the components of the electronic device housed in the casing.", "This field of through-contacts constitutes an internal field of connection points providing the electrical interconnection between the printed circuit board 4, 5, without relation with the field of connection points with the environment external to in the casing, taking the form of the pins of the semi-connector 3 fixed to the back of the casing.", "Should the components mounted on the printed circuit boards 4, 5 have smaller heights than the connectors 16-36, 15-37, 41-43, 42-44, then the space imposed by the height of the connectors between the printed circuit boards 4, 5 and the shielding plate 6′ can be occupied by an auxiliary printed circuit board 45, 46 placed in a mezzanine position by means of spacers 47, 48.These auxiliary boards 45, 46 may be connected to their carrier boards by wire connections or by flexible printed circuits.", "FIG.", "4 gives a view, in perspective, of another exemplary embodiment of the interconnecting module shown schematically in FIG.", "1.This exemplary embodiment is close to the one described here above with reference to FIG.", "2.It differs from that embodiment, however, in the shape of the contour of the side panels 32′ and 33′ which is no longer rectangular but has a large notch 50, 51.The two side panels 32′ and 33′ are folded and placed flat against each other so that their notches 50, 51 are not superimposed by allow the underlying face of the other side panel to be seen.", "As above, at a position transferred so as to be before the printed circuit boards housed in the casing, the two side panels 32′ and 33′ reproduce the field of connection points with the external environment of the casing embodied at the central panel 31 by the rear ends of the pins of the semi-connector which is fixed to the back of the casing, its body being seen at 20 in FIG.", "4.This transferred field of connection points is linked to the field of connection points with the external environment borne by the central panel 31, by means of electrical connection tracks divided between the two side panels 32′, 33′ and the flexible printed circuits 34, 35 joining them to the longitudinal edges of the central panel 31.This transferred field of connection points is again embodied by through-contacts whose ends act as pins for the nestable semi-connectors 52 to 58 mounted on the two faces of the side panels 32′ 33′.", "However, unlike the embodiment of FIG.", "2, the part of the transferred field of connection points borne by one of the side panels, 32′ or 33′ respectively, and the matching part of the transferred field of connection points borne by the other side panel, 33′ or 32′ respectively are no longer interlocked but geographically separated.", "In the block formed by the side panels 32′ and 33′, which are folded and placed flat against each other, the part of the transferred field of connection points borne by a side panel 32′ or 33′ takes up the place occupied by the notch of the other panel and vice versa.", "Thus, in FIG.", "4, the part of the transferred field of connection points borne by the side panel 32′ is embodied by the through-contacts constituting the pins of the semi-connectors 52 to 55 placed at the notch 50 of the side panel 33′ while the matching part of the transferred field of connection points borne by the side panel 33′ is embodied by the through-contacts constituting the pins of the semi-connectors 56 to 58 placed at the level of the notch 51 of the side panel 32′.", "This assembly enables the semi-connectors 52 to 58 of the transferred field of connection points to be mounted individually on each side panel 32′ and 33′ and not on the block constituted by the two side panels folded and placed flat against each other.", "This has the advantage of eliminating the problems raised by control over the thickness of the block constituted by the two side panels folded and placed flat against each other during the mounting of the semi-connectors by automatic machines which carry out the installation of the accurate positioning elements of the semi-connectors and the force-fitting of the through-contacts forming their pins.", "By contrast, it is necessary to provide for thickness shims 59, 60 to bring about uniformity in the height of all the semi-connectors accessible by one and the same face of the block constituted by the two side panels 32′, 33′ folded and placed flat against each other.", "It has been assumed, throughout the above description, that the entire field of connection points with the external environment external to the casing was relayed at the transferred field of connection points borne by the side panels of the interconnecting module.", "It goes without saying that these are connection points which may be of interest to one of the devices whose components are borne by the printed circuit boards housed in the casing.", "A certain number of connection points with the environment external to the housing, which are not of concern to these boards, are not relayed to the transferred field of contacts but directly wired to other circuits, often accessory circuits, mounted outside these printed circuit boards." ] ]	Patent_10451667
[ [ "Dispensing Apparatus", "A dispensing apparatus (1) including a combination of a tube (2), cap (4) and plunger (3).", "The tube is arranged for carrying a collapsible sausage (6) and includes longitudinally extending ribs (8) projecting inwardly of the tube and defining longitudinal grooves (7) therebetween, the grooves having an annular dimension (A) greater than the distance (B) between adjacent ribs.", "The cap includes: a coupling portion (20) for mounting the cap to the tube; an outlet (21) through which contents of the collapsible sausage carried within the tube arc dispensed; and a chamber (22) into which the sausage is adapted to be collapsed under action of the plunger within the tube.", "The plunger itself has a main body (11) with radial fins (10) projecting therefrom for receipt in the grooves of the tube, between adjacent ribs, the fins being dimensioned to engage lateral edges of the ribs, in a point contact manner." ], [ "1.A tube for carrying a collapsible sausage and dispensing contents therefrom, the tube having longitudinally extending ribs projecting inwardly of the tube and defining longitudinal grooves therebetween, the grooves having an annular dimension greater than the distance between adjacent ribs.", "2.A tube as claimed in claim 1, wherein the ribs and grooves extend substantially the entire length of the tube.", "3.A tube as claimed in claim 1 or 2, wherein ends of the tube are provided with inwardly directed stop surfaces for capturing a plunger therebetween.", "4.The ends of the tube are provided with an inwardly directed lip for engaging with and securing a cap relative thereto.", "5.A cap for use with the above tube, the cap having: a coupling portion for mounting the cap to the tube; an outlet through which contents of the collapsible sausage carried within the tube are dispensed; and a chamber into which the sausage is adapted to be collapsed under action of a plunger within the tube.", "6.A cap as claimed in claim 5, wherein the coupling portion includes a spline formed on an outer surface of the cap for receipt in an associated groove of the tube.", "7.A cap as claimed in claim 5 or 6, wherein the cap includes a well, provided within the chamber for receipt of an end clip of the sausage.", "8.A cap as claimed in any one of claims 5 to 7, wherein the cap includes piercing structure projecting from a base of the cap for puncturing the sausage.", "9.A cap as claimed in claim 8, wherein the piercing structure is provided in the well, adjacent the outlet.", "10.A cap as claimed in claims 8 or 9, wherein the piercing structure includes two teeth positioned toward either side of the outlet.", "11.A cap as claimed in claim 10, wherein one of tho teeth projects a lesser distance from the base of the cap and includes a vane directed away from an other of the teeth, so as to tension the sausage away from a puncture zone and facilitate propagation of a tear in the sausage.", "12.A cap as claimed in any one of claims 8 to 11, wherein the cap includes a shroud arranged adjacent the well, an a side of the outlet opposite the piercing structure, the shroud serving to impart a turning moment at an end of the sausage so as to deflect the end clip of the sausage into the well at a location offset from the outlet.", "13.A cap as claimed in claim 12, wherein the shroud is formed by a series of arc-shaped ribs.", "14.A cap as claimed in any one of claims 6 to 13, wherein the cap is provided with a rebate on an exterior surface thereof for engagement with a complimentary lip of the tube, for securing the cap relative to the tube.", "15.A plunger for use in the above described tube, the plunger having a main body with radial fins projecting therefrom for receipt in the grooves of the tube, between adjacent ribs, the fins being dimensioned to engage lateral edges of the ribs, in a point contact manner.", "16.A plunger as claimed in claim 15, wherein the body of the plunger includes a central bulb for driving the sausage into a cap coupled to the tube.", "17.A plunger as claimed in claim 16, wherein the plunger includes a reduced dimension region between the bulb and the fins.", "18.A plunger as claimed in claim 17, wherein the bulb includes a bore for receipt of an end clip of the sausage.", "19.A plunger as claimed in any one of claims 16 to 18, wherein the plunger is symmetric and reversible.", "20.A dispensing apparatus including: a tube, as claimed in any one of claims 1 to 4; a cap, as claimed in any one of claims 6 to 14, and a plunger, as claimed in any one of claims 15 to 19." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>It is known to dispense contents of a collapsible sausage from a caulking gun arrangement or the like which carries a tube, with a plunger.", "The plunger is intended to be reversible within the tube for subsequent dispensing of contents from another sausage, in a reverse direction.", "The known arrangement is, however, considered to be inadequate insofar as the plunger tends to got jammed and replacement of the sausage can be somewhat messy." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with the present invention, there is provided a tube for carrying a collapsible sausage and dispensing contents therefrom, the tube having longitudinally extending ribs projecting inwardly of the tube and defining longitudinal grooves therebetween, the grooves having an annular dimension greater than the distance between adjacent ribs.", "Preferably, the ribs and grooves extend substantially the entire length of the tube.", "Preferably, ends of the tube arc provided with inwardly directed stop surfaces for capturing a plunger therebetween.", "Preferably, ends of the tube are provided with an inwardly directed lip for engaging with and securing a cap relative thereto.", "In another aspect, there is provided a cap for use with the above tube, the cap having: a coupling portion for mounting the cap to the tube; an outlet through which contents of the collapsible sausage carried within the tube are dispensed; and a chamber into which the sausage is adapted to be collapsed under action of a plunger within the tube.", "Preferably, the coupling portion includes a spline formed on an outer surface of the cap for receipt in an associated groove of the tube.", "Preferably, the cap includes a well, provided within the chamber for receipt of an end clip of the sausage.", "Preferably, the cap includes piercing structure projecting from a base of the cap for puncturing the sausage.", "Preferably, the piercing structure is provided in the well, adjacent the outlet.", "Preferably, the piercing structure includes two teeth positioned toward either side of the outlet.", "More preferably, one of the teeth projects a lesser distance from the base of the cap and includes a vane directed away from an other of the teeth, so as to tension the sausage away from a puncture zone and facilitate propagation of a tear in the sausage.", "Preferably, the cap includes a shroud arranged adjacent the well, on a side of the outlet opposite the piercing structure, the shroud serving to impart a turning moment at an end of the sausage so as to deflect the end clip of the sausage into the well at a location offset from the outlet.", "Preferably, the shroud is formed by a series of arc-shaped ribs.", "Preferably, the cap is provided with a rebate on an exterior surface thereof for engagement with a complimentary lip of the tube, for securing the cap relative to the tube.", "In another aspect, there is provided a plunger for use in the above described tube, the plunger having a main body with radial fins projecting therefrom for receipt in the grooves of the tube, between adjacent ribs, the fins being dimensioned to engage lateral edges of the ribs, in a point contact manner.", "Preferably, the body of the plunger includes a central bulb for driving the sausage into a cap coupled so the tube.", "Preferably, the plunger includes a reduced dimension region between the bulb and the fins.", "Preferably, the bulb includes a bore for receipt of an end clip of the sausage.", "Preferably, the plunger is symmetric and reversible.", "In another aspect, there is provided a dispensing apparatus including a combination of the tube, cap and plunger, as described above." ], [ "FIELD OF THE INVENTION The present invention relates to a dispensing apparatus particularly an apparatus for dispensing contents from a collapsible sausage.", "BACKGROUND OF THE INVENTION It is known to dispense contents of a collapsible sausage from a caulking gun arrangement or the like which carries a tube, with a plunger.", "The plunger is intended to be reversible within the tube for subsequent dispensing of contents from another sausage, in a reverse direction.", "The known arrangement is, however, considered to be inadequate insofar as the plunger tends to got jammed and replacement of the sausage can be somewhat messy.", "OBJECT OF THE INVENTION The present invention seeks to address the above inadequacies.", "SUMMARY OF THE INVENTION In accordance with the present invention, there is provided a tube for carrying a collapsible sausage and dispensing contents therefrom, the tube having longitudinally extending ribs projecting inwardly of the tube and defining longitudinal grooves therebetween, the grooves having an annular dimension greater than the distance between adjacent ribs.", "Preferably, the ribs and grooves extend substantially the entire length of the tube.", "Preferably, ends of the tube arc provided with inwardly directed stop surfaces for capturing a plunger therebetween.", "Preferably, ends of the tube are provided with an inwardly directed lip for engaging with and securing a cap relative thereto.", "In another aspect, there is provided a cap for use with the above tube, the cap having: a coupling portion for mounting the cap to the tube; an outlet through which contents of the collapsible sausage carried within the tube are dispensed; and a chamber into which the sausage is adapted to be collapsed under action of a plunger within the tube.", "Preferably, the coupling portion includes a spline formed on an outer surface of the cap for receipt in an associated groove of the tube.", "Preferably, the cap includes a well, provided within the chamber for receipt of an end clip of the sausage.", "Preferably, the cap includes piercing structure projecting from a base of the cap for puncturing the sausage.", "Preferably, the piercing structure is provided in the well, adjacent the outlet.", "Preferably, the piercing structure includes two teeth positioned toward either side of the outlet.", "More preferably, one of the teeth projects a lesser distance from the base of the cap and includes a vane directed away from an other of the teeth, so as to tension the sausage away from a puncture zone and facilitate propagation of a tear in the sausage.", "Preferably, the cap includes a shroud arranged adjacent the well, on a side of the outlet opposite the piercing structure, the shroud serving to impart a turning moment at an end of the sausage so as to deflect the end clip of the sausage into the well at a location offset from the outlet.", "Preferably, the shroud is formed by a series of arc-shaped ribs.", "Preferably, the cap is provided with a rebate on an exterior surface thereof for engagement with a complimentary lip of the tube, for securing the cap relative to the tube.", "In another aspect, there is provided a plunger for use in the above described tube, the plunger having a main body with radial fins projecting therefrom for receipt in the grooves of the tube, between adjacent ribs, the fins being dimensioned to engage lateral edges of the ribs, in a point contact manner.", "Preferably, the body of the plunger includes a central bulb for driving the sausage into a cap coupled so the tube.", "Preferably, the plunger includes a reduced dimension region between the bulb and the fins.", "Preferably, the bulb includes a bore for receipt of an end clip of the sausage.", "Preferably, the plunger is symmetric and reversible.", "In another aspect, there is provided a dispensing apparatus including a combination of the tube, cap and plunger, as described above.", "BRIEF DESCRIPTION OF THE DRAWINGS The invention is described, by way of non-limiting example only, with reference to the drawings, in which: FIG.", "1 is an exploded sectional view of a dispensing apparatus; FIG.", "2 is a view of the apparatus of FIG.", "1, showing a sausage in a collapsed state; FIG.", "3 is an end view of the plunger of the apparatus of FIG.", "1; FIG.", "4 is a partial cross-sectional view of the plunger received in the tube; FIG.", "5 is a cross-sectional view of an alternative form of plunger; FIG.", "6 is a cross-sectional view of a cap of the apparatus of FIG.", "1; FIG.", "7 is an end view of the cap of FIG.", "6; FIG.", "8 is a cross-sectional view of an alternative cap for use with the apparatus of FIG.", "1; and FIG.", "9 is an end view of the cap of FIG.", "8.DETAILED DESCRIPTION Referring firstly to FIG.", "1, an apparatus 1 is shown as comprising a tube 2, a plunger 3 and an end cap 4 with a nozzle 5 fitted thereto.", "The tube is arranged to carry a collapsible sausage 6 and the plunger is in use driven along the tube 2 by a piston of a dispenser (not shown) such as a caulking gun, or the like, so as to collapse the sausage 6 and dispense the contents thereof from the nozzle 5.The sausage is shown in a collapsed condition in FIG.", "2.In order to ensure smooth and reliable travel of the plunger 3, the tube 2 is provided with a plurality of grooves 7, between adjacent longitudinal extending ribs 8.The plunger 3 has corresponding fins 10 projecting radially from a main body 11, as shown at FIG.", "3, for receipt in the grooves 7.The interconnection between the plunger 3 and grooves 7 is shown in more detail in FIG.", "4, wherein it can be seen that an annular dimension “A” of the grooves is greater than the distance “B” between lateral edges 12.The fins 10 are dimensioned to engage with the tube 2 only along the lateral edges 12, thereby establishing a point contact form of connection which has the effect of minimising friction between the plunger 3 and the tube 2.To maximise efficiency of dispensing contents from the sausage 6, the main body 11 of the plunger 3 is provided with a central bulb 13, for driving the sausage 6 into the cap 4.The bulb 13 is preferably formed with a bore 14 for receipt of an end clip 15 of the sausage 6.In an alternative form, the plunger 3 may have a reduced dimension region 16, as shown in FIG.", "5, which allows the collapsed sausage material, which is forced down over the bulb 13 to be scooped back toward the direction of travel “C” of the plunger, as indicated by arrow “D” so that the sausage does not jam between the plunger 3 and the tube 2.As can be seen from the drawings, the plunger 3 is symmetrically formed so as to allow for reversibility.", "For that purpose, the ends of the tube 2 may be provided with inwardly directed stop surfaces (not shown) for capturing the plunger 3 therebetween or, alternatively, the cap may itself be utilised to provide an end of travel stop for the plunger.", "The construction of the end cap 4 itself is shown in more detail in FIGS.", "6 and 7.The cap 4 includes a coupling portion 20, for mounting the cap 4 to the tube 2, an outlet 21 through which contents of the sausage 6 are dispensed and a chamber 22, into which the sausage is adapted to be collapsed, under action of the plunger 3.The coupling portion 20 may be provided with a spline 23 on an outer surface 24 of the cap 4 for receipt in an associated groove 7 of the tube 2.The spline 23 serves to prevent the cap 4 rotating relative to the tube 2 when the nozzle 5 is removed or attached to screw thread 25.The cap also includes a well 26 to receive an end clip 27 of the sausage 6 and a piercing structure 28 which projects from a base 29 of the cap for puncturing the sausage 6.The structure 28 is formed of two identical teeth 31, located adjacent and toward opposite sides of the outlet 21, which is in turn located off-centre of the cap to ensure the clip 27, which is received in the well 26, does not block the flow of the sausage contents therethrough.", "The off-centre arrangement of the outlet also provides an advantage in that a user can more readily dispense material into corners or otherwise awkward to access locations.", "An alternative cap conduction is shown in FIGS.", "8 and 9, where like reference numerals designate like parts to those shown in FIGS.", "6 and 7.Instead of having piercing structure formed of two identical teeth 31, however, one of the teeth 32 projects from the base 29 by a lesser extent than the other of the teeth 33 and is provided with a vane 34, so as to tension the sausage away from a puncture zone and facilitate propagation of a tear in the sausage.", "The cap 4 of FIGS.", "8 and 9 also includes a shroud 35, on a side of the outlet 21 opposite the piercing structure.", "The shroud serves to impart a turning moment at an end of the sausage, as it is driven into the cap 4, so as to deflect the and clip 27 into the well 26, at a location offset from the outlet 21.The shroud is formed of a series of annularly arranged arc-shaped ribs 36.The ribs 36 also serve to further tension the sausage in the vicinity of the piercing structure 28 and assist in reducing residue material within the sausage after a dispensing operation.", "FIG.", "8 also shows the cap 4 as including a rebate 37 on the exterior surface 24, which is arranged for engagement with a complimentary lip which may be provided in the tube for securing the cap 4 to the tube 2.As mentioned above, the cap 4 may serve as an end of travel stop for the plunger 3 either by the plunger engaging directly with an end wall 30 of the portion 20 or by the plunger 3 compacting sausage 6 against the structure 28, as well as the shroud 35.In either case, once the contents of the sausage have been fully dispensed, the sausage may be conveniently removed from the tube by uncoupling the cap 4 from the tube, with the collapsed sausage still held within the chamber 22, for the sausage to be discarded.", "Another full sausage can then be inserted in the tube and the rap 4 reattached to the tube at the opposite end thereof, for another dispensing operation.", "The invention has been described by way of non-limiting example only and many modifications and variations may be made thereto without departing from the spirit and scope of the invention as described." ] ]	Patent_10451671
[ [ "Compositions for oral use", "An oral composition comprising trifluoromethionine as an effective component is provided as a useful oral composition for preventing and/or treating intraoral diseases such as bad breath, periodontal disease, alveolar pyorrhea, etc., which is safe even if daily used." ], [ "1.An oral composition, which comprises trifluoromethionine as an effective component.", "2.An oral composition according to claim 1, which is a preparation for preventing and/or treating bad breath.", "3.An oral composition according to claim 1, which is a preparation for preventing and/or treating alveolar pyorrhea.", "4.An oral composition according to claim 1, which is a preparation for preventing and/or treating periodontal disease." ], [ "<SOH> BACKGROUND ART <EOH>Recently, there has been a growing interest in prevention and treatment of intraoral diseases such as bad breath, alveolar pyorrhea, periodontal disease, etc.", "Most important causative substances of bad breath are so-called volatile sulfides such as hydrogen sulfide, methylmercaptan and dimethyl sulfide, among which methylmercaptan is known to be produced by many causative organisms of periodontisis and thus has been watched with interest as an indicator of periodontisis and also as one of pathogenic factors of periodontisis.", "The methylmercaptan is produced from methionine by L-methionine-α-deamino-γ-mercaptomethane-lyase(METase) as an enzyme contained in bacteria.", "Among causative organisms of periodontal disease, well known organisms capable of efficiently producing methylmercaptan include bacteria of genus Porphyromonas such as Porphyromonas gingivalis, etc., bacteria of genus Fusobacterium such as Fusobacterium nucleatum, etc.", "Thus, it can be presumed that prevention and treatment of the above-mentioned intraoral diseases can be effectively attained by inhibiting propagation of these bacteria, and the ordinary antibacterial active substances have a possibility to kill the bacteria, thereby effectively attaining prevention and treatment of bad breath, etc.", "However, the ordinary antibacterial active substances can kill even intraoral indigenous bacteria free from pathogenicity, and thus are not recommendable from the viewpoint of preventing fixation of harmful exogenetic bacteria.", "Thus, it has been presumed useful to find substances having a bactericidal effect or a growth inhibiting effect selectively on the methylmercaptan-producing organisms, which have been presumed to cause the above-mentioned intraoral diseases." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 is a diagram showing results of antibacterial effect tests of trifluoromethionine as an effective component of the present oral composition on the strain Porphyromonas gingivalis W83.FIG.", "2 is a diagram showing results of antibacterial effect tests of trifluoromethionine as an effective component of the present oral composition on the strain Porphyromonas gingivalis ATCC33277.FIG.", "3 is a diagram showing results of antibacterial effect tests of trifluoromethionine as an effective component of the present oral composition on the strain Porphyromonas gingivalis M1217.FIG.", "4 is a diagram showing results of antibacterial effect tests of trifluoromethionine as an effective component of the present oral composition on the strain Fusobacterium nucleatum ATCC10953.FIG.", "5 is a diagram showing results of antibacterial effect tests of trifluoromethionine as an effective component of the present oral composition on the strain Actinobacillus actinomycetemcomitans Y4.FIG.", "6 is a diagram showing results of antibacterial effect tests of trifluoromethionine as an effective component of the present oral composition on the strain Escherichia coil BL21.detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to a composition for preventing and/or treating intraoral diseases such as bad breath, alveolar pyorrhea, periodontal disease, etc., and more particularly to a composition comprising trifluoromethionine as an effective component for preventing and/or treating intraoral diseases such as bad breath, alveolar pyorrhea, periodontal disease, etc.", "BACKGROUND ART Recently, there has been a growing interest in prevention and treatment of intraoral diseases such as bad breath, alveolar pyorrhea, periodontal disease, etc.", "Most important causative substances of bad breath are so-called volatile sulfides such as hydrogen sulfide, methylmercaptan and dimethyl sulfide, among which methylmercaptan is known to be produced by many causative organisms of periodontisis and thus has been watched with interest as an indicator of periodontisis and also as one of pathogenic factors of periodontisis.", "The methylmercaptan is produced from methionine by L-methionine-α-deamino-γ-mercaptomethane-lyase(METase) as an enzyme contained in bacteria.", "Among causative organisms of periodontal disease, well known organisms capable of efficiently producing methylmercaptan include bacteria of genus Porphyromonas such as Porphyromonas gingivalis, etc., bacteria of genus Fusobacterium such as Fusobacterium nucleatum, etc.", "Thus, it can be presumed that prevention and treatment of the above-mentioned intraoral diseases can be effectively attained by inhibiting propagation of these bacteria, and the ordinary antibacterial active substances have a possibility to kill the bacteria, thereby effectively attaining prevention and treatment of bad breath, etc.", "However, the ordinary antibacterial active substances can kill even intraoral indigenous bacteria free from pathogenicity, and thus are not recommendable from the viewpoint of preventing fixation of harmful exogenetic bacteria.", "Thus, it has been presumed useful to find substances having a bactericidal effect or a growth inhibiting effect selectively on the methylmercaptan-producing organisms, which have been presumed to cause the above-mentioned intraoral diseases.", "DISCLOSURE OF THE INVENTION An object of the present invention is to provide an oral composition comprising a substance having a bactericidal effect or growth-inhibiting effect selectively on the above-mentioned causative organisms of intraoral diseases as an effective component.", "In preparing METase-deficient strains of the above-mentioned intraoral organisms, the present inventors have found that the METase-deficient strains have no ability of producing methylmercaptan from methionine, showing that only METase takes part in the synthesis of methylmercaptan.", "METase is possessed by substantially all the organisms, but not by mammals including human beings.", "Thus, it can be presumed that a preventive and treating preparation of high safety can be provided for intraoral diseases by using a substance having a growth-inhibiting effect on the causative organisms of intraoral diseases, aiming at the METase as a target.", "As a result of investigations of growth-inhibiting effects on methylmercaptan-producing bacteria, using trifluoromethionine as a methionine derivative from the above-mentioned viewpoint, the present inventors have found that trifluoromethionine has a remarkable growth-inhibiting effect.", "That is, the present invention provides an oral composition, which comprises trifluoromethionine as an effective component.", "According to a preferable mode of the present invention, the present oral composition is used in prevention and/or treatment of bad breath, alveolar pyorrhea, and/or periodontal disease.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a diagram showing results of antibacterial effect tests of trifluoromethionine as an effective component of the present oral composition on the strain Porphyromonas gingivalis W83.FIG.", "2 is a diagram showing results of antibacterial effect tests of trifluoromethionine as an effective component of the present oral composition on the strain Porphyromonas gingivalis ATCC33277.FIG.", "3 is a diagram showing results of antibacterial effect tests of trifluoromethionine as an effective component of the present oral composition on the strain Porphyromonas gingivalis M1217.FIG.", "4 is a diagram showing results of antibacterial effect tests of trifluoromethionine as an effective component of the present oral composition on the strain Fusobacterium nucleatum ATCC10953.FIG.", "5 is a diagram showing results of antibacterial effect tests of trifluoromethionine as an effective component of the present oral composition on the strain Actinobacillus actinomycetemcomitans Y4.FIG.", "6 is a diagram showing results of antibacterial effect tests of trifluoromethionine as an effective component of the present oral composition on the strain Escherichia coil BL21.BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below: The present oral composition comprises trifluoromethionine as an effective component.", "Trifluoromethionine per se is a well known compound and can be prepared according to a process, for example, as disclosed in H. Duewel, E. Daub, V. Robinson, and J. F. Honek: Biochemistry, 36:3404-3416(1997).", "The form of the present oral composition is not particularly limited, and any form in common use for oral application is acceptable.", "That is, the forms of the present oral composition include, for example, troche, tooth powder, tooth paste, tooth washes (dental linses), mouth washes, gargles, cream, gel, pasta, spray, foam, etc.", "and further include edible forms such as candy, chewing gum, etc.", "To make the present oral composition into the above-mentioned forms, the present oral composition can contain carriers and additives in common use for oral compositions besides the above-mentioned effective component.", "Carriers or additives for use in the present oral composition include, for example, an abrasive, a demulcent, a moistening agent, a thickening agent, a surfactant, a perfume, a sweetening agent, an antiseptic, a preservative, a coloring agent, a pH controlling agent, and other effective compounds, and also a solvent, etc.", "The abrasive includes, for example, silica-bases abrasives such as precipitated silica, silica gel, aluminosilicate, zirconosilicate, etc., calcium secondary phosphate dihydrate and anhydrous calcium secondary phosphate, calcium hydrogen phosphate, insoluble sodium metaphosphate, potassium metaphosphate, calcium pyrophosphate, magnesium tertiary phosphate, calcium carbonate, aluminum hydroxide, alumina, magnesium carbonate, zeolite, silicic acid anhydride, silicic acid hydrate, aluminum silicate, zirconium silicate, bentonite, zeolite, aluminum oxide, aluminum hydroxide, synthetic resin-based abrasives, and their mixtures.", "The demulcent includes, for example, sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, alginates, carragheenan, gum arabic, polyvinyl alcohol, xanthane gum, gum tragacanth, starch, sodium polyacrylate, Carbopol, gum guar, gum arabic, gelatin, etc.", "The moistening agent includes, for example, polyethylene glycol, propylene glycol, sorbitol, glycerin, maltitol, xylitol, etc.", "The thickening agent includes, for example, glycerin, sorbitol, propylene glycol, polyethylene glycol, xylitol, maltitol, lactitol etc.", "The surfactant is used as a forming agent or a stabilizer for lipophilic substances.", "The surfactant includes, for example, anionic, nonionic, cationic and amphoteric surfactants, and specifically includes sodium alkylsulfate, alkylphosphoric acid ester, sodium alkylbenzenesulfonate, sodium N-acylsarcosinate, N-acylglutamates, polyoxyethylene hardened castor oil, polyoxyethylene-polypropylene block copolymer (Pluronic type surfactant), sucrose fatty acid esters, alkylglycosides, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, alkyldimethylamine oxide, alkylbetaine, polyoxyethylene hardened castor oil, alkylolamide polyglycerin fatty acid esters, etc.", "The perfume includes, for example, natural perfumes such as spearmint oil, peppermint oil, wintergreen oil, sassafras oil, clove oil, sage oil, eucalyptus oil, majorana oil, cinnamon oil, thyme oil, mentha oil, orange oil, wintergreen oil, lemon oil, orange oil, etc, and synthetic perfumes such as 1-menthol, anethole, carvone, eugenol, limonene, thymol, methyl salicylate, etc.", "The sweetening agent includes, for example, saccharin, saccharin sodium, dextrose, xylitol, stevioside, neohesperidyldihydrochalcone, perillartine, p-methoxycinnamic aldehyde, glycyrrhizinates, aspartame (aspartylphenylalanine methyl ester), Stevia extract etc.", "The antiseptic includes, for example, benzoic acid, sodium benzoate, parahydroxybenzoic acid ester, salicylic acid, sodium salicylate, sorbic acid, isopropylmethylphenol, etc.", "The preservative includes, for example, methylparaben, propylparaben, benzoate, sodium benzoate, paraoxybenzoic acid ester, titanium dioxide, etc.", "The bactericide includes, for example, chlorohexidines, quaternary ammonium salts, triclosan, alkyldiaminoethylglycin hydrochloride, etc.", "Water soluble fluoride includes, for example, sodium fluoride, sodium monofluorophosphate, etc.", "The coloring agent includes, for example, Blue No.", "1, Yellow No.", "4, titanium dioxide, etc.", "The pH controlling agent includes, for example, citric acid and its salts, phosphoric acid and its salts, malic acid and its salts, gluconic acid and its salts, maleic acid and its salts, aspartic acid and its salts, succinic acid and its salts, glucuronic acid and its salts, fumaric acid and its salts, glutamic acid and its salts, adipic acid and its salts, hydrochloric acid, alkali metal hydroxides, etc.", "Other effective components for use in the present invention include, for example, so far well known antibacterial agents, bad breath-preventing agents, etc., and specifically chlorophyll compounds, sodium chloride, vitamin C, vitamin E, nicotinic acid ester, allantoin chlorohydroxyaluminum, azulene, lysozyme chloride, hinokitiol, glycyrrhizic acid, dipotassium glycyrrhizinate, protease, fluorides such as sodium fluoride, potassium fluoride, ammonium fluoride, stannous fluoride, sodium monofluorophosphate, etc., water-soluble phosphoric acid compounds such as potassium salt, sodium salt, etc.", "of orthophosphoric acid, allantoin chlorohydroxyaluminum, hinokitiol, ascorbic acid, lysozyme chloride, glycyrrhizic acid and its salts, sodium chloride, tranexamic acid, ε-aminocaproic acid, d1-tocopherol acetate, azulene, glycyrrhetic acid, sodium copper chlorophyllin, copper gluconate, aluminum lactate, strontium chloride, potassium nitrate, berberine, hydroxamic acid and its derivatives, sodium tripolyphosphate, zeolite, dextranase, mutanase, amylase, methoxyethylene, maleic anhydride copolymer, polyvinylpyrrolidone, epidihydrocholesterin, dihydrocholesterol, benzethonium chloride, trichlorocarbanilide, zinc citrate, and crude drug extracts such as extracts of Japanese angelica roots, phellodendron barks, clove, rosemary, scutellaria roots, safflower etc.", "It is preferable in view of the gist of the present invention to use the above-mentioned other effective components in such an amount as not to kill the intraoral indigenous bacteria and as admitted to be safe even in ordinary common use.", "The solvent includes, for example, water, ethanol, etc.", "The present oral composition can be prepared by mixing trifluoromethionine as an effective component or a trifluoromethionine-producing compound with the above-mentioned carriers and additives by a well known method.", "Kinds and mixing proportions of the above-mentioned carriers and additives to be contained in the present oral composition are not particularly limited, but can be selected by those skilled in the art as desired in view of the individual forms of the above-mentioned oral composition.", "For example, in the case of a liquid composition such as tooth wash, mouth wash, etc., approximately 0.01-10% of a surfactant, 1-30% of a moistening agent and 50-95% of a solvent can be contained.", "In the case of a paste composition such as tooth paste, cream, etc., approximately 10-75% of an abrasive, 0.5-10% of a demulcent, and 10-85% of a moistening agent and water can be contained.", "A perfume, a sweetening agent, etc.", "can be contained usually in a range of approximately 0.01-5%.", "A pH of the present oral composition is preferably approximately 4-11, particularly 5-10.Content of trifluoromethionine as an effective component in the present oral composition is not particularly limited, but usually approximately 0.01-1% by weight, preferably 0.05-0.5% by weight, more preferably 0.1-0.2% by weight, on the basis of the composition.", "It is presumed that trifluoromethionine as an effective component of the present invention is converted to trifluoromethylmercaptan by METase of intraoral bacteria and propagation of intraoral bacteria is controlled by the trifluoromethylmercaptan.", "Trifluoromethylmercaptan is in a gaseous state at the ordinary temperatures, and thus is not retained in the body, assuring a high safety.", "It should be understood that the present invention is not restricted by any specific theory.", "EXAMPLES The present invention will be described in detail below, referring to Examples, which should be understood to be not restrictive of the present invention.", "Example 1 Antibacterial activity of trifluoromethionine on causative organism of periodontal disease Preparation Example Trifluoromethionine (TFM) prepared by Wako Pure Chemicals Industries, Ltd. according to a method disclosed in H. Duewel, E. Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404-3416(1997) was used.", "In all of the following Examples, the same TFM was used.", "Antibacterial Test 3 ml of GAM broth (Nissui Medical) containing 5 μg/ml of hemin and 1 μg/ml of menadione was separately incubated with one platinum loop each of subcultures of strains Porphyromonas gingivalis W83, ATCC33277 and M1217, and strain Fusobacterium nucleatum ATCC10953, followed by incubation at 37° C. under anaerobic conditions (10% CO2, 10% H2, 80% N2) overnight.", "The same fresh medium as above was inoculated with the resulting culture in such an amount that an absorbance at 550 nm of the inoculated medium could be about 0.15, followed by incubation under the same condition as above.", "Furthermore, the above-mentioned strains were incubated in the above-mentioned medium further containing 0.1 mM or 1.0 mM trifluoromethionine.", "Absorbance at 550 nm of the individual cultures was measured with time to evaluate propagation of bacteria through the turbidity of the medium.", "Results are shown in FIG.", "1 to 4 (A to D), where the results of using the trifluoromethionine-free medium are shown by □ curve, and those of using media containing 0.1 mM and 1.0 mM trifluoromethionine, respectively, are shown by ▪ and ● curves.", "Strain, Actinobacillus actinomycetemcomitans Y4, an intraoral bacteria, was incubated in a THY broth (containing 30 g of trypticase soy broth [BBL Microbiology Systems, Cockeysville, Md.]", "and 10 g of yeast extract [Difco] per liter) in the same manner as above except that the incubation was conducted in the presence of 5% CO2.Likewise, the strain was incubated in the same medium further containing 0.1 mM or 1.0 mM trifluoromethionine to evaluate propagation of the bacteria.", "Furthermore, strain Escherichia coli BL 21 was incubated in 2×TY broth (containing 16 g of bactotrypton (Difco), 10 g of yeast extract (Difco) and 5 g of sodium chloride per liter) in the same manner and also the strain was likewise incubated in the same medium further containing 0.1 mM or 1.0 mM trifluoromethionine to evaluate propagation of the bacteria.", "The results are shown in FIGS.", "5 and 6 (E and F), respectively, where the results of using the trifluoromethionine-free medium are shown by curve □, and the results of using the media containing 0.1 mM and 1.0 mM trifluoromethionine, respectively, are shown by curves ▪ and ●.", "As is evident from the results shown in FIGS.", "1 to 6, trifluoromethionine failed to show a significant antibacterial effect on enterobacteria Escherichia coli, but showed remarkable antibacterial effects on intraoral bacteria Porphyromonas gingivalis, Fusobacterium nucleatum and Actinobacllus actinomycetemcomitans.", "Thus, trifluoromethionine as an effective component of the present oral composition can specifically inhibit propagation of intraoral bacteria including methylmercaptan-producing ones, and are effective for prevention and treatment of intraoral diseases such as bad breath, periodontal disease, etc.", "and furthermore it seems that trifluoromethionine is incapable of killing intraoral nonpathogenic indigenous bacteria.", "Example 2 Antibacterial activity of trifluoromethionine on causative bacteria of periodontal disease (determination of minimum growth-inhibiting concentration) 3 ml of GAM broth (Nissui Medical) containing 5 μg/ml of hemin and 1 μg/ml of menadione was separately inoculated with one platinum loop each of subcultures of causative bacteria of periodontal disease, strains Porphyromonas gingivalis W83, W50, ATCC49417 and ATCC33277, strain Actinobacillus actinomycetemcomitans Y4, and strains Fusobacterium nucleatum ATCC10953 and ATCC25586, followed by incubation at 37° C. under anaerobic conditions (10% CO2, 10% H2 and 80% N2) overnight.", "Then, the resulting cultures were diluted to 10−8, 10−6, 10−4 and 10−3 to make inoculating bacterial solutions, respectively.", "Agar media containing 400, 200, 100, 50, 25, 10 and 5 μg/ml of TFM, respectively, were prepared and streaked, about 2 cm long, with a platinum loop each of the above-prepared inoculating bacterial solutions, followed by incubation under the same anaerobic conditions as above at 37° C. for 48-72 hours to evaluate growth of the bacteria.", "Minimum concentration at which the growth was clearly inhibited by visual observation was made a minimum growth-inhibiting concentration.", "Streptococcus sobrinus MT8246, Streptococcus sanuis ST160, Streptococcus mutans Xc and Streptococcus salivarius HHT were likewise incubated in Brain Heart Infusion (Difco) in the same manner as above except that the incubation was conducted in the presence of 5% CO2 to evaluate the growth.", "Minimum growth-inhibiting concentrations of TFM determined above for the individual bacteria are summarized in the following Table.", "TABLE 1 Minimum growth- inhibiting Strain concentration P. gingivalis W83 5 μg/ml W50 5 μg/ml ATCC 49417 5 μg/ml ATCC 33277 5 μg/ml A. actinomycetemcomitans Y4 10 μg/ml F. nucleatuium ATCC 10953 25 μg/ml ATCC 25586 25 μg/ml S. sobrinus MT8246 >400 μg/ml S. sanguis ST160 >400 μg/ml S. mutans Xc >400 μg/ml S. salivarius HHT >400 μg/ml It can be seen from the foregoing results that TFM shows selective antibacterial activity on the causative bacteria of periodontal disease.", "Example 3 Determination of minimum growth-inhibiting concentration of trifluoromethionine on standard strains: Minimum growth-inhibiting concentrations of trifluoromethionine and other antibacterial drugs on standard strains were determined by an agar plate dilution method according to the standard method of Japanese Society of Chemotherapy.", "Preparation of Drug Plates Drug solutions at a concentration 10 times as high as the maximum applicable concentration were prepared as original solutions of test drugs given in Table 2 (as to solvents used for this purpose, reference should be made also to Table 2).", "Serial two-fold dilution series of the original solutions were prepared at desired stages, and 1 ml each of the diluted drug solutions was added to 9 ml of media given in Table 3 to prepare drug plates.", "Concentration range of the individual drugs used are as follows: trifluoromethionine: 400-0.025 μg/ml (15 stages), tetracycline: 100-0.025 μg/ml (13 stages), and clarithromycin: 100-0.025 μg/ml (13 stages).", "Preparation of Inoculating Bacterial Solution, Incubation of Bacteria and Determination of Growth 1) Bacterial solutions stocked at −80° C. were inoculated on agar media (as to media used, reference should be made to Table 3), and then incubated at 37° C. overnight.", "When desired, subculturing was conducted once more on the same agar media (by overnight incubation at 37° C.).", "2) Colonies growing on the agar media were sampled by a platinum loop, suspended in liquid media (reference should be made to Table 3) and statically incubated at 37° C. for 18 hours (preculturing).", "3) Preculturing was continued up to McFarland 0.5 (McFarland 0.5=about 108 CFU/ml), and then the culture broths were diluted with Muellar Hinton Broth to make 106 CFU/ml.", "5 μl each of the diluted solutions were inoculated on the above-mentioned drug plates by an inoculator Microplanter.", "“McFarland” is a turbidity indicator in common use in the microbiological field, and an E. coli ATCC 25922 suspension at the same turbidity as McFarland 0.5 shows 1-2×108 CFU/ml.", "Thus, bacterial solutions at about 108 CFU/ml can be easily prepared by use of McFarland 0.5.Bacterial solutions at such a turbidity are commercially available, but can be easily prepared.", "McFarland 0.5 can be obtained by adding 0.5 ml of an aqueous 0.048 mol/l barium chloride solution to 99.5 ml of 1% v/v (0.18 mol/l) sulfuric acid.", "4) The individual bacteria were incubated at 37° C. for 18-20 hours and then the growth was determined in the same manner as in Example 2 to obtain minimum growth-inhibiting concentrations.", "Minimum growth-inhibiting concentrations (MIC) determined above are shown in Table 4.TABLE 2 Solvent for Solvent for Drug original solution dilution Trifluoromethionine Distilled water Distilled water Tetracycline Distilled water Distilled water Clarithromycin Methanol 0.1M, pH6.5 phosphate buffer TABLE 3 Agar medium Liquid medium Medium for for for determination Strain used preculturing preculturing of MIC Staphylococcus HIA MHB MHA aureus Staphylococcus HIA MHB MHA epidermidis Streptococcus sheep blood MHB + MHA + 5% Defibrinated pneumoniae agar medium 5% LHB horse blood Streptococcus sheep blood MHB + MHA + 5% Defibrinated pyogenes agar medium 5% LHB horse blood Streptococcus sheep blood MHB + MHA + 5% Defibrinated agalactiae agar medium 5% LHB horse blood Enterococcus HIA MHB MHA faecalis Enterococcus HIA MHB MHA faecium Micrococcus HIA MHB MHA luteus Bacillus HIA MHB MHA subtilis Moraxella sheep blood MHB + MHA + 5% Defibrinated (Branhamella) agar medium 5% LHB horse blood catarrhalis Pseudomonas HIA MHB MHA aeruginosa Escherichia HIA MHB MHA coli Haemophilus chocolate MHB + 5% horse influenzae agar 5% Fildes chocolate Serratia HIA MHB MHA marcescens Klebsiella HIA MHB MHA pneumoniae HIA: Heart Infusion Agar, MHB: Muellar Hinton Broth, LHB: Lysed Horse Blood, MHA: Muellar Hinton Agar TABLE 4 MIC (μg/ml) Species Strain TFM CAM TC Gram- Staphylococcus ATCC6538P >400 0.20 0.20 positive aureus ATCC29247 >400 50 0.78 cocci Staphylococcus ATCC12228 >400 0.20 50 epidermidis ATCC29886 >400 0.39 0.39 Enterococcus ATCC29247 >400 1.56 25 faecalis Enterococcus ATCC19434 >400 1.56 0.20 faecium Micrococcus ATCC9341 >400 0.05 0.78 luteus Streptococcus ATCC49619 >400 0.05 0.10 pneumoniae ATCC6301 >400 0.05 0.10 ATCC6302 >400 0.10 0.20 ATCC6303 >400 0.05 0.10 ATCC6306 >400 0.10 0.20 Streptococcus ATCC12385 >400 0.10 0.20 pyogenes ATCC12353 >400 0.10 0.20 ATCC12344 >400 0.05 0.20 Streptococcus ATCC13813 >400 0.05 0.20 agalactiae Gram- Bacillus ATCC6633 >400 0.10 0.20 positive subtilis bacillus Gram- Moraxella ATCC25238 100 0.10 0.20 negative (Branhamella) ATCC25240 25 0.20 0.20 cocci catarrhalis ATCC43617 100 0.20 0.20 Gram- Pseudomonas ATCC27853 >400 >100 12.5 negative aeruginosa bacilli Escherichia ATCC25922 >400 50 1.56 coli ATCC35218 >400 25 1.56 Serratia ATCC13880 >400 >100 100 marcescens Klebsiella ATCC700603 >400 >100 50 pneumoniae Haemophilus ATCC33391 50 6.25 0.78 influenzae ATCC33533 100 3.13 0.39 ATCC49247 50 6.25 25 ATCC49766 25 6.25 0.78 CAM: Clarithromycin, TC: Tetracycline It is evident from the foregoing results that TFM shows selective antibacterial activities on causative organism of periodontal disease.", "Example 4 Preparation Example 1.Tooth paste The following components (parts by weight) were mixed together according to the ordinary procedure to prepare a tooth paste: Calcium hydrogen phosphate 45.0 Silicic acid anhydride 2.0 Carboxymethylcellulose 1.2 sodium Glycerin 7.0 Sorbitol 15.0 Saccharin sodium 0.2 Methylparaben 0.2 Sodium lauryl sulfate 1.5 Allantoin 0.1 chlorohydroxyaluminum Perfume 0.9 Trifluoromethionine 0.1 Purified water balance Total 100.0 2.Tooth wash The following components (parts by weight) were mixed together according to the ordinary procedure to prepare a tooth wash: Silicic acid anhydride 7.5 Carboxymethylcellulose 0.8 sodium Glycerin 3.0 Sorbitol 30.0 Saccharin sodium 0.3 Methylparaben 0.1 Sucrose fatty acid ester 1.0 Sodium fluoride 0.2 Trifluoromethionine 0.1 Purified water balance Total 100.0 3.Mouth wash The following components (parts by weight) were mixed together according to the ordinary procedure to prepare a mouth wash: Xylitol 10.0 Ethanol 4.0 Polyoxyethylene hardened 0.2 castor oil Myristic acid 0.1 L-arginine 0.09 Perfume 0.15 Coloring agent trace amount Trifluoromethionine 0.1 Purified water balance Total 100.0 INDUSTRIAL UTILITY The present oral composition can effectively prevent and/or treat intraoral diseases such as bad breath, alveolar pyorrhea, periodontal disease, etc., and also the present oral composition can be daily used for prevention of bad breath, periodontal disease, etc.", "without killing intraoral non-pathogenic indigenous bacteria." ] ]	Patent_10451678
[ [ "Sulphur-containing amphiphilic agents for the transfer of biologically active molecules into cells", "The present invention relates to amphiphilic, cationic sulpho-substituted phosphatidyl ethanolamine analogs and their salts, which are capable of complexing biopolymers such as DNA, RNA, oligonucleotides, ribozymes, proteins and peptides and to infiltrate them into eukaryotic cells.", "Particularly suitable are compounds which are derived from 1,2-dioleoyl-3-sn-phosphatidyl ethanolamine (DOPE) and in which the phosphoric acid ester group of DOPE is replaced by an isosteric group CH2—SO—CH2 or CH2—S(O)2—CH2.", "Because of their property of forming aggregates with biologically active molecules, such as for example DNA or RNA, there compounds are particularly suitable for application in gene therapy, but also for diagnostic purposes." ], [ "1.Sulfur-containing amphiphiles of the general formula I: in which R1 denotes a straight or branched chain, saturated or unsaturated alkyl or acyl residue with 10-24 carbon atoms, a denotes a group O—R2 or CH2—O—R2, in which R2 has the meaning given for R1 and may be the same as R1 or different from R1, where with the presence of a steric center, the methine carbon atom connected to A can be present in R- or S-configuration or racemic, X denotes a group Y denotes a group N+R3R4R5Z− or a group NR3R4, where R3-R5, independently of each other, denote hydrogen, an alkyl group with 1-4 carbon atoms, a group —(CH2)i—OH, or a group —(CH2)i—NH2 with i=2-6 and Z− denotes a pharmaceutically acceptable anion, and where m and n, independently of each other, denote an integer 1-6.2.Compounds according to claim 1, wherein the residue R1 is an acyl residue from the group of lauroyl, myristoyl, palmitoyl, steroyl, oleoyl, linoloyl, or linoleoyl, m=2 and n=3.3.Compounds according to claim 1 or 2, wherein R1 is a lauroyl, myristoyl or oleoyl residue, the group A is a lauroyloxy, myristoyl or oleoyl, m=2 and n=3, X is a —SO— or —SO2— group, and Y is an —NH3+Z− group or an —NH2 group.", "4.Compounds according to one of claims 1-3, wherein the pharmaceutically acceptable anion is an ion from the group of halide, acetate, trifluoroacetate, mesylate, besylate, phosphate, tartrate, or citrate.", "5.Use of a reagent for the transfer of biologically active, anionic macromolecules into eukaryotic cells for pharmaceutical or diagnostic purposes, wherein aggregates are formed from the reagent, which contains at least one compound of claims 1-4, where in addition further lipoid compounds may be admixed in different proportions, with the biologically active, anionic macromolecules, and these aggregates are brought into contact with the cells in vivo or in vitro.", "6.Use of a reagent according to claim 5, wherein as the biologically active, anionic macromolecules, DNA, RNA, antisense DNA, antisense RNA, oligonucleotides, ribozymes, peptides or proteins are concerned.", "7.Use of a reagent according to claim 5 or 6, wherein the additionally admixed lipoid compounds belong to the phospholipids or steroid classes, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine being particularly suitable.", "8.Use of a reagent according to one of claims 5-7, wherein the reagent is present as a dispersion in aqueous media or as a solution in a solvent miscible with water, and in the case of an aqueous dispersion cryoprotective media from the group of lactose, trehalose, sucrose, glucose, fructose, galactose, maltose, mannitol or polyethylene glycol can be dissolved at the same time.", "9.Use of a reagent according to one of claims 5-8, wherein the biologically active anionic macromolecules may be present as complexes with polycationic molecules from the group of spermine, spermidine, protamine sulfate, histone H1, histone H2A, histone H2B, histone H3, histone H4, HMG1 or HMG17 protein.", "10.Use of a reagent according to one of claims 5-9, wherein the aggregates formed from the lipids and the anionic macromolecules are stored in lyophilized form, and are rehydrated in a suitable aqueous medium before use." ], [ "SPECIFICATION The present invention relates to cationic sulpho-substituted phosphatidyl ethanolamine analogs and their salts, which are capable of infiltrating biologically active macromolecules (particularly DNA and RNA) into eukaryotic cells.", "In the last ten years, the transfer of biopolymers, particularly of DNA, RNA and oligonucleotides, into eukaryotic cells has developed into a fundamental field of work in molecular biology and molecular medicine (P. A. Martin, S. M. Thomas; Human Gene Therapy 9 (1998) 87-114).", "Of particular interest here are above all gene therapeutic approaches, but also diagnostic medicine.", "Methods partially established heretofore for the insertion of DNA into eukaryotic cells depend on infiltrating the DNA sequence concerned with the aid of replication deficient, recombinant retro-, adeno-, or adeno-associated viruses.", "However, these have the disadvantage that until now they have been nearly exclusively restricted to ex vivo applications, since the viral proteins sometimes lead to intense immune reactions, and replication-competent viruses cannot always be excluded.", "Retroviruses have the further disadvantage that they integrate unspecifically but firmly into the host genome and thus can potentially result in malignant mutations.", "Because of these properties, it is understandable that these methods set extremely high safety requirements and can only be carried out with great financial expense.", "Biophysical methods, such as, for example, the bombardment of cells with DNA-laden gold particles (“biolistics”), or electroporation, are likewise only usable ex vivo for obvious reasons.", "The long known calcium phosphate coprecipitation and the DEAE-dextran methods appear little suitable because of their low efficiency .", "A further method, developed in recent years, for the infiltration of biologically active macromolecules into eukaryotic cells uses cationic polymers such as polylysine, polyethylenimine, or PAMAM-dendrimeres.", "Polyanions such as DNA, RNA or oligonucleotides form aggregates with these by electrostatic mutual action and in this form are taken up by the cells, presumably by endocytosis.", "However, poly-L-lysine has to be modified beforehand by chemical reaction with receptor ligands (e.g., transferrin, glycoproteins) (E. Wagner et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 87 (1990) 3410-3414) or endosmolytic peptides (E. Wagner, Proc.", "Natl.", "Acad.", "Sci.", "USA 89 (1992) 7934-7938) in order to have sufficient efficiency.", "Therefore, because of their polymeric nature, compounds of this type are very heterogeneously composed mixtures of substances and can be produced in a defined form only at great expense.", "A further method, which has assumed great importance in recent years, is based on the ground-breaking work of Feigner et al.", "Here liposomal or even micellar structures are generated from cationic, lipid amphiphiles, pure or mixed with neutral phospholipids such as dioleoylphosphatidyl ethanolamine (DOPE) (Feigner et al., WO 91/16024).", "Due to electrostatic mutual action with anionic biopolymers (such as DNA or RNA), these form aggregates which are thereafter efficiently taken up by eukaryotic cells.", "DNA can thus be transported into the cell nucleus and then leads to the expression of the corresponding protein.", "Although the mechanism for this has until now not been elucidated, there is however agreement that the aggregates reach the cell interior by endocytotic processes by means of endosomes.", "So that the internalized biopolymers can reach other cell compartments (e.g., cytoplasm or cell nucleus), they must emerge from the endosomes, since they would otherwise be enzymatically decomposed in the lysosomes.", "In most cases, this is attained by the admixture of the phospholipid DOPE, which because of its particular conical geometry is capable of inducing inverted hexagonal liquid crystalline phases well below (about 10° C.) a physiological temperature of 37° C. (J. O. Rädler et al., Science 281 (1998) 78-81).", "These have a high tendency to fuse with double-layer structures (e.g., biological membranes, endosomal membranes).", "In an article by Szoka et al., it is postulated that by this process of fusion, anionic, cellular lipids neutralize the charge of the infiltrated cationic lipids and thus release the complexed DNA into the cytosol (Szoka et al., Biochemistry 35 (1996) 5616-5623).", "Since the first description by Felgner, numerous cationic amphiphiles, mostly empirically found, have been synthesized for the transfer of anionic macromolecules, such as e.g.", "DNA (A. D. Miller, Angew.", "Chem.", "110 (1998) 1862-1880; L. Huang, X. Gao, Gene Therapy 2 (1995) 710-722).", "The cationic amphiphiles known heretofore have the disadvantage that they cannot induce inverted hexagonal phases.", "They therefore have hardly any fusogenic properties, and auxiliary lipids such as, e.g., DOPE, have to be admixed.", "A few lipids, such as, for example, DOGS (J. P. Behr et al., Proc.", "Natl.", "Acad.", "Sci.", "86 (1989) 6982-6986), are not capable of forming double layered structures and are present in micellar structures.", "Here also auxiliary lipids have to be admixed in order to attain a high gene transfer efficiency.", "Most reagents described heretofore are therefore multi-component mixtures, and the auxiliary lipids used, e.g., DOPE, are sensitive to hydrolysis and oxidation.", "The production of the biologically active formulations is therefore connected with considerable expense from the pharmaceutical standpoint, since the quality of each individual component has to be ensured.", "Many of the known cationic lipids, such as, e.g., DOTMA or DELRIE, have the disadvantage that because of ether bonds they are metabolized only with difficulty and therefore show distinct cell-toxic properties.", "The invention thus has as its object to make available new amphiphiles for the transfer of biopolymers (particularly of DNA and RNA) into eukaryotic cells, which are easily metabolized and show little cell toxicity, are capable of inducing hexagonal phases and thus possess strongly fusogenic properties, and at the same time are cationically charged, can be produced easily and cost-favorably in large quantities, and which require as small as possible an admixture of (helper) lipids.", "A transfer of biomolecules, particularly of DNA and RNA, into eukaryotic cells (transfection) which is surprisingly efficient in regard to the said requirements can be attained by the use according to the invention of sulfur-containing amphiphiles of the general formula I, wherein R1 denotes a straight or branched chain, saturated or unsaturated alkyl or acyl residue with 10-24 carbon atoms, A denotes a group O—R2 or CH2—O—R2, in which R2 has the meaning given for R1 and may be the same as R2 or different, where with the presence of a steric center, the methine carbon atom connected to A can be present in R- or else S-configuration or racemic, X denotes a group Y denotes a group N+R3R4R5Z− or a group NR3R4, where R3-R5, independently of each other, denote hydrogen, an alkyl group with 1-4 carbon atoms, a group —(CH2)i—OH, or a group —(CH2)i—NH2 with i=2-6, and Z− denotes a pharmaceutically acceptable anion, and where m and n, independently of each other, denote an integer 1-6.Preferred here are such compounds in which the residue R1 is an alkyl or acyl residue from the group of laur(o)yl, myrist(o)yl, palmit(o)yl, stear(o)yl, ole(o)yl, lin(o)yl, and linole(o)yl, and m=2 and n=3.Most particularly suitable are molecules in which R1 is a lauroyl, myristoyl or oleoyl residue, the group A is a lauroyloxy, myristoyloxy or oleoyloxy residue, m=2 and n=3, X is a —SO— or —SO2— group, and Y is an —NH3+Z− group or —NH2.In a preferred embodiment, the pharmaceutically acceptable anion is an ion from group halide, acetate, trifluoroacetate, mesylate, besylate, phosphate, tartrate, or citrate.", "The lipids according to the invention are suitable for the transfer of biologically active macromolecules, particularly DNA and RNA, into eukaryotic cells.", "The lipids can be present in aqueous (liposomal) dispersion or as a solution in water, and at the same time other, already known lipids, such as phospholipids or membrane-associated steroids, can be admixed.", "The DNA (RNA) can be pre-complexed with polycations, particularly with protamine sulfate.", "In contrast to the transfection agents known heretofore, the compounds according to the invention have a series of decisive advantages.", "Molecules of the described kind are used which are structurally very close to phosphatidyl ethanola-mine lipids.", "The structural unit of the phosphatidyl ethanolamine is replaced in the lipids according to the invention by the isosteric groups Because of the structural analogy, they potentially have a conical molecular geometry and therefore possess strong membrane fusogenic properties.", "However, the negative charge of the phosphoric acid ester unit is avoided, so that the lipids according to the invention are positively charged.", "They can therefore enter into electrostatic mutual action with negatively charged biomolecules and complex these.", "A further advantage is that the sulfo-analog lipids, like other sulfoxides or sulfones (e.g., DMSO), possess a high ability to pass into membranes.", "In contrast to many heretofore known cationic lipids, the lipids according to the invention furthermore have ester bonds and are therefore easily metabolized and hence potentially non-toxic.", "The production of the compounds takes place from inexpensive starting chemicals using methods familiar to one skilled in the art (Methods of Organic Chemistry (Houben-Weyl), 4th.", "edition, Thieme Verlag (Stuttgart) 1952) and protective group techniques (T. Greene, P. Wuts; Protective Groups in Organic Synthesis, 2d.", "ed., John Wiley & Sons (New York) 1991).", "The course of the reaction is shown in FIG.", "1.Alternatively to this, the lipids according to the invention can also be synthesized according to the course of the reaction described in FIG.", "2.The gene transfer properties of the lipids according to the invention (lipid 9 (Sulfectin A) and lipid 10 (Sulfectin B)) were tested on various cell lines and were compared with the reagents known heretofore (FIGS.", "3, 4 and 5).", "A plasmid coded for β-galactosidase was used as the reporter system.", "In the trials, the lipids were also mixed with, among other things, the neutral auxiliary lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).", "With the lipids 9 and 10 it is possible to transfect all investigated cell lines with markedly higher efficiency than with the known lipids Lipofectin®, LipofectAMINE™ (Life Technologies, USA), DOTAP (Roche Diagnostics, Germany), and DAC-30 (Eurogentec, Belgium).", "The gene transfer efficiency of the lipids is compared and shown in FIGS.", "3-5.In comparison with the commercial lipids, the lipids according to the invention make possible a surprisingly high gene expression.", "Among others a β-galactosidase expression up to 10 times higher was found.", "This is the more surprising in that in most cases (IMR90 and F98 cells) no admixture of an a helper lipid was necessary.", "With all the commercial lipids, on the other hand, a portion of DOPE was indispensable for an efficient gene transfer.", "The high gene transfer efficiency of the pure lipid is also advantageous for the production of the gene transfer reagent, since the formulation step of the cationic lipid and auxiliary lipid is omitted.", "Problems which are attributable to the oxidation sensitivity of DOPE cannot arise.", "Furthermore the possibility does not exist that the gene transfer properties are negatively affected by lyso-DOPE (hydrolysis product).", "Although the reaction paths and reagents used, shown in the following examples and Figures, represent preferred embodiments, the scope of the invention is not to be limited thereby.", "EXAMPLE 1 Preparation of 2-(2,2-diethyl-1,3-dioxolan-4-yl)ethanol (1) In a 250-ml round flask, 10.61 g (0.1 mol) of 1,2,4-butanetriol are dissolved in 70 ml anhydrous THF [tetrahydrofuran] and treated with 16 ml (0.15 mol) 3-pentanone and 580 mg toluenesulfonic acid.", "Thereupon 19 g of freshly dried molecular sieve are added and the mixture is well shaken for 48 hours at room temperature.", "After this time, 5 g of Ambersep 900 OHΓ ion exchanger are added, shaken for 20 min, then filtered.", "The filtrate is freed from solvent on a rotary evaporator and the residue is taken up in 170 ml ethyl acetate.", "The solution is washed three times, each time with 15 ml saturated NaHCO3 solution, and three times, each time with 15 ml saturated NaCI solution.", "The organic phase is dried over Na2SO4, filtered, and freed from solvent on the rotary evaporator.", "After drying in vacuum, 5.90 g (34 mmol) of a viscous, colorless oil are obtained.", "EXAMPLE 2 Preparation of 2-(2,2-diethyl-1,3-dioxolan-4-yl)ethyl-1-tosylate (2) In a 250-ml round flask, 5.90 g (34 mmol) of compound 1 are dissolved in 50 ml anhydrous dichloromethane and treated with 9.5 ml of triethylamine.", "Then 7.15 g (37.5 mmol) of tosyl chloride dissolved in 100 ml of anhydrous dichloromethane are dropped in via a dropping funnel during 20 min with stirring at room temperature.", "The mixture is stirred for 24 h at room temperature and then treated with 557 μl (5,1 mmol) of N,N-dimethylethylenediamine (in order to destroy excess tosyl chloride).", "The solution is transferred to a separating funnel and then washed with water and dilute sodium chloride.", "Finally it is again washed once with 15 ml saturated NaHCO3 solution and once with 15 ml saturated NaCl solution.", "The organic phase is dried over Na2SO4, filtered, and freed from solvent on the rotary evaporator.", "After drying in vacuum, 10.21 g (31.2 mmol) of a viscous, very light yellow oil are obtained.", "EXAMPLE 3 Preparation of S-acetyl-2-(2,2-diethyl-1,3-dioxolan-4-yl)ethyl-1-thiol (3) In a 250 ml round flask, 6.0 g (18.3 mmol) of compound 2 are dissolved in 150 ml acetone and treated with 4.18 g (36.6 mmol) finely powdered potassium thioacetate.", "The mixture is stirred under argon for 72 h at room temperature and then filtered from the solid.", "The filtrate is freed from solvent on the rotary evaporator and the residue is taken up in 170 ml ethyl acetate.", "The solution is washed in a separating funnel six times, each with 40 ml of water, and then once with 40 ml of saturated NaCl solution.", "The organic phase is dried over Na2SO4, filtered, and freed from solvent on the rotary evaporator.", "After drying in vacuum, 4.10 g (17.6 mmol) of a reddish brown oil are obtained.", "EXAMPLE 4 Preparation of 3-{[2-(2,2-diethyl-1,3-dioxolan-4-yl)ethyl]thio}-propyl-1-tert-butylcarbamate (4) In a 25 ml side connection flask, 1.243 g (5.35 mmol) of compound 3 are placed under argon and are treated with 5.35 ml of a 1 M solution of sodium methanethiolate in anhydrous methanol.", "The mixture is stirred at room temperature for 30 min and the solvent is then removed in vacuum with slight heating.", "The remaining red-brown paste is further dried for 15 min in vacuum; the flask is then flushed with argon and the mass is dissolved in 5 ml anhydrous methanol.", "Thereupon a solution of 1.062 g (4.46 mmol) 3-(boc-amino)propyl bromide are added in 5 ml anhydrous methanol, and the mixture is stirred under argon for 4 hours at room temperature.", "The solvent is removed on the rotary evaporator and the residue is taken up in 150 ml ethyl acetate.", "The solution is washed four times each with 20 ml water and twice each with 15 ml saturated NaCl solution.", "The organic phase is dried over Na2SO4, filtered, and freed from solvent on the rotary evaporator.", "After drying in vacuum, 2.04 g of a reddish brown crude product are obtained.", "Subsequent chromatographic purification on 50 g silica gel 60 with hexane/acetic ester as eluent gave 1.55 g (4.46 mmol) of a colorless oil.", "EXAMPLE 5 Preparation of 3-{[2-(2,2-dietbyl-1,3-dioxolan-4-yl)ethyl]-sulfinyl}-propyl-1-tert-butylcarbamate (5) In a 50 ml round flask with an attached gas discharge tube, 648 mg of compound 4 are placed and treated with 169 μl of a 35% hydrogen peroxide solution.", "The mixture is heated for 15 h at 38° C. with strong stirring, after which a homogeneous phase forms.", "After this time, the residue is taken up in 50 ml of ethyl acetate and stirred with a little anhydrous Na2SO4.It is then filtered off, thoroughly washed with ethyl acetate, and the solvent is removed on the rotary evaporator.", "The subsequent chromatographic purification on 50 g silica gel 60 with dichloromethane/methanol (13:1) as eluent gave 594 mg (1.63 mmol) of a colorless, viscous oil.", "EXAMPLE 6 Preparation of 3-{[2-(2,2-diethyl-1,3-dioxolan-4-yl)ethyl]-sulfonyl}-propyl-1-tert-butylcarbamate (6) In a 50 ml round flask with an attached gas discharge tube, 352 mg of compound 5 are placed and treated with a solution of 158 mg of potassium permanganate in 2 ml of saturated NaHCO3 solution.", "The mixture is heated to 35-38° C. with stirring for 15 h. The residue is thereafter intensively stirred with 70 ml of ethyl acetate and then filtered off from the insoluble manganese dioxide.", "The organic phase is dried over Na2SO4, filtered, and freed from solvent on the rotary evaporator.", "The subsequent chromatographic purification of the crude product on 50 g silica gel 60 with dichloromethane/methanol (20:1) as eluent gave 301 mg (0.793 mmol) of a colorless, very viscous oil.", "EXAMPLE 7 Preparation of 3-[(3,4-dioleoyloxybutyl)-sulfonyl]propyl-1-tert-butylcarbamate (7) In a 10 ml side connection flask, 174 mg (459 μmol) of compound 6 are dissolved under argon in 1.5 ml anhydrous methanol.", "Over a period of 8 hours, six portions each of 100 μl of a solution of 23.2 μl BF3 etherate in 2 ml anhydrous MeOH are added with stirring at room temperature.", "Thereafter 100 mg of a dry Ambersep 900OHΓ ion exchanger are added and stirred for 5 min.", "After filtering, the solvent is removed on the rotary evaporator and the residue is purified by chromatography on 20 ml silica gel 60 with dichloromethane/methanol (10:1) as eluent.", "81 mg (260 μmol) of a colorless, very viscous oil are obtained.", "This is dissolved in 3.5 ml of anhydrous dichloromethane in a 10 ml found flask and treated with 111 μl of triethylamine.", "With stirring and cooling, 210 mg of oleoyl chloride (purity (GC) 99%) is slowly added via a syringe.", "Further, a catalytic amount of DMAP is added, and the mixture is stirred for 18 h at room temperature in a darkened flask.", "The solution is then transferred with 40 ml dichloromethane into a separating funnel, and is washed twice, each time with 5 ml of water slightly acidified with HCl, and once with 5 ml of water.", "It is then washed once again with water made slightly alkaline (NaHCO3), and the organic phase after drying over Na2SO4 is freed from solvent on the rotary evaporator.", "Subsequent chromatographic purification on 20 g silica gel 60 with hexane/ethyl acetate (2:1) as eluent gave 105 mg (125 μmol) of a colorless, viscous oil.", "EXAMPLE 8 Preparation of 3-[(3,4-dioleoyloxybutyl)-sulfinyl]propyl-1-tert-butylcarbamate (8) In a 10 ml side connection flask, 200 mg (550 μmol) of compound 5 are dissolved under argon in 1.5 ml of anhydrous methanol.", "Then over a period of 8 hours, six portions each of 100 μl of a solution of 27.6 μl BF3 etherate in 2 ml anhydrous MeOH are added with stirring at room temperature.", "After this, 120 mg of a dry Ambersep 900 OHΓ ion exchanger are added and stirred for 5 min.", "After filtering off, the solvent is removed on the rotary evaporator and the residue (182 mg) is purified by chromatography on 20 g silica gel 60 with dichloromethane/methanol (9:1) as eluent.", "66 mg (223 μmol) of a colorless, very viscous oil are obtained.", "In a round flask, this is dissolved in 3.5 ml of anhydrous dichloromethane and treated with 125 μl of triethylamine.", "With stirring and cooling, 250 mg of oleoyl chloride (purity (GC) 99%) are slowly added via a syringe.", "A further catalytic amount of DMAP is added and the mixture is stirred for 18 h at room temperature in the darkened flask.", "With 40 ml of dichloromethane, the solution is then transferred into a separating funnel and washed twice, each time with 5 ml of slightly HCl-acidified water, and once with 5 ml of water.", "Then after washing once again, with 5 ml slightly alkaline water (NaHCO3), the organic phase after drying over Na2SO4 is freed from solvent on the rotary evaporator.", "Subsequent chromatographic purification of the crude product on 20 g silica gel 60 with hexane/acetic ester (1:1) as eluent gave 64 mg (78 μmol) of a colorless, viscous oil.", "EXAMPLE 9 Preparation of 3-[(3,4-dioleoyloxybutyl)-sulfonyl]propyl-1-ammonium trifluoroacetate (9) (Sulfectin A) In a 10 ml side-connection flask, under argon, are placed 52 mg (62 μmol) of compound 7, dissolved in 600 μl of anhydrous dichloromethane.", "With cooling and stirring, 400 μl of trifluoroacetic acid are then added and the mixture is stirred for 30 min at room temperature.", "The solvent is then removed in high vacuum without supplying heat, and the residue is dried for 1 h in vacuum.", "The remaining lipid film is taken up twice with 1.5 ml anhydrous dichloromethane each time, and is freed from solvent in vacuum.", "In conclusion, this procedure is repeated once with 1 ml of methanol and the lipid film is finally dried for 3 h in high vacuum.", "52.4 mg (61.3 μmol) of a colorless lipid film are obtained.", "EXAMPLE 10 Preparation of 3-[(3,4-dioleoyloxybutyl)-sulfinyl]propyl-1-ammonium trifluoroacetate (10) (Sulfectin B) In a 10 ml side-connection flask, under argon, are placed 44 mg (54 μmol) of compound 8, dissolved in 600 μl of anhydrous dichloromethane.", "With cooling and stirring, 400 μl of trifluoroacetic acid are then added and the mixture is stirred for 30 min at room temperature.", "The solvent is then removed in high vacuum without supplying heat, and the residue is dried for 1 h in vacuum.", "The remaining lipid film is taken up twice with 1.5 ml anhydrous dichloromethane each time, and freed from solvent in vacuum.", "In conclusion, this procedure is repeated once with 1 ml methanol and the lipid film is finally dried for 3 h in high vacuum, and 45 mg (53.6 μmol) of a colorless lipid film are obtained.", "EXAMPLE 11 Liposomal Formulation of the Lipids According to the Invention for the Transfection Experiments Lipids 9 and 10 are dissolved in chloroform and dried to a lipid film in vacuum.", "If necessary, they are previously mixed with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in different mol percent ratios.", "The remaining solvent residues are removed in high vacuum.", "The lipid films are then rehydrated in sterile water, and the liposomes are generated by ultrasonic treatment.", "The total lipid concentration of the resulting dispersion is 1 mg/ml.", "EXAMPLE 12 Transfection of Adherent Cell Lines General: The cell lines used, HeLa (human cervical carcinoma), F98 (rat glioblastoma), IMR90 (human embryonic lung) are cultivated under standard conditions (according to ATCC or EACC data) at 37° C./5% CO2 (HeLa/MEM-medium, F98/DMEM medium, IMR90/DMEM medium).", "The commercial transfection reagents Lipofect-AMINE™, Lipofectin®, DOTAP and DAC-30 were used according to the manufacturer's data.", "EXAMPLE 13 Transfection with pUT 651 Plasmid pUT 651 plasmid (CAYLA, France) was used as the reporter gene; it codes for lacZ under control of a CMV promoter.", "On the day before transfection, 5,000-10,000 cells per well were seeded on a 96-well culture plate, so that for transfection they had a confluence of about 70%.", "The medium was replaced by 80 μl/well of fresh medium (10% FCS) shortly before the transfection.", "The lipoplexes are prepared on a separate 96-well plate, in that 2 μg of DNA in 40 μl serum-free medium is mixed with 2-20 μl of liposome dispersion in 40 μl of serum-free medium.", "After an incubation time of about 30 min at room temperature, 20 μl per well of the lipoplex dispersion was pipetted onto the cells.", "After a transfection time of 24 h, the medium is changed and the cells are incubated for a further 48 h. Thereafter the medium is removed and the cells are lysed with Triton X-100.The lysate is treated with chlorophenol red β-galactopyranoside (Roche Diagnostics, 1 mg/ml in HBSS) as a substrate for β-galactosidase, and the optical density at different intervals is determined with a microplate reader (Dynex) (measurement wavelength 540 nm, reference wavelength 630 nm)." ] ]	Patent_10451687
[ [ "Optical system having a holographic optical element", "An optical laser system wherein a holographic optical element (HOE) replaces a bulky feedback system comprising a large number of optical element.", "The feedback system is adjusted so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherence, and the optical properties of the optical elements are recorded into the HOE.", "When the feedback system is removed the HOE reproduces the properties of the optical elements of the feedback system.", "The laser system is compact in size, cheap to manufacture, has high mechanical stability, and is less fragile than ordinary feedback systems.", "The laser system may be used in environments, such as the printing industry, which normally do not permit an ordinary feedback system, e.g.", "due to mechanical vibrations or misalignment due to temperature variations.", "A number of centre frequencies may be multiplexed into the HOE.", "May be mass produced.", "Furthermore, a method of producing such an optical laser system." ], [ "1.An optical system for emission of an output light beam, wherein a holographic optical element reproduces the optical properties of a plurality of optical elements, the plurality of optical elements forming a feedback system being adapted to cooperate with a laser device to select a high temporal and/or spatial coherent state of the laser device.", "2.An optical system according to claim 1, wherein at least one of the plurality of optical elements is selected from the group consisting of: spatial filters, gratings, mirrors, Fabry Perot etalons, frequency filters.", "3.A method of producing an optical system for emission of an output beam, the method comprising the steps of: inserting a holographic recording material into an external cavity formed between a laser device and a feedback system, said feedback system comprising a plurality of optical elements, emitting, by means of the laser device, a first light beam, at least part of said first light beam illuminating at least part of the feedback system via said holographic recording material, adjusting the feedback system so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherence, recording a holographic optical element in the holographic recording material, developing the holographic optical element so that the holographic optical element is adapted to reproduce the optical properties of the plurality of optical element when said feedback system is removed, and removing the feedback system.", "4.A method according to claim 3, the method comprising the steps of, for each of the optical elements: adjusting the feedback system so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherency, recording a holographic optical element in the holographic recording material, repeating the adjusting and recording steps until the properties of eachof the plurality of optical elements has been recorded, and performing the development after the optical properties of all the optical elements have been recorded and removing the feedback system when the holographic optical element has been developed.", "5.A method according to claim 3 or 4, wherein at least one of the plurality of optical elements is selected from the group consisting of: spatial filters, gratings, mirrors, Fabry Perot etalons, frequency filters.", "6.A method according to any of claims 3-5, further comprising the step of positioning the holographic optical element in connection with a laser device, so that the holographic optical element and the laser device may cooperate to select a state having a high temporal and/or spatial coherency.", "7.A method according to any of claims 4-6, further comprising the step of multiplexing a plurality of centre frequencies into the holographic optical element.", "8.A method according to claim 7, the method further comprising the steps of, for each of the plurality of centre frequencies: adjusting the feedback system to emit a centre frequency feedback light beam so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherency, and so that a specific centre frequency is obtained, recording a holographic optical element in the holographic recording material, repeating the adjusting and recording steps until each of the plurality of centre frequencies has been recorded, and performing the development after all the centre frequencies have been recorded and removing the feedback system when the holographic optical element has been developed.", "9.A method according to any of claims 3-8, wherein the developing step is performed using a chemical or thermal fixing procedure.", "10.A method according to any of claims 3-9, wherein the laser system is a compact laser system.", "11.A compact laser system for emission of an output light beam, the system comprising: alaser device for emission of a first light beam, and a holographic optical element being illuminated by at least a part of the first light beam, thereby causing a feedback light beam to be emitted from the holographic optical element and being reinjected into the active gain medium of the laser device, whereby the laser device and the holographic optical element cooperate to select a high spatial and/or high temporal coherent state of the laser device, whereby the laser system is controlled to emit an output light beam having an improved spatial and/or temporal coherence.", "12.A laser system according to claim11, wherein the holographic optical element is adapted to reconstruct an original light beam from a feedback system.", "13.A laser system according to claim 12, wherein the feedback system comprises one or more optical elements selected from the group consisting of: spatial filters, gratings, lenses, mirrors, Fabry Perot etalons, frequency filters.", "14.A laser system according to any of claims 11-13, wherein the holographic optical element is adapted to, in coopreation with the laser device, select at least one centre frequency from the first light beam.", "15.A laser system according to any of claims 11-14, wherein the holographic optical element is adapted to, in cooperation with the laser device, select a plurality of centre frequencies, each centre frequency being multiplexed into the holographic optical element.", "16.A laser system according to any of claims 11-15, wherein the laser device comprises a laser array.", "17.A laser system according to any of claims 11-16, wherein the laser device comprises at least one laser selected from the group consisting of: broad area lasers, laser diode arrays, laser diode bars, stacked laser arrays.", "18.A method of generating an output light beam from a laser system comprising a laser device and a holographic optical element, the method comprising the steps of: emitting, by means of the laser device, a first light beam in such a way tha t at least part of the holographic optical element is illuminated by at least part of the first light beam, injecting, by means of the holographic optical element and in response to the first light beam, a feedback light beam into the laser device, and outputting, by means of the holographic optical element and in response to the first light beam, an output light beam from the laser system, said output light beam having an imptoved spatial and/or temporal coherence state.", "19.A method according to claim 18, wherein the feedback system comprises one or more optical elements selected from the groupt consisting of: spatial filters, gratings, lenses, mirrors, Fabry Perot etalons, frequency filters.", "21.A method according to any of claims 18-20, further comprising the steps of, by means of the holographic optical element in cooperation with the laser device, selecting at least one centre frequency from the first light beam.", "22.A method according to any of claim 18-21, further comprising the steps of, by menas of the holographic optical element in cooperation with the laser device, selecting at least one centre frequency from the first light beam.", "23.A method of producing a compact laser system for emission of an output light beam, the method comprising the steps of: inserting a holographic recording material into a laser cavity formed between a laser device and a feedback system, emitting, by means of te laser device, a first light beam, at least part of said first light beam illuminating at least part of the feedback system via said holographic recording material, adjusting the feedback system to emit a feedback light beam so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherency, recording a holographic optical element in the holographic recording material, developing the holographic optical element so that the holographic optical element is capable of reconstructing the feedback light beam from the feedback system when said feedback system is removed, and removing the feedback system.", "24.A method according to claim 23, further comprising the step of multiplexing a plurality of centre frequencies into the holographic optical element.", "25.A method according to claim 24, the method further comprising the steps of, for each of the plurality of centre frequencies: adjusting the feedback system to emit a centre frequency feedback light beam so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherency, and so that a specific centre frequency is obtained, recording a holographic optical element in the holographic recording material, repeating the adjusting and recording steps until each of the plurality of centre frequencies has been recorded, and performing the development after all the centre frequencies have been recorded and removing the feedback system when the holographic optical element has been developed.", "26.A method according to any of claims 23-25, wherein the developing step is performed using a chemical or thermal fixing procedure." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>From WO 98/56087, it is known to use phase conjugate feedback in a laser system in order to obtain a highly coherent, possibly single mode, output light beam.", "However, the laser system disclosed in WO 98/56087 requires a number of optical elements.", "Such optical elements are expensive, especially if one of the optical elements comprises an anisotropic ferroelectric crystal, such as a BaTiO 3 crystal.", "Furthermore, BaTiO 3 crystals have a phase transition near room temperature and consequently they are very fragile and therefore need to be handled with much care.", "In addition to this, using a large number of optical elements requires a precise alignment of the elements, and it also results in a bulky laser system which is sensitive to mechanical vibrations.", "The alignment may be difficult to obtain and especially to preserve outside of a laboratory, and, furthermore, the bulky laser system is not very convenient for the user.", "‘In Holographic optical head for compact disk applications’, Optical Engineering, Vol.", "28(6), pp 650-653, June 1989, is disclosed an optical head for a CD player based on the holographic optical element and laser/detector hybrid technology.", "The holographic optical elements disclosed in this document are adapted to replace a number of refractive elements, such as lenses, beamsplitters, and diffraction gratings, or optionally a simple mirror.", "The document further discloses a method of fabrication of computer-generated holographic optical elements.", "A number of references describe the use of a holographic optical element (HOE) for replacing one or more optical elements.", "However, none of these references disclose using a HOE for injecting the beam back into the laser in order to improve the spatial and/or temporal properties of the output beam.", "The HOE is rather used for compensating for ‘beam defects’, such as astigmatism, after the beam has been output from the optical system.", "WO 99/57579 discloses a method for designing and constructing miniature optical systems and devices employing light diffractive optical elements (DOEs) for modifying the size and shape of laser beams produced from commercial-grade laser diodes.", "The DOEs may be implemented as holographic optical elements (HOEs).", "The DOE compensates for ‘beam defects’, such as astigmatism, of a beam emitted from a laser system.", "The beam is not injected back into the laser.", "U.S. Pat.", "No.", "6,018,402 discloses the use of a holographic optical element (HOE) to reconstruct optical elements typically used to phaseencode an object beam emanating from a spatial light modulator (SLM).", "The HOE replaces the complicated phase mask and conventional four-F lens system arrangement typically used to phase-encode an amplitude-encoded object beam emanating from the SLM.", "Thus, the HOE is in this case used for converting and transforming laser light from one state to another.", "‘Recent studies of miniaturization of optical disk pickups in Japan’ by Hiroshi Nishihara, Proceedings of the SPIE—The International Society for Optical Engineering, 1990, USA, vol.", "1248, pages 88-95, XP001029989, describes methods for improving pickups for compact disk players.", "A holographic optical element may be used for improving the output power of the diode laser.", "Thus, the temporal and/or spatial properties of the beam are not affected by the HOE.", "It is, furthermore, known to produce holographic optical elements, e.g.", "for producing bright, sharp, three-dimensional images.", "In contrast to the applications of HOE and DOE mentioned above the present invention deals with the improvement of the spatial and temporal coherence of high power laser diodes when the HOE or DOE is used to feedback light into the active region of the high power laser diode." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide a laser system which is compact and cheap to manufacture, which is less fragile than known laser systems, and which is less sensitive to misalignment of the optical elements which may be caused, e.g., by mechanical vibrations, temperature changes, etc.", "It is a further object to provide methods of manufacturing a laser system having the properties described above.", "It is an even further object to provide an optical system having a simple optical component which provides a passive feedback to the laser system.", "It is an even further object to provide an optical system in which the output beam has been subject to losses which are substantially smaller than losses caused by known feedback systems.", "It is an even further object to provide a feedback system with a high mechanical stability.", "It is an even further object to provide an optical system which is capable of producing an output beam having better spatial and/or temporal properties than the output beam from known systems.", "It is a very important object of the invention to provide a diode laser system with high output power, in the range of 1 W to 1000 W, which can be focused to a small diffraction limited spot.", "According to a first aspect of the present invention there is provided an optical system for emission of an output light beam, wherein a holographic optical element reproduces the optical properties of a plurality of optical elements, the plurality of optical elements forming a feedback system being adapted to cooperate with a laser device to select a high temporal and/or spatial coherent state of the laser device.", "The output light beam is emitted from the optical system, i.e.", "it is available for other purposes.", "That is, the output light beam may be used as a source of electromagnetic radiation.", "Thus, the output light beam is an electromagnetic output beam, such as a light beam, an ultraviolet beam, a microwave beam, an X-ray beam, or any other suitable kind of electromagnetic beam.", "The optical properties of the optical elements may be any suitable kind of optical properties, such as refractive index, reflectivity, selection of, e.g., frequencies or spatial modes, frequency doubling, etc., depending on which kinds of optical elements are used.", "The plurality of optical elements which may be reproduced by the holographic optical element form a feedback system being adapted to cooperate with a laser device to select a high temporal and/or spatial coherent state of the laser device.", "The laser device, thus, being adapted to supply a first light beam to the optical system, and the laser device and the holographic optical element reproducing the optical properties of the optical system may then cooperate to select a high temporal and/or spatial coherent state of the laser device.", "The feedback system used to improve the coherence properties of laser systems may be an optical system reflecting at least a part of the first light beam emitted from the laser device back into the laser device.", "In cooperation, the feedback system and the laser device then force the laser device to emit laser radiation with a high temporal and/or spatial coherence.", "When a high temporal coherent state is selected, the output light beam of the system, thus, comprises radiation within a very narrow frequency range, and, preferably, the output light beam substantially comprises only a single frequency.", "When a high spatial coherent state is selected the output light beam may be focused to a small spot size of the order of a wavelength.", "This is an important property for a large number of applications.", "Preferably, at least one of the plurality of optical elements is selected from the group consisting of: spatial filters, gratings, mirrors, Fabry Perot etalons, frequency filters.", "It is a great advantage of the present invention that a HOE is used as a feedback system.", "Thus, a HOE is substantially less expensive than a large number of optical elements.", "Furthermore, the size of the entire optical system is substantially reduced, and the system is much more stable, e.g.", "with respect to vibrations, misalignments, etc.", "Thus, the HOE may be attached to the laser facet itself, thereby substantially improving the mechanical stability properties of the optical system.", "Also, the HOE provides a passive feedback system as opposed to the active feedback system provided by a feedback system comprising non-linear optical components.", "This is a great advantage because in comparison with the active feedback the passive feedback only uses low cost elements.", "Finally, a feedback system being represented by a HOE may introduce fewer losses than a feedback system comprising the actual optical components which the HOE represents.", "This is because it is possible to let the HOE reproduce other optical properties of the individual optical component without reproducing the loss characteristics of that component.", "In case the HOE represent a large number of optical components, this is a very important advantage since the entire system may introduce very heavy losses.", "A spatial filter may, e.g., be an aperture, a slit, a pinhole or any other suitable kind of spatial filter.", "The optical properties of a spatial filter which may be reproduced by the holographic optical element in this case preferably comprise selection of specific modes or frequencies.", "In case one of the optical elements is a grating, the optical properties to be reproduced by the holographic optical element preferably comprise a frequency selectivity.", "In case one of the optical elements is a mirror, the optical properties to be reproduced by the holographic optical element preferably comprise the reflective properties of the mirror, such as a frequency dependent reflectivity, i.e.", "the reflectivity of the mirror at a certain frequency, the tilt angle of the mirror, and/or any focusing properties of the mirror.", "The mirror may be a plane mirror, a parabolic mirror, a spherical mirror, a mirror with a spatial selectivity, or a mirror having any other suitable shape.", "A frequency filter may e.g.", "comprise a grating, such as a diffractive optical element, preferably in combination with a spatial filter, such as an aperture or a slit, or it may be a filter which transmits electromagnetic radiation within a specific frequency range and reflects or absorbs any other frequencies.", "It may comprise an interference filter, an absorbance filter, such as a semiconductor doped glass, an etalon, such as a Fabry Perot element, a prism etc.", "The frequency filter may be adapted to select a range of frequencies, preferably a narrow range, most preferably a single frequency.", "In case one of the optical elements is a frequency filter, the optical properties to be reproduced by the holographic optical element preferably comprise frequency selection, transmission/reflection/absorption properties, selection of modes, reflective properties, refractive index, transmission properties, reflectivity, interference properties (destructive and/or constructive), or any other suitable properties of the frequency filter.", "According to a second aspect the present invention further provides a method of producing an optical system for emission of an output light beam, the method comprising the steps of: inserting a holographic recording material into an external cavity formed between a laser device and a feedback system, said feedback system comprising a plurality of optical elements, emitting, by means of the laser device, a first light beam, at least part of said first light beam illuminating at least part of the feedback system via said holographic recording material, adjusting the feedback system so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherence, recording a holographic optical element in the holographic recording material, developing the holographic optical element so that the holographic optical element is adapted to reproduce the optical properties of the plurality of optical element when said feedback system is removed, and removing the feedback system.", "The holographic recording material may be any material with a photosensitive refractive index and/or absorption coefficient for example a dichromatic gelatine, a Silver Bromide (AgBr) solution, photo resist, and/or a photorefractive medium.", "The laser device may be a single laser, e.g.", "a gas laser, a semiconductor laser, a broad area laser, a superluminescent laser diode, a dye laser, a Nd-YAG laser, an argon ion laser, a titanium sapphire laser, an F-center laser, or any other suitable kind of laser.", "It may also be an array of lasers, said lasers being of any of the types mentioned above.", "The feedback system is defined above.", "Adjusting the feedback system may comprise adjusting one or more of the optical elements forming the feedback system in such a way that a state having a high temporal and/or spatial coherency is selected.", "The adjusting step may, furthermore, comprise alignment of the optical elements.", "Additionally or alternatively, the adjusting step may comprise adjusting one or more optical element(s) in such a way that, e.g., a certain frequency, a certain spatial mode, etc.", "is selected.", "This may, for example, be done in the following way.", "In a preferred embodiment of the invention, the feedback system comprises a reflector, the reflector being adapted to reflect at least a part of the first light beam emitted by the laser device back into the laser device.", "The free running laser emits a large number of spatial modes.", "By using spatial filtering for example in the Fourier plane, e.g.", "by means of one or more spatial filter(s), such as aperture(s), slit(s), pinhole(s), etc., the system is adjusted to emit laser light having a high temporal and/or spatial coherency.", "The feedback system may, furthermore, comprise a grating or an etalon so that the frequency of the first light beam may be tuned by tilting the grating or the etalon.", "It is, thus, possible to adjust the system to emit an output light beam having, e.g., a certain spatial mode, frequency, etc., depending on the optical elements being provided in the feedback system.", "When the feedback system has been adjusted so that the output light beam has the desired properties, a holographic optical element having these properties is recorded in the holographic recording material positioned between the laser device and the feedback system.", "When the holographic optical element is subsequently developed, it will thus be adapted to reproduce the optical properties of the elements in the feedback system.", "The feedback system may then be removed and the holographic optical element will act as the feedback system, i.e.", "the output beam will have the same desired properties which the feedback system was adjusted to provide.", "Of course, the recorded and developed holographic optical element itself may not afterwards be adjusted as the feedback system, thus, limiting the flexibility of the system.", "However, it is an advantage of the system according to the present invention that the system provided is substantially non-sensitive to misalignments due to vibrations, temperature variations, etc.", "It is a further advantage that a rather bulky, expensive, and fragile feedback system may be replaced by the compact, cheap, and reliable holographic optical element.", "These advantages makes the system very advantageous for commercial purposes.", "It is, thus, possible to use to system under conditions which are normally not suited for a feedback system comprising a large number of fragile optical components, e.g.", "in an environment introducing vibrations, temperature variations, a dirty environment, etc.", "It is possible to manufacture HOEs under ideal conditions and subsequently position the HOEs in optical systems under less ideal conditions.", "Thus, an ideal feedback system is provided even though the environment is not suited for such a feedback system.", "Furthermore, the price of the entire system will be sufficiently low to attract potential customers.", "The system may, thus, advantageously be used, e.g., in the printing industry, medical applications, or telecommunication.", "In a very preferred embodiment laser systems having a HOE replacing a feedback system may be mass produced by recording one HOE by the method described above, and subsequently reproduce this HOE.", "The reproduced HOEs may then be positioned in laser systems having similar properties.", "It should be noted that the HOE in each case should be positioned in the laser system in a position corresponding to the position in which the original HOE was recorded in order for the HOE to properly reproduce the feedback system.", "Such mass produced laser systems are very advantageous from a commercial point of view since they are very cheap to manufacture, and the price therefore will be acceptable for potential customers.", "The method may comprise the steps of, for each of the optical elements: adjusting the feedback system so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherency, recording a holographic optical element in the holographic recording material, repeating the adjusting and recording steps until the properties of each of the plurality of optical elements has been recorded, and performing the development after the optical properties of all the optical elements have been recorded and removing the feedback system when the holographic optical element has been developed.", "In this case, the adjusting and recording steps are performed for each optical element of the feedback system.", "Thus, each optical element is in turn adjusted to achieve a desired optical property of the optical element in question.", "This optical property is then recorded.", "In order to record the optical properties of all the optical elements into the same holographic optical element, the holographic optical recording material is not developed until all the optical properties have been recorded.", "At least one of the plurality of optical elements may be selected from the group consisting of: spatial filters, gratings, mirrors, Fabry Perot etalons, frequency filters.", "These optical elements and their optical properties have been described above.", "Preferably, the method further comprises the step of positioning the holographic optical element in connection with a laser device, so that the holographic optical element and the laser device may cooperate to select a state having a high temporal and/or spatial coherency.", "That is, the holographic optical element may preferably be used to replace the feedback system in order to provide a compact, cheap, and mechanically stable laser system as described above, and as will be further described below.", "The method may further comprise the step of multiplexing a plurality of centre frequencies into the holographic optical element.", "In an embodiment where the feedback system comprises a grating, this may be performed in the following way.", "The feedback system may be adjusted to select one centre frequency and a corresponding grating may be induced in the holographic optical element.", "The laser device may then be turned off and the grating be tilted to select a new centre frequency.", "When the laser device is turned on again, a new hologram with a new centre frequency may be written into the holographic optical recording material.", "By repeating this procedure for each centre frequency, a plurality of frequencies are written into the holographic recording material.", "When all the desired frequencies have been written into the holographic recording material, the holographic optical element is developed, so as to obtain a holographic optical element having all the desired centre frequencies multiplexed into it.", "Thus, the method may further comprise the steps of, for each of the plurality of centre frequencies: adjusting the feedback system to emit a centre frequency feedback light beam so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherency, and so that a specific centre frequency is obtained, recording a holographic optical element in the holographic recording material, repeating the adjusting and recording steps until each of the plurality of centre frequencies has been recorded, and performing the development after all the centre frequencies have been recorded and removing the feedback system when the holographic optical element has been developed.", "The developing step may be performed using a chemical or thermal fixing procedure.", "Alternatively, the holographic recording material may be of a kind which is ‘self-developing’.", "In this case the development is performed automatically and does not require an active act.", "This is known per se.", "The laser system may advantageously be a compact laser system.", "According to a third aspect the invention further provides a compact laser system for emission of an output light beam, the system comprising: a laser device for emission of a first light beam, and a holographic optical element being illuminated by at least a part of the first light beam, thereby causing a feedback light beam to be emitted from the holographic optical element and being reinjected into the active gain medium of the laser device, whereby the laser device and the holographic optical element cooperate to select a high spatial and/or high temporal coherent state of the laser device, whereby the laser system is controlled to emit an output light beam having an improved spatial and/or temporal coherence.", "As described above the laser device may be any suitable kind of laser device, such as a gas laser, a semiconductor laser, a superluminescent laser diode, a dye laser, a Nd-YAG laser, an argon ion laser, a titanium sapphire laser, an F-center laser, or any other suitable kind of laser.", "It may also be an array of lasers, said lasers being of any of the types mentioned above.", "The first light beam may be an electromagnetic beam, preferably a monochromatic electromagnetic beam.", "In case the laser device is a single laser, the first light may also be a coherent light beam.", "In case the laser device is an array of lasers or another laser device having a broad bandwidth gain medium, the first light beam will in most cases have a very low degree of coherence.", "The holographic optical element is illuminated by at least part of the first light beam.", "It may, of course, be illuminated by all of the first light beam.", "At least part of the holographic optical element may be illuminated, or all of the holographic optical element may be illuminated.", "The feedback light beam is emitted from the holographic optical element in response to the first light beam illuminating the holographic optical element.", "The feedback light beam may be an electromagnetic beam, such as an electromagnetic beam comprising frequencies within the visible frequency range, the ultraviolet frequency range, the infrared frequency range, the X-ray frequency range, or any other suitable frequency range.", "The feedback light beam may be e.g.", "a complete reflection of the first light beam.", "Alternatively, it may be a reflected part of the first light beam, such as a part defined by a specific frequency range, a specific polarisation, a specific spatial mode, etc.", "Alternatively, the feedback light beam may be a beam which is generated by the holographic optical element in response to the first light beam.", "The feedback light beam is reinjected into the active gain medium of the laser device.", "In this way the laser device and the holographic optical element cooperate to select a high spatial and/or temporal coherent state of the laser device.", "The output light beam from the system will then have a high spatial and/or temporal coherent state.", "In order to obtain a high power, the laser device is often an array of lasers as described above.", "As mentioned above, this will very often result in a first light beam having a low degree of coherence.", "Since the output light beam has a high spatial and/or temporal coherent state, it thus has an improved spatial and/or temporal coherence as compared to the first light beam being emitted from the laser device.", "Thereby, the compact laser system is adapted for improving the coherency of a high power laser beam.", "The holographic optical element may be adapted to reconstruct an original light beam from a feedback system.", "The feedback system may comprise a number of optical elements as described above, and it is preferably operated as described above.", "The holographic optical element may, thus, replace the bulky, expensive, and fragile optical elements of the feedback system.", "Since a holographic optical element is compact, cheap, and less fragile than most ordinary optical elements, the resulting laser system will also be compact, cheap, and less fragile than laser systems having an ordinary feedback system comprising a number of optical elements.", "Furthermore, the resulting laser system will not be subject to misalignments due to, e.g., vibrations or temperature variations to the same extend that an ordinary laser system is.", "The feedback system may comprise one or more optical elements selected from the group consisting of: spatial filters, gratings, lenses, mirrors, Fabry Perot etalons, frequency filters.", "Most of these optical elements as well as their optical properties have been described above.", "The original light beam from the feedback system which the holographic optical element is adapted to reconstruct, preferably comprises information relating to the optical properties of the optical elements of the feedback system.", "The holographic optical element is most preferably recorded in such a way that these optical properties may be reproduced by the holographic optical element.", "Thus, the output light beam from the compact laser system may be substantially identical to the output light beam from a more bulky laser system having an ordinary feedback system.", "In case one of the optical elements is a lens, the optical properties to be reproduced by the holographic optical element preferably comprise refractive index, reflectivity, including internal reflectivity, focal length, radius of curvature, or any other suitable optical properties of the lens.", "The lens may be an ordinary concave or convex lens, or it may be another kind of refractive optical element, such as a prism.", "The holographic optical element may be adapted to, in cooperation with the laser device, select at least one centre frequency from the first light beam.", "This corresponds to selecting a high temporal coherent state.", "However, the exact value of the centre frequency may also be selected in this embodiment.", "This may e.g.", "be obtained by recording the holographic optical element using a feedback system which may be tuned so as to select a specific frequency.", "The holographic optical element may be adapted to, in cooperation with the laser device, select a plurality of centre frequencies, each centre frequency being multiplexed into the holographic optical element.", "This may be obtained by consecutively tuning a system as described above to each of the plurality of centre frequencies, and recording and developing the holographic optical element in such a way that all the centre frequencies are multiplexed into the holographic optical element.", "This procedure will be further described below.", "The laser device may comprise a laser array, such as an array of diode lasers, gas lasers, semiconductor lasers, dye lasers, Nd-YAG lasers, argon ion lasers, or any other suitable kind of lasers.", "Alternatively, it may be a single laser as described above.", "The laser device may comprise at least one laser selected from the group consisting of: broad area lasers, laser diode arrays, laser diode bars, stacked laser arrays.", "Broad area lasers and laser diode arrays comprise a number of diode lasers arranged in a row.", "Laser diode bars also comprise a number of diode lasers arranged in a row.", "However, the lasers of a laser diode bar are spatially separated, so that the light sources may be considered as a number of discrete point sources.", "Stacked laser arrays comprise a number of laser diode bars being stacked, so as to form a two-dimensional array of diode lasers.", "The above-mentioned types of lasers all provide an output beam having a high power, but a low degree of coherence.", "For some applications, it may therefore be necessary to improve the coherency of the output beam.", "This may be done as previously described.", "According to a fourth aspect the invention further provides a method of generating an output light beam from a laser system, the laser system comprising a laser device and a holographic optical element, the method comprising the steps of: emitting, by means of the laser device, a first light beam in such a way that at least part of the holographic optical element is illuminated by at least part of the first light beam, injecting, by means of the holographic optical element and in response to the first light beam, a feedback light beam into the laser device, and outputting, by means of the holographic optical element and in response to the first light beam, an output light beam from the laser system, said output light beam having an improved spatial and/or temporal coherence state.", "The first light beam, the feedback light beam, as well as the output light beam may be electromagnetic beams as described above.", "All of, or at least part of, the holographic optical element may be illuminated by the first light beam, and it may be illuminated by all of, or at least part of, the first light beam.", "The feedback light beam may be a fully or a partial reflection of the first light beam, or it may be generated by the holographic optical element, as described above.", "The laser device and the holographic optical element cooperate to select a state having a high temporal and/or spatial coherence, so that the output light beam has an improved spatial and/or temporal coherence as compared to the first light beam which is initially emitted from the laser device.", "This has been described above.", "The holographic optical element may reconstruct an original light beam from a feedback system.", "This has already been described.", "The feedback system may comprise one or more optical elements selected from the group consisting of: spatial filters, gratings, lenses, mirrors, Fabry Perot etalons, frequency filters.", "These optical elements as well as their optical properties have been described above.", "The method may further comprise the step of, by means of the holographic optical element in cooperation with the laser device, selecting at least one centre frequency from the first light beam.", "As described above this corresponds to selecting a state having a high temporal coherence.", "However, a specific frequency is chosen in this case.", "The method may further comprise the step of, by means of the holographic optical element in cooperation with the laser device, selecting a plurality of centre frequencies, each centre frequency having previously been multiplexed into the holographic optical element.", "This has also been described above.", "According to a fifth aspect the invention further provides a method of producing a compact laser system for emission of an output light beam, the method comprising the steps of: inserting a holographic recording material into a laser cavity formed between a laser device and a feedback system, emitting, by means of the laser device, a first light beam, at least part of said first light beam illuminating at least part of the feedback system via said holographic recording material, adjusting the feedback system to emit a feedback light beam so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherency, recording a holographic optical element in the holographic recording material, developing the holographic optical element so that the holographic optical element is capable of reconstructing the feedback light beam from the feedback system when said feedback system is removed, and removing the feedback system.", "The laser device may be any suitable kind of laser device as described above.", "The feedback system preferably comprises a number of optical elements each having specific optical properties.", "The first light beam as well as the feedback light beam may be electromagnetic beams as described above.", "The adjusting step is preferably performed by adjusting each of the optical elements of the feedback system.", "This may comprise tilting gratings to the correct angle, e.g.", "in order to obtain a specific frequency, positioning spatial filters correctly, e.g.", "in order to obtain a specific spatial mode, aligning the optical elements, e.g.", "in order to optimise the throughput of the system, and/or it may comprise any other suitable kind of adjusting of the feedback system.", "The adjusting step results in that the laser device and the feedback system, by means of the feedback light beam, cooperate to select a state having a high temporal and/or spatial coherency.", "The adjusting may be performed using spatial filtering in the Fourier plan.", "The holographic recording material may comprise a dichromatic gelatine, a silver bromide (AgBr) solution, photo resist, and/or a photorefractive medium, such as a lithium niobate crystal.", "When the holographic optical recording material has been developed to form the holographic optical element, the holographic optical element is capable of reconstructing the feedback light beam from the feedback system because the optical properties of the optical elements of the feedback system have been recorded into the holographic optical element.", "When the feedback system is removed, the laser device and the holographic optical element may therefore be able to cooperate to select a state having a high temporal and/or spatial coherency.", "That is, the output light beam emitted from the laser system with the feedback system removed will be substantially identical to the output light beam emitted from the laser system with the feedback system present instead of the holographic optical element.", "Thus, as described above, the holographic optical element may replace the rather bulky, expensive, etc.", "feedback system, thereby providing a laser system which is compact, cheap, etc.", "The method may further comprise the step of multiplexing a plurality of centre frequencies into the holographic optical element.", "Thus, the method may further comprise the steps of, for each of the plurality of centre frequencies: adjusting the feedback system to emit a centre frequency feedback light beam so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherency, and so that a specific centre frequency is obtained, recording a holographic optical element in the holographic recording material, repeating the adjusting and recording steps until each of the plurality of centre frequencies has been recorded, and performing the development after all the centre frequencies have been recorded and removing the feedback system when the holographic optical element has been developed.", "This has already been described above.", "A laser system according to the present invention may be used for a number of various applications, such as frequency doubling, coupling of light into thin core or single mode laser fibres, material processing, the printing industry, biomedical application, and/or any other suitable applications in which a compact, low cost, and reliable laser system is useful.", "The first, second, third, fourth, and fifth aspects of the present invention may each be combined with one or more of the other aspects of the present invention." ], [ "FIELD OF THE INVENTION The present invention relates to an optical system, in particular a compact optical system comprising a holographic optical element and to the use of a compact optical system in a laser system so as to provide a compact laser system with good spatial and temporal coherence.", "BACKGROUND OF THE INVENTION From WO 98/56087, it is known to use phase conjugate feedback in a laser system in order to obtain a highly coherent, possibly single mode, output light beam.", "However, the laser system disclosed in WO 98/56087 requires a number of optical elements.", "Such optical elements are expensive, especially if one of the optical elements comprises an anisotropic ferroelectric crystal, such as a BaTiO3 crystal.", "Furthermore, BaTiO3 crystals have a phase transition near room temperature and consequently they are very fragile and therefore need to be handled with much care.", "In addition to this, using a large number of optical elements requires a precise alignment of the elements, and it also results in a bulky laser system which is sensitive to mechanical vibrations.", "The alignment may be difficult to obtain and especially to preserve outside of a laboratory, and, furthermore, the bulky laser system is not very convenient for the user.", "‘In Holographic optical head for compact disk applications’, Optical Engineering, Vol.", "28(6), pp 650-653, June 1989, is disclosed an optical head for a CD player based on the holographic optical element and laser/detector hybrid technology.", "The holographic optical elements disclosed in this document are adapted to replace a number of refractive elements, such as lenses, beamsplitters, and diffraction gratings, or optionally a simple mirror.", "The document further discloses a method of fabrication of computer-generated holographic optical elements.", "A number of references describe the use of a holographic optical element (HOE) for replacing one or more optical elements.", "However, none of these references disclose using a HOE for injecting the beam back into the laser in order to improve the spatial and/or temporal properties of the output beam.", "The HOE is rather used for compensating for ‘beam defects’, such as astigmatism, after the beam has been output from the optical system.", "WO 99/57579 discloses a method for designing and constructing miniature optical systems and devices employing light diffractive optical elements (DOEs) for modifying the size and shape of laser beams produced from commercial-grade laser diodes.", "The DOEs may be implemented as holographic optical elements (HOEs).", "The DOE compensates for ‘beam defects’, such as astigmatism, of a beam emitted from a laser system.", "The beam is not injected back into the laser.", "U.S. Pat.", "No.", "6,018,402 discloses the use of a holographic optical element (HOE) to reconstruct optical elements typically used to phaseencode an object beam emanating from a spatial light modulator (SLM).", "The HOE replaces the complicated phase mask and conventional four-F lens system arrangement typically used to phase-encode an amplitude-encoded object beam emanating from the SLM.", "Thus, the HOE is in this case used for converting and transforming laser light from one state to another.", "‘Recent studies of miniaturization of optical disk pickups in Japan’ by Hiroshi Nishihara, Proceedings of the SPIE—The International Society for Optical Engineering, 1990, USA, vol.", "1248, pages 88-95, XP001029989, describes methods for improving pickups for compact disk players.", "A holographic optical element may be used for improving the output power of the diode laser.", "Thus, the temporal and/or spatial properties of the beam are not affected by the HOE.", "It is, furthermore, known to produce holographic optical elements, e.g.", "for producing bright, sharp, three-dimensional images.", "In contrast to the applications of HOE and DOE mentioned above the present invention deals with the improvement of the spatial and temporal coherence of high power laser diodes when the HOE or DOE is used to feedback light into the active region of the high power laser diode.", "SUMMARY OF THE INVENTION It is an object of the present invention to provide a laser system which is compact and cheap to manufacture, which is less fragile than known laser systems, and which is less sensitive to misalignment of the optical elements which may be caused, e.g., by mechanical vibrations, temperature changes, etc.", "It is a further object to provide methods of manufacturing a laser system having the properties described above.", "It is an even further object to provide an optical system having a simple optical component which provides a passive feedback to the laser system.", "It is an even further object to provide an optical system in which the output beam has been subject to losses which are substantially smaller than losses caused by known feedback systems.", "It is an even further object to provide a feedback system with a high mechanical stability.", "It is an even further object to provide an optical system which is capable of producing an output beam having better spatial and/or temporal properties than the output beam from known systems.", "It is a very important object of the invention to provide a diode laser system with high output power, in the range of 1 W to 1000 W, which can be focused to a small diffraction limited spot.", "According to a first aspect of the present invention there is provided an optical system for emission of an output light beam, wherein a holographic optical element reproduces the optical properties of a plurality of optical elements, the plurality of optical elements forming a feedback system being adapted to cooperate with a laser device to select a high temporal and/or spatial coherent state of the laser device.", "The output light beam is emitted from the optical system, i.e.", "it is available for other purposes.", "That is, the output light beam may be used as a source of electromagnetic radiation.", "Thus, the output light beam is an electromagnetic output beam, such as a light beam, an ultraviolet beam, a microwave beam, an X-ray beam, or any other suitable kind of electromagnetic beam.", "The optical properties of the optical elements may be any suitable kind of optical properties, such as refractive index, reflectivity, selection of, e.g., frequencies or spatial modes, frequency doubling, etc., depending on which kinds of optical elements are used.", "The plurality of optical elements which may be reproduced by the holographic optical element form a feedback system being adapted to cooperate with a laser device to select a high temporal and/or spatial coherent state of the laser device.", "The laser device, thus, being adapted to supply a first light beam to the optical system, and the laser device and the holographic optical element reproducing the optical properties of the optical system may then cooperate to select a high temporal and/or spatial coherent state of the laser device.", "The feedback system used to improve the coherence properties of laser systems may be an optical system reflecting at least a part of the first light beam emitted from the laser device back into the laser device.", "In cooperation, the feedback system and the laser device then force the laser device to emit laser radiation with a high temporal and/or spatial coherence.", "When a high temporal coherent state is selected, the output light beam of the system, thus, comprises radiation within a very narrow frequency range, and, preferably, the output light beam substantially comprises only a single frequency.", "When a high spatial coherent state is selected the output light beam may be focused to a small spot size of the order of a wavelength.", "This is an important property for a large number of applications.", "Preferably, at least one of the plurality of optical elements is selected from the group consisting of: spatial filters, gratings, mirrors, Fabry Perot etalons, frequency filters.", "It is a great advantage of the present invention that a HOE is used as a feedback system.", "Thus, a HOE is substantially less expensive than a large number of optical elements.", "Furthermore, the size of the entire optical system is substantially reduced, and the system is much more stable, e.g.", "with respect to vibrations, misalignments, etc.", "Thus, the HOE may be attached to the laser facet itself, thereby substantially improving the mechanical stability properties of the optical system.", "Also, the HOE provides a passive feedback system as opposed to the active feedback system provided by a feedback system comprising non-linear optical components.", "This is a great advantage because in comparison with the active feedback the passive feedback only uses low cost elements.", "Finally, a feedback system being represented by a HOE may introduce fewer losses than a feedback system comprising the actual optical components which the HOE represents.", "This is because it is possible to let the HOE reproduce other optical properties of the individual optical component without reproducing the loss characteristics of that component.", "In case the HOE represent a large number of optical components, this is a very important advantage since the entire system may introduce very heavy losses.", "A spatial filter may, e.g., be an aperture, a slit, a pinhole or any other suitable kind of spatial filter.", "The optical properties of a spatial filter which may be reproduced by the holographic optical element in this case preferably comprise selection of specific modes or frequencies.", "In case one of the optical elements is a grating, the optical properties to be reproduced by the holographic optical element preferably comprise a frequency selectivity.", "In case one of the optical elements is a mirror, the optical properties to be reproduced by the holographic optical element preferably comprise the reflective properties of the mirror, such as a frequency dependent reflectivity, i.e.", "the reflectivity of the mirror at a certain frequency, the tilt angle of the mirror, and/or any focusing properties of the mirror.", "The mirror may be a plane mirror, a parabolic mirror, a spherical mirror, a mirror with a spatial selectivity, or a mirror having any other suitable shape.", "A frequency filter may e.g.", "comprise a grating, such as a diffractive optical element, preferably in combination with a spatial filter, such as an aperture or a slit, or it may be a filter which transmits electromagnetic radiation within a specific frequency range and reflects or absorbs any other frequencies.", "It may comprise an interference filter, an absorbance filter, such as a semiconductor doped glass, an etalon, such as a Fabry Perot element, a prism etc.", "The frequency filter may be adapted to select a range of frequencies, preferably a narrow range, most preferably a single frequency.", "In case one of the optical elements is a frequency filter, the optical properties to be reproduced by the holographic optical element preferably comprise frequency selection, transmission/reflection/absorption properties, selection of modes, reflective properties, refractive index, transmission properties, reflectivity, interference properties (destructive and/or constructive), or any other suitable properties of the frequency filter.", "According to a second aspect the present invention further provides a method of producing an optical system for emission of an output light beam, the method comprising the steps of: inserting a holographic recording material into an external cavity formed between a laser device and a feedback system, said feedback system comprising a plurality of optical elements, emitting, by means of the laser device, a first light beam, at least part of said first light beam illuminating at least part of the feedback system via said holographic recording material, adjusting the feedback system so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherence, recording a holographic optical element in the holographic recording material, developing the holographic optical element so that the holographic optical element is adapted to reproduce the optical properties of the plurality of optical element when said feedback system is removed, and removing the feedback system.", "The holographic recording material may be any material with a photosensitive refractive index and/or absorption coefficient for example a dichromatic gelatine, a Silver Bromide (AgBr) solution, photo resist, and/or a photorefractive medium.", "The laser device may be a single laser, e.g.", "a gas laser, a semiconductor laser, a broad area laser, a superluminescent laser diode, a dye laser, a Nd-YAG laser, an argon ion laser, a titanium sapphire laser, an F-center laser, or any other suitable kind of laser.", "It may also be an array of lasers, said lasers being of any of the types mentioned above.", "The feedback system is defined above.", "Adjusting the feedback system may comprise adjusting one or more of the optical elements forming the feedback system in such a way that a state having a high temporal and/or spatial coherency is selected.", "The adjusting step may, furthermore, comprise alignment of the optical elements.", "Additionally or alternatively, the adjusting step may comprise adjusting one or more optical element(s) in such a way that, e.g., a certain frequency, a certain spatial mode, etc.", "is selected.", "This may, for example, be done in the following way.", "In a preferred embodiment of the invention, the feedback system comprises a reflector, the reflector being adapted to reflect at least a part of the first light beam emitted by the laser device back into the laser device.", "The free running laser emits a large number of spatial modes.", "By using spatial filtering for example in the Fourier plane, e.g.", "by means of one or more spatial filter(s), such as aperture(s), slit(s), pinhole(s), etc., the system is adjusted to emit laser light having a high temporal and/or spatial coherency.", "The feedback system may, furthermore, comprise a grating or an etalon so that the frequency of the first light beam may be tuned by tilting the grating or the etalon.", "It is, thus, possible to adjust the system to emit an output light beam having, e.g., a certain spatial mode, frequency, etc., depending on the optical elements being provided in the feedback system.", "When the feedback system has been adjusted so that the output light beam has the desired properties, a holographic optical element having these properties is recorded in the holographic recording material positioned between the laser device and the feedback system.", "When the holographic optical element is subsequently developed, it will thus be adapted to reproduce the optical properties of the elements in the feedback system.", "The feedback system may then be removed and the holographic optical element will act as the feedback system, i.e.", "the output beam will have the same desired properties which the feedback system was adjusted to provide.", "Of course, the recorded and developed holographic optical element itself may not afterwards be adjusted as the feedback system, thus, limiting the flexibility of the system.", "However, it is an advantage of the system according to the present invention that the system provided is substantially non-sensitive to misalignments due to vibrations, temperature variations, etc.", "It is a further advantage that a rather bulky, expensive, and fragile feedback system may be replaced by the compact, cheap, and reliable holographic optical element.", "These advantages makes the system very advantageous for commercial purposes.", "It is, thus, possible to use to system under conditions which are normally not suited for a feedback system comprising a large number of fragile optical components, e.g.", "in an environment introducing vibrations, temperature variations, a dirty environment, etc.", "It is possible to manufacture HOEs under ideal conditions and subsequently position the HOEs in optical systems under less ideal conditions.", "Thus, an ideal feedback system is provided even though the environment is not suited for such a feedback system.", "Furthermore, the price of the entire system will be sufficiently low to attract potential customers.", "The system may, thus, advantageously be used, e.g., in the printing industry, medical applications, or telecommunication.", "In a very preferred embodiment laser systems having a HOE replacing a feedback system may be mass produced by recording one HOE by the method described above, and subsequently reproduce this HOE.", "The reproduced HOEs may then be positioned in laser systems having similar properties.", "It should be noted that the HOE in each case should be positioned in the laser system in a position corresponding to the position in which the original HOE was recorded in order for the HOE to properly reproduce the feedback system.", "Such mass produced laser systems are very advantageous from a commercial point of view since they are very cheap to manufacture, and the price therefore will be acceptable for potential customers.", "The method may comprise the steps of, for each of the optical elements: adjusting the feedback system so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherency, recording a holographic optical element in the holographic recording material, repeating the adjusting and recording steps until the properties of each of the plurality of optical elements has been recorded, and performing the development after the optical properties of all the optical elements have been recorded and removing the feedback system when the holographic optical element has been developed.", "In this case, the adjusting and recording steps are performed for each optical element of the feedback system.", "Thus, each optical element is in turn adjusted to achieve a desired optical property of the optical element in question.", "This optical property is then recorded.", "In order to record the optical properties of all the optical elements into the same holographic optical element, the holographic optical recording material is not developed until all the optical properties have been recorded.", "At least one of the plurality of optical elements may be selected from the group consisting of: spatial filters, gratings, mirrors, Fabry Perot etalons, frequency filters.", "These optical elements and their optical properties have been described above.", "Preferably, the method further comprises the step of positioning the holographic optical element in connection with a laser device, so that the holographic optical element and the laser device may cooperate to select a state having a high temporal and/or spatial coherency.", "That is, the holographic optical element may preferably be used to replace the feedback system in order to provide a compact, cheap, and mechanically stable laser system as described above, and as will be further described below.", "The method may further comprise the step of multiplexing a plurality of centre frequencies into the holographic optical element.", "In an embodiment where the feedback system comprises a grating, this may be performed in the following way.", "The feedback system may be adjusted to select one centre frequency and a corresponding grating may be induced in the holographic optical element.", "The laser device may then be turned off and the grating be tilted to select a new centre frequency.", "When the laser device is turned on again, a new hologram with a new centre frequency may be written into the holographic optical recording material.", "By repeating this procedure for each centre frequency, a plurality of frequencies are written into the holographic recording material.", "When all the desired frequencies have been written into the holographic recording material, the holographic optical element is developed, so as to obtain a holographic optical element having all the desired centre frequencies multiplexed into it.", "Thus, the method may further comprise the steps of, for each of the plurality of centre frequencies: adjusting the feedback system to emit a centre frequency feedback light beam so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherency, and so that a specific centre frequency is obtained, recording a holographic optical element in the holographic recording material, repeating the adjusting and recording steps until each of the plurality of centre frequencies has been recorded, and performing the development after all the centre frequencies have been recorded and removing the feedback system when the holographic optical element has been developed.", "The developing step may be performed using a chemical or thermal fixing procedure.", "Alternatively, the holographic recording material may be of a kind which is ‘self-developing’.", "In this case the development is performed automatically and does not require an active act.", "This is known per se.", "The laser system may advantageously be a compact laser system.", "According to a third aspect the invention further provides a compact laser system for emission of an output light beam, the system comprising: a laser device for emission of a first light beam, and a holographic optical element being illuminated by at least a part of the first light beam, thereby causing a feedback light beam to be emitted from the holographic optical element and being reinjected into the active gain medium of the laser device, whereby the laser device and the holographic optical element cooperate to select a high spatial and/or high temporal coherent state of the laser device, whereby the laser system is controlled to emit an output light beam having an improved spatial and/or temporal coherence.", "As described above the laser device may be any suitable kind of laser device, such as a gas laser, a semiconductor laser, a superluminescent laser diode, a dye laser, a Nd-YAG laser, an argon ion laser, a titanium sapphire laser, an F-center laser, or any other suitable kind of laser.", "It may also be an array of lasers, said lasers being of any of the types mentioned above.", "The first light beam may be an electromagnetic beam, preferably a monochromatic electromagnetic beam.", "In case the laser device is a single laser, the first light may also be a coherent light beam.", "In case the laser device is an array of lasers or another laser device having a broad bandwidth gain medium, the first light beam will in most cases have a very low degree of coherence.", "The holographic optical element is illuminated by at least part of the first light beam.", "It may, of course, be illuminated by all of the first light beam.", "At least part of the holographic optical element may be illuminated, or all of the holographic optical element may be illuminated.", "The feedback light beam is emitted from the holographic optical element in response to the first light beam illuminating the holographic optical element.", "The feedback light beam may be an electromagnetic beam, such as an electromagnetic beam comprising frequencies within the visible frequency range, the ultraviolet frequency range, the infrared frequency range, the X-ray frequency range, or any other suitable frequency range.", "The feedback light beam may be e.g.", "a complete reflection of the first light beam.", "Alternatively, it may be a reflected part of the first light beam, such as a part defined by a specific frequency range, a specific polarisation, a specific spatial mode, etc.", "Alternatively, the feedback light beam may be a beam which is generated by the holographic optical element in response to the first light beam.", "The feedback light beam is reinjected into the active gain medium of the laser device.", "In this way the laser device and the holographic optical element cooperate to select a high spatial and/or temporal coherent state of the laser device.", "The output light beam from the system will then have a high spatial and/or temporal coherent state.", "In order to obtain a high power, the laser device is often an array of lasers as described above.", "As mentioned above, this will very often result in a first light beam having a low degree of coherence.", "Since the output light beam has a high spatial and/or temporal coherent state, it thus has an improved spatial and/or temporal coherence as compared to the first light beam being emitted from the laser device.", "Thereby, the compact laser system is adapted for improving the coherency of a high power laser beam.", "The holographic optical element may be adapted to reconstruct an original light beam from a feedback system.", "The feedback system may comprise a number of optical elements as described above, and it is preferably operated as described above.", "The holographic optical element may, thus, replace the bulky, expensive, and fragile optical elements of the feedback system.", "Since a holographic optical element is compact, cheap, and less fragile than most ordinary optical elements, the resulting laser system will also be compact, cheap, and less fragile than laser systems having an ordinary feedback system comprising a number of optical elements.", "Furthermore, the resulting laser system will not be subject to misalignments due to, e.g., vibrations or temperature variations to the same extend that an ordinary laser system is.", "The feedback system may comprise one or more optical elements selected from the group consisting of: spatial filters, gratings, lenses, mirrors, Fabry Perot etalons, frequency filters.", "Most of these optical elements as well as their optical properties have been described above.", "The original light beam from the feedback system which the holographic optical element is adapted to reconstruct, preferably comprises information relating to the optical properties of the optical elements of the feedback system.", "The holographic optical element is most preferably recorded in such a way that these optical properties may be reproduced by the holographic optical element.", "Thus, the output light beam from the compact laser system may be substantially identical to the output light beam from a more bulky laser system having an ordinary feedback system.", "In case one of the optical elements is a lens, the optical properties to be reproduced by the holographic optical element preferably comprise refractive index, reflectivity, including internal reflectivity, focal length, radius of curvature, or any other suitable optical properties of the lens.", "The lens may be an ordinary concave or convex lens, or it may be another kind of refractive optical element, such as a prism.", "The holographic optical element may be adapted to, in cooperation with the laser device, select at least one centre frequency from the first light beam.", "This corresponds to selecting a high temporal coherent state.", "However, the exact value of the centre frequency may also be selected in this embodiment.", "This may e.g.", "be obtained by recording the holographic optical element using a feedback system which may be tuned so as to select a specific frequency.", "The holographic optical element may be adapted to, in cooperation with the laser device, select a plurality of centre frequencies, each centre frequency being multiplexed into the holographic optical element.", "This may be obtained by consecutively tuning a system as described above to each of the plurality of centre frequencies, and recording and developing the holographic optical element in such a way that all the centre frequencies are multiplexed into the holographic optical element.", "This procedure will be further described below.", "The laser device may comprise a laser array, such as an array of diode lasers, gas lasers, semiconductor lasers, dye lasers, Nd-YAG lasers, argon ion lasers, or any other suitable kind of lasers.", "Alternatively, it may be a single laser as described above.", "The laser device may comprise at least one laser selected from the group consisting of: broad area lasers, laser diode arrays, laser diode bars, stacked laser arrays.", "Broad area lasers and laser diode arrays comprise a number of diode lasers arranged in a row.", "Laser diode bars also comprise a number of diode lasers arranged in a row.", "However, the lasers of a laser diode bar are spatially separated, so that the light sources may be considered as a number of discrete point sources.", "Stacked laser arrays comprise a number of laser diode bars being stacked, so as to form a two-dimensional array of diode lasers.", "The above-mentioned types of lasers all provide an output beam having a high power, but a low degree of coherence.", "For some applications, it may therefore be necessary to improve the coherency of the output beam.", "This may be done as previously described.", "According to a fourth aspect the invention further provides a method of generating an output light beam from a laser system, the laser system comprising a laser device and a holographic optical element, the method comprising the steps of: emitting, by means of the laser device, a first light beam in such a way that at least part of the holographic optical element is illuminated by at least part of the first light beam, injecting, by means of the holographic optical element and in response to the first light beam, a feedback light beam into the laser device, and outputting, by means of the holographic optical element and in response to the first light beam, an output light beam from the laser system, said output light beam having an improved spatial and/or temporal coherence state.", "The first light beam, the feedback light beam, as well as the output light beam may be electromagnetic beams as described above.", "All of, or at least part of, the holographic optical element may be illuminated by the first light beam, and it may be illuminated by all of, or at least part of, the first light beam.", "The feedback light beam may be a fully or a partial reflection of the first light beam, or it may be generated by the holographic optical element, as described above.", "The laser device and the holographic optical element cooperate to select a state having a high temporal and/or spatial coherence, so that the output light beam has an improved spatial and/or temporal coherence as compared to the first light beam which is initially emitted from the laser device.", "This has been described above.", "The holographic optical element may reconstruct an original light beam from a feedback system.", "This has already been described.", "The feedback system may comprise one or more optical elements selected from the group consisting of: spatial filters, gratings, lenses, mirrors, Fabry Perot etalons, frequency filters.", "These optical elements as well as their optical properties have been described above.", "The method may further comprise the step of, by means of the holographic optical element in cooperation with the laser device, selecting at least one centre frequency from the first light beam.", "As described above this corresponds to selecting a state having a high temporal coherence.", "However, a specific frequency is chosen in this case.", "The method may further comprise the step of, by means of the holographic optical element in cooperation with the laser device, selecting a plurality of centre frequencies, each centre frequency having previously been multiplexed into the holographic optical element.", "This has also been described above.", "According to a fifth aspect the invention further provides a method of producing a compact laser system for emission of an output light beam, the method comprising the steps of: inserting a holographic recording material into a laser cavity formed between a laser device and a feedback system, emitting, by means of the laser device, a first light beam, at least part of said first light beam illuminating at least part of the feedback system via said holographic recording material, adjusting the feedback system to emit a feedback light beam so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherency, recording a holographic optical element in the holographic recording material, developing the holographic optical element so that the holographic optical element is capable of reconstructing the feedback light beam from the feedback system when said feedback system is removed, and removing the feedback system.", "The laser device may be any suitable kind of laser device as described above.", "The feedback system preferably comprises a number of optical elements each having specific optical properties.", "The first light beam as well as the feedback light beam may be electromagnetic beams as described above.", "The adjusting step is preferably performed by adjusting each of the optical elements of the feedback system.", "This may comprise tilting gratings to the correct angle, e.g.", "in order to obtain a specific frequency, positioning spatial filters correctly, e.g.", "in order to obtain a specific spatial mode, aligning the optical elements, e.g.", "in order to optimise the throughput of the system, and/or it may comprise any other suitable kind of adjusting of the feedback system.", "The adjusting step results in that the laser device and the feedback system, by means of the feedback light beam, cooperate to select a state having a high temporal and/or spatial coherency.", "The adjusting may be performed using spatial filtering in the Fourier plan.", "The holographic recording material may comprise a dichromatic gelatine, a silver bromide (AgBr) solution, photo resist, and/or a photorefractive medium, such as a lithium niobate crystal.", "When the holographic optical recording material has been developed to form the holographic optical element, the holographic optical element is capable of reconstructing the feedback light beam from the feedback system because the optical properties of the optical elements of the feedback system have been recorded into the holographic optical element.", "When the feedback system is removed, the laser device and the holographic optical element may therefore be able to cooperate to select a state having a high temporal and/or spatial coherency.", "That is, the output light beam emitted from the laser system with the feedback system removed will be substantially identical to the output light beam emitted from the laser system with the feedback system present instead of the holographic optical element.", "Thus, as described above, the holographic optical element may replace the rather bulky, expensive, etc.", "feedback system, thereby providing a laser system which is compact, cheap, etc.", "The method may further comprise the step of multiplexing a plurality of centre frequencies into the holographic optical element.", "Thus, the method may further comprise the steps of, for each of the plurality of centre frequencies: adjusting the feedback system to emit a centre frequency feedback light beam so that the laser device and the feedback system cooperate to select a state having a high temporal and/or spatial coherency, and so that a specific centre frequency is obtained, recording a holographic optical element in the holographic recording material, repeating the adjusting and recording steps until each of the plurality of centre frequencies has been recorded, and performing the development after all the centre frequencies have been recorded and removing the feedback system when the holographic optical element has been developed.", "This has already been described above.", "A laser system according to the present invention may be used for a number of various applications, such as frequency doubling, coupling of light into thin core or single mode laser fibres, material processing, the printing industry, biomedical application, and/or any other suitable applications in which a compact, low cost, and reliable laser system is useful.", "The first, second, third, fourth, and fifth aspects of the present invention may each be combined with one or more of the other aspects of the present invention.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 shows the recording of a feedback system into a holographic optical element, in FIG.", "2, the holographic optical element has been developed and the feedback system has been removed, FIG.", "3 shows an example of a feedback system comprising a laser diode array receiving an external feedback from a reflector, and FIG.", "4 shows an example of an output from a laser operating simultaneously at several wavelengths.", "DETAILED DESCRIPTION OF THE DRAWINGS In FIG.", "1, a laser diode array 1 and an external cavity formed between the laser diode array 1 and the feedback system 3 is shown.", "The laser array 1 receives a feedback signal from the feedback system 3.The laser beam 11 emitted from the laser array 1 forms a twin lobe structure in the plane of the far field, and one of the lobes, the lobe 5, illuminates the feedback system 3 whereas the other lobe 7 provides an output light beam.", "A holographic optical recording material 9 is inserted in the path of the laser beam 11 between the laser array 1 and the feedback system 3, preferably, the holographic optical recording material 9 is inserted proximate to the laser array 1 emitting the laser beam 11 so that the resulting laser system may be minimised as much as possible.", "It is, though, emphasised that the holographic optical recording material may be inserted just after the laser or anywhere in the feedback system.", "According to a preferred embodiment of the present invention, the feedback system 3 is a frequency selective feedback system adjusted to select a high spatial and/or a high temporal coherent state of the laser array 1.Having adjusted the laser system to inject a feedback signal into the laser array 1 to select a high spatial and/or high temporal coherent state of the laser array 1, the holographic optical recording material 9 is developed either spontaneously or by a chemical and/or thermal procedure.", "In FIG.", "2, the optical system comprising the developed holographic optical element 13 is shown.", "The feedback system 3 is removed and the developed holographic optical element 13 is inserted in the path of the laser beam 11 at the same position as the position on which the holographic optical recording material 9 was recorded.", "The holographic optical element 13 hereby reconstructs the original signal from the feedback system (now removed) which is injected into the laser array 1, and in the preferred embodiment mentioned above, the holographic optical element 13 and the laser array 1 will select a high spatial and/or temporal coherent state of the laser array 1 whereby the laser array 1 is adapted to emit a high spatial and/or high temporal coherent output light beam 7.The holographic optical element may be of a material such as dichromatic gelatines, silver bromide, photo resist, or a photo-refractive medium, such as a lithium niobate crystal.", "FIG.", "3 shows an example of a frequency selective feedback system according to the preferred embodiment mentioned above.", "In FIG.", "3, the entire laser beam 11 illuminates the holographic optical recording material.", "The laser array 30 used in the embodiment shown in FIG.", "3 is a SDL-2432 GaAlAs broad area laser with a threshold of 0.29 Amps and a maximum output power of 0.5 Watts at a drive current of 0.75 Amps.", "The lasing wavelength at 20.0° C. is 813.5 nm and the emitting junction 31 is 1×100 μm.", "The light emitted from the laser array 30 is collimated with a Thorlab C230TM-B lens 33 with an effective focal length of 4.5 mm and a numerical aperture of 0.55.A beam splitter 51 is inserted in the system to couple a part of the beam 55 out for beam diagnostics.", "A cylindrical lens 35 with a focal length of 150 mm is inserted to collimate the beam.", "Furthermore, an etalon may be provided causing the feedback to be frequency selective, the etalon may be a Fabry-Perot etalon with a finesse of approx.", "17.Alternatively, an etalon having a lower finesse, such as approx.", "2.6 may be used.", "The etalon, thus, improves the temporal coherence of the output beam.", "All lenses and etalons have a broad band anti-reflection coating (R<1 percent) in order to minimise the loss of the external cavity.", "Furthermore, the exit face of the laser may have antireflection coating to improve the influence from the feedback system.", "The external cavity is terminated by a grating 37 and a reflector 39, the reflector 39 being a mirror or in a preferred embodiment, a four-wave mixing non-linear medium, such as a GaAlAs crystal arranged in a self-pumped configuration.", "A spatial filter 41 is inserted in the lobe 45 in the plane of the pseudo far field 43, whereby only selected spatial modes of the lobe 45 are directed towards the grating and the reflector.", "In the other lobe 47, a mirror 49 is inserted for coupling an output light beam out of the system.", "To record this system into a holographic optical recording medium, the holographic optical recording medium is positioned in the laser beam 11 after the collimating lens 33.When the system is adjusted to emit an output light beam having a high spatial and/or temporal coherence, the holographic optical recording medium is developed and the feedback system comprising lenses 34, 35, beam splitter 51, mirror 49, spatial filter 41, grating 37 and reflector 39, may be removed.", "Hereby, the complex optical system shown in FIG.", "3 may be replaced by a single holographic optical element, whereby the size of the optical system is dramatically reduced.", "Furthermore, adjustment of gratings, spatial filters, etalons, etc, is avoided during operation or start-up of the system, since this adjustment is inherently present in the holographic optical element.", "The holographic optical element need only to be inserted at the same position in relation to the laser array or laser device as the position of holographic optical recording medium during recording of the medium.", "This reduces the overall cost and complexity of the system, and for example very fragile and expensive components, such as non-linear mediums, may be replaced by a low-cost and reliable holographic optical element, and the fragile and expensive mediums may be used to record a plurality of holographic optical elements in controlled environments.", "A further advantage of the described system is that all components may be integrated on a single chip.", "The frequency selective feedback system may, as described in connection with FIG.", "3, be a grating based feedback system comprising a mirror, a spatial filter, grating and lenses.", "In an alternative preferred embodiment, the frequency selective feedback system may comprise gratings, lenses and a spatial filter.", "In a still further preferred embodiment of the invention, the frequency selective feedback system comprises non-linear four-wave mixing in combination with gratings, spatial filters and frequency filters.", "The grating is a frequency filter which only refracts a limited number of frequencies which then interact with the reflector.", "By tilting the grating so that the feedback beam is adjusted to match the lasing wavelength of a spatial mode with high gain in the laser array, the frequency of the output beam is tuned.", "Similarly, an etalon in combination with a non-linear four-wave mixing medium passes only a limited number of frequencies so that, as the reflectivity of the non-linear four-wave mixing medium increases, the spectrum narrows down significantly.", "Single spatial mode operation can be achieved if the orientation of the etalon is adjusted so the wavelength for peak transmission match the lasing wavelengths of a spatial mode with high gain or centre frequencies of the laser array.", "Advantage of the possibility of tilting the grating 37 or the etalon (not shown in FIG.", "3) for selecting a specific lasing wavelength of the laser array may be taken when recording a holographic optical recording medium.", "By having the holographic optical recording medium positioned in the optical system, the tilting angle of the grating may be varied.", "At a first centre frequency, a first holographic grating according to the tilting angle of the grating may be recorded in the holographic optical recording medium.", "The laser array is then turned off and the grating is tilted to select a new centre frequency.", "When the laser is turned on, a new hologram with a new centre frequency, a new holographic grating, is written into the holographic optical recording medium.", "By using this procedure at a plurality of centre frequencies, a plurality of holographic gratings are written into the holographic optical recording medium.", "When a predetermined number of frequencies have been written into the material, the material is developed and afterwards the holographic optical element may be used to provide an output as illustrated in FIG.", "4, where a number of four centre frequencies has been written into the holographic optical recording medium before developing the medium.", "The configuration used to obtain the output shown in FIG.", "4 is a configuration according to the configuration shown in FIG.", "2.The possibility of multiplexing a number of centre frequencies into an holographic optical element enables the laser array or any other laser device to operate simultaneously at several wavelengths.", "This is an especially important feature in high capacity telecommunication systems." ] ]	Patent_10451724
[ [ "Device for combustion of a carbon containing fuel in a nitrogen free atmosphere and a method for operating said device", "The present invention relates to a device for combustion of a carbon containing fuel in a nitrogen free atmosphere, and a method for operating said device.", "The device may be integrated with a power generation plant (i.e.", "gas turbine(s)) to obtain an energy efficient process for generation of power with reduced emission of carbon dioxide and NOx to the atmosphere.", "Furthermore, the device may be integrated with a chemical plant performing endothermic reactions." ], [ "1-17.(Cancelled).", "18.A device for combustion of a carbon containing fuel in a nitrogen free atmosphere, wherein said device comprises a hollow shell having an inlet for conveying said fuel, an inlet for conveying a compressed oxygen containing gas stream, an outlet for discharging an oxygen depleted gas stream and an outlet for discharging a bleed stream; and said shell encloses one or more heat exchange modules arranged to heat the incoming compressed oxygen containing gas stream; one or more mixed conducting membrane modules arranged to separate oxygen from said oxygen containing gas stream resulting in an oxygen rich gas stream and said oxygen depleted gas stream; a first and possibly a second combustion chamber for combustion of said fuel having an inlet connected to said inlet for fuel to convey fuel to said chamber, an inlet connected to said heat exchange module(s) to convey hot oxygen rich gas to said chamber and an outlet connected to said membrane module(s) to convey exhaust gas from the combustion chamber to the membrane module; a pressure booster installed prior to the first combustion chamber; means (11,21) for connecting said heat exchanger module(s) and membrane module(s); means (7,23) for connecting said heat exchanger module(s) and said membrane module(s) to the inlet for the compressed oxygen containing gas stream and the outlet for the oxygen depleted gas stream and means for conveying a part of said exhaust gas stream directly to said heat exchange module(s) and back to said inlet from the combustion chamber.", "19.A device according to claim 18, wherein said heat exchange module(s), said membrane module(s), said means (7,11,21,23), an outlet for said hot oxygen rich gas stream, said outlet for discharged oxygen depleted gas stream, an inlet for said exhaust gas and said inlet for compressed oxygen containing gas stream are all installed in a pressure vessel (reactor).", "20.A device according to claim 18, wherein said heat exchange module(s), said membrane module(s), said second combustion chamber, said means (7,11,21,23), an outlet for said hot oxygen rich gas stream, said outlet for discharged oxygen depleted gas stream, an inlet for said exhaust gas and said inlet for compressed oxygen containing gas stream are all installed in a pressure vessel.", "21.A device according to claim 18, wherein said modules and said second combustion chamber are vertically interconnected one above the other.", "22.A device according to claim 18, wherein said modules are vertically interconnected one above the other.", "23.A device according to claim 18, wherein said membrane module is installed between two heat exchange modules.", "24.A device according to claim 18, wherein said second combustion chamber is installed between one of the heat exchange module and a membrane module.", "25.A device according to claim 19, wherein said outlet for said hot oxygen rich gas stream is connected to the inlet to the first combustion chamber and said inlet for said exhaust gas is connected to the outlet from the first combustion chamber.", "26.A device according to claim 18, wherein said pressure booster is a fan or a compressor.", "27.A device according to claim 18, wherein said heat exchange module(s) and said membrane module(s) comprise a multichannel monolithic structure.", "28.A method for operating a device according to claim 18, wherein said method comprises the following steps: a compressed oxygen containing gas stream is fed to a first heat exchange module where it is heated by means of heat generated by combustion of a fuel in a combustion chamber; said heated gas stream is fed to a mixed conducting membrane module(s) where most of the oxygen is separated from said gas stream and an oxygen depleted gas stream is obtained; a sweep gas is fed to said membrane module to pick up oxygen and the oxygen enriched sweep gas is further fed to a pressure booster; the pressurized sweep gas stream enters the combustion chamber where it is mixed with a fuel for combustion; and said oxygen depleted gas stream is fed to another heat exchange module for further heating before leaving said device.", "29.A method for operating a device according to claim 28, wherein said combustion product; the exhaust gas, is applied as sweep gas.", "30.A method for operating a device according to claim 28, wherein a part of the exhaust gas is taken out as a bleed stream to prevent accumulation of mass in the device.", "31.Use of a device and a method according to claim 18 in a plant for generation of power.", "32.Use of a device and a method according to claim 18 in a chemical plant performing an endothermic reaction." ], [ "The present invention relates to a device for combustion of a carbon containing fuel in a nitrogen free atmosphere and a method for operating said device.", "The device may be integrated with a power generation plant (i.e.", "gas turbine(s)) to obtain an energy efficient process for generation of power with reduced emission of carbon dioxide and NOx to the atmosphere.", "Furthermore, the device may be integrated with a chemical plant performing endothermic reactions.", "Conventional combustion processes, used for carbon containing fuels, will in addition to producing the main end products carbon dioxide and water (steam), generate a considerable amount of heat (heat of combustion).", "A conventional combustion reaction between e.g.", "methane and oxygen will generate approximately 804 KJ per mol methane: CH4+2O2—>CO2+2H2O When this combustion process is integrated with e.g.", "a power generation plant (i.e.", "gas turbines) or a chemical plant performing endothermic reactions, it is crucial that the total energy loss from the combustion process is as low as possible.", "Furthermore, due to the environmental aspects of CO2 and NOx it is crucial that the emission of these components to the atmosphere is considerably reduced compared to conventional processes.", "Conventional combustion processes produce an exhaust gas with a CO2-concentration between 3 and 15% dependent on the fuel and the combustion- and heat recovery process applied.", "The reason the concentration is this low is because air comprises about 78% by volume of nitrogen.", "In high-temperature combustion processes in air, nitrogen will react with oxygen and produce the environmental hazardous gas pollutant NOx.", "A reduction in the emission of carbon dioxide to the atmosphere makes it necessary to either separate the carbon dioxide from the exhaust gas, or raise the concentration in the exhaust gas to levels suitable for use in different chemical processes or for injection in e.g.", "a geological formation for long term deposition or for enhanced recovery of oil from an oil reservoir.", "CO2 can be removed from cooled exhaust gas, normally discharged at near atmospheric pressure, by means of several separation processes, e.g.", "chemical active separation processes, physical absorption processes, adsorption by molecular sieves, membrane separation and cryogenic techniques.", "Chemical absorption, for instance by means of alkanole amines, is considered as the most practical and economical method to separate CO2 from exhaust gas.", "These separation processes consume energy and require heavy and voluminous equipment.", "Applied in connection with a power generation process, these separation processes will reduce the power output with 10% or more.", "An increase of the concentration of CO2 in exhaust gas from a combustion reaction to levels suitable for use in different chemical processes or for injection in e.g.", "a geological formation for long term deposition or for enhanced recovery of oil from an oil reservoir is possible by burning the carbon containing fuel with pure oxygen instead of air.", "Commercial air separation methods (e.g.", "cryogenic separation or pressure swing absorption (PSA)) applied for producing pure oxygen require 250 to 300 KWh/ton oxygen produced.", "If these methods are used for supplying oxygen to a combustion process in a gas turbine cycle these methods will reduce the net power output from the gas turbine cycle by at least 20%.", "The expenses of producing oxygen in a cryogenic unit will increase the price of produced electric power substantially and may amount to as much as 50% of the cost of the electric power.", "However, a less energy demanding method than these separation methods is known from the European Patent Application 658 367-A2.The patent application describes an application of a mixed conducting membrane (MCM) integrated with a gas turbine system and where the membrane separates oxygen from a heated air stream.", "The mixed conducting membrane (MCM) is defined as a membrane made of materials with both ionic and electronic conductivity.", "The membrane selectively transports oxygen.", "The driving force through the membrane is proportional to the logarithmic relation between oxygen partial pressures; log (pO2(I)/pO2(II)), where (I) represents the oxygen delivering side (air) of the membrane and (II) represents the oxygen receiving side of the membrane.", "To keep a high transport rate (flux) of oxygen it is important to keep a low partial pressure on the oxygen receiving side.", "Thus, to further improve the efficiency of this membrane process, a sweep gas is applied to reduce the partial pressure of oxygen on the oxygen receiving side of the membrane and thereby increase the flux of oxygen through the membrane; as e.g.", "described in U.S. Pat.", "No.", "5,562,754 and NO-A-972632.To obtain practical applications of mixed conducting membranes (MCM) when applied as an oxygen supplier in a combustion process the following criteria are essential: a) The driving force of the oxygen transport through the membrane expressed as the logarithmic relation between oxygen partial pressures; log (pO2 (I)/pO2 (II)), has to be kept at a high level.", "b) The membrane has to operate at high temperature levels (>600° C.) to achieve a sufficient oxygen flux through the membrane.", "Thus air or any other gases in contact with the membrane must have a high temperature.", "To ensure that the driving force through the membrane is kept at a high level oxygen on the oxygen receiving side of the membrane has to be: i) transported away from the membrane surface, by applying a sweep gas, or ii) consumed by a chemical reaction (e.g.", "a combustion process) directly on the oxygen receiving side.", "This implies that the device which shall perform an energy efficient combustion in a nitrogen free atmosphere must be designed to operate under process conditions as mentioned above.", "There remains therefore a need for such a device and a method for operating said device that is not described in the prior art.", "The main object of the present invention was to provide a device effective to achieve combustion of a carbon containing fuel in a nitrogen free atmosphere.", "Another object of the present invention was to provide a device effective to achieve a combustion process resulting in an exhaust gas with a high concentration of CO2 and a low concentration of NOx.", "Furthermore, another object of the invention was to provide a method for operating said device.", "Yet another object of the invention was to provide a plant and a method for an energy efficient generation of power.", "Still yet another object of the invention was to provide a plant and a method for generation of power with reduced emission of carbon dioxide and NOx to the atmosphere.", "The inventors found that the described objects were fulfilled by utilizing a device where one or more mixed conducting membrane module(s), one or more heat exchange module(s) and one or more combustion chamber(s) were enclosed within a hollow shell (a pressure vessel) defining an enclosure.", "The device may further be integrated with gas turbine(s) in a plant for generation of power.", "The device may also be integrated with a chemical plant performing an endothermic reaction to supply necessary heat to the reaction.", "The mixed conducting membrane(s) (MCM) which is utilized in the device according to the present invention will at conditions described above (a) and b)) transport oxygen from an oxygen delivering gas (e.g.", "air) to an oxygen receiving gas.", "The oxygen receiving gas has a lower partial pressure of oxygen than the oxygen delivering gas.", "To the oxygen receiving gas a carbon rich fuel (e.g.", "natural gas) is added and a heat generating combustion reaction between oxygen and added fuel takes place.", "Combustion of natural gas with pure oxygen will produce an exhaust gas containing the two main products carbon dioxide and water (steam).", "According to the present invention the exhaust gas is utilized as the oxygen receiving gas.", "The oxygen rich gas stream (i.e.", "the oxygen enriched exhaust gas) is fed to the combustion chamber and applied as oxidant in the combustion reaction.", "Thus, a production of the environmental harmful NOx gas is avoided.", "The thermal energy produced by the combustion reaction is by means of heat exchanger(s) utilized to heat air fed to the MCM-module(s) as well as to heat oxygen depleted air leaving the MCM-module(s) before it may enter a power generation turbine or a chemical plant performing an endothermic reaction.", "Thus the air stream fed to the membrane is heated without producing CO2 or NOx, in the stream.", "In the combustion reaction almost all oxygen is consumed and thus the exhaust gas, now having a very low partial pressure of oxygen, can be recirculated to the MCM as a sweep gas picking up oxygen before entering the combustion chamber again.", "Thus we have a continuous combustion.", "From the exhaust gas a bleed stream has to be taken out to balance the added fuel and oxygen received to prevent accumulation of mass.", "This bleed gas leaving the device at elevated pressure and temperature could also be fed to a power generation system (turbine).", "In the turbine the pressure of the bleed gas is decreased and further cooled to condense almost all steam to water.", "Thus the gas flow will consist mainly of carbon dioxide.", "This carbon dioxide gas flow has to be compressed to a pressure that allows injecting in an underground reservoir, a reservoir that could be an aquafier layer or a gas or oil reservoir.", "These reservoirs should be qualified for ensuring long term deposition.", "As mentioned above the exhaust gas is utilized as a sweep gas to pick up oxygen in the membrane module(s) and transport oxygen to one or more combustion chambers where fuel is added.", "The heat generated in the exhaust gas should in an efficient way be transported to the air stream, and in such a way, that leakage between sweep gas and air is prevented or minimised to an acceptable level.", "Furthermore, the inventors found that by utilizing a multiplate or a multichannel structure as a MCM-module and/or as a heat exchange module a very efficient device was achieved.", "Multichannel structures are found to be the most advantageous due to the fact that they can be extruded in one piece (i.e.", "a monolith) and thus a large surface area in one piece is obtained.", "Most preferably both the heat exchange module(s) and the MCM-modules are made of a ceramic material that is able to withstand the present process conditions (atmosphere, temperature and pressure).", "Such structures, especially with channel diameter below 10 mm, give a very high surface area/unit volume.", "By preparing every second row of channels by inlet slots as described in U.S. Pat.", "No.", "4,271,110 a simplified manifold system to every second row of channels could be achieved and thus give a low leakage rate probability between the air side and the oxygen receiving side.", "To obtain a largest possible surface area for heat exchange and/or oxygen transfer (when utilized as a MCM-module), the channels should be very small and every air channel should be surrounded by (i.e.", "have common walls with) the other gas (i.e.", "sweep/exhaust gas).", "Such a configuration needs a very complicated system for leading the two gases (manifolding) to each adjacent channel.", "According to the present invention such multichannel monolithic structures are connected or linked together in such a way that the MCM-module is installed between two heat exchange modules.", "Furthermore, these modules are installed in a pressure vessel hereinafter defined as the reactor.", "Such a system will ensure that the MCM is able to operate at a defined temperature higher than the temperature in the air stream fed to the system and below the temperature of combustion (i.e.", "the exhaust gas temperature from the combustion chamber).", "Another important feature of the present invention is the flow pattern of the two gas streams.", "The first gas stream (the air stream) has a flow from inlet to outlet of the reactor that follows longitudinal to the direction of channels in the monolithic structures (i.e.", "heat exchangers and MCM).", "This means that the gas enters and leaves the open channels from the short ends and flows through an open room or closed structure that connects these ends.", "The second gas stream has a flow direction in and out of the side slots of the monolith, through bypass rooms or connectors to the side slot of the adjacent monolithic structures.", "These bypass rooms are surrounding the inner open room of the first gas.", "Such a flow system of the gases will allow one of the gases, here the second gas, to leak and fill all available space or “empty” room of the reactor.", "The requirement for a gas tight sealing is then reduced for the first gas only to be a sealing towards the second gas (not to the “empty” space of the reactor) located at the inner coupling connectors between the monolithic structures.", "This feature is very important because a controlled leakage of gas is necessary to build up and equalise the pressure inside the reactor house, and only one of the gases is allowed to leak to prevent mixing.", "This controlled and necessary leakage allows a flexible sealing of defined leakage rate for the bypassing connectors of the second gas.", "Flexibility to avoid thermal stress in connecting parts/monolithic structures is very important to prevent fatal cracks.", "By filling the reactor with gas of almost the same pressure as the gas inside the monolith channels only the outer pressure shell of the reactor has to withstand the absolute or total pressure of the process.", "The pressure on the monolith walls is then reduced to withstand the differential pressure between the two gases (Gas 1 and Gas 2 in FIG.", "3).", "The scope of the invention and its special features are as defined by the attached claims.", "The invention will be further explained and envisaged in the following figures.", "FIG.", "1 shows a sketch of one embodiment of the device according to the present invention including its functional parts as heat exchange module, MCM-module and combustion chamber.", "Also included is a pressure booster, here shown as a jet ejector driven by high pressure (HP) steam.", "In this embodiment the modules are all installed within the reactor.", "FIG.", "2 shows another embodiment of the device according to the present invention including the same functional parts as heat exchange module, MCM-module as well as a combustion chamber and a pressure booster, but in this embodiment the combustion occurs in a separate vessel connected to the reactor.", "The pressure booster is installed in the connecting pipes, preferably prior to the combustion chamber where the sweep gas has its lowest temperature.", "FIG.", "3 shows a sketch of a multichannel monolith structure utilized as a MCM-module and/or as a heat exchange module.", "FIG.", "4 shows one embodiment of the reactor with the different modules as well as the other functional components in the reactor.", "FIG.", "5 shows different shapes of connectors between the MCM- and the heat exchange modules as well as different methods applied for sealing of the connectors between the modules.", "FIG.", "6 shows one embodiment of the device according to the present invention where the combustion chamber is installed outside the reactor as well as some of the internal components taken out of the reactor for the purpose of better illustrating these individual components.", "FIG.", "7 shows a more detailed illustration of the whole device according to the present invention as well as the individual components of the reactor.", "FIG.", "8.1 shows one embodiment of a plant for generation of power where the device according to the present invention is integrated with gas turbines.", "FIG.", "8.2 shows another embodiment of a plant for generation of power where the device according to the present invention is integrated with gas turbines and where more than one reactor have a common combustion chamber.", "FIG.", "9.1 shows one embodiment of the device according to the present invention where each process stream is given a tag number according to Table 1.FIG.", "9.2 illustrates the internal flow path of the air in the reactor.", "FIG.", "1 shows a principal sketch of the device according to the present invention where the process streams and the important process units (H-01), (X-01), (H-02), (F-01) and (I-01) are shown.", "The units are all installed inside the reactor pressure shell which is in this example identical to the device shell.", "The figure shows that an oxygen containing gas stream (here air) is conducted trough a compressor.", "The compressed air stream (AN-030) is further fed to the heat exchange module (H-01) where it is heated (AN-050) before entering the mixed conducting membrane module (X-01) in which oxygen is separated from the air stream resulting in an oxygen depleted air stream (AL-010).", "The oxygen depleted air stream (AL-010) enters the heat exchanger (H-02) for further heating before leaving the device (AL-020).", "The depleted air stream (AL-020) may be fed to a power generation turbine or a chemical plant performing endothermic reactions.", "A sweep gas (EG-020) is fed to the MCM-module (X-01) and is picking up oxygen at the oxygen receiving side of the membrane and further transported through heat exchange module (H-01).", "The oxygen enriched gas stream (EGO-030) is then pressurized in a pressure booster (I-01) before entering the combustion chamber (F-01).", "The combustion chamber (F-01) where fuel (NG-010) is added and burned is in this example installed inside the reactor pressure shell.", "The combustion gas or exhaust (EG-010) is now almost oxygen free due to combustion in (F-01).", "A part of the hot combustion product or exhaust gas (EG-010) is taken out as a bleed stream (EG-040) to prevent accumulation of mass in the reactor while the rest of the product gas is fed to the heat exchange module (H-02) and heated to the operational temperature of the membrane.", "In the membrane module stream (EG-020) is acting as a sweep gas.", "The hot and oxygen enriched sweep gas stream (EGO-020) is fed to the heat exchange module (H-01) to heat the incoming gas stream (AN-030).", "The heated air stream (AN-050) is entering the MCM-module (X-01) at the operational temperature of the MCM-modules (X-01).", "A pressure booster (I-01) has to be installed to enhance circulation in the sweep gas loop and ensure a continuous combustion.", "In FIG.", "1 this is a jet pump driven by injection of high pressure (HP) steam.", "The jet pump has the advantage of no moving parts and might be built in a material (i.e.", "ceramic) that can withstand very high temperatures.", "For power generation the oxygen depleted gas stream (AL-020) and the bleed gas stream (EG-040) may be fed to gas turbines to generate power.", "The bleed gas (EG-040) containing the main combustion products (CO2+H2O) will have a high temperature (combustion gas temperature).", "To generate power directly from the stream a gas turbine capable of handling the CO2 and H2O mixture is needed.", "Another power generating alternative for this stream is to cool down the gas to a temperature <550° C. where a conventional steam turbine can be used.", "This can be done by injecting water to stream (EG-040) or heat exchange with the incoming “cold” air stream (AN-030).", "FIG.", "2 shows another embodiment of the device according to the present invention where the pressure booster (I-01) and the combustion chamber (F-01) are installed outside the reactor pressure shell but within the device shell.", "This feature contributes to simplify the construction of the device.", "The advantage of installing (I-01) and (F-01) outside the reactor is to facilitate the maintenance work and makes it possible to apply cooling apparatus.", "Thus a rotary pressure increasing machine can be used as a pressure booster (I-01) as envisaged in this figure.", "The flow path in this embodiment is the same as in the embodiment shown in FIG.", "1.The only difference is that no high pressure (HP) steam is injected (because no jet pump is used), but this will not amend the principle flow pattern.", "Injecting high pressure (HP) steam as shown in FIG.", "1 will reduce the net power generation efficiency of the process and thus a rotary machine as shown in FIG.", "2 is with respect to efficiency more advantageous.", "An external combustion chamber will also simplify the fuel (NG-010) injection system and makes it easier to upscale the device as will be shown in FIG.", "8.FIG.", "3 shows a multichannel monolith structure which, according to the present invention might preferably be utilized as both a heat exchange module and a membrane module.", "As mentioned above, such structures are advantageous mainly because of their simple way to be manufactured.", "However, the present invention is not restricted to application of such structures only and other configurations (e.g.", "plates) may be an alternative.", "According to FIG.", "3, using stream notation as in FIGS.", "1 and 9, Gas 1 represents gas streams (AN-030) and (AN-050) if the monolith structure is module (H-01), gas streams (AN-050) and (AL-010) if the monolith structure is module (X-01).", "If the monolith structure is module (H-02), then Gas 1 is gas streams (AL-010) and (AL-020).", "Gas 2 represents the gas streams (EGO-020) and (EGO-030) if the module is (H-01), the gas streams (EG-030) and (EGO-010/020) if the module is (X-01) and gas streams (EG-020) and (EG-030) if the module is (H-02).", "Gas 1 follows the straight path through the channels and is thus always fed in and let out from the open rows of channels at the monolith ends.", "Gas 2, normally the sweep gas, is always fed in and taken out from the open slots in the side wall of the monolith structures.", "Since these monolithic structures preferably will be made by extrusion, all channels will be of the same length.", "The inlet and the outlet slots of Gas 2 must be made after extrusion by machining every second column of channels as visualised on the figure.", "After machining down to the preferred depth the open row of channels (made by machining) has to be closed by a sealing in such a way that a sufficient opening area for the side slot is kept (inlet and outlet for Gas 2).", "The problem of preventing leakage in the manifold system of two different gases leading in and out of the multichannel monolithic structures is minimised by making these inlet and outlet slots as described and shown in FIG.", "3.According to the present invention a channel diameter below 10 mm is used.", "A diameter between 1 and 8 is preferred.", "FIG.", "4 shows one embodiment of the reactor as described in FIG.", "2, where the combustion chamber and the pressure booster are mounted outside the reactor shell.", "In the figure the connecting flanges for the inlet (EG) and the outlet (EGO) of the sweep gas stream as well as the inlet (AN) of the air stream and the outlet (AL) of the oxygen depleted air stream are shown.", "Inside the reactor the flow path of these streams is visualized by dotted lines.", "Heat exchanger (H-01), MCM-modules (X-01) and the outlet heat exchanger (H-02) are fixed together by the connectors between (H-019, (X-01) and (H-02).", "These connectors are preferably glass sealed, before installed in the reactor, to ensure no leakage and thus will be one whole part (i.e.", "sealed together).", "During heating this whole part has to be allowed to expand.", "This will be further described in FIG.", "7.FIG.", "5 shows alternative shapes for the connectors between the (X-01) and (H-01/H-02).", "Thus (H-01) and (X-01) as well as (X-01) and (H-02) could be connected and sealed to each other by different components as shown.", "The most important factor is to have a tight sealing without leakage between the inner gas (i.e.", "Gas 1 as described in FIG.", "3, preferably air) and the outer gas (i.e.", "Gas 2 described in FIG.", "3, preferably sweep gas).", "FIG.", "6 shows one embodiment of the device according to the illustration in FIG.", "2, where the combustion chamber (F-01), as well as the pressure booster (I-01) are installed outside the reactor.", "Fuel (NG) is injected in the low temperature zone prior to (I-01) to ensure a good mixing with the oxygen enriched sweep gas (EGO) before entering the combustion chamber (F-01).", "Due to a too low temperature the combustion, at least partly, might be enhanced by a catalyst.", "The sweep gas stream (EGO) leaving (H-01) is cooled down by the air stream (AN) and has its lowest temperature before (I-01).", "The pressure in the stream (EGO) is increased by means of (I-01) before entering the combustion chamber (F-01) outside the reactor.", "In (F-01) oxygen in stream (EGO) reacts with added fuel and a combustion is obtained.", "In the combustion nearly all oxygen is consumed.", "Thus the exhaust gas (EG), mainly containing the reaction products (CO2 and H2O), will have a low content of oxygen.", "(EG) enters the second heat exchanger (H-02) where it is heating the oxygen depleted air stream (AL) leaving the reactor.", "(EG) is thus somewhat cooled down by (AL) in (H-02) before it enters the membrane module(s) (X-01).", "In (X-01) (EG) acts as a sweep gas picking up oxygen transferred through the membrane wall from the air side.", "The oxygen enriched sweep gas leaving (X-01), now named (EGO), is then entering the first heat exchanger (H-01) where the air stream (AN) is heated and the stream (EGO) is cooled.", "Thus a cooled oxygen containing sweep gas (EGO) is now returning via (I-01) to (F-01) and thus an exhaust/sweep gas loop is obtained enhancing a continuous combustion.", "Either from the oxygen enriched sweep gas (EGO) or from the exhaust gas (EG) a bleed gas has to be taken out to prevent accumulation of mass in the sweep gas loop due to the oxygen transfer from the air and the addition of the fuel.", "Example of bleed gas outlet is shown in FIGS.", "8.1 and 9.1.Also shown in FIG.", "6 are some of the individual components of the reactor.", "FIG.", "7 shows a more detailed embodiment of the device according to the present invention.", "Reactor pressure vessel 1 contains the low temperature heat exchanger 9, the high temperature heat exchanger 19 and the MCM-modules 15.Thus all other parts are built up around these units 9, 15 and 19 which ensure good heat transfer (from sweep/exhaust gas to air) and oxygen transfer (from air to sweep gas).", "The parts 8, 14 and 18 are used to make a round shape at the outer wall of the heat exchangers and MCM-modules to ensure less complicated sealing.", "These parts could also be made with channels in such a way that they can be used as heat exchangers 8 and 18 or as MCM-modules 14.The individual parts 10, 11, 12 and 13 will fit together and make the connection between the low temperature heat exchanger 9 and the MCM-modules 15 as shown in FIG.", "5.3.In the same way the individual parts 16, 17, 20 and 21 will make the connection between the MCM-modules 15 and the high temperature heat exchanger 19.The coupling part 11 preferably will be glass sealed in both ends to 9 and 15 and part 21 respectively will be sealed to 15 and 19.Thus the material in 11 has to match the thermal expansion of both 9 and 15 and respectively the material in 21 has to match the thermal expansion of 15 and 19.One option is to extrude these connection parts 11 and 21 with a gradual change in composition of material such that the material in the end of 11 connected to 9 matches its thermal expansion, respectively the other end of 11 matches the thermal expansion of 14.In the same way 21 also could be made in such a way that thermal expansion is matching material in both 15 and 19 to prevent cracks.", "Also the inlet plenum room for air, unit 7 could be glass sealed to the low temperature heat exchanger 9 to ensure minimum leakage.", "Thus also the material in 7 has to match the thermal expansion of 9.In the same way the outlet plenum 23 for oxygen depleted air might be glass sealed to 18 and 19 and thus 23 has to be made of a material that matches 18 and 19 in thermal expansion.", "Part 7 is in the inlet end (incoming air) made of a round shape (pipe) to make it easier to fit into a flexible sealing 5.Respectively this is also done for the outlet plenum 23 (of the oxygen depleted high temperature air).", "Also here, in same way as inlet, a ring sealing, 24 is shown.", "For a vertical orientation as shown in FIG.", "7 a lower flexible sealing may not be necessary.", "This end could be fixed and thermal expansion allowed to take place in the upper end through the flexibility of seal 5.Thus, in at least one end, a sealing that allows expansion in the longitudinal direction has to be included.", "In the present invention this is solved by designing the inlet and/or outlet connectors 4 and 25 in a round shape (pipe end).", "Thus this makes it easier to have a flexible sealing.", "Flexible sealing rings 5 and 24 have to be made of a temperature resistant material (ceramic or metal).", "Also other flexible “pipe” sealing systems is possible.", "The inlet and outlet pipes 4 and 25 may have the same shape to simplify the fabrication.", "Inlet pipe 4 leads the air stream to the inlet plenum made up by 7 and made in such a way that flexible sealings 5 can be mounted.", "The inlet pipe 4 is most preferably made of a material that also acts as a thermal barrier or lining between the hot inlet air and the outer metal pipe connected to the pressure vessel shell.", "This is especially important for the outlet pipe 25 in the high temperature end.", "Also shown are parts 6 and 22 that act as a thermal barrier or lining between exhaust/sweep gas and the flanged inlet/outlet metal pipe of the pressure vessel.", "Also shown is the thermal barrier and insulation 3 between the high temperature inner parts and the outer metal wall or shell of the pressure vessel Keeping a low temperature (<500° C.) in the outer pressure shell will reduce heat loss and allow the pressure shell to be made of a common engineering material (i.e.", "carbon steel).", "By lowering the temperature, the thickness of the wall and thus also the total weight of the device is reduced.", "This is important for an offshore installation.", "Parts 3 are also made in a shape and of such a material that it can act as support for the inner parts.", "2 is a layer of a flexible material between the inner wall (pressure shell) and 3 allowing for some movement caused by thermal expansion.", "FIG.", "8.1 shows one embodiment of the device according to the present invention where the device is integrated with gas turbines.", "FIG.", "8.2 shows another embodiment of integrating the reactor with gas turbines where more than one reactor have a common combustion chamber.", "According to the present invention one or more reactor units can be coupled together and share a common combustion chamber as shown in FIG.", "8.1.This will allow multiple production of standard sized reactors and a cost efficient production by increasing total power output (upscaling) by integrating or coupling standard sized reactors together as shown in FIG.", "8.2.If for example the single device in the plant as shown in FIG.", "8.1 is producing 10 MW of power, the plant shown in FIG.", "8.2 having 6 reactors of the same size as a standard single reactor the plant will produce about 60 MW.", "Shown in FIG.", "8.1 are two different alternatives for discharging the bleed stream.", "One alternative (Alt.", "1) is to discharge a bleed stream from the cold part of the sweep gas loop.", "The bleed stream will have a temperature that allows it to be sent directly to a steam turbine.", "The bleed stream taken out as shown in alternative one contains oxygen and this process stream can thus be used for further heat generation in a nitrogen free atmosphere and further as a heat source in an endothermic process.", "In alternative two (Alt.", "2) a bleed stream is discharged after the combustion and thus it is almost oxygen free and at a high temperature level.", "If a steam turbine is to be used to enhance power generation from this stream, the temperature must be lowered, i.e.", "by injecting water.", "The bleed stream can be discharged anywhere in the sweep gas stream loop.", "Also shown in FIG.", "8.1 is that the inlet air pipe (from compressor to reactor) is longer than outlet lean air pipe (from reactor to turbine).", "This is found advantageous due to the higher temperature of the outlet oxygen lean air stream compared to the inlet air stream.", "FIG.", "9.1 shows the device according to the present invention with the flow direction of the different gas streams.", "The figure shows that an oxygen containing gas stream (AN-030), preferably a compressed air stream, is fed to the heat exchange module (H-01) where the gas stream is heated before entering the mixed conducting membrane module (X-01).", "Oxygen is transported through the membrane wall to be picked up by the sweep gas stream (EG-030).", "An oxygen enriched sweep gas stream leaves the module (X-01) now named (EGO-010).", "A part of the total fuel, (NG-030), is mixed with stream (EGO-010) in an additional combustion chamber (F-02) situated between (X-01) and (H-01) where the heat generated from this combustion will be supplied to heat exchanger (H-01) for heating incoming air.", "It has to be emphasized that the present invention will work without this combustion chamber (F-02) as explained in FIG.", "6.For this embodiment the sweep gas stream (EGO-020) entering the heat exchanger (H-01) will have somewhat higher temperature than the stream (EGO-010) and somewhat lower content of oxygen.", "The sweep gas stream (EGO-020) is then fed to the heat exchanger (H-01) for heating incoming air to the MCM-module (X-01).", "The sweep gas stream (EGO-030) leaving (H-01) has now its lowest temperature and is supplied to the main combustion chamber (F-01) outside the reactor where most of the fuel (NG-020) is burned.", "A pressure booster (I-01) is installed close to the inlet of the main combustion chamber (F-01).", "The pressure increase from (EGO-030) to (EGO-040) enhanced by the pressure booster (I-01) is to ensure circulation in the sweep/exhaust gas loop.", "A part of the hot exhaust gas (EG-040) is discharged as a bleed stream to prevent accumulation of mass in the exhaust/sweep gas loop.", "In principle the bleed gas stream (EG-040) can be discharged anywhere in the sweep gas circulation loop.", "For example it can be discharged in the cold end, from (EGO-030), and sent directly to a steam turbine.", "The exhaust gas (EG-020) is fed via the high temperature heat exchanger (H-02) to the membrane module (X-01).", "Acting as sweep gas, (EG-030) is receiving oxygen transported through the membrane from the air side and further transports the oxygen to the combustion chamber.", "Thus a closed loop with a continuous combustion of a carbon rich fuel with O2 in a CO2 and H2O rich atmosphere is obtained.", "FIG.", "9.2 shows how the plenum inlet and outlet 7 and 23 and heat exchangers (H-01) and (H-02) and the MCM-module (X-01) can be built into one sealed unit.", "This is to illustrate one important feature of the present invention which is the flow direction or flow paths of the two main streams air and sweep gas that contributes to minimize the leakage between air and sweep gas stream.", "The air stream has a straight flow and flows directly through the inner closed rooms between the heat exchangers (H-01) and (H-02) and the MCM-module (X-01), while the sweep gas stream flows in and out of the open side slots of (H-01), (X-01) and (H-02).", "To ensure a pressure build up inside the reactor the sweep gas should be allowed to fill the open space of the reactor.", "This will ensure that only outer reactor shells have to be designed for withstanding total pressure of the process.", "Table 1 below gives example of data for the process flows with numbers according to FIG.", "9.1.Inlet conditions for the air stream; 20 bar, 450° C. and 79 kg/s.", "Oxygen transport through membrane is 6.12 kg/s (membrane area is installed according to this).", "Fuel is added to match the stoichiometry of the combustion reaction.", "A further advantage will be to have a low pressure difference (<5 bar) between the air side and the sweep gas side, preferably with somewhat higher pressure on the sweep gas side.", "This will ensure, in case of leakage between the stream and the sweep gas stream, that the direction of leakage will be from the sweep gas side (CO2 and H2O) into the air side.", "This will be less harmful than if air leaks into the combustion loop (sweep gas), especially from an environmental point of view because in case of nitrogen (air) leakage into combustion (sweep gas loop) the NOx gas could be produced.", "Further, a low pressure difference between air and sweep gas side will allow designing with thinner walls in monoliths and thus better heat and oxygen (only X-01) transfer.", "This will also result in lower weight.", "Table 1 below gives example of data for the process flows with numbers according to FIG.", "9.1.Inlet conditions for the air stream; 20 bar, 450° C. and 79 kg/s.", "Oxygen transport through membrane is 6.12 kg/s (membrane area is installed according to this).", "Fuel is added to match the stoichiometry of the combustion reaction.", "TABLE 1 Stream tag no.", ": Components: AL-010 AL-020 AN-030 AN-050 EG-010 EG-020 Mole Flow kmol/sec CH4 0 0 0 0 0.005724 0.004866 C2+ 0 0 0 0 0 0 CO2 0.00090 0.00090 0.00090 0.00090 0.68614 0.58322 N2 2.123768 2.123768 2.123768 2.123768 0.006194 0.005265 O2 0.3784649 0.3784649 0.5697220 0.5697220 0 0 Ar 0.0254891 0.0254891 0.0254891 0.0254891 0 0 H2O 0.0120752 0.0120752 0.0120752 0.0120752 1.212909 1.030973 Total Flow kmol/sec 2.540698 2.540698 2.731956 2.731956 1.91097 1.624324 Total Flow kg/sec 72.88 72.88 79.00 79.00 52.31 44.47 Total Flow cum/sec 13.99 16.33 8.31 14.67 12.16 10.33 Temperature C. 1019 1221 453 1000 1256 1256 Pressure bar 19.60 19.40 20.00 19.80 20.05 20.05 Entholpy kJ/kmol 30140.18 36894.61 11691.54 29700.68 −240270 −240270 Entholpy kJ/kg 1050.729 1286.197 404.3137 1027.1 −8776.913 −8776.913 Entholpy kW 76577.1 93738.07 31940.78 81140.93 −459150 −390280 Entropy J/kmol-K 24739.17 29681.75 6514.543 25024.88 22507.69 22507.69 Entropy J/kg-K 862.4418 1034.747 225.2841 865.4033 822.1919 822.1919 Density kmol/cum 0.1815737 0.1555783 0.3286462 0.186165 0.1572019 0.1572019 Density kg/cum 5.208446 4.462768 9.503466 5.383335 4.303437 4.303437 Average MW 28.68503 28.68503 28.91701 28.91701 27.37522 27.37522 Stream tag no.", ": Components: EG-030 EG-040 EGO-010 EGO-020 EGO-030 Mole Flow kmol/sec CH4 0.004866 0.000859 0.004866 0 0 C2+ 0 0 0 0 0 CO2 0.58322 0.10292 0.58322 0.59091 0.59091 N2 0.005265 0.000929 0.005265 0.005290 0.005290 O2 0 0 0.1912572 0.1762761 0.1762761 Ar 0 0 0 0 0 H2O 1.030973 0.1819364 1.030973 1.045701 1.045701 Total Flow kmol/sec 1.624324 0.2866454 1.815581 1.818177 1.818177 Total Flow kg/sec 44.47 7.85 50.59 50.63 50.63 Total Flow cum/sec 8.96 1.82 9.85 10.47 5.93 Temperature C. 1050 1256 1028 1093 505 Pressure bar 20.00 20.05 19.80 19.80 19.75 Entholpy kJ/kmol −250840 −240270 −221900 −221700 −248760 Entholpy kJ/kg −9162.846 −8776.913 −7964.114 −7960.869 −8932.566 Entholpy kW −407440 −68872.29 −402870 −403080 −452280 Entropy J/kmol-K 15111.08 22507.69 17971.26 20163.23 −5530.105 Entropy J/kg-K 551.9985 822.1919 645.0031 724.0367 −198.5793 Density kmol/cum 0.1812482 0.1572019 0.1843344 0.1737103 0.3064727 Density kg/cum 4.96171 4.303437 5.135978 4.837547 8.53476 Average MW 27.37522 27.37522 27.86228 27.84835 27.84835 Stream tag no.", ": Components: EGO-040 NG-020 NG-030 OX-010 () Mole Flow kmol/sec CH4 0 0.069575 0.001946 0 C2+ 0 0.0009874728 0.00029685977 0 CO2 0.59091 0.00254 0.00007 0 N2 0.005290 0.000904 0.000025 0 O2 0.1762761 0 0 0.1912572 Ar 0 0 0 0 H2O 1.045701 5.02105E−005 1.40470E−006 0 Total Flow kmol/sec 1.818177 0.0836758 2.34093E−003 0.1912572 Total Flow kg/sec 50.63 1.68 0.05 6.12 Total Flow cum/sec 5.83 0.05 0.0015 0.97 Temperature C. 511 34 34 1000 Pressure bar 20.25 36.00 36.00 21.00 Entholpy kJ/kmol −248530 −87413.69 −87413.69 32362.44 Entholpy kJ/kg −8924.502 −4353.816 −4353.816 1011.364 Entholpy kW −451880 −7314.411 −204.6293 6189.548 Entropy J/kmol-K −5449.594 −122240 −122240 21762.67 Entropy J/kg-K −195.6882 −6088.488 −6088.488 680.109 Density kmol/cum 0.3120345 1.533792 1.533792 0.1975274 Density kg/cum 8.689646 30.7947 30.7947 6.32064 Average MW 27.84835 20.07749 20.07749 31.9988 () The oxygen flow (OX-010) is not a physical gas flow as shown in the table.", "Oxygen is transported through the membrane as oxygen ions and thus (OX-010) in Table 1 is for the purpose of calculation." ] ]	Patent_10451729
[ [ "Antiglare,anticlouding element", "An anti-glare and anti-fogging element 1 consisting of an anti-glare element 5 having an electrochromic cell 4 between a front transparent base plate 2 and a back transparent base plate 3 and an anti-fogging element 8 formed on the front transparent base plate 2, which is configured as an anti-glare and anti-fogging mirror 10 by laminating a reflection film 9 on the back of the back transparent base plate 3 and has a reflectivity of 25% or less at the time of glare prevention, wherein the electrochromic cell 4 is composed of a front electrode and a back electrode sandwiching a substance, which exhibits an electrochromic phenomenon, therebetween; the anti-fogging element 8 is composed of a transparent photocatalytic reaction substance film of TiO2 laminated on the front side of the front transparent base plate 2, which exhibits a photocatalytic reaction, and a transparent porous inorganic oxide film 7 laminated on the photocatalytic reaction substance film 6, which exhibits a hydrophilic property; the photocatalytic reaction substance film 6 is 100 to 170 nm in thickness; and the porous inorganic oxide film 7 is 10 to 50 nm in thickness." ], [ "1.An anti-glare and anti-fogging element consisting of an anti-glare element having an electrochromic cell between a front transparent base plate and a back transparent base plate and an anti-fogging element placed on said front transparent base plate, which is configured as an anti-glare and anti-fogging mirror by laminating a reflection film on the back of said back transparent base plate and has a reflectivity of 25% or less at the time of glare prevention, characterized in that said electrochromic cell is composed of a front electrode and a back electrode sandwiching a substance, which exhibits an electrochromic phenomenon, therebetween; said anti-fogging element is composed of a transparent photocatalytic reaction substance film of TiO2 laminated on the front side of said front base plate, which exhibits a photocatalytic reaction, and a transparent porous inorganic oxide film laminated on said photocatalytic reaction substance film, which exhibits a hydrophilic property; and said photocatalytic reaction substance film is 100 to 170 nm in thickness and said porous inorganic oxide film is 10 to 50 nm in thickness.", "2.The anti-glare and anti-fogging element according to claim 1, characterized in that resistant heating body is laminated on the back of the side where said reflection film of said anti-glare element is formed and said heating body is configured to conduct electricity.", "3.The anti-glare and anti-fogging element according to claim 1 or 2, characterized in that said anti-glare and anti-fogging mirror is configured as an automotive outer mirror." ], [ "<SOH> FIELD OF THE INVENTION <EOH>The present invention relates to anti-glare and anti-fogging elements for use in an automotive mirror, display device, a light control glass, and the like." ], [ "<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.", "1 illustrates a basic configuration of an anti-glare and anti-fogging element according to the present invention.", "FIG.", "2 illustrates a basic configuration of an anti-glare and anti-fogging element in which a reflection film is formed on the anti-glare and anti-fogging element according to the present invention shown in FIG.", "1 .", "FIG.", "3 illustrates a configuration of an anti-fogging element on a glass base plate upon measuring surface reflectivity.", "FIG.", "4 is a graph showing experimental data illustrating relationship between the thickness of TiO 2 film and SiO 2 film of an anti-fogging element and the surface reflectivity.", "FIG.", "5 is a graph showing experimental data of spectral reflectivity characteristics of an anti-fogging element when the thickness of an SiO 2 film on a TiO 2 film is changed.", "FIG.", "6 illustrates an anti-glare and anti-fogging element of Example 1 according to the present invention.", "FIG.", "7 illustrates an anti-glare and anti-fogging element of Example 2 according to the present invention.", "FIG.", "8 illustrates an anti-glare and anti-fogging element of Example 3 according to the present invention.", "FIG.", "9 illustrates an anti-glare and anti-fogging element of Example 4 according to the present invention.", "FIG.", "10 illustrates an anti-glare and anti-fogging element of Example 5 according to the present invention.", "FIG.", "11 illustrates an anti-glare and anti-fogging element of Example 6 according to the present invention.", "FIG.", "12 illustrates an anti-glare and anti-fogging element of Example 7 according to the present invention.", "FIG.", "13 illustrates an anti-glare and anti-fogging element of Example 8 according to the present invention.", "FIG.", "14 illustrates an anti-glare and anti-fogging element of Example 9 according to the present invention.", "FIG.", "15 illustrates an experimental example in regard to dazzling of a mirror with an anti-glare and anti-fogging element.", "FIG.", "16 is a plan top view of FIG.", "15 .", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention relates to anti-glare and anti-fogging elements for use in an automotive mirror, display device, a light control glass, and the like.", "DESCRIPTION OF THE PRIOR ART An anti-fogging element consisting of a photocatalytic reaction substance and a porous inorganic oxide film is already known (Japanese Patent Application Laid-open No.", "H10-36144).", "Also, an anti-fogging photocatalytic composition composed of a semiconductive photocatalytic substance and fine oxide particles dispersed in said semiconductive photocatalytic substance is known (Japanese Patent Application Laid-open No.", "H09-225303).", "Furthermore, an anti-glare element comprising a material in between which exhibits an electrochromic phenomenon causing reversible color change of the material upon application of a voltage is known.", "In such an anti-glare element, an anti-glare and anti-fogging element in which a transparent photocatalytic reaction substance, which exhibits a photocatalytic reaction, and a porous layer of a transparent inorganic oxide, which exhibits hydrophilicity, are laminated one on top of the other on the front side of one side of a base plate is already known (Japanese Patent Application Laid-open No.", "H10-347837).", "Although Japanese Patent Application Laid-open No.", "H10-36144 and Japanese Patent Application Laid-open No.", "H09-225303 disclose configurations which utilize a semiconductive photocatalytic substance in an anti-fogging element, they don't disclose any configuration which comprises a substance, which exhibits an electrochromic phenomenon, and an anti-glare element in combination.", "The invention according to Japanese Patent Application Laid-open No.", "H10-347837 is a combined configuration of an anti-fogging element comprising a photocatalytic reaction substance (TiO2 film) and a porous inorganic oxide (porous SiO2 film) and an anti-glare element comprising an in-between substance which exhibits an electrochromic phenomenon; however, an anti-glare function is not sufficiently taken into consideration in this invention.", "Namely, when an anti-fogging element is composed of an SiO2 film and a TiO2 film, its surface reflectivity greatly changes depending on the thickness of the film because of the high reflectivity of TiO2.The anti-glare effect is reduced when an anti-fogging element having such a film thickness as to generate high surface reflectivity, and an electrochromic anti-glare element are combined.", "An object of the present invention is to provide an anti-glare and anti-fogging element in which reduction of an anti-glare effect can be prevented even when an anti-fogging element comprising an SiO2 film and TiO2 film is combined with an anti-glare element having an electrochromic substance.", "DISCLOSURE OF THE INVENTION In order to solve the abovementioned problem, the present invention provides an anti-glare and anti-fogging element consisting of an anti-glare element having an electrochromic cell between a front transparent base plate and a back transparent base plate and an anti-fogging element formed on said front transparent base plate, which is configured as an anti-glare and anti-fogging mirror by laminating a reflection film on the back of said back transparent base plate and has a reflectivity of 25% or less at the time of glare prevention, characterized in that said electrochromic cell is composed of a front electrode and a back electrode sandwiching a substance, which exhibits an electrochromic phenomenon, therebetween; said anti-fogging element is composed of a transparent photocatalytic reaction substance film of TiO2 laminated on the front side of said front base plate, which exhibits a photocatalytic reaction, and a transparent porous inorganic oxide film laminated on said photocatalytic reaction substance film, which exhibits a hydrophilic property; said photocatalytic reaction substance film is 100 to 170 nm in thickness; and said porous inorganic oxide film is 10 to 50 nm in thickness.", "The abovementioned element may comprise a resistant heating body laminated on the back of the side where said reflection film of said anti-glare element is formed and said heating body is configured to conduct electricity.", "The abovementioned anti-glare and anti-fogging mirror can also be used as an automotive outer mirror.", "BRIEF DESCRIPTION OF DRAWINGS FIG.", "1 illustrates a basic configuration of an anti-glare and anti-fogging element according to the present invention.", "FIG.", "2 illustrates a basic configuration of an anti-glare and anti-fogging element in which a reflection film is formed on the anti-glare and anti-fogging element according to the present invention shown in FIG.", "1.FIG.", "3 illustrates a configuration of an anti-fogging element on a glass base plate upon measuring surface reflectivity.", "FIG.", "4 is a graph showing experimental data illustrating relationship between the thickness of TiO2 film and SiO2 film of an anti-fogging element and the surface reflectivity.", "FIG.", "5 is a graph showing experimental data of spectral reflectivity characteristics of an anti-fogging element when the thickness of an SiO2 film on a TiO2 film is changed.", "FIG.", "6 illustrates an anti-glare and anti-fogging element of Example 1 according to the present invention.", "FIG.", "7 illustrates an anti-glare and anti-fogging element of Example 2 according to the present invention.", "FIG.", "8 illustrates an anti-glare and anti-fogging element of Example 3 according to the present invention.", "FIG.", "9 illustrates an anti-glare and anti-fogging element of Example 4 according to the present invention.", "FIG.", "10 illustrates an anti-glare and anti-fogging element of Example 5 according to the present invention.", "FIG.", "11 illustrates an anti-glare and anti-fogging element of Example 6 according to the present invention.", "FIG.", "12 illustrates an anti-glare and anti-fogging element of Example 7 according to the present invention.", "FIG.", "13 illustrates an anti-glare and anti-fogging element of Example 8 according to the present invention.", "FIG.", "14 illustrates an anti-glare and anti-fogging element of Example 9 according to the present invention.", "FIG.", "15 illustrates an experimental example in regard to dazzling of a mirror with an anti-glare and anti-fogging element.", "FIG.", "16 is a plan top view of FIG.", "15.BEST MODE OF CARRYING OUT THE INVENTION Mode of embodiment of an anti-glare and anti-fogging element according to the present invention will be explained referring to the attached drawings.", "[Basic Configuration] FIG.", "1 is a schematic drawing illustrating a basic configuration of an anti-glare and anti-fogging element according to the present invention.", "This anti-glare and anti-fogging element 1 comprises a combination of an anti-glare element 5 composed of a front transparent base plate 2 and a back transparent base plate 3 sandwiching an electrochromic cell 4, which exhibits an electrochromic phenomenon, therebetween; and an anti-fogging element 8 composed of a transparent photocatalytic reaction substance film 6, which exhibits a photocatalytic reaction, and a transparent porous inorganic oxide film 7, which functions as a hydrophilic film, and the anti-fogging element 8 is laminated on the front side of the front transparent base plate 2 of the anti-glare element 5.In the electrochromic cell 4 in FIG.", "1, a substance changes its color by a coloring or decoloring reaction caused by electron transfer when a voltage is applied between a front electrode and a back electrode.", "This electrochromic cell is already being utilized for anti-glare mirrors such as automotive anti-glare mirrors and light control glasses.", "The porous inorganic oxide film 7 is composed of an SiO2 film.", "This SiO2 film is formed to be porous having numerous fine holes opening to the surface to exhibit a hydrophilic property.", "Namely, water attached to the surface of the porous inorganic oxide film 7 is transferred into the numerous fine holes by a capillary phenomenon and thinly spread on the surface to prevent water drop formation, which consequently provides an anti-fogging effect.", "The photocatalytic reaction substance film 6 is composed of a TiO2 film.", "Ultraviolet light incoming from the front side of the anti-fogging element 8 passes through the transparent porous inorganic oxide film 7 and is absorbed at the photocatalytic reaction substance film 6 to excite the photocatalytic reaction substance film 6, which generates electron-hole pairs in the photocatalytic reaction substance film 6.These electron-hole pairs move up to the porous inorganic oxide film 7 and react with air and water on the surface of the porous inorganic oxide film 7 to generate highly oxidative O2− (superoxide anions) and .OH (hydroxy radicals).", "They decompose or remove organic substances such as dirt stuck or packed on the surface or inside the fine holes of the porous inorganic oxide film 7 and thus prevent the reduction of the hydrophilicity of the surface of the anti-glare element 8.FIG.", "2 is a drawing showing an anti-glare and anti-fogging mirror 10 which is an applied example of this anti-glare and anti-fogging element.", "This anti-glare and anti-fogging mirror 10 comprises a reflection film 9 arranged on the back of the anti-glare and anti-fogging element 1.This anti-glare and anti-fogging mirror 10 is used, for example, as an automobile outer mirror.", "In this case, the anti-glare and anti-fogging mirror 10 functions as an ordinary mirror by keeping the electrochromic cell 4 in a decoloring state during daytime.", "On driving at night or in a tunnel, a voltage is applied to the electrochromic substance to color the electrochromic cell 4 so that the amount of light passing through the reflection film is reduced and thus the amount of light reflected by the anti-glare and anti-fogging mirror 10 can be reduced.", "As a result, the anti-glare and anti-fogging mirror 10 provides an anti-glare effect by reducing the amount of light reflected to the incident rays such as light cast by headlights of a car following behind.", "In this way, the anti-glare and anti-fogging mirror 10 exhibits an anti-glare function.", "At the same time, as mentioned above, the porous inorganic substance film 7 makes the surface of the anti-fogging element 8 hydrophilic to provide an anti-fogging effect and dirt or the like packed in fine holes of the porous inorganic substance film 7 is decomposed or removed by the action of the photocatalytic reaction substance film 6 so that the anti-fogging element 5 can retain its hydrophilic property and the resulting anti-fogging effect.", "The materials consisting of the anti-fogging element 8 have the following refraction index denoted n. Glass composing the transparent base plate: n=approx.", "1.5, TiO2 composing the photocatalytic reaction substance film 6: n=approx.", "2.3, and SiO2 composing the porous inorganic substance film: n=approx.", "1.4.The refraction index of TiO2 is higher than that of glass.", "FIG.", "4 is an experimental data showing the change in the surface reflectivity of TiO2 (n=approx.", "2.3) and SiO2 (n=approx.", "1.4) in different film thickness, in the range of visible wavelength.", "While the surface reflectivity of the glass base plate (n=approx.", "1.5) in the range of visible wavelength (380-780 nm) is about 4.2%, the surface reflectivity of the TiO2 film comprising the photocatalytic reaction substance film 6 greatly varies depending on the film thickness as shown in FIG.", "4.The surface reflectivity of the film of SiO, which has a refraction index close to that of glass, slightly varies depending on the film thickness.", "An anti-glare and anti-fogging element according to the present invention is characterized by a configuration having the following range of figures in order to attain a reflectivity of 25% or less, which is considered to prevent from feeling dazzled (reasons will be shown in an experiment described hereinafter) although there may be some variation among individuals in feeling, and a reflectivity of 24% or less for the anti-fogging element, further to provide a function as a photocatalyst and an excellent resistance to abrasion, and to reduce view angle dependence which disadvantageously changes a tone of the color depending on view angles.", "Namely, the thickness of the photocatalytic reaction substance film (TiO2 film) is limited within the range of 100 to 170 nm, and the thickness of the porous inorganic substance film (SiO2 film) is limited within the range of 10 to 50 nm.", "Significance of limiting figures within these ranges, in particular the critical significance of upper and lower limits of these ranges will be explained below.", "First, the lower limits of the ranges of the thickness of the photocatalytic reaction substance film and the porous inorganic substance film will be explained.", "The relation between the thickness of the photocatalytic reaction substance film 6 and the photocatalytic function is explained referring to the result of an experiment carried out by the present inventors, shown in the following Table 1.In this experiment, a TiO2 film 6 was laminated on a transparent glass base plate 2 as a photocatalytic reaction substance film and then an SiO2 film 7 was formed thereon as a porous inorganic substance film to construct an anti-fogging element (See FIG.", "3).", "Multiple anti-fogging elements, prepared in this way, each having a TiO2 film 6 of different film thickness as shown in Table 1 were used.", "The waterdrop contact angle of each anti-fogging element was evaluated by actually mounting it on a driving car for a period of 6 months, during which the car was subjected to automatic double-waxing car wash once a month.", "As shown in Table 1, when the thickness of the TiO2 film is 100 nm or more, the contact angle is less than 20 degrees and thus the hydrophilicity can be maintained to provide an anti-fogging effect.", "TABLE 1 Thickness of photocatalytic Waterdrop contact angle TiO2 film Start After 6 months 75 nm Less than 5 degrees 30-40 degrees 100 nm ↑ Less than 20 degrees 150 nm ↑ Less than 10 degrees 200 nm ↑ ↑ 300 nm ↑ ↑ The following Table 2 shows the result of an experiment in which porous SiO2 films each having a different film thickness were prepared and the surface of each film was rubbed 10,000 times in a reciprocating motion using a rubbing unit wrapped with a cloth at a load of 1 N/cm2, and then observed.", "As shown in Table 2, sufficient resistance to abrasion can be attained when the thickness of the porous SiO2 film is 10 nm or more.", "Therefore, a thickness of 10 nm or more is appropriate for the porous SiO2 film.", "TABLE 2 Thickness of porous SiO2 film Surface appearance 5 nm Abrasion observed 10 nm No abrasion observed.", "20 nm ↑ 30 nm ↑ From the results above, in an anti-fogging element comprising a TiO2 film 6 as an photocatalytic reaction substance film laminated on a transparent glass base plate and an SiO2 film 7 as an porous inorganic substance film formed thereon, the lower limit of the thickness of the photocatalytic reaction substance film (TiO2 film) was set to be 100 nm and the lower limit of the thickness of the porous inorganic substance film (SiO2 film) was set to be 10 nm.", "Next, the upper limits of the photocatalytic reaction substance film (TiO2 film) and the porous inorganic substance film (SiO2 film) are explained.", "First, the SiO2 film is to be explained.", "As the thickness of the SiO2 film increases, it becomes hard for electron-hole pairs generated in the photocatalytic reaction substance film (TiO2 film) to move through the hydrophilic porous inorganic substance film, which interferes with the rapid proceeding of the photocatalytic reaction.", "Taking this interference into consideration, in order to promote the rapid photocatalytic reaction, the SiO2 film have to be about 50 nm in thickness and thus the upper limit is set to be 50 nm.", "Further, it has been confirmed that the waterdrop contact angle is 20 degrees or less when the thickness of the SiO2 film is 50 nm or less.", "Next, the upper limit of the photocatalytic reaction substance film (TiO2 film) is to be explained.", "As explained referring to FIG.", "4, the surface reflectivity of the TiO2 film greatly varies depending on the thickness of the TiO2 film.", "Here, although the lower limit of the thickness of the TiO2 film is set to be 100 nm as mentioned above, the surface reflectivity is minimum at a thickness of about 120 nm and then gradually increases reaching its maximum at a thickness of about 180 nm, after which the reflectivity decreases and then increases again, as shown in the curves for the TiO2 films having a thickness of greater than 100 nm, in FIG.", "4.Although the reflectivity also decreases at a film thickness of near 240 nm and 370 nm, the reflectivity in itself is higher than that at a thickness of near 120 nm.", "Further, as the film thickness increases, the number of spectral peaks and troughs increases, which causes a view angle dependent problem such that the tone of the color changes depending on the view angle.", "Therefore, a thickness of near 120 nm is considered to be most appropriate.", "Since the anti-fogging element 8 comprises a double-layer configuration in which the porous inorganic substance film (SiO2 film) is laminated on the photocatalytic reaction substance film (TiO2 film), it is important to set the upper limit of the thickness of the TiO2 film by taking the laminated SiO2 film into consideration.", "As mentioned above, although the lower limit and the upper limit of the thickness of the SiO2 film are set to be 10 nm and 50 nm, respectively, while the refraction index of the SiO2 film is as low as about 1.4, the reflectivity of the anti-fogging element itself comprising the SiO2 film formed on the TiO2 film decreases as the thickness of the SiO2 film increases when the SiO2 film is laminated on the TiO2 film in the range of 10 nm to 50 nm in thickness.", "In this connection, FIG.", "5 shows spectral reflectivity characteristics of the anti-fogging elements obtained by measuring the spectral reflectivity of the anti-fogging elements in which the thickness of the photocatalytic reaction substance film (TiO2 film) is set to be 100 nm and the porous inorganic substance film (SiO2 film) is laminated thereon at a thickness of 10 nm, 30 nm or 50 nm.", "FIG.", "5 shows that the reflectivity of the anti-fogging element decreases as the thickness of the SiO2 film increases, which is similarly observed when the thickness of the TiO2 film is changed.", "Although the thickness of the TiO2 film has thus no effect on the surface reflectivity of the anti-fogging element, it is necessary to discuss the effect of the thickness of the TiO2 film on reflection of the entire anti-glare and anti-fogging element and on the surface reflectivity of the anti-fogging element, since the anti-glare and anti-fogging element according to the present invention comprises a combination of the electrochromic anti-glare element and the anti-fogging element.", "Accordingly, the present inventors carried out an experiment in which the thickness of the SiO2 film in the anti-glare and anti-fogging element was set to be 10 nm (the lower limit allowable in terms of abrasion and to attain the maximal reflectivity of the anti-fogging element) and the surface reflectivity of the anti-fogging element on the glass base plate and the reflectivity of the anti-glare and anti-fogging element at the time of glare prevention were confirmed by changing the thickness of the TiO2 film.", "In this case, the reflectivity of the anti-glare and anti-fogging element never be lower than the reflectivity of the anti-fogging element since the anti-fogging element is formed on the front side in the anti-glare and anti-fogging element comprising a combination of the electrochromic anti-glare element and the anti-fogging element according to the present invention.", "In short, however low the reflectivity of the anti-glare element may be, it never be lower than the reflectivity of the anti-fogging element.", "Accordingly, taking the efficiency and durability of an electrochromic anti-glare element into consideration, an electrochromic anti-glare element having a configuration, to be explained below, which supposedly provides a reflectivity as low as possible was prepared, and then the reflectivity of an anti-glare and anti-fogging element having the anti-fogging element formed on its surface was measured.", "(Here, although the upper limit of the reflectivity of the electrochromic anti-glare element at the time of glare prevention should be shown, not the upper limit but the lower limit was shown since the reflectivity varies depending on the configuration of the electrochromic anti-glare element and the running voltage).", "The abovementioned electrochromic anti-glare element, which supposedly provides a reflectivity as low as possible, used in this experiment is explained as follows.", "A configuration shown in FIG.", "14 was applied in this experiment.", "In the embodied configuration, an ITO film 200 nm in thickness is used as a transparent conductive film 13 laminated on a 1.1 mm-thick glass base plate 11.On the other hand, a Cr—Rh film 100 nm in thickness is laminated as an electrode/reflection film 14 on a back glass base plate 12.An electrochromic solution 28 is composed of propylene carbonate as a solvent, 1,1′-dibenzyl-4,4′-bipyridinium fluroborate as a reductive coloring agent, and 5,10-dihydro-5,10-dimethyl fenadine as an oxidative coloring agent.", "An applied voltage was 1.3 V. The result of the experiment is shown in Table 3.As shown in Table 3, the reflectivity of the anti-glare and anti-fogging element was confirmed to be less than 25%, which is considered to be the upper limit of the reflectivity to attain an anti-glare effect, when the anti-glare and anti-fogging element comprising the SiO2 film 10 nm in thickness and the TiO2 film 170 nm in thickness was combined with the electrochromic anti-glare element having the abovementioned configuration which supposedly provides a reflectivity as low as possible.", "Accordingly, the upper limit of the thickness of the TiO2 film is set to be 170 nm.", "TABLE 3 Surface reflectivity Reflectivity of anti-glare and Thickness of SiO2 Thickness of TiO2 of anti-fogging anti-fogging element at the film (nm) film (nm) element (%) time of glare prevention (%) 10 150 15.6 16.7 10 155 18.0 19.1 10 160 20.0 21.1 10 165 21.9 23.0 10 170 23.3 24.3 10 175 24.3 25.3 10 185 24.9 25.9 Consequently, the thickness of the photocatalytic reaction substance film (TiO2 film) of the anti-glare and anti-fogging element according to the present invention is limited to the range from 100 nm to 170 nm and the thickness of the porous inorganic substance film (SiO2 film) is limited to the range from 10 nm to 50 nm.", "Significance of limiting the ranges and critical significance of the upper and lower limits are as explained above.", "Further, as described above, generally, when eyeglasses are treated with a hydrophilic coating (anti-fogging treatment) without special consideration on the film thickness, the surface reflectivity increases, which results in enhancing incidental reflection to occasionally cause a user discomfort.", "However, such a problem will be solved if an anti-fogging element comprising a photocatalytic reaction substance film (TiO2 film) 100-170 nm in thickness and a porous inorganic substance film (SiO2 film) 10-50 nm in thickness as mentioned above is applied as the hydrophilic coating to the eyeglasses.", "Thus, the anti-fogging element has a potential use in such a field.", "EXAMPLE 1 FIG.", "6 shows an anti-glare and anti-fogging element of Example 1 according to the present invention.", "This anti-glare and anti-fogging element 10 has a basic configuration of an integrated combination of an anti-glare element 5 composed of a front transparent glass base plate 11 and a back transparent glass base plate 12 sandwiching an electrochromic cell 4 therebetween and an anti-fogging element 8 composed of a transparent photocatalytic reaction substance film 6 and a hydrophilic transparent porous inorganic oxide film 7, as mentioned above.", "The electrochromic cell 4 has a configuration in which a reductive coloring layer 15, an electrolyte 16, and an oxidative coloring layer 17 are arranged in order from the back between a front transparent electrode 13 and a back transparent electrode 14.The back transparent electrode 14 is formed in an L shape and connected to the front transparent electrode 13 formed on the same side as the front transparent electrode 13.This electrochromic cell 4 is tightly held in a U shape with a sealing material 19 as shown in the figure.", "A front clip electrode 20 is connected to the front transparent electrode 13 and further designed to hold together the front transparent electrode 13, the front glass base plate 11, the photocatalytic reaction substance film 6 and the porous inorganic oxide film 7.A back clip electrode 21 is connected to a front transparent electrode 18 (a part separated from the front transparent electrode 13) and further designed to hold together the front glass base plate 11, the photocatalytic reaction substance film 6 and the porous inorganic oxide film 7.A voltage is applied to the electrochromic cell 4 by the front clip electrode 20 and the back clip electrode 21.Transparent conductive films of ITO, SnO2, or the like are used for the transparent electrodes 13 and 14.A reductive coloring film of WO3, MoO3 or the like is used for the reductive coloring layer 15.A solid electrolyte film of Ta2O5 or the like is used for the electrolyte 16.An oxidative coloring film of IrOx, NiOx, Sn.IrOx, or the like is used for the oxidative coloring layer 17, Reactions in Example 1 in which the oxidative coloring layer 15 is a WO3 film, the electrolyte 16 is a Ta2O5 film, and the oxidative coloring layer 17 is an Sn.IrOx film are now to be explained.", "In the oxidative coloring layer 17, H2O is present as iridium hydroxide Ir(OH)n. Upon application of a voltage to the electrochromic cell 4 by the front clip electrode 20 and the back clip electrode 21, transmission of proton H+ to the Ta2O5 film and electron emission to the front transparent electrode 13 occur in the oxidative coloring layer 17, and then the following oxidation reaction (1) proceeds to color the oxidative coloring layer 17.Ir(OH)n→IrOx(OH)n-x (colored)+xH++xe− (1) On the other hand, in the reductive coloring layer 15, proton H+ in the Ta2O5 film is transmitted into the WO3 film, the electron from the terminal is injected into the WO3 film, and then the following reductive reaction (2) proceeds in the WO3 film to color the WO3 film.", "WO3+xH++xe−→HxWO3 (colored) (2) Furthermore, when the applied voltage is switched to the opposite direction or the electrodes 20 and 21 are short-circuited, the reductive reaction proceeds in the opposite direction of the reaction (1) above in the IrOx film to decolor the oxidative coloring layer 17, and the oxidative reaction proceeds in the opposite direction of the reaction (2) above in the WO3 film to decolor the reductive coloring layer 15.Further, the Ta2O5 film contains H2O, which is ionized by the abovementioned voltage application and is present also in the form of a proton H+ or an OH− ion to contribute the abovementioned coloring and decoloring reactions.", "In Example 1, generally, light can be transmitted through the anti-glare and anti-fogging element from the front side utilizing the abovementioned decolored state.", "And the light transmission can be reduced by applying a voltage between the clip electrodes 20 and 21 to generate the coloring state.", "The system can be used as a light control glass or display device.", "When a mirror (reflection film) is arranged on the back of the back glass base plate, the system functions as an ordinary mirror in the decolored state and as an anti-glare mirror in the colored state by reducing light transmission.", "EXAMPLE 2 FIG.", "7 shows an anti-glare and anti-fogging element 22 of Example 2 according to the present invention.", "This anti-glare and anti-fogging element 22 comprises an integrated combination of an anti-glare element 5 composed of a front transparent glass base plate 11 and a back transparent glass base plate 12 sandwiching an electrochromic cell 4 therebetween and an anti-fogging element 8 composed of a transparent photocatalytic reaction substance film 6 and a hydrophilic transparent porous inorganic oxide film 7, as in Example 1.The electrochromic cell 4 has a configuration in which an electrode protection layer 23, an electrolyte solution 24, and an electrochromic substance 25 are sandwiched in order from the back between a front transparent electrode 13 and a back transparent electrode 14, and the electrolyte solution 24 and the electrochromic substance 25 are tightly held with a sealing material 19.A front clip electrode 20 is connected to the front transparent electrode 13 and further designed to hold together the front transparent electrode 13, the back glass base plate 11, the photocatalytic reaction substance film 6 and the porous inorganic oxide film 7.A back clip electrode 21 is connected to the back transparent electrode 14 via the electrode protection layer 23 and further designed to hold together the electrode protection layer 23, the back transparent electrode 14, and the back glass base plate 12.Transparent conductive films of ITO, SnO2, or the like are used for the transparent electrodes.", "WO3, MoO3, IrOx, or the like is used for the electrochromic substance.", "The electrolyte solution comprises an electrolyte such as LiI and LiClO4, a solvent such as γ-butyrolactone and propylene carbonate, and an ultraviolet light absorbent such as benzophenone and cyanoacrylate.", "Here, a reaction in Example 2, in which the electrochromic substance 25 used is a reductive coloring material WO3 and the electrolyte used in the electrolyte solution is LiI, is to be explained.", "Upon application of a voltage between the clip electrodes 20 and 21, Li+ (lithium cation) is released from the electrolyte and an electron is injected from the power source, and then the following reaction (3) proceeds to color WO3 blue (color development).", "xLi++WO3+xe−→LixWO3 (colored blue) (3) Although this reaction (3) is reversible, this blue reductive state is maintained when the power source circuit is opened in the state of LixWO3.When a reverse voltage is applied or the electrodes are short-circuited, a reverse reaction proceeds in the opposite direction of the reaction (3) to cause decoloring.", "This Example 2 can be applied to a light control glass or display device and also used as an anti-glare and anti-fogging mirror by placing a mirror on the back of the back glass base plate 14; by setting the thickness of the SiO2 film and TiO2 film in the anti-fogging element to be 10-50 nm and 100-170 nm, respectively, an anti-fogging effect can be attained by the anti-fogging element 8 and an anti-glare effect can be more assured.", "EXAMPLE 3 FIG.", "8 shows an anti-glare and anti-fogging element 26 of the Example 3 according to the present invention.", "This anti-glare and anti-fogging element 26 is different from the anti-glare and anti-fogging element 22 of Example 2 in that the order of the arrangement of the electrode prevention layer 23, the electrolytic solution 24, and the electrochromic substance 25 is reversed, but its configuration and reaction are the same EXAMPLE 4 FIG.", "9 shows an anti-glare and anti-fogging element 27 of Example 4 according to the present invention.", "This anti-glare and anti-fogging element 27 comprises an integrated combination of an anti-glare element 5 composed of a front transparent glass base plate 11 and a back transparent glass base plate 12 sandwiching an electrochromic cell 4 therebetween and an anti-fogging element 8 composed a transparent photocatalytic reaction substance film 6 and a hydrophilic transparent porous inorganic oxide film 7, as in Example 1.The electrochromic cell 4 has a configuration in which an electrochromic solution 28 is filled between a front transparent electrode 13 and a back transparent electrode 14 and is tightly held with a sealing material 19.A front clip electrode 20 is connected to the front transparent electrode 13 and further designed to hold together the front transparent electrode 13, the back glass base plate 11, the photocatalytic reaction substance film 6 and the porous inorganic oxide film 7.A back clip electrode 21 is connected to the back transparent electrode 14 and further designed to hold together the back transparent electrode 14 and the back glass base plate 12.The electrolyte solution 28 comprises an electrochromic substance such as viologen, a solvent such as γ-butyrolactone and propylene carbonate, and an ultraviolet absorbent such as benzophenone and cyanoacrylate.", "Generally, the electrochromic solution is in a decolored state.", "Upon application of a voltage between the clip electrodes 20 and 21, viologen exhibits an oxidation-reduction-reaction, which results in coloring (color development).", "This Example 4 can be applied to a light control glass or display device and also be used as an anti-glare and anti-fogging mirror by placing a mirror on the back of the back glass base plate 12.By setting the thickness of the SiO2 film and TiO2 film in the anti-fogging element to be 10-50 nm and 100-170 nm, respectively, an anti-fogging effect can be attained by the anti-fogging element 8 and an anti-glare effect can be more assured.", "EXAMPLE 5 FIG.", "10 shows an anti-glare and anti-fogging element 29 of Example 5.This anti-glare and anti-fogging element is an anti-glare and anti-fogging mirror mentioned in Example 2, in which an reflection film 30 of Cr, Al, Ag, Rh, Cr—Rh, or the like is formed on the back of a back glass base plate 12, a protection coat 31 is formed on further back of this film, and a back clip 21 holds together the layers between the back transparent electrode 14 and the protection coat 31, inclusive.", "The action of the system is the same as explained in Example 2.EXAMPLE 6 FIG.", "11 shows an anti-glare and anti-fogging element 32 of Example 6.This anti-glare and anti-fogging element 32 is an anti-glare and anti-fogging mirror mentioned in Example 3, in which an reflection film 30 of Cr, Al, Ag, Rh, Cr—Rh, or the like is formed on the back of a back glass base plate 12, a protection coat 31 is formed on further back of this film, and a back clip 21 holds together the layers between the back transparent electrode 14 and the protection coat 31, inclusive.", "The action of the system is the same as explained in Example 3.EXAMPLE 7 FIG.", "12 shows the anti-glare and anti-fogging element 33 of Example 7.This anti-glare and anti-fogging element 33 is an anti-glare and anti-fogging mirror, in which a reflection film 30 of Cr, Al, Ag, Rh, Cr—Rh or the like is formed on the back of the back glass base plate of Example 4, a protection coat 31 is formed on further back of this film, and a back clip 21 holds together the layers between the back transparent electrode 14 and the protection coat 31, inclusive.", "The action of the system is the same as explained in Example 4.EXAMPLE 8 FIG.", "13 shows an anti-glare and anti-fogging element (with a reflection film) 34 of Example 8.This anti-glare and anti-fogging element 34 is an anti-glare and anti-fogging mirror, in which the back transparent electrode 14 of Example 1 is made into an electrode and reflection film and this electrode and concurrently reflection film is composed of a metal film of Cr, Al, Ag, Rh, Cr—Rh, or the like.", "The action of the system is the same as explained in Example 1.EXAMPLE 9 FIG.", "14 shows an anti-glare and anti-fogging element 35 of Example 9.This anti-glare and anti-fogging element 35 has a configuration in which the back transparent electrode 14 of Example 4 is made into an electrode and concurrently reflection film and this electrode and concurrently reflection film is composed of a metal film of Cr, Al, Ag, Rh, Cr—Rh, or the like.", "The action of the system is the same as explained in Example 4.Further, in Examples 5 to 9, the anti-glare and anti-fogging element can be heated as a whole by a resistant heating body (not shown) furnished on the back of the protection coat 31 on the backside of the reflection film 30 or the back glass base plate 14 so that the water attached to the porous inorganic oxide film 7 can quickly evaporate and the anti-fogging effect can be further improved.", "In the present invention, the thickness of TiO2 and SiO2 films of the anti-fogging element was set as mentioned above on the assumption that dazzling can generally be avoided when the reflectivity of the anti-glare and anti-fogging element is controlled to be 25% or less.", "In order to verify that dazzling can generally be avoided when the reflectivity of the anti-glare and anti-fogging element is controlled to be 25% or less, an experiment was carried out to assess the degree of dazzling and the like experienced by subjects due to the reflection of the mirror during driving.", "This experimental example is to be explained as follows referring to FIG.", "15 and FIG.", "16.A front car 34 and a rear car 35 were arranged as shown in FIG.", "15 (a side view of the two cars aligned in front and behind) and FIG.", "16 (a top view of the two cars aligned in front and behind) so that a rearview mirror 37 on the right side of the front car 34 was irradiated by a headlight 36 of the rear car 35 coming in from the right behind or slightly from the right side (in this case the driver's seat is on the right side), which simulates a probable general condition that a driver 38 maximally feels dazzled at night, for example, while waiting for the traffic light to change.", "In this arrangement, the degree of dazzling was evaluated by irradiating the anti-fogging anti-glare element of the rearview mirror 37 with a headlight 36 from the back, at night in the absence of any other light source.", "The headlight used was a discharge valve headlight which utilizes an arc discharge as a luminous source or a halogen headlamp having a bulb filled with a halogen gas, and variations of the reflectivity of the anti-fogging and anti-glare element were 35%, 30%, 25%, 20%, and 15%.", "The degree of dazzling was confirmed by 10 subjects.", "The subjects looked straight at the rearview mirror 30 irradiated by the headlight 36 for 10 seconds and then looked aside from the rearview mirror to assess the degree of dazzling.", "The reflectivity with which none of the ten subjects felt dazzled was confirmed.", "The result of the evaluation is shown in the following Table 4.TABLE 4 Vision immediately Reflectivity of anti-fogging Degree of after straight and anti-glare element dazzling look at rearview mirror 35% Dazzled Blurred and out of focus 30% Slightly dazzled Slightly blurred 25% Not dazzled Clear 20% Not dazzled Clear 15% Not dazzled Clear As a result of this experiment, it was found that dazzling could be avoided when the reflectivity of the mirror with the anti-glare and anti-fogging element was 25%.", "POSSIBILITY OF INDUSTRIAL USE Since an anti-glare and anti-fogging element according to the present invention has the abovementioned configuration, it exhibits both anti-glare and anti-fogging effects and its anti-glare effect cannot be reduced because the reflectivity of the anti-glare and anti-fogging element as a whole can be reduced even at the moment of glare prevention.", "Accordingly, in particular, when used as an automotive outer mirror or room mirror, glare caused by lights of a car behind can be prevented.", "Furthermore, it can be used for various display devices, light control glasses or the like." ] ]	Patent_10451978
[ [ "Method and apparatus for unified storage of data for storage area network systems and network attached storage systems", "A unified data storage apparatus for storing data for both storage area network and network attached storage system architectures on the same storage medium.", "The storage medium (50) is partitioned for storage of logical disk volumes and files (58), and is further partitioned for storage of pointers to logical disk volumes (59)." ], [ "1.A data storage device, comprising: a first port; and a storage medium connected to the first port, comprising: a first portion that stores files; a second portion that stores logical disk volumes; and a third portion that stores pointers to the logical disk volumes.", "2.The data storage device as claimed in claim 1, wherein the storage medium comprises a hard disk.", "3.The data storage device as claimed in claim 1, wherein the storage medium comprises a redundant array of independent disks.", "4.The data storage device as claimed in claim 1, further comprising a file manager that manages the logical disk volumes and files stored on the storage medium.", "5.The data storage device as claimed in claim 1, further comprising a controller that controls read and write accesses to the storage medium.", "6.The data storage device as claimed in claim 5, wherein the storage device is attached to a network that includes at least one client device, and wherein the controller reads data from the storage medium or writes data to the storage medium according to a command received from at least one client device.", "7.The data storage device as claimed in claim 5, wherein the storage device is attached to a network that includes at least one applications server, and wherein the controller reads data from the storage medium or writes data to the storage medium according to a command received from the at least one applications server.", "8.A data storage device, comprising: a first port; a second port; and a storage medium connected to the first port and second port, comprising: a first portion that stores files; a second portion that stores logical disk volumes; and a third portion that stores pointers to the logical disk volumes.", "9.The data storage device as claimed in claim 8, wherein the storage medium comprises a hard disk.", "10.The data storage device as claimed in claim 8, wherein the storage medium comprises a redundant array of independent disks.", "11.The data storage device as claimed in claim 8, further comprising a file manager that manages the logical disk volumes and files stored on the storage medium.", "12.The data storage device as claimed in claim 8, further comprising a controller that controls read and write accesses to the storage medium.", "13.The data storage device as claimed in claim 12, wherein the storage device is attached to a network that includes at least one client device through the first port, and wherein the controller reads data from the storage medium or writes data to the storage medium according to a command received from at least one client device.", "14.The data storage device as claimed in claim 12, wherein the storage device is attached to a network that includes at least one applications server through the second port, and wherein the controller reads data from the storage medium or writes data to the storage medium according to a command received from the at least one applications server.", "15.A computer network, comprising: a storage area network comprising at least one applications server; a network attached storage system comprising: at least one client device; and at least one file server; and a storage device connected to the storage area network and the network attached storage system, the storage device comprising a storage medium having a first portion assigned to files for the at least one client device, a second portion assigned to logical disk volumes for the at least one applications server, and a third portion assigned to pointers to the logical disk volumes.", "16.A storage system responsive to service requests from client devices of a network attached storage system and to applications servers of a storage area network, the storage system comprising: a first interface to the network attached storage system; a second interface to the storage area network; and a storage medium comprising: a first portion assigned to store files for the client devices of the network attached storage system; a second portion assigned to store logical disk volumes for the applications servers of the storage area network; and a third portion assigned to store pointers to the logical disk volumes.", "17.The storage system as claimed in claim 16, wherein the storage medium comprises a plurality of disk drives.", "18.The storage system as claimed in claim 17, wherein the first portion is a selected disk drive from the plurality of disk drives.", "19.The storage system as claimed in claim 17, wherein the second portion is a selected disk drive from the plurality of disk drives.", "20.The storage system as claimed in claim 17, wherein the third portion is a selected disk drive from the plurality of disk drives.", "21.The storage system as claimed in claim 16, further comprising a controller that processes data requests from the client devices of the network attached storage system and the applications servers of the storage area network.", "22.The storage system as claimed in claim 21, wherein the controller stores a file to or retrieves a file from the first portion of the storage medium in response to a command from a client device.", "23.The storage system as claimed in claim 21, wherein the controller stores a logical disk volume to or retrieves a logical disk volume from the second portion of the storage medium in response to a command from an applications server.", "24.The storage system as claimed in claim 23, wherein the controller updates the third portion of the storage medium in response to a command from an applications server to retrieve or store a logical disk volume.", "25.A data storage device, comprising: a first storage medium assigned to files for client devices of a network attached storage system; a second storage medium assigned to logical disk volumes for applications servers of a storage attached network; and a third storage medium for pointers the logical disk volumes for the applications servers of the storage attached network.", "26.The data storage device as claimed in claim 25, wherein the first, second and third storage mediums comprise at least one disk drive.", "27.The data storage device as claimed in claim 25, wherein the at least one disk drive comprises a plurality of disk drives.", "28.A storage device, the storage device comprising: a first interface for connecting to a network attached storage system; a second interface for connecting to a storage area network; a storage medium comprising: a first portion assigned to store files for the client devices of the network attached storage system; a second portion assigned to store logical disk volumes for the applications servers of the storage area network; and a third portion assigned to store pointers to the logical disk volumes; and a controller connected to the first and second interface, wherein the controller manages read and write access to the storage medium.", "29.A data storage device, comprising: a first port; and a storage means connected to the first port for storing files and logical disk volumes, the storage means comprising: a first portion that stores the files; a second portion that stores the logical disk volumes; and a third portion that stores pointers to the logical disk volumes.", "30.The data storage device as claimed in claim 29, wherein the storage means comprises a hard disk.", "31.The data storage device as claimed in claim 29, wherein the storage means comprises a redundant array of independent disks.", "32.The data storage device as claimed in claim 29, further comprising file manager means for managing the logical disk volumes and files stored on the storage means.", "33.The data storage device as claimed in claim 29, further comprising a controller means for controlling read and write accesses to the storage means.", "34.The data storage device as claimed in claim 33, wherein the storage means is attached to a network that includes at least one client means, and wherein the controller means reads data from the storage means or writes data to the storage means according to a command received from at least one client means.", "35.The data storage device as claimed in claim 33, wherein the storage device is attached to a network that includes at least one applications server, and wherein the controller means reads data from the storage means or writes data to the storage means according to a command received from the at least one applications server.", "36.A storage system responsive to service requests from client means of a network attached storage system and to applications servers of a storage area network, the storage system comprising: a first interface means for connecting to the network attached storage system; a second interface means for connecting to the storage area network; and a storage means for storing files and logical disk volumes, the storage means comprising: a first portion assigned to store the files for the client means of the network attached storage system; a second portion assigned to store the logical disk volumes for the applications servers of the storage area network; and a third portion assigned to store pointers to the logical disk volumes.", "37.The storage system as claimed in claim 36, further comprising controller means for processing data requests from the client means of the network attached storage system and the applications servers of the storage area network.", "38.A storage device, the storage device comprising: a first interface means for connecting to a network attached storage system; a second interface means for connecting to a storage area network; a storage means for storing files and logical disk volumes, the storage means comprising: a first portion assigned to store files for the client means of the network attached storage system; a second portion assigned to store logical disk volumes for the applications servers of the storage area network; and a third portion assigned to store pointers to the logical disk volumes; and a controller means connected to the first and second interface for controlling read and write access to the storage means.", "39.A method of storing data from a plurality of storage systems on a storage medium, the method comprising: storing data for client devices of a network attached storage system on a first portion of the storage medium; storing data for applications servers of a storage area network on a second portion of the storage medium; and storing pointers to data for applications servers on a third portion of the storage medium.", "40.The method of storing data as claimed in claim 39, the method further comprising the reading or writing of data from the first portion of the storage medium in response to a command from a client device.", "41.The method of storing data as claimed in claim 39, the method further comprising the reading or writing of data from the second portion of the storage medium in response to a command from an applications server.", "42.The method of storing data as claimed in claim 39, the method further comprising updating pointers stored on the third portion of the storage medium in response to a command from an applications server to read or write data from the second portion of the storage medium.", "43.A computer system adapted to storing data from a plurality of storage systems on a storage medium, the computer system comprising: a processor; a memory comprising software instructions adapted to enable the computer system to perform the steps of: storing data for client devices of a network attached storage system on a first portion of the storage medium; storing data for applications servers of a storage area network on a second portion of the storage medium; and storing pointers to data for applications servers on a third portion of the storage medium.", "44.The computer system adapted to storing data from a plurality of storage systems on a storage medium as claimed in claim 43, wherein the software instructions are further adapted to enable the computer system to read or write data from the first portion of the storage medium in response to a command from a client device.", "45.The computer system adapted to storing data from a plurality of storage systems on a storage medium as claimed in claim 43, wherein the software instructions are further adapted to enable the computer system to read or write data from the second portion of the storage medium in response to a command from an applications server.", "46.The computer system adapted to storing data from a plurality of storage systems on a storage medium as claimed in claim 43, wherein the software instructions are further adapted to enable the computer system to update pointers stored on the third portion of the storage medium in response to a command from an applications server to read or write data from the second portion of the storage medium.", "47.A computer program product for storing data from a plurality of storage systems on a storage medium, the computer program product comprising: software instructions for enabling a computer system to perform predetermined operations, and a computer readable medium bearing the software instructions; the predetermined operations comprising: storing data for client devices of a network attached storage system on a first portion of the storage medium; storing data for applications servers of a storage area network on a second portion of the storage medium; and storing pointers to data for applications servers on a third portion of the storage medium.", "48.An executable program for a computer system for storing data from a plurality of storage systems on a storage medium, the executable program comprising: a first executable code portion which, when executed on a computer system, stores data for client devices of a network attached storage system on a first portion of the storage medium; a second executable code portion which, when executed on a computer system, stores data for applications servers of a storage area network on a second portion of the storage medium; and a third executable code portion which, when executed on a computer system, stores pointers to data for applications servers on a third portion of the storage medium.", "49.A method for handling data requests from a plurality of data requesters to a storage device, the method comprising: receiving the data request; determining if the data request originates from an application server of a storage attached network or a client device; if the data request originated from an application server and requests a logical disk volume, the method further comprises: determining if the requested logical disk volume is stored on the storage device; and retrieving the logical disk volume using a pointer to the logical disk volume; and if the data request originated from the client device and requests a file, the method further comprises retrieving the requested file from the storage device.", "50.A method for handling data write requests from a plurality of data writers to a storage device, the method comprising: receiving the data write request; determining if the data write request originates from an application server of a storage attached network or a client device; if the data write request originated from an application server and requests to write a logical disk volume to the storage device, the method further comprises: determining if the logical disk volume is already stored on the storage device; determining if the storage device has space for storing the logical disk volume; writing the logical disk volume to the storage device; and updating a pointer to the logical disk volume; and if the data write request originated from a client and requests to write a file to the storage device, the method further comprises writing the requested file to the storage device." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Technical Field of the Invention This invention is related to a method and apparatus for storing data used by Storage Area Networks and Network Attached Storage systems on the same storage medium.", "In particular, the storage of data for these two network architectures reduces the number of storage assets required for a network.", "The invention is embodied in a method for storing data from both Storage Area Network and Network Attached Storage architectures on the same storage medium, and a data storage apparatus for storing data received from both Storage Area Network and Network Attached Storage architectures.", "The invention is also embodied in a computer system that stores data received from both Storage Area Network and Network Attached Storage architectures on the same storage medium, and a computer program product that stores data from both Storage Area Network and Network Attached Storage architectures.", "2.Description of the Related Art There will now be provided a discussion of various topics to provide a proper foundation for understanding the invention.", "In order for a client device to be able to access to multiple servers running different operating systems, either the client device supports the file sharing protocol of each operating system or the server supports the file sharing protocol of each client device.", "Software that adds this capability is very common and allows interoperability between Windows®, Macintosh®, NetWare® and UNIX platforms.", "TABLE 1 lists several common operating systems and their transport and file sharing protocols for networking environments.", "TABLE 1 Operating Transport File Sharing System Protocol Protocol DOS NETBIOS SMB WINDOWS NETBEUI SMB, CIFS NETWARE IPX NCP MACINTOSH APPLETALK AFP UNIX TCP/IP NFS A Storage Area Network (SAN) system is a back-end network that uses peripheral channels to connect storage devices.", "Typically, the peripheral channels are Small Computer System Interface (SCSI), Serial Storage Architecture (SSA), Enterprise Systems Connection (ESCON) and Fibre Channel.", "SAN devices are usually dedicated high-bandwidth systems that handle traffic between servers and storage assets.", "Data objects on a SAN system are sets of logical disk volumes above which higher level object semantics can be implemented on specific application servers.", "Both centralized SANs and distributed SANs are currently used.", "A centralized SAN ties multiple hosts into a single storage system.", "The storage system is usually a Redundant Array of Independent Disks (RAID) device with large amounts of cache and redundant power supplies.", "Typically, this centralized storage architecture ties a server cluster together for fault tolerance (i.e., if one server fails, another server can take over).", "Centralized SAN also provides simplified sharing of data between multiple servers, and further provides multiple servers the capability to perform the work on the shared data.", "Referring to FIG.", "1 , a centralized SAN system is illustrated.", "The applications servers 1 , 2 and the mainframe computer 6 are connected to the disk array 4 via several peripheral channels 8 - 10 .", "As described above, the peripheral channels may use SCSI, SSA, ESCON or Fibre Channel protocols to transfer data between the disk array 4 and the applications servers 1 , 2 .", "A distributed SAN system connects multiple hosts with multiple storage systems.", "Referring to FIG.", "2 , a distributed SAN system is illustrated.", "Several applications servers 1 - 3 are connected to a switch 7 , which is also connected to several disk arrays 4 , 5 .", "The switch 7 handles the transfer of data between the multiple disk arrays 4 , 5 and the applications servers 1 - 3 via the peripheral channels 8 - 12 .", "Of course, SAN systems are not limited to only using disk arrays for data storage.", "For example, a distributed SAN system could be simultaneously connected to both single disk storage systems and disk array storage systems.", "In addition, a distributed SAN system can be constructed from hubs (which connect to the storage devices via loops), or a combination of hubs and switches.", "Referring to FIG.", "3 , the data path of data objects transferred between an applications server 15 and the disk storage 18 will be described.", "As noted above, data objects transferred in a SAN system are logical disk volumes.", "When a data request is received at the disk storage 18 for an identified logical disk volume, the disk storage 18 sends out the volume over peripheral channel 20 into the SAN network 19 .", "When the logical disk volume arrives at the applications server 15 , the file manager 17 handles the high-level object semantics necessary to supply the requested data to the software application 16 .", "A Network Attached Storage (NAS) system is connected to a front-end communications network just like a file server.", "Typically, the communications protocol is Ethernet, TCP/IP or FTP, but other lesser-used protocols are not excluded.", "A NAS system does not rely upon a complete operating system for its functionality.", "Instead, a slimmed-down micro-kernel targeted for file management is used.", "Traditional Local Area Network (LAN) protocols such as NFS (UNIX), SMB/CIFS (DOS/Windows) and NCP (NetWare) are examples of slimmed-down operating systems used for file management on a NAS system.", "Devices in a NAS system typically attach to a LAN and allow sets of users to retrieve and share files that may span over multiple operating system environments.", "Referring to FIG.", "4 , a NAS system is illustrated.", "Several clients 21 - 22 are connected to a hub 25 .", "The hub 25 is connected to a NAS server 23 .", "The NAS server 23 communicates with a disk array 24 to retrieve data for the clients 21 - 22 or to store data for the clients 21 - 22 .", "LAN channels 26 - 28 realize connections between the NAS server 23 , the hub 25 and the clients 21 - 22 .", "Referring to FIG.", "5 , the data path of data objects transferred between a client 33 and the disk storage 32 will be described.", "Typically, a NAS system exports higher level objects (i.e., files) to the LAN for use by the client systems attached to the LAN.", "A request for a file stored on the NAS server 30 is received from the NAS network 35 .", "The file manager 31 searches the disk storage 32 for the file, and if located, outputs the file to the NAS network 35 over the LAN channel 36 .", "When the file arrives at the client 33 , the software application 34 is able to manipulate the file.", "An advantage of the NAS system is that adding or removing a NAS system is like adding or removing any network node.", "In general, a SAN system (e.g., a channel-attached storage system) must be brought down in order to reconfigure it.", "Another advantage of a NAS system is that application servers are not involved with management functions, such as volume management, and can access the stored data as files.", "However, NAS systems are subject to the erratic behavior and overhead of the network." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention has been made in view of the above circumstances and to overcome the above problems and limitations of the prior art.", "Additional aspects and advantages of the invention will be set forth in part in the description that follows and in part will be obvious from the description, or may be learned by practice of the invention.", "The aspects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.", "A first aspect of the present invention is a data storage device comprising a first port connected to a storage area network to applications servers and client devices.", "The data storage device further comprises a storage medium having a first portion that stores files, a second portion that stores logical disk volumes and a third portion that stores pointers to the logical disk volumes.", "A second aspect of the present invention is data storage device comprising first and second ports.", "The data storage device further includes a storage medium connected to the first port and second port, and the storage medium has a first portion that stores files, a second portion that stores logical disk volumes and a third portion that stores pointers to the logical disk volumes.", "A third aspect of the present invention is a computer network that includes a storage area network having applications servers, and a network attached storage system having client devices and file servers.", "The computer network also includes a storage device connected to the storage area network and the network attached storage system.", "The storage device includes a storage medium having a first portion assigned to files for the client devices, a second portion assigned to logical disk volumes for the applications servers, and a third portion assigned to pointers to the logical disk volumes.", "A fourth aspect of the present invention is a storage system responsive to service requests from client devices of a network attached storage system and to applications servers of a storage area network.", "The storage system includes a first interface to the network attached storage system, a second interface to the storage area network and a storage medium.", "The storage medium comprises a first portion assigned to store files for the clients of the network attached storage system, a second portion assigned to store logical disk volumes for the applications servers of the storage area network, and a third portion assigned to store pointers to the logical disk volumes.", "A fifth aspect of the present invention is a data storage device that includes a first storage medium assigned to files for client devices of a network attached storage system.", "The data storage device further comprises a second storage medium assigned to logical disk volumes for the applications servers of a storage attached network, and a third storage medium for pointers the logical disk volumes for the applications servers of a storage attached network.", "A sixth aspect of the present invention is a storage device including a first interface for connecting to a network attached storage system and a second interface for connecting to a storage area network.", "The storage device further includes a storage medium having a first portion assigned to store files for the client devices of the network attached storage system, a second portion assigned to store logical disk volumes for the applications servers of the storage area network and a third portion assigned to store pointers to the logical disk volumes.", "The storage device also includes a controller connected to the first and second interface for controlling read and write access to the storage medium.", "A seventh aspect of the present invention is a data storage device having a first port and storage means connected to the first port.", "The storage means includes a first portion that stores the files, a second portion that stores the logical disk volumes and a third portion that stores pointers to the logical disk volumes.", "An eighth aspect of the present invention is a storage system responsive to service requests from client means of a network attached storage system and to applications servers of a storage area network.", "The storage device has a first interface means and second means for connecting to the networks.", "The storage device has storage means for storing files and logical disk volumes, and storage means is partitioned into a first portion assigned to store the files for the client means, a second portion assigned to store the logical disk volumes for the applications servers and a third portion assigned to store pointers to the logical disk volumes.", "A ninth aspect of the present invention is a storage device having a first and second interface means for connecting to network attached storage systems and to storage area networks.", "The storage device includes a storage means for storing files and logical disk volumes, and the storage means is partitioned into a first portion assigned to store files for the client means, a second portion assigned to store logical disk volumes for the applications servers and a third portion assigned to store pointers to the logical disk volumes.", "The storage device also includes a controller means connected to the first and second interface for controlling read and write access to the storage means.", "A tenth aspect of the present invention is a method of storing data from a plurality of storage systems on a storage medium.", "The method comprises storing data for client devices of a network attached storage system on a first portion of the storage medium, storing data for applications servers of a storage area network on a second portion of the storage medium, and storing pointers to data for applications servers on a third portion of the storage medium.", "A eleventh aspect of the present invention is a computer system adapted to storing data from a plurality of storage systems on a storage medium.", "The computer system comprises a processor and a memory comprising software instructions adapted to enable the computer system to store data for client devices of a network attached storage system on a first portion of the storage medium.", "The instructions are further adapted to enable the computer system to store data for applications servers of a storage area network on the second portion of a storage medium, and to store pointers to data for applications servers on a third portion of the storage medium.", "A twelfth aspect of the present invention is a computer program product for storing data from a plurality of storage systems on a storage medium.", "The computer program product comprises software instructions for enabling a computer system to perform predetermined operations, and a computer readable medium bearing the software instructions.", "The predetermined operations comprise storing data for client devices of a network attached storage system on a first portion of the storage medium, storing data for applications servers of a storage area network on a second portion of the storage medium, and storing pointers to data for applications servers on a third portion of the storage medium.", "A thirteenth aspect of the present invention is a executable program for a computer system for storing data from a plurality of storage systems on a storage medium.", "The executable program comprises a first executable code portion which, when executed on the computer system, stores data for client devices of a network attached storage system on a first portion of the storage medium.", "The executable program further comprises a second executable code portion which, when executed on the computer system, stores data for applications servers of a storage area network on a second portion of the storage medium.", "The executable program further comprises a third executable code portion which, when executed on the computer system, stores pointers to data for applications servers on a third portion of the storage medium.", "A fourteenth aspect of the present invention is a method for handling data requests from a plurality of data requestors to a storage device.", "The method comprises receiving the data request and determining if the data request originates from an application server of a storage attached network or a client device.", "If the data request originated from an application server and requests a volume, the method further comprises determining if the requested volume is stored on the storage device, and retrieving the volume using a pointer to the volume.", "If the data request originated from the client device and requests a file, the method further comprises retrieving the requested file from the storage device.", "A fifteenth aspect of the present invention is a method for handling data write requests from a plurality of data writers to a storage device.", "The method comprises receiving the data write request and determining if the data write request originates from an application server of a storage attached network or a client device.", "If the data write request originated from an application server and requests to write a volume to the storage device, the method further comprises determining if the volume is already stored on the storage device, determining if the storage device has space for storing the volume, writing the volume to the storage device, and updating a pointer to the volume.", "If the data write request originated from a client and requests to write a file to the storage device, the method further comprises writing the requested file to the storage device.", "The above aspects and advantages of the invention will become apparent from the following detailed description and with reference to the accompanying drawing figures." ], [ "BACKGROUND OF THE INVENTION 1.Technical Field of the Invention This invention is related to a method and apparatus for storing data used by Storage Area Networks and Network Attached Storage systems on the same storage medium.", "In particular, the storage of data for these two network architectures reduces the number of storage assets required for a network.", "The invention is embodied in a method for storing data from both Storage Area Network and Network Attached Storage architectures on the same storage medium, and a data storage apparatus for storing data received from both Storage Area Network and Network Attached Storage architectures.", "The invention is also embodied in a computer system that stores data received from both Storage Area Network and Network Attached Storage architectures on the same storage medium, and a computer program product that stores data from both Storage Area Network and Network Attached Storage architectures.", "2.Description of the Related Art There will now be provided a discussion of various topics to provide a proper foundation for understanding the invention.", "In order for a client device to be able to access to multiple servers running different operating systems, either the client device supports the file sharing protocol of each operating system or the server supports the file sharing protocol of each client device.", "Software that adds this capability is very common and allows interoperability between Windows®, Macintosh®, NetWare® and UNIX platforms.", "TABLE 1 lists several common operating systems and their transport and file sharing protocols for networking environments.", "TABLE 1 Operating Transport File Sharing System Protocol Protocol DOS NETBIOS SMB WINDOWS NETBEUI SMB, CIFS NETWARE IPX NCP MACINTOSH APPLETALK AFP UNIX TCP/IP NFS A Storage Area Network (SAN) system is a back-end network that uses peripheral channels to connect storage devices.", "Typically, the peripheral channels are Small Computer System Interface (SCSI), Serial Storage Architecture (SSA), Enterprise Systems Connection (ESCON) and Fibre Channel.", "SAN devices are usually dedicated high-bandwidth systems that handle traffic between servers and storage assets.", "Data objects on a SAN system are sets of logical disk volumes above which higher level object semantics can be implemented on specific application servers.", "Both centralized SANs and distributed SANs are currently used.", "A centralized SAN ties multiple hosts into a single storage system.", "The storage system is usually a Redundant Array of Independent Disks (RAID) device with large amounts of cache and redundant power supplies.", "Typically, this centralized storage architecture ties a server cluster together for fault tolerance (i.e., if one server fails, another server can take over).", "Centralized SAN also provides simplified sharing of data between multiple servers, and further provides multiple servers the capability to perform the work on the shared data.", "Referring to FIG.", "1, a centralized SAN system is illustrated.", "The applications servers 1,2 and the mainframe computer 6 are connected to the disk array 4 via several peripheral channels 8-10.As described above, the peripheral channels may use SCSI, SSA, ESCON or Fibre Channel protocols to transfer data between the disk array 4 and the applications servers 1,2.A distributed SAN system connects multiple hosts with multiple storage systems.", "Referring to FIG.", "2, a distributed SAN system is illustrated.", "Several applications servers 1-3 are connected to a switch 7, which is also connected to several disk arrays 4,5.The switch 7 handles the transfer of data between the multiple disk arrays 4,5 and the applications servers 1-3 via the peripheral channels 8-12.Of course, SAN systems are not limited to only using disk arrays for data storage.", "For example, a distributed SAN system could be simultaneously connected to both single disk storage systems and disk array storage systems.", "In addition, a distributed SAN system can be constructed from hubs (which connect to the storage devices via loops), or a combination of hubs and switches.", "Referring to FIG.", "3, the data path of data objects transferred between an applications server 15 and the disk storage 18 will be described.", "As noted above, data objects transferred in a SAN system are logical disk volumes.", "When a data request is received at the disk storage 18 for an identified logical disk volume, the disk storage 18 sends out the volume over peripheral channel 20 into the SAN network 19.When the logical disk volume arrives at the applications server 15, the file manager 17 handles the high-level object semantics necessary to supply the requested data to the software application 16.A Network Attached Storage (NAS) system is connected to a front-end communications network just like a file server.", "Typically, the communications protocol is Ethernet, TCP/IP or FTP, but other lesser-used protocols are not excluded.", "A NAS system does not rely upon a complete operating system for its functionality.", "Instead, a slimmed-down micro-kernel targeted for file management is used.", "Traditional Local Area Network (LAN) protocols such as NFS (UNIX), SMB/CIFS (DOS/Windows) and NCP (NetWare) are examples of slimmed-down operating systems used for file management on a NAS system.", "Devices in a NAS system typically attach to a LAN and allow sets of users to retrieve and share files that may span over multiple operating system environments.", "Referring to FIG.", "4, a NAS system is illustrated.", "Several clients 21-22 are connected to a hub 25.The hub 25 is connected to a NAS server 23.The NAS server 23 communicates with a disk array 24 to retrieve data for the clients 21-22 or to store data for the clients 21-22.LAN channels 26-28 realize connections between the NAS server 23, the hub 25 and the clients 21-22.Referring to FIG.", "5, the data path of data objects transferred between a client 33 and the disk storage 32 will be described.", "Typically, a NAS system exports higher level objects (i.e., files) to the LAN for use by the client systems attached to the LAN.", "A request for a file stored on the NAS server 30 is received from the NAS network 35.The file manager 31 searches the disk storage 32 for the file, and if located, outputs the file to the NAS network 35 over the LAN channel 36.When the file arrives at the client 33, the software application 34 is able to manipulate the file.", "An advantage of the NAS system is that adding or removing a NAS system is like adding or removing any network node.", "In general, a SAN system (e.g., a channel-attached storage system) must be brought down in order to reconfigure it.", "Another advantage of a NAS system is that application servers are not involved with management functions, such as volume management, and can access the stored data as files.", "However, NAS systems are subject to the erratic behavior and overhead of the network.", "SUMMARY OF THE INVENTION The invention has been made in view of the above circumstances and to overcome the above problems and limitations of the prior art.", "Additional aspects and advantages of the invention will be set forth in part in the description that follows and in part will be obvious from the description, or may be learned by practice of the invention.", "The aspects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.", "A first aspect of the present invention is a data storage device comprising a first port connected to a storage area network to applications servers and client devices.", "The data storage device further comprises a storage medium having a first portion that stores files, a second portion that stores logical disk volumes and a third portion that stores pointers to the logical disk volumes.", "A second aspect of the present invention is data storage device comprising first and second ports.", "The data storage device further includes a storage medium connected to the first port and second port, and the storage medium has a first portion that stores files, a second portion that stores logical disk volumes and a third portion that stores pointers to the logical disk volumes.", "A third aspect of the present invention is a computer network that includes a storage area network having applications servers, and a network attached storage system having client devices and file servers.", "The computer network also includes a storage device connected to the storage area network and the network attached storage system.", "The storage device includes a storage medium having a first portion assigned to files for the client devices, a second portion assigned to logical disk volumes for the applications servers, and a third portion assigned to pointers to the logical disk volumes.", "A fourth aspect of the present invention is a storage system responsive to service requests from client devices of a network attached storage system and to applications servers of a storage area network.", "The storage system includes a first interface to the network attached storage system, a second interface to the storage area network and a storage medium.", "The storage medium comprises a first portion assigned to store files for the clients of the network attached storage system, a second portion assigned to store logical disk volumes for the applications servers of the storage area network, and a third portion assigned to store pointers to the logical disk volumes.", "A fifth aspect of the present invention is a data storage device that includes a first storage medium assigned to files for client devices of a network attached storage system.", "The data storage device further comprises a second storage medium assigned to logical disk volumes for the applications servers of a storage attached network, and a third storage medium for pointers the logical disk volumes for the applications servers of a storage attached network.", "A sixth aspect of the present invention is a storage device including a first interface for connecting to a network attached storage system and a second interface for connecting to a storage area network.", "The storage device further includes a storage medium having a first portion assigned to store files for the client devices of the network attached storage system, a second portion assigned to store logical disk volumes for the applications servers of the storage area network and a third portion assigned to store pointers to the logical disk volumes.", "The storage device also includes a controller connected to the first and second interface for controlling read and write access to the storage medium.", "A seventh aspect of the present invention is a data storage device having a first port and storage means connected to the first port.", "The storage means includes a first portion that stores the files, a second portion that stores the logical disk volumes and a third portion that stores pointers to the logical disk volumes.", "An eighth aspect of the present invention is a storage system responsive to service requests from client means of a network attached storage system and to applications servers of a storage area network.", "The storage device has a first interface means and second means for connecting to the networks.", "The storage device has storage means for storing files and logical disk volumes, and storage means is partitioned into a first portion assigned to store the files for the client means, a second portion assigned to store the logical disk volumes for the applications servers and a third portion assigned to store pointers to the logical disk volumes.", "A ninth aspect of the present invention is a storage device having a first and second interface means for connecting to network attached storage systems and to storage area networks.", "The storage device includes a storage means for storing files and logical disk volumes, and the storage means is partitioned into a first portion assigned to store files for the client means, a second portion assigned to store logical disk volumes for the applications servers and a third portion assigned to store pointers to the logical disk volumes.", "The storage device also includes a controller means connected to the first and second interface for controlling read and write access to the storage means.", "A tenth aspect of the present invention is a method of storing data from a plurality of storage systems on a storage medium.", "The method comprises storing data for client devices of a network attached storage system on a first portion of the storage medium, storing data for applications servers of a storage area network on a second portion of the storage medium, and storing pointers to data for applications servers on a third portion of the storage medium.", "A eleventh aspect of the present invention is a computer system adapted to storing data from a plurality of storage systems on a storage medium.", "The computer system comprises a processor and a memory comprising software instructions adapted to enable the computer system to store data for client devices of a network attached storage system on a first portion of the storage medium.", "The instructions are further adapted to enable the computer system to store data for applications servers of a storage area network on the second portion of a storage medium, and to store pointers to data for applications servers on a third portion of the storage medium.", "A twelfth aspect of the present invention is a computer program product for storing data from a plurality of storage systems on a storage medium.", "The computer program product comprises software instructions for enabling a computer system to perform predetermined operations, and a computer readable medium bearing the software instructions.", "The predetermined operations comprise storing data for client devices of a network attached storage system on a first portion of the storage medium, storing data for applications servers of a storage area network on a second portion of the storage medium, and storing pointers to data for applications servers on a third portion of the storage medium.", "A thirteenth aspect of the present invention is a executable program for a computer system for storing data from a plurality of storage systems on a storage medium.", "The executable program comprises a first executable code portion which, when executed on the computer system, stores data for client devices of a network attached storage system on a first portion of the storage medium.", "The executable program further comprises a second executable code portion which, when executed on the computer system, stores data for applications servers of a storage area network on a second portion of the storage medium.", "The executable program further comprises a third executable code portion which, when executed on the computer system, stores pointers to data for applications servers on a third portion of the storage medium.", "A fourteenth aspect of the present invention is a method for handling data requests from a plurality of data requestors to a storage device.", "The method comprises receiving the data request and determining if the data request originates from an application server of a storage attached network or a client device.", "If the data request originated from an application server and requests a volume, the method further comprises determining if the requested volume is stored on the storage device, and retrieving the volume using a pointer to the volume.", "If the data request originated from the client device and requests a file, the method further comprises retrieving the requested file from the storage device.", "A fifteenth aspect of the present invention is a method for handling data write requests from a plurality of data writers to a storage device.", "The method comprises receiving the data write request and determining if the data write request originates from an application server of a storage attached network or a client device.", "If the data write request originated from an application server and requests to write a volume to the storage device, the method further comprises determining if the volume is already stored on the storage device, determining if the storage device has space for storing the volume, writing the volume to the storage device, and updating a pointer to the volume.", "If the data write request originated from a client and requests to write a file to the storage device, the method further comprises writing the requested file to the storage device.", "The above aspects and advantages of the invention will become apparent from the following detailed description and with reference to the accompanying drawing figures.", "BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate the invention and, together with the written description, serve to explain the aspects, advantages and principles of the invention.", "In the drawings, FIG.", "1 illustrates a conventional centralized SAN system with several servers and a disk array; FIG.", "2 illustrates a conventional distributed SAN system with several servers and multiple disk arrays; FIG.", "3 illustrates how data objects are passed from disk storage to the application on a conventional SAN system; FIG.", "4 illustrates a conventional NAS system with several clients and a NAS server; FIG.", "5 illustrates how data objects are passed from disk storage to the application on a conventional NAS system; FIG.", "6 illustrates a computer network according to an aspect of the invention, with an NAS server, client devices, a storage device and applications servers attached to a LAN; FIG.", "7 illustrates a data storage device according to an aspect of the invention; FIG.", "8 illustrates a SAN network with applications servers and a LAN network with a NAS server and client devices, with a storage device attached to both the SAN network and the LAN network; FIG.", "9 illustrates a data storage device according to another aspect of the invention; FIG.", "10 illustrates the partitioning of the storage medium for handling logical disk volumes and files, according to an aspect of the invention; FIG.", "11 illustrates the basic process flow for storing data from either an application server or a client device; FIG.", "12 illustrates the basic process flow for handling a data request from either an application server or a client device; and FIGS.", "13A-13B illustrate the basic process flow for handling a data storage request from either an application server or a client device.", "DETAILED DESCRIPTION OF THE INVENTION Prior to describing the aspects of the invention, some details concerning the prior art will be provided to facilitate the reader's understanding of the invention and to set forth the meaning of various terms.", "As used herein, the term “computer system” encompasses the widest possible meaning and includes, but is not limited to, standalone processors, networked processors, mainframe processors, and processors in a client/server relationship.", "The term “computer system” is to be understood to include at least a memory and a processor.", "In general, the memory will store, at one time or another, at least portions of executable program code, and the processor will execute one or more of the instructions included in that executable program code.", "As used herein, the term “embedded computer system” includes, but is not limited to, an embedded central processor and memory bearing object code instructions.", "Examples of embedded computer systems include, but are not limited to, personal digital assistants, cellular phones and digital cameras.", "In general, any device or appliance that uses a central processor, no matter how primitive, to control its functions can be labeled has having an embedded computer system.", "The embedded central processor will execute one or more of the object code instructions that are stored on the memory.", "The embedded computer system can include cache memory, input/output devices and other peripherals.", "As used herein, the terms “predetermined operations,” the term “computer system software” and the term “executable code” mean substantially the same thing for the purposes of this description.", "It is not necessary to the practice of this invention that the memory and the processor be physically located in the same place.", "That is to say, it is foreseen that the processor and the memory might be in different physical pieces of equipment or even in geographically distinct locations.", "As used herein, the terms “media,” “medium” or “computer-readable media” include, but is not limited to, a diskette, a tape, a compact disc, an integrated circuit, a cartridge, a remote transmission via a communications circuit, or any other similar medium useable by computers.", "For example, to distribute computer system software, the supplier might provide a diskette or might transmit the instructions for performing predetermined operations in some form via satellite transmission, via a direct telephone link, or via the Internet.", "Although computer system software might be “written on” a diskette, “stored in” an integrated circuit, or “carried over” a communications circuit, it will be appreciated that, for the purposes of this discussion, the computer usable medium will be referred to as “bearing” the instructions for performing predetermined operations.", "Thus, the term “bearing” is intended to encompass the above and all equivalent ways in which instructions for performing predetermined operations are associated with a computer usable medium.", "Therefore, for the sake of simplicity, the term “program product” is hereafter used to refer to a computer-readable medium, as defined above, which bears instructions for performing predetermined operations in any form.", "As used herein, a “redundant array of independent disks” (RAID) is a disk subsystem that increases performance and/or provides fault tolerance.", "RAID is a set of two or more hard disks and a specialized disk controller that contains the RAID functionality.", "A detailed description of the aspects of the invention will now be given referring to the accompanying drawings.", "As described earlier, in a SAN network, the applications servers are connected to the mass storage devices through the SAN network.", "In a NAS system, the client servers are connected to the storage devices on a LAN.", "It is not uncommon for both types of networks to comprise a complete system.", "For example, the application servers a SAN system (which are connected to the SAN network) may be also connected to a LAN in order to communicate amongst themselves.", "In another example, in a NAS system, a separate SAN network may be connected behind the NAS server.", "In this architecture, the SAN network is used to connect several storage devices to the NAS server (or sometimes more than one NAS server).", "Referring to FIG.", "6, a network incorporating the present invention is illustrated.", "The exemplary network comprises several applications servers 40-42, several client devices 43-44, a data storage device 45 and a NAS server 46.A LAN network 47 interconnects all the components.", "The data storage device 45 comprises an access port 48 (not shown) connected to the NAS server 46.The data storage device 45 further comprises a storage medium 50 (not shown) that is connected to the access port 48, thereby allowing the applications servers 40-42 and the client devices 43-44 access to the storage medium 50.The NAS server 46 uses the storage device 45 with file types of accesses, as in a normal NAS system.", "The application servers 40-42 use the LAN network 47 to access the storage device 45 with volume types of accesses.", "The SAN volume partitions and pointers on the storage medium are used to support these accesses.", "Typically, the storage medium 50 is a hard disk or a RAID device, although other types of storage mediums (e.g., optical disks, tape storage, semiconductor memory) could be used as well.", "The storage device 45 as shown in the network above will now be described in more detail.", "Referring to FIG.", "7, in order to access the data stored on the storage medium 50, the data storage device 45 further comprises a controller 49 that accesses the storage medium according to commands received from a client device through the access port 48.The bus 51 handles the command and data traffic between the controller 49, the storage medium 50 and the access port 48.The controller 49 reads data (e.g., files) from the storage medium 50 and writes data to the storage medium 50 in accordance with commands received through the storage area network 46 from a client device.", "In addition, the controller 49 accesses the storage medium according to commands received from an applications server through the access port 48.The controller 49 reads a volume from the storage medium 50 or writes volume to the storage medium 50 in accordance with commands received via the NAS server, and through the LAN (not shown in this Fig, but shown in FIG.", "6) from an applications server.", "The data storage device 45 also comprises a file manager to manage the volumes and files stored on the storage medium 50 (i.e, read files, write files, change attributes such as read/write access, etc.).", "As will be described below, the storage medium 50 comprises a NAS file partition 57 assigned for storing files for the clients of the network attached storage system, a SAN volume partition 58 assigned for storing volumes for the applications servers of the storage area network, and a SAN pointer partition 59 assigned for storing pointers to the volumes.", "Referring to FIG.", "8, another network incorporating the present invention is illustrated.", "The exemplary network comprises several applications servers 40-42, several client devices 43-44, a data storage device 60 and a NAS server 46.A LAN network 47 interconnects the client devices 43-44 and the NAS server 46.A SAN network 56 interconnects the applications servers 40-42.The data storage device 60 comprises access ports 54,55 (not shown) connected to the LAN network 47 and the SAN network 56, respectively.", "The data storage device 60 further comprises a storage medium 50 that is connected to the access ports 54,55, thereby allowing the applications servers 40-42 and the client devices 43-44 access to the storage medium 50.Typically, the storage medium 50 is a hard disk or a RAID device, although other types of storage mediums (e.g., optical disks, tape storage, semiconductor memory) could be used as well.", "The storage device 60 as shown in the network above will now be described in more detail.", "Referring to FIG.", "9, the data storage device 60 is connected to the clients of a network attached storage system (not shown) and to the applications servers of a storage area network (not shown).", "The data storage device 60 further comprises a NAS access port 54 to interface with the network attached storage system, and a SAN access port 55 to interface with the storage area network.", "As will be described below, the storage medium 50 comprises a NAS file partition 57 assigned for storing files for the clients of the network attached storage system, a SAN volume partition 58 assigned for storing volumes for the applications servers of the storage area network, and a SAN pointer partition 59 assigned for storing pointers to the volumes.", "The data storage device 60 also comprises a controller 49 to process data requests from the clients of the network attached storage system and the applications servers of the storage area network.", "The controller 49 stores a file to or retrieves a file from the NAS file partition 57 of the storage medium 50 in response to a command from a client device.", "In addition, the controller 49 stores a volume to or retrieves a volume from the SAN volume partition 58 of the storage medium 50 in response to a command from an applications server.", "The controller 49 also updates the SAN pointer partition 59 of the storage medium 50 in response to a command from an applications server to retrieve or store a volume on the storage medium 50.The NAS bus 52 handles the command and data traffic between the controller 49, the storage medium 50 and the NAS access port 54.The SAN bus 53 handles the command and data traffic between the controller 49, the storage medium 50 and the SAN access port 55.Referring to FIG.", "10, the storage medium 50 comprises NAS file partition 57 that is assigned to store files for the client devices 43-44.The storage medium 50 also comprises a SAN volume partition 58 that is assigned to store volumes for the applications servers 40-42.As shown in FIG.", "10, multiple volumes 58a-58n are stored in the SAN volume partition 58.These volumes 58a-58n are forwarded to the applications servers 40-42 when requested.", "The storage medium 50 further comprises a SAN pointer partition 59 assigned to store pointers for the volumes 58a-58n for the applications servers 40-42.In a multiple storage medium environment, partitioning as illustrated in FIG.", "10 is duplicated on each storage medium in the multiple storage medium environment.", "For example, if you have eight hard disk drives, each hard disk drive will have a dedicated NAS file partition 57, a SAN volume partition 58 and a SAN pointer partition 59.The storage medium 50 is a hard disk or a RAID device, although other types of storage mediums (e.g., optical disks, semiconductor memory, tape storage) could be used as well.", "If a plurality of hard disks are used as the storage medium, the NAS file partition 57 can simply be assigned to one or more of the disks comprising the plurality of disk drives.", "Likewise, the SAN volume partition 58 and the SAN pointer partition 59 can be assigned to one or more of the disk drives comprising the plurality of disk drives.", "Referring to FIG.", "11, another aspect of the invention will now be described.", "Since the present invention stores data that is assigned to both client devices of a network attached storage system and the application servers of a storage area network, the data storage device must sort through the different types of data and store them in their proper partitions.", "At S100, a determination is made of what type of data management request is being made (i.e., store data, retrieve data, change attributes, etc.)", "and from which type of network the data management request originates.", "Based on that determination, at S110, if the data management request originated from a NAS client device, than at S120, the data storage device 50 performs the requested data management function on the NAS file partition 57.If the data management request was not from a NAS client, the method shifts to S130.At S130, if the data management request was from an applications server of the storage area network, at S140, the data storage device 50 performs the requested data management function on the SAN volume partition 58.Once the requested data management function is complete, at S150, the data storage device 50 updates the SAN pointer partition 59 to reflect any changes made to the SAN volume partition 58.At S160, if the data management request unidentifiable, an error message is output.", "Another embodiment of the present invention described above is a computer system that is adapted to storing data from a plurality of storage systems on a storage medium.", "The computer system comprises a processor and a memory that comprises software instructions adapted to enable the computer system to perform various functions.", "Aside from the functions that are associated with an operating system, the memory comprises software instructions adapted to store data for client devices of a network attached storage system on a first portion of the storage medium.", "This first portion of the storage medium would be the NAS file partition 57 on a storage medium 50 in a storage device 45.The software instructions on the memory are also adapted to store data for applications servers of a storage area network on a second portion of a storage medium.", "This second portion of the storage medium would be the SAN volume partition 58 on a storage medium 50 in a storage device 45.The software instructions on the memory are also adapted to store pointers to data for applications servers on a third portion of a storage medium.", "This third portion of the storage medium would be the SAN pointer partition 59 on a storage medium 50 in a storage device 45.To execute the functions described above with respect to storing data on the storage medium, the software instructions are further adapted to enable the computer system to read or write data from the first portion of the storage medium in response to a command from a client device.", "Again, this first portion of the storage medium would be the NAS file partition 57 on a storage medium 50 in a storage device 45.The software instructions are also adapted to enable the computer system to read or write data from the second portion of the storage medium in response to a command from an applications server.", "Again, this second portion of the storage medium would be the SAN volume partition 58 on a storage medium 50 in a storage device 45.In addition, the software instructions are further adapted to enable the computer system to update pointers stored on die third portion of the storage medium in response to a command from an applications server to read or write data from the second portion of the storage medium.", "Again, this third portion of the storage medium would be the SAN pointer partition 59 on a storage medium 50 in a storage device 45.Another embodiment of the present invention described above is a computer program product for storing data from a plurality of storage systems on a storage medium.", "The computer program product comprises software instructions for enabling a computer system to perform predetermined operations, and a computer readable medium bearing the software instructions.", "The computer readable medium can be any one of the mediums described above.", "The predetermined operations comprise software instructions adapted to store data for client devices of a network attached storage system on a first portion of the storage medium.", "This first portion of the storage medium would be the NAS file partition 57 on a storage medium 50 in a storage device 45.The predetermined operations further comprise software instructions adapted to store data for applications servers of a storage area network on a second portion of a storage medium.", "This second portion of the storage medium would be the SAN volume partition 58 on a storage medium 50 in a storage device 45.The predetermined operations further comprise software instructions adapted to store pointers to data for applications servers on a third portion of a storage medium.", "This third portion of the storage medium would be the SAN pointer partition 59 on a storage medium 50 in a storage device 45.To execute the functions described above with respect to storing data on the storage medium, the predetermined operations are software instructions further adapted to enable the computer system to read or write data from the first portion of the storage medium in response to a command from a client device.", "Again, this first portion of the storage medium would be the NAS file partition 57 on a storage medium 50 in a storage device 45.The predetermined operations are software instructions further adapted to enable the computer system to read or write data from the second portion of the storage medium in response to a command from an applications server.", "Again, this second portion of the storage medium would be the SAN volume partition 58 on a storage medium 50 in a storage device 45.In addition, the predetermined operations are software instructions further adapted to enable the computer system to update pointers stored on the third portion of the storage medium in response to a command from an applications server to read or write data from the second portion of the storage medium.", "Again, this third portion of the storage medium would be the SAN pointer partition 59 on a storage medium 50 in a storage device 45.Another embodiment of the present invention described above is an executable program for a computer system for storing data from a plurality of storage systems on a storage medium.", "The executable program comprises a first executable code portion which, when executed on the computer system, stores data for client devices of a network attached storage system on a first portion of the storage medium.", "This first portion of the storage medium would be the NAS file partition 57 on a storage medium 50 in a storage device 45.The executable program comprises a second executable code portion which, when executed on the computer system, stores data for applications servers of a storage area network on a second portion of a storage medium.", "This second portion of the storage medium would be the SAN volume partition 58 on a storage medium 50 in a storage device 45.The executable program comprises a third executable code portion which, when executed on the computer system, stores pointers to data for applications servers on a third portion of a storage medium.", "This third portion of the storage medium would be the SAN pointer partition 59 on a storage medium 50 in a storage device 45.Referring to FIG.", "12, another aspect of the invention is illustrated.", "The present invention must handle data requests from a plurality of data requesters, and provides a method for handling such data requests.", "At S200, a request for data retrieval is received at the data storage device.", "At S210, a determination is made whether the request for data retrieval originated from a NAS client device or a SAN applications server.", "At S220, if the request for data retrieval originated from a NAS client device, the method shifts to S230, where a file manager is used to process the request for data retrieval.", "If the request for data retrieval did not originate from a NAS client device, the method shifts to S240.At S240, if the request for data retrieval originated from a SAN applications server, the method shifts to S260, where the pointer list is reviewed to determine if the requested logical disk volume is present on the data storage device.", "If the request for data retrieval did not originate from a SAN applications server, the method shifts to S250 and an error message is output.", "At S260, if the requested logical volume is present, the method shifts to S280, where the request for data retrieval is processed.", "If the requested logical volume is not present, the method shifts to S290, where the requestor is notified that the requested logical disk volume is unavailable.", "Referring to FIGS.", "13A-13B, another aspect of the invention is illustrated.", "The present invention must handle data writing requests from a plurality of data writers, and provides a method for handling such data writing requests.", "At S300, a request for data storage is received at the data storage device.", "At S310, a determination is made whether the request for data storage originated from a NAS client device or a SAN applications server.", "At S320, if the request for data storage originated from a NAS client device, the method shifts to S330, where a file manager is used to process the request for data storage.", "If the request for data storage did not originate from a NAS client device, the method shifts to S340.At S340, if the request for data storage originated from a SAN applications server, the method shifts to S350, where the size of the logical disk volume to be stored is determined.", "If the request for data storage did not originate from a SAN applications server, the method shifts to S430 and an error message is output.", "At S360, if there is insufficient disk space to store the logical disk volume, at S370, the data storage requestor is notified that the data storage device lacks sufficient storage space.", "If there is sufficient storage space on the data storage device, at S380, a determination is made whether the logical disk volume is replacing a logical disk volume that is currently mounted on the data storage device.", "At S390, if a currently mounted logical disk volume is to be replaced, the method shifts to S400, where the new logical disk volume replaces the currently mounted logical disk volume.", "Subsequent to the replacement, as S440, the pointers to the logical disk volumes stored on the data storage device are updated.", "If the new logical volume does not replace a currently mounted logical volume, the method shifts to S410.At S410, a determination is made whether the new logical volume is to be stored on the data storage device.", "At S420, if the new logical volume is to be stored, the method shifts to S430 wherein the new logical volume is stored on the data storage device.", "At S440, the pointers to the logical disk volumes stored on the data storage device are updated.", "The foregoing description of the aspects of the invention has been presented for purposes of illustration and description.", "It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention.", "The principles of the invention and its practical application were described in order to explain the to enable one skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated.", "Thus, while only certain aspects of the invention have been specifically described herein, it will be apparent that numerous modifications may be made thereto without departing from the spirit and scope of the invention.", "Further, acronyms are used merely to enhance the readability of the specification and claims.", "It should be noted that these acronyms are not intended to lessen the generality of the terms used and they should not be construed to restrict the scope of the claims to the embodiments described therein." ] ]	Patent_10465631
[ [ "Apparatus and method for preparing cerium oxide nanoparticles", "This invention provides a method for preparing cerium oxide nanoparticles with a narrow size distribution.", "The cerium oxide nanoparticles obtained by the method of the invention are nearly all crystalline.", "The method comprises providing a first aqueous solution comprising cerium nitrate and providing a second aqueous solution comprising hexamethylenetetramine.", "The first and second aqueous solutions are mixed to form a mixture, and the mixture is maintained at a temperature no higher than about 320° K to form nanoparticles.", "The nanoparticles that are formed are then separated from the mixture.", "A further aspect of the present invention is an apparatus for preparing cerium oxide nanoparticles.", "The apparatus comprises a mixing vessel having a first compartment for holding a first aqueous solution comprising cerium nitrate and a second compartment for holding a second aqueous solution comprising hexamethylenetetramine.", "The mixing vessel has a retractable partition separating the first and second compartments.", "When the retractable partition is retracted, rapid mixing of the first aqueous solution with the second aqueous solution takes place to form a mixture, and the mixture is maintained at a temperature no higher than about 320° K to form nanoparticles therein." ], [ "1.A method for preparing cerium oxide nanoparticles comprising the steps of: (a) providing a first aqueous solution comprising cerium nitrate; (b) providing a second aqueous mixture comprising hexamethylenetetramine; (c) rapidly mixing the first aqueous solution with the second aqueous solution to form a mixture; (d) maintaining the mixture at a temperature no higher than about 320° K to form nanoparticles in the mixture; and (e) separating the nanoparticles formed in the mixture from the mixture.", "2.The method of claim 1, wherein the first aqueous solution is provided in a first compartment of a mixing vessel, and the second aqueous solution is provided in a second compartment of the mixing vessel, the first and second compartments being separated by retractable partition, and wherein the mixing step comprises retracting the retractable partition of the mixing vessel to cause mixing of the first aqueous solution with the second aqueous solution to form the mixture.", "3.The method of claim 1, wherein one of the first and second aqueous solutions is provided in a mixing vessel having at least one inlet, and the other of the first and second aqueous solutions is pumped into the mixing vessels through the at least one inlet to cause rapid mixing of the first and second aqueous solutions to form the mixture.", "4.The method of claim 1, wherein the first aqueous solution has a concentration of cerium nitrate in the range of about 0.005 M to about 0.04 M. 5.The method of claim 1, wherein the second aqueous solution has a concentration of hexamethylenetetramine in the range of about 0.05 M to about 1.5 M. 6.The method of claim 5, wherein the concentration of hexamethylenetetramine is in the range of about 0.5 M to about 1.5 M. 7.The method of claim 1, further comprising the step of siring the mixture while the mixture is maintained at a temperature no higher than about 320° K. 8.The method of claim 2, wherein the mixing vessel includes a mechanical stirrer and the mixture is stirred with the mechanical stirrer.", "9.The method of claim 8, wherein the mechanical stirrer comprises a vertical stirring rod having a plurality of stirring elements attached thereto.", "10.The method of claim 9, wherein the vertical stirring rod has a first aperture for fitting a first inlet feed for introducing the first aqueous solution and a second aperture for fitting a second inlet feed for introducing the second aqueous solution, wherein the first inlet feed leads into the first compartment and the second inlet feed leads into the second compartment of the mixing vessel when the retractable partition is in place.", "11.The method of claim 10, wherein the vertical stirring rod further includes an outlet drain for the removal of liquid from the mixing vessel.", "12.The method of claim 1, wherein the mixture is maintained at a temperature no higher than about 320° K for a time period between about 2 hours and about 24 hours.", "13.The method of claim 12, wherein the time period is between about 5 hours and about 24 hours.", "14.The method of claim 12, wherein the time period is between about 12 hours and about 24 hours.", "15.The method of claim 1, further comprising the step of sintering the nanoparticles separated in step (e) in air at a temperature in the range of about 400° C. to about 800° C. 16.The method of claim 1, wherein step (e) comprises centrifuging the mixture to separate the nanoparticles from the mixture.", "17.The method of claim 2 or 3, wherein the step of separating the nanoparticles from the mixture comprises the steps of positioning the mixing vessel inside a centrifuge and centrifuging the mixture in the mixing vessel.", "18.The method of claim 1, wherein the cerium oxide nanoparticles include single crystalline cerium oxide nanoparticles.", "19.The method of claim 2, wherein retracting the retractable partition takes place in about 0.1 seconds to about 5 seconds.", "20.The method of claim 1, 7, or 12, wherein the mixture is maintained at a temperature of about 300° K to form nanoparticles in the mixture.", "21.A method for preparing cerium oxide nanoparticles, comprising the steps of: (a) providing a first aqueous solution comprising cerium nitrate in a mixing vessel; (b) rapidly adding a second aqueous solution comprising hexamethylenetetramine to the first aqueous solution in the mixing vessel to form a mixture; and (c) maintaining the mixture at a temperature no higher than about 320° K to form the nanoparticles in the mixture; and (d) separating the nanoparticles formed in step (c) from the mixture.", "22.The method of claim 21, wherein the mixing vessel has at least one inlet, and step (b) comprises rapidly pumping the second aqueous solution through the at least one inlet into the mixing vessel containing the first solution to form the mixture.", "23.A method for preparing cerium oxide nanoparticles, comprising the steps of: (a) providing a first aqueous solution comprising hexamethylenetetramine in a mixing vessel; (b) rapidly adding a second aqueous solution comprising cerium nitrate to the first aqueous solution in the mixing vessel to form a mixture; (c) maintaining the mixture at a temperature no higher than about 320° K to form the nanoparticles in the mixture; and (d) separating the nanoparticles formed in step (c) from the mixture.", "24.The method of claim 23, wherein the mixing vessel has at least one inlet, and step (b) comprises rapidly pumping the second aqueous solution through the inlet into the mixing vessel containing the first solution to form the mixture.", "25.The method of claim 21 or 23, wherein the step of separating the nanoparticles from the mixture comprises centrifuging the mixture.", "26.The method of claim 21 or 23, wherein the step of separating the nanoparticles from the mixture comprises positioning the mixing vessel inside a centrifuge, and centrifuging the mixture in the mixing vessel.", "27.The method of claim 21 or 23, wherein the mixture is maintained at a temperature of about 300° K to form nanoparticles in the mixture.", "28.An apparatus for the preparation of cerium oxide nanoparticles, comprising a mixing vessel having (a) a first compartment for holding a first aqueous solution comprising cerium nitrate; (b) a second compartment for holding a second aqueous solution comprising hexamethylenetetramine; and (c) a retractable partition separating the first and second compartments, wherein when the retractable partition is retracted, rapid mixing of the first and second aqueous solutions takes place to form a mixture, and the mixture in the mixing vessel is maintained at a temperature no higher than 3200K to form nanoparticles therein.", "29.The apparatus of claim 28, wherein the retractable partition is a vertical partition.", "30.The apparatus of claim 28, wherein the mixing vessel further comprises a mechanical stirrer for stirring the mixture.", "31.The apparatus of claim 30, wherein the mechanical stirrer comprises a rotatable vertical stirring rod having a plurality of siring elements attached thereto.", "32.The apparatus of claim 31, wherein the vertical stirring rod has a first aperture for fitting a first inlet feed and a second aperture for fitting a second inlet feed, wherein the first aqueous solution is provided through the first inlet feed into the first compartment and the second aqueous solution is provided through the second inlet feed into the second compartment.", "33.The apparatus of claim 32, wherein the vertical stirring rod further comprises an outlet drain for the removal of liquid from the mixing vessel.", "34.The apparatus of claim 28, further comprising a centrifuge, wherein the mixing vessel is adapted to be positioned inside the centrifuge.", "35.The apparatus of claim 28, wherein the mixing vessel is maintained at a temperature of about 300° K to form nanoparticles therein.", "36.A method for preparing cerium oxide nanoparticles containing a metal selected from the group consisting of platinum, gold, palladium, copper and nickel, wherein the method comprises the steps of: (a) providing a first aqueous solution comprising cerium nitrate; (b) providing a second aqueous solution comprising hexamethylenetetramine; (c) mixing the first and second aqueous solutions to form a mixture; (d) maintaining the mixture at a temperature no higher than about 320° K, to form nanoparticles therein; (e) separating the nanoparticles formed in step (d) from the mixture; and (f) soaking the nanoparticles separated from the mixture in a solution comprising ions or complex ions of the metal.", "37.The method of claim 36, wherein the metal comprises platinum, and step (f) comprises soaking the nanoparticles in a solution comprising chloroplatinic acid.", "38.The method of claim 36, wherein the mixture is maintained at a temperature of about 300° K to form nanoparticles therein." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The invention is directed to a method and apparatus for the preparation of nanoparticles of cerium oxide.", "In particular, the invention is directed to a method and apparatus for the preparation of cerium oxide nanoparticles having a narrow size distribution.", "2.Background Information Cerium oxide in the form of fine particles is useful as a catalyst for polymerization, for reforming fuels, and for abating polluting gas in automobile exhaust systems.", "The catalyst acts as an oxygen pressure regulator in the reduction of NO X to molecular nitrogen, the oxidation of hydrocarbons and carbon monoxide to water and carbon dioxide, and the conversion of H 2 S to H 2 and S. Cerium oxide has been used as a catalyst-component for the recombination of hydrogen and oxygen to water for sealed car batteries, which extends battery life.", "The oxide is a good ionic conductor and has been used as an electrolyte material for a solid oxide fuel cells and gas sensors, as discussed, for example, by Steele, B. C. H., Solids State Ionics , Vol.", "12 (1984), p. 391.The oxide has a high dielectric constant and a high refractive index (useful for optical coatings), and can be used as an insulating layer on semiconductor substrates.", "Cerium oxide is also of interest as a catalyst in vehicle emissions systems, as discussed in Yao, Y. F. and Kummer, J. T., Journal of Catalysis, Vol.", "103 (1987), p. 307, and has also found use as a solid oxide fuel cell electrolyte material, as shown in Mogesen, M., Sammes, N. M. and Tompsett, G. A., Solid State Ionics , Vol.", "172 (2000), p. 63; in gas sensors, as described in Lampe, U., Gerblinger, J. and Meixner, H., Sensors and Actuators B—Chemical , Vol.", "7 (1992), p. 787; in optical coatings, as described in Haas, G., Ramsey, J.", "B. and Thun, R., Journal of the Optical Society of America , Vol.", "48 (1957), p. 324; in high-T c superconductor structures, as discussed in Walkenhorst, A., Schmitt, M., Adrian, H. and Petersen, K., Applied Physics Letters , Vol.", "64 (1994), p. 1871; and silicon-on-insulator structures and high storage capacitor devices, as shown by Chikyow, T., Bedair, S. M., Tye, L. and El-Masry, N. A., Applied Physics Letters , Vol.", "65 (1994), p. 1030.Because of the relative hardness of the material, cerium oxide nanoparticles are also useful as an abrasive for fine polishing of surfaces of certain materials, such as quartz and silicon.", "Some applications may benefit from using monodispersed cerium oxide nanoparticles, due to either possibly new properties when such particles are nanodimensional or the greater control in uniform structures.", "A sub-micron scale cerium oxide powder has been prepared and used to decrease the sintering temperature from 1500° C. to 1200° C., as described by Chen, P. L. and Chen, I. W., Journal of the American Ceramic Society , Vol.", "76 (1993), p. 1577; however, there has been no report of any method for preparing cerium oxide nanoparticles having dimensions smaller than about 14.5 nm, and no report of cerium oxide particles having monodispersity.", "The electrical conductivity of multi-dispersed nanoparticles of cerium oxide prepared by a vacuum technique has been investigated by Chiang, Y. M., Lavik, E. B., Kosacki, I., Tuller, H. L. and Ying, J. Y., Electroceramics , Vol.", "1 (1997), p. 7.The vacuum sputtering technique used by Chiang et al.", "usually yields cerium oxide particles of a large size distribution, which makes it very difficult to test and sort out particle-size effects on the catalytic process for certain reactions.", "Tsunekawa, S., Sahara, R, Kawazoe, Y. and Ishikawa, K, Applied Surface Science , Vol.", "152 (1999), p. 53; and Tsunekawa, S., Ishikawa, K, Li, Z. Q., Kawazoe, Y. and Kasuya, Y., Physics Review Letters , Vol.", "85 (2000), p. 3440, both claim to have prepared mono-sized nanoparticles of cerium oxide and reported lattice expansions with decreasing particle-size in a few nanosized cerium oxide particles.", "Both papers suggest that a decrease in the size of cerium oxide nanoparticles is accompanied by a significant increase in the lattice parameter.", "Neither paper, however, discloses the method of preparation of cerium oxide nanoparticles or the apparatus used.", "The foregoing discussion shows that there is a need in the art for an efficient method and apparatus for preparing significant quantities of cerium oxide nanoparticles with a relatively narrow size distribution." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The aforementioned need is substantially met by the present invention, which in one aspect is a method for preparing cerium oxide nanoparticles.", "The method comprises providing a first aqueous solution comprising cerium nitrate and providing a second aqueous solution comprising hexamethylenetetramine.", "The first and second aqueous solutions are mixed to form a mixture, and the mixture is maintained at a temperature no higher than about 320° K to form nanoparticles therein.", "The nanoparticles that are formed are then separated from the mixture.", "Another aspect of the present invention is a method for preparing cerium oxide nanoparticles, where a first aqueous solution comprising cerium nitrate is provided in a first compartment of a mixing vessel and a second aqueous solution comprising hexamethylenetetramine is provided in a second compartment of the mixing vessel, the first and second compartments being separated by a retractable partition.", "The retractable partition is retracted to allow the first and second aqueous solutions to mix so as to form a mixture, and the mixture is maintained at a temperature no higher than about 320° K to form nanoparticles.", "The nanoparticles that are formed are then separated from the mixture.", "Still another aspect of the present invention is a method for preparing cerium oxide nanoparticles, where a first aqueous solution comprising one of cerium nitrate and hexamethylenetetramine is provided in a mixing vessel having at least one inlet.", "A second aqueous solution comprising the other one of cerium nitrate and hexamethylenetetramine is pumped into the mixing vessel through the at least one inlet so as to cause mixing of the first and second aqueous solutions to form a mixture.", "The mixture is maintained at a temperature no higher than about 320° K to form nanoparticles.", "The nanoparticles that are formed are then separated from the mixture.", "A further aspect of the present invention is an apparatus for preparing cerium oxide nanoparticles.", "The apparatus comprises a mixing vessel having a first compartment for holding a first aqueous solution comprising cerium nitrate and a second compartment for holding a second aqueous solution comprising hexamethylenetetramine.", "The mixing vessel has a retractable partition separating the first and second compartments.", "When the retractable partition is retracted, rapid mixing of the first aqueous solution with the second aqueous solution takes place to form a mixture, and the mixture is maintained in a mixing vessel at a temperature no higher than about 320° K to form nanoparticles therein.", "The method and apparatus of the invention have the advantage of being usable to prepare cerium oxide in a quantity which is limited only by the size of the mixing vessel.", "We have prepared up to about 70 gm of nanoparticles per batch.", "This is a very large amount when compared to the scale of nanoparticle synthesis of the prior art.", "By providing for a fast reaction rate and controlling the reaction time, cerium oxide nanoparticles can be prepared within the desired size distribution.", "The method and mixing vessel also have the advantage of providing cerium oxide nanoparticles which are crystalline.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1 is a graphical plot showing the variation of cerium oxide particle size as a function of the time of mixing of the reactants at 300° K FIGS.", "2A-2C depict a side view and two top views, respectively, of one embodiment of the mixing vessel according to the present invention.", "FIG.", "3 depicts a side view of another embodiment of the mixing vessel according to the present invention.", "FIG.", "4 are graphical plots showing the variation of the light absorption spectrum of the reaction mixture with the cerium oxide particle size at different stages of the reaction.", "FIG.", "5 is a graphical plot showing lattice parameter versus particle size of cerium oxide for a particle size less than 80 nm.", "FIG.", "6 depicts a high resolution TEM image of cerium oxide nanoparticles showing (111) planes with 0.3 nm spacing.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "CROSS-REFERENCE TO RELATED APPLICATION This application claims priority from U.S.", "Provisional Application Ser.", "No.", "60/289,062, filed May 7, 2001, U.S.", "Provisional Application Ser.", "No.", "60/302,562, filed Jul.", "2, 2001, and U.S.", "Provisional Application Ser.", "No.", "60/333,451, filed Nov. 27, 2001, which are all incorporated herein by reference in their entirety.", "BACKGROUND OF THE INVENTION 1.Field of the Invention The invention is directed to a method and apparatus for the preparation of nanoparticles of cerium oxide.", "In particular, the invention is directed to a method and apparatus for the preparation of cerium oxide nanoparticles having a narrow size distribution.", "2.Background Information Cerium oxide in the form of fine particles is useful as a catalyst for polymerization, for reforming fuels, and for abating polluting gas in automobile exhaust systems.", "The catalyst acts as an oxygen pressure regulator in the reduction of NOX to molecular nitrogen, the oxidation of hydrocarbons and carbon monoxide to water and carbon dioxide, and the conversion of H2S to H2 and S. Cerium oxide has been used as a catalyst-component for the recombination of hydrogen and oxygen to water for sealed car batteries, which extends battery life.", "The oxide is a good ionic conductor and has been used as an electrolyte material for a solid oxide fuel cells and gas sensors, as discussed, for example, by Steele, B. C. H., Solids State Ionics, Vol.", "12 (1984), p. 391.The oxide has a high dielectric constant and a high refractive index (useful for optical coatings), and can be used as an insulating layer on semiconductor substrates.", "Cerium oxide is also of interest as a catalyst in vehicle emissions systems, as discussed in Yao, Y. F. and Kummer, J. T., Journal of Catalysis, Vol.", "103 (1987), p. 307, and has also found use as a solid oxide fuel cell electrolyte material, as shown in Mogesen, M., Sammes, N. M. and Tompsett, G. A., Solid State Ionics, Vol.", "172 (2000), p. 63; in gas sensors, as described in Lampe, U., Gerblinger, J. and Meixner, H., Sensors and Actuators B—Chemical, Vol.", "7 (1992), p. 787; in optical coatings, as described in Haas, G., Ramsey, J.", "B. and Thun, R., Journal of the Optical Society of America, Vol.", "48 (1957), p. 324; in high-Tc superconductor structures, as discussed in Walkenhorst, A., Schmitt, M., Adrian, H. and Petersen, K., Applied Physics Letters, Vol.", "64 (1994), p. 1871; and silicon-on-insulator structures and high storage capacitor devices, as shown by Chikyow, T., Bedair, S. M., Tye, L. and El-Masry, N. A., Applied Physics Letters, Vol.", "65 (1994), p. 1030.Because of the relative hardness of the material, cerium oxide nanoparticles are also useful as an abrasive for fine polishing of surfaces of certain materials, such as quartz and silicon.", "Some applications may benefit from using monodispersed cerium oxide nanoparticles, due to either possibly new properties when such particles are nanodimensional or the greater control in uniform structures.", "A sub-micron scale cerium oxide powder has been prepared and used to decrease the sintering temperature from 1500° C. to 1200° C., as described by Chen, P. L. and Chen, I. W., Journal of the American Ceramic Society, Vol.", "76 (1993), p. 1577; however, there has been no report of any method for preparing cerium oxide nanoparticles having dimensions smaller than about 14.5 nm, and no report of cerium oxide particles having monodispersity.", "The electrical conductivity of multi-dispersed nanoparticles of cerium oxide prepared by a vacuum technique has been investigated by Chiang, Y. M., Lavik, E. B., Kosacki, I., Tuller, H. L. and Ying, J. Y., Electroceramics, Vol.", "1 (1997), p. 7.The vacuum sputtering technique used by Chiang et al.", "usually yields cerium oxide particles of a large size distribution, which makes it very difficult to test and sort out particle-size effects on the catalytic process for certain reactions.", "Tsunekawa, S., Sahara, R, Kawazoe, Y. and Ishikawa, K, Applied Surface Science, Vol.", "152 (1999), p. 53; and Tsunekawa, S., Ishikawa, K, Li, Z. Q., Kawazoe, Y. and Kasuya, Y., Physics Review Letters, Vol.", "85 (2000), p. 3440, both claim to have prepared mono-sized nanoparticles of cerium oxide and reported lattice expansions with decreasing particle-size in a few nanosized cerium oxide particles.", "Both papers suggest that a decrease in the size of cerium oxide nanoparticles is accompanied by a significant increase in the lattice parameter.", "Neither paper, however, discloses the method of preparation of cerium oxide nanoparticles or the apparatus used.", "The foregoing discussion shows that there is a need in the art for an efficient method and apparatus for preparing significant quantities of cerium oxide nanoparticles with a relatively narrow size distribution.", "SUMMARY OF THE INVENTION The aforementioned need is substantially met by the present invention, which in one aspect is a method for preparing cerium oxide nanoparticles.", "The method comprises providing a first aqueous solution comprising cerium nitrate and providing a second aqueous solution comprising hexamethylenetetramine.", "The first and second aqueous solutions are mixed to form a mixture, and the mixture is maintained at a temperature no higher than about 320° K to form nanoparticles therein.", "The nanoparticles that are formed are then separated from the mixture.", "Another aspect of the present invention is a method for preparing cerium oxide nanoparticles, where a first aqueous solution comprising cerium nitrate is provided in a first compartment of a mixing vessel and a second aqueous solution comprising hexamethylenetetramine is provided in a second compartment of the mixing vessel, the first and second compartments being separated by a retractable partition.", "The retractable partition is retracted to allow the first and second aqueous solutions to mix so as to form a mixture, and the mixture is maintained at a temperature no higher than about 320° K to form nanoparticles.", "The nanoparticles that are formed are then separated from the mixture.", "Still another aspect of the present invention is a method for preparing cerium oxide nanoparticles, where a first aqueous solution comprising one of cerium nitrate and hexamethylenetetramine is provided in a mixing vessel having at least one inlet.", "A second aqueous solution comprising the other one of cerium nitrate and hexamethylenetetramine is pumped into the mixing vessel through the at least one inlet so as to cause mixing of the first and second aqueous solutions to form a mixture.", "The mixture is maintained at a temperature no higher than about 320° K to form nanoparticles.", "The nanoparticles that are formed are then separated from the mixture.", "A further aspect of the present invention is an apparatus for preparing cerium oxide nanoparticles.", "The apparatus comprises a mixing vessel having a first compartment for holding a first aqueous solution comprising cerium nitrate and a second compartment for holding a second aqueous solution comprising hexamethylenetetramine.", "The mixing vessel has a retractable partition separating the first and second compartments.", "When the retractable partition is retracted, rapid mixing of the first aqueous solution with the second aqueous solution takes place to form a mixture, and the mixture is maintained in a mixing vessel at a temperature no higher than about 320° K to form nanoparticles therein.", "The method and apparatus of the invention have the advantage of being usable to prepare cerium oxide in a quantity which is limited only by the size of the mixing vessel.", "We have prepared up to about 70 gm of nanoparticles per batch.", "This is a very large amount when compared to the scale of nanoparticle synthesis of the prior art.", "By providing for a fast reaction rate and controlling the reaction time, cerium oxide nanoparticles can be prepared within the desired size distribution.", "The method and mixing vessel also have the advantage of providing cerium oxide nanoparticles which are crystalline.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1 is a graphical plot showing the variation of cerium oxide particle size as a function of the time of mixing of the reactants at 300° K FIGS.", "2A-2C depict a side view and two top views, respectively, of one embodiment of the mixing vessel according to the present invention.", "FIG.", "3 depicts a side view of another embodiment of the mixing vessel according to the present invention.", "FIG.", "4 are graphical plots showing the variation of the light absorption spectrum of the reaction mixture with the cerium oxide particle size at different stages of the reaction.", "FIG.", "5 is a graphical plot showing lattice parameter versus particle size of cerium oxide for a particle size less than 80 nm.", "FIG.", "6 depicts a high resolution TEM image of cerium oxide nanoparticles showing (111) planes with 0.3 nm spacing.", "DETAILED DESCRIPTION OF THE INVENTION Referring to FIG.", "2B, there is shown a top view of an exemplary embodiment of a mixing vessel 30 separated into two sealed compartments 32 and 33 by a retractable partition 28 comprising two foldable panels 26 and 27.One of the compartments 32 and 33 contains an aqueous solution of cerium nitrate (Ce(NO3)3) at a concentration in the range of about 0.0037 M to 0.04 M, while the other of the compartments 32 and 33 contains an aqueous solution of hexamethylenetetramine at a concentration of about 0.5 M to about 1.5 M, preferably in the range of about 0.5 M to about 1.5 M. Turning to FIG.", "2C, when the retractable partition 28 of the mixing vessel 30 is retracted by folding the two panels 26 and 27 against the inner wall of the mixing vessel 30 the aqueous solution of cerium nitrate and the aqueous solution of hexamethylenetetramine rapidly mixed to form a mixture.", "The mixture is maintained in the mixing vessel 30 at a temperature no higher than 320° K., preferably at 300° K, to form nanoparticles therein.", "Initial rapid mixing enables the nanoparticles to nucleate at approximately the same time.", "Following nucleation, the nanoparticles all grow at the same rate in the course of the reaction, thereby ensuring monodispersity of the nanoparticles.", "Referring to FIG.", "2A, while the mixture is maintained in the mixing vessel 30 at a temperature no higher than about 320° K, it is being continuously stirred with a mechanical stirrer 31 built into the mixing vessel 30.The mechanical stirrer 31 comprises a vertical rotatable rod 20 having a plurality of horizontal stirring rods 25 attached thereto.", "The exemplary mixing vessel of FIGS.", "2A, 2B and 2C will be described in greater detail hereinbelow.", "The stirring of the mixture is performed for a period between about 2 hours and about 24 hours, preferably between about 5 hours and about 24 hours.", "By controlling the reaction to such a time interval, batches of approximately spherical cerium oxide particles of a size ranging from 3 nm to about 12 nm in diameter are obtained.", "FIG.", "1 is a graphical plot showing the increase in particle size with increasing reaction time.", "In another exemplary embodiment of the present invention, either one of the two aqueous solutions comprising cerium nitrate and hexamethylenetetramine, respectively, may be placed in a mixing vessel, and the other one of the aqueous solutions is poured into the mixing vessel to cause mixing of the two aqueous solutions to form a mixture.", "The mixture is maintained in the mixing vessel 30 at a temperature no higher than about 320° K to form nanoparticles therein.", "The nanoparticles formed in the mixture are then separated from the mixture.", "The nanoparticles obtained by the method of the invention have a cerium to oxygen ratio corresponding to the formula CeO2—Y, where y ranges from 0 to 0.5.Advantageously, the value of y is 0.For at least some nanoparticles, X-ray absorption spectroscopy measurements indicate the presence of both tetravalent and trivalent cerium ions.", "Accordingly, for these nanoparticles, the value of y in the above formula is greater than 0.At least some tetravalent cerium is present in most nanoparticles, since at least some of the Ce3+ ions are oxidized to Ce4+ ions by oxygen in the air.", "Accordingly, the value of y is typically less than 0.5, which would correspond to pure Ce2O3, and ranges from 0 to about 0.35.The nanoparticles are monodispersed, wherein the term as used herein is intended to mean particles in which the full width at half maximum (FWHM) of the size distribution plot for a batch of 100 or more particles is less than +/−15% of the median size.", "The nanoparticle size increases with increasing length of the reaction time.", "Larger cerium oxide nanoparticles are obtained when the sting of the mixture is carried out for about 12 to about 24 hours.", "After completion of the maintaining and sting, the resulting mixture is centrifuged to separate nanoparticles of CeO2-y, where y ranges from 0 to about 0.35, from the reaction mixture.", "The nanoparticles separated by centrifugation may be sintered to obtain larger nanoparticles.", "To this end, The larger cerium oxide nanoparticles after centrifugation are sintered in air at temperatures ranging between about 400° and about 800° C. for a period of time between about 8 and about 16 hours.", "The sintering temperature is ramped up at a rate of about 100° C./hr, and the temperature is preferably maintained at its maximum value for about 30 minutes.", "Thereafter, the sintering temperature is ramped down at a rate of about −100° C./hr, until the initial temperature is reached.", "Without wishing to be bound by any theory or mechanism, it is believed that hexamethylenetetramine hydrolyzes slowly to produce formaldehyde and ammonia.", "Ammonia then reacts with water to become ammonium hydroxide.", "As a result, the pH increases to a value of about 8.Since cerium oxide contains cerium in an oxidation state of +4, which is a Lewis acid, an increase in pH favors the formation of cerium oxide.", "As cerium oxide starts to form, the pH of the mixture drops to 6.5 after 200 minutes and reaches a steady state value of about 6.1 after about 1000 minutes for the reaction of a 0.5 M solution of hexamethylenetetramine in water with a solution of 0.0375 M cerium nitrate in water.", "The method of the invention may be used to prepare cerium oxide nanoparticles which contain additional components, including the incorporation of metals and other materials.", "These components can be incorporated onto, or into, the nanoparticles.", "For example, platinum may be impregnated onto the cerium oxide nanoparticles obtained by the procedure described above by soaking the cerium oxide nanoparticles in a solution of chloroplatinic acid, H2PtCl6, followed by drying.", "The soaking step may be preceded by a centrifugation step which separates the nanoparticles from the mixture in which they are produced.", "Similarly, gold, palladium, copper or nickel may be incorporated onto the cerium oxide nanoparticles by soaking the nanoparticles in solutions comprising ions or complex ions of the respective metals.", "Referring to FIG.", "2A, in an exemplary embodiment of the apparatus of the invention, the mixing vessel 30 comprises an inlet for each solution of the reactants and at least one outlet for removal of liquid from the vessel 30.In a particularly advantageous embodiment of the mixing vessel, the vessel 30 comprises a mechanical stirrer 31.The stirrer 31 comprises a vertical rod 20 which can spin about its axis when driven by a drive motor (not shown) coupled thereto.", "Rod 20 is advantageously coated with a chemically inert layer such as TEFLON.® Rod 20, which is positioned along the vertical axis of the cylindrical mixing vessel 30, bears a plurality of horizontal stirring rods 25 and contains apertures 21, 22, 23, for fitting inlet feeds (not shown) for water, a solution of cerium nitrate and a solution of hexamethylenetetramine, respectively, and an outlet drain 24 for removal of liquid from the mixing vessel 30.To prevent contamination of the reactants, the apertures 21, 22, and 23 only allow flow into the mixing vessel 30, and the outlet drain 24 only allows flow out of the vessel 30.The vessel 30 also includes a retractable partition 28.As shown in FIGS.", "2B and 2C, which are top views of the mixing vessel 30, the retractable partition 28 consists of two retractable elements 26 and 27.The retractable elements 26 and 27 are foldable against the inner wall of the mixing vessel 30.Before mixing occurs, the two retractable elements 26 and 27 unfold from the wall of the vessel 30 and extend to the center rod 20 of the stirrer 31, as shown in FIG.", "2B.", "Each of panels 26 and 27 of retractable partition 28 is of a width sufficient for the panel to reach the center rod 20 and form a seal with the center rod 20, and has a height (not shown in the figure) that is greater than the height of the center rod 20.Each of the panels 26 and 27 also forms a seal with the bottom and inner side wall of the mixing vessel 30 when the partition 28 is closed, thereby defining two sealed compartments 32 and 33.The cerium nitrate solution is provided through an inlet feed fitted to aperture 22 to compartment 32, while the hexamethylenetetrmine solution is provided through an inlet feed fitted to aperture 23 to compartment 33.Accordingly, before the retractable panels 26 and 27 are folded against the inner wall of the mixing vessel 30, compartment 32 contains only the aqueous solution of cerium nitrate and compartment 33 contains only the aqueous solution of hexamethylenetetramine.", "To start the reaction, the retractable panels 26 and 27 are folded against the inner wall of the mixing vessel 30 to enable rapid mixing of the two solutions, as shown in FIG.", "2C.", "Each of the foldable panels 26 and 27 includes a layer that is made from a chemically inert “shape memory” alloy.", "When the panels 26 and 27 are folded, the layer is “trained” by heating to conform to the shape of the cylindrical wall of the vessel 30.Advantageously, the chemically inert “shape memory” alloy is Nitinol.", "The Nitinol layer may be heated by a heating coil (not shown in FIG.", "2C) plated onto a thin, soft plastic layer adjacent to the Nitinol layer.", "FIG.", "3 shows a side view of the mixing vessel 30 that includes a tight cover 40 with a seal (not shown) to prevent spills during the mixing of the reactants and during the centrifugation which may follow maintaining and stirring of the mixture, as is further discussed herein.", "The mixing vessel 30 in the embodiment of FIG.", "3 is transparent to light and vertical separations 29 of the horizontal stirring rods 25 define regions of the mixture in which nanoparticles forming in such regions may be studied by light absorption.", "This technique enables monitoring of the size of the nanoparticles in the regions of the mixture defined by the separations 29 of the horizontal stirring rods 25 so as to allow monitoring of the reaction progress.", "An additional light absorption region 38 is defined by the vertical separation between the center 41 of the cover 40 and the top 42 of the vertical rod 20.Both the center 41 of the cover 40 and the top 42 of the vertical rod 20 remain stationary during the spinning of the vertical rod 20.The mixing vessel 30 may be made of a material transparent to light, such as quartz.", "Alternatively, the mixing vessel may contain windows of a material transparent to light which allow light to reach the absorption regions 29 and 38.When the retractable partition is retracted, the respective solutions in the first and second compartments 32 and 33 of the mixing vessel 30 mix and nanoparticles of cerium oxide begin to form.", "At the appropriate time, which depends on the desired particle size as discussed above, the mixture may be centrifuged to separate the particles from the mixture.", "In an advantageous embodiment of the invention, the mixing vessel can also serve as a centrifuging vessel.", "The removal of the retractable partition ensures that a thorough initial mixing takes place in a short time.", "Preferably, the time for fully retracting the partition is between 0.1 and 5 seconds.", "Without wishing to be bound by any theory, it is believed that during the first mixing of the two solutions, the nucleation of the CeO2 particles occurs.", "After the first mixing, the chemical driving force and the corresponding concentrations are too low for the generation of new surfaces required for further nucleation of particles, but are still sufficient for continuous growth of the existing nucleated particles.", "The monodispersity of the resulting nanoparticles is believed to be the result of the initial nucleation and subsequent growth at a uniform rate.", "Advantageously, the stirrer 31 stirs the mixture at a rate of about 50 to about 300 rpm for the duration of the reaction, which depends on the desired particle size as discussed above.", "The string rate depends on the viscosity of the mixture.", "The reaction may also proceed without stirring when the total volume of the reaction mixture is about 20 ml or less.", "A reaction on this scale may be conducted by mixing the two solutions in a vial of methyl methacrylate and the progress of the reaction may be monitored by measuring the light absorption of the mixture in the vial at different reaction times.", "FIG.", "4 shows plots of light absorption versus photon energy measured in about 5 ml of a reaction mixture in a plastic vial for different reaction times.", "As the reaction time increases, the maximum in the light absorption spectrum shifts to lower photon energies.", "The shift to lower is photon energies is consistent with an increase in the size of the nanoparticles absorbing the radiation, as is discussed in Brus, L. E., “Quantum crystals and nonlinear optics,” Applied Physics A, Vol.", "53 (1991), p. 456, herein incorporated by reference in its entirety.", "In an another embodiment of the invention, the mixing vessel (not shown) does not have a retractable partition as shown in FIG.", "2, but has one or more detachable liners (not shown) made from a chemically inert material, such as TEFLON® or polyethylene, which adhere to the inner wall of the vessel.", "In this embodiment, either one of the two solutions is first placed in a mixing vessel.", "The second solution is then pumped into the mixing vessel containing the first solution through a plurality of inlets (not shown) which are distributed throughout the mixing vessel.", "Advantageously, the second solution is pumped at high pressure to ensure initial rapid and thorough mixing and nucleation of the nanoparticles at approximately the same time.", "The nanoparticles all grow at the same rate and thereby achieve monodispersity.", "Upon completion of the reaction, the reaction mixture may be centrifuged using the mixing vessel as the centrifuging vessel.", "Centrifugation drives the majority of the particles onto the detachable liners covering the inner wall of the mixing vessel.", "These nanoparticles may then be obtained by detaching the liners from the inner wall of the mixing vessel.", "In another advantageous embodiment of the invention, the mixing vessel is positioned inside a centrifuge.", "The suspension that results from the formation of the cerium oxide nanoparticles in the aqueous medium can be centrifuged at 10,000 rpm or higher to separate the particles and the supernatant when the particles reach a desired size.", "The time required for separation by centrifugation depends on the particle size.", "For example, for 8 nm particles, the centrifugation time for complete separation is around 50 minutes for a 10 cm radius centrifuge spinning at 10,000 rpm.", "In general, the time required is readily calculated from standard centrifugation equations for separating particles from a liquid suspension, as are known to persons of ordinary skill in the art.", "The process of separation is effective in the case of cerium oxide particles due to the substantial difference in the densities of the supernatant (ρ≅1 gm/cm3) and the CeO2-y particles (ρ≅7.2 gm/cm3).", "Over 99% of the cerium oxide nanoparticles obtained by the method of the invention are single crystals, and the cerium oxide particle diameter may be measured by X-ray diffraction.", "X-ray diffraction experiments may be performed using a diffractometer of model Scintag X2 with Cu Kα irradiation under the same conditions, including the same scan rate (0.025 degree/step, 5 s/step).", "The lattice parameter a is determined from fitting the x-ray diffraction peak position.", "The mean particle diameter χ0 is determined from the peak width using the Scherrer formula, χ0=0.94λ/B cosθB, where λ is the wavelength of the Cu Kα1 line, θB is the angle between the incident beam and the reflecting lattice planes, and B is the width (in radians) of the diffraction peak.", "The size dispersion is approximately gaussian with a full width at the 1/e2 points, Δχ, which is 44% of the mean diameter.", "The lattice parameter decreases with increasing particle size for particle sizes smaller than about 25 nm, as shown by the curve in FIG.", "5.The lattice parameter of micron-scale cerium oxide particles (not shown in FIG.", "5) available from Alfa Aesar is 5.4087 Å.", "The lattice parameter of 6.1 nm particles is 5.4330 Å, representing a 0.45% increase when compared to the lattice parameter of micron-scale cerium oxide particles.", "The size of the particles may also be measured from high resolution transmission electron micrographs (TEM).", "The full width at half maximum (FWHM) of the size distribution peak for a batch of 100 or more particles of cerium oxide is found to be less than +/−15% of the median size.", "The lattice images of over 50 synthesized nanoparticles indicate that over 99% of the synthesized individual nanoparticles are single crystals.", "As an example, reference is made to FIG.", "6, in which two sets of <111> planes are imaged from an octahedral particle with the electron beam parallel to the two <110> edges of the octahedron.", "The particle size can be then obtained by multiplying the number of <111> plane images 61 by the distance 62 between successive <111> planes.", "Imaging the <111> atomic planes for a particle of known size provided a value of the distance between successive <111> planes of 0.3 nm.", "The size of any particle may be then measured by multiplying this value by the number of <111> plane images for that particle.", "The particle sizes obtained from TEM agree with the particle sizes measured by X-ray diffraction data.", "Table 1 shows a summary of the values of mean particle diameter χ0 and lattice parameters a for different sample pellets prepared under different preparation conditions using varying reaction times and sintering temperatures.", "Sample pellet A is a reference 5 μm pellet available from Alfa Aesar.", "While sintering is not a required step for the preparation of the nanoparticles according to the method of the invention, sintering may be used to obtain larger nanoparticles than the nanoparticles obtained after centrifugation but before sintering.", "Samples B-D were maintained at the sintering temperatures shown in Table 1 for 30 minutes.", "TABLE 1 Mixing Time Sintering Sample (hrs.)", "Temperature (° C.) χ0 a (nm) A N/a — ˜5 μm 0.54087 B 12 850 ˜25 nm 0.54087 C 12 700 ˜15 nm 0.54131 D 12 600 ˜10 nm 0.54152 E 8 not sintered ˜7.4 nm 0.54285 F 5 not sintered ˜6.1 nm 0.54330 Example: Preparation of Cerium Oxide Nanoparticles 750 ml of an aqueous solution of 0.00375 M of Ce(NO3)3 was placed in the first compartment of a mixing vessel having two compartments separated by a retractable partition, and 750 ml of an aqueous solution of 0.5 M hexamethylenetetramine was placed in the second compartment of the mixing vessel.", "The partition was retracted and the resulting mixture was constantly stirred at about 300° K for 12 hours to yield CeO2-y nanoparticles of about 12 nm in diameter, where 0<y<0.35.CeO2-y nanoparticles were obtained with about 30% yield, which represents a very high yield for cerium oxide nanoparticle preparation.", "The average nanoparticle growth rate observed was of one monolayer per 15 minutes.", "It should be understood that various changes and modifications to the exemplary embodiments described herein will be apparent to those skilled in the art without departing from the spirit and scope of this invention, which is defined by the appended claims." ] ]	Patent_10465942
[ [ "Circuit board router apparatus and method thereof", "A circuit board router (10) and method thereof.", "De-paneling of printed circuit boards (62) off a panel 860) is efficiently increased by a router (40) which is positioned at a location above the panel (60).", "A fixture positions the panel 860) below the router (40) on a base (16).", "A controller (64) activates a first drive mechanism (20), a second drive mechanism (26), and a third drive mechanism (32) to guide an X-arm (18), a Y-arm (24) and a Z-arm (10), respectively.", "The router (40), located on the Z-arm (30), moves downward to engage a router bit (42) to the panel (60) to depanel the printed circuit board (62) from the panel.", "A fixture chip (72), which has a preprogrammed pattern of the panel (60), is embedded inside the fixture (58).", "A radio frequency transmitter (80) transmits the pattern to a radio frequency receiver (82) that relays the pattern to the controller (64)." ], [ "1.A circuit board router device for depaneling a printed circuit board comprising: a frame removably supporting a fixture adapted to hold a circuit board panel; a router assembly coupled to the frame, the router assembly including an X-arm that moves a router spindle along the X axis, a Y-arm that moves the router spindle along the Y axis, and a Z-arm that moves the router spindle along the Z axis, wherein the router spindle is operatively coupled to the Z-arm; a router bit affixed to the router spindle; a controller operationally associated with a first drive mechanism that drives the X-arm, a second drive mechanism that drives the Y-arm, and a third drive mechanism that drives the Z-arm, wherein the controller positions the router bit in accordance with positioning coordinates with respect to the fixture; an input/output device operatively associated with the frame, the input/output device including display capability for displaying diagnostic information relating to the router device, and further including data input capability for accepting programming information for the controller; and an information-carrying device in communication with the controller, the information-carrying device including pattern information identifying a router pattern to be utilized in depaneling circuit boards.", "2.The circuit board router device of claim 1, wherein the information-carrying device is an integrated circuit device imbedded inside said fixture.", "3.The circuit board router device of claim 1, wherein the fixture further comprises indicia identifying the pattern associated with the information-carrying device.", "4.The circuit board router device of claim 1, wherein the pattern information from the information-carrying device is communicated to the controller by an information transmitter.", "5.The circuit board router device of claim 4, wherein the information transmitter is a radio frequency transmitter associated with the fixture.", "6.The circuit board router device of claim 1, further comprising an information receiver that receives pattern information from the information-carrying device.", "7.The circuit board router device of claim 6, wherein the information receiver is a radio frequency receiver coupled to the controller.", "8.The circuit board router device of claim 1, wherein the information-carrying device accepts pattern information programmed through the input/output device.", "9.The circuit board router device of claim 8, wherein the input/output device is a touch-screen display.", "10.The circuit board router device of claim 8, wherein the input/output device includes an associated keyboard adapted to receive router pattern information.", "11.The circuit board router device of claim 1, wherein the information-carrying device is a disk drive, and pattern information is downloaded from the disk drive to the controller.", "12.The circuit board router device of claim 1, wherein the information-carrying device is a CD-ROM drive, and pattern information is downloaded from the CD-ROM drive to the controller.", "13.The circuit board router device of claim 1, further comprising a camera operationally associated therewith, wherein the camera is utilized to identify locations on the PCB panel to be routed, and a set of parameters are input to the controller for each location so identified to establish router pattern information.", "14.The circuit board router device of claim 13, wherein the parameters include thickness data of the PCB, X-Y traverse speed data and X-Y cut speed data of the router bit, lower Z height data associated with the cut, upper Z height for router bit travel to the subsequent location, speed of the router spindle, and diameter of the router bit.", "15.A method for operating a circuit board router device for depaneling a printed circuit board, the method comprising the steps of: (a) providing a frame removably supporting a fixture adapted to hold a circuit board panel to be routed; (b) providing a router assembly coupled to the frame, the router assembly including an X-arm that moves a router spindle along the X axis, a Y-arm that moves the router spindle along the Y axis, and a Z-arm that moves the router spindle along the Z axis, wherein the router spindle is operatively coupled to the Z-arm; (c) providing a router bit affixed to the router spindle; (d) providing a controller operationally associated with a first drive mechanism that drives the X-arm, a second drive mechanism that drives the Y-arm, and a third drive mechanism that drives the Z-arm, wherein the controller positions the router bit in accordance with positioning coordinates with respect to the fixture; (e) providing an input/output device operatively associated with the frame, the input/output device including display capability for displaying diagnostic information relating to the router device, and further including data input capability for accepting programming information for the controller; and (f) providing an information-carrying device in communication with the controller, the information-carrying device including pattern information identifying a router pattern to be utilized in depaneling circuit boards.", "16.The method in accordance with claim 15, further comprising the steps of: (g) providing a camera utilized to identify locations on the PCB panel to be routed; and (h) inputting a set of parameters to the controller for each location so identified to establish router pattern information.", "17.The method in accordance with claim 16, wherein the step (h) of inputting a set of parameters further comprises the steps of: (h1) calibrating the camera to the router spindle location; (h2) entering thickness data of the PCB into the controller; (h3) entering X-Y traverse speed data and X-Y cut speed data of the router bit into the controller; (h4) entering lower Z height data for the cut and the upper Z height for the travel to a subsequent location into the controller; and (h5) entering speed of the router spindle and diameter of the router bit into the controller." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>In the industry, printed circuit boards (PCB's) when manufactured are assembled by affixing a plurality of PCB's to a panel.", "By affixing a plurality of PCB's to a panel, substantial savings of time, material and money have been obtained as handling a plurality of PCB's simplifies and speeds up the automated processing of the PCB's.", "As the commercial demands of PCB's in the electronics industry increases, the plurality of PCB's assembled on a single panel require more efficient handling by the processing equipment.", "An important consideration in the processing of the PCB's is the removal of the individual PCB from the panel for further processing or installation into the finished product, such as a computer or other electronic equipment.", "Efficiently removing the PCB's from the panel allows more panels to be processed, resulting in economic gain.", "Removing the PCB's from the panel is referred to as “depaneling” or “liberating” the PCB's from the panel.", "Methods presently used in the industry to depanel each individual PCB from the interconnected PCB's in the panel have typically included shearing, routing, break-away methods of routed tabs, scoring, perforation, and various punch and die techniques.", "Routing employs cutting rout slots in the panel around individual PCB's to define the perimeter of the individual PCB.", "As such, the routing leaves support tabs around the perimeter for holding the individual boards in place.", "Such tabs are then cut, broken, or routed to remove each board.", "Scoring utilizes grooving lines along portions of individual board perimeters.", "Such score lines are then used as weak areas to separate the board by breaking the PCB from the panel along the score lines.", "In addition, various perforations have been used to define the perimeters of the individual boards.", "Breaking along the lines of perforation is then used to depanel the individual boards.", "Other methods of depaneling include punch and die techniques wherein a custom made die is used to punch each individual board out of the panel.", "These methods of depaneling contain deficiencies, however.", "The present scorers reduce the rigidity of the panel.", "Accordingly, the panels are prone to sagging during further processing after one of the PCB's is separated.", "As a result of the sagging, the subsequent PCB's are not as accurately processed.", "Perforation and scoring yield very poor quality edges.", "Accordingly, the edges cannot be held to close tolerances.", "Additionally, the punch and die method requires expensive tooling as the punch and die is custom made with respect to the panel.", "Thus, panels having different configurations require different punches and dies.", "Additionally, the tooling needs to be replaced with each new panel, requiring further downtime of the punch and die.", "Thus, a need exists for a high volume and high speed depaneling of PCB's from panels containing a plurality of PCB's.", "A need also exists for a router which enables damage free depaneling of the PCB from the panel.", "Further, a need exists for a router that depanels the PCB from a location above the PCB.", "Additionally, a need exists for a router that can be programmed to read a panel configuration and depanel the PCB without changing any tooling.", "Devices are known in the industry that accept a panel of PC boards and depanel the individual PC boards.", "U.S. Pat.", "No.", "5,894,648, issued to Hill, discloses a depaneling apparatus that removes the individual PCB from the panel and automatically positions the separated PCB to a registration area.", "The depaneler then automatically moves the PCB from the registration area to a subsequent processing station.", "In this depaneler, the PCB is depaneld by a router that cuts the PCB from underneath the panel.", "This depaneler contains deficiencies, however.", "Design constraints of an assembly line may not allow the routing mechanism to be underneath the panel.", "Further, locating the router under the area where the panel is to be processed limits access to the router.", "Thus, during maintenance or breakdowns, more time is needed to access the router, resulting in less operation time and increased maintenance costs.", "Further, in some assemblies, it may not be practical to automatically move the separated PCB to a further processing station.", "Further, the depaneler requires a loading track to position the panel for routing, which may not be practical with regard to the allowable workspace.", "Another approach is disclosed in U.S. Pat.", "No.", "4,742,615 issued to Lopez, which recites a routing method and apparatus.", "This device, however, positions the router underneath the panel and routs from below, which may be impractical due to workspace limitations.", "Further, this device can only rout one predetermined set of panels as opposed to adapting to rout panels with different configurations.", "Another approach is disclosed in U.S. Pat.", "No.", "5,067,229 issued to Nakamura, which recites a cutting device for electronic components.", "This cutting device also cuts from underneath the panel.", "Further, the device requires an identification pattern consisting of eight sections of coated and non-coated sections of the panel in order for the device to sense which type of panel is to be processed, adding to the complexity and cost of the cutting device." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>In one embodiment, the present invention comprises a circuit board router device to depanel a printed circuit board.", "The router device comprises a base positioned on top of a frame.", "Attached to the base is a router assembly.", "The router assembly comprises an X-arm to move the router spindle in the X axis, a Y-arm to move the router spindle in the Y axis, and a Z-arm to move the router spindle in the Z axis.", "The router assembly further comprises a router bit held by the spindle.", "The router spindle is attached to the Z-arm to engage a panel from a location above the panel.", "The panel holds a plurality of PCB's as a single unit.", "Tabs connect the PCB's to the panel in which the panel is positioned within a fixture.", "The fixture is placed underneath the router spindle to engage a router bit against the tabs to depanel the PCB's from the panel.", "The routed tabs and panel are discarded through a base aperture located on the base.", "The illustrated embodiment further comprises a controller to control a first drive mechanism, a second drive mechanism, and a third drive mechanism which drive the X-arm, the Y-arm and the Z-arm, respectively, to proper coordinates above the fixture.", "A display screen is connected to the frame, which is capable of programming the controller.", "The display screen is also capable of displaying diagnostic information of the router device.", "In one embodiment, the router device is pre-programmable to rout a particular pattern of the panel.", "In this embodiment, a fixture chip is embedded within the fixture in which the fixture has an identifying mark to indicate which pattern of panel is being positioned in the fixture.", "The fixture chip mates with a radio frequency transmitter which relays the programmed pattern of the fixture chip to a radio frequency receiver.", "The radio frequency receiver in turns relays the programmed pattern to the controller." ], [ "FIELD OF THE INVENTION The present invention relates to a router device.", "In particular, the present invention relates to a router device that automatically depanels individual printed circuit boards from a panel.", "BACKGROUND OF THE INVENTION In the industry, printed circuit boards (PCB's) when manufactured are assembled by affixing a plurality of PCB's to a panel.", "By affixing a plurality of PCB's to a panel, substantial savings of time, material and money have been obtained as handling a plurality of PCB's simplifies and speeds up the automated processing of the PCB's.", "As the commercial demands of PCB's in the electronics industry increases, the plurality of PCB's assembled on a single panel require more efficient handling by the processing equipment.", "An important consideration in the processing of the PCB's is the removal of the individual PCB from the panel for further processing or installation into the finished product, such as a computer or other electronic equipment.", "Efficiently removing the PCB's from the panel allows more panels to be processed, resulting in economic gain.", "Removing the PCB's from the panel is referred to as “depaneling” or “liberating” the PCB's from the panel.", "Methods presently used in the industry to depanel each individual PCB from the interconnected PCB's in the panel have typically included shearing, routing, break-away methods of routed tabs, scoring, perforation, and various punch and die techniques.", "Routing employs cutting rout slots in the panel around individual PCB's to define the perimeter of the individual PCB.", "As such, the routing leaves support tabs around the perimeter for holding the individual boards in place.", "Such tabs are then cut, broken, or routed to remove each board.", "Scoring utilizes grooving lines along portions of individual board perimeters.", "Such score lines are then used as weak areas to separate the board by breaking the PCB from the panel along the score lines.", "In addition, various perforations have been used to define the perimeters of the individual boards.", "Breaking along the lines of perforation is then used to depanel the individual boards.", "Other methods of depaneling include punch and die techniques wherein a custom made die is used to punch each individual board out of the panel.", "These methods of depaneling contain deficiencies, however.", "The present scorers reduce the rigidity of the panel.", "Accordingly, the panels are prone to sagging during further processing after one of the PCB's is separated.", "As a result of the sagging, the subsequent PCB's are not as accurately processed.", "Perforation and scoring yield very poor quality edges.", "Accordingly, the edges cannot be held to close tolerances.", "Additionally, the punch and die method requires expensive tooling as the punch and die is custom made with respect to the panel.", "Thus, panels having different configurations require different punches and dies.", "Additionally, the tooling needs to be replaced with each new panel, requiring further downtime of the punch and die.", "Thus, a need exists for a high volume and high speed depaneling of PCB's from panels containing a plurality of PCB's.", "A need also exists for a router which enables damage free depaneling of the PCB from the panel.", "Further, a need exists for a router that depanels the PCB from a location above the PCB.", "Additionally, a need exists for a router that can be programmed to read a panel configuration and depanel the PCB without changing any tooling.", "Devices are known in the industry that accept a panel of PC boards and depanel the individual PC boards.", "U.S. Pat.", "No.", "5,894,648, issued to Hill, discloses a depaneling apparatus that removes the individual PCB from the panel and automatically positions the separated PCB to a registration area.", "The depaneler then automatically moves the PCB from the registration area to a subsequent processing station.", "In this depaneler, the PCB is depaneld by a router that cuts the PCB from underneath the panel.", "This depaneler contains deficiencies, however.", "Design constraints of an assembly line may not allow the routing mechanism to be underneath the panel.", "Further, locating the router under the area where the panel is to be processed limits access to the router.", "Thus, during maintenance or breakdowns, more time is needed to access the router, resulting in less operation time and increased maintenance costs.", "Further, in some assemblies, it may not be practical to automatically move the separated PCB to a further processing station.", "Further, the depaneler requires a loading track to position the panel for routing, which may not be practical with regard to the allowable workspace.", "Another approach is disclosed in U.S. Pat.", "No.", "4,742,615 issued to Lopez, which recites a routing method and apparatus.", "This device, however, positions the router underneath the panel and routs from below, which may be impractical due to workspace limitations.", "Further, this device can only rout one predetermined set of panels as opposed to adapting to rout panels with different configurations.", "Another approach is disclosed in U.S. Pat.", "No.", "5,067,229 issued to Nakamura, which recites a cutting device for electronic components.", "This cutting device also cuts from underneath the panel.", "Further, the device requires an identification pattern consisting of eight sections of coated and non-coated sections of the panel in order for the device to sense which type of panel is to be processed, adding to the complexity and cost of the cutting device.", "OBJECTS OF THE INVENTION It is therefore an object of the present invention to provide an apparatus that enables damage-free depaneling of a printed circuit board from a panel.", "Another object of the present invention is to depanel the printed circuit board from a location above the printed circuit board.", "Another object of the present invention is to provide an automatic program to program the routing pattern into a controller based upon the panel placed in the device.", "Another object of the present invention is to provide a method of damage-free depaneling of a printed circuit board from a panel.", "Still further objects and advantages will become apparent from a consideration of the following descriptions and drawings.", "SUMMARY OF THE INVENTION In one embodiment, the present invention comprises a circuit board router device to depanel a printed circuit board.", "The router device comprises a base positioned on top of a frame.", "Attached to the base is a router assembly.", "The router assembly comprises an X-arm to move the router spindle in the X axis, a Y-arm to move the router spindle in the Y axis, and a Z-arm to move the router spindle in the Z axis.", "The router assembly further comprises a router bit held by the spindle.", "The router spindle is attached to the Z-arm to engage a panel from a location above the panel.", "The panel holds a plurality of PCB's as a single unit.", "Tabs connect the PCB's to the panel in which the panel is positioned within a fixture.", "The fixture is placed underneath the router spindle to engage a router bit against the tabs to depanel the PCB's from the panel.", "The routed tabs and panel are discarded through a base aperture located on the base.", "The illustrated embodiment further comprises a controller to control a first drive mechanism, a second drive mechanism, and a third drive mechanism which drive the X-arm, the Y-arm and the Z-arm, respectively, to proper coordinates above the fixture.", "A display screen is connected to the frame, which is capable of programming the controller.", "The display screen is also capable of displaying diagnostic information of the router device.", "In one embodiment, the router device is pre-programmable to rout a particular pattern of the panel.", "In this embodiment, a fixture chip is embedded within the fixture in which the fixture has an identifying mark to indicate which pattern of panel is being positioned in the fixture.", "The fixture chip mates with a radio frequency transmitter which relays the programmed pattern of the fixture chip to a radio frequency receiver.", "The radio frequency receiver in turns relays the programmed pattern to the controller.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a front elevation view of the circuit board router embodying principles of the invention; FIG.", "2 is a side elevation view of the router of FIG.", "1 embodying principles of the invention; FIG.", "3 is a perspective detail view of the circuit board router embodying principles of the invention; FIG.", "4 is a perspective view of the router of FIG.", "3 embodying principles of the invention; FIG.", "5 is a plan view of the fixture and populated panel of an embodiment of the invention; FIG.", "6 is a front elevation view of the circuit board router embodying principles of the present invention; FIG.", "7 is a perspective view of FIG.", "6 embodying principles of the present invention; FIG.", "8 is a detail view of the router spindle and router bit of an embodiment of the present invention; FIG.", "9 is a perspective detail view of FIG.", "8 embodying principles of the present invention; and FIG.", "10 is a plan view of the radio frequency transmitter and receiver embodying principles of the present invention.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring now to the accompanying drawings, a router device 10 of the type generally contemplated can either be electrically, pneumatically or hydraulically operated dependant upon the force necessary to carry out the depaneling process required.", "The router device 10 comprises a frame 12 supported by supports or stanchions 14.The supports 14 may be adjustable to accommodate for different heights of the frame 12, or to accommodate different levels of a floor 15.The frame 12 is open underneath to allow access within the router device 10.The frame 12, however, may be closed to protect the area within the frame 12.Further, the frame 12 has an access space 56 to hold electrical equipment.", "As shown in the illustrated embodiment of FIG.", "1, the access space 56 includes a shelf with a handle 57.In other embodiments, the access space 56 may comprise a drawer.", "Positioned on top of the frame 12 is a base 16 which is of a generally square shape as shown in the illustrated embodiment of FIGS.", "1 and 2.The base 16 is flat to provide a bed 36 or workspace.", "Attached to the base 16 is a router assembly 17.The router assembly 17 comprises an X-arm 18 to move in an X axis, a Y-arm 24 to move in the Y axis, a Z-arm 30 to move in the Z axis, a router spindle 40, and a router bit 42.A router assembly mount 19 positions the router assembly 17 above the base 16, as shown in FIGS.", "1 and 2.A panel 60 containing four sets of a printed circuit board (PCB) 62 is shown in FIG.", "5.The panel 60 as known in the art holds each PCB 62 together as a single unit.", "Tabs 63 define the edges of the PCB's 62.Tabs 63 connect each PCB 62 to the panel 60 and are depaneled during the routing process to release each PCB 62 from the panel 60.Panel 60 becomes debris after the PCB 62 has been routed.", "Four sets of PCB's 62 are shown as exemplary, but one skilled in the art will recognize that the panel 60 may be comprised of any number of PCB's 62.The panel 60 is capable of being attached to a fixture 58, which serves as a support tray.", "The fixture 58 is removably attached on the base 16 as shown in FIG.", "6.In the illustrated embodiment, the Y-arm 24 is attached to and below the X-arm 18, while the Z-arm 30 is attached to the X-arm 18.The X-arm is attached perpendicular to the Y-arm 24.Accordingly, the Z-arm 30 is attached perpendicular to the X-arm 18.The X-arm 18 is attached to the Y-arm by a first mount 27, while the Z-arm 30 is also attached to the X-arm 18 by a second mount 31.The X-arm 18 and the Y-arm 24 are reciprocally moveable back and forth along the respective X and Y axes while the Z-arm 30 is reciprocally moveable up and down along the Z axis.", "As configured, any movement of the Y-arm 24 will result in movement of the X-arm 18 and the Z-arm 30 in the direction of the Y axis.", "Further, any movement of the X-arm 18 will result in movement of the Z-arm 30 along the X axis.", "It will be known to those in the art that the X-arm 18 and the Y-arm 24 directions are chosen arbitrarily, as rotating the base 16 by ninety degrees would result in the proper reciprocating movement along the proper denoted axis.", "The first mount 27 is driven along the Y axis on the Y-arm 24 by a first drive mechanism 20.In the illustrated embodiment, the first drive mechanism 20 is a motor.", "The first drive mechanism 20 may also comprise other drive mechanisms, such as (but not limited to), jack screws, springs, gears, and magnets.", "The first mount 27 travels in the direction along the Y-arm 24 along a first guide 22 illustrated as a track.", "The first guide 22 may however include, but is not limited to, a rail, a conveyor, a channel, a magnetic strip, an optical field, and a rack and pinion.", "The second mount 31 is driven along the X axis on the X-arm 18 by a second drive mechanism 26.In the illustrated embodiment, the second drive mechanism 26 is a motor.", "The second drive mechanism 26 may also comprise other drive mechanisms, such as, but not limited to, jack screws, springs, gears, and magnets.", "The second mount 31 travels in the direction along the X-arm 18 along a second guide 28 illustrated as a track.", "The second guide 28 may however include, but is not limited to, a rail, a conveyor, a channel, a magnetic strip, an optical field and a rack and pinion.", "The third mount 33, which carries the router spindle 40, is driven along the Z axis on the Z-arm 30 by a third drive mechanism 32.In the illustrated embodiment, the third drive mechanism 32 is a motor.", "The third drive mechanism 32 may also comprise other drive mechanisms, such as but not limited to, jack screws, springs, gears, and magnets.", "The third mount 33 travels in the direction along the Z-arm 30 along a third guide 34 illustrated as a track.", "The third guide 34 may however include, but is not limited to, a rail, a conveyor, a channel, a magnetic strip, an optical field and a rack and pinion.", "The router spindle 40 is attached to the third mount 31 to engage the panel 60 from a location positioned above the panel 60.The router spindle 40 is lowered along the Z-arm 30 to engage the panel 60.The router spindle 40 rotates the router bit 42 to depanel the PCB 62 by routing the tabs 63 from the PCB 62.A camera 68 is also attached to the Z-arm 30 by a camera mount 70 to aid a controller 64 during the depaneling process.", "In the illustrated embodiment, the camera 68 is connected to the controller 64 by flex tubing in order for the camera 68 to be flexible for panels 60 with different configurations.", "Additionally, at least one base aperture 44 is positioned through the base 16 in order for the routed panel 60 and tabs 63, which are now debris, to be discarded through the base aperture 44.Additionally, a vacuum system (not shown) may also be connected adjacent the router bit 42 in order to collect the debris.", "An access cover 52 covers most of the area above the base 16.To allow visual operation and access of the router assembly 17, an access cover aperture 54 is positioned on the front of the access cover 52.The access cover aperture 52 is configured to allow access from the back of the access cover 52.The access cover 52 may be removably fixed to the base 16.As shown in FIGS.", "3 and 4, the controller 64 communicates with the first drive mechanism 20, the second drive mechanism 26, and the third drive mechanism 32 to coordinate the operations of the router spindle 40.The controller 64 further provides on-line programming and diagnostic information of the router device 10.The controller 64 may be a computer or similar device.", "The connections from the controller 64, such as the electrical wires, are understood by those in the art.", "As shown in FIG.", "1, a control panel 65, which is attached to the frame 12, can signal the controller 64 to activate.", "Additionally, a display screen 66 is provided which is capable of programming the controller 64 via a touch screen format.", "The display screen 66 can also display the current operation status of the router device 10.The display screen 66 is mounted on a display support 50 as shown in FIG.", "3.The display support 50 may be adjustable to provide different viewing angles and different viewing heights of the display screen 66.The display support 50 is attached to a vertical member 45, which offsetably connects to the frame 12 by a bracket 49, as shown in FIGS.", "3 and 4.Attached to the top of the vertical member 45 is a signal 46 that indicates the status of the router device 10.The signal 46 may send visual signals, such as illuminating lights, to depict different stages of operation.", "For example, one light may indicate that the router device 10 is in operation, while another light may indicate that the router device 10 is not in operation.", "Further, the signal 46 may send audio signals, such as different tones, to indicate the operation status of the router device 10.As shown in FIG.", "5, a particular pattern exists between the PCB's 62 to be depaneld.", "As this pattern may vary according to the particular panel 60 disposed within the fixture 58, the present invention is adapted to automatically program the controller 64 to rout to the particular pattern of the panel 60.A fixture chip 72 is fixed into the fixture 58 in which the fixture 58 has an identifying mark 75 displayed to indicate the fixture chip 72 as shown in FIG.", "10.The fixture chip 72 is preprogrammed with the particular pattern of the panel 60.Thus, for example, if the panel 60 has a routing pattern #1, the fixture chip 72 is preprogrammed with routing pattern #1.Accordingly, the identifying mark 75 would indicate routing pattern #1.The fixture chip 72 has leads (not shown) that mate with a cable 76 as shown in FIG.", "10.The cable 76 connects to a radio frequency transmitter 80 that is positioned on the base 16.The radio frequency transmitter 80 transmits the routing pattern to a radio transmitting receiver 82 which is also positioned on the base 16.The radio transmitting receiver 82 transmits the routing pattern to the controller 64 to activate the routing spindle 40 on the panel 60.In an alternative embodiment, the fixture 58 has a chip (not shown) that communicates with a sensor (not shown) located on the base 16 that the fixture has been placed on the base 16.The sensor (not shown) then communicates to the controller 64 to start the depaneling process.", "Thus, in this alternative embodiment, placing the fixture 58 on the base 16 causes the operation to start.", "During operation, the access cover 52 is opened and the fixture 58 is placed on the base 16.The panel 60, which is populated with PCB's 62, is placed into the fixture 58.During manual operation, the fixture 58 is placed over the base aperture 44 but under the router assembly 17.The camera 68 is then calibrated to the router spindle 40 location.", "Next, the thickness data of the PCB 62 is entered into the controller 64 along with the X-Y traverse speed data and the X-Y cut speed data of the router bit 42.The router bit 42 may be replaced according to the thickness data.", "Next, the lower Z height data is entered to the controller 64 for the cut and the upper Z height for the travel data is also entered into the controller 64.Further, the speed of the router spindle 40 is entered into the controller 64 along with the diameter of the router bit 42.The required data may be entered via a keyboard (not shown) or via the display screen 66.Alternatively, the required data may be downloaded from a source such as a floppy disc or CD ROM into the controller 64.Further, the fixture 58 may contain the fixture chip 72.In this embodiment, the fixture chip 72 transmits the required data of the panel 60 via the radio frequency transmitter 80 to the radio frequency receiver 82 via the cable 76.The radio frequency receiver 82 transmits the required date to the controller 64 to start the depaneling process.", "In an alternative embodiment, the camera 68 is “taught” proper start and stop cut locations on the panel 60.In this embodiment, the camera 68 is positioned to a start location and the start location is recorded by the controller 64.The camera 68 is then positioned to a stop location and the stop location is recorded by the controller 64.Each PCB 62 is mapped on the panel 60 according to the camera 68.In a further alternative embodiment, the router spindle 40 is manually moved to each start location and each stop location to map the pattern of the PCB's 62.The start and stop locations are known as a fiducial and teach location.", "This information is then recorded by the controller 64 to start the depaneling process.", "This method is continued to teach the controller 64 all cut locations.", "Thus, once the designated router program is selected to record the required data into the controller 64, the controller 64 positions the router spindle 40 to the correct location by activating the first drive mechanism 20, the second drive mechanism 26, and the third drive mechanism 32, which correctly positions the first mount 27 along the first guide 22, the second mount along the second guide 28, and the third mount 33 along the third guide 34, respectively.", "The router spindle 40 travels down the Z-arm 30 by the third mount 33 to engage the panel 60.The controller 64 then activates the router bit 42 to route the tabs 63 to free the PCB 62 from the panel 60.The controller 64 moves the router bit 42 to the next location until all PCB's 62 are free from the panel 60.During the depaneling operation, the vacuum system (not shown) may be activated to collect the debris of the tabs 63 and panel 60.After the depaneling is complete, the router bit 42 is terminated.", "At this time, the access cover 52 is lifted and the PCB's 62, which are now depaneled, are lifted out off the base 16.Any leftover tab 63 or panel 60 may be discarded through the base aperture 44.A new panel 60 is then place on the fixture 58 in order for the routing sequence to begin again.", "Although the foregoing detailed description of the present invention has been described by reference to various embodiments, and the best mode contemplated for carrying out the present invention has been herein shown and described, it will be understood that modifications or variations in the structure and arrangement of these embodiments, other than those specifically set forth herein, may be achieved by those skilled in the art, and that such modifications are to be considered as being within the overall scope of the present invention." ] ]	Patent_10465957
[ [ "Interface materials and methods of production and use thereof", "Layered interlace materials described herein comprise at least one crosslinkable thermal interface component and at least one compliant fibrous interface component coupled to the thermal interface component.", "A method of forming layered interface materials comprises: a) providing a crosslinkable thermal interface component; b) providing a compliant fibrous interface component; and c) physically coupling the thermal interface component and the compliant fibrous interface component.", "At least one additional layer, including a substrate layer, can be coupled to the layered interface material." ], [ "1.A layered interface material, comprising: at least one crosslinkable thermal interface component; and at least one compliant fibrous interface component coupled to the thermal interface component.", "2.The layered interface material of claim 1, wherein the at least one thermal interface component comprises at least one rubber compound, at least one amine resin and at least one thermally conductive filler.", "3.The layered interface material of claim 2, wherein the at least one thermal interface component further comprises at least one phase change material.", "4.The layered interface material of claim 2, wherein the at least one rubber compound comprises at least one terminal hydroxy group.", "5.The layered interface material of claim 2, wherein the at least one rubber compound comprises at least one saturated compound.", "6.The layered interface material of claim 4, wherein the at least one rubber compound further comprises at least one saturated compound.", "7.The layered interface material of claim 4, wherein the at least one rubber compound comprises hydrogenated polyalkyldiene mono-ol, hydrogenated polyalkyldiene diol, or a combination or mixture thereof.", "8.The layered interface material of claim 7, wherein the hydrogenated polyalkyldiene mono-ol comprises hydrogenated polybutadiene mono-ol.", "9.The layered interface material of claim 7, wherein the hydrogenated polyalkyldiene diol comprises hydrogenated polybutadiene diol.", "10.The layered interface material of claim 2, wherein the at least one amine resin comprises a melamine resin.", "11.The layered interface material of claim 10, wherein the melamine resin comprises an alkylated melamine resin.", "12.The layered interface material of claim 11, wherein the alkylated melamine resin comprises butylated melamine resin.", "13.The layered interface material of claim 2, wherein the at least one thermally conductive filler comprises a metal powder, a boron nitride compound or a combination thereof.", "14.The layered interface material of claim 13, wherein the metal powder comprises aluminum powder, silver powder, copper powder or a combination thereof.", "15.The layered interface material of claim 3, wherein the at least one phase change material comprises a wax.", "16.The layered interface material of claim 15, wherein the wax comprises a paraffin wax.", "17.The layered interface material of claim 2, further comprising at least one catalytic material.", "18.The layered interface material of claim 3, further comprising at least one catalytic material.", "19.The layered interface material of claim 1, wherein the compliant fibrous interface component comprises a plurality of flocked thermally conductive fibers.", "20.The layered interface material of claim 19, wherein the plurality of flocked thermally conductive fibers are embedded in an adhesive material.", "21.The layered material of claim 20, wherein the plurality of flocked thermally conductive fibers are embedded in a substantially vertical orientation with portions of the plurality of fibers extending out of the adhesive material.", "22.The layered material of claim 21, wherein the plurality of flocked thermally conductive fibers comprise an encapsulant material disposed between the portions of the plurality of fibers, wherein the plurality of fibers extends out of the encapsulant material.", "23.The layered material of claim 19, wherein the plurality of flocked thermally conductive fibers comprises carbon, graphite, metal, ceramic, conductive polymer, diamond or a combination thereof.", "24.The layered material of claim 23, wherein the plurality of flocked thermally conductive fibers comprises carbon.", "25.The layered material of claim 19, wherein the plurality of flocked thermally conductive fibers comprises a length of at least about 0.0005 inches.", "26.The layered material of claim 25, wherein the plurality of flocked thermally conductive fibers comprises a length of at least about 0.001 inches.", "27.The layered material of claim 26, wherein the plurality of flocked thermally conductive fibers comprises a length of at least about 0.01 inches.", "28.The layered material of claim 27, wherein the plurality of flocked thermally conductive fibers comprises a length of at least about 0.1 inches.", "29.The layered material of claim 28, wherein the plurality of flocked thermally conductive fibers comprises a length of at least about 1 inch.", "30.The layered material of claim 19, wherein the plurality of flocked thermally conductive fibers comprises a fiber diameter of at least about 3 microns.", "31.The layered material of claim 30, wherein the plurality of flocked thermally conductive fibers comprises a fiber diameter of at least about 30 microns.", "32.The layered material of claim 32, wherein the plurality of flocked thermally conductive fibers comprises a fiber diameter of at least about 300 microns.", "33.The layered material of claim 19, wherein the encapsulant comprises a gel material.", "34.The layered material of claim 33, wherein the gel material comprises a silicon gel, a spray gasket material or a combination thereof.", "35.The layered material of claim 19, wherein the plurality of thermally conductive fibers comprises a thermal conductivity of at least about 25 W/mK.", "36.A layered component comprising the layered interface material of claim 1.37.An electronic component comprising the layered interface material of claim 1.38.A layered component comprising the layered interface material of claim 2.39.An electronic component comprising the layered interface material of claim 2.40.A layered component comprising the layered interface material of claim 3.41.An electronic component comprising the layered interface material of claim 3.42.A tape comprising the layered interface material of claim 3.43.A method of producing a layered interface material, comprising: providing a crosslinkable thermal interface component; providing a compliant fibrous interface component; and physically coupling the thermal interface component and the compliant fibrous interface component.", "44.A method of forming the crosslinkable thermal interface component of claim 43, comprising: providing at least one saturated rubber compound; providing at least one amine resin; crosslinking the at least one saturated rubber compound and the at least one amine resin to form a crosslinked rubber-resin mixture; adding at least one thermally conductive filler to the crosslinked rubber-resin mixture; and adding a wetting agent to the crosslinked rubber-resin mixture.", "45.The method of claim 44, further comprising adding at least one phase change material to the crosslinked rubber-resin mixture.", "46.A liquid thermal interface composition formed by the method of claim 45.47.A solid thermal interface composition formed by the method of claim 45.48.A tape comprising the thermal interface component of claim 45.49.A method of forming the compliant fibrous interface component of claim 43, comprising: providing thermally conductive fibers having a length; providing a substrate; applying adhesive to the substrate; flocking the fibers to the substrate; embedding the fibers into the adhesive with a portion of the fibers extending out of the adhesive; curing the adhesive; disposing a curable encapsulant between the fibers extending out of the adhesive and beneath the free ends of the fibers; compressing the fibers with encapsulant between the fibers into the adhesive; and curing the encapsulant while under compression." ], [ "<SOH> BACKGROUND <EOH>Electronic components are used in ever increasing numbers of consumer and commercial electronic products.", "Examples of some of these consumer and commercial products are televisions, personal computers, internet servers, cell phones, pagers, palm-type organizers, portable radios, car stereos, or remote controls.", "As the demand for these consumer and commercial electronics increases, there is also a demand for those same products to become smaller, more functional, and more portable for consumers and businesses.", "As a result of the size decrease in these products, the components that comprise the products must also become smaller.", "Examples of some of those components that need to be reduced in size or scaled down are printed circuit or wiring boards, resistors, wiring, keyboards, touch pads, and chip packaging.", "Components, therefore, are being broken down and investigated to determine if there are better building materials and methods that will allow them to be scaled down to accommodate the demands for smaller electronic components.", "In layered components, one goal appears to be decreasing the number of the layers while at the same time increasing the functionality and durability of the remaining layers.", "This task can be difficult, however, given that several of the layers and components of the layers should generally be present in order to operate the device.", "Also, as electronic devices become smaller and operate at higher speeds, energy emitted in the form of heat increases dramatically.", "A popular practice in the industry is to use thermal grease, or grease-like materials, alone or on a carrier in such devices to transfer the excess heat dissipated across physical interfaces.", "Most common types of thermal interface materials are thermal greases, phase change materials, and elastomer tapes.", "Thermal greases or phase change materials have lower thermal resistance than elastomer tape because of the ability to be spread in very thin layers and provide intimate contact between adjacent surfaces.", "Typical thermal impedance values range between 0.6-1.6° C. cm 2 /w.", "However, a serious drawback of thermal grease is that thermal performance deteriorates significantly after thermal cycling, such as from 65° C. to 150° C., or after power cycling when used in VLSI chips.", "It has also been found that the performance of these materials deteriorates when large deviations from surface planarity causes gaps to form between the mating surfaces in the electronic devices or when large gaps between mating surfaces are present for other reasons, such as manufacturing tolerances, etc.", "When the heat transferability of these materials breaks down, the performance of the electronic device in which they are used is adversely affected.", "Thus, there is a continuing need to: a) design and produce thermal interface materials and layered materials that meet customer specifications while minimizing the size of the device and number of layers; b) produce more efficient and better designed materials and/or components with respect to the compatibility requirements of the material, component or finished product; and c) develop reliable methods of producing desired thermal interface materials and layered materials and components comprising contemplated thermal interface and layered materials." ], [ "<SOH> SUMMARY <EOH>Layered interface materials described herein comprise at least one crosslinkable thermal interface component and at least one compliant fibrous interface component coupled to the thermal interface component.", "A method of forming contemplated layered interface materials comprises: a) providing a crosslinkable thermal interface component; b) providing a compliant fibrous interface component; and c) physically coupling the thermal interface component and the compliant fibrous interface component.", "At least one additional layer, including a substrate layer, can be coupled to the layered interface material.", "A constituent of layered interface materials described herein comprises at least one crosslinkable thermal interface component that is produced by combining at least one rubber compound, at least one amine resin and at least one thermally conductive filler.", "This contemplated thermal interface component takes on the form of a liquid or “soft gel”.", "The gel state is brought about through a crosslinking reaction between the at least one rubber compound composition and the at least one amine resin composition.", "More specifically, the amine resin is incorporated into the rubber composition to crosslink the primary hydroxyl groups on the rubber compounds thus forming the soft gel phase.", "Therefore, it is contemplated that at least some of the rubber compounds will comprise at least one terminal hydroxyl group.", "Amine or amine-based resins are added or incorporated into the rubber composition or mixture and/or combination of rubber compounds primarily to facilitate a crosslinking reaction between the amine resin and the primary or terminal hydroxyl groups on at least one of the rubber compounds.", "The crosslinking reaction between the amine resin and the rubber compounds leads to a “soft gel” phase to the mixture, instead of a liquid state.", "Once the thermal interface component composition that comprises at least one rubber compound, at least one amine resin, and at least one thermally conductive filler has been prepared, the composition must be compared to the needs of the electronic component, vendor, or electronic product to determine whether a phase change material is needed to change some of the physical properties of the composition.", "Phase change materials are useful in thermal interface component applications because they store and release heat as they oscillate between solid and liquid form.", "A phase change material gives off heat as it changes to a solid state, and as it returns to a liquid, it absorbs heat.", "The phase change temperature is the melting temperature at which the heat absorption and rejection takes place.", "A method for forming the crosslinkable thermal interface components disclosed herein comprises a) providing at least one saturated rubber compound, b) providing at least one amine resin, c) crosslinking the at least one saturated rubber compound and the at least one amine resin to form a crosslinked rubber-resin mixture, d) adding at least one thermally conductive filler to the crosslinked rubber-resin mixture, and e) adding a wetting agent to the crosslinked rubber-resin mixture.", "This method can also further comprise adding at least one phase change material to the crosslinked rubber-resin mixture.", "A contemplated thermal interface component can be provided as a dispensable liquid paste to be applied by dispensing methods and then cured as desired.", "It can also be provided as a highly compliant, cured, elastomer film or sheet for pre-application on interface surfaces or on other materials, such as heat sinks, substrates, and/or a compliant fibrous interface material or component.", "It can further be provided and produced as a soft gel or liquid that can be applied to surfaces by any suitable dispensing method.", "Even further, the material can be provided as a tape that can be applied directly to interface surfaces, substrates, compliant fibrous interface materials or component and/or electronic components.", "Compliant fibrous interface components comprise a plurality of thermally conductive fibers, at least one encapsulant, and at least one optional adhesive material.", "Suitable thermally conductive fibers comprise diamond fibers, conductive polymer fibers, carbon fibers, graphite fibers and metal fibers, such as copper fibers and aluminum fibers.", "The thermally conductive fibers are cut to a particular length, usually depending on the needs/specifications of the customer or vendor, e.g.", "from at least about 0.0005 inches to at least about 1 inch.", "Thermally conductive fibers may also be cut to at least about 0.001 inches, to at least about 0.01 inches and/or to at least about 0.1 inches.", "Thermally conductive fibers may have a fiber diameter of at least about 3 microns, of at least about 30 microns and/or at least about 300 microns.", "Conductive fibers having a fiber diameter of at least about 10 microns are presently preferred.", "Applications of the contemplated layered interface materials, thermal interface components and compliant fibrous interface components described herein comprise incorporating the materials into a layered material, a layered component, an electronic component, a semiconductor component, a finished electronic product or a finished semiconductor product.", "Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention." ], [ "FIELD OF THE INVENTION The field of the invention is interface materials in electronic components, semiconductor components and other related layered materials applications.", "BACKGROUND Electronic components are used in ever increasing numbers of consumer and commercial electronic products.", "Examples of some of these consumer and commercial products are televisions, personal computers, internet servers, cell phones, pagers, palm-type organizers, portable radios, car stereos, or remote controls.", "As the demand for these consumer and commercial electronics increases, there is also a demand for those same products to become smaller, more functional, and more portable for consumers and businesses.", "As a result of the size decrease in these products, the components that comprise the products must also become smaller.", "Examples of some of those components that need to be reduced in size or scaled down are printed circuit or wiring boards, resistors, wiring, keyboards, touch pads, and chip packaging.", "Components, therefore, are being broken down and investigated to determine if there are better building materials and methods that will allow them to be scaled down to accommodate the demands for smaller electronic components.", "In layered components, one goal appears to be decreasing the number of the layers while at the same time increasing the functionality and durability of the remaining layers.", "This task can be difficult, however, given that several of the layers and components of the layers should generally be present in order to operate the device.", "Also, as electronic devices become smaller and operate at higher speeds, energy emitted in the form of heat increases dramatically.", "A popular practice in the industry is to use thermal grease, or grease-like materials, alone or on a carrier in such devices to transfer the excess heat dissipated across physical interfaces.", "Most common types of thermal interface materials are thermal greases, phase change materials, and elastomer tapes.", "Thermal greases or phase change materials have lower thermal resistance than elastomer tape because of the ability to be spread in very thin layers and provide intimate contact between adjacent surfaces.", "Typical thermal impedance values range between 0.6-1.6° C. cm2/w.", "However, a serious drawback of thermal grease is that thermal performance deteriorates significantly after thermal cycling, such as from 65° C. to 150° C., or after power cycling when used in VLSI chips.", "It has also been found that the performance of these materials deteriorates when large deviations from surface planarity causes gaps to form between the mating surfaces in the electronic devices or when large gaps between mating surfaces are present for other reasons, such as manufacturing tolerances, etc.", "When the heat transferability of these materials breaks down, the performance of the electronic device in which they are used is adversely affected.", "Thus, there is a continuing need to: a) design and produce thermal interface materials and layered materials that meet customer specifications while minimizing the size of the device and number of layers; b) produce more efficient and better designed materials and/or components with respect to the compatibility requirements of the material, component or finished product; and c) develop reliable methods of producing desired thermal interface materials and layered materials and components comprising contemplated thermal interface and layered materials.", "SUMMARY Layered interface materials described herein comprise at least one crosslinkable thermal interface component and at least one compliant fibrous interface component coupled to the thermal interface component.", "A method of forming contemplated layered interface materials comprises: a) providing a crosslinkable thermal interface component; b) providing a compliant fibrous interface component; and c) physically coupling the thermal interface component and the compliant fibrous interface component.", "At least one additional layer, including a substrate layer, can be coupled to the layered interface material.", "A constituent of layered interface materials described herein comprises at least one crosslinkable thermal interface component that is produced by combining at least one rubber compound, at least one amine resin and at least one thermally conductive filler.", "This contemplated thermal interface component takes on the form of a liquid or “soft gel”.", "The gel state is brought about through a crosslinking reaction between the at least one rubber compound composition and the at least one amine resin composition.", "More specifically, the amine resin is incorporated into the rubber composition to crosslink the primary hydroxyl groups on the rubber compounds thus forming the soft gel phase.", "Therefore, it is contemplated that at least some of the rubber compounds will comprise at least one terminal hydroxyl group.", "Amine or amine-based resins are added or incorporated into the rubber composition or mixture and/or combination of rubber compounds primarily to facilitate a crosslinking reaction between the amine resin and the primary or terminal hydroxyl groups on at least one of the rubber compounds.", "The crosslinking reaction between the amine resin and the rubber compounds leads to a “soft gel” phase to the mixture, instead of a liquid state.", "Once the thermal interface component composition that comprises at least one rubber compound, at least one amine resin, and at least one thermally conductive filler has been prepared, the composition must be compared to the needs of the electronic component, vendor, or electronic product to determine whether a phase change material is needed to change some of the physical properties of the composition.", "Phase change materials are useful in thermal interface component applications because they store and release heat as they oscillate between solid and liquid form.", "A phase change material gives off heat as it changes to a solid state, and as it returns to a liquid, it absorbs heat.", "The phase change temperature is the melting temperature at which the heat absorption and rejection takes place.", "A method for forming the crosslinkable thermal interface components disclosed herein comprises a) providing at least one saturated rubber compound, b) providing at least one amine resin, c) crosslinking the at least one saturated rubber compound and the at least one amine resin to form a crosslinked rubber-resin mixture, d) adding at least one thermally conductive filler to the crosslinked rubber-resin mixture, and e) adding a wetting agent to the crosslinked rubber-resin mixture.", "This method can also further comprise adding at least one phase change material to the crosslinked rubber-resin mixture.", "A contemplated thermal interface component can be provided as a dispensable liquid paste to be applied by dispensing methods and then cured as desired.", "It can also be provided as a highly compliant, cured, elastomer film or sheet for pre-application on interface surfaces or on other materials, such as heat sinks, substrates, and/or a compliant fibrous interface material or component.", "It can further be provided and produced as a soft gel or liquid that can be applied to surfaces by any suitable dispensing method.", "Even further, the material can be provided as a tape that can be applied directly to interface surfaces, substrates, compliant fibrous interface materials or component and/or electronic components.", "Compliant fibrous interface components comprise a plurality of thermally conductive fibers, at least one encapsulant, and at least one optional adhesive material.", "Suitable thermally conductive fibers comprise diamond fibers, conductive polymer fibers, carbon fibers, graphite fibers and metal fibers, such as copper fibers and aluminum fibers.", "The thermally conductive fibers are cut to a particular length, usually depending on the needs/specifications of the customer or vendor, e.g.", "from at least about 0.0005 inches to at least about 1 inch.", "Thermally conductive fibers may also be cut to at least about 0.001 inches, to at least about 0.01 inches and/or to at least about 0.1 inches.", "Thermally conductive fibers may have a fiber diameter of at least about 3 microns, of at least about 30 microns and/or at least about 300 microns.", "Conductive fibers having a fiber diameter of at least about 10 microns are presently preferred.", "Applications of the contemplated layered interface materials, thermal interface components and compliant fibrous interface components described herein comprise incorporating the materials into a layered material, a layered component, an electronic component, a semiconductor component, a finished electronic product or a finished semiconductor product.", "Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1 shows a graph of displacement v. pressure for an embodiment of a compliant fibrous interface component.", "FIG.", "2 shows a graph of thermal resistance v. pressure for an embodiment of a compliant fibrous interface component.", "FIG.", "3 shows a graph of thickness v. thermal resistance for an embodiment of a compliant fibrous interface component.", "FIG.", "4 shows a graph of cycle v. thermal resistance for an embodiment of a compliant fibrous interface component.", "FIGS.", "5A-C show a method of producing a preferred embodiment of the compliant fibrous interface component of the present invention.", "FIG.", "6 shows a preferred embodiment of the compliant fibrous interface component of the present invention.", "FIGS.", "7A-B show several preferred embodiments of the layered interface material of the present invention.", "DETAILED DESCRIPTION A suitable interface material or component should conform to the mating surfaces (“wets” the surface), possess a low bulk thermal resistance and possess a low contact resistance.", "Bulk thermal resistance can be expressed as a function of the material's or component's thickness, thermal conductivity and area.", "Contact resistance is a measure of how well a material or component is able to make contact with a mating surface, layer or substrate.", "The thermal resistance of an interface material or component can be shown as follows: Θinterface=t/kA+2Θcontact Equation 1 where Θ is the thermal resistance, t is the material thickness, k is the thermal conductivity of the material A is the area of the interface The term “t/kA” represents the thermal resistance of the bulk material and “2Θcontact” represents the thermal contact resistance at the two surfaces.", "A suitable interface material or component should have a low bulk resistance and a low contact resistance, i.e.", "at the mating surface.", "Many electronic and semiconductor applications require that the interface material or component accommodate deviations from surface flatness resulting from manufacturing and/or warpage of components because of coefficient of thermal expansion (CTE) mismatches.", "A material with a low value for k, such as thermal grease, performs well if the interface is thin, i.e.", "the “t” value is low.", "If the interface thickness increases by as little as 0.002 inches, the thermal performance can drop dramatically.", "Also, for such applications, differences in CTE between the mating components causes the gap to expand and contract with each temperature or power cycle.", "This variation of the interface thickness can cause pumping of fluid interface materials (such as grease) away from the interface.", "Interfaces with a larger area are more prone to deviations from surface planarity as manufactured.", "To optimize thermal performance, the interface material should be able to conform to non-planar surfaces and thereby lower contact resistance.", "Optimal interface materials and/or components possess a high thermal conductivity and a high mechanical compliance, e.g.", "will yield elastically when force is applied.", "High thermal conductivity reduces the first term of Equation 1 while high mechanical compliance reduces the second term.", "The layered interface materials and the individual components of the layered interface materials described herein accomplish these goals.", "When properly oriented, the thermally conductive fibers of the compliant fibrous interface component described herein will span the distance between the mating surfaces thereby allowing a continuous high conductivity path from one surface to the other surface.", "If the fibers are flexible and able to move in its tip region, better contact can be made with the surface.", "This contact will result in an excellent degree of surface contact and will minimize the contact resistance of the interface material.", "Layered interface materials described herein comprise at least one crosslinkable thermal interface component and at least one compliant fibrous interface component coupled to the thermal interface component.", "A method of forming contemplated layered interface materials comprises: a) providing a crosslinkable thermal interface component; b) providing a compliant fibrous interface component; and c) physically coupling the thermal interface component and the compliant fibrous interface component.", "At least one additional layer may be coupled with the layered interface material described herein.", "The at least one additional layer can comprise another interface material, a surface, a substrate, an adhesive or any other suitable layer.", "Crosslinkable Thermal Interface Component A contemplated crosslinkable thermal interface component is produced by combining at least one rubber compound, at least one amine resin and at least one thermally conductive filler.", "This contemplated interface material takes on the form of a liquid or “soft gel”.", "As used herein, “soft gel” means a colloid in which the disperse phase has combined with the continuous phase to form a viscous “jelly-like” product.", "The gel state or soft gel state of the thermal interface component is brought about through a crosslinking reaction between the at least one rubber compound composition and the at least one amine resin composition.", "More specifically, the amine resin is incorporated into the rubber composition to crosslink the primary hydroxyl groups on the rubber compounds, thus forming the soft gel phase.", "Therefore, it is contemplated that at least some of the rubber compounds will comprise at least one terminal hydroxyl group.", "As used herein, the phrase “hydroxyl group” means the univalent group —OH occurring in many inorganic and organic compounds that ionize in solution to yield OH radicals.", "Also, the “hydroxyl group” is the characteristic group of alcohols.", "As used herein, the phrase “primary hydroxyl groups” means that the hydroxyl groups are in the terminal position on the molecule or compound.", "Rubber compounds contemplated herein may also comprise additional secondary, tertiary, or otherwise internal hydroxyl groups that could also undergo a crosslinking reaction with the amine resin.", "This additional crosslinking may be desirable depending on the final gel state needed for the product or component in which the gel is to be incorporated.", "It is contemplated that the rubber compounds could be “self-crosslinkable” in that they could crosslink intermolecularly with other rubber compounds or intramolecularly with themselves, depending on the other components of the composition.", "It is also contemplated that the rubber compounds could be crosslinked by the amine resin compounds and perform some self-crosslinking activity with themselves or other rubber compounds.", "In preferred embodiments, the rubber compositions or compounds utilized can be either saturated or unsaturated.", "Saturated rubber compounds are preferred in this application because they are less sensitive to thermal oxidation degradation.", "Examples of saturated rubbers that may be used are ethylene-propylene rubbers (EPR, EPDM), polyethylene/butylene, polyethylene-butylene-styrene, polyethylene-propylene-styrene, hydrogenated polyalkyldiene “mono-ols” (such as hydrogenated polybutadiene mono-ol, hydrogenated polypropadiene mono-ol, hydrogenated polypentadiene mono-ol), hydrogenated polyalkyldiene “diols” (such as hydrogenated polybutadiene diol, hydrogenated polypropadiene diol, hydrogenated polypentadiene diol) and hydrogenated polyisoprene.", "However, if the compound is unsaturated, it is most preferred that the compound undergo a hydrogenation process to rupture or remove at least some of the double bonds.", "As used herein, the phrase “hydrogenation process” means that an unsaturated organic compound is reacted with hydrogen by either a direct addition of hydrogen to some or all of the double bonds, resulting in a saturated product (addition hydrogenation), or by rupturing the double bond entirely, whereby the fragments further react with hydrogen (hydrogenolysis).", "Examples of unsaturated rubbers and rubber compounds are polybutadiene, polyisoprene, polystyrene-butadiene and other unsaturated rubbers, rubber compounds or mixtures/combinations of rubber compounds.", "As used herein, the term “compliant” encompasses the property of a material or a component that is yielding and formable, especially at about room temperature, as opposed to solid and unyielding at room temperature.", "As used herein, the term “crosslinkable” refers to those materials or compounds that are not yet crosslinked.", "As used herein, the term “crosslinking” refers to a process in which at least two molecules, or two portions of a long molecule, are joined together by a chemical interaction.", "Such interactions may occur in many different ways including formation of a covalent bond, formation of hydrogen bonds, hydrophobic, hydrophilic, ionic or electrostatic interaction.", "Furthermore, molecular interaction may also be characterized by an at least temporary physical connection between a molecule and itself or between two or more molecules.", "More than one rubber compound of each type may be combined to produce a crosslinkable thermal interface component; however, it is contemplated that in the preferred thermal interface component, at least one of the rubber compounds or constituents will be a saturated compound.", "Olefin-containing or unsaturated thermal interface components, with appropriate thermal fillers, exhibit a thermal capability of less than 0.5 cm2° C./w.", "Unlike thermal grease, thermal performance of the thermal interface component will not degrade after thermal cycling or flow cycling in IC devices because liquid olefins and liquid olefin mixtures (such as those comprising amine resins) will crosslink to form a soft gel upon heat activation.", "Moreover, when applied as a thermal interface component, it will not be “squeezed out” as thermal grease does in use and will not display interfacial delamination during thermal cycling.", "Amine or amine-based resins are added or incorporated into the rubber composition or mixture of rubber compounds primarily to facilitate a crosslinking reaction between the amine resin and the primary or terminal hydroxyl groups on at least one of the rubber compounds.", "The crosslinking reaction between the amine resin and the rubber compounds produces a “soft gel” phase in the mixture, instead of a liquid state.", "The degree of crosslinking between the amine resin and the rubber composition and/or between the rubber compounds themselves will determine the consistency of the soft gel.", "For example, if the amine resin and the rubber compounds undergo a minimal amount of crosslinking (10% of the sites available for crosslinking are actually used in the crosslinking reaction) then the soft gel will be more “liquid-like”.", "However, if the amine resin and the rubber compounds undergo a significant amount of crosslinking (40-60% of the sites available for crosslinking are actually used in the crosslinking reaction and possibly there is a measurable degree of intermolecular or intramolecular crosslinking between the rubber compounds themselves) then the gel would become thicker and more “solid-like”.", "Amine and amino resins are those resins that comprise at least one amine substituent group on any part of the resin backbone.", "Amine and amino resins are also synthetic resins derived from the reaction of urea, thiourea, melamine or allied compounds with aldehydes, particularly formaldehyde.", "Typical and contemplated amine resins are primary amine resins, secondary amine resins, tertiary amine resins, glycidyl amine epoxy resins, alkoxybenzyl amine resins, epoxy amine resins, melamine resins, alkylated melamine resins, and melamine-acrylic resins.", "Melamine resins are particularly useful and preferred in several contemplated embodiments described herein because a) they are ring-based compounds, whereby the ring contains three carbon and three nitrogen atoms, b) they can combine easily with other compounds and molecules through condensation reactions, c) they can react with other molecules and compounds to facilitate chain growth and crosslinking, d) they are more water resistant and heat resistant than urea resins, e) they can be used as water-soluble syrups or as insoluble powders dispersible in water, and f) they have high melting points (greater than 325° C. and are relatively non-flammable).", "Alkylated melamine resins, such as butylated melamine resins, propylated melamine resins, pentylated melamine resins hexylated melamine resins and the like, are formed by incorporating alkyl alcohols during the resin formation.", "These resins are soluble in paint and enamel solvents and in surface coatings.", "Thermal filler particles to be dispersed in the thermal interface component or mixture should advantageously have a high thermal conductivity.", "Suitable filler materials include metals, such as silver, copper, aluminum, and alloys thereof; and other compounds, such as boron nitride, aluminum nitride, silver coated copper, silver-coated aluminum, conductive polymers and carbon fibers.", "Combinations of boron nitride and silver or boron nitride and silver/copper also provide enhanced thermal conductivity.", "Boron nitride in amounts of at least 20 wt % and silver in amounts of at least about 60 wt % are particularly useful.", "Preferably, fillers with a thermal conductivity of greater than about 20 and most preferably at least about 40 w/m° C. can be used.", "Optimally, it is desired to have a filler of not less than about 80 w/m° C. thermal conductivity.", "As used herein, the term “metal” means those elements that are in the d-block and f-block of the Periodic Chart of the Elements, along with those elements that have metal-like properties, such as silicon and germanium.", "As used herein, the phrase “d-block” means those elements that have electrons filling the 3d, 4d, 5d, and 6d orbitals surrounding the nucleus of the element.", "As used herein, the phrase “f-block” means those elements that have electrons filling the 4f and 5f orbitals surrounding the nucleus of the element, including the lanthanides and the actinides.", "Preferred metals include indium, silver, copper, aluminum, tin, bismuth, gallium and alloys thereof, silver coated copper, and silver coated aluminum.", "The term “metal” also includes alloys, metal/metal composites, metal ceramic composites, metal polymer composites, as well as other metal composites.", "As used herein, the term “compound” means a substance with constant composition that can be broken down into elements by chemical processes.", "Of special efficacy is a filler comprising a particular form of carbon fiber referred to as “vapor grown carbon fiber” (VGCF), such as is available from Applied Sciences, Inc., Cedarville, Ohio.", "VGCF, or “carbon micro fibers”, are highly graphized types by heat treatment (thermal conductivity=1900 w/m° C.).", "Addition of about 0.5 wt.", "% carbon micro fibers provides significantly increased thermal conductivity.", "Such fibers are available in varying lengths and diameters; namely, 1 millimeter (mm) to tens of centimeters (cm) length and from under 0.1 to over 100 μm in diameter.", "One useful form of VGCF has a diameter of not greater than about 110 m and a length of about 50 to 100 μm, and possess a thermal conductivity of about two or three times greater than with other common carbon fibers having diameters greater than 5 μm.", "It is difficult to incorporate large amounts of VGCF in polymer systems and interface components and systems, such as the hydrogenated rubber and resin combination already discussed.", "When carbon microfibers, e.g.", "(about 1 μm, or less) are added to the polymer they do not mix well, primarily because a large amount of fiber must be added to the polymer to obtain any significant beneficial improvement in thermal conductivity.", "However, we have discovered that relatively large amounts of carbon microfibers can be added to polymer systems that have relatively large amounts of other conventional fillers.", "A greater amount of carbon microfibers can be added to the polymer when added with other fibers, which can be added alone to the polymer, thus providing a greater benefit with respect to improving thermal conductivity of the thermal interface component.", "Desirably, the ratio of carbon microfibers to polymer is in the range of 0.05 to 0.50 by weight.", "Once the thermal interface component that comprises at least one rubber compound, at least one amine resin, and at least one thermally conductive filler has been prepared, the composition must be compared to the needs of the electronic component, vendor, or electronic product to determine if an additional phase change material is needed to change some of the physical properties of the composition.", "Specifically, if the needs of the component or product require that the composition or interface material be in a “soft gel” form or a somewhat liquid form, then an additional phase change material may not need to be added.", "However, if the component, layered material or product requires that the composition or material be more like a solid, then at least one phase change material should be added.", "Phase-change materials that are contemplated herein comprise waxes, polymer waxes or mixtures thereof, such as paraffin wax.", "Paraffin waxes are a mixture of solid hydrocarbons having the general formula CnH2n+2 and having melting points in the range of about 20° C. to 100° C. Examples of some contemplated melting points are about 45° C. and 60° C. Thermal interface components that have melting points in this range are PCM45 and PCM60HD—both manufactured by Honeywell Electronic Materials.", "Polymer waxes are typically polyethylene waxes, polypropylene waxes, and have a range of melting points from about 40° C. to 160° C. PCM45 comprises a thermal conductivity of about 3.0 W/mK, a thermal resistance of about 0.25° C.cm2/W (0.0038° C.cm2/W), is typically applied at a thickness of about 0.0015 inches (0.04 mm) and comprises a typical softness of about 5 to 30 psi (plastically flow under).", "Typical characteristics of PCM45 are a) a super high packaging density—over 80%, b) a conductive filler, c) extremely low thermal resistance, and as mentioned earlier d) about a 45° phase change temperature.", "PCM60HD comprises a thermal conductivity of about 5.0 W/mK, a thermal resistance of about 0.17° C.cm21W (0.0028° C.cm2/W), is typically applied at a thickness of about 0.0015 inches (0.04 mm) and comprises a typical softness of about 5 to 30 psi (plastically flow under).", "Typical characteristics of PCM45 are a) a super high packaging density—over 80%, b) a conductive filler, c) extremely low thermal resistance, and as mentioned earlier d) about a 60° phase change temperature.", "TM350 (a thermal interface component not comprising a phase change material and manufactured by Honeywell Electronic Materials) comprises a thermal conductivity of about 3.0 W/mK, a thermal resistance of about 0.25° C.cm2/W (0.0038° C.cm2/W), is typically applied at a thickness of about 0.0015 inches (0.04 mm) and comprises a typical softness of about 5 to 30 psi plastically flow under).", "Typical characteristics of TM350 are a) a super high packaging density—over 80%, b) a conductive filler, c) extremely low thermal resistance, d) about a 125° curing temperature, and e) dispensable non-silicone-based thermal gel.", "Phase change materials are useful in thermal interface component applications because they store and release heat as they oscillate between solid and liquid form.", "As a phase change material changes to a solid state, it gives off heat.", "As it returns to a liquid, it absorbs heat.", "The phase change temperature is the melting temperature at which the heat absorption and rejection takes place.", "Paraffin-based phase change materials, however, have several drawbacks.", "On their own, they can be very fragile and difficult to handle.", "They also tend to squeeze out of a gap from the device in which they are applied during thermal cycling, very much like grease.", "The rubber-resin modified paraffin polymer wax system described herein avoids these problems and provides significantly improved ease of handling, is capable of being produced in flexible tape or solid layer form, and does not pump out or exude under pressure.", "Although the rubber-resin-wax mixtures may have the same or nearly the same temperature, their melt viscosity is much higher and they do not migrate easily.", "Moreover, the rubber-wax-resin mixture can be designed to be self-crosslinking, which ensures elimination of the pump-out problem in certain applications.", "Examples of contemplated phase change materials are malenized paraffin wax, polyethylene-maleic anhydride wax, and polypropylene-maleic anhydride wax.", "The rubber-resin-wax mixtures will functionally form at a temperature between about 50 to 150° C. to form a crosslinked rubber-resin network.", "It is also advantageous to incorporate additional fillers, substances or particles, such as filler particles, wetting agents or antioxidants into the thermal interface component.", "Substantially spherical filler particles can be added to the thermal interface component to maximize packing density.", "Additionally, substantially spherical shapes or the like will provide some control of the thickness during compaction.", "Typical particle sizes useful for fillers in the rubber material may be in the range of about 1-20 μm with a maximum of about 100 μm.", "Dispersion of filler particles can be facilitated by addition of functional organometallic coupling agents or “wetting” agents, such as organosilane, organotitanate, organozirconium, etc.", "Organotitanate acts a wetting enhancer to reduce paste viscosity and to increase filler loading.", "An organotitanate that can be used is isopropyl triisostearyl titanate.", "The general structure of organotitanate is RO—Ti(OXRY) where RO is a hydrolyzable group, and X and Y are binder functional groups.", "Antioxidants may also be added to inhibit oxidation and thermal degradation of the cured rubber gel or solid thermal-interface component.", "Typical useful antioxidants include Irganox 1076, a phenol type or Irganox 565, an amine type, (at 0.01% to about 1 wt.", "%), available from Ciba Giegy of Hawthorne, N.Y.", "Typical cure accelerators include tertiary amines such as didecylanethylamine, (at 50 ppm—0.5 wt.", "%).", "At least one catalyst may also be added to the thermal interface component in order to promote a crosslinking or chain reaction between the at least one rubber compound, the at least one amine resin, the at least one phase change material, or all three.", "As used herein, the term “catalyst” means that substance or condition that notably affects the rate of a chemical reaction without itself being consumed or undergoing a chemical change.", "Catalysts may be inorganic, organic, or a combination of organic groups and metal halides.", "Although they are not substances, light and heat can also act as catalysts.", "In contemplated embodiments, the catalyst is an acid.", "In preferred embodiments, the catalyst is an organic acid, such as carboxylic, acetic, formic, benzoic, salicylic, dicarboxylic, oxalic, phthalic, sebacic, adipic, oleic, palmitic, stearic, phenylstearic, amino acids and sulfonic acid.", "A method for forming the crosslinkable thermal interface components disclosed herein comprises a) providing at least one saturated rubber compound, b) providing at least one amine resin, c) crosslinking the at least one saturated rubber compound and the at least one amine resin to form a crosslinked rubber-resin mixture, d) adding at least one thermally conductive filler to the crosslinked rubber-resin mixture, and e) adding a wetting agent to the crosslinked rubber-resin mixture.", "This method can also further comprise adding at least one phase change material to the crosslinked rubber-resin mixture.", "As discussed herein, liquid and solid thermal interface components can be formed using the contemplated method, along with tapes, electronic components, semiconductor components, layered materials and electronic and semiconductor products.", "The contemplated thermal interface component can be provided as a dispensable liquid paste to be applied by dispensing methods (such as screen printing) and then cured as desired.", "It clan also be provided as a highly compliant, cured, elastomer film or sheet for pre-application on interface surfaces, such as heat sinks.", "It can further be provided and produced as a soft gel or liquid that can be applied to surfaces by any suitable dispensing method.", "Even further, the thermal interface component can be provided as a tape that can be applied directly to interface surfaces or electronic components.", "To illustrate several embodiments of the thermal interface components, a number of examples were prepared by mixing the components described in Examples A through F. As indicated in the tables, the properties of the compositions including viscosity, product form, thermal impedance, modulus of elasticity, and thermal conductivity are also reported.", "The examples shown include one or more of the optional additions, e.g., antioxidant, wetability enhancer, curing accelerators, viscosity reducing agents and crosslinking aids.", "The amounts of such additions may vary but, generally, they may be usefully present in the following approximate amounts (in wt.", "%): filler up to 95% of total (filler plus rubbers); wetability enhancer 0.1 to 1% (of total); antioxidant 0.01 to 1% (of total); curing accelerator 50 ppm—0.5% (of total); viscosity reducing agents 0.2-15%; and crosslinking aids 0.1-2%.", "It should be noted the addition at least about 0.5% carbon fiber significantly increases thermal conductivity.", "Composition (by wt %) A B C D E F Hydrogenated polybutylene mono-ol 7.5 6.3 10 11.33 5 18 Hydrogenated polybutadiene diol none none 2 none none none Paraffin Wax 3.1 2.2 none none none none Alkylated melamine resin (butylated) 1.7 0.4 1.33 2 1 4 Organotitanate 1.5 1.0 6.67 6.67 4 8 Sulfonic Acid Catalyst 0.1 none none none none none Phenolic Antioxidant 0.1 0.1 none none none none Aluminum powder 86 90 80 80 none none Silver Powder none none none none 90 none Boron Nitride none none none none none 70 Product Form Tape Tape Liquid Liquid Liquid Liquid Thermal Impedance (oC.", "cm2/w) 0.25 0.18 0.25 0.25 0.3 0.35 Thermal conductivity (w · m/oC.)", "3.0 5.0 2.8 2.8 2.3 2.0 Modulus of Elasticity, Pa 300000 270000 500000 300000 280000 270000 Viscosity, Pa · s N/A N/A 200 160 150 220 Compliant Fibrous Interface Component Compliant fibrous interface components, such as those described herein, comprise a plurality of thermally conductive fibers, an encapsulant, and an optional adhesive material.", "Examples of compliant fibrous interface components can be found in U.S. patent application Ser.", "No.", "09/193,415; U.S. patent application Ser.", "No.", "09/103,416 and U.S. patent application Ser.", "No.", "09/333,564—all of which are incorporated herein by reference in their entirety.", "Also, compliant fibrous interface components, such as those described herein, are those manufactured by Honeywell Electronic Materials under the tradename of GELVET.", "GELVET comprises a thermal conductivity of about 30.0 W/mK, a thermal resistance of about 0.68° C.cm2/W (0.0010° C.cm2/W), is typically applied at a thickness of about 0.012 to 0.100 inches (0.3-2.5 mm) and comprises a typical softness of about greater than 25% of deflection under 10 psi.", "Typical characteristics of GELVET are a) thickness variable over a wide range, b) compliance to geometric and thermal mismatch, c) very high thermal conductivity, and d) reliable over long-term use of the component.", "FIGS.", "1-4 show several performance measurements of the GELVET component, including displacement v. pressure (FIG.", "1), thermal resistance v. pressure (FIG.", "2), thickness v. thermal resistance (FIG.", "3), and cycle v. thermal resistance (FIG.", "4).", "Suitable thermally conductive fibers comprise diamond fibers, conductive polymer fibers, carbon fibers, graphite fibers and metal fibers, such as copper fibers and aluminum fibers.", "The thermally conductive fibers are cut to a particular length, e.g.", "from at least about 0.0005 inches to at least about 1 inch.", "Thermally conductive fibers contemplated herein may also be cut to at least about 0.001 inches, to at least about 0.01 inches and/or to at least about 0.1 inches.", "Thermally conductive fibers contemplated herein may have a fiber diameter of at least about 3 microns, of at least about 30 microns and/or at least about 300 microns.", "Conductive fibers having a fiber diameter of at least about 10 microns are presently preferred.", "Suitable thermally conductive fibers have a thermal conductivity of at least about 25 W/mK.", "Some suitable fibers are those available from Amoco identified as K-1100, K-800, P-120, P-100, P-70 and T50; as well as fibers available from Toray designated as M46J and M46JB.", "Thermally conductive fibers disclosed herein can be cleaned, if necessary, to remove any coatings present on the fibers.", "Some commercially available fibers are sold with a coating applied to the surface, which is preferably removed by cleaning the fibers.", "One method of cleaning thermally conductive fibers is by heating the fibers in air to burn off the coating, i.e.", "sizing.", "However, chemical cleaning methods can also be used.", "A method of forming the compliant fibrous interface component comprises: a) providing thermally conductive fibers having a length; b) providing a substrate; c) applying adhesive to the substrate; d) flocking the fibers to the substrate; e) embedding the fibers into the adhesive with a portion of the fibers extending out of the adhesive; f) curing the adhesive; disposing a curable encapsulant between the fibers extending out of the adhesive and beneath the free ends of the fibers; g) compressing the fibers with encapsulant between the fibers into the adhesive; and h) curing the encapsulant while under compression.", "To produce a compliant fibrous interface component, a first adhesive is applied to a substrate.", "The adhesive may comprise any suitable material, but will likely comprise a low stress adhesive, for example, an adhesive comprising an epoxy compound (e.g.", "Eccobond 281 from Grace Specialty Polymers), although cyanate ester adhesive, BMI, silicones, organosilicones, gels and spray gasket materials are also useful.", "The fibers are flocked to the substrate, thereby embedding the fibers in the adhesive, as shown in FIG.", "5A, for example, by electroflocking.", "Electroflocking is a well-known procedure whereby two plates, separated by some distance, are charged to opposite polarity.", "The procedure is described generically by Bolgen (Bolgen, Stig W., “Flocking Technology”, Journal of Coated Fabrics, Volume 21, page 123, 1991); and specifically for electroflocking of carbon fibers by Shigematsu in “Application of Electrostatic Flocking to Thermal Control Coating”, Proceedings of the 14th International Symposium on Space Technology and Science, 1984, page 583; and by Kato in “Formation of a Very Low-Reflectance Surface by Electrostatic Flocking”, Proceedings of the 4th European Symposium on Space Environmental and Control Systems, 1991, page 565.The disclosures of these articles are expressly incorporated herein by reference in their entirety.", "In the electroflocking process, fibers on one plate pick up that plate's charge and become attracted to the opposite plate.", "They embed in the adhesive when they hit the opposite plate.", "If they do not stick initially, fibers bounce back and forth between plates until they become embedded in the adhesive, escape the electric field or the charge on the plates is removed.", "The fiber structure that results is aligned with respect to the electric field lines, i.e.", "has a substantially vertical orientation and has a velvet-like appearance.", "Mechanical flocking involves passing an adhesive-coated object over a series of rapidly rotating rollers or beater bars, which cause the substrate to vibrate.", "Fibers are fed onto the substrate by gravity from a hopper.", "The vibrations produced by the rollers or beater bars orient the fibers and drive them into the adhesive.", "Excess fiber is removed, leaving a fiber structure with substantially vertical orientation.", "Pneumatic flocking uses an airstream to deliver fibers to an adhesive coated surface.", "While in flight, fibers align themselves in the direction of the airflow and embed in the adhesive in an oriented manner.", "Different flocking methods may be used alone, or in combination with one another, e.g.", "pneumatic/electrostatic flocking.", "With this combination method, an airstream containing fibers is directed through a nozzle.", "At the exit of the nozzle, a charge orients the fibers with respect to the electric field lines.", "The fiber structure that results is also aligned, i.e.", "has substantial vertical orientation, but may be denser, more uniform or produced more rapidly than when either method is used alone.", "The flocked fibers are seated into the adhesive with a portion of their lengths extending from the adhesive layer referred to as “free fiber tips”.", "After flocking, a downward force (compression) is applied to the free fiber tips to seat the fibers in the adhesive and to minimize the distance between the fiber tips embedded in the adhesive and the surface substrate to which the adhesive is applied, as shown in FIGS.", "5B and 5C.", "The adhesive is then cured by any suitable method, including self-curing, heat curing and/or infrared curing.", "Oftentimes, heating for about 30 minutes at about 150° C. may be used for curing, depending on the adhesive and curing conditions.", "As shown in FIG.", "6, an encapsulant, 30, for example a gel such as GE RTV6166 dielectric gel available from General Electric Corporation is introduced to fill space between fibers 32 leaving free fiber tips 34 extending from the gel.", "This process can be done by stenciling uncured gel onto the fibers or applying the gel to the givers and letting the gel soak or wick in to the fibers.", "In contemplated embodiments, the gels will spontaneously wet the fibers and will wick in to the fiber structure.", "The gel may or may not include a thermally conductive filler material.", "A release liner, e.g.", "waxy or silicone-coated paper, may be placed on top of the fibers and uncured gel to prevent the cured gel/fiber material from sticking to a clamping fixture, and to provide protection to the interface material during shipping or subsequent handling.", "The interface material with uncured gel between the fibers is compressed to less than the nominal cut fiber length and clamped in place to this compressed height.", "For example, if the fiber is about 0.020 inches long, adhesive cured gel is introduced then clamped to a height of about 0.017 inches before curing the gel, which holds the fiber at this height while the gel is cured.", "The gel is then cured, e.g.", "thermally cured, while under compression.", "Heating generally accelerates curing and is desirable to create a contemplated free fiber tip structure.", "Both the compression and thermal cure aid in creating the free fiber tip structure.", "The thermal cure is beneficial since the CTE of the gel is higher than that of the fibers and the gel will shrink more than the fibers upon cooling to room temperature, thereby exposing more fiber tips.", "In producing the interface material, the adhesive curing may be delayed to coincide with the curing of the gel.", "In one embodiment, the fibers are seated at the same time as the gel and the adhesive are cured.", "As indicated, compression is beneficial, and curing under compression is beneficial, because the gel will maintain the cured thickness and the fibers can spring back somewhat to stick up from the gel.", "Cohesion of the gel to the fibers is not strong enough to keep the fibers from assuming their original position prior to curing.", "Curing results in the free fiber tips which are desirable for enhanced thermal contact with the adjacent surface(s).", "A method of forming contemplated layered interface materials comprises: a) providing a crosslinkable thermal interface component; b) providing a compliant fibrous interface component; and c) physically coupling the thermal interface component and the compliant fibrous interface component.", "The crosslinkable thermal interface components and the compliant fibrous interface components can be individually prepared and provided by using the methods previously described herein.", "The two components are then physically coupled to produce a layered interface material.", "As used herein, the term “interface” means a couple or bond that forms the common boundary between two parts of matter or space.", "An interface may comprise a physical attachment or physical couple of two parts of matter or components or a physical attraction between two parts of matter or components, including bond forces such as covalent and ionic bonding, and non-bond forces such as Van der Waals, electrostatic, coulombic, hydrogen bonding and/or magnetic attraction.", "The two components, as described herein, may also be physically coupled by the act of applying one component to the surface of the other component.", "The layered interface material may then be applied to a substrate, another surface, or another layered material, as shown in FIGS.", "7A and 7B.", "The electronic component 100 comprises a layered interface material 110, a substrate layer 120 and an additional layer 130.The layered interface material 110 comprises a compliant fibrous interface component 112 and a thermal interface component 114.Substrates contemplated herein may comprise any desirable substantially solid material.", "Particularly desirable substrate layers would comprise films, glass, ceramic, plastic, metal or coated metal, or composite material.", "In preferred embodiments, the substrate comprises a silicon or germanium arsenide die or wafer surface, a packaging surface such as found in a copper, silver, nickel or gold plated leadframe, a copper surface such as found in a circuit board or package interconnect trace, a via-wall or stiffener interface (“copper” includes considerations of bare copper and it's oxides), a polymer-based packaging or board interface such as found in a polyimide-based flex package, lead or other metal alloy solder ball surface, glass and polymers such as polyimide.", "The “substrate” may even be defined as another polymer material when considering cohesive interfaces.", "In more preferred embodiments, the substrate comprises a material common in the packaging and circuit board industries such as silicon, copper, glass, and another polymer.", "Additional layers of material may be coupled to the layered interface materials in order to continue building a layered component or printed circuit board.", "It is contemplated that the additional layers will comprise materials similar to those already described herein, including metals, metal alloys, composite materials, polymers, monomers, organic compounds, inorganic compounds, organometallic compounds, resins, adhesives and optical wave-guide materials.", "A layer of laminating material or cladding material can be coupled to the layered interface materials depending on the specifications required by the component.", "Laminates are generally considered fiber-reinforced resin dielectric materials.", "Cladding materials are a subset of laminates that are produced when metals and other materials, such as copper, are incorporated into the laminates.", "(Harper, Charles A., Electronic Packaging and Interconnection Handbook, Second Edition, McGraw-Hill (New York), 1997.)", "Spin-on layers and materials may also be added to the layered interface materials or subsequent layers.", "Spin-on stacked films are taught by Michael E. Thomas, “Spin-On Stacked Films for Low keff Dielectrics”, Solid State Technology (July 2001), incorporated herein in its entirety by reference.", "Applications of the contemplated thermal interface components, layered interface materials and compliant fibrous interface components described herein comprise incorporating the materials and/or components into another layered material, an electronic component or a finished electronic product.", "Electronic components, as contemplated herein, are generally thought to comprise any layered component that can be utilized in an electronic-based product.", "Contemplated electronic components comprise circuit boards, chip packaging, separator sheets, dielectric components of circuit boards, printed-wiring boards, and other components of circuit boards, such as capacitors, inductors, and resistors.", "Electronic-based products can be “finished” in the sense that they are ready to be used in industry or by other consumers.", "Examples of finished consumer products are a television, a computer, a cell phone, a pager, a palm-type organizer, a portable radio, a car stereo, and a remote control.", "Also contemplated are “intermediate” products such as circuit boards, chip packaging, and keyboards that are potentially utilized in finished products.", "Electronic products may also comprise a prototype component, at any stage of development from conceptual model to final scale-up/mock-up.", "A prototype may or may not contain all of the actual components intended in a finished product, and a prototype may have some components that are constructed out of composite material in order to negate their initial effects on other components while being initially tested.", "Thus, specific embodiments and applications of interface materials have been disclosed.", "It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein.", "The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims.", "Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context.", "In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced." ] ]	Patent_10465968
[ [ "Casing", "The invention relates to casing for portable communication devices and light guides for such devices.", "The casing (2) has a light guide for over-laying a display located in the display region (4) to deliver front lighting to the display and extending across the input region (5), the light guide providing a recess in the input region for operation of a key of the key arrangement." ], [ "1.A casing for a portable communication device having an operating face for providing a key arrangement in an input region for user operation of the device and a display region, the casing comprising: a light guide extending across the input region, the light guide providing a recess in the input region for operation of a key of the key arrangement and a light permeable layer arranged on the lightguide defining the outer surface of the casing in the input region.", "2.A casing according to claim 1 wherein there are a plurality of keys in the key arrangement.", "3.A casing according to claim 1 wherein an opaque layer is located between the light guide and the light permeable layer.", "4.A casing according to claim 1 wherein the light permeable layer comprises the light guide.", "5.A casing according to claim 1 wherein the light permeable layer provides the front face of the device across the input and display regions.", "6.A casing according to claim 1 wherein the light permeable layer is transparent.", "7.A casing according to claim 1 wherein the light permeable layer is translucent.", "8.A casing according to claim 1 wherein the light guide is a flexible material.", "9.A casing according to claim 1 wherein the light permeable layer is arranged on the light guide in segments with lateral discontinuities.", "10.A casing according to claim 9 wherein the lateral discontinuities extend across the full lateral extent of the substrate.", "11.A casing according to claim 9 wherein the laterally extending discontinuities are shaped to receive keys of the key arrangement.", "12.A casing according to claim 1 wherein the light permeable layer and the light guide have the same optical qualities.", "13.A casing according to claim 1 wherein the light permeable layer and the light guide are the same colour.", "14.A casing according to claim 1 wherein the light permeable layer is a material brittle with respect to the material of the light guide.", "15.A casing according to claim 1 wherein the light permeable layer is a precious stone.", "16.A casing according to claim 1 wherein the light permeable layer is formed from sapphire or diamond or glass.", "17.A casing according to claim 1 wherein the light guide provides a lip that protrudes beyond the extent of the light permeable layer.", "18.A casing according to claim 17 wherein the front face is retained in place by gripping the lip between elements of a casing of the portable device.", "19.A casing according to claim 1 wherein light sources are provided along opposing sides of the light guide in the input region.", "20.A casing according to claim 1 wherein the face is longer longitudinally than it is laterally.", "21.A casing according to claim 1 wherein a light source is provided proximate the display region.", "22.A casing according to claim 21 wherein the light source proximate the display region is a light pipe.", "23.A casing according to claim 22 wherein the light pipe is fed with light from light sources located along the edge of the light guide.", "24.A portable communication device comprising a casing in accordance with claim 1.25.A portable communication device according to claim 24 wherein the communication device is a radio telephone.", "26.A casing for a portable communication device having an operational face for providing a key arrangement in an input region for user operation of the device and a display region, the device comprising: a light guide for overlaying a display located in the display region to deliver front lighting for the display; and a light guide extending across the input region to deliver lighting to the key arrangement, the light guide providing a recess in the input region for operation of a key of the key arrangement.", "27.", "(canceled) 28.", "(canceled)" ], [ "The invention relates to casings for portable communication devices and light guides for such devices.", "Electronic devices typically have a display region in which information is provided to the user and an input region which the user uses to operate the device.", "Both these regions require lighting when the ambient light level is low and sometimes at other times to improve the usability of the device.", "In many electronic devices the display region includes a liquid crystal display (LCD) that is typically back lit to improve the visibility of the material displayed.", "For back lighting, a light guide is positioned behind the LCD to guide light to the whole surface.", "Light Emitting Diodes (LED) are generally positioned along a side of the light guide to deliver light across the whole of the LCD.", "LCDs are sensitive and easily damaged so a protective layer is typically provided over the top of the display distanced by an air gap from the LCD to allow some give in the system if the front of the display receives a blow or is stressed or broken in some way.", "The input region of an electronic device typically includes an array of keys used to input information.", "These keys will typically be provided on one or more key mats.", "It is sometimes desirable to illuminate the keys so that the device can be operated when ambient light is not sufficient.", "In these circumstances the legends associated with the keys are illuminated.", "These may be present on the keys themselves or on an opaque casing adjacent the associated key.", "In either instance a transparent light guide is generally provided behind the key mat to convey light to the legends.", "In accordance with one aspect of the present invention there is provided a casing for a portable communication device having an operating face providing a key arrangement in an input region for user operation of the device and a display region, the casing comprising: a light guide for overlaying a display located in the display region to deliver front lighting to the display and extending across the input region, the light guide providing a recess in the input region for operation of a key of the key arrangement.", "By providing an optically unitary light guide across both the input and display regions, the light can be more effectively utilised than with separate light guides for the display and input regions.", "Light from the display region illuminates the input region.", "The resultant effect is unusual and attractive and provides scope for design variation that provide additional advantages.", "The key arrangement may comprise a single input element such as a key or an array of such elements.", "In embodiments of the invention a light permeable layer may define the outer surface of the device.", "The light guide itself may be formed from a composite layer.", "To increase the options for the materials of the light guide a layer may be arranged on a substrate in segments.", "The segmented layer may also act as a light guide.", "By providing a flexible substrate and arranging the segments on the substrate so that there are gaps that extend across the substrate, the material of the segmented layer can be more brittle or less flexible than the substrate.", "By providing these lateral discontinuities in the segmented layer some of the design constraints for the material selected for the segmented layer are removed.", "The light guide may define the front surface of the device across the input region.", "The characteristics of a material for wear and appearance may then take precedence over flexibility when a discontinuous additional layer is provided.", "The lateral extending discontinuities or gaps can be arranged to receive keys of the key arrangement.", "When the face is longer longitudinally than it is laterally the layer is likely to bow along its longitudinal axis.", "The lateral discontinuities act as hinges to prevent brittle or fragine materials failing catastrophically.", "In doing this, the impression of a seamless front can be achieved without foregoing the lateral discontinuities that provide flexibility along a longitudinal axis.", "The keys or other user input elements may be individually mounted on the device, with the substrate providing apertures to allow motion of the key for operating the device.", "The gaps between the second layer segments receiving the key heads and restricting their rotation.", "The materials for the optically seamless light guide can be selected with a degree of freedom, the materials may be transparent.", "The segmented layer and the substrate may have the same optical qualities.", "They may be the same colour.", "When the face of the device is optically permeable, light sources located behind the light permeable face will be visible.", "In embodiments of the invention the light sources are provided at opposing sides of the light guide in the input region.", "These can then be used to provide diffuse light across the light guide.", "For mounting a composite light guide having brittle or fragile compontent a lip that protrudes beyond the extent of the segmented layer may be provided for mounting in a casing of the communications device.", "In accordance with another aspect of the present invention there is provided a casing for a portable communication device having an operational face for providing a key arrangement in an input region for user operation of the device and a display region, the casing comprising: a light guide for overlaying a display located in the display region to deliver front lighting for the display; and a light guide extending across the input region to deliver lighting to the key arrangement, the light guide providing a recess in the input region for operation of a key of the key arrangement.", "The casing may be a conventional one part casing or a clam shell, or other two or more part arrangement, where the user input elements or keys may be located on a different face to a display.", "In such two part arrangements generally respective casing portions are fixed such that one is movable relative to the other.", "The movement may be rotational or lateral.", "Embodiments of the invention will now be described in more detail with reference to FIGS.", "1 to 21 of the accompanying drawings of which: FIG.", "1 is a perspective view of a communication device showing one embodiment of the invention; FIG.", "2 shows front, rear, sides and top views of the communication device of FIG.", "1; FIG.", "3 is a schematic representation of a communication device suitable for embodiments of the present invention; FIG.", "4 is an exploded view of the face of a communication device of one embodiment of the invention without keys; FIG.", "5 is a view of one layer of a light guide; FIGS.", "6a and 6b are sections through the communication device of FIG.", "1 in the display region; FIG.", "7 is a perspective view of a side frame element and located rails for constructing a casing FIG.", "8 is a perspective view of the rear of the device with the battery cover removed; FIGS.", "9a and 9b are sections through the communication device of FIG.", "1 in the battery region; FIG.", "10 is a perspective view of the rear of the device with the battery cover in place; FIGS.", "11a and 11b are sections showing other embodiments of the invention; FIG.", "12 is a perspective view of part of the input region of the device illustrating the location of legends associated with keys; FIG.", "13 a longitudinal section through the communication device; FIG.", "14 is a schematic section through the light guide illustrating the surfaces available for carrying the legends; FIGS.", "15 and 16 are perspective views of a partially constructed input region of the device; FIG.", "17 is a perspective view of keys of the device including their actuation mechanisms; FIG.", "18 illustrates some of the keys illustrated in FIG.", "17, with the key tips removed; FIGS.", "19a and 19b are front and rear perspective views of the outer shell of a bezel respectively; FIGS.", "20a and 20b are a graph showing force against distance for a key such as that illustrated in FIGS.", "17 and 18, and a conventional key using a dome respectively; and FIG.", "21 is a bottom plan view of the device of FIG.", "1 (with the chin face protector omitted).", "The embodiment illustrated in FIG.", "1 is a handheld communication device 1 with a precious retainable casing 2.The casing 2 of the device has been designed to be customizable to individual taste with precious materials.", "To enable individual devices to be created, the number of external parts that the casing is formed from is relatively large in contrast to existing communications devices.", "The casing 2 has an optically permeable front face 3 providing a seamless transition from a display region 4 to an input region 5 that includes a key array 6.The key array 6 includes a first group of alphanumeric keys 7, for entering telephone numbers, writing text messages (SMS), writing names (associated with phone numbers), etc.", "Each of the twelve alphanumeric keys 7 is provided with a figure “0-9” or a sign “#” or “*”, respectively.", "In an alpha mode each key is associated with a number of letters and special signs used in text editing.", "The key array 6 additionally comprises two soft keys 8, 9, two call handling keys 10, 11, two scroll keys 12, and an on/off key 13.The functionality of the soft keys 8, 9 may depend on the state of the communication device and position within the menu accessed using the scroll keys 12.The current functionality of the soft keys 8, 9 can be shown in separate fields in the display region 4 just above the respective keys 8, 9.The two call handling keys 10, 11 are used for establishing a call or a conference call, terminating a call or rejecting an incoming call.", "The two direction keys or scroll keys 12, in the handset illustrated are placed centrally on the front surface of the communication device between the display region 4 and the group of alphanumeric keys 7 perform a scrolling function.", "The seamless face 3 is formed from an optically seamless light guide 14 providing light to illuminate the display region 4 and key legends 15 located on the light guide 14 and associated with individual keys of the key array 6.The front face 3 is overlaid with a pillow 16 providing apertures 17 to channel sound from a loud speaker 18 and providing an appropriate feel for an element of the device that will be located close to the user's ear.", "The front face 3 is surrounded by a bezel 19 that can be made from a precious metal.", "This acts to protect the edges of the light guide 14 and can help in some embodiments in securing the elements of the casing 2 together.", "The keys of the key array in this particular embodiment are arranged to provide particular sensory guidance to the user.", "Aspects of the design also allow the light guide 14 to be made from a wide range of materials including those that are brittle and so need to be carefully handled and protected from shattering.", "The casing 2 is formed from the front face 3 surrounded by the bezel 19, a side frame 20 and a back cover 21.The interface between the side frame 20 and the bezel 19 and the side frame 20 and the rear cover 21 are shielded by rails 22, 23 in this embodiment.", "The rails 22, 23 hide raw edges from view and exposure to ensure that the device 1 is both long lasting and elegant.", "The construction of the casing 2 enables the casing 2 to be opened with ease to update the engine 24 inside when desired.", "The casing 2 is also designed to allow the device to accommodate different sized and shaped engines 24, which may be necessary through its life.", "Other elements of the design will be discussed in greater detail with reference to the later drawings.", "By way of introduction, the device will be briefly discussed with reference to its functional elements.", "The communication device 1, includes the key array 6, a display 25, an antenna 26, an ear piece speaker 18, a polyphonic speaker 27, and a microphone 28.The communication device 1 is adapted for communication via a wireless telecommunication network, e.g.", "a wireless network.", "However, the communication device could also have been designed for a cordless network.", "FIG.", "3 shows schematically and functionally elements of the communication device 1.The microphone 28 records the user's speech, and the analogue signals formed thereby are A/D converted by an A/D converter before the speech is encoded in an audio part 29.The encoded speech signal is transferred to processor 30.The processor 30 may support software in the phone.", "The processor 30 also forms the interface to peripheral units of the apparatus.", "These may include a Random Access Memory (RAM) 31 and a Flash Read Only Memory (ROM) 32, a SIM card 33, the display 25, and the key array 6, and perhaps a browser application 34, and a location module 35.The browser application 34 can be used to request and receive information from the Internet.", "The location module 35 enables the terminal 1 to determine its current position.", "The processor 30 communicates with a transceiver 36, e.g.", "a circuit, which is adapted to send and receive messages in a telecommunication network.", "The telecommunications network may be a GSM network, but the invention may also be applied in connection with other networks, such as other kinds of wireless networks and various forms of cordless phone systems or in dual band phones accessing sets of these systems or networks.", "The audio part 29 speech-decodes the signal, which is transferred from the processor 30 to the earpiece 18 via a D/A converter.", "As discussed above, the front face 3 is optically permeable and acts as a light guide.", "The seamless light guide, passing light over the display region 4 as well as providing light to illuminate the key legends 15 reduces the number of lighting elements required to illuminate the device and provides a seamless transition from one area of the front of the device to another.", "By using this technique for delivering light and also allowing the light guide 14 to form the front surface of the device 1 as in the embodiment disclosed, the device is visually striking, there being no visible transition from the display region 4 to the input region 5 where the key array 6 is located.", "In other embodiments the light guide for the display and the input regions are separate.", "The embodiment illustrated in FIG.", "1 has a light permeable front face 3 that can be formed from sapphire or a similar precious stone.", "As soon as a mineral such as sapphire is used in place of other more flexible light permeable materials such as plastics, additional problems are introduced associated with the characteristics of the material.", "In designing a durable device having, for example, a brittle material for a front piece 3 there are many difficulties.", "In one manifestation of the embodiment illustrated in FIGS.", "1, 2 and 4 to 6, the light guide 14 is an oprtically seamless composite layer.", "As can be seen most clearly in FIG.", "4, a plastics layer 37 that may be formed from perspex or other transparent and/or translucent materials runs the entire length of the front face 3.There is a cut-out 38 for accommodating the ear piece speaker 18 that is optimized for use close to the ear.", "There is another cut-out accommodating a light pipe diffuser 39.This is located under the pillow 16 and extends across the width of the device 1.By placing the light pipe 39 under the pillow 16, a bright area on the front face is avoided and the light visible in the light guide will be diffuse.", "LEDs 40, 41 are located at each side of the device at each end of the pipe 39.Light from the LEDs 40, 41 is channelled through the pipe 39 and diffused.", "The light pipe 39 in this embodiment, is coated with reflective material so that light is channelled through the surface 42 that runs along the top of the LCD display 25.Embodiments without the coating provide some of the advantages.", "The diffused light is accordingly channelled into and along the major axis of the light guide 14 to provide substantially uniform lighting for the LCD 25.In this way light is pumped into the light guide 14 along the major axis from above the LCD 25.The light from the light pipe 39 illuminates the display region 4 and provides lighting for the input region 5.As illustrated in FIG.", "15, additional LEDs 43 or other lighting elements are positioned along the edges of the perspex layer or sheet 37 in the input region 5 to further illuminate the legends 15 for the keys of the array 6.The surface of extending portions 78 of the Perspex layer or sheet 37 is shaped to channel light from adjacent top firing LEDs 67 or the like towards the upper surface of the main portion of the layer or sheet 37.This assists in providing substantially uniform lighting for the input region.", "The additional LEDs 43 or the like are shielded from view by the bezel 19 to avoid light spots being visible and encouraging a uniform diffuse light across the light guide.", "The LCD 25 itself is bonded to the rear surface of the perspex sheet 37 with an energy absorbing adhesive sheet 45.Using this technique and ensuring that there are no air gaps between the front of the LCD 25 and the perspex sheet 37, provides a robust solution for mounting the LCD 25.The surface of the LCD 25 that is delicate and generally needs a protective layer distanced from it is protected by the perspex layer 37 adhered to one of its faces.", "The drivers 44 for the LCD 25 can be seen in FIG.", "5.In the embodiment illustrated the perspex layer 37 includes apertures 48 for locating individual keys and allowing the key shafts 49 to travel to make contact with a printed circuit board 50 located within the casing 2 to register user input.", "An independently inventive key layout, suitable for use with the general concept discussed, will be described in greater detail below.", "As can be seen most clearly from FIG.", "4, the second layer of the composite light guide 51 is made in a number of sections 52-58.This is to allow for the choice of brittle materials to be used for the second layer 51.Sapphire has properties including its scratch resistance and optical clarity that make it suitable for use in this context.", "The light guide provides a suitable external surface to the device.", "Other materials such as glass, ceramic materials or other minerals and precious stones could, however, also be used.", "The optical benefits of a light guide extending across the display and input regions providing diffuse light that is utilized across the entire face are achieved even if an opaque ceramic layer is provided over the light guide.", "Arrangements for the second layer similar to those disclosed in concept in the illustrated embodiment are not necessary when the materials are not brittle.", "Alternative arrangements will, however, be apparent to the skilled man for accommodating brittle materials in this context.", "As mentioned above sapphire, in common with ceramic materials, is brittle.", "When considering how to introduce such a material into a casing 2 that is to be long lasting and durable, problems arise.", "This is particularly the case when the device 1 will inevitably be subjected to knocks and is likely to be dropped many times in its long existence.", "All brittle parts are bonded to a carrier formed from e.g.", "plastic or titanium in order to better absorb shock.", "In the embodiment described, the sapphire is formed in individual sections 52-58 for ease of manufacture.", "Each of the pieces is adhered to the perspex layer 37 using a UV curing adhesive based on hybrid acrylic technology.", "In order to increase the durability of the composite light guide 14 and strengthen it against damage, the sections 52-58 are adhered to a more flexible perspex layer 37 leaving gaps 59 extending across the entire width.", "These gaps 59 act as hinges to allow the composite layer 14 to be relatively flexible along its major axis.", "In the embodiment illustrated, the layout of the key array 6 has been designed with this in mind.", "The alpha numeric keys 7 are arranged in groups of three extending across the whole width of the second layer of the composite 51.The function keys or soft key 8, 9 are also arranged in two groups of three.", "The soft keys 8, 9 and the top scroll key 12a follows the line of the alpha numeric keys 7.The call handling keys 10, 11 and the bottom scroll key 12b, which is displaced from the line of the others, defines the lower edge of one of the elements 57 and the upper edge of another of the elements 56.This makes the second row of keys 10, 11, 12b distinctive whilst still avoiding problems in manufacture of the elements 52-58.The specific key array 6 illustrated has other properties and advantages that will be discussed later.", "Although the embodiment described is designed with a brittle material such as sapphire in mind, the resultant arrangement could be formed from any number of other materials including plastics that provide the required optical characteristics.", "Similarly different key arrangements could be used without departing from individually inventive concepts that are disclosed.", "When using a brittle material for a front face light guide 14 problems are also encountered when connecting the front face 3 to the rest of the casing elements.", "In the illustrated embodiment the edges of the brittle elements 52-58 are protected by the bezel 19 that sits around the whole of the face.", "The bezel 19, in this embodiment, is formed from a bezel support 60 overlaid with a 0.5 mm thick metal sheet, the bezel cover 61.The metal sheet 61 is bonded to the bezel support 60, that may be formed from plastic, with an adhesive such as a two part epoxy adhesive or a two part acrylic adhesive.", "As the metal sheet 61 is relatively thin this keeps the weight of the casing 2 down and also allows relatively precious metals such as titanium or gold to be used at relatively lower cost.", "The bezel 19 could, however, be a single part and/or be formed from a single material for example titanium.", "The transparent face 3 in this particular embodiment is securely held in place by a robust mechanism.", "The perspex layer 37 to which the elements 52-55 are bonded extends beyond the area covered by the elements to provide a lip or edge 62 around the whole of the composite light guide 14.This edge 62 is used to secure the front face 3 in position as it is tucked under the bezel 19 that defines the perimeter of the front face and squeezed between the bezel 19 and the side frame 20.As can be seen in FIG.", "1, the bezel 19 is punctuated with front face protectors 63 whose function will be described in more detail later.", "These front face protectors 63, in this particular embodiment play a role in holding the casing elements together.", "The front face protectors 63 have a tip part 64 that extends over the bezel 19 and a shaft 65 that passes through the bezel 19 and into the plastic side frame 20.The shaft 65 may be tightened onto the frame 20 using a nut 95.In this case the nut stops the front face protectors from falling out of the device.", "In one embodiment the shafts 65 are screwed into the tips 64 at one end and into the side frame element 20 at the other although other manners of connection could be employed.", "The shaft 65 and tip 64 could also be one piece rather than two.", "The bezel 19 and the perspex layer 37 of the transparent face 2 are captured by the front face protector 63 and side frame element 20 as can be seen in FIG.", "9b.", "The front face protectors 63 accordingly grip the transparent face 3 securely between the side frame 20 and the bezel 19.The front face protectors 63 are only located next to the key array 6.In order to secure the transparent face 3 around its perimeter additional screws 66 are added to draw the bezel 19 towards the side frame 20 and squeeze the transparent face 3 in place.", "In one embodiment, the screws 66 thread through the side frame 20 and tap into the bezel support 60.Once the bezel support 60 is secured to the side frame 20 the outer shell 61 of the bezel 19 can be adhered to the support 60 to hide the tapped screw ends 66.Alternatively the bezel 19 is stuck to the support first (a sub-assembly) and this sub-assembly is then screwed to the frame.", "As can be seen in the figures, although the more flexible transparent substrate 37 is overlaid by the bezel 19 secured in position by the front face protectors 63 and other connectors, the sapphire is maintained spaced from the bezel 19 so that it is protected from chipping or other damage as a result of contact.", "The edges of the sapphire elements do not protrude above the edge of the bezel 19 to provide further protection for the more vulnerable edges.", "In the arrangement described, the transparent face 3 is gripped in position between the bezel 19 and the side frame 20.The more flexible substrate 37 could, however receive screws or other fasteners to located it relative to the other frame members without damaging the more brittle sapphire layer.", "To reduce the ingress of dirt or fluids a sealant 69 is located between the bezel 19 and the transparent face 3.One way in which this can be introduced is to paint a seal on the underside of the bezel 19 which when dry is compressed between the transparent light guide 14 and the bezel 19 when the bezel 19 and the side frame 20 are pulled together.", "As can be seen in FIG.", "6 or 9, a first rail 22 is located in the interface between the side frame element 20 and the front face 3.The rail 22 hides the discontinuity between the parts from sight and protects vulnerable edges to enhance the durability of the device 1.The rail 22 may be a stamped metal piece ‘T’ shaped in section extending around the perimeter of the side frame 20.The stem of the ‘T’ 68 is trapped between the side frame member 20 and the bezel 19 as these two pieces are drawn together on assembly.", "The stem of the T has apertures 70 that sit over corresponding projections 71 on the side frame 20 member to prevent it from being withdrawn from between the two parts when in position.", "The top of the T acts as a carrier plate to which an additional extruded piece 72 is soldered or otherwise adhered to provide a seamless finish.", "The stem could instead be formed from a plastics material in which case an adhesive could be used to attach the extrusion to the stem.", "The side frame member 20 in this particular embodiment is a plastic support 73 with an exterior cover 74.The cover 74 may again be a metal sheet or perhaps a wood veneer or another layer such as leather.", "The bonding agent most suitable for adhering the layer to the plastic frame member 73 will differ depending upon the materials that need to be adhered.", "For metal to plastic a suitable adhesive is a 2 part epoxy resin or 2 part acrylic adhesive.", "For metal to metal the adhesive would be a reactive polyurethane film or a two part epoxy resin.", "Similarly the back cover 21 in this embodiment may have a plastic frame and be covered with for example a leather, wood or perhaps metal veneer.", "With the flexible veneer materials such as leather, it is preferable for the material to be taut on the frame.", "This is achieved during the veneering process using conventional techniques.", "The transparent face 3 provides a seamless transition between the display 4 and the input regions 5.In the display region 4, the LCD 25 is located behind the transparent face 3 and in the input region 5 the key mechanism 75 lies behind it.", "In one embodiment described, the key mechanism 75 can be seen through the front face 3.However, in other embodiments the inner surface 76 of the perspex substrate 37 may be coated with an opaque material to prevent the inside of the device being seen.", "Alternative methods of obscuring the inner workings of the device from view such as introducing additional opaque layers or obscuring the view in other ways will be apparent to the skilled man.", "One such option is to provide an opaque layer that may carry etchings providing legends between the perspect layer 37 and the second layer 51.For embodiments where, for example, the key operating mechanism 75 can be viewed through the front face 3, it may be desirable to obscure the pcb 50 from view.", "The constructed casing 2 provides a housing for the printed circuit board 50 holding the engine components 24, and a battery 79.When the device 1 is a radio telephone, a SIM card holder 80 is provided to accommodate a SIM card 81.For operation under some radio protocols this will clearly not be necessary.", "Returning briefly to the front of the device, located above the display region 4 is the pillow 16 so named because it designed for the user to rest his or her ear against when making a telephone call.", "The pillow 16 overlays the ear piece 18 of the device.", "As described previously there is a cut-out in the perspex substrate 37 of the light guide 14 to accommodate the ear piece 18, the pillow 16, in the illustrated embodiment is adhered to the perspex substrate 37 covering the ear piece 18.The pillow 16 provides suitable apertures 17 to channel sound to the user.", "The perspex and sapphire light guide 14 is relatively cold to the touch, the material of the pillow 16 can be chosen to be a material that inherently warmer to the touch and less prone to marking than a transparent shiny surface.", "Materials that are thought to be particularly suitable are ceramics or wood and leather veneers.", "The shape of the pillow 16 obscures part of the LCD 25, providing opposing triangular sections 82, 83 that are used to indicate battery capacity and signal strength respectively.", "To complete the structure, the casing 2 has a rear cover 21 and internal compartments 84, 85 suitable for retaining the engine 24 the battery 79 and in this embodiment a SIM card 81.The device illustrated, in common with many other radio telephones, advantageously allows access to the battery compartment 85 as the battery 79 may periodically need replacement.", "In order for the engine 24 to be protected during this activity, the battery 79 is retained in a compartment providing connectors to the engine 24.Along side the battery compartment 85 in this embodiment is the SIM card holder 80.Under protocols where a SIM card 81 is used to hold subscription information, the user may wish to remove the SIM card 81.For this reason it is convenient for the back cover 21 to be removable to expose both the battery 79 and the SIM card 81.The battery compartment 85 and SIM card holder 80 are provided by an internal casing element 87 that is secured to the side frame 20.This may be formed from plastic or one of many other suitable materials.", "In one embodiment the compartments are formed from stamped metal sheet which may be stainless steel.", "The battery contacts 90 protrude into the battery cavity 85 to provide a simple way of connecting the battery 79 to the engine 24.The battery connection will typically be have a power connector 91, a ground 92 and two signal connectors 93, 94.In the embodiment illustrated, the power 91a,b,c and ground 92a,b,c connectors are divided into three.", "This reduces the resistance between the battery 79 and the engine 24 as the leads are in parallel, it also reduces the chance of power being lost to the engine 24 as a result of a harmonic resonance frequency of the connector being reached as at all times there is likely to be at least part of each three part connector completing the circuit between the engine 24 and the battery 79.The casing 2 is completed by assembling the back cover 21 to the side frame 20.In the illustrated embodiment, the back cover 21 is formed from three pieces 96, 97, 98.A first piece 96 overlays the antenna location.", "In this embodiment it provides an aperture 99 in which the polyphonic speaker 27 is located to provide for polyphonic sound.", "This additional speaker 27 has not been optimised for use in close proximity to the ear as has the speaker 18 located beneath the pillow 16.This allows it to be more effective as a handsfree speaker and enables a greater range of ring tones to be utilised.", "As the first piece 96 also covers the antenna its dielectric constant is relevant.", "In a device of this type where the casing 2 is to survive generations of engines 24, the materials from which is formed are important.", "They should be hardwearing and durable, retaining their attributes for years longer than is currently required.", "For this reason ceramics have been considered for the back cover 21 for some embodiments of the invention.", "With ceramics, however, despite having many desirable characteristics, for locations near an antenna relevant for devices containing such an element, the dielectric constant of the material interferes with the desired transmission pattern.", "To provide many of their desirable characteristics without the dielectric constant becoming too high, the rear cover piece 96 can be formed from a material with a lower dielectric constant with a suitable depth layer of deposited on it.", "The rear casing element 96 covering the antenna is again screwed to the side frame element 20 and in the process captures the second rail 23 similar in construction to the first rail 22 between the side frame 20 and the back cover 21.Again the rail 23 is provided with apertures 70 that are positioned over corresponding projections 71 on the side frame 20 to more securely hold the rail 23 in position when the back cover 21 is attached.", "A second rear casing element 97 covers the battery compartment 85 and the SIM card holder 80.This element is releasably secured to the other casing elements using screws 99 to allow relatively easy access to the SIM card 81 and battery 79.In a preferred embodiment a quarter turn of a screw 100 releases the cover element 97 to allow it to be removed.", "The same action can disconnect the battery 79 to allow the SIM card 81 to be removed.", "The second rear casing element 97 is attached to the side frame 20 in a similar manner to that described in relation to the first rear casing element.", "The third element of the rear casing 98 covers the polyphonic speaker 27.This may be only relatively thin and formed from a precious material such as gold.", "It will be provided with a suitable aperture 102 to channel sound from the polyphonic speaker 27 that may be use in hands free mode and for providing an audible ring tone.", "In the embodiment described the front 3 and rear casing elements 96, 97, 98 are secured to the side frame 20.A silicon sealant 69 similar to that provided on the underside of the bezel 19 may be extruded around the inside of the back cover 21 to prevent unwanted ingress of dirt and fluid.", "The skilled man will realise that the number of parts and the manner in which they are connected can be changed without departing from the several inventive concepts described.", "For example, in other embodiments the casing may be held together using other techniques and in other ways.", "As well as obscuring exposed edges, the rails 22, 23 provide additional elements for customization as they can be formed from a variety of different materials, in particular different types of precious metal for example gold or platinum.", "They also act to protect vulnerable edges of veneered frames from exposure to potentially damaging forces.", "In doing this unsightly edges are obscured from view.", "This provides quality and durability in keeping with the overall concept of a retainable casing for a communications device.", "Regardless of the material of the veneer, this is a convenient arrangement for securing all the elements in place for a durable finish.", "In an alternative embodiment illustrated in FIG.", "4b, instead of providing rails 22, 23 to protect exposed edges, the adjacent edges themselves have returns, which in the illustrated embodiment are turned inward 105, 106 and held in close proximity.", "The returns are of curved cross-section in the illustrated embodiment.", "This arrangement also provides the advantage of protecting the exposed edges and keeps unsightly discontinuities from view.", "A casing with an optically permeable front face provides new opportunities.", "In a particular embodiment, described with reference to FIGS.", "12 and 14 legends 15 associated with the keys of the key array 6 are located on both the front 37a and rear 37b surfaces of the perspex layer 37.With communications devices becoming ever more sophisticated, it is common for each key provided to have a number of different functions.", "It is useful to be able to label visually the different possible functions associated with a key.", "For the alpha numeric keys 7 in particular, each key is associated with a single digit and a group of letters or symbols.", "Ideally these should be displayed clearly.", "Historically the keys have held the associated legends.", "As devices have become smaller this has, however, becomes more difficult, it being an especial problem to distinguish between the different legends for respective modes of operation as the oletters and numbers are so small.", "In the illustrated embodiment, the legends 15 for associated keys are located on respective surfaces of the light guide 14 which, as the device is tilted, move together or apart depending upon the angle from which they are viewed.", "This provides a visual distinction between the legends 15 on the different surfaces which can be used to distinguish between the modes of operation.", "As the front face is optically permeable, the legends appear to float in or above the device.", "By spacing the legends along an axis perpendicular to the third dimension, the thickness of the front face, the front face is given some prominence, thus an additional dimension has been added to the normally opaque front face.", "In the embodiment illustrated the legends 15 are located on the outermost and innermost surfaces of the perspex layer 37a, 37b.", "The legends 15 appear to be floating in the light permeable layer.", "The manner in which the legend 15 is located on the layer is a matter of choice for the skilled man.", "One method is to print the legend on to the surface using an etched plate filled with ink.", "Another way may be by using PVD (Physical Vapour Deposition) techniques to deposit the ‘printing’ directly onto the perspex layer.", "Other techniques for fixing legends could be used including attaching labels.", "In the embodiment discussed the legends 15a 15b are located on opposing surfaces of the perspex layer 37a 37b.", "This enables a single element, the perspex layer 37, to be printed instead of each of the sapphire or other pieces 52-58.Problems encountered with ink wearing off when printing techniques are used are also avoided by protecting the surfaces containing the printing with the second layer.", "In other embodiments and to increase the depth of spacing of the legends and hence the separations possible when viewed at different angles it may be desirable to place the legends on the innermost 37a and outermost surfaces 51a of the composite.", "In order to fix the legend 15 printed on the outer surface 51a where it may encounter wear, a coating or other protective layer may be provided over the surface.", "This may not be necessary if PVD techniques are used to produce the legend 15 as such techniques produce a bond abetween the deposited layer and the substrate that is more har wearing.", "In still other embodiments the legend 15 could be provided on the underside 51b of the sapphire elements and on the innermost layer of the perspex substrate 37b.", "Again by avoiding the external surfaces of the transparent layer the problem of wear of the legends 15 is largely avoided.", "The outermost legend is protected by the top layer 51 of the composite light guide 14 and the innermost legend 15 is not accessible.", "In sandwiching the legends between two optically permeable layers 37, 51 which of the two sandwiching layers carries the legend is largely immaterial.", "The properties of the respective materials, if indeed they are different, and other factors can be taken into account in deciding which layer 37b, 51b actually carries the legend.", "With a composite optically permeable substrate, the more layers that are provided, the more visually distinguishable legends can be accommodated.", "For example with two composite layers there are three available planes for legends, with three layers, four planes and so on.", "If all the legends were placed adjacent a single key, the visual distinction may cause difficulty with large numbers of layers being used to distinguish functionalities.", "Other embodiments could use the different planes for legends at different locations on the device so that one set of keys has a legend in a first plane, a second set of keys in a second plane and so on.", "To accentuate the illusion of the legends floating, or to increase the distinction between the two layers, the thickness of the optically permeable layer between the respective layers carrying the legends can be increased.", "In the same way these features can be reduced by decreasing the thichkness between the planes carrying the legends.", "The ink or paint can be chosen by the skilled man to provide a number of effects.", "The ink may, for example, be required to pearlesce or fluoresce, or be black, white, or one or more of a wide range of colours.", "As the legend is located on a light guide, fluorescent and pearlescent materials enhance the visibility of the legends.", "Instead of techniques that add ink or other visible materials to the surface merely etching the layers may be sufficient to make the legends visible if the illumination is adequate.", "Embodiments illustrating a light guide overlaying both display and input regions have been described with reference to an illustrated embodiments in the context of other independently inventive features claimed in copending applications.", "The skilled man will realise that many alterations to the specific features disclosed can be made without departing from the scope of the invention.", "In particular the materials of the the light guide are not restricted to those described and the light guide, as discussed may be concealed beneath an opaque layer.", "The input region of the device will now be described in more detail, primarily with regard to FIGS.", "9 and 15 to 21.The input region 5 comprises a key array 6, as described above.", "In this embodiment, the key array is made up of a plurality of individual keys 7-13.Each of these individual keys comprises a key tip 64 and shaft 49 extending from substantially the centre of the key tip 64, together with an upper bearing 103, O ring 107, circlet 108, spring plate 110 and lower bearing 109 positioned respectively along the shaft 49a from the key tip 64.The spring plate 110 is supported by a spring plate support 114 provided on the PCB 118.The spring plate comprises a main body, and a tongue 111 formed from a single piece of sheet metal.", "The tongue has been formed by stamping an inner portion of the sheet, so that two sides and an end of the tongue are free from the remaining main body of the sheet.", "The spring plate also has a portion or portions stamped out to provide an aperture for the key shaft 49 and spring plate support 114.One end 113 of the spring plate comprises contacts for contacting respective contact regions on the PCB.", "As will be appreciated by a person skilled in the art, a single contact could be used for this purpose, but the provision of two contacts provides greater reliability.", "The spring plate support 114 comprises three members upstanding from the PCB 118.These members may form part of a unitary structure, or may be separate elements.", "The first member comprises a recess 115 dimensioned to receive one end 112 of the spring plate 110 and hold that end 112 in position.", "The second member of the support 114 comprises a lip 116 extending towards the first member.", "This lip 116 is provided to restrict the upward movement of the other end 113 of the spring plate 110.The third member of the support 114 comprises a recess 117 for receiving the end of the tongue.", "This third member is positioned relative to the second member, such that the tongue of the spring plate 110 has to be flexed in order for the end of the tongue to correspond with its recess 117.Optionally, the spring plate support may comprise a fourth member comprising a lip extending towards the second member.", "In this case, the PCB contact regions are extended to the surface of this lip, so as to bring them closer to the spring plate contacts.", "Alternatively the contact region bearing lip may be formed as part of the second member itself, or eliminated altogether.", "As mentioned above, in the present embodiment, the individual keys are grouped in threes, their tips extending across the whole width of the second layer of the composite 51.This facilitates manufacture of the brittle elements 52 to 58.The tips of the alphamumeric keys 7, soft keys 8, 9 and top scroll key 12a are in alignment with those of the other keys in their group.", "In the remaining group of keys, however, the tip of the bottom scroll key 12b is displaced from the line of the tips of the other two keys in its group, namely the call handling keys 10, 11.In any event, in each group the edges of adjacent key tips complement each other, and are closely spaced.", "This eliminates the need for the composite 51, or other filler material, to extend between the key tips.", "It also has the advantage of simplifying the overall appearance of the input region of the device to the user.", "Further properties and advantages of the specific key array 6 will be discussed below.", "As can be seen in particular from FIGS.", "17 and 18, the spring plates 110 and supports 114 of adjacent alphanumeric keys 7 are positioned perpendicular to each other.", "This provides a geometrically simple solution to the problem that the spring plates cannot be positioned in alignment with the keys themselves.", "One cause of this problem in the embodiment illustrated is that the spring plate of each outer key is longer than the average length of its corresponding key tip, and this extra length cannot be accommodated elsewhere.", "This is primarily because the spring plate of each central key is only minimally smaller than the average length of the corresponding key tip, and the keys are closely abutted (there is only a gap of about 0.245 between the keys) so that there is insufficient space to allow for the extra length.", "Furthermore, whilst the key shaft 49 of each key is substantially central to the key tip 64, the key shaft aperture of the spring plate 110 is off-centre.", "This exacerbates the problem for the outer keys, and even results in the spring plate of each centre key not being able to be accommodated in the space under its corresponding key tip.", "The keys may be constructed, and the input region of the device assembled as follows.", "Upper bearings are inserted into the apertures 48 of the perspex layer of the device.", "Ruby bearings are preferably selected for this purpose, for three main reasons.", "Firstly, ruby is very hard wearing, and will thus be able to handle multiple operations of the keys over a substantial period of time.", "Secondly, the upper bearings of this device are larger in diameter than the corresponding widths of the key tips, which means that they will be visible in situations in which the front face of the device is transparent (e.g.", "second layer 51 may be sapphire).", "Hence, advantage may be taken of the fact that a ruby is an attractive jewel, which the user will be pleased to see.", "Thirdly, the use of a hard material such as ruby will provide greater accuracy of fit of the key in the device, as opposed to using a resilient material, such as PFTE.", "Lower bearings 109 are provided in a titanium plate which is fixed to the rear of the PCB 118.The inner and outer diameter of the lower bearings 109 are smaller than the upper bearings 103, but their centres are aligned.", "They are also preferably made of different material—the material of the lower bearings having shock absorbing qualities such as PFTE.", "The key shafts 49 are machined, to have a first portion 49a of appropriate thickness to pass through the inner diameter of the upper bearings 103 and a second reduced diameter portion 49 to pass through the inner diameter of the lower bearings 109.Shaft 49 and circlet 108 may be machined from a single piece of metal, or the circlet 108 may be subsequently attached to the shaft 49.They are preferably formed of stainless steel.", "Further, an O ring 107 is provided adjacent the circlet 108, on the thicker diameter portion 49a of the shaft 49, to provide a water seal.", "Both the circlet 108 and the O ring have an outer diameter smaller than that of the upper bearing, so that they are not visible to the user when in situ.", "However, the circlet has an outer diameter sufficiently larger than the inner diameter of the upper bearing, so as to prevent the key from falling out of the device.", "The key tips are crafted into the desired shape from a desired material, which may be a metal such as gold, platinum, silver, or stainless steel.", "They may also bear precious stones.", "For example a key tip or tips may be diamond encrusted, or have a precious stone set in it.", "Once a key shaft has been passed through an upper bearing, a key tip is joined to it.", "This may be achieved using conventional braising techniques.", "The spring plate supports 114 are provided on the PCB 118.Each spring plate support 114 is preferably of unitary structure, manufactured from lightweight metal such as aluminium.", "The supports may then be soldered onto the PCB using conventional techniques.", "The spring plate supports 114 are positioned to hold the spring plates 110 so that the centres of their shaft apertures align with those of the lower bearings 109.Contact regions for the spring plate contacts are provided on the PCB (or the lip of the second or fourth member of the support as described above).", "The spring plates 110 themselves are provided by stamping sheet metal, such as beryllium cooper with gold flash or the like.", "The plates 110 are affixed to the supports 114 by positioning one end 113 of the spring plate under the lip 116 of the second member of the support 114, fitting the other end 112 of the spring plate in the recess 115 of the first member of the support 114, flexing the tongue so that its end corresponds to the recess 117 of the third member of the support 114, and positioning the end of the tongue in that recess.", "When in position, the free end 113 of the spring plate 110 is naturally biased upwards toward the restraining lip 116.The dimensions of various components of the keys and their relative positions are important in smooth key operation.", "The distance between the upper and lower bearings has been maximised by placing them either side of the spring plate switching mechanism and passing the shaft through the spring plate.", "In such a position, the bearings hold the key straight when it is operated, thereby avoiding contact with neighbouring key tips or the need to place keys further apart to prevent such contact.", "This, in turn, results in good switching functionality and feel.", "Also, the dimensions of the keys are such that the lower surface of each key tip 64 contacts the surface of the perspex layer 37 before excess pressure can be applied to the spring plate 110.In the present embodiment, the circlet 108 is positioned along the shaft such that it gently rests on the spring plate when the keys are in their normal non-depressed state.", "Also, the thicker portion of the shaft is of an appropriate length that, when in the normal state, there is a gap between the lower surface of the key tips and the surface of the perspex layer 37 which is the same as, or only slightly greater than the distance the circlet 108 has to travel to cause the spring plate contacts to contact the contact regions.", "This overcomes a potential problem of pressure being applied to the spring plate if the switch itself provides the end stop for the motion.", "The dimensions of the keys are also important for the external appearance of the device.", "Preferably, the key tips are of a thickness that they protrude from the surface of the second layer 51 of the composite, at least when the keys are in the aforementioned normal state.", "When the composite is substantially transparent, this will give the impression of floating keys, and add to the three dimensional effect mentioned earlier concerning the key legends 15.Furthermore, the key tips should be sufficiently deep to be partially sunk into the second layer of the composite, and preferably have at least two opposing substantially flat sides which correspond with sides of the second layer to prevent lateral rotation of the keys.", "A gap of the order of 0.1 mm is achieved in the present device between the keys and second layer of the composite: a gap insignificant to the human eye and suitable for assisting in the prevention of lateral rotation of the keys.", "Lateral rotation is further hindered in the present embodiment, by virtue of the provision of mirrored slanting of the sides of adjacent keys.", "In order to ensure the correct relative spacings of elements of the key, the perspex layer 37, PCB 118, and titanium plate 77 are clamped together.", "In this embodiment, the clamping is provided by the front face protectors 63, and by the provision of additional tapped bosses in projections 68 of the perspex plate and associated fixing means.", "Consequently, once the keys have been positioned, the perspex layer, PCB and titanium plate can be clamped together using the bosses and fixing means, thereby holding the keys in place and forming a manageable module.", "This module may then be readily installed in the device using the front face protectors as described above with reference to FIG.", "9B.", "Operation of the keys of the device will now be described.", "As the user applies pressure to a key tip, the shaft moves downwards, travelling through the bearings and shaft aperture of the spring plate.", "This results in the circlet applying pressure to the part of the main body of the spring plate which defines the shaft aperture.", "Continued pressure on the key tip will then cause the circlet to apply an increasing force to this part of the spring plate, causing the main body to deform around the circlet.", "Eventually, this deformation will cause the tongue to overcentre, resulting in the free end 113 of the spring plate 110 flicking from its naturally biased position (upwards towards the restraining lip 116) to a second position, in which the spring plate contacts contact the contact regions on the PCB.", "An electrical signal is consequently sent to the processor indicative of actuation of that key.", "This arrangement gives a distinct click providing a clear quality indication to the user that the key has been actuated.", "As the user removes pressure from the key tip, the circlet, in turn, removes pressure from the spring plate 110.The tongue promptly returns to its normal position, and the free end of the spring plate flicks up to its naturally biased position, breaking the contact.", "FIG.", "20a is a graph illustrating the force against distance profile for a key such as that illustrated in FIGS.", "17 and 18.This profile improves the tactility of the key over, for example, a typical keydome arrangement, which had a fairly flat profile as can be seen in FIG.", "20b.", "When using a conventional key dome type arrangement, the user has to apply a constant force until the point where the key actuates.", "As a result, he does not get a tactile indication that he is nearing the position when actuation is likely to occur.", "In contrast, when using the device illustrated in FIGS.", "17 and 18, the user can realise the fact that he is nearing the position when actuation is likely to occur as he is having to increase the force applied for a given travel of the key.", "Furthermore, the user is informed when actuation takes place, and again when deactuation takes place, by respective clicking sounds provided by the key.", "The central V shaped key tips of the embodiment illustrated in FIG.", "1 enable the user to determine the central vertical axis of the device both by sight and by touch.", "This is made even easier by the pillow 16 being provided with an apex.", "Consequently the user can quickly locate a desired central key.", "The apexes of each central key tip also identify the mid point along the length of the key tip, the point from which the key shaft 49 extends.", "Hence, they facilitate more accurate depression of the key.", "This, in turn, may assist in the prevention of contact with neighbouring key tips or the need to place keys further apart to prevent such contact.", "Likewise, in this embodiment, the combination of adjacent outer key tips and front face protectors forming a V shape enable the user to determine the position of the vertical axes to one side of which the outer keys lie.", "Consequently, the user can quickly locate a desired outer key.", "The key location process is facilitated in this embodiment as the outer key tips extend to the interface between the second layer of the composite 51 and the bezel 19.The front face of the device illustrated in FIG.", "1 is protected by the pillow and front face protectors, 63.The front face 3 is slightly convex, with the highest points lying along its central longitudinal axis.", "Hence, ordinarily, if placed face down, the device would rest on this axis, resulting in scratches to its surface.", "Clearly, this is not acceptable, particularly when the second layer of composite is sapphire or the like.", "The device illustrated in FIG.", "1 has been designed to avoid this problem.", "The pillow 16 and face protector 63a prevent the device resting on the second layer of composite.", "Also, as mentioned above, in the preferred embodiment the key tips protrude slightly from the surface.", "Hence, the central key tips too may protect the second layer of composite from damage.", "However, preferably the pillow 16 and chin front face protector 63a are raised sufficiently above the front surface, that the device does not rest on the central key tips either, so as to protect them from damage too.", "The device is also designed so that the front face is protected if the edge of the face is knocked.", "As can be seen from the plan view of the device, depicted in FIG.", "21, the bezel front face protectors 63b protrude beyond the surface of the second layer of composite along the interface with the bezel, thereby protecting the second layer of composite from damage in that region.", "They also reduce the likelihood of damage to the bezel due to knocks.", "Moreover, they protrude further than the adjacent key tips, hence protecting those key tips from damage too.", "One further benefit of the front face protectors 63, particularly the bezel is that they are dimensioned so as to prevent the keys from being accidentally actuated if, for example, the device was placed face down.", "That is, the top surface of the front face protector tips should either be at the same level or higher than the top surface of a key tip (e.g.", "as in the relationship between the protectors 63b and the adjacent outer key tips) or, if they are at a lower level than the top surface of a key tip, the distance between the top surface of the protector and key tips must be smaller than the distance the key needs to travel in order for the spring plate contacts to contact the PCB contact region for actuation the key.", "Aspects of the invention have been discussed with reference to a radio telephone function.", "It will be clear to the skilled man that these aspects apply equally to other portable communications devices supporting in addition or as an alternative other functions, such as, amongst others electronic diaries, and electronic notepads.", "The present invention includes any novel feature or combination of features disclosed herein either explicitly or any generalisation thereof irrespective of whether or not it relates to the claimed invention or mitigates any or all of the problems addressed.", "In view of the foregoing description it will be evident to a person skilled in the art that various modifications may be made within the scope of the invention." ] ]	Patent_10465983
[ [ "Device and method for analyzing ion channels in membranes", "The present invention relates to devices and methods for analyzing ion channels in membranes.", "The invention is characterized by a biochip comprising a substrate in which openings are provided in the form of an M×N matrix for receiving therein a cell membrane including at least one ion channel (I) or an artificial lipid membrane (Me), wherein M≧1 and N≧1." ], [ "1.A biochip (1; 2; 3) for analyzing ion channels, comprising a substrate (10; 20; 30) in which openings (19; 29; 39) are provided in the form of an M×N matrix for receiving therein a cell membrane (Me) including at least one ion channel (1) or an artificial lipid membrane including at least one ion channel, wherein M≧1 and N≧1.2.A biochip (1; 2; 3) according to claim 1, wherein the surface of the biochip has in the area of each opening a means for improving the contact between the cell membrane and the biochip, said means being provided on the receiving side.", "3.A biochip according to claim 2, wherein the means for improving the contact is implemented in the form of a patterning of the surface.", "4.A biochip according to claim 3, wherein said patterning is provided in the form of one or a plurality of rings which is or which are arranged around each opening, or in the form of one or a plurality of squares or rectangles which is or which are arranged around each opening.", "5.A biochip according to claim 1, wherein each opening is substantially circular.", "6.A biochip according to claim 1, wherein the substrate comprises a base portion (10; 20; 30) which has a first thickness (d1) and window portions (11; 21; 31) which are formed in said base portion and which have a second thickness (d2), each opening being provided in a respective window portion.", "7.A biochip according to claim 1, wherein the substrate comprises a semiconductor material, such as GaAs, Si or AlGaAs, or an insulator, such as a glass or quartz, or polymers, such as polydimethyl-siloxane (PDMS).", "8.A biochip according to claim 6, wherein the substrate comprising the base portion and the window portions formed in said base portion consists of one material.", "9.A biochip according to claim 1, wherein electrodes are provided on one or on both sides of the substrate.", "10.A biochip according to claim 9, wherein the electrodes are implemented such that they are adapted to have applied thereto a temporally constant electromagnetic field and/or a high-frequency alternating electromagnetic field.", "11.A biochip according to claim 1, wherein planar waveguides are integrated in the biochip for applying high-frequency alternating fields.", "12.A biochip according to claim 1, wherein interdigital electrodes are provided on the biochip for generating surface-acoustic waves.", "13.A biochip according to claim 1, wherein active and/or passive components are integrated on the substrate.", "14.A biochip according to claim 13, wherein said active and/or passive components comprise a field effect amplifier means for preamplifying measuring signals.", "15.A biochip according to claim 1, wherein an optical near-field means is provided for observing the ion channel or the ion channels.", "16.A biochip according to claim 15, wherein the optical near-field means comprise scanning probe means.", "17.A biochip according to claim 1, wherein microfluid channels are provided for on-chip perfusion.", "18.A biochip according to claim 1, wherein the biochip has applied thereto a layer of flexible, non-conductive polymer on the receiving side, said layer comprising at least two openings through which at least the openings in the substrate are exposed.", "19.A biochip according to claim 1, wherein the surface on the receiving side is hydrophobic.", "20.A biochip according to claim 1, wherein channels extending parallel to the substrate surface are provided in or above said substrate surface.", "21.A method of producing a biochip for analyzing ion channels comprising a substrate in which openings are formed, in the form of an M×N matrix, for receiving therein a cell membrane including at least one ion channel or an artificial lipid membrane including at least one ion channel, wherein M≧1 and N≧1, said method comprising the steps of: providing a substrate, forming at least one window portion in said substrate, and forming an opening in each window portion.", "22.A method according to claim 21, wherein each window portion is formed by means of wet- or dry-etching methods.", "23.A method according to claim 21, wherein each window portion is formed by means of laser thinning or by means of hot shaping.", "24.A method according to claim 21, wherein each opening is formed by means of laser thinning or ion track etching.", "25.A method according to claim 21, wherein each opening is formed by means of dry-etching methods or by means of a focused ion beam.", "26.A method according to claim 21, comprising the following additional step: local or non-local heat treatment of the substrate for improving the contact with a cell membrane.", "27.A method of analyzing ion channels in membranes, said method comprising the steps of: providing a biochip according to claim 1, applying one or a plurality of singulated cells in an aqueous suspension to the biochip, positioning not more than one cell on one opening.", "28.A method according to claim 27, wherein the cells are applied with the aid of at least one pipette or cannula.", "29.A method according to claim 28, wherein the ion channel currents are measured with the aid of electrodes integrated in each pipette or cannula.", "30.A method of analyzing ion channels in membranes, said method comprising the steps of: providing a biochip according to claim 20, flushing one or a plurality of singulated cells in an aqueous suspension into the biochip via the channels extending parallel to the substrate surface, positioning not more than one cell on one opening.", "31.A method according to claim 27, wherein, for positioning each cell, a vacuum is applied at the side of an opening located opposite the receiving side.", "32.A method according to claim 27, wherein, for positioning each cell, an electric direct voltage and/or alternating voltage is/are applied perpendicularly to the substrate surface.", "33.A method according to claim 27, wherein surface-acoustic waves are used for positioning each cell.", "34.A method according to claim 27, wherein, for positioning each cell, mechanical, chemical, electric, magnetic or electromechanical gradients or fields are applied through the opening.", "35.A method according to claim 27, wherein, for positioning each cell, additional cells or particles are added on the receiving side.", "36.A method according to claim 27, said method comprising the following additional step: detecting each cell on an opening by measuring at least one electric parameter of said opening.", "37.A method according to claim 27, said method comprising the following additional step: electrophysiological characterization of each cell.", "38.A method according to claim 27, wherein active substances are applied or de-applied by flushing in or sucking off a solution.", "39.A device for analyzing ion channels in membranes, comprising: a first biochip according to claim 1, and a second biochip provided with a means for positioning cells relative to the openings of said first biochip, wherein the respective surfaces on the receiving side are located in opposed relationship with and at a fixed or variable distance from one another.", "40.device according to claim 39, wherein the cell positioning means comprises a means for generating surface waves.", "41.A device according to claim 39, wherein the biochips are supported in direct contact with one another and wherein the surface of the second biochip located on the receiving side has integrated therein fluid channels which extend parallel to the surface and which are open towards said surface.", "42.A measuring probe (4), comprising a biochip (1; 2; 3) according to claim 1, a holding device (45) having a central cavity or a plurality of cavities which communicate with the aperture or the apertures of the biochip (1; 2; 3) and provided on the side of the substrate that is located opposite to the side where the membrane (M) is applicable, wherein the opening of the holding device facing away from the substrate is implemented such that an electrode means (43) can be inserted therein.", "43.A measuring probe according to claim 42, wherein the holding device consists of glass or polycarbonate.", "44.A measuring probe according to claim 42, wherein the holding device is adapted to be screw-fastened to the biochip.", "45.A measuring probe according to claim 42, wherein sealing means are provided between the holding device and the biochip.", "46.A measuring probe according to claim 42, wherein the holding device is adhesively attached to the substrate.", "47.A measuring probe according to claim 42, wherein the electrode means is adapted to be screwed into the glass tube.", "48.A measuring probe according to claim 47, wherein sealing means are provided between the holding device and the electrode means.", "49.A measuring probe according to claim 42, wherein a vacuum-generating means (46) is provided in said glass tube." ], [ "<SOH> FIELD OF THE INVENTION <EOH>The present invention relates to devices and methods for analyzing ion channels in membranes, in particular devices and methods for executing the so-called patch clamp technique with the aid of a biochip, especially for use in high throughput processes." ], [ "FIELD OF THE INVENTION The present invention relates to devices and methods for analyzing ion channels in membranes, in particular devices and methods for executing the so-called patch clamp technique with the aid of a biochip, especially for use in high throughput processes.", "PRIOR ART Ion channels are membrane proteins which serve as switchable pores for a flow of current.", "Ion channels, which are the smallest excitable biological structures, especially constitute the fundamental switching elements of the nervous system.", "It follows that the equipment of a neurocyte with ion channels of different types essentially determines the neurocyte's role in the processing of information in the brain.", "This applies, by the way, also to non-neuron excitable cells in a similar manner, e.g.", "to those of the cardiac muscle and its stimulus conduction systems.", "Switching processes in ion channels are analyzed for obtaining e.g.", "information on possible malfunctions and their elimination by means of drugs and the like.", "For analyzing ion channels in cell membranes with respect to their switching processes, i.e.", "their opening and closing mechanisms, the patch clamp method is used in the prior art.", "For this purpose, so-called patch clamp pipettes consisting of glass are used.", "Such a pipette is shown in FIG.", "5.This pipette comprises an opening 59 having a diameter of approx.", "1 μm.", "In addition, the pipette comprises a pipette shaft 58 in which an electrode 53 is provided.", "For analyzing an ion channel, a membrane patch is sucked up by means of such a pipette filled with an electrolyte so that a close contact will be established between the membrane and the glass.", "In this way, a very high sealing resistance of an order of magnitude of >1 GΩ is obtained.", "This permits measurement of very small ion currents, down to a few 100 fA, through the membrane.", "The known device is, however, disadvantageous insofar as it is not suitable for simultaneously analyzing a large number of substances or the effect of a substance on a large number of different (e.g.", "genetically modified) ion channels.", "The known device is therefore not suitable for high throughput analyzing.", "Hence, this device is can be used for substance screening in the pharmaceutical industry only to a very limited extent.", "Another disadvantage of the known device is that the time scale on which the opening and closing mechanisms in the ion channels take place is accessible only to a very limited extent with this device consisting of a glass pipette, an electrode and an amplifier.", "This has the effect that, when this device is used for the patch clamp method, the bandwidth will be limited to less than 100 kHz.", "For analyzing the opening and closing mechanisms in ion channels, time scales corresponding to a bandwidth of >1 MHz would, however, be desirable.", "It is therefore the object of the present invention to provide a device for analyzing ion channels in cell membranes, which is suitable for high throughput processes, e.g.", "for use in the pharmaceutical industry, and/or which exhibits an improved signal-to-noise ratio and an improved timing resolution.", "DESCRIPTION OF THE INVENTION This object is achieved by a biochip for analyzing ion channels, comprising a substrate in which openings are provided in the form of an M×N array for receiving therein a cell membrane including at least one ion channel or for receiving therein an artificial lipid membrane including at least one ion channel, wherein M≧1 and N≧1.When such a biochip is used, the use of a pipette whose comparatively long shaft leads to a high stray capacitance can be dispensed with.", "The critical geometrical parameters can, however, be optimized from the very beginning, and this has the effect that the signal-to-noise ratio will be improved markedly in comparison with the prior art whereby the timing resolution will be improved.", "This applies to biochips with a single opening, i.e.", "for M=N=1 as well as to biochips with a plurality of openings, i.e.", "M>1 and/or N>1.Due to the plurality of openings for receiving therein membranes including ion channels, it will additionally be possible to parallelize the patch clamp technique in the case of M>1 and/or N>1, whereby M×N measurements can be carried out simultaneously with one chip.", "In this case it will be particularly advantageous to adapt the shape of this M×N array to the geometry of the 96, 384 or 1536 cuvette plates used as a standard in the pharmaceutical industry.", "These cuvette plates can be inserted into automatic pipetting devices by means of which substances can advantageously be applied to the biochip described here.", "A special advantage is that, by means of automatic pipetting devices or by other arrays of pipettes or cannulae which are arranged in a fixed mode relative to one another, solutions or cells can be taken simultaneously from a plurality of cuvettes of the standard cuvette plates and applied to the biochip, since the arrangement of the pipettes or cannulae relative to one another can be maintained for applying the solutions or cells to the biochip.", "In addition, membranes which have been applied to the biochip according to the present invention will, in comparison with the known device, be much more easily accessible due to the geometry of the biochip.", "This offers a much better possibility of observing the membranes and of manipulating them chemically and/or mechanically and/or electrically.", "According to an advantageous embodiment of the above-described biochip, the surface has in the area of each opening a means for improving the contact with the cell membrane, said means being provided on the receiving side of the respective opening and being used for guaranteeing improved adhesion of the membrane to the biochip in the area of the aperture (opening).", "Also the electrical sealing resistance can be increased in this way.", "In accordance with an advantageous further development, the means for improving the contact can be implemented in the form of a patterning of the surface.", "For this purpose, the patterning can be provided in the form of one or a plurality of rings which is or which are arranged around each opening, or in the form of one or a plurality of squares or rectangles which is or which are arranged around each opening.", "The patterning can especially be provided in the form of a depression in the surface of the biochip, said depression being arranged concentrically around and in closely spaced relationship with the opening and having a diameter which is many times larger than the diameter of the opening so that the edge of the opening projects upwards beyond the surrounding biochip level.", "This has the effect that a cell membrane will be dented by the edge of the opening whereby the contact between the biochip and the membrane will be improved.", "Each opening can have length and width dimensions in the range of 10 μm to 10 nm.", "The number of ion channels observed can be adjusted in this way.", "In addition, a smaller opening will also reduce the membrane area and thus the capacitance and this will improve the measurement resolution still further.", "The biochip according to the present invention is also excellently suitable for forming artificial lipid membranes (artificial lipid bilayer) on the opening, this formation taking place in analogy with the known black lipid or lipid bilayer method.", "This permits ion channels to be analyzed by fusing vesicles, which include ion channels, with the artificial lipid bilayer.", "Due to the fact that the size of the aperture is small in comparison with the known bilayer method (in known devices the size of the aperture is normally >100 μm) and due to the resultant small capacitance, the signal-to-noise ratio can be improved.", "In accordance with a preferred embodiment of the above-described biochip, each opening can be substantially circular.", "Such circular shapes can easily be implemented in the bio-chip.", "If a simple implementation is not necessary, also other shapes can be chosen for the cross-sections of the openings.", "According to a preferred further development of all the above-described biochips, the substrate can comprises a base portion which has a first thickness and a window portion or a plurality of window portions which is/are formed in said base portion and which has/have a second thickness, an opening being provided in each of the respective window portions.", "The thickness of the base portion can here especially range from 1 mm to 100 μm and the thickness of the window portion can range from 1 μm to 50 μm.", "This further development guarantees that the mechanical stability of the substrate will be preserved, whereas the length of the aperture (at right angles to the cross-section of the opening) and thus also the electric access resistance will remain as small as possible.", "In addition, this further development can be used for producing apertures with diameters of 10 μm down to less than 1 μm with the aid of a dry-etching step, laser ablation or latent ion track etching.", "On the basis of this further development it will also be possible to fill the aperture more easily with the electrolytic solution and to establish an electric contact therewith.", "The depression formed on the lower surface of the biochip by local thinning permits a simple application of solutions by means of a pipette; due to capillary forces, said solutions penetrate into the aperture and fill said aperture.", "In accordance with an advantageous further development of all the above-described bio-chips, the substrate can comprise a semiconductor material, such as GaAs, Si or AlGaAs, or an insulator, such as glass or quartz, or polymers, such as polycarbonate, acrylic glass or polydimethylsiloxane (PDMS).", "A large number of advantages, in particular a simple production by means of a process technology perfected for the respective material, can be achieved by these materials.", "According to an advantageous further development, the substrate comprising the base portion and the window portions formed in said base portion consists of one material.", "The production process of the biochip can be simplified in this way.", "When a substrate consisting of a semiconductor material, in particular of Si, GaAs or AlGaAs, is used, a passivating and insulating layer can be provided, said layer being applied to one surface or to both surfaces of the substrate.", "This insulating layer can especially consist of SiO2, Si3N4, glass or polymers, and of multi-layer systems in which these materials are combined with one another and/or with the above-mentioned semiconductors and/or with metals, and have thicknesses of 50 nm up to several μm.", "By means of these materials, a sealing resistance of a few GΩ can be realized, this kind of sealing resistance being necessary for measuring currents in the pA range.", "In the production of this embodiment, the insulating layer can also fulfil the function of an etch stop layer and, in the case of anisotropic etching of the semiconductor it can lead to the formation of a window portion in which only the insulating layer is still present.", "The aperture can then be defined lithographically and the self-supporting insulating layer can be applied by dry-etching processes.", "As a further advantageous alternative, polymers, such as polydimethylsiloxane (PDMS), can be used as a substrate material.", "When the above-described biochip is produced from PDMS, a 3D negative template (mould) is used, which has the inverted structure of the desired biochip.", "The PDMS is first viscous and, after having been mixed with a curing agent, it is cast into the mould and cured with or without heating (approx.", "60 to 100 ° C.).", "The flexible biochip can then be released from the mould; said release can be carried out more easily when the mould has been coated with silanes previously.", "For the production of this embodiment a chemical modification of the surfaces (especially oxidation in the plasma incinerator or also other suitable methods) will be advantageous.", "Moreover, all the surfaces of the biochip may be provided with additional insulating and passivating layers of the above-mentioned materials and they may have chemical modifications (silanization, oxidation).", "According to a preferred further development of all the above-described biochips, electrodes can be provided on one or on both sides of the substrate.", "In particular, electrodes consisting e.g.", "of gold, silver or of other suitable metals can be applied directly to the chip by means of vapour deposition.", "This will simplify the test set-up, since the electrodes are already fixedly integrated on the biochip and since the step of applying and adjusting the electrodes can therefore be dispensed with.", "In addition, when this arrangement is used, and in particular when the electrodes are arranged such that the distance between said electrodes and the membrane is only a few μm, the parasitic capacitances and resistances can be reduced still further, and this will lead to another improvement of the signal-to-noise ratio.", "Whether a biochip with integrated electrodes on one or on both sides of the substrate is used can be determined in dependence upon the test to be carried out.", "Electrodes which are particularly suitable for this purpose are Ag/AgCl electrodes.", "These electrodes have the advantage that an electrode polarization, which would corrupt the measurement results, will be avoided.", "Furthermore, additional electrodes can be integrated so that high-frequency alternating electromagnetic fields can be applied via the aperture.", "In particular by applying a high-frequency alternating field in the range of MHz to GHz, the dynamics of the ion channels (conformation changes, ion permeation and ligand binding) can be influenced and analyzed.", "For applying such high-frequency fields, the use of antenna structures (e.g.", "the bow tie antenna known from the field of high-frequency technology) will be particularly suitable.", "An effective coupling of the electromagnetic field to the ion channel can be achieved in this way.", "An advantageous alternative is the integration of planar waveguides (so-called strip lines) for high-frequency alternating fields.", "The electrodes can have a width of 40 nm and they can be arranged at a distance of only a few nm from the opening so as to optimize coupling in of the power of the alternating fields.", "When a substrate is used which comprises a base portion having a first thickness and one or a plurality of window portions formed in said base portion and having a second thickness, Ag/AgCl electrodes in the form of wires or sintered capsules (pellets) can be introduced in this recess, whereby the aperture will be electrically contacted as well.", "For mechanically manipulating cells or liquids on the biochip, interdigital electrodes can be provided on the biochip for generating surface-acoustic waves with the aid of which cells or liquids can be positioned relative to the aperture of the biochip.", "In particular, surface acoustic waves can keep the cells in motion so that they will not adhere to the chip; this would make it impossible to suck them into the aperture or to cause them to move into said aperture in some other way.", "According to a preferred further development of the above-described biochips, not only electrodes but also electrically and/or optically active and/or passive components can be integrated on the substrate.", "This results in a further structural simplification of the test set-up.", "Especially also the signal paths can be kept short in this way, and this will again have an advantageous effect on the signal-to-noise ratio.", "The biochips may, for example, comprise integrated field effect transistor means for preamplifying measuring signals.", "The electrodes, the electrically and/or optically active and/or passive components can be integrated on the substrate in an advantageous manner, if desired on the etch stop layer and the insulating layer, respectively.", "In accordance with further preferred embodiments, optical near-field means for observing the ion channel or the ion channels can be provided in all the above-described biochips.", "The possibility of using near-field means results from the geometry-dependent easy accessibility of a membrane on the biochip.", "Hence, especially all scanning probe methods, such as scanning force microscopy (AFM), scanning near-field optical microscopy (SNOM) and scanning tunneling microscopy (STM), can be used easily for observing the membranes.", "On the basis of the geometry-dependent easy accessibility, also other image-forming methods, such as scanning electron microscopy (REM), confocal fluorescence microscopy (also in combination with SNOM), fluorescence spectroscopy, optical microscopy or individual photon detection, can be used.", "In particular biochips consisting of glass or polydimethylsiloxane (PDMS) are suitable for fluorescence tests, since the substrate has here a weak fluorescent background.", "In accordance with an advantageous embodiment, microfluid channels can be provided in the above-described biochips for on-chip perfusion.", "According to a particularly advantageous further development of all the hitherto described biochips, the biochip has applied thereto a layer of flexible, non-conductive polymer on the receiving side, said layer comprising at least two openings through which at least the openings in the substrate are exposed.", "It follows that the area of an opening in the polymer layer is at least as large as the area of an opening in the substrate.", "The layer is preferably 10 μm to 5 mm thick and consists e.g.", "of PDMS.", "The openings may, for example, be produced by punching.", "Through these openings in the flexible polymer, whose diameter can be e.g.", "10-5000 μm, individual areas resembling cuvettes are defined on the biochip on the receiving side; these cuvette-like areas serve to receive liquid therein and the substrate of the biochip including at least one aperture is exposed in said areas on the receiving side.", "A particularly advantageous aspect of this arrangement is that the individual apertures are thus also electrically separated from one another on the receiving side.", "Each opening in the polymer layer may, for example, expose precisely one aperture and part of the substrate surrounding said aperture.", "Alternatively, also a plurality of apertures can be exposed by on opening in the polymer layer; in this case, a cuvette encloses a plurality of apertures.", "PDMS is particularly suitable as a substrate for these cuvettes, since it has good adhesive properties with respect to glass and quartz as well as with respect to the other above-mentioned substrates which can be used for designing the biochip, and since it is biocompatible.", "Alternatively, the substrate surface of the biochip can be rendered hydrophobic by treatment with chemicals so that solution drops deposited on the receiving side on top of the apertures will rest on said apertures with a steep contact angle and remain reliably separated from one another.", "This has the effect that, without the aid of any additional structure a liquid compartment will be formed, which is effective as a cuvette as well.", "According to another particularly advantageous embodiment of all the above-described bio-chips, channels extending parallel to the substrate surface are provided in or above said substrate surface.", "Alternatively, these channels are formed directly as trenches in the surface of the substrate and are open at the top.", "According to another advantageous alternative, the biochip is, on the receiving side, provided with a PDMS layer or any other substrate which is adherent to the biochip and through which trenches extend that are open towards the surface of the biochip substrate including the aperture.", "These trenches may especially have diameters and depths between 5 and 500 μm.", "By applying the layer containing these trenches to the biochip, said trenches become fluid channels which are closed by the substrate surface of the biochip.", "In accordance with a specially preferred embodiment, these trenches are designed in such a way that they extend in a cross-shaped or star-shaped pattern towards and away from the apertures.", "In accordance with a specially preferred embodiment of this further development, these channels are furthermore dimensioned such that cells contained in a liquid flowing through said channels will move either individually (one after the other) or in some other arrangement through said channels.", "Hence, such channels are suitable for moving cells horizontally to the chip surface from the periphery of the biochip accurately over and across the apertures in such a way that, when a vacuum is applied through an aperture, this will immediately have the effect that the respective cell on top of said aperture will be sucked in.", "The above-described biochips can be produced in a simple way.", "Fundamentally, the following steps are common to all methods: providing a substrate, forming one or a plurality of window portions in said substrate, and forming one opening per window portion.", "In the case of a biochip on the basis of a semiconductor substrate with an insulating layer, it will be advantageous to use the following method for forming the window portion: an insulating layer, which is provided on the upper and on the lower side and which is resistant to the wet-chemical etching method (especially KOH), is removed on the lower side in a lithographically defined area by a dry-etching step, whereby the semiconductor substrate will be exposed directly in this area.", "The following wet-chemical etch step (especially KOH) then causes, by anisotropic etching, the formation of an etch trench having the form of an inverse pyramid.", "If the primary exposed substrate surface is sufficiently large, this etch trench can extend up to the opposite side, but due to the insulating layer provided on said opposite side, which is resistant to the wet-chemical etchant and acts therefore as an etch-stop layer, the trench will remain closed on one side in any case.", "This permits a precise implementation of a rectangular window portion in a very simple manner, the area of said window portion depending on the area of the substrate exposed on the lower side in the first step.", "Layers which proved to be advantageous as an etch-stop or insulating layer are especially an Si3Nx layer, preferably an Si3N4 layer, an SiO2 layer, or Si3Nx/SiO2 multi-layer systems.", "Finally, the opening itself can be formed in the window portion by optical lithography and a dry-etching step.", "This method is suitable for comparatively large openings (≧1 μm).", "If smaller openings, i.e.", "openings down to a size of 10 nm, are to be provided, the opening can be formed e.g.", "by electron-beam lithography and a dry-etching step.", "According to a preferred alternative, the opening can be formed by means of a focussed ion beam.", "When the biochip is implemented on a glass substrate or on a quartz substrate, an isotropic HF etching method can be used for defining the window portion by local thinning of the glass substrate.", "Likewise, the window portion can alternatively be formed by ablation with a laser having a suitable wavelength or by hot shaping (hot pressing).", "The actual opening can be formed in the window by lithography in combination with a dry-etching step on the one hand.", "In the case of these substrate materials, the aperture can also be produced by etching by means of a latent track of a single high-energy ion which has passed through the thinned window area.", "On the other hand, it also possible to form, according to a preferred embodiment, the aperture in the thinned window portion by ablation with a laser having a suitable wavelength.", "For this purpose, it will be particularly advantageous to use an excimer laser having a wavelength in the ultraviolet region.", "Especially when the substrate in the window portion has previously been thinned to a thickness between 10 and 50 μm, apertures having a diameter of less than 10 μm down to less than 1 μm can be produced by irradiation with laser light.", "According to a preferred embodiment of all the above-described biochips, the substrate surface, the edge of the aperture or the inner wall of the aperture can be treated by local heating, e.g.", "by a laser having a suitable wavelength, (so-called tempering), so as to make said substrate surface, said edge of the aperture or said inner wall of the aperture more suitable for close contact with a cell membrane, smooth them, by way of example, or modify the chemical structure of the substrate in a suitable way.", "This can also be done by non-local heating of the whole biochip.", "The temperatures reached during local or non-local heating may be lower as well as higher than the melting point of the respective substrate.", "When the biochip is made from PDMS no etch step will be carried out, since a moulding process is here used, i.e.", "the window portion as well as the openings are transferred from a 3D negative template.", "The etching methods and the lithography methods described are, however, used for producing the negative template.", "All the above-described advantageous further developments can be used for biochips with an opening (M=N=1) as well as for biochips with a plurality of openings (M>1 and/or N>1).", "All the above-described biochips can be used not only for the conventional analyzation of ion channels in membranes but also for a great variety of other purposes.", "The opening or the openings of the biochip can have incorporated therein subareas of the cell membrane of cells (e.g.", "cells isolated from tissues or primary cultures, and cell lines, which express certain ion channels).", "For this purpose, it will be advantageous to position first one cell per aperture.", "In order to do so, singulated (non-coherent) cells in an aqueous suspension are applied to the biochip, the aperture being already filled with an electrolytic solution.", "According to an advantageous embodiment, cells are applied with the aid of at least one pipette or cannula.", "This can be done automatically, e.g.", "by means of electronically controlled xyz motors.", "In a preferred embodiment, a separate pipette or cannula is provided for each aperture.", "According to a further particularly advantageous arrangement, these pipettes or cannulae include integrated electrodes which are suitable for measuring the ion current through ion channels and which are in electric contact with the cuvette and consequently the aperture via the electrolytic solution contained in the pipette or cannula.", "Providing such measuring electrodes on the chip substrate on the receiving side is then no longer necessary.", "If the biochip is provided with channels extending parallel to the substrate surface, as has been described hereinbefore, one or a plurality of singulated cells can be flushed into the biochip through these channels where they can be positioned on a respective opening.", "For positioning a cell on the aperture, a vacuum can be applied from the aperture side located opposite the receiving side so that the resultant flow of fluid will move a cell onto the aperture.", "Alternatively or additionally a constant electric field can be applied via the aperture.", "This will promote the formation of a tight contact between cell and biochip.", "Again alternatively or additionally, direct voltage or alternating voltage fields can be applied through suitable electrodes provided on the biochip; by means of these voltage fields, cells are electrophoretically or dielectrophoretically moved towards the aperture or held in position on said aperture.", "Again alternatively or additionally, surface-acoustic waves produced by further electrodes can be used for positioning cells or liquid drops containing cells on the aperture.", "Again alternatively or additionally, further mechanical, chemical (e.g.", "osmotic or oncotic), electric, magnetic or electromechanical gradients or fields can be applied through the aperture so as to move cells directly or indirectly towards the aperture.", "Alternatively or additionally, further cells or other particles or solutions can be added on the receiving side so as to position cells on the aperture; due to their specific weight or due to other properties, these further cells or particles or solutions will move the cells mechanically and/or by other forces towards the receiving-side surface of the biochip and or towards the aperture and/or fix them there.", "Preferably, all the above-described methods for positioning a cell on an aperture are also used for fixing the cell on the aperture.", "Preferably, an electrophysiological characterization of each cell is carried out by means of the above-described biochips.", "In analogy with the so-called whole-cell voltage clamp known from the field of patch clamp technology, it is also possible to establish contact with the interior of a whole cell via the aperture.", "It will be advantageous to do this by abruptly reducing the pressure in the aperture (suction pulse) for a short time (duration: preferably 10 ms to 10 s, amplitude: preferably −10 to −1000 mmHg), by applying an electric voltage pulse (duration: preferably 0.1 to 1000 ms, amplitude preferably 100 mV to 10 V) or by adding a pore-forming agent (e.g.", "gramicidin or nystatin) for perforating the membrane portion located in the aperture.", "The presence of a cell on top of the aperture can be detected by measuring the conductance or the high-frequency impedance or other electric parameters of the aperture.", "Subsequently, the suction pulse can be triggered, for example.", "According to an advantageous embodiment, an application or de-application of active substances is carried out by flushing in or sucking off a solution.", "Flushing in or sucking off can be effected by pipettes or cannulae.", "If fluid channels exist, they can be used for flushing in or sucking off.", "The application or de-application of active agents can take place prior to or during a measurement.", "Furthermore, all the above-described biochips can be provided with devices which are arranged on the lower side located opposite the receiving side and which permit the simple application of a negative pressure or of an excess pressure relative to the upper side (i.e.", "a pressure gradient through the apertures).", "These devices can be implemented e.g.", "as hollow chambers in a flexible polymer substrate (e.g.", "PDMS), which are filled with liquid and which are located below each of the respective openings and window portions; these hollow chambers are connected to the respective apertures and through said apertures with the upper side of the biochip and their volume can be reduced in size by pressure applied from outside and generated by a mechanical device, and re-enlarged by reducing said pressure.", "The application of a pressure gradient through the apertures can also take place through micro-fluid channels and hose systems communicating with these channels.", "According to another preferred embodiment, one of the biochips described can also be combined with a further second biochip provided with a means for positioning cells relative to the openings of said first biochip, the respective surfaces on the receiving side being located in opposed relationship with and at a fixed or variable distance from one another.", "This combination can be established e.g.", "by a fixed or a flexible connection of the two biochips in such a way that their respective receiving-side surfaces are opposed to one another and are e.g.", "either separated by a gap of 10-1000 μm width or in direct contact with each other.", "It will be advantageous when the means for positioning cells of the second biochip comprises a means for generating surface waves.", "If the biochips are supported in direct contact with each other, the receiving-side surface of the second biochip is provided with fluid channels which extend preferably parallel to the surface and which are open towards the surface.", "In this case, the cells can be flushed in through these fluid channels.", "The biochips according to the present invention can also be used in a measuring probe comprising a glass tube provided on the side of the substrate which is located opposite to the side where the membrane is applicable, the opening of the glass tube facing away from the substrate being implemented such that an electrode can be inserted therein.", "This offers the possibility of moving an electrode in an ionic solution towards the opening for analyzing the ion channel or the ion channels.", "Alternatively, a holding device consisting of polycarbonate or of some other material apart from glass can be provided instead of a glass tube; this holding device can be provided with a central cavity or a plurality of cavities, which communicate with the aperture or the apertures of the biochip and to which the biochip is adhesively attached or fixed in some other way, an electrode or a plurality of electrodes in an ionic solution being adapted to be inserted in said holding device.", "At these cavities communicating with the apertures of the biochip, means can again be provided, which permit the application of an excess pressure or of a negative pressure so that cells originating from a suspension applied on the receiving side can be kept away from or sucked into the aperture.", "In particular, the biochip and the device including the cavities can have provided between them a layer of a flexible polymer substrate (e.g.", "PBMS) so as to guarantee a tight seal.", "In this way, a means is obtained in which the above described biochip can easily be integrated.", "Especially, it will also easily be possible to integrate this measuring probe in known patch clamp set-ups, especially in upright and inverted optical microscopes and measurement sites for optical and mechanical scanning probe methods.", "In accordance with an advantageous embodiment, the opening of the glass tube or of the holding device facing away from the substrate can, in such a measuring probe, be implemented such that an electrode means can be screwed into said opening.", "In this kind of arrangement, the electrode means can be replaced rapidly and can, moreover, be reused.", "This arrangement is suitable for use e.g.", "with a biochip having integrated electrodes only on the upper side of the chip.", "The measuring probe can, in an expedient manner, also be sold together with the screw-in electrode.", "In such an arrangement it will be expedient to provide sealing means, e.g.", "O-rings, between the opening of the glass tube and the screw-in electrode so as to retain the electrolyte in the glass tube or holding device.", "According to an advantageous embodiment, the glass tube or the holding device is adapted to be adhesively attached to the substrate or to be screw-fastened to the substrate making use of a sealing ring.", "A simple and tight connection between the glass tube and the substrate can be guaranteed in this way.", "Fastening by means of screwing according to the second alternative additionally leads to a simple re-usability of the biochip, since it permits aggressive cleaning of the biochip.", "The above-described measuring probes can advantageously be implemented in such a way that they comprise a means for generating a vacuum in the glass tube or the holding device.", "With the aid of this means, a membrane patch of a cell, which is also in solution, can be defined by the usual suction technique.", "This means that all the steps required for carrying out an analysis of ion channels can be executed at a single device.", "This leads to improved handling properties of the device.", "In the following, special embodiments of the present invention will be explained making reference to the drawing enclosed, in which: FIG.", "1a shows a sectional view of a first embodiment of a biochip according to the present invention; FIG.", "1b shows a top view of said first embodiment of a biochip according to the present invention; FIG.", "1c shows a top view of a modification of the first embodiment of a biochip according to the present invention; FIG.", "2 shows a second embodiment of the biochip according to the present invention; FIG.", "3 shows a third embodiment of the biochip according to the present invention; FIG.", "4 shows an embodiment of the measuring probe according to the present invention; and FIG.", "5 shows a pipette for analyzing ion channels according to the prior art.", "FIG.", "1a and 1b show a first embodiment 1 of a biochip according to the present invention.", "This biochip comprises a substrate formed with an opening 19 for receiving therein a cell membrane which comprises at least one ion channel.", "In the present case, the biochip is shown with M=N=1.The substrate comprises a base portion 10 with a first thickness d, and a window portion 11 with a second thickness d2 in which the opening 19 is provided.", "The thickness of the base portion 10 ranges from 1 mm to 100 μm and the thickness of the window portion ranges from 1 μm to 50 nm.", "The window portion has an area of a few 10 μm2 to 0.1 mm2.The opening 19 is substantially circular and has a diameter which ranges from 10 μm to 10 nm.", "The size of the opening is determined by the number of ion channels which are to be analyzed in a cell membrane.", "The biochip 1 consists of a (0001) quartz (Z cut) in which the window portion 11 is first formed by an anisotropic wet-chemical etch step.", "The etchant used for this purpose is HF.", "Depending on the size of the desired opening, said opening is formed in the last step by optical lithography and a dry-etching step or by electron-beam lithography and a dry-etching step.", "Furthermore, the surface of the biochip according to FIG.", "1 is, in the area of the opening, provided with a means for improving the contact between the biochip and the cell membrane.", "In the present case, this means is formed by patterning the surface.", "For this purpose, annular raised portions 15 are provided, which are arranged around the opening.", "These raised portions have the effect that the membrane with an ion channel to be analyzed will be dented, whereby improved adhesion will be achieved due to a hydraulic effect and the electrical sealing resistance will be increased.", "The patterning in the biochip according to FIGS.", "1a and 1b is only exemplary.", "It is especially also possible to use other forms of raised portions, e.g.", "one or a plurality of squares or rectangles, which is or which are arranged around each opening.", "One of these alternatives is shown in FIG.", "1c.", "FIG.", "2 shows a second embodiment of a biochip according to the present invention.", "Also this biochip comprises a substrate 20, 21 formed with an opening 29 for receiving therein a cell membrane which comprises at least one ion channel.", "Also in the case of the biochip shown in FIG.", "2, M=N=1.The geometrical shape and the dimensions of the biochip 2 correspond to those of the biochip 1 shown in FIG.", "1.In order to avoid repetitions, reference is only made to the relevant description of FIG.", "1 in this connection.", "The reference numerals of corresponding parts differ from one another only with respect to their first figure.", "The substrate of the biochip 2 comprises a base portion 20, which is again made from quartz, and an etch-stop layer in which the window portion 21 is formed.", "This etch-stop layer consists of Si3Nx, preferably of Si3N4.A characteristic feature of biochip 2, in comparison with biochip 1 of FIG.", "1, is that it can be produced by a simplified method.", "The substrate 20 has first applied thereto an etch-stop film.", "Subsequently, the window portion 21 is formed from the opposite side up to said etch-stop layer, said window portion being formed by an anisotropic wet-chemical HF etch step.", "Finally, the opening is formed preferably by one of the methods described in connection with the first embodiment.", "FIG.", "3 shows a first embodiment of a biochip 3 according to the present invention.", "With regard to the geometrical dimensions and the structural design, the biochip 3 essentially corresponds to the structural design of the biochips described in FIGS.", "1 and 2 so that reference is here once more made to the description of these chips in order to avoid repetitions.", "The reference numerals of corresponding parts differ from one another only with respect to their first figure.", "In contrast to the biochips shown in these FIGS.", "1 and 2, the base portion 30 of the substrate consists of a semiconductor material, e.g.", "(100)-Si.", "This semiconductor material has applied thereto an insulating layer in which the window portion 31 is formed.", "The insulating layer 31 additionally serves as an etch-stop layer in the production process.", "The production process is therefore similar to that of the biochip produced according to FIG.", "2.In the embodiment shown, this layer consists of Si3N4.In particular, the insulating and etch-stop layer is first applied to the silicon base portion 30 by means of a PECVD method.", "Following this, the window portion 31 is formed in the substrate from the opposite side, said window portion being formed by an anisotropic wet-chemical KOH etch step.", "In so doing, etching is executed up to the etch-stop layer.", "Depending on the desired size of the opening, said opening can then be formed, in a manner corresponding to the above-described embodiments, by optical lithography or electron-beam lithography and a dry-etching step.", "In the last step, the electrodes 32 and 33, which consist here of Ag/AgCl, are applied to the upper and to the lower surface of the substrate.", "In FIG.", "3 it is also shown how a membrane Me with an ion channel I has been introduced in the opening 39.For the subsequent measurement, which will be described in detail with reference to FIG.", "4, an electrolytic liquid 34 must be provided on top of the membrane and the electrode 32 as well as in the etch trench.", "FIG.", "1 to 3 each show biochips with M=N=1.It goes without saying that the statements made hereinbefore also apply to biochips with substrates in which a plurality of openings is provided.", "These openings can be provided in the form of an M×N array.", "In such an array they can be arranged regularly or such that the individual rows are displaced relative to one another.", "The biochips shown in FIG.", "1 to 3 only represent preferred embodiments of the present invention and should not be regarded as a limitation of said invention.", "Hence, a large number of other embodiments, which are not shown, is possible.", "It is, for example, not necessary that the opening is circular.", "It may have different cross-sections, depending on the respective requirements to be satisfied.", "In addition, various materials can be used for forming the biochips.", "It will, for example, be possible to use glass instead of the quartz, and, instead of the silicon, a different semiconductor material, e.g.", "GaAs, may be used.", "Especially in the case of a substrate consisting of a semiconductor material, but not exclusively in the case of such substrates, the surfaces of the substrate may be coated with a passivating layer.", "Furthermore, different kinds of electrodes can be used, e.g.", "electrodes which are suitable for generating an electromagnetic field in the area of the ion channel.", "In addition, electrically and/or optically active and/or passive components can be integrated.", "on the substrate.", "Likewise, various methods which are known to a sufficient extent from the field of semiconductor technology can, in dependence upon the respective materials used, be employed for producing the biochips.", "FIG.", "4 shows a measuring probe according to one embodiment of the present invention.", "This measuring probe includes a substrate comprising a base portion 40 and a window portion 41 in which an opening 49 is formed.", "In addition, a first electrode 42 is arranged on the substrate.", "Below the substrate 40, a holding device 45 is secured in position, which is provided with a central cavity communicating with the opening 49 and which is followed by an electrode 43 with a holder.", "In addition, the measuring probe comprises a means for generating a vacuum in the holding device, said means being designated by reference numeral 46.In addition to the embodiment of the measuring probe which is shown here and which should.", "not be regarded as a limitation of the present invention, further modifications are possible.", "For example, arbitrary ones of the biochips according to the present invention can be used as biochips.", "In particular the dimensions are then determined by the respective field of use, i.e.", "especially by the number of the channels to be analyzed.", "The holding device may e.g.", "secured to the substrate by means of an adhesive.", "The electrode means including the holder can be implemented such that it can be screwed into the holding device from below.", "Furthermore, a sealing ring can be provided between the opening of the holding device and the electrode that can be screwed in.", "In the following, it will be described how ion currents through the ion channel can be measured by the present measuring probe.", "The cell membrane is first applied to the substrate in an electrolytic solution.", "By actuating the vacuum generating means 46, the membrane including the ion channel is sucked into the opening.", "The measuring probe contains an electrolytic solution 44 as well.", "Finally, the current flowing through the ion channel can be measured via the two electrodes 42 and 43." ] ]	Patent_10466018
[ [ "Mammalian tribbles signaling pathways and methods and reagents related thereto", "The invention provides methods and reagents for modulating mitogen activated protein kinase pathways using mammalian tribbles homologs (htrb)." ], [ "1.An isolated htrb-1 encoding nucleic acid comprising a nucleotide sequence which is at least about 90% identical to the nucleotide sequence set forth in SEQ ID No.", "1 or the complement thereof.", "2.The nucleic acid of claim 1, wherein the nucleic acid comprises a nucleotide sequence at least about 95% identical to the nucleotide sequence set forth in SEQ ID No.", "1 or the complement thereof.", "3.The nucleic acid of claim 1, wherein the nucleic acid comprises a nucleotide sequence at least about 99% identical to the nucleotide sequence set forth in SEQ ID No.", "1 or the complement thereof.", "4.The nucleic acid of claim 1, wherein the nucleic acid comprises a nucleotide sequence at least about 95% identical to the nucleotide sequence set forth in SEQ ID No.", "1 or the complement thereof, and encodes an AP-1 inhibitory activity.", "5.The nucleic acid of claim 1, which hybridize to an htrb-1 ORF encoding nucleic acid corresponding to nucleotides 282 to 1400 of SEQ ID No.", "1.6.The isolated nucleic acid of claim 1, which further encodes an htrb polypeptide that is at least about 75% identical to the htrb-1 polypeptide sequence set forth in SEQ ID No.2.7.The isolated nucleic acid of claim 6, which further encodes an AP-1 inhibitory activity.", "8.The isolated nucleic acid of claim 1, wherein the nucleic acid encodes an htrb bioactivity selected from the group consisting of: an inhibition of IL-8 basal expression, an inhibition of AP-1 transcriptional activation, an inhibition of MEKK-1 kinase signaling, an inhibition of MKK-7 kinase signaling, a cellular hypertrophy-promoting activity, an activation of ERK kinase signaling, and an inhibition of JNK kinase signaling.", "9.An isolated nucleic acid comprising a nucleotide sequence that hybridizes under stringent conditions to a htrb-1 nucleotide sequence selected from the group consisting of: nucleotides 283 to 730 of SEQ ID No.", "1; nucleotides 1 to 729 of SEQ ID No.", "1; and nucleotides 1500 to 1916 of SEQ ID No.", "1.10.The nucleic acid of claim 9, which further encodes an htrb polypeptide that is at least about 50% identical to the htrb-1 polypeptide sequence set forth in SEQ ID No.", "2.11.The nucleic acid of claim 10, which further encodes an htrb-1 bioactivity.", "12.The nucleic acid of claim 9, which further includes at least 25 contiguous nucleotides that are identical to said htrb nucleotide sequence.", "13.An isolated nucleic acid that encodes the htrb-1 polypeptide sequence set forth in SEQ ID No.", "2.14.An isolated polypeptide comprising a polypeptide sequence of at least 10 contiguous amino acids from the htrb-1 sequence spanning amino acid residues 1 to 150 of SEQ ID No.", "2.15.The polypeptide of claim 14 comprising at least 20 contiguous amino acids from the htrb-1 sequence spanning amino acid residues 1 to 150 of SEQ ID No.", "2.16.An isolated polypeptide comprising an amino acid sequence that is at least 70% identical to the htrb-1 polypeptide sequence set forth in SEQ ID No.", "2.17.The polypeptide of claim 16, wherein the polypeptide sequence is at least 80% identical to the htrb-1 polypeptide sequence set forth in SEQ ID No.", "2.18.The polypeptide of claim 16, wherein the polypeptide sequence is at least 90% identical to the htrb-1 polypeptide sequence set forth in SEQ ID No.", "2.19.The polypeptide of claim 13, having at least one htrb-1 bioactivity.", "20.The polypeptide of claim 19, wherein the htrb-1 bioactivity is selected from the group consisting of: an inhibition of IL-8 basal expression, an inhibition of AP-1 transcriptional activation, an inhibition of MEKK-1 kinase signaling, an inhibition of MKK-7 kinase signaling, a cellular hypertrophy-promoting activity, an activation of ERK kinase signaling, and an inhibition of JNK kinase signaling.", "21.An isolated htrb-1 polypeptide having the sequence set forth in SEQ ID No.", "2.22.A method of modulating an AP-1 mediated inflammatory signal in a cell comprising providing the cell with a htrb agonist or antagonist.", "23.The method of claim 22 wherein the htrb agonist or antagonist is a htrb polypeptide, a htrb peptidomimetic or a htrb nucleic acid.", "24.The method of claim 23 wherein the htrb agonist or antagonist is selected from the group consisting of: htrb-1, htrb-1ΔN, htrb-1ΔC, htrb-1ΔNΔC, htrb-3, an htrb-1 5′ UTR and N-terminal variable region antisense construct, an htrb-3 5′ UTR and N-terminal variable region antisense construct, an htrb-1 3′UTR sense construct.", "25.The method claim 22, wherein the AP-1 mediated inflammatory signal is selected from the group consisting of: a TNF induced inflammatory signal, and an interleukin induced inflammatory signal.", "26.A method of inhibiting an AP-1 mediated inflammatory signal in a cell comprising contacting the cell with an htrb polypeptide of of claim 14.27.The method of claim 26, wherein the htrb polypeptide is selected from the group consisting of: htrb-1, htrb-1ΔN, htrb-1ΔC, htrb-1ΔNΔC, htrb-3, htrb-3ΔN, htrb-3ΔC, and htrb-3ΔNΔC.", "28.A method of activating an ERK-mediated signal in a cell comprising providing the cell with an htrb agonist activity.", "29.", "(canceled) 30.The method of claim 28, wherein the hrtb agonist activity is provided by a htrb polypeptide selected from the group consisting of: htrb-1, htrb-1ΔN, htrb-1ΔC, htrb-1ΔNΔC, htrb-3, htrb-3ΔN, htrb-3ΔC, and htrb-3ΔNΔC.", "31.The method of claim 28, wherein the ERK-mediated signal is selected from the group consisting of: an AP-1-mediated gene activation signal, an estrogen receptor-mediated gene activation signal, an FGF induced signal, and a PMA induced signal.", "32.A method of identifying an interleukin regulatory gene comprising: (a) transfecting a mammalian reporter cell comprising an interleukin gene reporter with a low-complexity pool of a mammalian cDNA vector library; (b) screening the transfected reporter cell for positive clones by identifying transfected cells with either an increase or decrease in the interleukin gene reporter activity relative to the mammalian reporter cell transfected with the vector alone; and (c) identifying the interleukin regulatory gene from the positive clones by retransfecting the low complexity pool from said positive clones and sequencing the cDNA inserts from the positive clones obtained upon retransfection, thereby identifying an interleukin regulatory gene.", "33.The method of claim 32, wherein the interleukin gene reporter is selected from the group consisting of: an IL-1A gene reporter, an IL-1B gene reporter, an IL-1RN gene reporter, and IL-8 gene reporter.", "34.The method of claim 32, wherein the mammalian cell is selected from the group consisting of: a HeLa cell, an NIH 3T3 cell, a Raw cell, a peripheral blood lymphocyte.", "35.The method of claim 32, wherein the mammalian cDNA library is selected from the group consisting of: a PBMC library, a HeLa cell library, a PMA-induced mammalian cell library, and a cytokine-induced mammalian cell library.", "36.A method of identifying the gene targets of an interleukin regulatory gene in an inflammatory signaling network comprising: (a) expressing an interleukin regulatory gene clone, comprising an interleukin regulatory gene cDNA and an expression vector, in a population of mammalian cells; (b) isolating a population of nucleic acids representing expressed genes from said cells; (c) determining the gene expression profile of the interleukin regulatory gene expressing cells by microarray analysis of the population of nucleic acids representing expressed genes from said cells; and (d) comparing the gene expression pattern of mRNA expression from the cells transfected with the interleukin regulatory gene clone with that obtained by transfecting the vector alone in order to identify genes, other than the said interleukin regulatory gene, which are either up-regulated or down-regulated in the interleukin regulatory gene expressing cells, thereby identifying the gene targets of an interleukin regulatory gene in an inflammatory signaling network.", "37.", "(canceled) 38.The method of claim 36, wherein the mammalian cell is selected from the group consisting of: a HeLa cell, an NIH 3T3 cell, a Raw cell, a peripheral blood lymphocyte.", "39.The method of claim 36, wherein the population of nucleic acids representing expressed genes is an mRNA population.", "40.The method of claim 36, wherein the population of nucleic acids representing expressed genes is a cDNA population.", "41.The method of claim 36, wherein the microarray analysis provides a gene transcription profile or gene expression fingerprint." ], [ "<SOH> 1.BACKGROUND OF THE INVENTION <EOH>The function of immune and inflammatory genes play a central role in the pathology of many diseases including rheumatoid arthritis, inflammatory bowel disorder, psoriasis, and Alzheimer's disease.", "There is evidence to suggest that these immune and inflammatory genes function as a complex network of interdependent signaling components.", "These signaling components mediate signaling events which take place both extracellularly (e.g.", "through the action of various cytokines such as the interleukins) and intracellulary (e.g.", "through the action of signal transducing kinases and transcriptional regulators such as AP-1 and NF-B).", "A variety of means exist for regulating inflammatory responses involved in disease processes.", "For example, aspirin (salicylic acid) inhibit activation of NF-kB by blocking I-kB kinase, a key enzyme in NF-kB activation.", "Sulfasalazine and gold compounds also inhibit NF-kB activation.", "Glucocorticoids suppress expression of inflammatory genes by binding glucocorticoid receptors involved in NF-kB activation.", "Such drugs are commonly used to regulate inflammatory diseases such as rheumatoid arthritis.", "Nevertheless there is a need to develop drugs with particular anti-inflammatory specificities that are particularly adapted to controlling certain aberrant inflammatory processes while allowing nonpathological inflammatory processes to continue without interference.", "Indeed, a variety of anti-inflammatory medicaments would benefit the development of optimized drug treatments for specific patients with particular needs (see Davies & Skjodt (2000) Clin Pharmoacokinet 38: 377-92).", "Furthermore, continuing advances in understanding the molecular mechanisms of inflammation will benefit the development of more effective, more specific and less toxic drugs to control inflammatory diseases.", "An understanding of the relationships between the genes comprising this network would provide a broad array of drug targets for the control of autoimmune and inflammatory disease processes.", "Molecular agonists and antagonists could be designed to act alone or in concert at one or more points in this gene network in order to effect control of the disease process.", "A large body of work has recently been focused on signalling networks triggered by proinflammatory cytokines, bacterial cell walls and shear stress.", "In general, two major signalling cascades are activated by these stimuli.", "The major intracellular signaling cascades involved in immune and inflammatory gene network regulation are the mitogen activated protein kinase (MAPK, or stress kinase) cascade and the IkB kinase cascade as well as the JAK/STAT signal transduction pathway (see e.g.", "Rivest et al.", "(2000) Proc Soc Exp Biol Med 223: 22-38).", "Activation of NF-kB is thought to be mediated primarily via I-kB kinase (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also termed Jun kinases or JNKs).", "IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma.", "The latter protein is essential for activation of the IKKs, but its mechanism of action is not known, although the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells, may prove informative (see Leonardi et al.", "(2000) PNAS, USA 97: 10494-9).", "When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter.", "Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs.", "CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules and CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.", "Individual stress kinase mediated pathways behave differently in different cell types.", "Specificity may be achieved in part by cell type specific expression of certain pathway components such as CIKS.", "It is important that all components contributing to the regulation and specificity of these mitogen activated protein kinase signalling pathways be identified as each represents a target for regulation of this important class of inflammatory signalling events." ], [ "<SOH> 2.SUMMARY OF THE INVENTION <EOH>The invention is based in part upon the cloning and identification of certain mammalian htrbs genes and encoded htrbs proteins which function as inhibitors of particular stress kinase pathways.", "The htrbs genes are mammalian homologs of the Drosophila tribbles gene, which coordinates mitosis with morphogenesis and cell fate determination in fruit fly development (see Mata et al.", "(2000) Cell 101: 511-22; and Grosshans & Wieschaus (2000) Cell 101: 523-31).", "The invention provides a human homolog of the Drosophila tribbles gene which has been termed htrb-1, for human tribbles homologue-1 (also known as homo SKIP1 (Gen Bank Accession NO.", "AF250310).", "The htrb-1 inhibits basal but not induced activity of the cytokine responsive interleukin-1 (IL-8) gene reporter, which is responsive to both NF-kB and AP-1 induction through binding sites for these transcriptional activators present in the IL-8 promoter.", "The htrb-1 gene of the invention specifically represses AP-1 but not NF-kB or JAK/STAT mediated transcriptional induction.", "Therefore the htrb-1 gene of the invention provides a convenient and a specific tool for modulating stress kinase-induced pathways.", "In preferred embodiments, the invention provides an isolated htrb-1 encoding nucleic acid comprising a nucleotide sequence which is at least about 90% identical to the nucleotide sequence set forth in SEQ ID No.", "1 or the complement thereof and more preferably at least about 95% or 99% identical.", "In certain embodiments, the nucleic acid of the invention further an AP-1 activation inhibitory activity.", "The invention further provides isolated nucleic acid which encodes an htrb polypeptide, such as a polypeptide that is at least about 75% identical to the htrb-1 polypeptide sequence set forth in SEQ ID No.", "2, and which, preferably, further encodes an AP-1 inhibitory activity.", "In certain preferred embodiments, the isolated nucleic acid of the invention encodes an htrb bioactivity such as an IL-8 basal expression inhibitory activity, and AP-1 transcriptional activation inhibitory activity, an MEKK-1 kinase signaling inhibitory activity, an MKK-7 kinase signaling inhibitory activity, an ERK kinase signaling inhibitory activity, a JNK kinase signaling inhibitory activity, or a cellular hypertrophy-promoting activity.", "Preferred nucleic acids of the invention include isolated nucleic acid with a nucleotide sequence that hybridizes under stringent conditions to certain particular htrb-1 nucleotide sequences such as: nucleotides 1 to 448 of SEQ ID No.", "1; nucleotides 1 to 729 of SEQ ID No.", "1; and nucleotides 1500 to 1916 of SEQ ID No.", "1.The invention further provides isolated htrb polypeptides which include a polypeptide sequence of at least 10, and, more preferably, 20 or 30 contiguous amino acids from the htrb-1 sequence spanning amino acid residues 1 to 150 of SEQ ID No.", "2.Preferably the htrb polypeptides of the invention are at least about 70%, and, more preferably 80, 90 95 or 99% identical to the htrb-1 sequence set forth in SEQ ID No.", "2.In preferred embodiments, the htrb polypeptide encodes an htrb-1 bioactivity such as an ability to: inhibit IL-8 basal expression, inhibit AP-1 transcriptional activation, inhibit MEKK-1 kinase signaling, inhibit MKK-7 kinase signaling, inhibit ERK kinase signaling, inhibit JNK kinase signaling, or promote cellular hypertrophy.", "In particularly preferred embodiments, the invention provides methods of modulating an AP-1 mediated inflammatory signal in a cell by providing the cell with a htrb agonist or antagonist.", "The htrb agonist or antagonist can be an htrb polypeptide, an htrb peptidomimetic or an htrb nucleic acid.", "Preferred htrb nucleic acid agonist or antagonists of the invention include htrb-1, htrb-1 N htrb-1 C, htrb-1 N C, htrb-3, an htrb-1 5′ UTR and N-terminal variable region antisense construct, an htrb-3 5′ UTR and N-terminal variable region antisense construct, and an htrb-1 3′UTR sense construct.", "Preferred htrb polypeptide agonist or antagonists of the invention include htrb-1, htrb-1 N htrb-1 C, htrb-1 N C, htrb-3, htrb-3 N htrb-3 C, and htrb-3 N C. The method of the invention may be used to inhibit an AP-1 mediated inflammatory signal such as a TNF induced inflammatory signal, or an interleukin induced inflammatory signal.", "The method of the invention further provides a method of activating an ERK-mediated signal in a cell by providing the cell with an htrb agonist activity.", "The ERK-mediated signal may be, for example, an AP-1-mediated gene activation signal, an estrogen receptor-mediated gene activation signal, an FGF induced signal, or a PMA induced signal.", "A particularly preferred embodiment of the invention provides a method of identifying an interleukin regulatory gene by a particular cloning process.", "The process of the invention includes: (1) transfecting a mammalian reporter cell comprising an interleukin gene or inflammatory gene reporter with a low-complexity pool of a mammalian cDNA vector library; (2) screening the transfected reporter cell for positive clones by identifying transfected cells with either an increase or decrease in the interleukin gene reporter activity relative to the mammalian reporter cell transfected with the vector alone; and (3) identifying the interleukin regulatory gene from the positive clones by retransfecting the low complexity pool from said positive clones and sequencing the cDNA inserts from the positive clones obtained upon retransfection, so as to identify an interleukin regulatory gene.", "The interleukin or inflammatory gene reporter may be an IL-1A gene reporter, an IL-1B gene reporter, an IL-1RN gene reporter, or an IL-8 gene reporter.", "Preferred cells for use in this aspect of the invention include mammalian cells such as HeLa cells, NIH 3T3 cells, Raw cells, or peripheral blood lymphocytes.", "Preferred libraries for use in this aspect of the invention includes mammalian cDNA libraries such as PBMC libraries, HeLa cell libraries, PMA-induced mammalian cell libraries, or a mammalian cell library constructed from another cytokine-induced mammalian cell such as an IL-5, TGF-beta, interferon-alpha, or IL-12 induced mammalian cell.", "In preferred embodiments of this aspect of the invention, an interative doing process is used to derive still other inflammatory regulatory network genes.", "This process of the invention involves: first expressing an interleukin regulatory gene clone, comprising an interleukin regulatory gene cDNA and an expression vector, in a population of mammalian cells; next isolating a population of nucleic acids representing expressed genes from said cells; determining the gene expression profile of the interleukin regulatory gene expressing cells by microarray analysis of the population of nucleic acids representing expressed genes from said cells; and then comparing the gene expression pattern of mRNA expression from the cells transfected with the interleukin regulatory gene clone with that obtained by transfecting the vector alone in order to identify genes, other than the said interleukin regulatory gene, which are either up-regulated or down-regulated in the interleukin regulatory gene expressing cells, so as to identify the other gene targets of the interleukin regulatory gene making up the inflammatory signaling network.", "In preferred embodiments the method assesses altered expression of responsive genes by microarray analysis such as gene transcription profiling or gene expression fingerprinting." ], [ "1.BACKGROUND OF THE INVENTION The function of immune and inflammatory genes play a central role in the pathology of many diseases including rheumatoid arthritis, inflammatory bowel disorder, psoriasis, and Alzheimer's disease.", "There is evidence to suggest that these immune and inflammatory genes function as a complex network of interdependent signaling components.", "These signaling components mediate signaling events which take place both extracellularly (e.g.", "through the action of various cytokines such as the interleukins) and intracellulary (e.g.", "through the action of signal transducing kinases and transcriptional regulators such as AP-1 and NF-B).", "A variety of means exist for regulating inflammatory responses involved in disease processes.", "For example, aspirin (salicylic acid) inhibit activation of NF-kB by blocking I-kB kinase, a key enzyme in NF-kB activation.", "Sulfasalazine and gold compounds also inhibit NF-kB activation.", "Glucocorticoids suppress expression of inflammatory genes by binding glucocorticoid receptors involved in NF-kB activation.", "Such drugs are commonly used to regulate inflammatory diseases such as rheumatoid arthritis.", "Nevertheless there is a need to develop drugs with particular anti-inflammatory specificities that are particularly adapted to controlling certain aberrant inflammatory processes while allowing nonpathological inflammatory processes to continue without interference.", "Indeed, a variety of anti-inflammatory medicaments would benefit the development of optimized drug treatments for specific patients with particular needs (see Davies & Skjodt (2000) Clin Pharmoacokinet 38: 377-92).", "Furthermore, continuing advances in understanding the molecular mechanisms of inflammation will benefit the development of more effective, more specific and less toxic drugs to control inflammatory diseases.", "An understanding of the relationships between the genes comprising this network would provide a broad array of drug targets for the control of autoimmune and inflammatory disease processes.", "Molecular agonists and antagonists could be designed to act alone or in concert at one or more points in this gene network in order to effect control of the disease process.", "A large body of work has recently been focused on signalling networks triggered by proinflammatory cytokines, bacterial cell walls and shear stress.", "In general, two major signalling cascades are activated by these stimuli.", "The major intracellular signaling cascades involved in immune and inflammatory gene network regulation are the mitogen activated protein kinase (MAPK, or stress kinase) cascade and the IkB kinase cascade as well as the JAK/STAT signal transduction pathway (see e.g.", "Rivest et al.", "(2000) Proc Soc Exp Biol Med 223: 22-38).", "Activation of NF-kB is thought to be mediated primarily via I-kB kinase (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also termed Jun kinases or JNKs).", "IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma.", "The latter protein is essential for activation of the IKKs, but its mechanism of action is not known, although the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells, may prove informative (see Leonardi et al.", "(2000) PNAS, USA 97: 10494-9).", "When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter.", "Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs.", "CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules and CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.", "Individual stress kinase mediated pathways behave differently in different cell types.", "Specificity may be achieved in part by cell type specific expression of certain pathway components such as CIKS.", "It is important that all components contributing to the regulation and specificity of these mitogen activated protein kinase signalling pathways be identified as each represents a target for regulation of this important class of inflammatory signalling events.", "2.SUMMARY OF THE INVENTION The invention is based in part upon the cloning and identification of certain mammalian htrbs genes and encoded htrbs proteins which function as inhibitors of particular stress kinase pathways.", "The htrbs genes are mammalian homologs of the Drosophila tribbles gene, which coordinates mitosis with morphogenesis and cell fate determination in fruit fly development (see Mata et al.", "(2000) Cell 101: 511-22; and Grosshans & Wieschaus (2000) Cell 101: 523-31).", "The invention provides a human homolog of the Drosophila tribbles gene which has been termed htrb-1, for human tribbles homologue-1 (also known as homo SKIP1 (Gen Bank Accession NO.", "AF250310).", "The htrb-1 inhibits basal but not induced activity of the cytokine responsive interleukin-1 (IL-8) gene reporter, which is responsive to both NF-kB and AP-1 induction through binding sites for these transcriptional activators present in the IL-8 promoter.", "The htrb-1 gene of the invention specifically represses AP-1 but not NF-kB or JAK/STAT mediated transcriptional induction.", "Therefore the htrb-1 gene of the invention provides a convenient and a specific tool for modulating stress kinase-induced pathways.", "In preferred embodiments, the invention provides an isolated htrb-1 encoding nucleic acid comprising a nucleotide sequence which is at least about 90% identical to the nucleotide sequence set forth in SEQ ID No.", "1 or the complement thereof and more preferably at least about 95% or 99% identical.", "In certain embodiments, the nucleic acid of the invention further an AP-1 activation inhibitory activity.", "The invention further provides isolated nucleic acid which encodes an htrb polypeptide, such as a polypeptide that is at least about 75% identical to the htrb-1 polypeptide sequence set forth in SEQ ID No.", "2, and which, preferably, further encodes an AP-1 inhibitory activity.", "In certain preferred embodiments, the isolated nucleic acid of the invention encodes an htrb bioactivity such as an IL-8 basal expression inhibitory activity, and AP-1 transcriptional activation inhibitory activity, an MEKK-1 kinase signaling inhibitory activity, an MKK-7 kinase signaling inhibitory activity, an ERK kinase signaling inhibitory activity, a JNK kinase signaling inhibitory activity, or a cellular hypertrophy-promoting activity.", "Preferred nucleic acids of the invention include isolated nucleic acid with a nucleotide sequence that hybridizes under stringent conditions to certain particular htrb-1 nucleotide sequences such as: nucleotides 1 to 448 of SEQ ID No.", "1; nucleotides 1 to 729 of SEQ ID No.", "1; and nucleotides 1500 to 1916 of SEQ ID No.", "1.The invention further provides isolated htrb polypeptides which include a polypeptide sequence of at least 10, and, more preferably, 20 or 30 contiguous amino acids from the htrb-1 sequence spanning amino acid residues 1 to 150 of SEQ ID No.", "2.Preferably the htrb polypeptides of the invention are at least about 70%, and, more preferably 80, 90 95 or 99% identical to the htrb-1 sequence set forth in SEQ ID No.", "2.In preferred embodiments, the htrb polypeptide encodes an htrb-1 bioactivity such as an ability to: inhibit IL-8 basal expression, inhibit AP-1 transcriptional activation, inhibit MEKK-1 kinase signaling, inhibit MKK-7 kinase signaling, inhibit ERK kinase signaling, inhibit JNK kinase signaling, or promote cellular hypertrophy.", "In particularly preferred embodiments, the invention provides methods of modulating an AP-1 mediated inflammatory signal in a cell by providing the cell with a htrb agonist or antagonist.", "The htrb agonist or antagonist can be an htrb polypeptide, an htrb peptidomimetic or an htrb nucleic acid.", "Preferred htrb nucleic acid agonist or antagonists of the invention include htrb-1, htrb-1 N htrb-1 C, htrb-1 N C, htrb-3, an htrb-1 5′ UTR and N-terminal variable region antisense construct, an htrb-3 5′ UTR and N-terminal variable region antisense construct, and an htrb-1 3′UTR sense construct.", "Preferred htrb polypeptide agonist or antagonists of the invention include htrb-1, htrb-1 N htrb-1 C, htrb-1 N C, htrb-3, htrb-3 N htrb-3 C, and htrb-3 N C. The method of the invention may be used to inhibit an AP-1 mediated inflammatory signal such as a TNF induced inflammatory signal, or an interleukin induced inflammatory signal.", "The method of the invention further provides a method of activating an ERK-mediated signal in a cell by providing the cell with an htrb agonist activity.", "The ERK-mediated signal may be, for example, an AP-1-mediated gene activation signal, an estrogen receptor-mediated gene activation signal, an FGF induced signal, or a PMA induced signal.", "A particularly preferred embodiment of the invention provides a method of identifying an interleukin regulatory gene by a particular cloning process.", "The process of the invention includes: (1) transfecting a mammalian reporter cell comprising an interleukin gene or inflammatory gene reporter with a low-complexity pool of a mammalian cDNA vector library; (2) screening the transfected reporter cell for positive clones by identifying transfected cells with either an increase or decrease in the interleukin gene reporter activity relative to the mammalian reporter cell transfected with the vector alone; and (3) identifying the interleukin regulatory gene from the positive clones by retransfecting the low complexity pool from said positive clones and sequencing the cDNA inserts from the positive clones obtained upon retransfection, so as to identify an interleukin regulatory gene.", "The interleukin or inflammatory gene reporter may be an IL-1A gene reporter, an IL-1B gene reporter, an IL-1RN gene reporter, or an IL-8 gene reporter.", "Preferred cells for use in this aspect of the invention include mammalian cells such as HeLa cells, NIH 3T3 cells, Raw cells, or peripheral blood lymphocytes.", "Preferred libraries for use in this aspect of the invention includes mammalian cDNA libraries such as PBMC libraries, HeLa cell libraries, PMA-induced mammalian cell libraries, or a mammalian cell library constructed from another cytokine-induced mammalian cell such as an IL-5, TGF-beta, interferon-alpha, or IL-12 induced mammalian cell.", "In preferred embodiments of this aspect of the invention, an interative doing process is used to derive still other inflammatory regulatory network genes.", "This process of the invention involves: first expressing an interleukin regulatory gene clone, comprising an interleukin regulatory gene cDNA and an expression vector, in a population of mammalian cells; next isolating a population of nucleic acids representing expressed genes from said cells; determining the gene expression profile of the interleukin regulatory gene expressing cells by microarray analysis of the population of nucleic acids representing expressed genes from said cells; and then comparing the gene expression pattern of mRNA expression from the cells transfected with the interleukin regulatory gene clone with that obtained by transfecting the vector alone in order to identify genes, other than the said interleukin regulatory gene, which are either up-regulated or down-regulated in the interleukin regulatory gene expressing cells, so as to identify the other gene targets of the interleukin regulatory gene making up the inflammatory signaling network.", "In preferred embodiments the method assesses altered expression of responsive genes by microarray analysis such as gene transcription profiling or gene expression fingerprinting.", "3.BRIEF DESCRIPTION OF THE FIGURES FIG.", "1 shows htrb-1 mediated repression of basal, but not IL-1 beta- or TNF alpha- activated, expression of IL-8 reporter (panel A); the specific repression of AP-1 activation (panel B) but not NF-kB activation (panel C) by htrb-1; a comparison of htrb-1 and htrb-3 mediated repression of AP -1 (panel E); the repression of MEKK-1 mediated AP-1 activation (squares, horizontal axis) by htrb-1 (triangles); and htrb-3 repression of p38-mediated activation.", "FIG.", "2 shows an alignment of htrb-1, htrb-3 and related tribbles gene sequences FIG.", "3 shows expression of antisense htrb-1 and htrb-3 RNA inhibits stress kinase activation.", "FIG.", "4 shows that htrb-1 inhibits MEKK-1 and MKK7 mediated AP-1 activation.", "FIG.", "5 shows that htrb-1 and htrb-3 alter c-Jun and ERK phosphorylation kinetics.", "FIG.", "6 shows deletion analysis of htrb-1.FIG.", "7 shows that htrb genes are expressed and act in a tissue-specific manner.", "FIG.", "8 shows the detection of htrb-1 and htrb-3 expression by confocal microscopy.", "FIG.", "9 shows the titration of sense vs. antisense htrb-3.FIG.", "10 shows the nucleic acid (panel A) and polypeptide sequence of htrb-1 (GenBank Accession No.", "AF250310).", "FIG.", "11 shows the nucleic acid (panel A) and polypeptide sequence of htrb-3 (GenBank Accession No.", "AF250311).", "Table 1 shows that htrb-3 inhibits AP-1, but not NF-kB, induction by a variety of cytokines.", "4.DETAILED DESCRIPTION OF THE INVENTION 4.1.General The invention is based in part upon the cloning and identification of certain mammalian htrbs genes and encoded htrbs proteins which function as inhibitors of particular stress kinase pathways.", "The htrbs genes are mammalian homologs of the Drosophila tribbles gene, which coordinates mitosis with morphogenesis and cell fate determination in fruit fly development (see Mata et al.", "(2000) Cell 101: 511-22; and Grosshans & Wieschaus (2000) Cell 101: 523-31).", "The invention provides a human homolog of the Drosophila tribbles gene which has been termed htrb-1, for human tribbles homologue-1.The htrb-1 gene is also known as the human SKIP1 gene and the nucleic acid sequence (SEQ ID NO.", "1) and corresponding polypeptide sequence (SEQ ID NO.", "2) are described in GenBank Accession NO.", "AF250310 and shown in FIG.", "10.SEQ ID NO.", "1 nucleotides 282 to 1400 correspond to the ORF of the htrb-1 gene which encode the htrb-1 polypeptide (SEQ ID NO.", "2).", "The htrb-1 gene inhibits basal but not induced activity of the cytokine responsive interleukin-1 (IL-8) gene reporter, which is responsive to both NF-kB and AP-1 induction through binding sites for these transcriptional activators present in the IL-8 promoter.", "The htrb-1 gene of the invention specifically represses AP-1 but not NF-kB or JAK/STAT mediated transcriptional induction.", "Therefore the htrb-1 gene of the invention provides a convenient and a specific tool for modulating stress kinase-induced pathways.", "Mitogen Activated Protein Kinase cascades are activated by a wide variety of extracellular stimuli.", "However, the individual pathways respond differently in different cell types.", "This specificity is achieved partially by cell type specific expression of certain pathway components.", "The instant invention provides a family of mammalian tribbles homologs (htrbs) which influence the activity of MAPK pathways.", "Expression of these proteins is tightly regulated (Mayumi-Matsuda, K. et al.", "(1999) Biochemical and Biophysical Research Communications 258:260-64; Wilkin, F. et al.", "(1997) European Journal of Biochemistry 248:660-68).", "Overexpression of htrbs or suppression of endogenous levels leads to the inhibition of a stress kinase responsive reporter, suggesting that these proteins represent a rate-limiting component of MAPK pathways.", "The mechanism of htrb action involves control of phosphorylation of extracellular signal regulated kinases (ERKs), which is strongly potentiated by elevated htrb levels.", "In addition, optimal activation of ERK, JNK and p38 responsive reporters by upstream kinases is achieved in the presence of different amounts of htrb, suggesting that selective activation of individual MAPK pathways occurs by regulation of htrb expression.", "There is an increasing body of experimental evidence, supported by mathematical models, (Levchenko, A. et al.", "(2000) Proc.", "Natl.", "Acad.", "Sci.", "USA 97:5818-23), to suggest, that regulation of the expression levels of scaffolding components is an effective way to limit the amplitude of signalling responses (e.g.", "Cacace, A.M. et al.", "(1999) Molecular and Cellular Biology 19:229-40; Yasuda, J. et al.", "(1999) Mol.", "Cell.", "Biol.", "19:7245-54).", "Based on our observations we suggest a model where htrb proteins are scaffolds, regulating the dynamics of MAPK mediated cellular responses.", "The invention further provides htrb-2 and htrb-3 genes, which are 41% and 51% identical, respectively, to the htrb-1 gene.", "The htrb-3 gene is also known as the human SKIP3 gene and the nucleic acid sequence (SEQ ID NO.", "3) and corresponding polypeptide sequence (SEQ ID NO.", "4) are described in GenBank Accession NO.", "AF250311 and shown in FIG.", "11.Nucleotides 1 to 1083 of SEQ ID NO.", "3 correspond to the ORF of htrb-3 and, accordingly, encode the htrb-3 polypeptide corresponding to SEQ ID No.", "4.The invention still further provides methods of using these compositions to control inflammatory signaling.", "The invention further provides methods for the cloning and identification of other genes that are components of an inflammatory signaling network.", "In preferred embodiments, this method includes screening of low-complexity pools of cDNA for genes which effect an increase or a decrease in the basal or induced levels of an interleukin gene reporter.", "In certain embodiments, the method of the invention involves expression screening for gene products which modulate the activity of a cytokine responsive reporter construct, such as an interleukin 8 (IL-8) reporter construct.", "Interleukin (IL)-8 is a C-X-C chemokine that plays an important role in acute inflammation through its G protein-coupled receptors CXCR1 and CXCR2.In particularly preferred embodiments, further components of the gene network are cloned and identified by an iterative process involving micro-array analysis with the interleukin regulatory genes thus identified.", "The goals of these methods of biotechnological business include the identification of a broad array of drug targets for the control of autoimmune and inflammatory disease processes.", "A further goal includes the design of molecular agonists and antagonists which may act alone or in concert at any one or more points in the inflammatory gene network to effect control of an autoimmune or inflammatory disease process.", "4.2.Definitions For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below.", "The term “aberrant activity”, as applied to an activity of a polypeptide such as htrb, refers to an activity which differs from the activity of the wild-type or native polypeptide or which differs from the activity of the polypeptide in a healthy subject.", "An activity of a polypeptide can be aberrant because it is stronger than the activity of its native counterpart.", "Alternatively, an activity can be aberrant because it is weaker or absent relative to the activity of its native counterpart.", "An aberrant activity can also be a change in an activity.", "For example an aberrant polypeptide can interact with a different target peptide.", "A cell can have an aberrant htrb activity due to overexpression or underexpression of the gene encoding htrb.", "The term “agonist”, as used herein, is meant to refer to an agent that mimics or upregulates (e.g.", "potentiates or supplements) an htrb bioactivity.", "An htrb agonist can be a wild-type htrb protein or derivative thereof having at least one bioactivity of the wild-type htrb , e.g.", "an AP-1 activation inhibitory activity.", "An htrb therapeutic can also be a compound that upregulates expression of an htrb gene or which increases at least one bioactivity of an htrb protein.", "An agonist can also be a compound which increases the interaction of an htrb polypeptide with another molecule, e.g, an IL-1 type I or type II receptor.", "Such an agonist may, for example, increase the binding of htrb to a MAPK, thereby promoting an htrb -dependent blocking of a MAPK mediated inflammatory signal.", "Alternatively, an htrb agonist may increase the binding of htrb to an MAPK.", "The term “allele”, which is used interchangeably herein with “allelic variant” refers to alternative forms of a gene or portions thereof.", "Alleles occupy the same locus or position on homologous chromosomes.", "When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele.", "When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene.", "Alleles of a specific gene can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides.", "Frequently occurring sequence variations include transition mutations (i.e.", "purine to purine substitutions and pyrimidine to pyrimidine substitutions, e.g.", "A to G or C to T), transversion mutations (i.e.", "purine to pyrimidine and pyrimidine to purine substitutions, e.g.", "A to T or C to G), and alteration in repetitive DNA sequences (e.g.", "expansions and contractions of trinucleotide repeat and other tandem repeat sequences).", "An allele of a gene can also be a form of a gene containing a mutation.", "The term “allelic variant of a polymorphic region of an htrb gene” refers to a region of an htrb gene having one or several nucleotide sequences found in that region of the gene in other individuals.", "“Antagonist” as used herein is meant to refer to an agent that downregulates (e.g.", "suppresses or inhibits) at least one htrb bioactivity.", "An htrb antagonist can be a compound which inhibits or decreases the interaction between an htrb protein and another molecule, e.g., a MAPK.", "An antagonist can also be a compound that down-regulates expression of an htrb gene or which reduces the amount of htrb protein present.", "The htrb antagonist can be a dominant negative form of an htrb polypeptide, e.g., a form of an htrb polypeptide which is capable of interacting with a target peptide.", "The htrb antagonist can also be a nucleic acid encoding a dominant negative form of an htrb polypeptide, an htrb antisense nucleic acid, or a ribozyme capable of interacting specifically with an htrb RNA.", "Yet other htrb antagonists are molecules which bind to an htrb polypeptide and inhibit its action.", "Such molecules include peptides, e.g., forms of htrb target peptides which do not have biological activity, and which inhibit binding to htrb target molecules, such as an AP-1 transcription factor.", "The term “antibody” as used herein is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includes fragments thereof which are also specifically reactive with a vertebrate, e.g., mammalian, protein.", "Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies.", "Thus, the term includes segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein.", "Nonlimiting examples of such proteolytic and/or recombinant fragments include Fab, F(ab′)2, Fab′, Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H] domain joined by a peptide linker.", "The scFv's may be covalently or non-covalently linked to form antibodies having two or more binding sites.", "The subject invention includes polyclonal, monoclonal, or other purified preparations of antibodies and recombinant antibodies.", "A disease, disorder, or condition “associated with” or “characterized by” an aberrant expression of a nucleic acid refers to a disease, disorder, or condition in a subject which is caused by, contributed to by, or causative of an aberrant level of expression of a nucleic acid.", "As used herein the term “bioactive fragment of an htrb polypeptide” refers to a fragment of a full-length htrb polypeptide, wherein the fragment specifically mimics or antagonizes the activity of a wild-type htrb polypeptide.", "The bioactive fragment preferably is a fragment capable of interacting with a MAPK.", "“Biological activity” or “bioactivity” or “activity” or “biological function”, which are used interchangeably, for the purposes herein means an effector or antigenic function that is directly or indirectly performed by an htrb polypeptide (whether in its native or denatured conformation), or by any subsequence thereof.", "Biological activities include binding to a target peptide, e.g., a MAPK, preferably an ERK.", "An htrb bioactivity can be modulated by directly affecting an htrb polypeptide.", "Alternatively, an htrb bioactivity can be modulated by modulating the level of an htrb polypeptide, such as by modulating expression of an htrb gene.", "The term “biomarker” refers a biological molecule, e.g., a nucleic acid, peptide, hormone, etc., whose presence or concentration can be detected and correlated with a known condition, such as a disease state.", "“Cells”, “host cells” or “recombinant host cells” are terms used interchangeably herein.", "It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell.", "Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.", "A “chimeric polypeptide” or “fusion polypeptide” is a fusion of a first amino acid sequence encoding one of the subject htrb polypeptides with a second amino acid sequence defining a domain (e.g.", "polypeptide portion) foreign to and not substantially homologous with any domain of an htrb polypeptide.", "A chimeric polypeptide may present a foreign domain which is found (albeit in a different polypeptide) in an organism which also expresses the first polypeptide, or it may be an “interspecies”, “intergenic”, etc.", "fusion of polypeptide structures expressed by different kinds of organisms.", "In general, a fusion polypeptide can be represented by the general formula X-htrb-Y, wherein htrb represents a portion of the polypeptide which is derived from an htrb polypeptide, and X and Y are independently absent or represent amino acid sequences which are not related to an htrb sequence in an organism, including naturally occurring mutants.", "A “delivery complex” shall mean a targeting means (e.g.", "a molecule that results in higher affinity binding of a gene, protein, polypeptide or peptide to a target cell surface and/or increased cellular or nuclear uptake by a target cell).", "Examples of targeting means include: sterols (e.g.", "cholesterol), lipids (e.g.", "a cationic lipid, virosome or liposome), viruses (e.g.", "adenovirus, adeno-associated virus, and retrovirus) or target cell specific binding agents (e.g.", "ligands recognized by target cell specific receptors).", "Preferred complexes are sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell.", "However, the complex is cleavable under appropriate conditions within the cell so that the gene, protein, polypeptide or peptide is released in a functional form.", "As is well known, genes may exist in single or multiple copies within the genome of an individual.", "Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same activity.", "The term “DNA sequence encoding an htrb polypeptide” may thus refer to one or more genes within a particular individual.", "Moreover, certain differences in nucleotide sequences may exist between individual organisms, which are called alleles.", "Such allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a polypeptide with the same biological activity.", "The term “equivalent” is understood to include nucleotide sequences encoding functionally equivalent polypeptides.", "Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will, therefore, include sequences that differ from the nucleotide sequence of the nucleic acids shown in, for example, SEQ ID Nos.", "1 and 3, due to the degeneracy of the genetic code.", "The term “haplotype” as used herein is intended to refer to a set of alleles that are inherited together as a group (are in linkage disequilibrium) at statistically significant levels (pcorr<0.05).", "As used herein, the phrase “an htrb haplotype” refers to a haplotype in the htrb loci which may include polymorphic variations of htrb gene sequences.", "“Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules.", "Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison.", "When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position.", "A degree of homology or similarity or identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences.", "A degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences.", "A degree of homology or similarity of amino acid sequences is a function of the number of amino acids, i.e.", "structurally related, at positions shared by the amino acid sequences.", "An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the htrb sequences of the present invention.", "As used herein, the term “htrb ” refers to a mammalian homolog of a Drosophila tribbles gene, or an equivalent thereof.", "As used herein, the term htrb is used interchangeably with the term “SKIP)” gene or protein, a second name for htrb based upon its stress kinase inhibitory activity (i.e.", "SKIP from Stress Kinase Inhibitor Protein, wherein htrb-1 is used interchangably with SKIP-1).", "Further as used herein, the htrb-3 gene is also referred to as the SKIP-3 gene.", "The term “htrb nucleic acid” refers to a nucleic acid encoding an htrb protein, such as nucleic acids having SEQ ID Nos.", "1 or 3 or fragments thereof, a complement thereof, and derivatives thereof.", "The terms “htrb polypeptide” and “htrb protein” are intended to encompass polypeptides comprising the amino acid sequence shown as SEQ ID No.", "1 or 3 et al.", "or fragments thereof, and homologs thereof and include agonist and antagonist polypeptides.", "The term “htrb binding partner” or “htrb BP” refers to various cell proteins which bind to an htrb protein.", "The term “htrb therapeutic” refers to various forms of htrb polypeptides, as well as peptidomimetics, nucleic acids, or small molecules, which can modulate at least one activity of an htrb polypeptide, e.g., interaction with an htrb receptor interaction with and/or an htrb coreceptor, by mimicking or potentiating (agonizing) or inhibiting (antagonizing) the effects of a naturally-occurring htrb polypeptide.", "An htrb therapeutic which mimics or potentiates the activity of a wild-type htrb polypeptide is an “htrb agonist”.", "Conversely, an htrb therapeutic which inhibits the activity of a wild-type htrb polypeptide is an “htrb antagonist”.", "“Increased risk” refers to a statistically higher frequency of occurrence of the disease or condition in an individual carrying a particular polymorphic allele in comparison to the frequency of occurrence of the disease or condition in a member of a population that does not carry the particular polymorphic allele.", "The term “interact” as used herein is meant to include detectable relationships or association (e.g.", "biochemical interactions) between molecules, such as interaction between protein-protein, protein-nucleic acid, nucleic acid-nucleic acid, and protein-small molecule or nucleic acid-small molecule in nature.", "The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule.", "For example, an isolated nucleic acid encoding one of the subject htrb polypeptides preferably includes no more than 10 kilobases kb) of nucleic acid sequence which naturally immediately flanks the htrb gene in genomic DNA, more preferably no more than 5 kb of such naturally occurring flanking sequences, and most preferably less than 1.5 kb of such naturally occurring flanking sequence.", "The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.", "Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.", "The term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.", "A “knock-in” transgenic animal refers to an animal that has had a modified gene introduced into its genome and the modified gene can be of exogenous or endogenous origin.", "A “knock-out” transgenic animal refers to an animal in which there is partial or complete suppression of the expression of an endogenous gene (e.g, based on deletion of at least a portion of the gene, replacement of at least a portion of the gene with a second sequence, introduction of stop codons, the mutation of bases encoding critical amino acids, or the removal of an intron junction, etc.).", "In preferred embodimbents, the “knock-out” gene locus corresponding to the modified endogenous gene no longer encodes a functional polypeptide activity and is said to be a “null” allele.", "Accordingly, knock-out transgenic animals of the present invention include those carrying one htrb gene null mutation, such as htrb null allele heterozygous animals, and those carrying two htrb gene null mutations, such as htrb null allele homozygous animals.", "A “knock-out construct” refers to a nucleic acid sequence that can be used to decrease or suppress expression of a protein encoded by endogenous DNA sequences in a cell.", "In a simple example, the knock-out construct is comprised of a gene, such as the htrb gene, with a deletion in a critical portion of the gene so that active protein cannot be expressed therefrom.", "Alternatively, a number of termination codons can be added to the native gene to cause early termination of the protein or an intron junction can be inactivated.", "In a typical knock-out construct, some portion of the gene is replaced with a selectable marker (such as the neo gene) so that the gene can be represented as follows: htrb 5′/neo/htrb 3′, where htrb 5′ and htrb 3′, refer to genomic or cDNA sequences which are, respectively, upstream and downstream relative to a portion of the htrb gene and where neo refers to a neomycin resistance gene.", "In another knock-out construct, a second selectable marker is added in a flanking position so that the gene can be represented as: htrb/neo/htrb/TK, where TK is a thymidine kinase gene which can be added to either the htrb 5′ or the htrb 3′ sequence of the preceding construct and which further can be selected against (i.e.", "is a negative selectable marker) in appropriate media.", "This two-marker construct allows the selection of homologous recombination events, which removes the flanking TK marker, from non-homologous recombination events which typically retain the TK sequences.", "The gene deletion and/or replacement can be from the exons, introns, especially intron junctions, and/or the regulatory regions such as promoters.", "“Linkage disequilIbrium” refers to co-inheritance of two alleles at frequencies greater than would be expected from the separate frequencies of occurrence of each allele in a given control population.", "The expected frequency of occurrence of two alleles that are inherited independently is the frequency of the first allele multiplied by the frequency of the second allele.", "Alleles that co-occur at expected frequencies are said to be in “linkage equilibrium”.", "The cause of linkage disequilibrium is often unclear.", "It can be due to selection for certain allele combinations or to recent admixture of genetically heterogeneous populations.", "In addition, in the case of markers that are very tightly linked to a disease gene, an association of an allele (or group of linked alleles) with the disease gene is expected if the disease mutation occurred in the recent past, so that sufficient time has not elapsed for equilibrium to be achieved through recombination events in the specific chromosomal region.", "When referring to allelic patterns that are comprised of more than one allele, a first allelic pattern is in linkage disequilibrium with a second allelic pattern if all the alleles that comprise the first allelic pattern are in linkage disequilibrium with at least one of the alleles of the second allelic pattern.", "An example of linkage disequilibrium is that which occurs between the alleles at the IL-1RN (+2018) and IL-1RN (VNTR) polymorphic sites.", "The two alleles at IL-1RN (+2018) are 100% in linkage disequilibrium with the two most frequent alleles of IL-1RN (VNTR), which are allele 1 and allele 2.The term “marker” refers to a sequence in the genome that is known to vary among individuals.", "For example, the IL-1RN gene has a marker that consists of a variable number of tandem repeats (VNTR).", "The term “modulation” as used herein refers to both upregulation (i.e., activation or stimulation (e.g., by agonizing or potentiating)) and downregulation (i.e.", "inhibition or suppression (e.g., by antagonizing, decreasing or inhibiting)).", "The term “mutated gene” refers to an allelic form of a gene, which is capable of altering the phenotype of a subject having the mutated gene relative to a subject which does not have the mutated gene.", "If a subject must be homozygous for this mutation to have an altered phenotype, the mutation is said to be recessive.", "If one copy of the mutated gene is sufficient to alter the genotype of the subject, the mutation is said to be dominant.", "If a subject has one copy of the mutated gene and has a phenotype that is intermediate between that of a homozygous and that of a heterozygous subject (for that gene), the mutation is said to be co-dominant.", "The “non-human animals” of the invention include mammalians such as rodents, non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.", "Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse, though transgenic amphibians, such as members of the Xenopus genus, and transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation.", "The term “chimeric animal” is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant gene is expressed in some but not all cells of the animal.", "The term “tissue-specific chimeric animal” indicates that one of the recombinant htrb genes is present and/or expressed or disrupted in some tissues but not others.", "As used herein, the term “nucleic acid” refers to polynucleotides or oligonucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).", "The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.", "The term “nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID No.", "x” refers to the nucleotide sequence of the complementary strand of a nucleic acid strand having SEQ ID No.", "x.", "The term “complementary strand” is used herein interchangeably with the term “complement”.", "The complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non-coding strand.", "When referring to double stranded nucleic acids, the complement of a nucleic acid having SEQ ID No.", "x refers to the complementary strand of the strand having SEQ ID No.", "x or to any nucleic acid having the nucleotide sequence of the complementary strand of SEQ ID No.", "x.", "When referring to a single stranded nucleic acid having the nucleotide sequence SEQ ID No.", "x, the complement of this nucleic acid is a nucleic acid having a nucleotide sequence which is complementary to that of SEQ ID No.", "x.", "The nucleotide sequences and complementary sequences thereof are always given in the 5′ to 3′ direction.", "The term “percent identical” refers to sequence identity between two amino acid sequences or between two nucleotide sequences.", "Identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison.", "When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position.", "Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences.", "Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences.", "Various alignment algorithms and/or programs may be used, including FASTA, BLAST, or ENTREZ.", "FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings.", "ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.", "In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.", "Other techniques for alignment are described in Methods in Enzymology, vol.", "266: Computer Methods for Macromolecular Sequence Analysis (1996), ed.", "Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA.", "Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences.", "The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments.", "See Meth.", "Mol.", "Biol.", "70: 173-187 (1997).", "Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences.", "An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer.", "MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer.", "This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors.", "Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.", "Databases with individual sequences are described in Methods in Enzymology, ed.", "Doolittle, supra.", "Databases include Genbank, EMBL, and DNA Database of Japan (DDBJ).", "Preferred nucleic acids have a sequence at least 70%, and more preferably 80% identical and more preferably 90% and even more preferably at least 95% identical to an nucleic acid sequence of a sequence shown in one of SEQ ID Nos.", "of the invention.", "Nucleic acids at least 90%, more preferably 95%, and most preferably at least about 98-99% identical with a nucleic sequence represented in one of SEQ ID Nos: 1-2 are of course also within the scope of the invention.", "In preferred embodiments, the nucleic acid is mammalian.", "In comparing a new nucleic acid with known sequences, several alignment tools are available.", "Examples include PileUp, which creates a multiple sequence alignment, and is described in Feng et al., J. Mol.", "Evol.", "(1987)25:351-360.Another method, GAP, uses the alignment method of Needleman et al., J. Mol.", "Biol.", "(1970) 48:443-453.GAP is best suited for global alignment of sequences.", "A third method, BestFit, functions by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman, Adv.", "Appl.", "Math.", "(1981) 2:482-489.The term “polymorphism” refers to the coexistence of more than one form of a gene or portion (e.g., allelic variant) thereof.", "A portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a “polymorphic region of a gene”.", "A polymorphic region can be a single nucleotide, the identity of which differs in different alleles.", "A polymorphic region can also be several nucleotides long.", "A “polymorphic gene” refers to a gene having at least one polymorphic region.", "As used herein, the term “promoter” means a DNA sequence that regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in cells.", "The term encompasses “tissue specific” promoters, i.e.", "promoters, which effect expression of the selected DNA sequence only in specific cells (e.g.", "cells of a specific tissue).", "The term also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.", "The term also encompasses non-tissue specific promoters and promoters that constitutively express or that are inducible (i.e.", "expression levels can be controlled).", "The term “propensity to disease,” also “predisposition” or “susceptibility” to disease or any similar phrase, means that certain htrb locus polymorphic alleles are hereby discovered to be associated with or predictive of a particular disease.", "The alleles are thus over-represented in frequency in individuals with disease as compared to healthy individuals.", "Thus, these alleles can be used to predict disease even in pre-symptomatic or pre-diseased individuals.", "The terms “protein”, “polypeptide” and “peptide” are used interchangeably herein when referring to a gene product.", "The term “recombinant protein” refers to a polypeptide of the present invention which is produced by recombinant DNA techniques, wherein generally, DNA encoding an htrb polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.", "Moreover, the phrase “derived from”, with respect to a recombinant htrb gene, is meant to include within the meaning of “recombinant protein” those proteins having an amino acid sequence of a native htrb polypeptide, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions (including truncation) of a naturally occurring form of the polypeptide.", "“Small molecule” as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5 kD and most preferably less than about 4 kD.", "Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules.", "Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention to identify compounds that modulate an htrb bioactivity.", "As used herein, the term “specifically hybridizes” or “specifically detects” refers to the ability of a nucleic acid molecule of the invention to hybridize to at least approximately 6, 12, 20, 30, 50, 100, 150, 200, 300, 350, 400 or 425 consecutive nucleotides of a vertebrate, preferably an htrb gene.", "“Transcriptional regulatory sequence” is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.", "In preferred embodiments, transcription of one of the htrb genes is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended.", "It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring forms of htrb polypeptide.", "As used herein, the term “transfection” means the introduction of a nucleic acid, e.g., via an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.", "“Transformation”, as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of an htrb polypeptide or, in the case of anti-sense expression from the transferred gene, the expression of a naturally-occurring form of the htrb polypeptide is disrupted.", "As used herein, the term “transgene” means a nucleic acid sequence (encoding, e.g., one of the htrb polypeptides, or an antisense transcript thereto) which has been introduced into a cell.", "A transgene could be partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).", "A transgene can also be present in a cell in the form of an episome.", "A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.", "A “transgenic animal” refers to any animal, preferably a non-human mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.", "The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.", "The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.", "This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.", "In the typical transgenic animals described herein, the transgene causes cells to express a recombinant form of one of the htrb polypeptides, e.g.", "either agonistic or antagonistic forms.", "However, transgenic animals in which the recombinant htrb gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below.", "Moreover, “transgenic animal” also includes those recombinant animals in which gene disruption of one or more htrb genes is caused by human intervention, including both recombination and antisense techniques.", "The term “treating” as used herein is intended to encompass curing as well as ameliorating at least one symptom of the condition or disease.", "The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.", "One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.", "Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.", "Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”.", "In general, expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids” which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.", "In the present specification, “plasmid” and “vector” are used interchangeably as the plasmid is the most commonly used form of vector.", "However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.", "The term “wild-type allele” refers to an allele of a gene which, when present in two copies in a subject results in a wild-type phenotype.", "There can be several different wild-type alleles of a specific gene, since certain nucleotide changes in a gene may not affect the phenotype of a subject having two copies of the gene with the nucleotide changes.", "4.3.Nucleic Acids of the Present Invention The invention provides htrb nucleic acids, homologs thereof, and portions thereof.", "Preferred nucleic acids have a sequence at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, and more preferably 85% homologous and more preferably 90% and more preferbly 95% and even more preferably at least 99% homologous with a nucleotide sequence of an htrb gene, e.g., such as a sequence shown in one of SEQ ID Nos: 1 or 3 or complement thereof of the htrb nucleic acids having the GenBank Accession Nos.", ": htrb-1 (AF250310) and htrb-3 (AF250311).", "Nucleic acids at least 90%, more preferably 95%, and most preferably at least about 98-99% identical with a nucleic sequence represented in one of SEQ ID Nos.", "1 or 3 or complement thereof are of course also within the scope of the invention.", "In preferred embodiments, the nucleic acid is mammalian and in particularly preferred embodiments, includes all or a portion of the nucleotide sequence corresponding to the coding region of the nucleic acid set forth in SEQ ID No.", "1 or 3 which correspond to the htrb-1 and htrb-3 ORF respectively.", "The invention further provides an evolutionarily conserved nucleic acid sequence found in the 3′ UTR (untranslated region) of the htrb transcript encoded by both mammalian homologs of the htrb gene, which is described in further detail in the examples which follow.", "The invention also pertains to isolated nucleic acids comprising a nucleotide sequence encoding htrb polypeptides, variants and/or equivalents of such nucleic acids.", "The term equivalent is understood to include nucleotide sequences encoding functionally equivalent htrb polypeptides or functionally equivalent peptides having an activity of an htrb protein such as described herein.", "Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitution, addition or deletion, such as allelic variants; and will, therefore, include sequences that differ from the nucleotide sequence of the htrb gene shown in SEQ ID Nos.", "1, 2, 3, or 4 due to the degeneracy of the genetic code.", "Preferred nucleic acids are vertebrate htrb nucleic acids.", "Particularly preferred vertebrate htrb nucleic acids are mammalian.", "Regardless of species, particularly preferred htrb nucleic acids encode polypeptides that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to an amino acid sequence of a vertebrate htrbprotein.", "In one embodiment, the nucleic acid is a cDNA encoding a polypeptide having at least one bio-activity of the subject htrb polypeptide.", "Preferably, the nucleic acid includes all or a portion of the nucleotide sequence corresponding to the nucleic acid of SEQ ID Nos.", "1 or 3.Still other preferred nucleic acids of the present invention encode an htrb polypeptide which is comprised of at least 2, 5, 10, 25, 50, 100, 150 or 200 amino acid residues.", "For example, such nucleic acids can comprise about 50, 60, 70, 80, 90, or 100 base pairs.", "Also within the scope of the invention are nucleic acid molecules for use as probes/primer or antisense molecules (i.e.", "noncoding nucleic acid molecules), which can comprise at least about 6, 12, 20, 30, 50, 60, 70, 80, 90 or 100 base pairs in length.", "Another aspect of the invention provides a nucleic acid which hybridizes under stringent conditions to a nucleic acid represented by SEQ ID Nos.", "1 or 3 or complement thereof or the nucleic acids having ATCC Designation No.", "XXXXXX or No.", "XXXXXX.", "Appropriate stringency conditions which promote DNA hybridization, for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6 or in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989).", "For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or temperature and salt concentration may be held constant while the other variable is changed.", "In a preferred embodiment, an htrb nucleic acid of the present invention will bind to one of SEQ ID Nos.", "1 or 3 or complement thereof under moderately stringent conditions, for example at about 2.0×SSC and about 40° C. In a particularly preferred embodiment, an htrb nucleic acid of the present invention will bind to one of SEQ ID Nos.", "1 or 3 or complement thereof under high stringency conditions.", "In another particularly preferred embodiment, an htrb nucleic acid sequence of the present invention will bind to a portion of one of SEQ ID Nos.", "1 or 3 that corresponds to the htrb ORF nucleic acid sequences, under high stringency conditions.", "Nucleic acids having a sequence that differs from the nucleotide sequences shown in one of SEQ ID Nos.", "1 or 3 of the invention or complement thereof due to degeneracy in the genetic code are also within the scope of the invention.", "Such nucleic acids encode functionally equivalent peptides (i.e., peptides having a biological activity of an htrb polypeptide) but differ in sequence from the sequence shown in the sequence listing due to degeneracy in the genetic code.", "For example, a number of amino acids are designated by more than one triplet.", "Codons that specify the same amino acid, or synonyms (for example, CAU and CAC each encode histidine) may result in “silent” mutations which do not affect the amino acid sequence of an htrb polypeptide.", "However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject htrb polypeptides will exist among mammals.", "One skilled in the art will appreciate that these variations in one or more nucleotides (e.g., up to about 3-5% of the nucleotides) of the nucleic acids encoding polypeptides having an activity of an htrb polypeptide may exist among individuals of a given species due to natural allelic variation.", "4.3.1 Probes and Primers The nucleotide sequences determined from the cloning of htrb genes from mammalian organisms will further allow for the generation of probes and primers designed for use in identifying and/or cloning other htrb homologs in other cell types, e.g., from other tissues, as well as htrb homologs from other mammalian organisms.", "For instance, the present invention also provides a probe/primer comprising a substantially purified oligonucleotide, which oligonucleotide comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least approximately 12, preferably 25, more preferably 40, 50 or 75 consecutive nucleotides of sense or anti-sense sequence selected from SEQ ID Nos.", "1 or 3 of the invention.", "For instance, primers based on the nucleic acid represented in SEQ ID Nos.", "1 or 3 can be used in PCR reactions to clone htrb polypeptide encoding genes.", "In preferred embodiments, the htrb primers are designed so as to optimize specificity and avoid secondary structures which affect the efficiency of priming.", "Optimized PCR primers of the present invention are designed so that “upstream” and “downstream” primers have approximately equal melting temperatures such as can be estimated using the formulae: Tm=81.5 C−16.6(log10 [Na+])+0.41(% G+C)−0.63 (% formamide)−(600/length); or Tm(C)=2(A/T)+4(G/C).", "Optimized htrb primers may also be designed by using various programs, such as “Primer3” provided by the Whitehead Institute for Biomedical Research at http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi.", "In preferred embodiments, the htrb probes and primers can be used to detect htrb locus polymorphisms which occur within and surrounding the htrb gene sequence.", "Genetic variations within the htrb locus may be associated with the likelihood of the development of a number of human diseases and conditions, such as inflammatory and autoimmune diseases in which htrb encoded polypeptides play an important etiological role.", "Accordingly the invention provides probes and primers for htrb locus polymorphisms, including polymorphisms associated with the human and mouse htrb gene.", "PCR primers of the invention include those which flank an htrb human polymorphism and allow amplification and analysis of this region of the genome.", "Analysis of polymorphic allele identity may be conducted, for example, by direct sequencing or by the use of allele-specific capture probes or by the use of molecular beacon probes.", "Alternatively, the polymorphic allele may allow for direct detection by the creation or elimination of a restriction endonuclease recognition site(s) within the PCR product or after an appropriate sequence modification is designed into at least one of the primers such that the altered sequence of the primer, when incorporated into the PCR product resulting from amplification of a specific htrb polymorphic allele, creates a unique restriction site in combination with at least one allele but not with at least one other allele of that polymorphism.", "htrb polymorphisms corresponding to variable number of tandem repeat (VNTR) polymorphisms may be detected by the electrophoretic mobility and hence size of a PCR product obtained using primers which flank the VNTR.", "Still other htrb polymorphisms corresponding to restriction fragment length polymorphisms (RFLPs) may be detected directly by the mobility of bands on a Southern blot using appropriate htrb locus probes and genomic DNA or cDNA obtained from an appropriate sample organism such as a human or a non-human animal.", "Likewise, probes based on the subject htrb sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins, for use, e.g, in prognostic or diagnostic assays (further described below).", "The invention provides probes which are common to alternatively spliced variants of the htrb transcript, such as those corresponding to at least 12 consecutive nucleotides complementary to a sequence found in any of SEQ ID Nos.", "1 or 3 of the invention.", "In addition, the invention provides probes which hybridize specifically to alternatively spliced forms of the htrb transcript.", "Probes and primers can be prepared and modified, e.g., as previously described herein for other types of nucleic acids.", "4.3.2 Antisense, Ribozyme and Triplex Techniques Another aspect of the invention relates to the use of the isolated nucleic acid in “antisense” therapy.", "As used herein, “antisense” therapy refers to administration or in situ generation of oligonucleotide molecules or their derivatives which specifically hybridize (e.g., bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding one or more of the subject htrb proteins so as to inhibit expression of that protein, e.g., by inhibiting transcription and/or translation.", "The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.", "In general, “antisense” therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.", "An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an htrb protein.", "Alternatively, the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of an htrb gene.", "Such oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and are therefore stable in vivo.", "Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat.", "Nos.", "5,176,996; 5,264,564; and 5,256,775).", "Additionally, general approaches to constructing oligomers useful in anti sense therapy have been reviewed, for example, by Van der Krol et al.", "(1988) BioTechniques 6:958-976; and Stein et al.", "(1988) Cancer Res 48:2659-2668.With respect to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the −10 and +10 regions of the htrb nucleotide sequence of interest, are preferred.", "Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to htrb mRNA.", "The antisense oligonucleotides will bind to the htrb mRNA transcripts and prevent translation.", "Absolute complementarity, although preferred, is not required.", "In the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.", "The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid.", "Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be).", "One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.", "Oligonucleotides that are complementary to the 5′ end of the mRNA, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation.", "However, sequences complementary to the 3′ untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well.", "(Wagner, R. 1994.Nature 372:333).", "Therefore, oligonucleotides complementary to either the 5′ or 3′ untranslated, non-coding regions of an htrb gene could be used in an antisense approach to inhibit translation of endogenous htrb mRNA.", "Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon.", "Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could also be used in accordance with the invention.", "Whether designed to hybridize to the 5′, 3′ or coding region of htrb mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably less than about 100 and more preferably less than about 50, 25, 17 or 10 nucleotides in length.", "Regardless of the choice of target sequence, it is preferred that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression.", "It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides.", "It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein.", "Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide.", "It is preferred that the control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.", "The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.", "The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.", "The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc.", "Natl.", "Acad.", "Sci.", "84:648-652; PCT Publication No.", "W088/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No.", "W089/10134, published Apr.", "25, 1988), hybridization-triggered cleavage agents.", "(See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents.", "(See, e.g., Zon, 1988, Pharm.", "Res.", "5:539-549).", "To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.", "The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxytiethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.", "The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.", "The antisense oligonucleotide can also contain a neutral peptide-like backbone.", "Such molecules are termed peptide nucleic acid (PNA)-oligomers and are described, e.g., in Perry-O'Keefe et al.", "(1996) Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 93:14670 and in Eglom et al.", "(1993) Nature 365:566.One advantage of PNA oligomers is their ability to bind to complementary DNA essentially independently from the ionic strength of the medium due to the neutral backbone of the DNA.", "In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.", "In yet a further embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide.", "An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gautier et al., 1987, Nucl.", "Acids Res.", "15:6625-6641).", "The oligonucleotide is a 2′-0-methylribonucleotide (Inoue et al., 1987, Nucl.", "Acids Res.", "15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett.", "215:327-330).", "Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).", "As examples, phosphorothioate oligonucleotides maybe synthesized by the method of Stein et al.", "(1988, Nucl.", "Acids Res.", "16:3209), methylphosphonate olgonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 85:7448-7451), etc.", "While antisense nucleotides complementary to the htrb coding region sequence can be used, those complementary to the transcribed untranslated region and to the region comprising the initiating methionine are most preferred.", "The antisense molecules can be delivered to cells which express htrb in vivo.", "A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.", "However, it may be difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation on endogenous mRNAs in certain instances.", "Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter.", "The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous htrb transcripts and thereby prevent translation of the htrb mRNA.", "For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA.", "Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.", "Such vectors can be constructed by recombinant DNA technology methods standard in the art.", "Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.", "Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells.", "Such promoters can be inducible or constitutive and can include but not be limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al, 1982, Nature 296:39-42), etc.", "Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site.", "Alternatively, viral vectors can be used which selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systematically).", "Ribozyme molecules designed to catalytically cleave htrb mRNA transcripts can also be used to prevent translation of htrb mRNA and expression of htrb (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225 and U.S. Pat.", "No.", "5,093,246).", "While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy htrb mRNAs, the use of hammerhead ribozymes is preferred.", "Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA.", "The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′.", "The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.For example, there are a number of potential hammerhead ribozyme cleavage sites within the nucleotide sequence of human htrb-1 and htrb-3.Preferably the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the htrb mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.", "The ribozymes of the present invention also include RNA endoribonucleases (hereinafter “Cech-type ribozymes”) such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-433; published International patent application No.", "WO88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216).", "The Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.", "The invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in an htrb gene.", "As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.)", "and should be delivered to cells which express the htrb gene in vivo.", "A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous htrb messages and inhibit translation.", "Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.", "Endogenous htrb gene expression can also be reduced by inactivating or “knocking out” the htrb gene or its promoter using targeted homologous recombination.", "(E.g., see Smithies et al., 1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al., 1989 Cell 5:313-321; each of which is incorporated by reference herein in its entirety).", "For example, a mutant, non-functional htrb (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous htrb gene (either the coding regions or regulatory regions of the htrb gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express htrb in vivo.", "Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the htrb gene.", "Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive htrb (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra).", "However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.", "Alternatively, endogenous htrb gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the htrb gene (i.e., the htrb promoter and/or enhancers) to form triple helical structures that prevent transcription of the htrb gene in target cells in the body.", "(See generally, Helene, C. 1991, Anticancer Drug Des., 6(6):569-84; Helene, C., et al., 1992, Ann.", "N.Y. Acad.", "Sci., 660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15).", "Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription are preferably single stranded and composed of deoxyribonucleotides.", "The base composition of these oligonucleotides should promote triple helix formation via Hoogsteen base pairing rules, which generally require sizable stretches of either purines or pyrimidines to be present on one strand of a duplex.", "Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix.", "The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.", "In addition, nucleic acid molecules may be chosen that are purine-rich, for example, containing a stretch of G residues.", "These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in CGC triplets across the three strands in the triplex.", "Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called “switchback” nucleic acid molecule.", "Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizable stretch of either purines or pyrimidines to be present on one strand of a duplex.", "Antisense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules.", "These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis.", "Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.", "Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.", "Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.", "Moreover, various well-known modifications to nucleic acid molecules may be introduced as a means of increasing intracellular stability and half-life.", "Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.", "4.3.3.Vectors Encoding htrb Proteins and htrb Expressing Cells The invention further provides plasmids and vectors encoding an htrb protein, which can be used to express an htrb protein in a host cell.", "The host cell may be any prokaryotic or eukaryotic cell.", "Thus, a nucleotide sequence derived from the cloning of mammalian htrb proteins, encoding all or a selected portion of the full-length protein, can be used to produce a recombinant form of an htrb polypeptide via microbial or eukaryotic cellular processes.", "Ligating the polynucleotide sequence into a gene construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial) cells, are standard procedures well known in the art.", "Vectors that allow expression of a nucleic acid in a cell are referred to as expression vectors.", "Typically, expression vectors used for expressing an htrb protein contain a nucleic acid encoding an htrb polypeptide, operably linked to at least one transcriptional regulatory sequence.", "Regulatory sequences are art-recognized and are selected to direct expression of the subject htrb proteins.", "Transcriptional regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).", "In one embodiment, the expression vector includes a recombinant gene encoding a peptide having an agonistic activity of a subject htrb polypeptide, or alternatively, encoding a peptide which is an antagonistic form of an htrb protein.", "Suitable vectors for the expression of an htrb polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.", "A number of vectors exist for the expression of recombinant proteins in yeast.", "For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al.", "(1983) in Experimental Manipulation of Gene Expression, ed.", "M. Inouye Academic Press, p. 83, incorporated by reference herein).", "These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.", "In addition, drug resistance markers such as ampicillin can be used.", "In an illustrative embodiment, an htrb polypeptide is produced recombinantly utilizing an expression vector generated by sub-cloning the coding sequence of one of the htrb genes represented in SEQ ID Nos.", "1 or 3.The preferred mammalian expression vectors contain both prokaryotic sequences, to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.", "The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.", "Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.", "Alternatively, derivatives of viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.", "The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art.", "For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.", "by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.In some instances, it may be desirable to express the recombinant htrb polypeptide by the use of a baculovirus expression system.", "Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the β-gal containing pBlueBac III) When it is desirable to express only a portion of an htrb protein, such as a form lacking a portion of the N-terminus, i.e.", "a truncation mutant which lacks the signal peptide, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed.", "It is well known in the art that a methionine at the N-terminal position can be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP).", "MAP has been cloned from E. coli (Ben-Bassat et al.", "(1987) J. Bacteriol.", "169:751-757) and Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al.", "(1987) PNAS 84:2718-1722).", "Therefore, removal of an N-terminal methionine, if desired, can be achieved either in vivo by expressing htrb derived polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e.g., procedure of Miller et al., supra).", "Moreover, the gene constructs of the present invention can also be used as part of a gene therapy protocol to deliver nucleic acids encoding either an agonistic or antagonistic form of one of the subject htrb proteins.", "Thus, another aspect of the invention features expression vectors for in vivo or in vitro transfection and expression of an htrb polypeptide in particular cell types so as to reconstitute the function of, or alternatively, abrogate the function of htrb in a tissue.", "This could be desirable, for example, when the naturally-occurring form of the protein is misexpressed or the natural protein is mutated and less active.", "In addition to viral transfer methods, non-viral methods can also be employed to cause expression of a subject htrb polypeptide in the tissue of an animal.", "Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.", "In preferred embodiments, non-viral targeting means of the present invention rely on endocytic pathways for the uptake of the subject htrb polypeptide gene by the targeted cell.", "Exemplary targeting means of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.", "In other embodiments transgenic animals, described in more detail below could be used to produce recombinant proteins.", "4.4.Polypeptides of the Present Invention The present invention makes available isolated htrb polypeptides which are isolated from, or otherwise substantially free of other cellular proteins.", "The term “substantially free of other cellular proteins” (also referred to herein as “contaminating proteins”) or “substantially pure or purified preparations” are defined as encompassing preparations of htrb polypeptides having less than about 20% (by dry weight) contaminating protein, and preferably having less than about 5% contaminating protein.", "Functional forms of the subject polypeptides can be prepared, for the first time, as purified preparations by using a cloned gene as described herein.", "Preferred htrb proteins of the invention have an amino acid sequence which is at least about 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, or 95% identical or homologous to an amino acid sequence of SEQ ID No.", "2 or 4.Even more preferred htrb proteins comprise an amino acid sequence of at least 10, 20, 30, or 50 residues which is at least about 70, 80, 90, 95, 97, 98, or 99% homologous or identical to an amino acid sequence of SEQ ID Nos.", "2 or 4.Such proteins can be recombinant proteins, and can be, e.g., produced in vitro from nucleic acids comprising a nucleotide sequence set forth in SEQ ID Nos.", "1 or 3, or another nucleic acid of the invention or homologs thereof.", "For example, recombinant polypeptides preferred by the present invention can be encoded by a nucleic acid, which is at least 85% homologous and more preferably 90% homologous and most preferably 95% homologous with a nucleotide sequence set forth in a SEQ ID Nos.", "1 or 3 of the invention.", "Polypeptides which are encoded by a nucleic acid that is at least about 98-99% homologous with the sequence of SEQ ID No.", "1 or 3 of the invention are also within the scope of the invention.", "In a preferred embodiment, an htrb protein of the present invention is a mammalian htrb protein.", "In a particularly preferred embodiment an htrb protein is set forth as SEQ ID No.", "2 or SEQ ID No.", "4.In particularly preferred embodiments, an htrb protein has an htrb bioactivity.", "It will be understood that certain post-translational modifications, e.g., phosphorylation and the like, can increase the apparent molecular weight of the htrb protein relative to the unmodified polypeptide chain.", "The invention also features protein isoforms encoded by splice variants of the present invention.", "Such isoforms may have biological activities identical to or different from those possessed by the htrb proteins specified by Nos.", "2 or 4.Such isoforms may arise, for example, by alternative splicing of one or more htrb gene transcripts.", "htrb polypeptides preferably are capable of functioning as either an agonist or antagonist of at least one biological activity of a wild-type (“authentic”) htrb protein of the appended sequence listing.", "The term “evolutionarily related to”, with respect to amino acid sequences of htrb proteins, refers to both polypeptides having amino acid sequences which have arisen naturally, and also to mutational variants of human htrb polypeptides which are derived, for example, by combinatorial mutagenesis.", "Full length proteins or fragments corresponding to one or more particular motifs and/or domains or to arbitrary sizes, for example, at least 5, 10, 20, 25, 50, 75 and 100, amino acids in length are within the scope of the present invention.", "For example, isolated htrb polypeptides can be encoded by all or a portion of a nucleic acid sequence shown in any of SEQ ID Nos.", "1 or 3.Isolated peptidyl portions of htrb proteins can be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides.", "In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.", "For example, an htrb polypeptide of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length.", "The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as either agonists or antagonists of a wild-type (e.g., “authentic”) htrb protein.", "An htrb polypeptide can be a membrane bound form or a soluble form.", "A preferred soluble htrb polypeptide is a polypeptide which does not contain a hydrophobic signal sequence domain.", "Such proteins can be created by genetic engineering by methods known in the art.", "The solubility of a recombinant polypeptide may be increased by deletion of hydrophobic domains, such as predicted transmembrane domains, of the wild type protein.", "In general, polypeptides referred to herein as having an activity (e.g., are “bioactive”) of an htrb protein are defined as polypeptides which include an amino acid sequence encoded by all or a portion of the nucleic acid sequences shown in one of SEQ ID No.", "1 or 3 and which mimic or antagonize all or a portion of the biological/biochemical activities of a naturally occurring htrb protein.", "Examples of such biological activity include a region of conserved structure referred to as the htrb conserved domain (see FIG.", "6A, htrb NC construct).", "Other biological activities of the subject htrb proteins will be reasonably apparent to those skilled in the art.", "According to the present invention, a polypeptide has biological activity if it is a specific agonist or antagonist of a naturally-occurring form of an htrb protein.", "Assays for determining whether a compound, e.g, a protein, such as an htrb protein or variant thereof, has one or more of the above biological activities include those assays, well known in the art, which are used for assessing htrb agonist and htrb antagonist activities.", "For example, the ability of recombinant htrb polypeptide to block activation of an AP-1 reporter construct.", "In contrast, the ability of recombinant htrb polypeptides to interfere with cytokine induced activation of interleukin-8 gene expression is indicative of htrb antagonist activity.", "Other preferred proteins of the invention are those encoded by the nucleic acids set forth in the section pertaining to nucleic acids of the invention.", "In particular, the invention provides fusion proteins, e.g., htrb immunoglobulin fusion proteins.", "Such fusion proteins can provide, e.g., enhanced stability and solubility of htrb proteins and may thus be useful in therapy.", "Fusion proteins can also be used to produce an immunogenic fragment of an htrb protein.", "For example, the VP6 capsid protein of rotavirus can be used as an immunologic carrier protein for portions of the htrb polypeptide, either in the monomeric form or in the form of a viral particle.", "The nucleic acid sequences corresponding to the portion of a subject htrb protein to which antibodies are to be raised can be incorporated into a fusion gene construct which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising htrb epitopes as part of the virion.", "It has been demonstrated with the use of immunogenic fusion proteins utilizing the Hepatitis B surface antigen fusion proteins that recombinant Hepatitis B virions can be utilized in this role as well.", "Similarly, chimeric constructs coding for fusion proteins containing a portion of an htrb protein and the poliovirus capsid protein can be created to enhance immunogenicity of the set of polypeptide antigens (see, for example, EP Publication No: 0259149; and Evans et al.", "(1989) Nature 339:385; Huang et al.", "(1988) J. Virol.", "62:3855; and Schlienger et al.", "(1992) J. Virol.", "66:2).", "The Multiple antigen peptide system for peptide-based immunization can also be utilized to generate an immunogen, wherein a desired portion of an htrb polypeptide is obtained directly from organo-chemical synthesis of the peptide onto an oligomeric branching lysine core (see, for example, Posnett et al.", "(1988) JBC 263:1719 and Nardelli et al.", "(1992) J. Immunol.", "148:914).", "Antigenic determinants of htrb proteins can also be expressed and presented by bacterial cells.", "In addition to utilizing fusion proteins to enhance immunogenicity, it is widely appreciated that fusion proteins can also facilitate the expression of proteins, and accordingly, can be used in the expression of the htrb polypeptides of the present invention.", "For example, htrb polypeptides can be generated as glutathione-S-transferase (GST-fusion) proteins.", "Such GST-fusion proteins can enable easy purification of the htrb polypeptide, as for example by the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds.", "Ausubel et al.", "(N.Y.: John Wiley & Sons, 1991)).", "Additionally, fusion of htrb polypeptides to small epitope tags, such as the FLAG or hemagluttinin tag sequences, can be used to simplify immunological purification of the resulting recombinant polypeptide or to facilitate immunological detection in a cell or tissue sample.", "Fusion to the green fluorescent protein, and recombinant versions thereof which are known in the art and available commercially, may further be used to localize htrb polypeptides within living cells and tissue.", "The present invention further pertains to methods of producing the subject htrb polypeptides.", "For example, a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding the subject polypeptides can be cultured under appropriate conditions to allow.", "expression of the peptide to occur.", "Suitable media for cell culture are well known in the art.", "The recombinant htrb polypeptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such peptide.", "In a preferred embodiment, the recombinant htrb polypeptide is a fusion protein containing a domain which facilitates its purification, such as GST fusion protein.", "Moreover, it will be generally appreciated that, under certain circumstances, it may be advantageous to provide homologs of one of the subject htrb polypeptides which function in a limited capacity as one of either an htrb agonist (mimetic) or an htrb antagonist, in order to promote or inhibit only a subset of the biological activities of the naturally-occurring form of the protein.", "Thus, specific biological effects can be elicited by treatment with a homolog of limited function, and with fewer side effects relative to treatment with agonists or antagonists which are directed to all of the biological activities of naturally occurring forms of htrb proteins.", "Homologs of each of the subject htrb proteins can be generated by mutagenesis, such as by discrete point mutation(s), or by truncation.", "For instance, mutation can give rise to homologs which retain substantially the same, or merely a subset, of the biological activity of the htrb polypeptide from which it was derived.", "Alternatively, antagonistic forms of the protein can be generated which are able to inhibit the flnction of the naturally occurring form of the protein, such as by competitively binding to an htrb receptor.", "The recombinant htrb polypeptides of the present invention also include homologs of the wildtype htrb proteins, such as versions of those protein which are resistant to proteolytic cleavage, as for example, due to mutations which alter ubiquitination or other enzymatic targeting associated with the protein.", "htrb polypeptides may also be chemically modified to create htrb derivatives by forming covalent or aggregate conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like.", "Covalent derivatives of htrb proteins can be prepared by linking the chemical moieties to functional groups on amino acid sidechains of the protein or at the N-terminus or at the C-terminus of the polypeptide.", "Modification of the structure of the subject htrb polypeptides can be for such purposes as enhancing therapeutic or prophylactic efficacy, stability (e.g., ex vivo shelf life and resistance to proteolytic degradation), or post-translational modifications (e.g., to alter phosphorylation pattern of protein).", "Such modified peptides, when designed to retain at least one activity of the naturally-occurring form of the protein, or to produce specific antagonists thereof, are considered functional equivalents of the htrb polypeptides described in more detail herein.", "Such modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.", "The substitutional variant may be a substituted conserved amino acid or a substituted non-conserved amino acid.", "For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e.", "isosteric and/or isoelectric mutations) will not have a major effect on the biological activity of the resulting molecule.", "Conservative replacements are those that take place within a family of amino acids that are related in their side chains.", "Genetically encoded amino acids can be divided into four families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine.", "In similar fashion, the amino acid repertoire can be grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3) aliphatic=glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine and methionine.", "(see, for example, Biochemistry, 2nd ed., Ed.", "by L. Stryer, WH Freeman and Co.: 1981).", "Whether a change in the amino acid sequence of a peptide results in a functional htrb homolog (e.g., functional in the sense that the resulting polypeptide mimics or antagonizes the wild-type form) can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the wild-type protein, or competitively inhibit such a response.", "Polypeptides in which more than one replacement has taken place can readily be tested in the same manner.", "This invention further contemplates a method for generating sets of combinatorial mutants of the subject htrb proteins as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g., homologs).", "The purpose of screening such combinatorial libraries is to generate, for example, novel htrb homologs which can act as either agonists or antagonist, or alternatively, possess novel activities all together.", "Thus, combinatorially-derived homologs can be generated to have an increased potency relative to a naturally occurring form of the protein.", "In one embodiment, the variegated htrb libary of htrb variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene htrb library.", "For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential htrb sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of htrb sequences therein.", "There are many ways by which such libraries of potential htrb homologs can be generated from a degenerate oligonucleotide sequence.", "Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector.", "The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential htrb sequences.", "The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al.", "(1981) Recombinant DNA, Proc 33 rd Cleveland Sympos.", "Macromolecules, ed.", "A G Walton, Amsterdam: Elsevier pp 273-289; Itakura et al.", "(1984) Annu.", "Rev.", "Biochem.", "53:323; Itakura et al.", "(1984) Science 198:1056; Ike et al.", "(1983) Nucleic Acid Res.", "11:477.Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al.", "(1990) Science 249:386-390; Roberts et al.", "(1992) PNAS 89:2429-2433; Devlin et al.", "(1990) Science 249: 404-406; Cwirla et al.", "(1990) PNAS 87: 6378-6382; as well as U.S. Pat.", "Nos.", "5,223,409, 5,198,346, and 5,096,815).", "Likewise, a library of coding sequence fragments can be provided for an htrb clone in order to generate a variegated population of htrb fragments for screening and subsequent selection of bioactive fragments.", "A variety of techniques are known in the art for generating such 1, including chemical synthesis.", "In one embodiment, a library of coding sequence fragments can be generated by (i) treating a double stranded PCR fragment of an htrb coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule; (ii) denaturing the double stranded DNA; (iii) renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products; (iv) removing single stranded portions from reformed duplexes by treatment with S1 nuclease; and (v) ligating the resulting fragment library into an expression vector.", "By this exemplary method, an expression library can be derived which codes for N-terminal, C-terminal and internal fragments of various sizes.", "A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a certain property.", "Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of htrb homologs.", "The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting libraries of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.", "Each of the illustrative assays described below are amenable to high through-put analysis as necessary to screen large numbers of degenerate htrb sequences created by combinatorial mutagenesis techniques.", "Combinatorial mutagenesis has a potential to generate very large libraries of mutant proteins, e.g., in the order of 1026 molecules.", "Combinatorial libraries of this size may be technically challenging to screen even with high throughput screening assays.", "To overcome this problem, a new technique has been developed recently, recrusive ensemble mutagenesis (REM), which allows one to avoid the very high proportion of non-functional proteins in a random library and simply enhances the frequency of functional proteins, thus decreasing the complexity required to achieve a useful sampling of sequence space.", "REM is an algorithm which enhances the frequency of functional mutants in a library when an appropriate selection or screening method is employed (Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan et al., 1992, Parallel Problem Solving from Nature, 2., In Maenner and Manderick, eds., Elsevir Publishing Co., Amsterdam, pp.", "401-410; Delgrave et al., 1993, Protein Engineering 6(3):327-331).", "The invention also provides for reduction of the htrb proteins to generate mimetics, e.g., peptide or non-peptide agents, such as small molecules, which are able to disrupt binding of an htrb polypeptide of the present invention with a molecule, e.g.", "target peptide.", "Thus, such mutagenic techniques as described above are also usefull to map the determinants of the htrb proteins which participate in protein-protein interactions involved in, for example, binding of the subject htrb polypeptide to a target peptide.", "To illustrate, the critical residues of a subject htrb polypeptide which are involved in molecular recognition of its receptor can be determined and used to generate htrb derived peptidomimetics or small molecules which competitively inhibit binding of the authentic htrb protein with that moiety.", "By employing, for example, scanning mutagenesis to map the amino acid residues of the subject htrb proteins which are involved in binding other proteins, peptidomimetic compounds can be generated which mimic those residues of the htrb protein which facilitate the interaction.", "Such mimetics may then be used to interfere with the normal function of an htrb protein.", "For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al.", "in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffinan et al.", "in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al.", "in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al.", "(1986) J Med Chem 29:295; and Ewenson et al.", "in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), b-turn dipeptide cores (Nagai et al.", "(1985) Tetrahedron Lett 26:647; and Sato et al.", "(1986) J Chem Soc Perkin Trans 1:1231), and b-aminoalcohols (Gordon et al.", "(1985) Biochem Biophys Res Commun126:419; and Dann et al.", "(1986) Biochem Biophys Res Commun 134:71).", "4.5.Anti-htrb Antibodies and Uses Therefor Another aspect of the invention pertains to an antibody specifically reactive with a mammalian htrb protein, e.g., a wild-type or mutated htrb protein.", "For example, by using immunogens derived from an htrb protein, e.g., based on the cDNA sequences, anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed.", "by Harlow and Lane (Cold Spring Harbor Press: 1988)).", "A mammal, such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., a mammalian htrb polypeptide or an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein as described above).", "Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.", "An immunogenic portion of an htrb protein can be administered in the presence of adjuvant.", "The progress of immunization can be monitored by detection of antibody titers in plasma or serum.", "Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.", "In a preferred embodiment, the subject antibodies are immunospecific for antigenic determiinants of an htrb protein of a mammal, e.g., antigenic determinants of a protein set forth in SEQ ID No.", "2 or 4 or closely related homologs (e.g., at least 90% homologous, and more preferably at least 94% homologous).", "Following immunization of an animal with an antigenic preparation of an htrb polypeptide, anti-htrb antisera can be obtained and, if desired, polyclonal anti-htrb antibodies isolated from the serum.", "To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.", "Such techniques are well known in the art, and include, for example, the hybridoma technique originally developed by Kohler and Milstein ((1975) Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar et al., (1983) Inmunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp.", "77-96).", "Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a mammalian htrb polypeptide of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells.", "In one embodiment anti-human htrb antibodies specifically react with the protein encoded by a nucleic acid having SEQ ID No.", "1 or 3.The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with one of the subject mammalian htrb polypeptides.", "Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies.", "For example, F(ab)2 fragments can be generated by treating antibody with pepsin.", "The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments.", "The antibody of the present invention is further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for an htrb protein conferred by at least one CDR region of the antibody.", "In preferred embodiments, the antibody further comprises a label attached thereto and able to be detected, (e.g., the label can be a radioisotope, fluorescent compound, enzyme or enzyme co-factor).", "Anti-htrb antibodies can be used, e.g., to monitor htrb protein levels in an individual for determining, e.g., whether a subject has a disease or condition associated with an aberrant htrb protein level, or allowing determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder.", "The level of htrb polypeptides may be measured from cells in bodily fluid, such as in blood samples.", "Another application of anti-htrb antibodies of the present invention is in the immunological screening of cDNA libraries constructed in expression vectors such as λgt11, λgt18-23, λZAP, and λORF8.Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins.", "For instance, λgt11 will produce fusion proteins whose amino termini consist of β-galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide.", "Antigenic epitopes of an htrb protein, e.g., other orthologs of a particular htrb protein or other paralogs from the same species, can-then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-htrb antibodies.", "Positive phage detected by this assay can then be isolated from the infected plate.", "Thus, the presence of htrb homologs can be detected and cloned from other animals, as can alternate isoforms (including splice variants) from humans.", "4.6.Transgenic Animals The invention further provides for transgenic animals, which can be used for a variety of purposes, e.g., to identify htrb therapeutics.", "Transgenic animals of the invention include non-human animals containing a heterologous htrb gene or fragment thereof under the control of an htrb promoter or under the control of a heterologous promoter.", "Accordingly, the transgenic animals of the invention can be animals expressing a transgene encoding a wild-type htrb protein or fragment thereof or variants thereof, including mutants and polymorphic variants thereof.", "Such animals can be used, e.g., to determine the effect of a difference in amino acid sequence of an htrb protein from the sequence set forth in SEQ ID Nos.", "2 or 4, such as a polymorphic difference.", "These animals can also be used to determine the effect of expression of an htrb protein in a specific site or for identifying htrb therapeutics or confirming their activity in vivo.", "In one aspect, the invention provides transgenic non-human organisms and cell lines for use in the in vivo screening and evaluation of drugs or other therapeutic regimens useful in the treatment of inflammatory disorders.", "In one embodiment, the invention is a transgenic animal with a targeted disruption in an interleukin-1 gene.", "In particular, the gene is the htrb gene.", "The animal may be chimeric, heterozygotic or homozygotic for the disrupted gene.", "Homozygotic knock-out htrb mammals provide a model for studying inflammatory conditions, such as rheumatoid arthritis, inflammatory bowel disorder, Type I diabetes, psoriasis, osteoporosis, nephropathy in diabetes mellitus, alopecia areata, Graves disease, systemic lupus erythematosus, lichen sclerosis, ulcerative colitis, coronary artery disease, arteritic disorders, diabetic retinopathy, low birth weight, pregnancy complications, severe periodontal disease, psoriasis and insulin dependent diabetes, but is particularly characterized by arteritic lesions.", "The targeted disruption may be anywhere in the gene, subject only to the requirement that it inhibit production of functional htrb protein.", "In a preferred embodiment, the disruption removes the entire htrb coding sequence such as that of htrb-1 contained in SEQ ID No.", "1.The transgenic animal maybe of any species (except human), but is preferably a mammal.", "In a preferred embodiment, the non-human animal comprising a targeted disruption in the htrb gene, wherein said targeted disruption inhibits production of wild-type htrb polypeptide so that the phenotype of a non-human mammal homozygous for the targeted disruption is characterized by an altered inflammatory response.", "In another aspect, the invention features a cell or cell line, which contains a targeted disruption in the htrb gene.", "In a preferred embodiment, the cell or cell line is an undifferentiated cell, for example, a stem cell, embryonic stem cell, oocyte or embryonic cell.", "Yet in a further aspect, the invention features a method of producing a non-human mammal with a targeted disruption in an htrb gene.", "For example, an htrb knock-out construct can be created with a portion of the htrb gene having an internal portion of said htrb gene replaced by a marker.", "The knock-out construct can then be transfected into a population of embryonic stem m(ES) cells.", "Transfected cells can then be selected as expressing the marker.", "The transfected ES cells can then be introduced into an embryo of an ancestor of said mammal.", "The embryo can be allowed to develop to term to produce a chimeric mammal with the knock-out construct in its germline.", "Breeding said chimeric mammal will produce a heterozygous mammal with a targeted disruption in the htrb gene.", "Homozygotes can be generated by crossing heterozygotes.", "In another aspect, the invention features htrb knock-out constructs, which can be used to generate the animals described above.", "In one embodiment, the htrb construct can comprise a portion of the htrb gene, wherein an internal portion of said htrb gene is replaced by a selectable marker.", "Preferably, the marker is the neo gene and the portion of the htrb gene is at least 2.5 kb long or 7.0 or 9.5 kb long (including the replaced portion and any htrb flanking sequences).", "The internal portion preferably covers at least a portion of an exon and in some embodiments it covers all of the exons which encode an htrb polypeptide.", "In still another aspect, the invention features methods for testing agents for effectiveness in treating and/or preventing an inflammatory condition.", "In one embodiment, the method can employ the transgenic animal or cell lines, as described above.", "For example, a test agent can be administered to the transgenic animal and the ability of the agent to ameliorate the inflammatory condition can be scored as having effectiveness against said inflammatory condition.", "Any inflammatory condition with an htrb component can be tested using these mammals, but in particular, conditions characterized by arteritic lesions are studied.", "The method may also be used to test agents that are effective against inflammatory proteins and their downstream components.", "The transgenic animals can also be animals containing a transgene, such as reporter gene, under the control of an htrb promoter or fragment thereof.", "These animals are useful, e.g., for identifying htrb drugs that modulate production of htrb, such as by modulating htrb gene expression.", "An htrb gene promoter can be isolated, e.g., by screening of a genomic library with an htrb cDNA fragment and characterized according to methods known in the art.", "In a preferred embodiment of the present invention, the transgenic animal containing said htrb reporter gene is used to screen a class of bioactive molecules known as steroid hormones for their ability to modulate htrb expression.", "In a more preferred embodiment of the invention, the steroid hormones screened for htrb expression modulating activity belong to the group known as androgens.", "In a still more preferred embodiment of the invention, the steroid hormone is testosterone or a testosterone analog.", "Yet other non-human animals within the scope of the invention include those in which the expression of the endogenous htrb gene has been mutated or “knocked out”.", "A “knock out” animal is one carrying a homozygous or heterozygous deletion of a particular gene or genes.", "These animals could be useful to determine whether the absence of htrb will result in a specific phenotype, in particular whether these mice have or are likely to develop a specific disease, such as high susceptibility to heart disease or cancer.", "Furthermore these animals are useful in screens for drugs which alleviate or attenuate the disease condition resulting from the mutation of the htrb gene as outlined below.", "These animals are also useful for determining the effect of a specific amino acid difference, or allelic variation, in an htrb gene.", "That is, the htrb knock out animals can be crossed with transgenic animals expressing, e.g., a mutated form or allelic variant of htrb, thus resulting in an animal which expresses only the mutated protein and not the wild-type htrb protein.", "In a preferred embodiment of this aspect of the invention, a transgenic htrb knock-out mouse, carrying the mutated htrb locus on one or both of its chromosomes, is used as a model system for transgenic or drug treatment of the condition resulting from loss of htrb expression.", "Methods for obtaining transgenic and knockout non-human animals are well known in the art.", "Knock out mice are generated by homologous integration of a “knock out” construct into a mouse embryonic stem cell chromosome which encodes the gene to be knocked out.", "In one embodiment, gene targeting, which is a method of using homologous recombination to modify an animal's genome, can be used to introduce changes into cultured embryonic stem cells.", "By targeting a htrb gene of interest in ES cells, these changes can be introduced into the gernlines of animals to generate chimeras.", "The gene targeting procedure is accomplished by introducing into tissue culture cells a DNA targeting construct that includes a segment homologous to a target htrb locus, and which also includes an intended sequence modification to the htrb genomic sequence (e.g., insertion, deletion, point mutation).", "The treated cells are then screened for accurate targeting to identify and isolate those which have been properly targeted.", "Gene targeting in embryonic stem cells is in fact a scheme contemplated by the present invention as a means for disrupting a htrb gene function through the use of a targeting transgene construct designed to undergo homologous recombination with one or more htrb genomic sequences.", "The targeting construct can be arranged so that, upon recombination with an element of a htrb gene, a positive selection marker is inserted into (or replaces) coding sequences of the gene.", "The inserted sequence functionally disrupts the htrb gene, while also providing a positive selection trait.", "Exemplary htrb targeting constructs are described in more detail below.", "Generally, the embryonic stem cells (ES cells ) used to produce the knockout animals will be of the same species as the knockout aninal to be generated.", "Thus for example, mouse embryonic stem cells will usually be used for generation of knockout mice.", "Embryonic stem cells are generated and maintained using methods well known to the skilled artisan such as those described by Doetschman et al.", "(1985) J. Embryol.", "Exp.", "87:27-45).", "Any line of ES cells can be used, however, the line chosen is typically selected for the ability of the cells to integrate into and become part of the germ line of a developing embryo so as to create germ line transmission of the knockout construct.", "Thus, any ES cell line that is believed to have this capability is suitable for use herein.", "One mouse strain that is typically used for production of ES cells, is the 129J strain.", "Another ES cell line is murine cell line D3 (American Type Culture Collection, catalog no.", "CKL 1934) Still another preferred ES cell line is the WW6 cell line (Ioffe et al.", "(1995) PNAS 92:7357-7361).", "The cells are cultured and prepared for knockout construct insertion using methods well known to the skilled artisan, such as those set forth by Robertson in: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed.", "IRL Press, Washington, D.C. [1987]); by Bradley et al.", "(1986) Current Topics in Devel.", "Biol.", "20:357-371); and by Hogan et al.", "(Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1986]).", "A knock out construct refers to a uniquely configured fragment of nucleic acid which is introduced into a stem cell line and allowed to recombine with the genome at the chromosomal locus of the gene of interest to be mutated.", "Thus a given knock out construct is specific for a given gene to be targeted for disruption.", "Nonetheless, many common elements exist among these constructs and these elements are well known in the art.", "A typical knock out construct contains nucleic acid fragments of not less than about 0.5 kb nor more than about 10.0 kb from both the 5′ and the 3′ ends of the genomic locus which encodes the gene to be mutated.", "These two fragments are separated by an intervening fragment of nucleic acid which encodes a positive selectable marker, such as the neomycin resistance gene (neoR).", "The resulting nucleic acid fragment, consisting of a nucleic acid from the extreme 5′ end of the genomic locus linked to a nucleic acid encoding a positive selectable marker which is in turn linked to a nucleic acid from the extreme 3′ end of the genomic locus of interest, omits most of the coding sequence for htrb or other gene of interest to be knocked out.", "When the resulting construct recombines homologously with the chromosome at this locus, it results in the loss of the omitted coding sequence, otherwise known as the structural gene, from the genomic locus.", "A stem cell in which such a rare homologous recombination event has taken place can be selected for by virtue of the stable integration into the genome of the nucleic acid of the gene encoding the positive selectable marker and subsequent selection for cells expressing this marker gene in the presence of an appropriate drug (neomycin in this example).", "Variations on this basic technique also exist and are well known in the art.", "For example, a “knock-in” construct refers to the same basic arrangement of a nucleic acid encoding a 5′ genomic locus fragment linked to nucleic acid encoding a positive selectable marker which in turn is linked to a nucleic acid encoding a 3′ genomic locus fragment, but which differs in that none of the coding sequence is omitted and thus the 5′ and the 3′ genomic fragments used were initially contiguous before being disrupted by the introduction of the nucleic acid encoding the positive selectable marker gene.", "This “knock-in” type of construct is thus very useful for the construction of mutant transgenic animals when only a limited region of the genomic locus of the gene to be mutated, such as a single exon, is available for cloning and genetic manipulation.", "Alternatively, the “knock-in” construct can be used to specifically eliminate a single functional domain of the targetted gene, resulting in a transgenic animal which expresses a polypeptide of the targetted gene which is defective in one function, while retaining the function of other domains of the encoded polypeptide.", "This type of “knock-in” mutant frequently has the characteristic of a so-called “dominant negative” mutant because, especially in the case of proteins which homomultimerize, it can specifically block the action of (or “poison”) the polypeptide product of the wild-type gene from which it was derived.", "In a variation of the knock-in technique, a marker gene is integrated at the genomic locus of interest such that expression of the marker gene comes under the control of the transcriptional regulatory elements of the targeted gene.", "A marker gene is one that encodes an enzyme whose activity can be detected (e.g., b-galactosidase), the enzyme substrate can be added to the cells under suitable conditions, and the enzymatic activity can be analyzed.", "One skilled in the art will be familiar with other useful markers and the means for detecting their presence in a given cell.", "All such markers are contemplated as being included within the scope of the teaching of this invention.", "As mentioned above, the homologous recombination of the above described “knock out” and “knock in” constructs is very rare and frequently such a construct inserts nonhomologously into a random region of the genome where it has no effect on the gene which has been targeted for deletion, and where it can potentially recombine so as to disrupt another gene which was otherwise not intended to be altered.", "Such nonhomologous recombination events can be selected against by modifying the abovementioned knock out and knock in constructs so that they are flanked by negative selectable markers at either end (particularly through the use of two allelic variants of the thymidine kinase gene, the polypeptide product of which can be selected against in expressing cell lines in an appropriate tissue culture medium well known in the art—i.e.", "one containing a drug such as 5-bromodeoxyaridine).", "Thus a preferred embodiment of such a knock out or knock in construct of the invention consist of a nucleic acid encoding a negative selectable marker linked to a nucleic acid encoding a 5′ end of a genomic locus linked to a nucleic acid of a positive selectable marker which in turn is linked to a nucleic acid encoding a 3′ end of the same genomic locus which in turn is linked to a second nucleic acid encoding a negative selectable marker Nonhomologous recombination between the resulting knock out construct and the genome will usually result in the stable integration of one or both of these negative selectable marker genes and hence cells which have undergone nonhomologous recombination can be selected against by growth in the appropriate selective media (e.g.", "media containing a drug such as 5-bromodeoxyuridine for example).", "Simultaneous selection for the positive selectable marker and against the negative selectable marker will result in a vast enrichment for clones in which the knock out construct has recombined homologously at the locus of the gene intended to be mutated.", "The presence of the predicted chromosomal alteration at the targeted gene locus in the resulting knock out stem cell line can be confirmed by means of Southern blot analytical techniques which are well known to those familiar in the art.", "Alternatively, PCR can be used.", "Each knockout construct to be inserted into the cell must first be in the linear form.", "Therefore, if the knockout construct has been inserted into a vector (described infra), linearization is accomplished by digesting the DNA with a suitable restriction endonuclease selected to cut only within the vector sequence and not within the knockout construct sequence.", "For insertion, the knockout construct is added to the ES cells under appropriate conditions for the insertion method chosen, as is known to the skilled artisan.", "For example, if the ES cells are to be electroporated, the ES cells and knockout construct DNA are exposed to an electric pulse using an electroporation machine and following the manufacturer's guidelines for use.", "After electroporation, the ES cells are typically allowed to recover under suitable incubation conditions.", "The cells are then screened for the presence of the knock out construct as explained above.", "Where more than one construct is to be introduced into the ES cell, each knockout construct can be introduced simultaneously or one at a time.", "After suitable ES cells containing the knockout construct in the proper location have been identified by the selection techniques outlined above, the cells can be inserted into an embryo.", "Insertion may be accomplished in a variety of ways known to the skilled artisan, however a preferred method is by microinjection.", "For microinjection, about 10-30 cells are collected into a micropipet and injected into embryos that are at the proper stage of development to permit integration of the foreign ES cell containing the knockout construct into the developing embryo.", "For instance, the transformed ES cells can be microinjected into blastocytes.", "The suitable stage of development for the embryo used for insertion of ES cells is very species dependent, however for mice it is about 3.5 days.", "The embryos are obtained by perfusing the uterus of pregnant females.", "Suitable methods for accomplishing this are known to the skilled artisan, and are set forth by, e.g., Bradley et al.", "(supra).", "While any embryo of the right stage of development is suitable for use, preferred embryos are male.", "In mice, the preferred embryos also have genes coding for a coat color that is different from the coat color encoded by the ES cell genes.", "In this way, the offspring can be screened easily for the presence of the knockout construct by looking for mosaic coat color (indicating that the ES cell was incorporated into the developing embryo).", "Thus, for example, if the ES cell line carries the genes for white fur, the embryo selected will carry genes for black or brown fur.", "After the ES cell has been introduced into the embryo, the embryo may be implanted into the uterus of a pseudopregnant foster mother for gestation.", "While any foster mother may be used, the foster mother is typically selected for her ability to breed and reproduce well, and for her ability to care for the young.", "Such foster mothers are typically prepared by mating with vasectomized males of the same species.", "The stage of the pseudopregnant foster mother is important for successful implantation, and it is species dependent.", "For mice, this stage is about 2-3 days pseudopregnant.", "Offspring that are born to the foster mother may be screened initially for mosaic coat color where the coat color selection strategy (as described above, and in the appended examples) has been employed.", "In addition, or as an alternative, DNA from tail tissue of the offspring may be screened for the presence of the knockout construct using Southern blots and/or PCR as described above.", "Offspring that appear to be mosaics may then be crossed to each other, if they are believed to carry the knockout construct in their germ line, in order to generate homozygous knockout animals.", "Homozygotes may be identified by Southern blotting of equivalent amounts of genomic DNA from mice that are the product of this cross, as well as mice that are known heterozygotes and wild type mice.", "Other means of identifying and characterizing the knockout offspring are available.", "For example, Northern blots can be used to probe the mRNA for the presence or absence of transcripts encoding either the gene knocked out, the marker gene, or both.", "In addition, Western blots can be used to assess the level of expression of the htrb gene knocked out in various tissues of the offspring by probing the Western blot with an antibody against the particular htrb protein, or an antibody against the marker gene product, where this gene is expressed.", "Finally, in situ analysis (such as fixing the cells and labeling with antibody) and/or FACS (fluorescence activated cell sorting) analysis of various cells from the offspring can be conducted using suitable antibodies to look for the presence or absence of the knockout construct gene product.", "Yet other methods of making knock-out or disruption transgenic animals are also generally known.", "See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).", "Recombinase dependent knockouts can also be generated, e.g.", "by homologous recombination to insert target sequences, such that tissue specific and/or temporal control of inactivation of a htrb-gene can be controlled by recombinase sequences (described infra).", "Animals containing more than one knockout construct and/or more than one transgene expression construct are prepared in any of several ways.", "The preferred manner of preparation is to generate a series of mammals, each containing one of the desired transgenic phenotypes.", "Such animals are bred together through a series of crosses, backcrosses and selections, to ultimately generate a single animal containing all desired knockout constructs and/or expression constructs, where the animal is otherwise congenic (genetically identical) to the wild type except for the presence of the knockout construct(s) and/or transgene(s) .", "A htrb transgene can encode the wild-type form of the protein, or can encode homologs thereof, including both agonists and antagonists, as well as antisense constructs.", "In preferred embodiments, the expression of the transgene is restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, cis-acting sequences that control expression in the desired pattern.", "In the present invention, such mosaic expression of a htrb protein can be essential for many forms of lineage analysis and can additionally provide a means to assess the effects of, for example, lack of htrb expression which might grossly alter development in small patches of tissue within an otherwise normal embryo.", "Toward this and, tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns.", "Moreover, temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.", "Genetic techniques, which allow for the expression of transgenes can be regulated via site-specific genetic manipulation in vivo, are known to those skilled in the art.", "For instance, genetic systems are available which allow for the regulated expression of a recombinase that catalyzes the genetic recombination of a target sequence.", "As used herein, the phrase “target sequence” refers to a nucleotide sequence that is genetically recombined by a recombinase.", "The target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing recombinase activity.", "Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of one of the subject htrb proteins.", "For example, excision of a target sequence which interferes with the expression of a recombinant htrb gene, such as one which encodes an antagonistic homolog or an antisense transcript, can be designed to activate expression of that gene.", "This interference with expression of the protein can result from a variety of mechanisms, such as spatial separation of the htrb gene from the promoter element or an internal stop codon.", "Moreover, the transgene can be made wherein the coding sequence of the gene is flanked by recombinase recognition sequences and is initially transfected into cells in a 3′ to 5′ orientation with respect to the promoter element.", "In such an instance, inversion of the target sequence will reorient the subject gene by placing the 5′ end of the coding sequence in an orientation with respect to the promoter element which allow for promoter driven transcriptional activation.", "The transgenic animals of the present invention all include within a plurality of their cells a transgene of the present invention, which transgene alters the phenotype of the “host cell” with respect to regulation of cell growth, death and/or differentiation.", "Since it is possible to produce transgenic organisms of the invention utilizing one or more of the transgene constructs described herein, a general description will be given of the production of transgenic organisms by referring generally to exogenous genetic material.", "This general description can be adapted by those skilled in the art in order to incorporate specific transgene sequences into organisms utilizing the methods and materials described below.", "In an illustrative embodiment, either the cre/loxP recombinase system of bacteriophage P1 (Lakso et al.", "(1992) PNAS 89:6232-6236; Orban et al.", "(1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.", "(1991) Science 251:1351-1355; PCT publication WO 92/15694) can be used to generate in vivo site-specific genetic recombination systems.", "Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxp sequences.", "loxp sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination.", "The orientation of loxp sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al.", "(1984) J. Biol.", "Chem.", "259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxp sequences are oriented as inverted repeats.", "Accordingly, genetic recombination of the target sequence is dependent on expression of the Cre recombinase.", "Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added agents.", "This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element.", "Thus, the activation expression of a recombinant htrb protein can be regulated via control of recombinase expression.", "Use of the cre/loxP recombinase system to regulate expression of a recombinant htrb protein requires the construction of a transgenic animal containing transgenes encoding both the Cre recombinase and the subject protein.", "Animals containing both the Cre recombinase and a recombinant htrb gene can be provided through the construction of “double” transgenic animals.", "A convenient method for providing such animals is to mate two transgenic animals each containing a transgene, e.g., a htrb gene and recombinase gene.", "One advantage derived from initially constructing transgenic animals containing a htrb transgene in a recombinase-mediated expressible format derives from the likelihood that the subject protein, whether agonistic or antagonistic, can be deleterious upon expression in the transgenic animal.", "In such an instance, a founder population, in which the subject transgene is silent in all tissues, can be propagated and maintained.", "Individuals of this founder population can be crossed with animals expressing the recombinase in, for example, one or more tissues and/or a desired temporal pattern.", "Thus, the creation of a founder population in which, for example, an antagonistic htrb transgene is silent will allow the study of progeny from that founder in which disruption of htrb mediated induction in a particular tissue or at certain developmental stages would result in, for example, a lethal phenotype.", "Similar conditional transgenes can be provided using prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the htrb transgene.", "Exemplary promoters and the corresponding trans-activating prokaryotic proteins are given in U.S. Pat.", "No.", "4,833,080.Moreover, expression of the conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the trans-activating protein, e.g.", "a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner.", "By this method, an htrb transgene could remain silent into adulthood until “turned on” by the introduction of the trans-activator.", "In an exemplary embodiment, the “transgenic non-human animals” of the invention are produced by introducing transgenes into the germline of the non-human animal.", "Embryonal target cells at various developmental stages can be used to introduce transgenes.", "Different methods are used depending on the stage of development of the embryonal target cell.", "The specific line(s) of any animal used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness.", "In addition, the haplotype is a significant factor.", "For example, when transgenic mice are to be produced, strains such as C57BL/6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, Me.).", "Preferred strains are those with H-2b, H-2d or H-2q haplotypes such as C57BL/6 or DBA/1.The line(s) used to practice this invention may themselves be transgenics, and/or may be knockouts (i.e., obtained from animals which have one or more genes partially or completely suppressed) .", "In one embodiment, the transgene construct is introduced into a single stage embryo.", "The zygote is the best target for micro-injection.", "In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1-2pl of DNA solution.", "The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al.", "(1985) PNAS 82:4438-4442).", "As a consequence, all cells of the transgenic animal will carry the incorporated transgene.", "This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.", "Normally, fertilized embryos are incubated in suitable media until the pronuclei appear.", "At about this time, the nucleotide sequence comprising the transgene is introduced into the female or male pronucleus as described below.", "In some species such as mice, the male pronucleus is preferred.", "It is most preferred that the exogenous genetic material be added to the male DNA complement of the zygote prior to its being processed by the ovum nucleus or the zygote female pronucleus.", "It is thought that the ovum nucleus or female pronucleus release molecules which affect the male DNA complement, perhaps by replacing the protamines of the male DNA with histones, thereby facilitating the combination of the female and male DNA complements to form the diploid zygote.", "Thus, it is preferred that the exogenous genetic material be added to the male complement of DNA or any other complement of DNA prior to its being affected by the female pronucleus.", "For example, the exogenous genetic material is added to the early male pronucleus, as soon as possible after the formation of the male pronucleus, which is when the male and female pronuclei are well separated and both are located close to the cell membrane.", "Alternatively, the exogenous genetic material could be added to the nucleus of the sperm after it has been induced to undergo decondensation.", "Sperm containing the exogenous genetic material can then be added to the ovum or the decondensed sperm could be added to the ovum with the transgene constructs being added as soon as possible thereafter.", "Introduction of the transgene nucleotide sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.", "Following introduction of the transgene nucleotide sequence into the embryo, the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both.", "In vitro incubation to maturity is within the scope of this invention.", "One common method in to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.", "For the purposes of this invention a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism.", "Generally, the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete or gametes.", "Thus, the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism.", "Generally, a euploid zygote is preferred.", "If an aneuploid zygote is obtained, then the number of chromosomes should not vary by more than one with respect to the euploid number of the organism from which either gamete originated.", "In addition to similar biological considerations, physical ones also govern the amount (e.g., volume) of exogenous genetic material which can be added to the nucleus of the zygote or to the genetic material which forms a part of the zygote nucleus.", "If no genetic material is removed, then the amount of exogenous genetic material which can be added is limited by the amount which will be absorbed without being physically disruptive.", "Generally, the volume of exogenous genetic material inserted will not exceed about 10 picoliters.", "The physical effects of addition must not be so great as to physically destroy the viability of the zygote.", "The biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.", "The number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur.", "Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1,000-20,000 copies of the transgene construct, in order to insure that one copy is functional.", "As regards the present invention, there will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences.", "Any technique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, nuclear membrane or other existing cellular or genetic structures.", "The exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection.", "Microinjection of cells and cellular structures is known and is used in the art.", "Reimplantation is accomplished using standard methods.", "Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct.", "The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.", "Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method.", "Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene.", "Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product.", "Typically, DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene.", "Alternatively, the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.", "Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like.", "Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.", "Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal.", "Where mating with a partner is to be performed, the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both.", "Alternatively, the partner may be a parental line.", "Where in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both.", "Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.", "The transgenic animals produced in accordance with the present invention will include exogenous genetic material.", "As set out above, the exogenous genetic material will, in certain embodiments, be a DNA sequence which results in the production of a htrb protein (either agonistic or antagonistic), and antisense transcript, or a htrb mutant.", "Further, in such embodiments the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.", "Retroviral infection can also be used to introduce transgene into a non-human animal.", "The developing non-human embryo can be cultured in vitro to the blastocyst stage.", "During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).", "Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds.", "(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).", "The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al.", "(1985) PNAS 82:6927-6931; Van der Putten et al.", "(1985) PNAS 82:6148-6152).", "Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al.", "(1987) EMBO J.", "6:383-388).", "Alternatively, infection can be performed at a later stage.", "Virus or virus-producing cells can be injected into the blastocoele (Jahner et al.", "(1982) Nature 298:623-628).", "Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal.", "Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring.", "In addition, it is also possible to introduce transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al.", "(1982) supra).", "A third type of target cell for transgene introduction is the embryonal stem cell (ES).", "ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al.", "(1981) Nature 292:154-156; Bradley et al.", "(1984) Nature 309:255-258; Gossler et al.", "(1986) PNAS 83: 9065-9069; and Robertson et al.", "(1986) Nature 322:445-448).", "Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction.", "Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal.", "The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.", "For review see Jaenisch, R. (1988) Science 240:1468-1474.4.7.Screening Assays for htrb Therapeutics The invention further provides screening methods for identifying htrb therapeutics, e.g., for treating and/or preventing the development of diseases or conditions caused by, or contributed to by an abnormal htrb activity or which can benefit from a modulation of an htrb activity or protein level.", "Examples of such diseases, conditions or disorders such as those involving the inflammatory response inluding without limitation: rheumatoid arthritis, inflammatory bowel disorder, Type I diabetes, psoriasis, osteoporosis, nephropathy in diabetes mellitus, alopecia areata, Graves disease, systemic lupus erythematosus, lichen sclerosis, ulcerative colitis, coronary artery disease, arteritic disorders, diabetic retinopathy, low birth weight, pregnancy complications, severe periodontal disease, psoriasis and insulin dependent diabetes, but is particularly characterized by arteritic lesions, cancer e.g., cancers involving the growth of steroid hormone-responsive tumors (e.g.", "breast, prostate, or testicular cancer), vascular diseases or disorders (e.g.", "thrombotic stroke, ischemic stroke, as well as peripheral vascular disease resulting from atherosclerotic and thrombotic processes), cardiac disorders (e.g., myocardial infarction, congestive heart failure, unstable angina and ishemic heart disease); and cardiovascular system diseases and disorders (e.g.", "those resulting from hypertension, hypotension, cardiomyocyte hypertrophy and congestive heart failure) or other diseases conditions or disorders which result from aberrations or alterations of htrb-dependent processes.", "An htrb therapeutic can be any type of compound, including a protein, a peptide, peptidomimetic, small molecule, and nucleic acid.", "A nucleic acid can be, e.g., a gene, an antisense nucleic acid, a ribozyme, or a triplex molecule.", "An htrb.therapeutic of the invention can be an agonist or an antagonist.", "Preferred htrb agonists include htrb proteins or derivatives thereof which mimic at least one htrb activity, e.g., fibroblast growth fa,ctor receptor binding or heparin sulfate binding.", "Other preferred agonists include compounds which are capable of increasing the production of an htrb protein in a cell, e.g., compounds capable of up-regulating the expression of an htrb gene, and compounds which are capable of enhancing an htrb activity and/or the interaction of an htrb protein with another molecule, such as a target peptide.", "Preferred htrb antagonists include htrb proteins which are dominant negative proteins, which, e.g., are capable of binding to fibroblast growth factor receptors, but not heparin sulfate.", "Other preferred antagonists include compounds which decrease or inhibit the production of an htrb protein in a cell and compounds which are capable of downregulating expression of an htrb gene, and compounds which are capable of downregulating an htrb activity and/or interaction of an htrb protein with another molecule.", "In another preferred embodiment, an htrb antagonist is a modified form of a target peptide, which is capable of interacting with the FGFR binding domain of an htrb protein, but which does not have biological activity, e.g., which is not itself a cell surface receptor.", "The invention also provides screening methods for identifying htrb therapeutics which are capable of binding to an htrb protein, e.g., a wild-type htrb protein or a mutated form of an htrb protein, and thereby modulate the growth factor activity of htrb or otherwise cause the degradation of htrb.", "For example, such an htrb therapeutic can be an antibody or derivative thereof which interacts specifically with an htrb protein (either wild-type or mutated).", "Thus, the invention provides screening methods for identifying htrb agonist and antagonist compounds, comprising selecting compounds which are capable of interacting with an htrb protein or with a molecule capable of interacting with an htrb protein such as an FGF receptor and/or heparin sulfate and/or a compound which is capable of modulating the interaction of an htrb protein with another molecule, such as a receptor and/or heparin sulfate.", "In general, a molecule which is capable of interacting with an htrb protein is referred to herein as “htrb binding partner”.", "The compounds of the invention can be identified using various assays depending on the type of compound and activity of the compound that is desired.", "In addition, as described herein, the test compounds can be further tested in animal models.", "Set forth below are at least some assays that can be used for identifying htrb therapeutics.", "It is within the skill of the art to design additional assays for identifying htrb therapeutics.", "4.7.1.Cell-Free Assays Cell-free assays can be used to identify compounds which are capable of interacting with an htrb protein or binding partner, to thereby modify the activity of the htrb protein or binding partner.", "Such a compound can, e.g., modify the structure of an htrb protein or binding partner and thereby effect its activity.", "Cell-free assays can also be used to identify compounds which modulate the interaction between an htrb protein and an htrb binding partner, such as a target peptide.", "In a preferred embodiment, cell-free assays for identifying such compounds consist essentially in a reaction mixture containing an htrb protein and a test compound or a library of test compounds in the presence or absence of a binding partner.", "A test compound can be, e.g., a derivative of an htrb binding partner, e.g., a biologically inactive target peptide, or a small molecule.", "Accordingly, one exemplary screening assay of the present invention includes the steps of contacting an htrb protein or functional fragment thereof or an htrb binding partner with a test compound or library of test compounds and detecting the formation of complexes.", "For detection purposes, the molecule can be labeled with a specific marker and the test compound or library of test compounds labeled with a different marker.", "Interaction of a test compound with an htrb protein or fragment thereof or htrb binding partner can then be detected by determining the level of the two labels after an incubation step and a washing step.", "The presence of two labels after the washing step is indicative of an interaction.", "An interaction between molecules can also be identified by using real-time BIA (Biomolecular Interaction Analysis, Pharmacia Biosensor AB) which detects surface plasmon resonance (SPR), an optical phenomenon.", "Detection depends on changes in the mass concentration of macromolecules at the biospecific interface, and does not require any labeling of interactants.", "In one embodiment; a library of test compounds can be immobilized on a sensor surface, e.g., which forms one wall of a micro-flow cell.", "A solution containing the htrb protein, functional fragment thereof, htrb analog or htrb binding partner is then flown continuously over the sensor surface.", "A change in the resonance angle as shown on a signal recording, indicates that an interaction has occurred.", "This technique is further described, e.g., in BIAtechnology Handbook by Pharmacia.", "Another exemplary screening assay of the present invention includes the steps of (a) forming a reaction mixture including: (i) an htrb polypeptide, (ii) an htrb binding partner, and (iii) a test compound; and (b) detecting interaction of the htrb and the htrb binding protein.", "The htrb polypeptide and htrb binding partner can be produced recombinantly, purified from a source, e.g., plasma, or chemically synthesized, as described herein.", "A statistically significant change (otentiation or inhibition) in the interaction of the htrb and htrb binding protein in the presence of the test compound, relative to the interaction in the absence of the test compound, indicates a potential agonist (mimetic or potentiator) or antagonist (inhibitor) of htrb bioactivity for the test compound.", "The compounds of this assay can be contacted simultaneously.", "Alternatively, an htrb protein can first be contacted with a test compound for an appropriate amount of time, following which the htrb binding partner is added to the reaction mixture.", "The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.", "Moreover, a control assay can also be performed to provide a baseline for comparison.", "In the control assay, isolated and purified htrb polypeptide or binding partner is added to a composition containing the htrb binding partner or htrb polypeptide, and the formation of a complex is quantitated in the absence of the test compound.", "Complex formation between an htrb protein and an htrb binding partner may be detected by a variety of techniques.", "Modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled htrb proteins or htrb binding partners, by immunoassay, or by chromatographic detection.", "Typically, it will be desirable to immobilize either htrb or its binding partner to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.", "Binding of htrb to an htrb binding partner, can be accomplished in any vessel suitable for containing the reactants.", "Examples include microtitre plates, test tubes, and micro-centrifuge tubes.", "In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.", "For example, glutathione-S-transferase/htrb (GST/htrb) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)", "or glutathione derivatized microtitre plates, which are then combined with the htrb binding partner, e.g.", "an 35S-labeled htrb binding partner, and the test compound, and the mixture incubated under conditions conducive to complex formation, e.g.", "at physiological conditions for salt and pH, though slightly more stringent conditions may be desired.", "Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g.", "beads placed in scintilant), or in the supernatant after the complexes are subsequently dissociated.", "Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of htrb protein or htrb binding partner found in the bead fraction quantitated from the gel using standard electrophoretic techniques such as described in the appended examples.", "Other techniques for immobilizing proteins on matrices are also available for use in the subject assay.", "For instance, either htrb or its cognate binding partner can be immobilized utilizing conjugation of biotin and streptavidin.", "For instance, biotinylated htrb molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).", "Alternatively, antibodies reactive with htrb can be derivatized to the wells of the plate, and htrb trapped in the wells by antibody conjugation.", "As above, preparations of an htrb binding protein and a test compound are incubated in the htrb presenting wells of the plate, and the amount of complex trapped in the well can be quantitated.", "Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the htrb binding partner, or which are reactive with htrb protein and compete with the binding partner; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity.", "In the instance of the latter, the enzyme can be chemically conjugated or provided as a fusion protein with the htrb binding partner.", "To illustrate, the htrb binding partner can be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of polypeptide trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g.", "3,3′-diamino-benzadine terahydrochloride or 4-chloro-1-napthol.", "Likewise, a fusion protein comprising the polypeptide and glutathione-S-transferase can be provided, and complex formation quantitated by detecting the GST activity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).", "For processes which rely on immunodetection for quantitating one of the proteins trapped in the complex, antibodies against the protein, such as anti-htrb antibodies, can be used.", "Alternatively, the protein to be detected in the complex can be “epitope tagged” in the form of a fusion protein which includes, in addition to the htrb sequence, a second polypeptide for which antibodies are readily available (e.g.", "from commercial sources).", "For instance, the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety.", "Other useful epitope tags include myc-epitopes (e.g., see Ellison et al.", "(1991) J Biol Chem 266:21150-21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharmacia, N.J.).", "Cell-free assays can also be used to identify compounds which interact with an htrb protein and modulate an activity of an htrb protein.", "Accordingly, in one embodiment, an htrb protein is contacted with a test compound and the catalytic activity of htrb is monitored.", "In one embodiment, the abililty of htrb to bind a target molecule is determined.", "The binding affinity of htrb to a target molecule can be determined according to methods known in the art.", "4.7.2.Cell Based Assays In addition to cell-free assays, such as described above, htrb proteins as provided by the present invention, facilitate the generation of cell-based assays, e.g., for identifying small molecule agonists or antagonists.", "In one embodiment, a cell expressing an htrb receptor protein on the outer surface of its cellular membrane is incubated in the presence of a test compound alone or in the presence of a test compound and an htrb protein and the interaction between the test compound and the htrb receptor protein or between the htrb protein (preferably a tagged htrb protein) and the htrb receptor is detected, e.g., by using a microphysiometer (McConnell et al.", "(1992) Science 257:1906).", "An interaction between the htrb receptor protein and either the test compound or the htrb protein is detected by the microphysiometer as a change in the acidification of the medium.", "This assay system thus provides a means of identifying molecular antagonists which, for example, function by interfering with htrb—htrb receptor interactions, as well as molecular agonist which, for example, function by activating an htrb receptor.", "Cell based assays can also be used to identify compounds which modulate expression of an htrb gene, modulate translation of an htrb mRNA, or which modulate the stability of an htrb mRNA or protein.", "Accordingly, in one embodiment, a cell which is capable of producing htrb, e.g., a choriocarcinoma cell line such as JEG-3, is incubated with a test compound and the amount of htrb produced in the cell medium is measured and compared to that produced from a cell which has not been contacted with the test compound.", "The specificity of the compound vis a vis htrb can be confirmed by various control analysis, e.g., measuring the expression of one or more control genes.", "Compounds which can be tested include small molecules, proteins, and nucleic acids.", "In particular, this assay can be used to determine the efficacy of htrb antisense molecules or ribozymes.", "In another embodiment, the effect of a test compound on transcription of an htrb gene is determined by transfection experiments using a reporter gene operatively linked to at least a portion of the promoter of an htrb gene.", "A promoter region of a gene can be isolated, e.g., from a genomic library according to methods known in the art.", "The reporter gene can be any gene encoding a protein which is readily quantifiable, e.g, the luciferase or CAT gene.", "Such reporter gene are well known in the art.", "In preferred embodiments, the invention provides cell-based assays employing the choriocarcinoma cell line JEG-3.Analysis of this cell line has shown that it produces a 17 kDa htrb polypeptide which is immunoprecipitated with rabbit anti-htrb polyclonal antiserum.", "Accordingly, this cell line can be adapted for screening assays for agents which up-regulate or down-regulate the expression of the htrb gene or otherwise affect the steady-state level of an htrb polypeptide(s) or the efficiency of an htrb polypeptide post-translational activity such as an htrb proteolytic processing event, htrb glycosylation, htrb phosphorylation or htrb secretion.", "Other cell-based screening assays which can be employed in the method of the present invention are known or would be apparent to one of skill in the art.", "For example, htrb agonists and antagonists may be identified by their ability to affect downstream AP-1 dependent transcriptional activation.", "AP-1 dependent activation occurs by multiple mechanisms.", "This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.", "4.8.Predictive Medicine The invention further features predictive medicines, which are based, at least in part, on the identity of the novel htrb genes and alterations in the genes and related pathway genes, which affect the expression level and/or function of the encoded htrb protein in a subject.", "For example, information obtained using the diagnostic assays described herein (alone or in conjunction with information on another genetic defect, which contributes to the same disease) is useful for diagnosing or confirming that a symptomatic subject (e.g.", "a subject symptomatic for inflammatory rheumatoid arthritis), has a genetic defect (e.g.", "in an htrb gene or in a gene that regulates the expression of an htrb gene), which causes or contributes to the particular disease or disorder.", "Alternatively, the information (alone or in conjunction with information on another genetic defect, which contributes to the same disease) can be used prognostically for predicting whether a non-symptomatic subject is likely to develop a disease or condition, which is caused by or contributed to by an abnormal htrb activity or protein level in a subject.", "Based on the prognostic information, a doctor can recommend a regimen (e.g.", "diet or exercise) or therapeutic protocol, useful for preventing or prolonging onset of the particular disease or condition in the individual.", "In addition, knowledge of the particular alteration or alterations, resulting in defective or deficient htrb genes or proteins in an individual (the htrb genetic profile), alone or in conjunction with information on other genetic defects contributing to the same disease (the genetic profile of the particular disease) allows customization of therapy for a particular disease to the individual's genetic profile, the goal of “pharmacogenomics”.", "For example, an individual's htrb genetic profile or the genetic profile of a disease or condition, to which htrb genetic alterations cause or contribute, can enable a doctor to 1) more effectively prescribe a drug that will address the molecular basis of the disease or condition; and 2) better determine the appropriate dosage of a particular drug.", "For example, the expression level of htrb proteins, alone or in conjunction with the expression level of other genes, known to contribute to the same disease, can be measured in many patients at various stages of the disease to generate a transcriptional or expression profile of the disease.", "Expression patterns of individual patients can then be compared to the expression profile of the disease to determine the appropriate drug and dose to administer to the patient.", "The ability to target populations expected to show the highest clinical benefit, based on the htrb or disease genetic profile, can enable: 1) the repositioning of marketed drugs with disappointing market results; 2) the rescue of drug candidates whose clinical development has been discontinued as a result of safety or efficacy limitations, which are patient subgroup-specific; and 3) an accelerated and less costly development for drug candidates and more optimal drug labeling (e.g.", "since the use of htrb as a marker is useful for optimizing effective dose).", "These and other methods are described in further detail in the following sections.", "4.8.1.Prognostic and Diagnostic Assays The present methods provide means for determining if a subject has (diagnostic) or is at risk of developing (prognostic) a disease, condition or disorder that is associated with an aberrant htrb activity, e.g., an aberrant level of htrb protein or an aberrant htrb bioactivity.", "Examples of such diseases, conditions or disorders include without limitation: inflammatory diseases including rheumatoid arthritis, inflammatory bowel disorder, psoriasis, lupus erythematosus, ulcerative colitis and alopecia areata as well as diabetic nephropathy; cancers involving the growth factor cytokines or steroid hormone-responsive tumors (e.g.", "breast, prostate, or testicular cancer); vascular diseases or disorders (e.g.", "thrombotic stroke, ischemic stroke, as well as peripheral vascular disease resulting from atherosclerotic and thrombotic processes); cardiac disorders (e.g.", "myocardial infarction, unstable angina and ishemic heart disease); cardiovascular system diseases and disorders (e.g.", "those resulting from hypertension, hypotension, cardiomyocyte hypertrophy and congestive heart failure) wound healing; limb regeneration; neurological damage or disease (e.g.", "that associated with Alzheimer's disease, Parkinson's disease, AIDS-related complex, or cerebral palsy); or other diseases conditions or disorders which result from aberrations or alterations of htrb-dependent processes including: collateral growth and remodeling of cardiac blood vessels, angiogenesis, cellular transformation through autocrine or paracrine mechanisms, chemotactic stimulation of cells (e.g.", "endothelial), neurite outgrowth of neuronal precursor cell types (e.g.", "PC12 phaeochromoctoma), maintenance of neural physiology of mature neurons, proliferation of embryonic mesenchyme and limb-bud precursor tissue, mesoderm induction and other developmental processes, stimulation of collagenase and plasminogen activator secretion, tumor vascularization, as well as tumor invasion and metastasis.", "Accordingly, the invention provides methods for determining whether a subject has or is likely to develop, a disease or condition that is caused by or contributed to by an abnormal htrb level or bioactivity, for example, comprising determining the level of an htrb gene or protein, an htrb bioactivity and/or the presence of a mutation or particular polymorphic variant in the htrb gene.", "In one embodiment, the method comprises determining whether a subject has an abnormal mRNA and/or protein level of htrb, such as by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, immunoprecipitation, Western blot hybridization, or immunohistochemistry.", "According to the method, cells are obtained from a subject and the htrb protein or mRNA level is determined and compared to the level of htrb protein or mRNA level in a healthy subject.", "An abnormal level of htrb polypeptide or mRNA level is likely to be indicative of an aberrant htrb activity.", "In another embodiment, the method comprises measuring at least one activity of htrb.", "For example, the affinity of htrb for heparin, can be determi+ned, e.g., as described herein.", "Similarly, the constant of affinity of an htrb protein of a subject with a binding partner (e.g.", "an IL-1 type I or type II receptor) can be determined.", "Comparison of the results obtained with results from similar analysis performed on htrb proteins from healthy subjects is indicative of whether a subject has an abnormal htrb activity.", "In preferred embodiments, the methods for determining whether a subject has or is at risk for developing a disease, which is caused by or contributed to by an aberrant htrb activity is characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of (i) an alteration affecting the integrity of a gene encoding an htrb polypeptide, or (ii) the mis-expression of the htrb gene.", "For example, such genetic alterations can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from an htrb gene, (ii) an addition of one or more nucleotides to an htrb gene, (iii) a substitution of one or more nucleotides of an htrb gene, (iv) a gross chromosomal rearrangement of an htrb gene, (v) a gross alteration in the level of a messenger RNA transcript of an htrb gene, (vii) aberrant modification of an htrb gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild type splicing pattern of a messenger RNA transcript of an htrb gene, (viii) a non-wild type level of an htrb polypeptide, (ix) allelic loss of an htrb gene, and/or (x) inappropriate post-translational modification of an htrb polypeptide.", "As set out below, the present invention provides a large number of assay techniques for detecting alterations in an htrb gene.", "These methods include, but are not limited to, methods involving sequence analysis, Southern blot hybridization, restriction enzyme site mapping, and methods involving detection of absence of nucleotide pairing between the nucleic acid to be analyzed and a probe.", "These and other methods are further described infra.", "Specific diseases or disorders, e.g., genetic diseases or disorders, are associated with specific allelic variants of polymorphic regions of certain genes, which do not necessarily encode a mutated protein.", "Thus, the presence of a specific allelic variant of a polymorphic region of a gene, such as a single nucleotide polymorphism (“SNP”), in a subject can render the subject susceptible to developing a specific disease or disorder.", "Polymorphic regions in genes, e.g, htrb genes, can be identified, by determining the nucleotide sequence of genes in populations of individuals.", "If a polymorphic region, e.g., SNP is identified, then the link with a specific disease can be determined by studying specific populations of individuals, e.g, individuals which developed a specific disease, such as congestive heart failure, hypertension, hypotension, or a cancer (e.g.", "a cancer involving growth of a steroid responsive tumor or tumors).", "A polymorphic region can be located in any region of a gene, e.g., exons, in coding or non coding regions of exons, introns, and promoter region.", "It is likely that htrb genes comprise polymorphic regions, specific alleles of which may be associated with specific diseases or conditions or with an increased likelihood of developing such diseases or conditions.", "Thus, the invention provides methods for determining the identity of the allele or allelic variant of a polymorphic region of an htrb gene in a subject, to thereby determine whether the subject has or is at risk of developing a disease or disorder associated with a specific allelic variant of a polymorphic region.", "In an exemplary embodiment, there is provided a nucleic acid composition comprising a nucleic acid probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of an htrb gene or naturally occurring mutants thereof, or 5′ or 3′ flanking sequences or intronic sequences naturally associated with the subject htrb genes or naturally occurring mutants thereof.", "The nucleic acid of a cell is rendered accessible for hybridization, the probe is contacted with the nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected.", "Such techniques can be used to detect alterations or allelic variants at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.", "A preferred detection method is allele specific hybridization using probes overlapping the mutation or polymorphic site and having about 5, 10, 20, 25, or 30 nucleotides around the mutation or polymorphic region.", "In a preferred embodiment of the invention, several probes capable of hybridizing specifically to allelic variants, such as single nucleotide polymorphisms, are attached to a solid phase support, e.g., a “chip”.", "Oligonucleotides can be bound to a solid support by a variety of processes, including lithography.", "For example a chip can hold up to 250,000 oligonucleotides.", "Mutation detection analysis using these chips comprising oligonucleotides, also termed “DNA probe arrays” is described e.g., in Cronin et al.", "(1996) Human Mutation 7:244.In one embodiment, a chip comprises all the allelic variants of at least one polymorphic region of a gene.", "The solid phase support is then contacted with a test nucleic acid and hybridization to the specific probes is detected.", "Accordingly, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridization experiment.", "In certain embodiments, detection of the alteration comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e.g.", "U.S. Pat.", "Nos.", "4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligase chain reaction (LCR) (see, e.g., Landegran et al.", "(1988) Science 241:1077-1080; and Nakazawa et al.", "(1994) PNAS 91:360-364), the latter of which can be particularly useful for detecting point mutations in the htrb gene (see Abravaya et al.", "(1995) Nuc Acid Res 23:675-682).", "In a merely illustrative embodiment, the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to an htrb gene under conditions such that hybridization and amplification of the htrb gene (if present) occurs, and (iv) detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.", "It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.", "Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al., 1990, Proc.", "Natl.", "Acad.", "Sci.", "USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., 1989, Proc.", "Natl.", "Acad.", "Sci.", "USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art.", "These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.", "In a preferred embodiment of the subject assay, mutations in, or allelic variants, of an htrb gene from a sample cell are identified by alterations in restriction enzyme cleavage patterns.", "For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis.", "Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat.", "No.", "5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.", "In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the htrb gene and detect mutations by comparing the sequence of the sample htrb with the corresponding wild-type (control) sequence.", "Exemplary sequencing reactions include those based on techniques developed by Maxim and Gilbert (Proc.", "Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al (1977) Proc.", "Nat.", "Acad.", "Sci 74:5463).", "It is also contemplated that any of a variety of automated sequencing procedures may be utilized when performing the subject assays (Biotechniques (1995) 19:448), including sequencing by mass spectrometry (see, for example PCT publication WO 94/16101; Cohen et al.", "(1996) Adv Chromatogr 36:127-162; and Griffin et al.", "(1993) Appl Biochem Biotechnol 38:147-159).", "It will be evident to one skilled in the art that, for certain embodiments, the occurrence of only one, two or three of the nucleic acid bases need be determined in the sequencing reaction.", "For instance, A-track or the like, e.g., where only one nucleic acid is detected, can be carried out.", "In a further embodiment, protection from cleavage agents (such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine) can be used to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNA heteroduplexes (Myers, et al.", "(1985) Science 230:1242).", "In general, the art technique of“mismatch cleavage” starts by providing heteroduplexes formed by hybridizing (labelled) RNA or DNA containing the wild-type htrb sequence with potentially mutant RNA or DNA obtained from a tissue sample.", "The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands.", "For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digest the mismatched regions.", "In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions.", "After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation.", "See, for example, Cotton et al (1988) Proc.", "Natl Acad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymol.", "217:286-295.In a preferred embodiment, the control DNA or RNA can be labeled for detection.", "In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in htrb cDNAs obtained from samples of cells.", "For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.", "(1994) Carcinogenesis 15:1657-1662).", "According to an exemplary embodiment, a probe based on an htrb sequence, e.g., a wild-type htrb sequence, is hybridized to a cDNA or other DNA product from a test cell(s).", "The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like.", "See, for example, U.S. Pat.", "No.", "5,459,039.In other embodiments, alterations in electrophoretic mobility will be used to identify mutations or the identity of the allelic variant of a polymorphic region in htrb genes.", "For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al.", "(1989) Proc Natl.", "Acad.", "Sci USA 86:2766, see also Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79).", "Single-stranded DNA fragments of sample and control htrb nucleic acids are denatured and allowed to renature.", "The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.", "The DNA fragments may be labelled or detected with labelled probes.", "The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.", "In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al.", "(1991) Trends Genet 7:5).", "In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985) Nature 313:495).", "When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.", "In a further embodiment, a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).", "Examples of other techniques for detecting point mutations or the identity of the allelic variant of a polymorphic region include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension.", "For example, oligonucleotide primers may be prepared in which the known mutation or nucleotide difference (e.g., in allelic variants) is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al.", "(1986) Nature 324:163); Saiki et al (1989) Proc.", "Natl Acad.", "Sci USA 86:6230).", "Such allele specific oligonucleotide hybridization techniques may be used to test one mutation or polymorphic region per reaction when oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations or polymorphic regions when the oligonucleotides are attached to the hybridizing membrane and hybridized with labelled target DNA.", "Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention.", "Oligonucleotides used as primers for specific amplification may carry the mutation or polymorphic region of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al (1989) Nucleic Acids Res.", "17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238.In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al (1992) Mol.", "Cell Probes 6:1).", "It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) ?roc.", "Natl.", "Acad.", "Sci USA 88:189).", "In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.", "In another embodiment, identification of the allelic variant is carried out using an oligonucleotide ligation assay (OLA), as described, e.g., in U.S. Pat.", "No.", "4,998,617 and in Landegren, U. et al., Science 241:1077-1080 (1988).", "The OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target.", "One of the oligonucleotides is linked to a separation marker, e.g,.", "biotinylated, and the other is detectably labeled.", "If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate.", "Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand.", "Nickerson, D. A. et al.", "have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.) 87:8923-8927 (1990).", "In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.", "Several techniques based on this OLA method have been developed and can be used to detect specific allelic variants of a polymorphic region of an htrb gene.", "For example, U.S. Pat.", "No.", "5,593,826 discloses an OLA using an oligonucleotide having 3′-amino group and a 5′-phosphorylated oligonucleotide to form a conjugate having a phosphoramidate linkage.", "In another variation of OLA described in Tobe et al.", "((1996) Nucleic Acids Res 24: 3728), OLA combined with PCR permits typing of two alleles in a single microtiter well.", "By marking each of the allele-specific primers with a unique hapten, i.e.", "digoxigenin and fluorescein, each OLA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase.", "This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors.", "The invention further provides methods for detecting single nucleotide polymorphisms in an htrb gene.", "Because single nucleotide polymorphisms constitute sites of variation flanked by regions of invariant sequence, their analysis requires no more than the determination of the identity of the single nucleotide present at the site of variation and it is unnecessary to determine a complete gene sequence for each patient.", "Several methods have been developed to facilitate the analysis of such single nucleotide polymorphisms.", "In one embodiment, the single base polymorphism can be detected by using a specialized exonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R. (U.S. Pat.", "No.", "4,656,127).", "According to the method, a primer complementary to the allelic sequence immediately 3′ to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human.", "If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridized primer.", "Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection.", "Since the identity of the exonuclease-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymorphic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction.", "This method has the advantage that it does not require the determination of large amounts of extraneous sequence data.", "In another embodiment of the invention, a solution-based method is used for determining the identity of the nucleotide of a polymorphic site.", "Cohen, D. et al.", "(French Patent No.", "2,650,840; PCT Appln.", "No.", "WO91/02087).", "As in the Mundy method of U.S. Pat.", "No.", "4,656,127, a primer is employed that is complementary to allelic sequences immediately 3′ to a polymorphic site.", "The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.", "An alternative method, known as Genetic Bit Analysis or GBA™ is described by Goelet, P. et al.", "(PCT Appln.", "No.", "92/15712).", "The method of Goelet, P. et al.", "uses mixtures of labeled terminators and a primer that is complementary to the sequence 3′ to a polymorphic site.", "The labeled terminator that is incorporated is thus determined by, and complementary to, the nucleotide present in the polymorphic site of the target molecule being evaluated.", "In contrast to the method of Cohen et al.", "(French Patent 2,650,840; PCT Appln.", "No.", "WO91/02087) the method of Goelet, P. et al.", "is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.", "Recently, several primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (Komher, J. S. et al., Nucl.", "Acids.", "Res.", "17:7779-7784 (1989); Sokolov, B. P., Nucl.", "Acids Res.", "18:3671 (1990); Syvanen, A.", "-C., et al., Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.) 88:1143-1147 (1991); Prezant, T. R. et al., Hum.", "Mutat.", "1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal.", "Biochem.", "208:171-175 (1993)).", "These methods differ from GBA TM in that they all rely on the incorporation of labeled deoxynucleotides to discriminate between bases at a polymorphic site.", "In such a format, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms that occur in runs of the same nucleotide can result in signals that are proportional to the length of the run (Syvanen, A.", "-C., et al., Amer.J.", "Hum.", "Genet.", "52:46-59 (1993)).", "For mutations that produce premature termination of protein translation, the protein truncation test (PT) offers an efficient diagnostic approach (Roest, et.", "al., (1993) Hum.", "Mol.", "Genet.", "2:1719-21; van der Luijt, et.", "al., (1994) Genomics 20:1-4).", "For PTT, RNA is initially isolated from available tissue and reverse-transcribed, and the segment of interest is amplified by PCR.", "The products of reverse transcription PCR are then used as a template for nested PCR amplification with a primer that contains an RNA polymerase promoter and a sequence for initiating eukaryotic translation.", "After amplification of the region of interest, the unique motifs incorporated into the primer permit sequential in vitro transcription and translation of the PCR products.", "Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of translation products, the appearance of truncated polypeptides signals the presence of a mutation that causes premature termination of translation.", "In a variation of this technique, DNA (as opposed to RNA) is used as a PCR template when the target region of interest is derived from a single exon.", "The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid, primer set; and/or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an htrb polypeptide.", "Any cell type or tissue may be utilized in the diagnostics described below.", "In a preferred embodiment a bodily fluid, e.g., blood, is obtained from the subject to determine the presence of a mutation or the identity of the allelic variant of a polymorphic region of an htrb gene.", "A bodily fluid, e.g, blood, can be obtained by known techniques (e.g.", "venipuncture).", "Alternatively, nucleic acid tests can be performed on dry samples (e.g.", "hair or skin).", "For prenatal diagnosis, fetal nucleic acid samples can be obtained from maternal blood as described in International Patent Application No.", "WO91/07660 to Bianchi.", "Alternatively, amniocytes or chorionic villi may be obtained for performing prenatal testing.", "When using RNA or protein to determine the presence of a mutation or of a specific allelic variant of a polymorphic region of an htrb gene, the cells or tissues that may be utilized must express the htrb gene.", "Preferred cells for use in these methods include cardiac cells (see Examples).", "Alternative cells or tissues that can be used, can be identified by determining the expression pattern of the specific htrb gene in a subject, such as by Northern blot analysis.", "Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary.", "Nucleic acid reagents may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G. J., 1992, PCR in situ hybridization: protocols and applications, Raven Press, NY).", "In addition to methods which focus primarily on the detection of one nucleic acid sequence, profiles may also be assessed in such detection schemes.", "Fingerprint profiles may be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT-PCR.", "Antibodies directed against wild type or mutant htrb polypeptides or allelic variants thereof, which are discussed above, may also be used in disease diagnostics and prognostics.", "Such diagnostic methods, may be used to detect abnormalities in the level of htrb polypeptide expression, or abnormalities in the structure and/or tissue, cellular, or subcellular location of an htrb polypeptide.", "Structural differences may include, for example, differences in the size, electronegativity, or antigenicity of the mutant htrb polypeptide relative to the normal htrb polypeptide.", "Protein from the tissue or cell type to be analyzed may easily be detected or isolated using techniques which are well known to one of skill in the art, including but not limited to western blot analysis.", "For a detailed explanation of methods for carrying out Western blot analysis, see Sambrook et al, 1989, supra, at Chapter 18.The protein detection and isolation methods employed herein may also be such as those described in Harlow and Lane, for example, (Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety.", "This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection.", "The antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of htrb polypeptides.", "In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention.", "The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.", "Through the use of such a procedure, it is possible to determine not only the presence of the htrb polypeptide, but also its distribution in the examined tissue.", "Using the present invention, one of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.", "Often a solid phase support or carrier is used as a support capable of binding an antigen or an antibody.", "Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.", "The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.", "The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.", "Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.", "Alternatively, the surface may be flat such as a sheet, test strip, etc.", "Preferred supports include polystyrene beads.", "Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.", "One means for labeling an anti-htrb polypeptide specific antibody is via linkage to an enzyme and use in an enzyme immunoassay (EIA) (Voller, “The Enzyme Linked Immunosorbent Assay (ELISA)”, Diagnostic Horizons 2:1-7, 1978, Microbiological Associates Quarterly Publication, Walkersville, Md.", "; Voller, et al., J. Clin.", "Pathol.", "31:507-520 (1978); Butler, Meth.", "Enzymol.", "73:482-523 (1981); Maggio, (ed.)", "Enzyme Inmunoassay, CRC Press, Boca Raton, Fla., 1980; Ishikawa, et al., (eds.)", "Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981).", "The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.", "Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.", "The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme.", "Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.", "Detection may also be accomplished using any of a variety of other immunoassays.", "For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect fingerprint gene wild type or mutant peptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein).", "The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.", "It is also possible to label the antibody with a fluorescent compound.", "When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence.", "Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.", "The antibody can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series.", "These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).", "The antibody also can be detectably labeled by coupling it to a chemiluminescent compound.", "The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.", "Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.", "Likewise, a bioluminescent compound may be used to label the antibody of the present invention.", "Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction.", "The presence of a bioluminescent protein is determined by detecting the presence of luminescence.", "Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.", "Moreover, it will be understood that any of the above methods for detecting alterations in a gene or gene product or polymorphic variants can be used to monitor the course of treatment or therapy.", "4.8.2.Pharmacogenomics Knowledge of the particular alteration or alterations, resulting in defective or deficient htrb genes or proteins in an individual (the htrb genetic profile), alone or in conjunction with information on other genetic defects contributing to the same disease (the genetic profile of the particular disease) allows a customization of the therapy for a particular disease to the individual's genetic profile, the goal of “pharmacogenomics”.", "For example, subjects having a specific allele of an htrb gene may or may not exhibit symptoms of a particular disease or be predisposed of developing symptoms of a particular disease.", "Further, if those subjects are symptomatic, they may or may not respond to a certain drug, e.g., a specific htrb therapeutic, but may respond to another.", "Thus, generation of an htrb genetic profile, (e.g., categorization of alterations in htrb genes which are associated with the development of a particular disease), from a population of subjects, who are symptomatic for a disease or condition that is caused by or contributed to by a defective and/or deficient htrb gene and/or protein (an htrb genetic population profile) and comparison of an individual's htrb profile to the population profile, permits the selection or design of drugs that are expected to be safe and efficacious for a particular patient or patient population (i.e., a group of patients having the same genetic alteration).", "For example, an htrb population profile can be performed, by determining the htrb profile, e.g., the identity of htrb genes, in a patient population having a disease, which is caused by or contributed to by a defective or deficient htrb gene.", "Optionally, the htrb population profile can further include information relating to the response of the population to an htrb therapeutic, using any of a variety of methods, including, monitoring: 1) the severity of symptoms associated with the htrb related disease, 2) htrb gene expression level, 3) htrb mRNA level, and/or 4) htrb protein level.", "and (iii) dividing or categorizing the population based on the particular genetic alteration or alterations present in its htrb gene or an htrb pathway gene.", "The htrb genetic population profile can also, optionally, indicate those particular alterations in which the patient was either responsive or non-responsive to a particular therapeutic.", "This information or population profile, is then useful for predicting which individuals should respond to particular drugs, based on their individual htrb profile.", "In a preferred embodiment, the htrb profile is a transcriptional or expression level profile and step (i) is comprised of determining the expression level of htrb proteins, alone or in conjunction with the expression level of other genes, known to contribute to the same disease.", "The htrb profile can be measured in many patients at various stages of the disease.", "Pharmacogenomic studies can also be performed using transgenic animals.", "For example, one can produce transgenic mice, e.g., as described herein, which contain a specific allelic variant of an htrb gene.", "These mice can be created, e.g, by replacing their wild-type htrb gene with an allele of the human htrb gene.", "The response of these mice to specific htrb therapeutics can then be determined.", "4.8.3.Monitoring of Effects of htrb Therapeutics During Clinical Trials The ability to target populations expected to show the highest clinical benefit, based on the htrb or disease genetic profile, can enable: 1) the repositioning of marketed drugs with disappointing market results; 2) the rescue of drug candidates whose clinical development has been discontinued as a result of safety or efficacy limitations, which are patient subgroup-specific; and 3) an accelerated and less costly development for drug candidates and more optimal drug labeling (e.g.", "since the use of htrb as a marker is useful for optimizing effective dose).", "The treatment of an individual with an htrb therapeutic can be monitored by determining htrb characteristics, such as htrb protein level or activity, htrb mRNA level, and/or htrb transcriptional level.", "This measurements will indicate whether the treatment is effective or whether it should be adjusted or optimized.", "Thus, htrb can be used as a marker for the efficacy of a drug during clinical trials.", "In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a preadministration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an htrb protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the htrb protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the htrb protein, mRNA, or genomic DNA in the preadministration sample with the htrb protein, IRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.", "For example, increased administration of the agent may be desirable to increase the expression or activity of htrb to higher levels than detected, i.e., to increase the effectiveness of the agent.", "Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of htrb to lower levels than detected, i.e., to decrease the effectiveness of the agent.", "Cells of a subject may also be obtained before and after administration of an htrb therapeutic to detect the level of expression of genes other than htrb, to verify that the htrb therapeutic does not increase or decrease the expression of genes which could be deleterious.", "This can be done, e.g., by using the method of transcriptional profiling.", "Thus, mRNA from cells exposed in vivo to an htrb therapeutic and mRNA from the same type of cells that were not exposed to the htrb therapeutic could be reverse transcribed and hybridized to a chip containing DNA from numerous genes, to thereby compare the expression of genes in cells treated and not treated with an htrb-therapeutic.", "If, for example an htrb therapeutic turns on the expression of a proto-oncogene in an individual, use of this particular htrb therapeutic may be undesirable.", "4.9.htrb Therapeutics and Methods of Treatment The present invention provides for both prophylactic and therapeutic methods of treating a subject having or likely to develop a disorder associated with aberrant interleukin-1 expression or activity, e.g., inflammation or autoimmune disorders.", "The cytokines interleukin-1 (IL-1) and tumour necrosis factor (TNF) are important mediators of inflammatory responses, and appear to play a central role in the pathogenesis of many chronic inflammatory diseases.", "It is now well documented that their biological activities in vivo are sufficient to reproduce local inflammation and matrix catabolism by attracting and activating white blood cells to tissues, and stimulating their secretion of other lymphocytotropic cytokines and catabolic enzymes.", "Higher production of these cytokines have also been associated with response to infection, where local induction of IL-1 and TNF facilitates the elimination of the microbial invasion.", "Classic studies however also report that in some infectious conditions very high levels of monocytic cytokines are produced, which activate a cascade of concomitant events such as tissue catabolism, vascular reactivity and hyper-coagulation with damaging effects on the host.", "For example, cytokines function throughout development and may be of particular importance in the development and function of the human placenta (reviewed in Jokhi et al.", "(1997) Cytokine 9: 126-37).", "A variety of cytokines have been demonstrated at the placental-uterine interface, but the exact cellular sources of production have not yet been identified due to the complex tissue topography of the implantation site.", "The expression of the cytokines EGF, interleukin 1 beta (IL-1 beta), IL-2, IL-3, interferon alpha (IFN-alpha), IFN-gamma, tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta 1 (TGF-beta 1) have been assayed from cells isolated from the placenta and decidua.", "Furthermore, the expression of the cytokine receptors IGF-1r, PDGF-r alpha/beta, IL-1rII, IL-6r, IL-7r, IFN-gamma r, TNF-rp80 and endoglin by placental and uterine cells has been assessed by both immunohistological and flow cytometric methods.", "These studies reveal a complex array of cytokine activities at the human placental-uterine interface.", "The pro-inflammatory cytokines IL-1, IL-6 and tumor necrosis factor-alpha (TNFa) appear to function in the link between prenatal intrauterine infection (IUI) and neonatal brain damage.", "Furthermore, maternal IUI increases the risk of preterm delivery, which in turn is associated with an increased risk of intraventricular hemorrhage, neonatal white matter damage, and subsequent cerebal palsy (Dammann et al.", "(1997) Pediatr Res 42: 1-8).", "IL-1, IL-6, and TNFa have been found associated with IUI, preterm birth, neonatal infections, and neonatal brain damage.", "The presence of such cytokines in the three relevant maternal/fetal compartments (uterus, fetal circulation, and fetal brain) and their potential ability of the cytokines to cross boundaries (both placental and the blood-brain barrier) between these compartments suggests their potential role in intraventricular hemorrhage, neonatal white matter damage during prenatal maternal infection.", "Therefore interrupting the proinflammatory cytokine cascade mediated by IL-1 might prevent later disability in those born near the end of the second trimester.", "Interleukin-1 beta (IL-1 β) is present in normal amniotic fluid and is produced by human placental macrophages.", "The amount of IL-1 β detected in the second trimester amniotic fluid has been shown to exhibit a threefold increase with the onset of labor.", "IL-1 β is a potent stimulator of the synthesis of prostaglandins by decidua and by amnion.", "High levels of the prostaglandins PGE2 and PGF2α in the amniotic fluid have been associated with preterm labor and intraamniotic infection.", "This may be explained by the fact that amnion from women with preterm labor and histologic chorioamnionitis produced more PGE2 than amnion from women without placental inflammation.", "Such elevated levels of PGE2 have been associated with premature low birth weight (PBLW) even in the absence of clinical or subclinical genitourinary tract infection and indeed the majority of PLBW deliveries may be caused by an infection of unknown origin.", "IL-1 was the first cytokine implicated in the onset of labor in the presence of infection.", "IL-1 is produced in vitro by human decidua in response to bacterial products.", "In patients with preterm labor and bacteria in the amniotic cavity, amniotic fluid IL-1 bioactivity and concentrations are elevated.", "Placental necrosis and fetal resorption can be induced in rats by the injection of recombinant human IL-1 β on day 12 of gestation.", "Furthermore, both the amniotic fluid IL-1 β concentration and bioactivity are elevated during labor compared to controls.", "In addition, IL-1 β is known to stimulate prostaglandin production by amnion and decidua in vitro.", "Accordingly, htrb therapeutics of the present invention include those which antagonize interleukin-1 dependent disorders of the human placental including intraventricular hemorrhage, neonatal white matter damage and subsequent cerebal palsy, and the occurrence of premature low birth weight deliveries.", "4.9.1.Prophylactic Methods In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant htrb expression or activity by administering to the subject an agent which modulates htrb expression or at least one htrb activity.", "Subjects at risk for such a disease can be identified by a diagnostic or prognostic assay, e.g., as described herein.", "Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the htrb aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.", "Depending on the type of htrb aberrancy, for example, a htrb agonist or htrb antagonist agent can be used for treating the subject prophylactically.", "The prophylactic methods are similar to therapeutic methods of the present invention and are further discussed in the following subsections.", "4.9.2.Therapeutic Methods In general, the invention provides methods for treating a disease or condition which is caused by or contributed to by an aberrant htrb activity comprising administering to the subject an effective amount of a compound which is capable of modulating an htrb activity.", "Among the approaches which may be used to ameliorate disease symptoms involving an aberrant htrb activity are, for example, antisense, ribozyme, and triple helix molecules described above.", "Examples of suitable compounds include the antagonists, agonists or homologues described in detail herein.", "4.9.3.Effective Dose Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining The Ld50 (The Dose Lethal To 50% Of The Population) And The Ed50 (the dose therapeutically effective in 50% of the population).", "The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic induces are preferred.", "While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.", "The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.", "The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.", "The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.", "For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays.", "A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.", "Such information can be used to more accurately determine useful doses in humans.", "Levels in plasma may be measured, for example, by high performance liquid chromatography.", "4.9.4.Formulation and Use Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.", "Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, injection, inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.", "For such therapy, the compounds of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration.", "Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous.", "For injection, the compounds of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.", "In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use.", "Lyophilized forms are also included.", "For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate).", "The tablets may be coated by methods well known in the art.", "Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.", "Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).", "The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.", "Preparations for oral administration may be suitably formulated to give controlled release of the active compound.", "For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.", "For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.", "In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.", "Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.", "The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.", "Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.", "The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.", "Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.", "The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such, as cocoa butter or other glycerides.", "In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.", "Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.", "Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.", "Other suitable delivery systems include microspheres which offer the possiblity of local noninvasive delivery of drugs over an extended period of time.", "This technology utilizes microspheres of precapillary size which can be injected via a coronary chatheter into any selected part of the e.g.", "heart or other organs without causing inflammation or ischemia The administered therapeutic is slowly released from these microspheres and taken up by surrounding tissue cells (e.g.", "endothelial cells).", "Systemic administration can also be by transmucosal or transdermal means.", "For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.", "Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives, in addition, detergents may be used to facilitate permeation.", "Transmucosal administration may be through nasal sprays or using suppositories.", "For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.", "A wash solution can be used locally to treat an injury or inflammation to accelerate healing.", "In clinical settings, a gene delivery system for the therapeutic htrb gene can be introduced into a patient by any of a number of methods, each of which is familiar in the art.", "For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.", "In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.", "For example, the gene delivery vehicle can be introduced by catheter (see U.S. Pat.", "No.", "5,328,470) or by stereotactic injection (e.g., Chen et al.", "(1994) PNAS 91: 3054-3057).", "An htrb gene, such as any one of the sequences represented in the group consisting of SEQ ID Nos.", "1 or 3 or the htrb alternative 5′ ends or a sequence homologous thereto can be delivered in a gene therapy construct by electroporation using techniques described, for example, by Dev et al.", "((1994) Cancer Treat Rev 20:105-115).", "The pharmaceutical preparation of the gene therapy construct or compound of the invention can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle or compound is imbedded.", "Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.", "The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.", "The pack may for example comprise metal or plastic foil, such as a blister pack.", "The pack or dispenser device may be accompanied by instructions for administration.", "4.10.Kits The invention further provides kits for use in diagnostics or prognostic methods or for treating a disease or condition associated with an aberrant htrb protein.", "The invention also provides kits for determining which htrb therapeutic should be administered to a subject.", "The invention encompasses kits for detecting the presence of htrb mRNA or protein in a biological sample or for determining the presence of mutations or the identity of polymorphic regions in an htrb gene.", "For example, the kit can comprise a labeled compound or agent capable of detecting htrb protein or mRNA in a biological sample; means for determining the amount of htrb in the sample; and means for comparing the amount of htrb in the sample with a standard.", "The compound or agent can be packaged in a suitable container.", "The kit can further comprise instructions for using the kit to detect htrb mRNA or protein.", "In one embodiment, the kit comprises a pharmaceutical composition containing an effective amount of an htrb antagonist therapeutic and instruction for use in treating or preventing hypertension.", "In another embodiment, the kit comprises a pharmaceutical composition comprising an effective amount of an htrb agonist therapeutic and instructions for use in treating insect bites.", "Generally, the kit comprises a pharmaceutical composition comprising an effective amount of an htrb agonist or antagonist therapeutic and instructions for use as an analgesic.", "For example, the kit can comprise a pharmaceutical composition comprising an effective amount of an htrb agonist therapeutic and instructions for use as an analgesic.", "Yet other kits can be used to determine whether a subject has or is likely to develop a disease or condition associated with an aberrant htrb activity.", "Such a kit can comprise, e.g., one or more nucleic acid probes capable of hybridizing specifically to at least a portion of an htrb gene or allelic variant thereof, or mutated form thereof 4.11.Additional Uses for htrb Proteins and Nucleic Acids The htrb nucleic acids of the invention can further be used in the following assays.", "In one embodiment, the human htrb nucleic acid having SEQ ID No.", "1 or a portion thereof, or a nucleic acid which hybridizes thereto can be used to determine the precise chromosomal localization of an htrb gene within the IL-1 locus.", "Furthermore, the htrb gene can also be used as a chromosomal marker in genetic linkage studies involving genes other than htrb.", "Chromosomal localization of a gene can be performed by several methods well known in the art.", "For example, Southern blot hybridization or PCR mapping of somatic cell hybrids can be used for determining on which chromosome or chromosome fragment a specific gene is located.", "Other mapping strategies that can similarly be used to localize a gene to a chromosome or chromosomal region include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.", "Furthermore, fluorescence in situ hybridization (FISH) of a nucleic acid, e.g., an htrb nucleic acid, to a metaphase chromosomal spread is a one step method that provides a precise chromosomal location of the nucleic acid.", "This technique can be used with nucleic acids as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.", "Such techniques are described, e.g, in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).", "Using such techniques, a gene can be localized to a chromosomal region containing from about 50 to about 500 genes.", "If the htrb gene is shown to be localized in a chromosomal region which cosegregates, i.e., which is associated, with a specific disease, the differences in the cDNA or genomic sequence between affected and unaffected individuals are determined.", "The presence of a mutation in some or all of the affected individuals but not in any normal individuals, will be indicative that the mutation is likely to be causing or contributing to the disease.", "The present invention is further illustrated by the following examples which should not be construed as limiting in any way.", "The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application are hereby expressly incorporated by reference.", "The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.", "Such techniques are explained fully in the literature.", "See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed.", "by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al.", "U.S. Pat.", "No: 4,683,195; Nucleic Acid Hybridization(B. D. Hames & S. J. Higgins eds.", "1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds.", "1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.", "154 and 155 (Wu et al.", "eds.", "), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Acadernic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).", "4.12 Detecting Gene Expression and Microarray Transcription Profiling The invention provides for iterative methodologies for determining inflammatory gene regulatory pathways by: (1) cloning a first gene regulating a given inflammatory gene; (2) using that first inflammatory regulatory gene in an expression profiling procedure to isolate other inflammatory pathway genes whose expression is altered by over or under-expression of the first gene; and (3) still further determining the expression profile of a cell over or under expressing each of the other inflammatory pathway genes to isolate still other inflammatory pathway genes whose expression is altered by over or under-expression of each of the “other” inflammatory pathway genes.", "Many different methods are known in the art for measuring gene expression.", "Classical methods include quantitative RT-PCR, Northern blots and ribonuclease protection assays.", "Such methods may be used to examine expression of individual genes as well as entire gene clusters.", "However, as the number of genes to be examined increases, the time and expense may become prohibitive.", "Large scale detection methods allow faster, less expensive analysis of the expression levels of many genes simultaneously.", "Such methods typically involve an ordered array of probes affixed to a solid substrate.", "Each probe is capable of hybridizing to a different set of nucleic acids.", "In one method, probes are generated by amplifying or synthesizing a substantial portion of the coding regions of various genes of interest.", "These genes are then spotted onto a solid support.", "mRNA samples are obtained, converted to cDNA, amplified and labeled (usually with a fluorescence label).", "The labeled cDNAs are then applied to the array, and cDNAs hybridize to their respective probes in a manner that is linearly related to their concentration.", "Detection of the label allows measurement of the amount of each cDNA adhered to the array.", "Many methods for performing such DNA array experiments are well known in the art.", "Exemplary methods are described below but are not intended to be limiting.", "Arrays are often divided into microarrays and macroarrays, where microarrays have a much higher density of individual probe species per area.", "Microarrays may have as many as 1000 or more different probes in a 1 cm2 area.", "There is no concrete cut-off to demarcate the difference between micro- and macroarrays, and both types of arrays are contemplated for use with the invention.", "However, because of their small size, microarrays provide great advantages in speed, automation and cost-effectiveness.", "Microarrays are known in the art and consist of a surface to which probes that correspond in sequence to gene products (e.g., cDNAs, mRNAs, oligonucleotides) are bound at known positions.", "In one embodiment, the microarray is an array (i.e., a matrix) in which each position represents a discrete binding site for a product encoded by a gene (e.g., a protein or RNA), and in which binding sites are present for products of most or almost all of the genes in the organism's genome.", "In a preferred embodiment, the “binding site” (hereinafter, “site”) is a nucleic acid or nucleic acid analogue to which a particular cognate cDNA can specifically hybridize.", "The nucleic acid or analogue of the binding site can be, e.g., a synthetic oligomer, a full-length cDNA, a less-than full length cDNA, or a gene fragment.", "Although in a preferred embodiment the microarray contains binding sites for products of all or almost all genes in the target organism's genome, such comprehensiveness is not necessarily required.", "Usually the microarray will have binding sites corresponding to at least 100 genes and more preferably, 500, 1000, 4000 or more.", "In certain embodiments, the most preferred arrays will have about 98-100% of the genes of a particular organism represented.", "In other embodiments, the invention provides customized microarrays that have binding sites corresponding to fewer, specifically selected genes.", "Microarrays with fewer binding sites are cheaper, smaller and easier to produce.", "In particular, the invention provides microarrays customized for the determination of graft status.", "In preferred embodiments customized microarrays comprise binding sites for fewer than 4000, fewer than 1000, fewer than 200 or fewer than 50 genes, and comprise binding sites for at least 2, preferably at least 3, 4, 5 or more genes of any of clusters A, B, C, D, E, F or G. Preferably, the microarray has binding sites for genes relevant to testing and confirming a biological network model of interest.", "Several exemplary human microarrays are publically available.", "The Affymetrix GeneChip HUM 6.8K is an oligonucleotide array composed of 7,070 genes.", "A microarray with 8,150 human cDNAs was developed and published by Research Genetics (Bittner et al., 2000, Nature 406:443-546).", "The probes to be affixed to the arrays are typically polynucleotides.", "These DNAs can be obtained by, e.g., polymerase chain reaction (PCR) amplification of gene segments from genomic DNA, cDNA (e.g., by RT-PCR), or cloned sequences.", "PCR primers are chosen, based on the known sequence of the genes or cDNA, that result in amplification of unique fragments (i.e.", "fragments that do not share more than 10 bases of contiguous identical sequence with any other fragment on the microarray).", "Computer programs are useful in the design of primers with the required specificity and optimal amplification properties.", "See, e.g., Oligo p1 version 5.0 (National Biosciences).", "In the case of binding sites corresponding to very long genes, it will sometimes be desirable to amplify segments near the 3′ end of the gene so that when oligo-dT primed CDNA probes are hybridized to the microarray, less-than-full length probes will bind efficiently.", "Random oligo-dT priming may also be used to obtain cDNAs corresponding to as yet unknown genes, known as ESTs.", "Certain arrays use many small oligonucleotides corresponding to overlapping portions of genes.", "Such oligonucleotides may be chemically synthesized by a variety of well known methods.", "Synthetic sequences are between about 15 and about 500 bases in length, more typically between about 20 and about 50 bases.", "In some embodiments, synthetic nucleic acids include non-natural bases, e.g., inosine.", "As noted above, nucleic acid analogues may be used as binding sites for hybridization.", "An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et al., 1993, PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules, Nature 365:566-568; see also U.S. Pat.", "No.", "5,539,083).", "In an alternative embodiment, the binding (hybridization) sites are made from plasmid or phage clones of genes, cDNAs (e.g., expressed sequence tags), or inserts therefrom (Nguyen et al., 1995, Differential gene expression in the murine thymus assayed by quantitative hybridization of arrayed cDNA clones, Genomics 29:207-209).", "In yet another embodiment, the polynucleotide of the binding sites is RNA.", "The nucleic acids or analogues are attached to a solid support, which may be made from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, or other materials.", "A preferred method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al., 1995, Science 270:467-470.This method is especially useful for preparing microarrays of cDNA.", "(See also DeRisi et al., 1996, Nature Genetics 14:457-460; Shalon et al., 1996, Genome Res.", "6:639-645; and Schena et al., 1995, Proc.", "Natl.", "Acad.", "Sci.", "USA 93:10539-11286).", "Each of the aforementioned articles is incorporated by reference in its entirety for all purposes.", "A second preferred method for making microarrays is by making high-density oligonucleotide arrays.", "Techniques are known for producing arrays containing thousands of oligonucleotides complementary to defined sequences, at defined locations on a surface using photolithographic techniques for synthesis in situ (see, Fodor et al., 1991, Science 251:767-773; Pease et al., 1994, Proc.", "Natl.", "Acad.", "Sci.", "USA 91:5022-5026; Lockhart et al., 1996, Nature Biotech 14:1675; U.S. Pat.", "Nos.", "5,578,832; 5,556,752; and 5,510,270, each of which is incorporated by reference in its entirety for all purposes) or other methods for rapid synthesis and deposition of defined oligonucleotides (Blanchard et al., 1996, 11: 687-90).", "When these methods are used, oligonucleotides of known sequence are synthesized directly on a surface such as a derivatized glass slide.", "Usually, the array produced is redundant, with several oligonucleotide molecules per RNA.", "Oligonucleotide probes can be chosen to detect alternatively spliced mRNAs.", "Other methods for making microarrays, e.g., by masking (Maskos and Southern, 1992, Nuc.", "Acids Res.", "20:1679-1684), may also be used.", "In principal, any type of array, for example, dot blots on a nylon hybridization membrane (see Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.", "), Vol.", "1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989, which is incorporated in its entirety for all purposes), could be used, although, as will be recognized by those of skill in the art, very small arrays will be preferred because hybridization volumes will be smaller.", "The nucleic acids to be contacted with the microarray may be prepared in a variety of ways.", "Methods for preparing total and poly(A)+RNA are well known and are described generally in Sambrook et al., supra.", "Labeled cDNA is prepared from mRNA by oligo dT-primed or random-primed reverse transcription, both of which are well known in the art (see e.g., Klug and Berger, 1987, Methods Enzymol.", "152:316-325).", "Reverse transcription may be carried out in the presence of a dNTP conjugated to a detectable label, most preferably a fluorescently labeled dNTP.", "Alternatively, isolated mRNA can be converted to labeled antisense RNA synthesized by in vitro transcription of double-stranded cDNA in the presence of labeled dNTPs (Lockhart et al., 1996, Nature Biotech.", "14:1675).", "The cDNAs or RNAs can be synthesized in the absence of detectable label and may be labeled subsequently, e.g., by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent.", "When fluorescent labels are used, many suitable fluorophores are known, including fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others (see, e.g., Kricka, 1992, Academic Press San Diego, Calif.).", "In another embodiment, a label other than a fluorescent label is used.", "For example, a radioactive label, or a pair of radioactive labels with distinct emission spectra, can be used (see Zhao et al., 1995, Gene 156:207; Pietu et al., 1996, Genome Res.", "6:492).", "However, use of radioisotopes is a less-preferred embodiment.", "Nucleic acid hybridization and wash conditions are chosen so that the population of labeled nucleic acids will specifically hybridize to appropriate, complementary nucleic acids affixed to the matrix.", "As used herein, one polynucleotide sequence is considered complementary to another when, if the shorter of the polynucleotides is less than or equal to 25 bases, there are no mismatches using standard base-pairing rules or, if the shorter of the polynucleotides is longer than 25 bases, there is no more than a 5% mismatch.", "Preferably, the polynucleotides are perfectly complementary (no mismatches).", "Optimal hybridization conditions will depend on the length (e.g., oligomer versus polynucleotide greater than 200 bases) and type (e.g., RNA, DNA, PNA) of labeled nucleic acids and immobilized polynucleotide or oligonucleotide.", "General parameters for specific (i.e., stringent) hybridization conditions for nucleic acids are described in Sambrook et al., supra, and in Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, which is incorporated in its entirety for all purposes.", "Non-specific binding of the labeled nucleic acids to the array can be decreased by treating the array with a large quantity of non-specific DNA—a so-called “blocking” step.", "When fluorescently labeled probes are used, the fluorescence emissions at each site of a transcript array can be, preferably, detected by scanning confocal laser microscopy.", "When two fluorophores are used, a separate scan, using the appropriate excitation line, is carried out for each of the two fluorophores used.", "Alternatively, a laser can be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see Shalon et al., 1996, Genome Research 6:639-645).", "In a preferred embodiment, the arrays are scanned with a laser fluorescent scanner with a computer controlled X-Y stage and a microscope objective.", "Sequential excitation of the two fluorophores is achieved with a multi-line, mixed gas laser and the emitted light is split by wavelength and detected with two photomultiplier tubes.", "Fluorescence laser scanning devices are described in Schena et al., 1996, Genome Res.", "6:639-645 and in other references cited herein.", "Alternatively, the fiber-optic bundle described by Ferguson et al., 1996, Nature Biotech.", "14:1681-1684, may be used to monitor mRNA abundance levels at a large number of sites simultaneously.", "Fluorescent microarray scanners are commercially available from Affymetrix, Packard BioChip Technologies, BioRobotics and many other suppliers.", "Signals are recorded, quantitated and analyzed using a variety of computer software.", "In one embodiment the scanned image is despeckled using a graphics program (e.g., Hijaak Graphics Suite) and then analyzed using an image gridding program that creates a spreadsheet of the average hybridization at each wavelength at each site.", "If necessary, an experimentally determined correction for “cross talk” (or overlap) between the channels for the two fluors may be made.", "For any particular hybridization site on the transcript array, a ratio of the emission of the two fluorophores is preferably calculated.", "The ratio is independent of the absolute expression level of the cognate gene, but is useful for genes whose expression is significantly modulated by drug administration, gene deletion, or any other tested event.", "According to the method of the invention, the relative abundance of an mRNA in two samples is scored as a perturbation and its magnitude determined (i.e., the abundance is different in the two sources of mRNA tested), or as not perturbed (i.e., the relative abundance is the same).", "As used herein, a difference between the two sources of RNA of at least a factor of about 25% (RNA from one source is 25% more abundant in one source than the other source), more usually about 50%, even more often by a factor of about 2 (twice as abundant), 3 (three times as abundant) or 5 (five times as abundant) is scored as a perturbation.", "Present detection methods allow reliable detection of difference of an order of about 2-fold to about 5-fold, but more sensitive methods are expected to be developed.", "Preferably, in addition to identifying a perturbation as positive or negative, it is advantageous to determine the magnitude of the perturbation.", "This can be carried out, as noted above, by calculating the ratio of the emission of the two fluorophores used for differential labeling, or by analogous methods that will be readily apparent to those of skill in the art.", "In one embodiment of the invention, transcript arrays reflecting the transcriptional state of a cell of interest are made by hybridizing a mixture of two differently labeled sets of cDNAs, to the microarray.", "One cell is a cell of interest, while the other is used as a standardizing control.", "The relative hybridization of each cell's cDNA to the microarray then reflects the relative expression of each gene in the two cell.", "For example, to assess gene expression in a variety of breast cancers, Perou et al.", "(2000, supra) hybridized fluorescently-labeled cDNA from each tumor to a microarray in conjunction with a standard mix of cDNAs obtained from a set of breast cancer cell lines.", "In this way, gene expression in each tumor sample was compared against the same standard, permitting easy comparisons between tumor samples.", "In preferred embodiments, the data obtained from such experiments reflects the relative expression of each gene represented in the microarray.", "Expression levels in different samples and conditions may be compared using a variety of statistical methods.", "A variety of statistical methods are available to assess the degree of relatedness in expression patterns of different genes.", "The statistical methods may be broken into two related portions: metrics for determining the relatedness of the expression pattern of one or more gene, and clustering methods, for organizing and classifying expression data based on a suitable metric (Sherlock, 2000, Curr.", "Opin.", "Immunol.", "12:201-205; Butte et al., 2000, Pacific Symposium on Biocomputing, Hawaii, World Scientific, p.418-29).", "In one embodiment, Pearson correlation may be used as a metric.", "In brief, for a given gene, each data point of gene expression level defines a vector describing the deviation of the gene expression from the overall mean of gene expression level for that gene across all conditions.", "Each gene's expression pattern can then be viewed as a series of positive and negative vectors.", "A Pearson correlation coefficient can then be calculated by comparing the vectors of each gene to each other.", "An example of such a method is described in Eisen et al.", "(1998, supra).", "Pearson correlation coefficients account for the direction of the vectors, but not the magnitudes.", "In another embodiment, Euclidean distance measurements may be used as a metric.", "In these methods, vectors are calculated for each gene in each condition and compared on the basis of the absolute distance in multidimensional space between the points described by the vectors for the gene.", "In a further embodiment, the relatedness of gene expression patterns may be determined by entropic calculations (Butte et al.", "2000, supra).", "Entropy is calculated for each gene's expression pattern.", "The calculated entropy for two genes is then compared to determine the mutual information.", "Mutual information is calculated by subtracting the entropy of the joint gene expression patterns from the entropy for calculated for each gene individually.", "The more different two gene expression patterns are, the higher the joint entropy will be and the lower the calculated mutual information.", "Therefore, high mutual information indicates a non-random relatedness between the two expression patterns.", "The different metrics for relatedness may be used in various ways to identify clusters of genes.", "In one embodiment, comprehensive pairwise comparisons of entropic measurements will identify clusters of genes with particularly high mutual information.", "In preferred embodiments, expression patterns for two genes are correlated if the normalized mutual information score is greater than or equal to 0.7, and preferably greater than 0.8, greater than 0.9 or greater than 0.95.In alternative embodiments, a statistical significance for mutual information may be obtained by randomly permuting the expression measurements 30 times and determining the highest mutual information measurement obtained from such random associations.", "All clusters with a mutual information higher than can be obtained randomly after 30 permutations are statistically significant.", "In a further embodiment, expression patterns for two genes are correlated if the correlation coefficient is greater than or equal to 0.8, and preferably greater than 0.85, 0.9 or, most preferably greater than 0.95.In another embodiment, agglomerative clustering methods may be used to identify gene clusters.", "In one embodiment, Pearson correlation coefficients or Euclidean metrics are determined for each gene and then used as a basis for forming a dendrogram.", "In one example, genes were scanned for pairs of genes with the closest correlation coefficient.", "These genes are then placed on two branches of a dendrogram connected by a node, with the distance between the depth of the branches proportional to the degree of correlation.", "This process continues, progressively adding branches to the tree.", "Ultimately a tree is formed in which genes connected by short branches represent clusters, while genes connected by longer branches represent genes that are not clustered together.", "The points in multidimensional space by Euclidean metrics may also be used to generate dendrograms.", "In yet another embodiment, divisive clustering methods may be used.", "For example, vectors are assigned to each gene's expression pattern, and two random vectors are generated.", "Each gene is then assigned to one of the two random vectors on the basis of probability of matching that vector.", "The random vectors are iteratively recalculated to generate two centroids that split the genes into two groups.", "This split forms the major branch at the bottom of a dendrogram.", "Each group is then further split in the same manner, ultimately yielding a fully branched dendrogram.", "In a further embodiment, self-organizing maps (SOM) may be used to generate clusters.", "In general, the gene expression patterns are plotted in n-dimensional space, using a metric such as the Euclidean metrics described above.", "A grid of centroids is then placed onto the n-dimensional space and the centroids are allowed to migrate towards clusters of points, representing clusters of gene expression.", "Finally the centroids represent a gene expression pattern that is a sort of average of a gene cluster.", "In certain embodiments, SOM may be used to generate centroids, and the genes clustered at each centroid may be further represented by a dendrogram.", "An exemplary method is described in Tamayo et al., 1999, PNAS 96:2907-12.Once centroids are formed, correlation must be evaluated by one of the methods described supra.", "In another aspect, the invention provides probe sets.", "Preferred probe sets are designed to detect expression of multiple genes and provide information about the status of a graft.", "Preferred probe sets of the invention comprise probes that are useful for the detection of at least two genes belonging to gene clusters A, B, C, D, E, F or G. Particularly preferred probe sets will comprise probes useful for the detection of at least three, at least four or at least five genes belonging to gene clusters A, B, C, D, E, F or G. Certain probe sets may additionally comprise probes that are useful for the detection of one or more genes of gene cluster H. Probe sets of the invention do not comprise probes useful for the detection of more than 10,000 gene transcripts, and preferred probe sets will comprise probes useful for the detection of fewer than 4000, fewer than 1000, fewer than 200, and most preferably fewer than 50 gene transcripts.", "Probe sets of the invention are particularly useful because they are smaller and cheaper than probe sets that are intended to detect as many genes as possible in a particular genome.", "The probe sets of the invention are targeted at the detection of gene transcripts that are informative about transplant status.", "Probe sets of the invention may comprise a large or small number of probes that detect gene transcripts that are not informative about transplant status.", "Such probes are useful as controls and for normalization.", "Probe sets may be a dry mixture or a mixture in solution.", "In preferred embodiments, probe sets of the invention are affixed to a solid substrate to form an array of probes.", "It is anticipated that probe sets may also be useful for multiplex PCR.", "5.EXAMPLES Example 1: Cloning and Identification of Inflammatory Pathway Genes In order to identify novel genes involved in proinflammatory cytokine signaling, we have developed an expression screen to isolate gene products having the capacity to modulate the activity of a cytokine responsive reporter (Kiss-Toth, E. et al.", "(2000) Journal of Immunological Methods 239:125-135).", "The promoter of the IL-8 chemokine gene regulating the expression of the firefly luciferase gene was used as a readout.", "The promoter fragment (−79 to +45: pIL8) contains an AP-1, a C/EBP and an NFκB site, a typical structure for inflammatory signal responsive promoters.", "We have also found that the level of secretion of human IL-8 into the medium in this system is correlated with the activity of the reporter systems; thus the reporters are a valid surrogate measure of the activity of the endogenous gene.", "We have isolated both repressors and activators of signaling and have derived a lower bound estimate of the number of gene products that play a role in controlling transcription of the IL-8 gene.", "30 pools of ca 300 clones from an oligo-dT primed human peripheral blood mononuclear cell cDNA expression library in pCDM8 were screened in vivo against the reporter pIL-8—firefly luciferase with an internal control pTK (HSV Thymidine Kinase promoter)—Renilla luciferase, using a dual luciferase assay in HeLa cells.", "On pool (p) was positive, with a markedly elevated ratio of pIL-8/pTK activity compared to the other 29.We analyzed this and one negative pool in a single cell pIL-8-EGFP transcription assay, as described previously.", "The positive pool was active (FIG.", "1B), with 5-10% of the cells in the transfected population showed induction of the reporter, compatible with the number expected to be productively transfected with one of the 300 clones in the pool.", "After pool breakdown, sequencing revealed that the active insert contained the entire coding region (ORF) for interferon-γ, in the sense orientation.", "This finding is compatible with reports that IFN-γ activates NF-κB.", "Further, despite the expected secretion of the cytokine, no paracrine “spreading” of the signal was seen in the GFP assay, and those cells that express it are apoptotic as judged by the fact that they are a small and bright and annexin V positive in two colour confocal fluorescence micrographs (data not shown).", "This is compatible with previous findings that IFN-γ can cause apoptosis, via up-regulation of FasL/Fas or Trail/Trail-R and that it induces NFκB activation, possibly synergistically after induction of TNF/TNFR family members.", "The data from the screen provide a lower limit estimate for the number of genes involved in controlling pIL-8 luc function.", "The hit rate is 1/8000 and the library contains approximately 5×106 independent clones, suggesting that the screen will detect about 600 clones encoding inhibitors or activators of pIL-8 luc transcription.", "The library is non-directional and oligo dT primed, with an average insert size of 2-3 kb.", "Clearly half the clones will be antisense and many will contain only 3′ UTRs.", "It is difficult to estimate what fraction of the clones n the library contain inserts encoding expressible proteins and indeed our results (see below) suggest that a full length coding region is not always required for the detection of relevant clones.", "Screening of a random primed human B-cell line (CB23) library for type II IL-1 receptor yielded an estimate of 1/300,000 for clones with a full length ORF.", "The frequency in our library is likely to be lower since it was prepared from a complex cell population, activated peripheral blood mononuclear cells, rather than a cell line and is oligo dT primed.", "Given this, and bearing in mind the SKIP result (see below), we estimate no more than 5 expressible clones for a rare mRNA (<1/25,000 in an clonal population), suggesting that a minimum of 100 distinct gene products, detectable by overexpression, play a role in regulating the IL-8 promoter.", "This number is reasonable, given the estimate of 40 genes on chromosome 3 in D. melanogaster, involved in controlling Dif and Dorsal translocation in fat body cells (see Wu & Anderson (1998) Nature 392: 93-7).", "This is likely only a subset of all components, since not all relevant gene products may alter activity when overexpressed.", "Indeed, we have found that IL-1RI and TLR4 do not activate pIL8, when over expressed in HeLa cells, unless exogenous ligand is added to the cultures.", "Two of the clones obtained were active in altering levels of IL-8 —one as an activator and the other as an inhibitor of IL-8 mediated transcription.", "The activator was a full length Re1A cDNA clone in the sense orientation; an expected finding given the structure of the IL-8 promoter.", "The inhibitor cDNA clone encodes NAK-1, the human homolog of the murine orphan steroid hormone receptor Nur77, in the sense orientation.", "Nur77/N10/NAK-1/NGFI-B (a member of the NR4 subfamily) was previously identified as an immediate-early gene induced by a variety of stimuli in PC12 cells and fibroblasts [it is expressed in many cell types after activation by mitogens, for example].", "A large body of literature implicates Nur77 and the closely related transcription factors NOR-1 and Nurr1 as playing a central role in apoptosis in many cell types, for example in T cell activation induced cell death (AICD) negative selection.", "Nur77/N10 transgenic mice show a dramatic reduction in both double and single positive thymocytes due to extensive early onset apoptosis.", "A recent report shows that Nur77 is one of a limited set of immediate early genes up-regulated (1 hour post-induction) in response to hen egg lysozyme stimulation of naive IgHEL B lymphocytes.", "Transient overexpression of NAK-1 inhibited both the basal and IL-1 stimulated activity of the IL-8 promoter.", "In addition, stimulation of AP-1, NFκB and CRE reporters was suppressed by NAK-1.It also blocked activation of a synthetic LHRE promoter.", "Thus the inhibitory activity of NAK-1 is broad, possibly a consequence of overexpression.", "However, it is not a general inhibitor of transcription or translation, since the activity of the internal control reporter was not inhibited.", "Nur77 has been shown to facilitate AICD by a mechanism, which is only partially understood, and our data suggest that NAK-1 can act as an inhibitor of cytokine signaling.", "Steroid hormone receptors can block transactivation by NFκB and the activity of Nur77/NAK-1 can be potentiated by heterodimerisation with Nor-1, Nurrl and RXRs.", "Retinoids have also been shown to block cytokine gene expression by acting through NFκB.", "Our data suggest that NAK-1/Nur77 may be pro-apoptotic due in part in part to blockade of survival signals such as NFκB.", "A second repressor clone encoded a 3′ UTR fragment in the sense orientation terminating in the poly-A tract.", "The sequence of the corresponding full-length transcript was deduced by searching human genomic and cDNA sequence databases.", "The partial sequence has been previously reported (GenBank AJ000480).", "The full length cDNA sequence was confirmed by sequencing ests obtained from the IMAGE collection.", "We have termed the gene product Stress Kinase Inhibitory Protein-1 (SKIP-1).", "Screening pools from the cDNA expression library made from PBMC (Hamann, J. et al.", "(1993) Journal of Immunology 150:4920-27) further led us to identify a cDNA of previously unknown function.", "A homologous Drosophila gene, tribbles, has been described recently (Grosshans, J. et al.", "(2000) Cell 101:523-31; Seher, T. C. et al.", "(2000) Curr.", "Biol.", "10:623-29; Mata, J. et al.", "(2000) Cell 101:511-22).", "Therefore we suggest the name human tribbles homologue-1 (htrb-1) for the isolated mammalian protein encoded by our clone.", "Example 2 Analysis of htrb-1 Repression Specificity FIG.", "1 A show the effect of htrb-1 overexpression on IL-8 reporter activity.", "HeLa cells were transfected with the IL-8 luciferase reporter with or without a htrb-1 expression construct.", "Black bars indicate reporter activation without htrb-1 cotransfection, while gray bars show the reporter activation in the presence of 50 ng htrb-1 expression construct.", "Cells were stimulated with proinflammatory cytokines and the reporter activity was determined.", "B-D htrb-1 is a specific inhibitor of stress kinase signalling.", "HeLa cells were transiently transfected with AP-1 (B), NFkB (C) and human growth hormone (D) signalling pathway specific transcription reporters and were activated in the presence (gray bars) or absence (black bars) of htrb-1 expression plasmid by a MEKK-1 expression construct (B, C) or human growth hormone (D).", "E pAP-1 luc was stimulated with V12 Ras expression plasmid and the effect of htrb-1 and htrb-3 co-expression was investigated.", "F AP-1 reporter (diamonds) was stimulated with MEKK-1 expression plasmid (squares) and the effect of htrb-1 overexpression (triangles) on the stimulation was studied.", "The activity of constitutively active TK-Rluc versus the inducible AP-1 luc was plotted.", "G The p38 responsive pFR-luc+pFA-CHOP reporter was stimulated with MEK-3 expression construct.", "htrb-3 was co-transfected with the reporter in the presence or absence of the stimulus, as indicated.", "Upon overexpression, htrb-1 was found to inhibit the basal activity of the IL-8 reporter, while the induction of this promoter by IL-1 and TNFalpha was not affected (FIG.", "1A).", "The IL-8 promoter fragment used in this assay contains binding sites for NFkB and AP-1 and most of the cytokine inducible activity is mediated through to the NFkB site, while the AP-1 site contributes significantly to the basal promoter activity (Mukaida, N. et al.", "(1994) Journal of Leukocyte Biology 56:554-58).", "To determine which signaling pathway is inhibited by htrb-1, a cDNA encoding the full length htrb-1 ORF was tested against transcription reporters containing multiple copies of NFkB or AP-1 binding sites.", "The reporters were activated by MEKK-1.Overexpression of the htrb-1 protein suppressed the activation of the AP-1 but not of the NFkB reporter (FIGS.", "1B and C) nor of a JAK/STAT pathway specific promoter (FIG.", "1D).", "These data suggest that the observed negative effect of htrb-1 on signaling is specific for stress kinase pathways.", "To confirm that the observed AP-1 inhibition is not restricted to MEKK-1 overexpression, V12 Ras was co-transfected with the AP-1 reporter in the absence or presence of htrb-1 or htrb-3 expression constructs (see below) (FIG.", "1E).", "Ras-mediated activation was also blocked by elevated htrb levels.", "The effect of htrb overexpression on the activity of the constitutive HSV-TK promoter (FIG.", "1F) and the possible involvement in activation of p38 mediated general stress responses (FIG.", "1G) were assessed.", "Our data show that the effect of htrb-1 on the inhibition of MEKK-1 mediated AP-1 activation was specific for the inducible reporter, while the activity of the TK-control reporter was not affected by the treatments (FIG.", "1F).", "Example 3 Identification and Analysis of other htrb Genes FIG.", "2 shows multiple alignment of htrb protein family.", "Searching public DNA databases revealed 3 human htrb genes and further est-s, encoding trb homologs in vertebrates and insects.", "Putative protein sequences of human htrb family and the identified trb homologs have been aligned by ClustalX, using default parameters.", "Positions of htrb mutations are indicated by arrows.", "Abbreviations: h- Homo sapiens, m- Mus musculus, r- Rattus norvegicus, c- Canis canis, b- Bos taurus, x- Xenopus laevis, o- Oncorhynchus mykiss, a- Aedes aegypti.", "Public database searching with the htrb-1 sequence identified two further human genes, htrb-2 and htrb-3, 41 and 51% identical to htrb-1 respectively.", "In addition, several putative trb gene products were found in vertebrate species and in insects (FIG.", "2).", "All these proteins share a central similar kinase-like domain.", "In addition, each possesses short N-terminal (approx.", "70-100 residues) and a C-terminal (approx.", "25 residues) domains which are neither closely related to any other sequence in the databases, nor to each other.", "A partial human htrb-1 sequence has been reported (Wilkin, F. et al.", "(1997) European Journal of Biochemistry 248:660-68).", "Canine trb-2 has been described to be a highly labile cytoplasmic phosphoprotein lacking kinase activity: the mRNA is up-regulated by mitogens (Wilkin, F. et al.", "(1997) European Journal of Biochemistry 248:660-68; Wilkin, F. et al.", "(1996) Journal of Biological Chemistry 271:28451-57).", "A rat homologue of htrb-3 has been identified as a novel kinase-like gene induced during neuronal cell death (Mayumi-Matsuda, K. et al.", "(1999) Biochemical and Biophysical Research Communications 258:260-64).", "A Drosophila homologue is tribbles, which regulates mitosis and morphogenesis by regulating string/CDC25 (Grosshans, J. et al.", "(2000) Cell 101:523-31; Seher, T. C. et al.", "Curr.", "Biol.", "10:623-29; Mata, J. et al.", "(2000) Cell 101:511-22; Rorth, P. et al.", "(2000) Mol.", "Cell 6:23-30).", "The trb kinase-like domain shows homology to protein serine-threonine kinases in general, and to calcium calmodulin kinases and SNF1 kinases in particular.", "However the trbs lack the active site lysine, and are predicted to be kinase dead, as shown for Canine trb-2 (Wilkin, F. et al.", "(1997) European Journal of Biochemistry 248:660-68) and tribbles (Grosshans, J. et al.", "(2000) Cell 101:523-31).", "To determine whether htrb-3 has transcriptional regulatory activities similar to those of htrb-1, the effect of htrb-3 expression on activation of the AP-1 reporter was investigated.", "The two mammalian homologs had similar activities (see FIGS.", "3A) and, furthermore, the observed AP-1 inhibition was not restricted to MEKK-1 overexpression, because V12 Ras mediated AP-1 activation was similarly affected by the expression of htrb-1 or htrb-3 expression constructs (FIG.", "1E).", "In addition, the basal activity of a p38 responsive reporter was not influenced by htrb-3 and the activation of these kinases was slightly inhibited by overexpressing htrb-3 (FIG.", "1G).", "To further investigate the effect of htrb expression levels on stress kinase signalling, an antisense construct, expressing the htrb-1 or htrb-3 5′UTR and the region corresponding to the N-terminal variable region in reverse orientation was co-transfected into HeLa cells with AP-1 or NFkB reporters and a MEKK-1 or NIK expression construct as an activator (FIG.", "3A, B).", "As observed with the full length sense cDNA, these constructs strongly inhibited AP-1 activation while having little effect on NFkB.", "FIG.", "3 shows expression of antisense htrb-1 and htrb-3 RNA inhibits stress kinase activation.", "Effects of cotransfected antisense htrb-1 or htrb-3 construct were measured on activation of AP-1 (A) and NFkB responsive reporters (B) by MEKK-1 or NIK expression plasmids, respectively.", "(C) HeLa cells were transfected with sense or antisense htrb-3 expression constructs, the cell size was measured by flow cytometry and the forward scatter was plotted.", "(D) Mock- and htrb-3 transfected HeLa cells were permeabilised, RNAse treated, and stained with propidium iodide.", "The DNA content of the cells was measured by flow cytometry.", "Example 4 Investigation of htrb Cell Cycle Regulation It has been shown that trb −/− genotype or overexpression of tribbles in Imaginal Disc Cells causes a G2 block and an increase in cell size (Grosshans, J. et al.", "(2000) Cell 101:523-31; Mata, J. et al.", "(2000) Cell 101:511-22).", "This effect was cell-type specific (Grosshans, J. et al.", "(2000) Cell 101:523-31; Seher, T. C. (2000) Curr.", "Biol.", "10:623-29).", "As htrbs are the closest known mammalian homologs to tribbles, the effect of htrb expression levels in HeLa cells on the cell size and cell cycle was investigated.", "As observed with tribbles, elevated or suppressed htrb-3 (and htrb-1, data not shown) levels resulted in an increase in the cell size (FIG.", "3C).", "In contrast, cell cycle distribution was not affected by overexpressed htrb-3 (FIG.", "3D).", "These observations suggest that vertebrate cell size may be controlled by trb proteins, just as Drosophila imaginal disc cell size is under the control of tribbles (Grosshans, J. et al.", "(2000) Cell 101:523-31; Mata, J. et al.", "(2000) Cell 101:511-22).", "Example 5 Analysis of htrb-1 Mediated Repression Table 1 shows that htrb-3 inhibits AP-1, but not NF-kB, induction by a variety of cytokines.", "10 nM final concentration of each cytokine and 50 ng/ml PMA were used to stimulate HeLa cells for 4 hrs.", "Activation is expressed as relative to the PBS control.", "Those with the capacity to activate AP-1 reporter were studied for the effect of htrb-3.Results are expressed as percent inhibition caused by the cotransfected htrb-3 construct.", "Abbreviations: IL- Interleukin, bFGF- Basic Fibroblast Growth Factor, EGF- Epidermal Growth Factor, Ins.", "like GF- Insulin-like Growth Factor, TGFbeta- Transforming Growth Factor Beta-1, M-CSF- Macrophage Colony Stimulating Factor, G-CSF- Granulocyte Colony Stimulating Factor, GM-CSF- Granulocyte Macrophage Colony Stimulating Factor, PDGF-BB-Platelet-derived Growth Factor-BB, LIF-Leukemia inhibitory Factor, PMA-Tetradecanoylphorbol Acetate.", "Overexpression of htrb-1 inhibits MEKK-1 mediated AP-1 but not NFkB activation in HeLa cells.", "A similar effect was seen when the AP-1 reporter was stimulated by PMA (FIG.", "4A, B) or by a panel of human cytokines (see Table 1).", "To characterise the possible site of htrb action, we tested whether increasing the dose of MEKK-1 could bypass the effect of htrb-3 (FIG.", "4C).", "Our data show that this is not the case, suggesting that htrb-3 interacts with a rate-limiting factor downstream of MEKK-1.According to our current understanding, MEKK-1 phosphorylates two downstream kinases, MKK4 (SEKI) and MKK7 (Foltz, I. N. et al.", "(1998) J. Biol.", "Chem.", "273:9344-51; Holland, P. M. et al.", "(1997) Journal of Biological Chemistry 272:24994-98).", "Activation of these kinases leads to phosphorylation of several transcription factors, including c-Jun and CREB2 via Jun kinases (Davis, R. J.", "(1999) Biochemical Society Symposia 64:1-12).", "To further characterise the point of action of htrb genes, expression constructs for MKK4 and MKK7 were cotransfected with or without of a htrb-1 expression plasmid (FIG.", "4E).", "MKK7-mediated AP-1 activation was inhibited upon co-expression of htrb-1.Furthermore, activation of AP-1 by PMA was potentiated by overexpressing MKK7 and this activity was blocked upon cotransfection of htrb-1 or htrb-3 (FIG.", "4F).", "The MKK4 construct used did not stimulate the AP-1 reporter (FIG.", "4E) nor did it potentiate PMA activation, under the conditions used (FIG.", "4F).", "To examine the possible involvement of MKK4 in htrb action, HeLa cells were transfected with htrb-1 or htrb-3 expression constructs, and stimulated with PMA (FIG.", "4D).", "The phosphorylation level of the endogenous MKK4 protein was not influenced either by PMA or by htrbs.", "In summary, our data suggest that MKK7 but not MKK4 contributes to AP-1 activation in this system and the inhibitory effect of htrbs is exerted at or downstream of MKK7.To test this, phosphorylation of an effector transcription factor, c-Jun was determined in cell extracts from PMA stimulated HeLa cells (FIG.", "5A).", "While the maximal level of activation was comparable, the kinetics of c-Jun phosphorylation were significantly altered upon htrb-3 overexpression, resulting in a more rapid down-regulation of the stress-kinase response.", "FIG.", "4 shows that htrb-1 inhibits MEKK-1 and MKK7 mediated AP-1 activation.", "HeLa cells were transfected with AP-1 reporter (diamonds) and constant doses (50 ng/well) of htrb-1 (triangles) or htrb-3 (squares) expression construct and stimulated with PMA.", "A Increasing dose of PMA was used to stimulate for 4 hrs.", "B The effect of 50 ng/ml PMA was followed for 6 hrs.", "C increasing amount of MEKK-1 expression plasmid (black bars) and constant doses (50 ng/well) of htrb-3 expression construct (gray bars) were cotransfected to study whether overexpression of MEKK-1 can bypass the htrb-3 inhibition.", "D HeLa cells were transfected with htrb-1 or htrb-3 expression constructs and stimulated 24 hrs post-transfection by 50 ng/ml PMA.", "pMKK4 levels were followed by western blotting equal amount of total cell lysates.", "E The activity of AP-1 reporter was measured in response to co-transfection of MKK4 or MKK7 expression constructs in the presence (gray bars) or absence (black bars) of htrb-1 expression plasmid.", "F HeLa cells transfected with AP-1 reporter, MKK4 or MKK7 expression plasmids +/− htrb-1 expression construct were stimulated with 2ng/ml PMA and the reporter activity was determined.", "The overall cellular response to an external MAPK activation stimulus depends on the extent to which individual MAPK pathways are activated.", "This is a precisely regulated process, which involves many factors, including the micro-environment, cell cycle state, co-stimulatory signals.", "To investigate whether other MAPK pathways are influenced by overexpression of htrbs, HeLa cells were co-transfected with htrb-3 expression constructs and ERK, JNK or p38 responsive reporters and stimulated by overexpressing upstream MAP kinases, which specifically activate these effector kinases.", "An increasing dose of htrb-3 was cotransfected (FIG.", "5B).", "As our data demonstrate, a low dose of htrb-3 was able to promote the activation of JNK and ERKs, while p38 activation was suppressed.", "High htrb doses (>20 ng) inhibited the activation of all MAPK pathways.", "Furthermore, the optimal htrb-3 dose for facilitating MAPK activation was different for ERKs and JNKs, suggesting that regulation of htrb levels might be a sensitive mechanism to change the balance between the activation of individual MAPK pathways.", "Example 6 htrb Potentiates ERK Activation The effect of htrb overexpression on ERK activation was studied.", "HeLa cells were activated with PMA and the level of phopshorylated ERKs was monitored by western blot (FIG.", "5C, D).", "Our results show that basal level of phospho-ERK was increased by htrb-3 and the maximal stimulation was strongly enhanced as a result of htrb-1 and htrb-3 overexpression.", "Similarly, increased ERK activity was detected in kinase assays, performed by using PMA activated cell extracts from HeLa cells, where htrb-1 or htrb-3 was overexpressed (FIG.", "5E).", "In contrast to the observed effect of htrb on JNK and ERK phosphorylation levels and kinase activity, p38 kinase activity was suppressed upon overexpressing these proteins (FIG.", "5F).", "In FIG.", "5(A) HeLa cells were transfected with htrb-1 or htrb-3 expression constructs and stimulated by 50 ng/ml PMA.", "Phospho-cJun levels were determined by western blotting equal amount of total cell lysates and quantitated by NIH Image.", "(B) pFR luc reporter was transfected into HeLa cells together with pFA-CHOP, activated by pMEK-3 (a p38 activator).", "(triangles) or with pFA2-Elk-1, activated by pMEK-1 (an ERK activator) (diamonds).", "AP-1 luc was stimulated by cotransfection with pMEKK-1 to activate the stress kinase cascade (squares).", "(C) HeLa cells were transfected and treated as on FIG.", "5(A).", "The total and phospho-ERK levels were determined by western blotting.", "(D) Phospho-ERK levels were quantitated: mock transfected (diamonds), htrb-1 co-transfected (squares), htrb-3 co-transfected (triangles).", "Kinase assays were performed on PMA stimulated cell extract to measure ERK (E) or p38 (F) activity.", "Example 7 Analysis of htrb Protein Domain Function Multiple alignment of trb proteins suggests the existence of three structurally distinct modules (FIG.", "2).", "To investigate the role of these domains in trb function, htrb-1 deletion mutants were generated lacking one or two domains (FIG.", "6A).", "The resulted constructs were expressed HeLa cells as GFP fusion proteins to determine the intracellular localisation of full-length htrb-1 and the deletion mutants, respectively.", "htrb-1 was found to be localised in the nucleus but excluded from the nucleoli.", "While deletion of the C-terminal variable domain had no effect on the intracellular distribution (not shown), deleting the N-terminal variable region yielded in a protein which was no longer preferentially localised in the nucleus (FIG.", "6B upper panels).", "Deletion mutants lacking both termini showed a uniform intracellular distribution (not shown).", "Similar intracellular localisation was found when full length or N-terminal deletion mutant htrb-3-GFP fusion proteins were expressed (FIG.", "6B lower panels).", "The relevance of the different htrb domains in stress kinase signalling was investigated in transient transfection assays.", "HeLa cells were transfected with the full length and the htrb-1 deletion constructs, with an MEKK-1 expression plasmid as an activator and the effect of htrb mutants on AP-1 activation were determined (FIG.", "6C).", "All htrb-1 deletion mutants showed strong inhibition of MEKK-1 mediated AP-1 activation, suggesting that overexpression of the htrb kinase-like domain is sufficient to inhibit stress kinase signalling.", "Although the overall protein sequence of htrbs is remarkably similar to tribbles, the proline-rich N-terminal domain, which is responsible for targeting htrb-1 and htrb-3 to the nucleus show the lowest level of homology.", "This raises the possibility that the mammalian homologs can be targeted to different cellular organelles than the Drosophila protein and/or interact with different proteins.", "FIG.", "6 shows deletion mutagenesis of htrb-1.", "(A) trbs share a highly conserved central domain and variable terminal regions.", "Deletion constructs of htrb-1 were generated lacking one or both variable domains and expressed as a GFP fusion protein.", "(B) HeLa cells were transfected with expression constructs, expressing the full length or truncated htrb-1 or htrb-3 forms.", "Truncated constructs lack the N-terminal variable region.", "24 hrs after transfection cells were fixed with 5% formaldehyde and counterstained with propidium iodide (PI).", "Confocal micrographs were taken by exciting PI and GFP.", "Micrographs were transferred to PowerMacintosh and overlaid.", "(C) Ability of htrb-1-GFP truncated mutants to inhibit MEKK-1 mediated AP-1 activation was tested in transiently transfected HeLa cells.", "We have further shown that an htrb-1 3′UTR construct alone is capable of increasing htrb-1 protein levels, because the steady state levels of expression of endogenous htrb-1 message are increased following overexpression of the htrb-1 3′UTR (data not shown).", "The mechanism of this regulation may involve a negative regulator of htrb-1 translation that is titrated away in the presence of excess 3′UTR RNA.", "Example 8 htrb Tissue Specificity of Expression and Cell Specificity of Function Although htrb proteins show high primary sequence homology to each other, their in vivo function may not be redundant.", "Specificity is often achieved by tissue specific expression of the individual family members (e.g.", "JNK or JIP genes).", "This possibility was tested by determining the mRNA expression profile of all three htrb genes in HeLa cells and quiescent human tissues.", "HeLa cells express all three htrb genes (FIG.", "7C).", "In contrast, all three htrb genes show restricted expression patterns suggesting tissue specific roles (FIG.", "7A).", "This hypothesis was confirmed by cotransfecting HeLa cells, NIH 3T3 fibroblasts and RAW 264.7 macrophages with htrb-1 or htrb-3 expression constructs and an AP-1 luciferase reporter, and activating them by overexpressing MEKK-1 (FIG.", "7B).", "While both htrb genes disrupted AP-1 activation in HeLa cells, only htrb-3 had an inhibitory effect in RAW cells.", "Activation of AP-1 in NIH 3T3 cells was not affected by overexpression of either of the htrbs.", "These results demonstrate that the downstream components of MEKK-1 mediated AP-1 activation and the observed inhibitory effect of htrbs on this signalling cascade are cell type and htrb family member specific.", "FIG.", "7 shows that htrb genes are expressed and act in a tissue-specific manner.", "A Multi-tissue PCR was performed to characterise the expression profile of htrb genes by using “Human Rapid-Scan panel” (OriGene) .", "The following tissues were screened: 1.Brain, 2.Heart, 3.Kidney, 4.Spleen, 5.Liver, 6.Colon, 7.Lung, 8.Small Intestine, 9.Muscle, 10.Stomach, 11.Testis, 12.Placenta, 13.Salivary Gland, 14.Thyroid Gland, 15.Adrenal Gland, 16.Pancreas, 17.Ovary, 18.Uterus, 19.Prostate, 20.Skin, 21.PBL, 22.Bone Marrow, 23.Fetal Brain, 24.Fetal Liver.", "B HeLa, NIH 3T3 and RAW 264.7 cells were transiently transfected with AP-1 reporter, MEKK-1 expression vector and htrb-1 or htrb-3 expression constructs.", "C Total RNA was purified from HeLa cells and expression of the htrb genes was tested by RT-PCR.", "D Working hypothesis for mechanism of the trb action.", "Example 9 htrb Scaffolding Function Scaffolding proteins have been demonstrated to be indispensable for the activation of the NFkB and MAPK activation cascades in yeast and mammals (Yamaoka, S. et al.", "(1998) Cell 93:1231-40; Rudolph, D. et al.", "(2000) Genes Dev.", "14:854-62; Harhaj, E. W. et al.", "(1999) Journal of Biological Chemistry 274:22911-14; Schaeffer, H. J. et al (1998) Science 281:1668-71; Whitmarsh, A. J. et al.", "(1998) Science 281:1671-74).", "Here we describe a family human homologs of Drosophila tribbles.", "Our data suggest that expression levels of htrb-1 and/or htrb-3 control the amplitude of MAPK activation.", "According to our hypothesis (FIG.", "7D), htrb proteins (“T”) are essential in the assembly of active MAPK complexes (“A”, and “B”).", "Overexpression or suppression of htrb levels results in the enrichment of inactive complexes and, in turn, reduced MAPK mediated activation.", "The proposed model of trb action (FIG.", "7D), explains both our observations and those in the Drosophila experiments (Grosshans, J. et al.", "(2000) Cell 101:523-31; Seher, T. C. et al.", "(2000) Curr.", "Biol.", "10:623-29; Mata, J. et al.", "(2000) Cell 101:511-22) (FIG.", "5B), since scaffolds can facilitate or inhibit signalling responses depending on their concentration (Ferrell, J. E. (2000) www.stke.org/cgi/content/fill/OC_sigtrans;2000/52/pel).", "FIG.", "8 showsthe detection of htrb-1 and htrb-3 expression by confocal microscopy.", "A HeLa cells were transiently transfected with htrb-3-GFP expression plasmid, in combination with antisense htrb-3 and antisense htrb-1 expression constructs.", "24 hrs later, confocal micrographs were taken using 10× air lenses and htrb-GFP expressing cells were detected by using NIH image (for details of the analysis see: Kiss-Toth, E. et al., Journal of Immunological Methods 239, 125-135 (2000)).", "The brightness vs. the area of the detected particles is plotted.", "Red spots indicate cells having the fluorescence above the background level, thus representing htrb-GFP expressors.", "B HeLa cells were transfected with plasmids expressing full length or truncated htrb-1-GFP proteins.", "Similarly to panel 8A, expressors were detected on a single cell level.", "FIG.", "9 shows the titration of sense vs. antisense htrb-3.HeLa cells were transfected with the indicated reporter and expression constructs and activated by either PMA (A) or co-transfection of MEKK-1 expression vector (B).", "Example 10 Materials and Methods The Human trb sequences reported in this paper have been deposited in the GenBank database: htrb-1 (AF250310), and htrb-3 (AF250311).", "SEQ ID No.", "1=complete cDNA sequence of htrb-1 SEQ ID No.", "2=htrb-1 polypeptide sequence SEQ ID No.", "3=htrb-3 polypeptide sequence SEQ ID No.", "4=htrb-3 polypeptide sequence.", "Plasmids, PCR: MKK4, MKK7 (Holland, P. M. et al.", "(1997) Journal of Biological Chemistry 272:24994-98), IL-8-luc (Wyllie, D. H. et al.", "(2000) Journal of Immunology 165:7125-32) and LHRE-TK-luc (Maamra, M. et al.", "(1999) Journal of Biological Chemistry 274:14791-98) were described earlier.", "V12 Ras was a kind gift of Dr. J.", "Downward.", "pAP-1 luc, pNF B luc, pFR luc, pFA-CHOP, pFA2-Elk-1, pMEKK-1 pMEK-1 and pMEK-3 were part of the PathDetect (Stratagene) signal transduction reporter system.", "htrb clones: Deletion constructs lacking the varible regions were made by PCR using the following primers: htrb-1 5′ deletion primer: 5′-CCGGATCCACCATGATCGCCGACTACCTGCTG-3′, 3′ deletion primer: 5′-CCGGTACCTTACGGCCGAAACCAGGGGTGCAGTAG-3′ htrb-3 5′ deletion primer: 5′-CCGGATCCATCCATCCATGATTGGGCCCTATGTCCTCCTGGAG-3′.", "PCR products were subcloned into PCR 2.1 TOPO (Invitrogen) and sequenced.", "For constructing GFP fusion proteins, htrb mutants were subcloned into pEGFPN1 or pEGFPN2 (Clontech), as appropriate.", "Cell cultures, transfections: HeLa (ECACC, 85060701) and NIH 3T3 cells were maintained in DMEM with 10% fetal calf serum (FCS) and penicillin-streptomycin.", "Raw cells were cultured in RPMI supplemented with 10% FCS and penicillin-streptomycin.", "Cells (1.5×104 per well) were seeded into 96-well tissue culture plates 24 h prior to transfection.", "Transfections were performed using SuperFect (Qiagen) according to the manufacturer's advice; each well received 500 ng of inducible reporter construct (pIL-8 luc, pAP-1 luc, pNFkB luc orpLHRE-TK luc) 100 ng of pTK-RLuc (Promega) for normalization of transfection efficiency, and 50 ng of htrb-1 or htrb-3 expression vectors under investigation, unless stated otherwise in the appropriate figure legend.", "500 ng pFR luc, 100 ng of pTK-RLuc and 10 ng pFA-CHOP or pFA2-ELk-1, 25 ng pMEK-1 or pMEK-3 plasmids were transfected to specifically activate p38 or ERK and to study the effect of htrb-3 on the activation.", "Sufficient pCDNA3.1 (Invitrogen) (“empty vector”) was added to keep the total DNA dose constant at 700 ng/well.", "2 hrs after transfection, cells were washed and 100 ul of fresh medium added.", "Triplicate wells were transfected for each treatment.", "Stimulations were performed for 4 hrs (unless indicated otherwise), twenty-four hours later.", "2nM IL-1beta or 10 ng/ml TNFalpha, 0.5 ug/ml human growth hormone, 50 ng/ml PMA or 10 nM of the other cytokines, listed on Table 1 was used (unless stated otherwise on the figure).", "Agonists were prepared and added as 10× stocks in 11 ul of PBS.", "Reporter levels were measured following 4 hours stimulation using the Dual-Luciferase system (Promega) as recommended by the manufacturer.", "Cytokines: IL-1beta was a kind gift from the Immunex Corporation.", "The other human cytokine preparations were kindly provided by Dr. Steve Poole, NIBSC.", "Western blotting: For detection of pJun, pERK and pMKK4, polyclonal antibodies were purchased from Sigma.", "Protein concentrations of cell lysates were determined and an equal amount of total protein was loaded in each lane.", "Kinase assays were performed by using the appropriate kits from New England Biolabs.", "Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein.", "Such equivalents are intended to be encompassed by the following claims.", "TABLE 1 A fold AP-1 fold NFkB Cytokine activation activation Interferon-α 3.35 ± 6% 1.23 ± 13% Interferon-γ 1.67 ± 5% 1.25 ± 6% IL-2 1.02 ± 17% 1.00 ± 34% IL-3 1.91 ± 7% 1.22 ± 11% IL-4 1.40 ± 26% 0.95 ± 8% IL-5 8.24 ± 8% 1.74 ± 2% IL-6 0.96 ± 11% 1.05 ± 6% IL-7 0.76 ± 24% 1.10 ± 6% IL-9 1.36 ± 16% 0.86 ± 2% IL-10 0.78 ± 5% 0.89 ± 5% IL-11 0.69 ± 8% 0.76 ± 9% IL-12 2.14 ± 11% 0.69 ± 13% IL-13 0.84 ± 10% 1.08 ± 21% IL-15 1.34 ± 10% 1.76 ± 7% bFGF 0.72 ± 14% 1.14 ± 9% EGF 0.84 ± 4% 1.28 ± 6% Ins.", "like GF 0.69 ± 16% 1.07 ± 3% TGF-β 13.37 ± 13% 6.61 ± 5% Oncostatin M 0.72 ± 18% 0.87 ± 6% M-CSF 1.52 ± 5% 1.20 ± 16% G-CSF 0.96 ± 14% 1.06 ± 15% GM-CSF 1.04 ± 15% 1.38 ± 21% PDGF-BB 9.06 ± 6% 0.76 ± 18% LIF 0.73 ± 7% 0.85 ± 10% Scatter factor 3.30 ± 8% 1.03 ± 4% PMA 10.37 ± 9% 5.16 ± 15% B AP-1 NFκB Cytokine inhibition inhibition Scatter factor 34% — IL-5 46% −1.5% TGF-β 30% 2% Interferon-α 31% — IL-12 16% — PDGF-BB 29% — PMA 39% 0.8%" ] ]	Patent_10466020
[ [ "Micromechanical flow sensor with tensile coating", "A sensor integrated on a semiconductor device (1), in particular a flow sensor, comprises a measuring element (2) on a membrane (5).", "In order to prevent a buckling of the membrane (5) a tensile coating (9) is applied.", "The coating covers the membrane, but it preferably leaves all the active electronic components integrated on the semiconductor chip (1) uncovered, such that their electrical properties are not affected." ], [ "1-15.", "(canceled) 16.A sensor with a semiconductor device, on which a measuring element and a circuit with active electronic components are integrated, wherein the measuring element is arranged on a membrane above an opening or recess of the semiconductor device, wherein a tensile coating is arranged on the semiconductor device for tautening the membrane, wherein the tensile coating leaves at least a part of the active components of the circuit uncovered.", "17.The sensor of claim 16 wherein the tensile coating leaves at least the active electronic components of the circuit uncovered.", "18.The sensor of claim 16 wherein the tensile coating extends of the membrane.", "19.The sensor of claim 16 wherein the active electronic components of the circuit comprise transistors.", "20.The sensor of claim 16 wherein the circuit is designed for processing signals of the measuring element.", "21.The sensor of claim 16 wherein the tensile coating extends beyond the membrane on at least two opposite sides.", "22.The sensor of claim 16 wherein the tensile stress of the coating is at least 100 MPa.", "23.The sensor of claim 16 wherein circuit parts are arranged on the membrane and wherein the tensile coating is arranged on the membrane and on the circuit parts.", "24.The sensor of claim 16 wherein a protective layer for protecting the electronic components is arranged on the sensor, wherein the protective layer is under compressive stress and wherein the protective layer does not extend over the membrane.", "25.The sensor of claim 24 wherein the protective layer and the tensile coating are of silicon nitride.", "26.The sensor of claim 16 wherein metallic structures are arranged on the semiconductor device and wherein the tensile coating is separated from the metallic structures by at least one separating layer.", "27.A method for producing a sensor having a semiconductor device on which a measuring element and a circuit with active electronic components are integrated, the measuring element being arranged on a membrane above an opening or recess of the semiconductor device, said method comprising the steps of applying a compressive protective layer on the semiconductor device, removing the compressive protective layer at least in a region of the membrane and applying the tensile coating at least in the region of the membrane.", "28.The method of claim 27 wherein, below the protective layer, a topmost metal layer is arranged and wherein, for removing the protective layer, the protective layer is etched off by a first etching agent, wherein the topmost metal layer acts as an etch stop, whereupon the topmost metal layer is removed by a second etching agent.", "29.Sensor of claim 16 wherein the circuit is designed for processing signals of the measuring element and not covered by the tensile coating.", "30.Sensor of claim 16 wherein the tensile coating extends beyond all sides of the membrane.", "31.Sensor of claim 16 wherein metallic structures are arranged on the semiconductor device and wherein the tensile coating is separated from the metallic structures by at least one separating layer of silicon oxide.", "32.A flow sensor comprising a semiconductor device with a recess or opening therein, a membrane above the recess or opening, a measuring element integrated on the semiconductor device, and arranged on the membrane a circuit with active electronic components integrated on the semiconductor device, a tensile coating for tautening the membrane, wherein the tensile coating leaves at least a part of the active components of the circuit uncovered.", "33.A sensor comprising a semiconductor device with a recess or opening therein, a membrane above the recess or opening, a measuring element integrated on the semiconductor device, and arranged on the membrane a circuit with active electronic components integrated on the semiconductor device, a tensile coating for tautening the membrane, wherein the tensile coating leaves at least a part of the active components of the circuit uncovered." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The invention relates to a sensor according to the preamble of claim 1 as well as to a method of its production.", "Sensors of this type are e.g.", "flow or temperature sensors, where at least a part of the measuring element is arranged on a membrane.", "This membrane has often a thickness of a few micrometers only and spans an opening or recess in the semiconductor device.", "Preferably, further active electronic components are integrated on the semiconductor device of sensors of this type, such as transistors for amplifiers or reference voltage sources.", "The membrane is usually formed by the layers deposited during the production of the circuit, wherein the semiconductor below the layers is etched away.", "The layers that are deposited in most of the conventional production processes, are, however, usually under compressive stress, i.e.", "pressure forces are acting within the plane of the layer, e.g.", "because the layers were applied at elevated temperatures and contracted less than the substrate while cooling down.", "The magnitude of the compressive stress depends on the manufacturing process and on the layer structure of the membrane.", "This compressive stress can lead to an undesired buckling of the membrane, which renders it mechanically unstable." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Hence, it is an object to provide a sensor of the type mentioned initially that avoids this problem.", "In order to prevent a buckling of the membrane, a tensile coating is applied on the membrane, This coating leaves at least part, preferably all, of the active electronic components integrated on the semiconductor device uncovered.", "As it has been found, the coating can otherwise lead to a change or degradation of the function of these components because it affects the electronic parameters of the semiconductor.", "Preferably, all active electronic components are therefore left uncovered by the tensile coating.", "The tensile coating covers preferably the whole membrane.", "In order to exert a pulling force suited for tightening the membrane, it should preferably extend beyond the membrane somewhat at least at two opposite sides.", "The invention is especially suited for being applied in integrated flow sensors." ], [ "CROSS REFERENCE TO RELATED APPLICATIONS This application claims the priority of Swiss patent application 0031/01, filed Jan. 10, 2001, the disclosure of which is incorporated herein by reference in its entirety.", "BACKGROUND OF THE INVENTION The invention relates to a sensor according to the preamble of claim 1 as well as to a method of its production.", "Sensors of this type are e.g.", "flow or temperature sensors, where at least a part of the measuring element is arranged on a membrane.", "This membrane has often a thickness of a few micrometers only and spans an opening or recess in the semiconductor device.", "Preferably, further active electronic components are integrated on the semiconductor device of sensors of this type, such as transistors for amplifiers or reference voltage sources.", "The membrane is usually formed by the layers deposited during the production of the circuit, wherein the semiconductor below the layers is etched away.", "The layers that are deposited in most of the conventional production processes, are, however, usually under compressive stress, i.e.", "pressure forces are acting within the plane of the layer, e.g.", "because the layers were applied at elevated temperatures and contracted less than the substrate while cooling down.", "The magnitude of the compressive stress depends on the manufacturing process and on the layer structure of the membrane.", "This compressive stress can lead to an undesired buckling of the membrane, which renders it mechanically unstable.", "SUMMARY OF THE INVENTION Hence, it is an object to provide a sensor of the type mentioned initially that avoids this problem.", "In order to prevent a buckling of the membrane, a tensile coating is applied on the membrane, This coating leaves at least part, preferably all, of the active electronic components integrated on the semiconductor device uncovered.", "As it has been found, the coating can otherwise lead to a change or degradation of the function of these components because it affects the electronic parameters of the semiconductor.", "Preferably, all active electronic components are therefore left uncovered by the tensile coating.", "The tensile coating covers preferably the whole membrane.", "In order to exert a pulling force suited for tightening the membrane, it should preferably extend beyond the membrane somewhat at least at two opposite sides.", "The invention is especially suited for being applied in integrated flow sensors.", "BRIEF DESCRIPTION OF THE DRAWINGS Further embodiments, advantages and applications of the invention are given in the dependent claims as well as in the now following description making reference to the drawings, wherein: FIG.", "1 is a top view of a flow sensor, wherein the components that lie below the tensile coating are shown in dashed lines, FIG.", "2 is a sectional view along line I-I of FIG.", "2, FIG.", "3 is a top view of the flow sensor where an additinoal protective layer is shown in addition to the tensile coating, FIG.", "4 is an example of a structure in a region of the protective layer and FIG.", "5 is an example of a structure in a region of the tensile coating.", "WAYS TO CARRY OUT THE INVENTION In FIGS.", "1 and 2 an embodiment of the invention in the form of a flow sensor is shown.", "It comprises a semi-conductor device 1, onto which a measuring element 2 and a circuit 3 are integrated.", "In semiconductor device 1 an opening or recess 4 has been etched out, which is covered by a thin membrane 5.A heating 6 is arranged on membrane 5.Two meandering thermopiles 7, 8 are provided symmetrically to heating 6, which act as temperature sensors.", "The orientation of the thermopiles 7, 8 and the heating 6 in respect to the flow direction of the medium to be measured is such that the medium first flows over first thermopile 7, then over heating 6, and finally over second thermopile 8.The measuring element 2 is covered by a tensile coating 9, which is under tensile stress and extends beyond membrane 5 on all sides or at least on two opposite sides of recess or opening 4.The overlap reaches at least sufficiently far in order to provide anchoring for the tensile coating 9 on semiconductor device 1 for receiving the tension.", "The tensile stress in tensile coating 9 is at least sufficiently large to exceed a compressive stress in membrane 5, which leads to a total tensile stress.", "Coating 9 therefore keeps membrane 5 tight and prevents or counteracts a buckling thereof.", "Tensile coating 9 can e.g.", "consist of a silicon oxide, silicon nitride or a polymer, in particular polyimide.", "Other possible materials are e.g.", "“Diamond Like Carbon” (DLC), polyether ether ketone (PEEK) or silicon.", "Silicon nitride has been found to be especially suited.", "The tensile stress in coating 9 can be controlled by means of known methods by suitable choice of the manufacturing parameters, see e.g.", "U.", "Munch et al., “Industrial Fabrication Technology for CMOS Infrared Sensor Arrays” in “Transducers '97, International conference on Solid State Sensors and Actuators”, IEEE 1997′, where it is described how, by suitable selection of the low frequency power and the pressure in a PECVD method, the tensile stress of a layer of silicon oxide nitride can be adjusted.", "A coating under tensile stress can also be manufactured by applying a coating material with a higher thermal expansion coefficient than silicon at elevated temperature onto semiconductor device 1.When cooling the device down, a tensile coating is generated inevitably.", "The tensile stress should be chosen sufficiently large such that it can compensate a possible compressive stress in membrane 5.Preferably, the tensile stress is at least 100 MPa.", "Photolithographic methods can be used for structuring or defining the spatial extension of tensile coating 9.A shadow mask can be used as well, or a lift-off technique can be applied, where an additional material layer below coating 9 is dissolved wherever coating 9 is to be removed.", "The general principle of operation of measuring element 2 is described in detail in “Scaling of Thermal CMOS Gas Flow Microsensors: Experiment and Simulation” by F. Mayer et al., in Proc.", "IEEE Micro Electro Mechanical Systems, (IEEE, 1996), pp.", "116ff.", "In particular, the temperatures over the thermopiles 7, 8 are measured for determining the mass flow over the sensor.", "The difference of these temperatures is a function of the mass flow.", "Circuit 3, which can e.g.", "be implemented in CMOS technology, is provided for the corresponding processing of the signals from the thermopiles 7, 8.It comprises amplifiers, A/D-converters with reference voltage sources, and a digital processing circuit with interface.", "For connecting circuit 3 with the exterior world, contact pads 10 are provided.", "As can be seen from FIG.", "1, tensile coating 9 only covers a part of semiconductor device 1, namely the part that is exposed to the medium to be measured.", "In particular, tensile coating 9 does not extend over circuit 3.Experiments have shown than mechanical stress caused by the tensile coating can affect the electrical parameters of semiconductor device 1, which can e.g.", "lead to a change of the properties of transistors, reference voltage sources, and other devices, in particular of active components and resistors.", "By not laying tensile coating 9 over these components, such a degradation can be avoided.", "This simplifies the manufacturing process because the known electrical parameters of the semiconductor can be used for modelling the circuit.", "Due to tensile coating 9 a buckling of the membrane can, as mentioned, be prevented.", "It also prevents or reduces a bending of membrane 5 if a pressure difference is applied over the same.", "In the above example, the invention has been described for a flow detector, but it can also be used in other applications: A membrane 5 of the type shown in FIG.", "2 can also be used in pressure sensors, where a pressure difference to be measured is applied over the membrane.", "In this case, tensile coating 9 can also be used for changing the sensitivity of the sensor.", "The higher the tensile stress and the elastic modulus in coating 9, the lower the sensitivity becomes.", "Further, the tensile coating 9 can be used for other types of sensors where a membrane of the type of FIG.", "2 is used, e.g.", "for infrared sensors.", "The tensile coating 9 can even be an active part of the sensor.", "Thus, it may consist of a material the dielectric or electric properties of which vary depending on a parameter to be measured.", "In a humidity sensor, a polymeric tensile coating, the dielectric constant or conductivity of which varies depending on current humidity, may e.g.", "be used.", "In a substance detector, tensile coating 9 can e.g.", "undergo chemical reactions with the substance to be measured, or its chemical potential or work function can change.", "Also the optical properties of the tensile coating can depend on a parameter to be measured.", "The tensile coating 9 can also have further functions.", "For example, it can in particular form an insulating layer that separates the components arranged on the membrane from the medium to be measured.", "It can e.g.", "serve as a passivation that prevents a damage of the components by acids or water.", "The layers of membrane 5 can be layers that are a result of the process for manufacturing circuit 3.Therefore, the mechanical properties, and in particular the tensility of these layers cannot be chosen freely.", "The additional tensile coating 9 allows it, however, to keep membrane 5 taut and to control its flexing properties independently from the used process.", "In the above described example the tensile coating is lying over membrane 5 as well as on the components arranged on the membrane.", "It can, however, also be arranged below membrane 5 or as a layer within membrane 5.In addition, electronic semiconductor components are often provided with a protective layer.", "This protective layer consists preferably of silicon nitride (Si3N4) and serves, in particular, for protecting the topmost metal layer from corrosion.", "In order to make the protective layer as tight as possible, it is, as a rule, compressive, i.e.", "it is under a compressive stress parallel to the semiconductor surface.", "In normal CMOS manufacturing processes it is applied to the device in a last step and covers the same substantially completely, with the exception of the contact pads 10.Such a protective layer can counteract the effect of tensile layer 9.Hence, it is preferably structured such that it, at least, does not extend over membrane 5.For this purpose, it can be left away in a region of membrane 5 or it can be removed before applying the tensile coating.", "A corresponding sensor is shown in FIG.", "3.It comprises a protective layer 12, which is under compressive stress and covers and protects at least circuit 3.The protective effect of protective layer 12 is, in general, better than the one of tensile coating 9 because the latter can tend to form holes and fractures because of its inherent tensile stress.", "Therefore, tensile coating 9 should not be applied directly on a metal layer (which corrodes easily).", "As a rule, several metal layers are provided in normal CMOS devices, as it is shown in FIG.", "4.In this example, the topmost metal layer 13 is covered by protective coating 12 and separated from the next to top metal layer 14 by means of a silicon oxide layer 15.Below the lower metal layer 14, further layers 16 may follow.", "If protective layer 12 is replaced by tensile coating 9, topmost metal layer 13 should be omitted, as it is shown in FIG.", "5.Hence, in the present case, no structures of topmost metal layer 13 should be provided in the area of coating 9.This ensures that, in the area of coating 9, all metal structures are protected by silicon oxide layer 15.Silicon oxide layer 15 therefore forms a separating layer between coating 9 and the metal structures of the device and protects the same from environmental influence.", "As mentioned above, protective layer 12 can be omitted in the area of membrane 5 or it can be removed prior to applying tensile coating 9.In the latter case, protective layer 9 has to be etched off in the area of membrane 5.During this, it should, however, be avoided that silicon oxide layer 15, by means of which the structures of lower metal layer 14 are to be protected, is damaged.", "As there are hardly any etching processes with a good selectivity between silicon oxide and silicon nitride, topmost metal layer 13 is preferably used as an etching stop when etching off protective layer 12.For this purpose, the latter is structured to extend over the whole membrane 5.Then the device is provided with coating 12.Now, coating 12 can be etched off in the area of the membrane by means of a first etching agent, wherein topmost metal layer 13 protects the next lower silicon nitride layer 15.Then topmost metal layer 13 can be removed in the area of membrane 5 by a metal specific second etching agent, again without impairing silicon oxide layer 14.Finally, coating 9 is applied to silicon oxide layer 14.The rule according to which coating 9 should not lie directly on a metal structure must also be observed in the area of so-called “scribe lines”.", "These are diffusion barriers that are formed by omitting, in an area, all layers with the exception of the metal layers.", "If a scribe line is arranged below coating 9, silicon layer 15 should be left over the scribe line.", "While the present application describes preferred embodiments of the invention, it is to be distinctly pointed out that the invention is not limited thereto and can also be carried out in different manner within the scope of the following claims." ] ]	Patent_10466026
[ [ "Graft copolymers and impact-resistant flame-retardant resin compositions containing the same", "The present invention provides a polyorganosiloxane-containing graft copolymer which is obtainable by polymerizing 0.5 to 10 parts by weight of a vinyl monomer (B) comprising 100 to 50% by weight of a polyfunctional monomer (b-1) containing two or more polymerizable unsaturated bonds in the presence of 40 to 90 parts of polyorganosiloxane particles, followed by further polymerizing 5 to 50 parts by weight of a vinyl monomer (C); A polyorganosiloxane-containing graft copolymer which is obtainable by polymerizing 0 to 10 parts by weight of a vinyl monomer (B) comprising 100 to 50% by weight of a polyfunctional monomer containing two or more polymerizable unsaturated bonds in the presence of 30 to 95 parts of a polyorganosiloxane in a latex form as obtained by seed polymerization using, as a seed polymer, a hydrophilic polymer capable of swelling in the corresponding organosiloxane, followed by further polymerizing 5 to 70 parts by weight of a vinyl monomer (C); a flame retardant which comprises said copolymer; and a resin composition which comprises said retardant and a thermoplastic resin." ], [ "1.A polyorganosiloxane-containing graft copolymer which is obtainable by polymerizing 0.5 to 10 parts by weight of a vinyl monomer (B) comprising 100 to 50% by weight of a polyfunctional monomer (b-1) containing two or more polymerizable unsaturated bonds and 0 to 50% by weight of another copolymerizable monomer (b-2), in the presence of 40 to 90 parts (as solid content) by weight of polyorganosiloxane particles (A1), followed by further polymerization of 5 to 50 parts by weight of a vinyl monomer (C), with the sum of (A1), (B) and (C) being 100 parts by weight.", "2.The polyorganosiloxane-containing graft copolymer according to claim 1, wherein the polyorganosiloxane particles (A1) have a volume average particle diameter of 0.008 to 0.6 μm.", "3.The polyorganosiloxane-containing graft copolymer according to claim 1, wherein the vinyl monomer (C) gives a polymer thereof having a solubility parameter of 9.15 to 10.15 (cal/cm3)1/2.4.The polyorganosiloxane-containing graft copolymer according to claim 1, wherein the polyorganosiloxane particles (A1) are in a latex form.", "5.The polyorganosiloxane-containing graft copolymer according to claim 1, wherein the vinyl monomer (C) comprises at least one monomer selected from the group consisting of aromatic vinyl monomers, vinyl cyanide monomers, (meth)acrylate ester monomers and carboxyl group-containing vinyl monomers.", "6.A flame retardant which comprises the polyorganosiloxane-containing graft copolymer according to claim 1.7.A flame retardant resin composition which comprises 0.1 to 30 parts by weight, per 100 parts by weight of a thermoplastic resin, of the flame retardant according to claim 6 as incorporated in the thermoplastic resin.", "8.The flame retardant resin composition according to claim 7, wherein the thermoplastic resin is a polycarbonate resin.", "9.A polyorganosiloxane-containing graft copolymer which is obtainable by polymerizing 0 to 10 parts by weight of a vinyl monomer (B) comprising 100 to 50% by weight of a polyfunctional monomer (b-1) containing two or more polymerizable unsaturated bonds and 0 to 50% by weight of another copolymerizable monomer (b-2), in the presence of 30 to 95 parts by weight (as solid content) of a polyorganosiloxane (A2) in a latex form obtainable by seed polymerization using, as a seed polymer, a hydrophilic polymer capable of swelling in the corresponding organosiloxane, followed by further polymerizing 5 to 70 parts by weight of a vinyl monomer (C), with the sum of (A2), (B) and (C) being 100 parts by weight.", "10.The polyorganosiloxane-containing graft copolymer according to claim 9, wherein the seed polymer has such a degree of hydrophilicity that the extraction rate of water-soluble components in dry seed polymer is 10 to 100% by weight, as determined after adding water, in an amount of 20 weight-times that of the seed polymer in a dry state, to the dry seed polymer, followed by stirring at 23° C. for 1 hour and wherein the seed polymer shows such a degree of swelling in the organosiloxane that the rate of swelling by volume as determined from the ratio between the latex particle diameter after stirring and the latex particle diameter before stirring is 3 to 50 times after adding the organosiloxane, in an amount 50 times by weight that of the dry seed polymer, to the seed polymer latex, followed by stirring at 23° C. for 1 hour.", "11.The polyorganosiloxane-containing graft copolymer according to claim 9, wherein the seed polymer has such a degree of hydrophilicity that the extraction rate of water-soluble components in dry seed polymer is 50 to 100% by weight, as determined after adding water, in an amount of 20 weight-times that of the seed polymer in a dry state, to the dry seed polymer, followed by stirring at 23° C. for 1 hour and wherein the seed polymer shows such a degree of swelling in the organosiloxane that the rate of swelling by volume as determined from the ratio between the latex particle diameter after stirring and the latex particle diameter before stirring is 3 to 15 times after adding the organosiloxane, in an amount 50 weight-times that of the dry seed polymer, to the seed polymer latex, followed by stirring at 23° C. for 1 hour.", "12.The polyorganosiloxane-containing graft copolymer according to claim 9, wherein the vinyl monomer (C) comprises at least one monomer selected from the group consisting of aromatic vinyl monomers, vinyl cyanide monomers, (meth)acrylate ester monomers and carboxyl group-containing vinyl monomers.", "13.A flame retardant which comprises the polyorganosiloxane-containing graft copolymer according to claim 9.14.A resin composition excellent in impact resistance and flame retardancy which comprises a thermoplastic resin and the flame retardant according to claim 13 as incorporated therein in an amount of 0.1 to 30 parts by weight per 100 parts by weight of the thermoplastic resin.", "15.The resin composition according to claim 14, wherein the thermoplastic resin is a polycarbonate resin.", "16.The polyorganosiloxane-containing graft copolymer according to claim 2, wherein the vinyl monomer (C) gives a polymer thereof having a solubility parameter of 9.15 to 10.15 (cal/cm3)1/2.17.The polyorganosiloxane-containing graft copolymer according to claim 2, wherein the polyorganosiloxane particles (A1) are in a latex form.", "18.The polyorganosiloxane-containing graft copolymer according to claim 3, wherein the polyorganosiloxane particles (A1) are in a latex form.", "19.The polyorganosiloxane-containing graft copolymer according to claim 2, wherein the vinyl monomer (C) comprises at least one monomer selected from the group consisting of aromatic vinyl monomers, vinyl cyanide monomers, (meth)acrylate ester monomers and carboxyl group-containing vinyl monomers.", "20.The polyorganosiloxane-containing graft copolymer according to claim 3, wherein the vinyl monomer (C) comprises at least one monomer selected from the group consisting of aromatic vinyl monomers, vinyl cyanide monomers, (meth)acrylate ester monomers and carboxyl group-containing vinyl monomers." ], [ "<SOH> BACKGROUND ART <EOH>Owing to their good impact resistance, heat resistance and electric characteristics, among others, thermoplastic resins, in particular polycarbonate resins, are widely used as materials of electric and electronic parts, OA (office automation) apparatus and instruments, and household utensils, or as building materials.", "Polycarbonate resins, though higher in flame retardancy as compared with polystyrene and other resins, are required to be highly flame retardant in particular in such fields as electric and electronic parts, OA apparatus and instruments and the like and, therefore, attempts have been made to improve their flame retardancy by adding various flame retardants.", "Thus, for instance, the addition of organohalogen compounds or organophosphorus compounds has so far been in wide practice.", "However, organohalogen compounds and organophosphorus compounds have a problem from the toxicity viewpoint.", "In particular, it is a drawback of organohalogen compounds that they generate a corrosive gas upon combustion thereof.", "Thus, the demand for halogen-free and phosphorus-free flame retardants has been increasing in recent years.", "The utilization of polyorganosiloxane compounds (also called silicones) as halogen-free and phosphorus-free flame retardants has been proposed.", "For example, Japanese Kokai Publication Sho-54-36365 describes that kneading of a monoorganopolysiloxane-based silicone resin with a non-silicone polymer gives a flame retardant resin.", "Japanese Kohyo Publication Hei-3-48947 describes that a mixture of a silicone resin and a salt of a metal of the group IIA provides thermoplastic resins with flame retardancy.", "Japanese Kokai Publication Hei-8-113712 describes a method of producing flame retardant resin compositions which comprises dispersing a silicone resin prepared by blending 100 parts by weight of a polyorganosiloxane with 10 to 150 parts by weight of a silica filler in thermoplastic resins.", "Japanese Kokai Publication Hei-10-139964 describes that flame retardant resin compositions are obtained by adding a solvent-soluble silicone resin having a weight average molecular weight of not less than 10,000 but not more than 270,000 to an aromatic ring-containing non-silicone resin.", "However, the silicone resins described in the above-cited publications are indeed effective in providing flame retardancy but their effects are still unsatisfactory.", "When the addition level is increased to fill up the shortage, a problem arises that the impact resistance of the resin composition decreases, making it difficult to obtain flame retardant resin composition balanced between flame retardancy and impact resistance.", "Japanese Kokai Publication 2000-17029 describes that when a composite rubber-based flame retardant produced by graft polymerization of a vinyl monomer onto a composite rubber composed of a polyorganosiloxane rubber and a polyalkyl (meth)acrylate rubber is incorporated in thermoplastic resins, flame retardant resin compositions can be obtained.", "Japanese Kokai Publication 2000-226420 describes that flame retardant resin compositions can be obtained by incorporating a polyorganosiloxane-based flame retardant produced by grafting a vinyl monomer onto composite particles consisting of an aromatic group-containing polyorganosiloxane and a vinyl polymer in thermoplastic resins.", "Japanese Kokai Publication 2000-264935 describes that flame retardant resin compositions can be obtained by incorporating, in thermoplastic resins, a polyorganosiloxane-containing graft copolymer prepared by graft copolymerization of a vinyl monomer onto polyorganosiloxane particles not larger than 0.2 μm in size.", "The flame retardant resin compositions described in the above-cited Japanese Kokai Publication 2000-17029, Japanese Kokai Publication 2000-226420 and Japanese Kokai Publication 2000-264935 all indeed show satisfactory levels of impact resistance but are unsatisfactory in flame retardancy.", "Thus, they have a problem that the flame retardancy-impact resistance balance is poor." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide a polyorganosiloxane-containing graft copolymer utilizable as a halogen-free and phosphorus-free flame retardant and excellent in flame retardancy and impact resistance improving effects as well as a flame-retardant resin composition excellent in flame retardancy and impact resistance using the graft copolymer mentioned above.", "The present inventors made intensive investigations concerning the above subject and, as a result, found that a specific polyorganosiloxane-containing graft copolymer is excellent in flame retardancy and impact resistance improving effects and that a flame-retardant resin composition excellent in flame retardancy and impact resistance can be obtained by incorporating the polyorganosiloxane-containing graft copolymer in a thermoplastic resin.", "Based on such findings, the present invention has now been completed.", "Thus, in accordance with a first aspect thereof, the present invention relates to: a polyorganosiloxane-containing graft copolymer which is obtainable by polymerizing 0.5 to 10 parts (parts by weight; hereinafter the same shall apply) of a vinyl monomer (B) comprising 100 to 50% (% by weight; hereinafter the same shall apply) of a polyfunctional monomer (b-1) containing two or more polymerizable unsaturated bonds and 0 to 50% of another copolymerizable monomer (b-2), in the presence of 40 to 90 parts of polyorganosiloxane particles (A1), followed by further polymerization of 5 to 50 parts of a vinyl monomer (C) [the sum of (A1), (B) and (C) being 100 parts] (claim 1 ); the polyorganosiloxane-containing graft copolymer according to claim 1 , wherein the polyorganosiloxane particles (A1) have a volume average particle diameter of 0.008 to 0.6 μm (claim 2 ); the polyorganosiloxane-containing graft copolymer according to claim 1 or 2 , wherein the vinyl monomer (C) gives a polymer thereof having a solubility parameter of 9.15 to 10.15 (cal/cm 3 ) 1/2 (claim 3 ); the polyorganosiloxane-containing graft copolymer according to any of claims 1 to 3 , wherein the polyorganosiloxane particles (A1) are in a latex form (claim 4 ); the polyorganosiloxane-containing graft copolymer according to any of claims 1 to 4 , wherein the vinyl monomer (C) comprises at least one monomer selected from the group consisting of aromatic vinyl monomers, vinyl cyanide monomers, (meth)acrylate ester monomers and carboxyl group-containing vinyl monomers (claim 5 ); a flame retardant which comprises a polyorganosiloxane-containing graft copolymer according to claim 1 (claim 6 ); a flame retardant resin composition which comprises 0.1 to 30 parts, per 100 parts of a thermoplastic resin, of a flame retardant according to claim 6 as incorporated in the thermoplastic resin (claim 7 ); and the flame retardant resin composition according to claim 7 , wherein the thermoplastic resin is a polycarbonate resin (claim 8 ).", "In accordance with a second aspect thereof, the invention relates to: a polyorganosiloxane-containing graft copolymer which is obtainable by polymerizing 0 to 10 parts by weight of a vinyl monomer (B) comprising 100 to 50% by weight of a polyfunctional monomer (b-1) containing two or more polymerizable unsaturated bonds and 0 to 50% by weight of another copolymerizable monomer (b-2), in the presence of 30 to 95 parts by weight (as solid content) of a polyorganosiloxane (A2) in a latex form obtainable by seed polymerization using, as a seed polymer, a hydrophilic polymer capable of swelling in the corresponding organosiloxane, followed by further polymerizing 5 to 70 parts by weight of a vinyl monomer (C) [the sum of (A2), (B) and (C) being 100 parts] (claim 9 ); the polyorganosiloxane-containing graft copolymer according to claim 9 , wherein the seed polymer has such a degree of hydrophilicity that the extraction rate of water-soluble components in dry seed polymer is 10 to 100% by weight, as determined after adding water, in an amount of 20 weight-times that of the seed polymer in a dry state, to the dry seed polymer, followed by stirring at 23° C. for 1 hour and wherein the seed polymer shows such a degree of swelling in the organosiloxane that the rate of swelling by volume as determined from the ratio between the latex particle diameter after stirring and the latex particle diameter before stirring is 3 to 50 times after adding the organosiloxane, in an amount 50 times by weight that of the dry seed polymer, to the seed polymer latex, followed by stirring at 23° C. for 1 hour (claim 10 ); the polyorganosiloxane-containing graft copolymer according to claim 9 , wherein the seed polymer has such a degree of hydrophilicity that the extraction rate of water-soluble components in dry seed polymer is 50 to 100% by weight, as determined after adding water, in an amount of 20 weight-times that of the seed polymer in a dry state, to the dry seed polymer, followed by stirring at 23° C. for 1 hour, and wherein the seed polymer shows such a degree of swelling in the organosiloxane that the rate of swelling by volume as determined from the ratio between the latex particle diameter after stirring and the latex particle diameter before stirring is 3 to 15 times after adding the organosiloxane, in an amount 50 weight-times that of the dry seed polymer, to the seed polymer latex, followed by stirring at 23° C. for 1 hour (claim 11 ); The polyorganosiloxane-containing graft copolymer according to any of claims 9 to 11 , wherein the vinyl monomer (C) comprises at least one monomer selected from the group consisting of aromatic vinyl monomers, vinyl cyanide monomers, (meth)acrylate ester monomers and carboxyl group-containing vinyl monomers (claim 12 ); a flame retardant which comprises the polyorganosiloxane-containing graft copolymer according to claim 9 (claim 13 ); a resin composition excellent in impact resistance and flame retardancy which comprises a thermoplastic resin and the flame retardant according to claim 13 as incorporated therein in an amount of 0.1 to 30 parts by weight per 100 parts by weight of the thermoplastic resin (claim 14 ); The resin composition according to claim 14 , wherein the thermoplastic resin is a polycarbonate resin (claim 15 ).", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to polyorganosiloxane-containing graft copolymers and impact-resistant, flame-retardant resin compositions containing the same.", "BACKGROUND ART Owing to their good impact resistance, heat resistance and electric characteristics, among others, thermoplastic resins, in particular polycarbonate resins, are widely used as materials of electric and electronic parts, OA (office automation) apparatus and instruments, and household utensils, or as building materials.", "Polycarbonate resins, though higher in flame retardancy as compared with polystyrene and other resins, are required to be highly flame retardant in particular in such fields as electric and electronic parts, OA apparatus and instruments and the like and, therefore, attempts have been made to improve their flame retardancy by adding various flame retardants.", "Thus, for instance, the addition of organohalogen compounds or organophosphorus compounds has so far been in wide practice.", "However, organohalogen compounds and organophosphorus compounds have a problem from the toxicity viewpoint.", "In particular, it is a drawback of organohalogen compounds that they generate a corrosive gas upon combustion thereof.", "Thus, the demand for halogen-free and phosphorus-free flame retardants has been increasing in recent years.", "The utilization of polyorganosiloxane compounds (also called silicones) as halogen-free and phosphorus-free flame retardants has been proposed.", "For example, Japanese Kokai Publication Sho-54-36365 describes that kneading of a monoorganopolysiloxane-based silicone resin with a non-silicone polymer gives a flame retardant resin.", "Japanese Kohyo Publication Hei-3-48947 describes that a mixture of a silicone resin and a salt of a metal of the group IIA provides thermoplastic resins with flame retardancy.", "Japanese Kokai Publication Hei-8-113712 describes a method of producing flame retardant resin compositions which comprises dispersing a silicone resin prepared by blending 100 parts by weight of a polyorganosiloxane with 10 to 150 parts by weight of a silica filler in thermoplastic resins.", "Japanese Kokai Publication Hei-10-139964 describes that flame retardant resin compositions are obtained by adding a solvent-soluble silicone resin having a weight average molecular weight of not less than 10,000 but not more than 270,000 to an aromatic ring-containing non-silicone resin.", "However, the silicone resins described in the above-cited publications are indeed effective in providing flame retardancy but their effects are still unsatisfactory.", "When the addition level is increased to fill up the shortage, a problem arises that the impact resistance of the resin composition decreases, making it difficult to obtain flame retardant resin composition balanced between flame retardancy and impact resistance.", "Japanese Kokai Publication 2000-17029 describes that when a composite rubber-based flame retardant produced by graft polymerization of a vinyl monomer onto a composite rubber composed of a polyorganosiloxane rubber and a polyalkyl (meth)acrylate rubber is incorporated in thermoplastic resins, flame retardant resin compositions can be obtained.", "Japanese Kokai Publication 2000-226420 describes that flame retardant resin compositions can be obtained by incorporating a polyorganosiloxane-based flame retardant produced by grafting a vinyl monomer onto composite particles consisting of an aromatic group-containing polyorganosiloxane and a vinyl polymer in thermoplastic resins.", "Japanese Kokai Publication 2000-264935 describes that flame retardant resin compositions can be obtained by incorporating, in thermoplastic resins, a polyorganosiloxane-containing graft copolymer prepared by graft copolymerization of a vinyl monomer onto polyorganosiloxane particles not larger than 0.2 μm in size.", "The flame retardant resin compositions described in the above-cited Japanese Kokai Publication 2000-17029, Japanese Kokai Publication 2000-226420 and Japanese Kokai Publication 2000-264935 all indeed show satisfactory levels of impact resistance but are unsatisfactory in flame retardancy.", "Thus, they have a problem that the flame retardancy-impact resistance balance is poor.", "SUMMARY OF THE INVENTION It is an object of the present invention to provide a polyorganosiloxane-containing graft copolymer utilizable as a halogen-free and phosphorus-free flame retardant and excellent in flame retardancy and impact resistance improving effects as well as a flame-retardant resin composition excellent in flame retardancy and impact resistance using the graft copolymer mentioned above.", "The present inventors made intensive investigations concerning the above subject and, as a result, found that a specific polyorganosiloxane-containing graft copolymer is excellent in flame retardancy and impact resistance improving effects and that a flame-retardant resin composition excellent in flame retardancy and impact resistance can be obtained by incorporating the polyorganosiloxane-containing graft copolymer in a thermoplastic resin.", "Based on such findings, the present invention has now been completed.", "Thus, in accordance with a first aspect thereof, the present invention relates to: a polyorganosiloxane-containing graft copolymer which is obtainable by polymerizing 0.5 to 10 parts (parts by weight; hereinafter the same shall apply) of a vinyl monomer (B) comprising 100 to 50% (% by weight; hereinafter the same shall apply) of a polyfunctional monomer (b-1) containing two or more polymerizable unsaturated bonds and 0 to 50% of another copolymerizable monomer (b-2), in the presence of 40 to 90 parts of polyorganosiloxane particles (A1), followed by further polymerization of 5 to 50 parts of a vinyl monomer (C) [the sum of (A1), (B) and (C) being 100 parts] (claim 1); the polyorganosiloxane-containing graft copolymer according to claim 1, wherein the polyorganosiloxane particles (A1) have a volume average particle diameter of 0.008 to 0.6 μm (claim 2); the polyorganosiloxane-containing graft copolymer according to claim 1 or 2, wherein the vinyl monomer (C) gives a polymer thereof having a solubility parameter of 9.15 to 10.15 (cal/cm3)1/2 (claim 3); the polyorganosiloxane-containing graft copolymer according to any of claims 1 to 3, wherein the polyorganosiloxane particles (A1) are in a latex form (claim 4); the polyorganosiloxane-containing graft copolymer according to any of claims 1 to 4, wherein the vinyl monomer (C) comprises at least one monomer selected from the group consisting of aromatic vinyl monomers, vinyl cyanide monomers, (meth)acrylate ester monomers and carboxyl group-containing vinyl monomers (claim 5); a flame retardant which comprises a polyorganosiloxane-containing graft copolymer according to claim 1 (claim 6); a flame retardant resin composition which comprises 0.1 to 30 parts, per 100 parts of a thermoplastic resin, of a flame retardant according to claim 6 as incorporated in the thermoplastic resin (claim 7); and the flame retardant resin composition according to claim 7, wherein the thermoplastic resin is a polycarbonate resin (claim 8).", "In accordance with a second aspect thereof, the invention relates to: a polyorganosiloxane-containing graft copolymer which is obtainable by polymerizing 0 to 10 parts by weight of a vinyl monomer (B) comprising 100 to 50% by weight of a polyfunctional monomer (b-1) containing two or more polymerizable unsaturated bonds and 0 to 50% by weight of another copolymerizable monomer (b-2), in the presence of 30 to 95 parts by weight (as solid content) of a polyorganosiloxane (A2) in a latex form obtainable by seed polymerization using, as a seed polymer, a hydrophilic polymer capable of swelling in the corresponding organosiloxane, followed by further polymerizing 5 to 70 parts by weight of a vinyl monomer (C) [the sum of (A2), (B) and (C) being 100 parts] (claim 9); the polyorganosiloxane-containing graft copolymer according to claim 9, wherein the seed polymer has such a degree of hydrophilicity that the extraction rate of water-soluble components in dry seed polymer is 10 to 100% by weight, as determined after adding water, in an amount of 20 weight-times that of the seed polymer in a dry state, to the dry seed polymer, followed by stirring at 23° C. for 1 hour and wherein the seed polymer shows such a degree of swelling in the organosiloxane that the rate of swelling by volume as determined from the ratio between the latex particle diameter after stirring and the latex particle diameter before stirring is 3 to 50 times after adding the organosiloxane, in an amount 50 times by weight that of the dry seed polymer, to the seed polymer latex, followed by stirring at 23° C. for 1 hour (claim 10); the polyorganosiloxane-containing graft copolymer according to claim 9, wherein the seed polymer has such a degree of hydrophilicity that the extraction rate of water-soluble components in dry seed polymer is 50 to 100% by weight, as determined after adding water, in an amount of 20 weight-times that of the seed polymer in a dry state, to the dry seed polymer, followed by stirring at 23° C. for 1 hour, and wherein the seed polymer shows such a degree of swelling in the organosiloxane that the rate of swelling by volume as determined from the ratio between the latex particle diameter after stirring and the latex particle diameter before stirring is 3 to 15 times after adding the organosiloxane, in an amount 50 weight-times that of the dry seed polymer, to the seed polymer latex, followed by stirring at 23° C. for 1 hour (claim 11); The polyorganosiloxane-containing graft copolymer according to any of claims 9 to 11, wherein the vinyl monomer (C) comprises at least one monomer selected from the group consisting of aromatic vinyl monomers, vinyl cyanide monomers, (meth)acrylate ester monomers and carboxyl group-containing vinyl monomers (claim 12); a flame retardant which comprises the polyorganosiloxane-containing graft copolymer according to claim 9 (claim 13); a resin composition excellent in impact resistance and flame retardancy which comprises a thermoplastic resin and the flame retardant according to claim 13 as incorporated therein in an amount of 0.1 to 30 parts by weight per 100 parts by weight of the thermoplastic resin (claim 14); The resin composition according to claim 14, wherein the thermoplastic resin is a polycarbonate resin (claim 15).", "DETAILED DISCLOSURE OF THE INVENTION First Aspect of the Invention The polyorganosiloxane-containing graft copolymer according to the first aspect of the invention is one obtainable by polymerizing 0.5 to 10 parts of a vinyl monomer (B) (hereinafter also referred to as “vinyl monomer B”), which comprises 100 to 50% of a polyfunctional monomer (b-1) (hereinafter also referred to as “polyfunctional monomer (b-1)”) containing two or more polymerizable unsaturated bonds and 0 to 50% of another copolymerizable monomer (b-2) (hereinafter also referred to as “copolymerizable monomer (b-2)”), in the presence of 40 to 90 parts (as solid content) of polyorganosiloxane particles (A1), followed by further polymerization of 5 to 50 parts of a vinyl monomer (C), with the sum of (A1), (B) and (C) amounting to 100 parts.", "The polyorganosiloxane particles (A1) preferably have a volume average particle diameter of not less than 0.008 μm, more preferably not less than 0.01 μm, as determined by light scattering method or electron microscopic observation.", "On the other hand, it is preferably not larger than 0.6 μm, more preferably not larger than 0.2 μm, most preferably not larger than 0.15 μm.", "It tends toward difficulty to obtain particles smaller in volume average particle diameter than 0.008 μm and, when such diameter exceeds 0.6 μm, the flame retardancy tends to deteriorate.", "The coefficient of variation in the particle diameter distribution (100× standard deviation/volume average particle diameter) (%) of the polyorganosiloxane particles is desirably controlled so that it may amount preferably to 10 to 100%, more preferably to 20 to 60%, since then the moldings produced from the resin composition containing the graft copolymer according to the first aspect of the invention can have a good surface appearance.", "From the flame retardancy and impact resistance viewpoint, the polyorganosiloxane particles (A1) preferably has a toluene-insoluble matter content (as determined by immersing 0.5 g of the particles in 80 ml of toluene at 23° C. for 24 hours) of not more than 95%, more preferably not more than 50%, most preferably not more than 20%.", "In the first aspect of the invention, the polyorganosiloxane particles (A1) include, within the meaning thereof, not only particles made of a polyorganosiloxane(s) alone but also modified polyorganosiloxane particles containing not more than 5% of another or other (co)polymers.", "Thus, the polyorganosiloxane particles (A1) may contain not more than 5% of polybutyl acrylate, a butyl acrylate-styrene copolymer and/or the like therein.", "As specific examples of the polyorganosiloxane particles (A1), there may be mentioned polydimethylsiloxane particles, polymethylphenylsiloxane particles, and dimethylsiloxane-diphenylsiloxane copolymer particles.", "These particle species may be used singly or two or more of them may be used in combination.", "The polyorganosiloxane particles (A1) can be obtained, for example, by polymerizing (1) an organosiloxane, (2) a bifunctional silane compound, (3) an organosiloxane and a bifunctional silane compound, (4) an organosiloxane and a vinylic polymerizable group-containing silane compound, (5) a bifunctional silane compound and a vinylic polymerizable group-containing silane compound or (6) an organosiloxane, a bifunctional silane compound and a vinylic polymerizable group-containing silane compound, and the like, and optionally further with an at least tri functional silane compound(s).", "The “bifunctional silane compound” is a silane compound having a total number of two of a hydroxyl group(s) and/or a hydrolyzable group(s) each bound to a silicon atom.", "The “at least trifunctional silane compound” means a silane compound having a total number of at least three of a hydroxyl group(s) and/or a hydrolyzable group(s) each bound to a silicon atom.", "The above-mentioned organosiloxane and bifunctional silane compound are components constituting the main skeleton of the polyorganosiloxane chain.", "As specific examples of the organosiloxane, there may be mentioned, among others, hexamethylcyclotrisiloxane (D3), octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), dodecamethylcyclohexasiloxane (D6), tetradecamethylcycloheptasiloxane (D7), and hexadecamethylcyclooctasiloxane (D8).", "As specific examples of the bifunctional silane compound, there may be mentioned diethoxydimethylsilane, dimethoxydimethylsilane, diphenyldimethoxysilane, diphenyldiethoxysilane, 3-chloropropylmethyldimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, heptadecafluorodecylmethyldimethoxysilane, trifluoropropylmethyldimethoxysilane, octadecylmethyldimethoxysilane, and the like.", "From the viewpoint of economical efficacy and flame retardancy, silane compounds or mixtures thereof comprising 70 to 100%, preferably 80 to 100%, of D4 or a mixture of D3 to D7 or a mixture of D3 to D8 and, as the rest, 0 to 30%, preferably 0 to 20% of diphenyldimethoxysilane, diphenyldiethoxysilane and/or the like are preferred among others.", "The above-mentioned vinylic polymerizable group-containing silane compound is component for introducing a vinylic polymerizable group into side chains and/or a terminus or termini of the copolymer by copolymerization with the above-mentioned organosiloxane, bifunctional silane compound, at least trifunctional silane compound and/or the like, and such vinylic polymerizable group serves as an active site for grafting in chemical binding with a vinyl (co)polymer formed from the vinyl monomer (B) or vinyl monomer (C) mentioned later herein.", "Furthermore, it is a component capable of forming a crosslink between such active sites for grafting in the manner of radical reaction induced by a radical polymerization initiator and thus capable of serving also as a crosslinking agent.", "On that occasion, the radical polymerization initiator may be the same one as can be used in the graft polymerization to be mentioned later herein.", "Even when crosslinking is caused by the radical reaction, grafting is still possible since such sites partly remain as active sites for grafting.", "As specific examples of the above-mentioned vinylic polymerizable group-containing silane compound, there may be mentioned, among others, γ-methacryloyloxypropyldimethoxymethylsilane, γ-methacryloyloxypropyltrimethoxysilane, γ-methacryloyloxypropyltriethoxysilane, γ-methacryloyloxypropyldiethoxymethylsilane, γ-acryloyloxypropyldimethoxymethylsilane, γ-acryloyloxypropyltrimethoxysilane and like (meth)acryloyloxy group-containing silane compounds, p-vinylphenyldimethoxymethylsilane, p-vinylphenyltrimethoxysilane and like vinylphenyl group-containing silane compounds, vinylmethyldimethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane and like vinyl group-containing silane compounds, mercaptopropyltrimethoxysilane, mercaptopropyldimethoxymethylsilane and like mercapto group-containing silane compounds.", "Among these, (meth)acryloyloxy group-containing silane compounds, vinyl group-containing silane compounds and mercapto group-containing silane compounds are preferred from the economical viewpoint.", "In cases where the above-mentioned vinylic polymerizable group-containing silane compound is of the trialkoxysilane type, it serves also as the at least trifunctional silane compound mentioned below.", "The at least trifunctional silane compound is used as a component for introducing a crosslinked structure into the polyorganosiloxane and providing the same with rubber elasticity as a result of copolymerization thereof with the above-mentioned organosiloxane, bifunctional silane compound and/or vinylic polymerizable group-containing silane compound, among others, namely as a crosslinking agent for the polyorganosiloxane.", "As specific examples, there may be mentioned tetraethoxysilane, methyltriethoxysilane, methyltrimethoxysilane, ethyltriethoxysilane, 3-glycidoxypropyltrimethoxysilane, heptadecafluorodecyltrimethoxysilane, trifluoropropyltrimethoxysilane, octadecyltrimethoxysilane and like tetrafunctional or trifunctional alkoxysilane compounds.", "Among these, tetraethoxysilane and methyltriethoxysilane are preferably used in view of the high efficiency of crosslinking attainable therewith.", "In the polymerization thereof, the above-mentioned organosiloxane, bifunctional silane compound, vinylic polymerizable group-containing silane compound, and at least trifunctional silane compound are generally used in proportions such that the organosiloxane and/or bifunctional silane compound (the ratio between the organosiloxane and bifunctional silane compound generally being 100/0 to 0/100 by weight, preferably 100/0 to 70/30 by weight) amounts to 50 to 99.9%, preferably 60 to 99.5%, the vinylic polymerizable group-containing silane compound to 0 to 40%, preferably 0.5 to 30%, and the at least trifunctional silane compound to 0 to 50%, preferably 0 to 39%.", "The vinylic polymerizable group-containing silane compound and at least trifunctional silane compound do not simultaneously amount to 0%, and at least one of them is preferably used in an amount of not less than 0.1%.", "When the proportion of the above-mentioned organosiloxane and/or bifunctional silane compound is too small, the resin compositions obtained by incorporating the resulting copolymer therein tend to become brittle.", "When the proportion is excessive, the amount of the vinylic polymerizable group-containing silane compound and/or at least trifunctional silane compound becomes excessively small and the effects of using these tend to be hardly produced.", "Furthermore, when the proportion of the above-mentioned vinylic polymerizable group-containing silane compound and/or at least trifunctional silane compound is too small, the flame retardancy-causing effect becomes unsatisfactory and, when it is excessive, the resin compositions obtained by incorporating the resulting copolymer therein tend to become brittle.", "The above-mentioned polyorganosiloxane particles (A1) are preferably produced, for example, by emulsion polymerization of the polyorganosiloxane-forming components, such as the organosiloxane, bifunctional silane compound or/and vinylic polymerizable group-containing silane compound, optionally with the at least trifunctional silane compound as added according to need.", "The above-mentioned emulsion polymerization may be carried out by emulsifying and dispersing the above polyorganosiloxane-forming components in water in the presence of an emulsifier by means of mechanical shearing and making the system acidic.", "When, in this case, emulsion droplets not smaller than several micrometers are prepared by means of mechanical shearing, the volume average particle diameter of the polyorganosiloxane particles (A1) obtained after polymerization can be controlled within the range of 0.02 to 0.6 μm by varying the amount of the emulsifier used.", "As for the coefficient of variation in particle diameter distribution (100×standard deviation/volume average particle diameter) (%), a value of 20 to 70% can be obtained.", "For producing polyorganosiloxane particles not larger than 0.1 μm with a narrow particle diameter distribution, the polymerization is preferably carried out in a multistage manner.", "For example, a 1 to 20% portion of an emulsion composed of emulsion droplets not smaller than several micrometers as obtained by emulsification, under mechanical shearing, of the above-mentioned polyorganosiloxane-forming components, water and the emulsifier is subjected in advance to emulsion polymerization under acidic conditions, the remaining portion of the emulsion is added and subjected to polymerization in the presence of the polyorganosiloxane particles obtained, which serve as seeds.", "It is possible to control, by adjusting the emulsifier amount, in a manner such that the thus-obtained polyorganosiloxane particles may have a volume average particle diameter of 0.02 to 0.1 μm with a coefficient of variation in particle diameter distribution of 10 to 60%.", "According to a more preferred procedure for the multistage polymerization, the multistage polymerization is carried out in the same manner using a vinyl (co)polymer obtained by ordinary emulsion polymerization of a vinyl monomer(s) (e.g.", "styrene, butyl acrylate, methyl methacrylate), which is(are) used on the occasion of graft polymerization mentioned later herein, in lieu of the polyorganosiloxane particles serving as seeds, whereby it is possible to control, by adjusting the emulsifier amount, in a manner such that the polyorganosiloxane (modified polyorganosiloxane) particles obtained may have a volume average particle diameter of 0.008 to 0.1 μm with a coefficient of variation in particle diameter distribution of 10 to 50%.", "The above-mentioned emulsion droplets not smaller than several micrometers can be prepared by using a high-speed stirrer, for example a Homomixer.", "In the above-mentioned emulsion polymerization, an emulsifier which will not lose its emulsifying ability under acidic conditions is used.", "As specific example, there may be mentioned alkylbenzenesulfonic acids, sodium alkylbenzenesulfonates, alkylsulfonic acids, sodium alkylsulfonates, sodium (di)alkyl sulfosuccinates, sodium polyoxyethylene nonylphenyl ether sulfonates, sodium alkylsulfates, and the like.", "These may be used singly or a combination of two or more may also be used.", "Among them, alkylbenzenesulfonic acids, sodium alkylbenzenesulfonates, alkylsulfonic acids, sodium alkylsulfonates, and sodium (di)alkyl sulfosuccinates are preferred in view of the relatively high emulsion stability of the emulsion.", "Particularly preferred are alkylbenzenesulfonic acids and alkylsulfonic acids, since they also serve as polymerization catalysts for the polyorganosiloxane-forming components.", "The acidic condition can be obtained by adding, to the system, an inorganic acid, such as sulfuric acid or hydrochloric acid, or an organic acid, such as an alkylbenzenesulfonic acid, an alkylsulfonic acid or trifluoroacetic acid.", "For avoiding corrosion of the production equipment and for attaining an adequate rate of polymerization, the pH is preferably adjusted to 1 to 3, more preferably to 1.0 to 2.5.For attaining an adequate rate of polymerization, the heating for the polymerization is carried out preferably at 60 to 120° C., more preferably 70 to 100° C. Under such an acidic condition, the Si—O—Si bonds forming the polyorganosiloxane skeleton are in a state of equilibrium between cleavage and formation, and this equilibrium varies depending on the temperature.", "Therefore, for stabilizing the polyorganosiloxane chain, the system is preferably neutralized with an aqueous solution of an alkali, such as sodium hydroxide, potassium hydroxide or sodium carbonate.", "Furthermore, as the temperature lowers, the above equilibrium shifts to the formation side, facilitating the formation of products with a high-molecular weight and a high degree of crosslinking, so that, for obtaining polymer having high-molecular weight or high degree of crosslinking, the system after carrying out the polymerization of the polyorganosiloxane-forming components at 60° C. or above is preferably cooled to room temperature or below and, after about 5 to 100 hours of standing, it is neutralized.", "The thus-obtained polyorganosiloxane particles (A1), when formed, for example, by polymerization of the organosiloxane and/or bifunctional silane compound, with or without further addition of the vinylic polymerizable group-containing silane compound, occur as a vinylic polymerizable group-containing polymer generally resulting from random copolymerization.", "In cases where the at least trifunctional silane compound is used in the copolymerization, the particles have a crosslinked network structure.", "When crosslinking is effected between vinylic polymerizable groups in the manner of radical reaction using such a radical polymerization initiator as used on the occasion of graft polymerization, which is mentioned later herein, the product has a crosslinked structure resulting from chemical bonding between vinylic polymerizable groups, with part of the vinylic polymerizable groups remaining unreacted.", "Graft polymerization of the vinyl monomer (B) and vinyl monomer (C) onto the polyorganosiloxane particles (A1) gives the polyorganosiloxane-containing graft copolymer.", "In the polymerization of the vinyl monomer (B) and vinyl monomer (C) in the presence of the polyorganosiloxane particles, there are also produced, as byproducts, the so-called free polymer molecules resulting from polymerization of the branch components (herein polymers of the vinyl monomer (B) and vinyl monomer (C)) alone without their grafting onto the stem component (herein the polyorganosiloxane particles (A1)).", "The product is thus obtained as a mixture of the graft copolymer and free polymer molecules.", "In accordance with the first aspect of the invention, these both species are collectively referred to as the graft copolymer.", "The above-mentioned graft copolymer structurally results from grafting of the vinyl monomer (B) onto the polyorganosiloxane particles (A1) and grafting of the vinyl monomer (C) not only onto the polyorganosiloxane particles (A1) but also onto the polymer molecules formed by the vinyl monomer (B), hence the free polymer content is low.", "The graft copolymer preferably has an acetone-insoluble matter content (as determined by immersing 1 g of the graft copolymer in 80 ml acetone at 23° C. for 48 hours) of not less than 80%, more preferably not less than 85% in view of good flame retardant effects obtainable in such a case.", "The above-mentioned vinyl monomer (B) is used for enhancing the flame retardant and impact resistance-improving effects.", "It comprises 100 to 50%, preferably 100 to 80%, more preferably 100 to 90%, of a polyfunctional monomer (b-1) containing two or more polymerizable unsaturated bonds and 0 to 50%, preferably 0 to 20%, more preferably 0 to 10%, of another copolymerizable monomer (b-2).", "When the proportion of the polyfunctional monomer (b-1) is too small, or when the copolymerizable monomer (b-2) is excessive, the graft copolymer finally obtained tends to become less effective in improving the impact resistance.", "The polyfunctional monomer (b-1) is a compound containing two or more polymerizable unsaturated bonds in the molecule.", "As specific examples thereof, there may be mentioned allyl methacrylate, triallyl cyanurate, triallyl isocyanurate, diallyl phthalate, ethylene glycol dimethacrylate, 1,3-butylene glycol dimethacrylate, and divinylbenzene.", "These may be used singly or two or more of them may be used in combination.", "Among them, the use of allyl methacrylate, in particular, is preferred from the economical and efficacy viewpoint.", "As specific examples of the copolymerizable monomer (b-2), there may be mentioned, among others, aromatic vinyl monomers such as styrene, α-methylstyrene, paramethylstyrene and parabutylstyrene, vinyl cyanide monomers such as acrylonitrile and methacrylonitrile, (meth)acrylate ester monomers such as methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, 2-ethylhexyl acrylate, glycidyl acrylate, hydroxyethyl acrylate, hydoxybutyl acrylate, methyl methacrylate, ethyl methacrylate, butyl methacrylate, lauryl methacrylate, glycidyl methacrylate and hydroxyethyl methacrylate, and carboxyl group-containing vinyl monomers such as itaconic acid, (meth)acrylic acid, fumaric acid and maleic acid.", "These may be used singly or two or more of them may be used in combination.", "The above-mentioned vinyl monomer (C) is a component to be used for obtaining the polyorganosiloxane-containing graft copolymer.", "It is also a component to be used for securing compatibility between the graft copolymer and a thermoplastic resin to thereby disperse the graft copolymer in the thermoplastic resin uniformly for the improvements in flame retardancy and impact resistance by incorporating the graft copolymer in the thermoplastic resin.", "Therefore, the vinyl monomer (C) is preferably selected so that a polymer of the vinyl monomer may preferably have a solubility parameter of not less than 9.15 (cal/cm3)1/2, more preferably not less than 9.17 (cal/cm3)1/2, still more preferably not less than 9.20 (cal/cm3)1/2.Also preferably, it is selected so that the solubility parameter in question may be not more than 10.15 (cal/cm3)1/2, more preferably not more than 10.10 (cal/cm3)1/2, still more preferably not more than 10.05 (cal/cm3)1/2.When the solubility parameter is outside the above range, the flame retardancy tends to decrease.", "The solubility parameter values are calculated using the small group parameters according to the group contribution method described in “Polymer Handbook”, 4th edition, published by John Wiley & Sons, Inc., 1999, Section VII, pages 682-685.For example, the value for poly (methyl methacrylate) (regarding the repeating unit molecular weight as 100 g/mole, and the density as 1.19 g/cm3) is 9.25 [(cal/cm3)1/2], for poly(butyl acrylate) (regarding the repeating unit molecular weight as 128 g/mole, and the density as 1.06 g/cm3) 8.97 [(cal/cm3)1/2], for poly(butyl methacrylate) (regarding the repeating unit molecular weight as 142 g/mole, and the density as 1.06 g/cm3) 9.47 [(cal/cm3)1/2], for polystyrene (regarding the repeating unit molecular weight as 104, and the density as 1.05 g/cm3) 9.03 [(cal/cm3)1/2], and for polyacrylonitrile (regarding the repeating unit molecular weight as 53, and the density as 1.18 g/cm3) 12.71 [(cal/cm3)1/2].", "Used as the density values for the respective polymers were those described in Ullman's Encyclopedia of Industrial Chemistry, published by VCH, 1992, volume A21, page 169.As for the solubility parameter δc of each copolymer, the value for the main component was employed when the copolymer weight fraction is less than 5% and, when that weight fraction is not less than 5%, it was supposed that the additivity rule based on the weight fractions can hold good.", "Thus, the solubility parameter δc can be calculated from the solubility parameters on of respective homopolymers of m vinyl monomer species constituting the copolymer in question and weight fraction Wn of that polymer according to the following equation (1): δ ⁢ ⁢ c = ∑ n = 1 n = m ⁢ δ ⁢ ⁢ nWn / ∑ n = 1 n = m ⁢ Wn ( 1 ) Thus, for example, the solubility parameter of a copolymer composed of 75% of styrene and 25% of acrylonitrile can be found to be 9.95 [(cal/cm3)1/2] by using the solubility parameter 9.03 [(cal/cm3)1/2] of polystyrene and the solubility parameter 12.71 [(cal/cm3)1/2] of polyacrylonitrile and using the equation (1).", "For the solubility parameter δs of a vinyl polymer obtained by carrying out polymerization in two or more stages while varying the vinyl monomer species in each stage, it was supposed that the additivity rule can hold good with respect to the values obtained by dividing the weights of the vinyl polymers obtained in the respective stages by the whole weight of the vinyl polymer finally obtained, namely the weight fractions.", "Thus, the value δs of the polymer polymerized in q stages can be calculated from the solubility parameter δi of the polymer obtained in each stage and the weight fraction Wi of that polymer according to the following equation (2): δ ⁢ ⁢ s = ∑ i = 1 i = q ⁢ δ ⁢ ⁢ iWi / ∑ i = 1 i = q ⁢ Wi ( 2 ) Thus, for example, when polymerization was carried out in two stages and, in stage 1, 50 parts of a copolymer composed of 75% of styrene and 25% of acrylonitrile was obtained and, in stage 2, 50 parts of a polymer of methyl methacrylate was obtained, the solubility parameter of the polymer obtained by such two-stage polymerization can be calculated as 9.60 [(cal/cm3)1/2] by using the solubility parameter value 9.95 [(cal/cm3)1/2] for the 75% styrene-25% acrylonitrile copolymer and the solubility parameter value 9.25 [(cal/cm3)1/2] for poly(methyl methacrylate) and using the equation (2).", "The above-mentioned vinyl monomer (C) includes the same ones as mentioned hereinabove as the other copolymerizable monomer (b-2) referring to the vinyl monomer (B).", "These may be used singly or two or more of them may be used in combination.", "The polyorganosiloxane-containing graft copolymer according to the first aspect of the invention can be obtained by polymerizing 0.5 to 10 parts (preferably not less than 1 part, more preferably not less than 2 parts, but preferably not more than 5 parts, more preferably not more than 4 parts) of the vinyl monomer (B) in the presence of 40 to 90 parts (as solid content) (preferably not less than 60 parts, but preferably not more than 80 parts, more preferably not more than 75 parts) of the above-mentioned polyorganosiloxane particles (A1) and further polymerizing 5 to 50 parts (preferably not less than 15 parts, more preferably not less than 21 parts, but preferably not more than 39 parts, more preferably not more than 38 parts) of the vinyl monomer (C) so that the total amount may become 100 parts.", "When the amount of the polyorganosiloxane particles (A1) is too small or too large, the flame retardant effect tends to decrease in either case.", "When the amount of the vinyl monomer (B) is too small, the flame retardant and impact resistance improving effects tend to decrease and, when it is excessive, the impact resistance improving effect tends to decrease.", "When the vinyl monomer (C) is too small or too large, the flame retardant effect tends to lower in either case.", "In carrying out above graft polymerization, the technique of ordinary seed emulsion polymerization can be applied.", "Thus, the above-mentioned vinyl monomer (B) and vinyl monomer (C) can be subjected to radical polymerization in the presence of polyorganosiloxane particles (A1).", "In the radical polymerization in question, the polyorganosiloxane particles (A1) are preferably in a latex form.", "Namely, it is preferred that the radical polymerization should be carried out in latex of the polyorganosiloxane particles (A1).", "The vinyl monomer (B) and vinyl monomer (C) each may be polymerized in one stage or in two or more stages.", "The above radical polymerization can be carried out by the method comprising thermally decomposing a radical polymerization initiator to thereby cause the reaction to proceed, or by the method comprising allowing the reaction to proceed in a redox system using a reducing system, for instance, without any particular restriction.", "As specific examples of the radical polymerization initiator, there may be mentioned organic peroxides such as cumene hydroperoxide, tert-butyl hydroperoxide, benzoyl peroxide, tert-butyl peroxyisopropyl carbonate, di-tert-butyl peroxide, tert-butyl peroxylaurate, lauroyl peroxide, succinic acid peroxide, cyclohexanone peroxide, and acetylacetone peroxide, inorganic peroxides such as potassium persulfate and ammonium persulfate, and azo compounds such as 2,2′-azobisisobutyronitrile and 2,2′-azobis-2,4-dimethylvaleronitrile, among others.", "Among these, organic peroxides and inorganic peroxides are particularly preferred because of their high reactivity.", "As the reducing agent to be used in the above-mentioned redox system, there may be mentioned such mixture as iron(II) sulfate/glucose/sodium pyrophosphate, iron(II) sulfate/dextrose/sodium pyrophosphate, and iron(II) sulfate/sodium formaldehyde sulfoxylate/ethylenediamine acetate, for instance.", "The above-mentioned radical polymerization initiator is used generally in an amount of 0.005 to 20 parts, preferably 0.01 to 10 parts, most preferably 0.04 to 5 parts, per 100 parts of the sum total of the vinyl monomer (B) and/or vinyl monomer (C) used or, in the case of multi-stage polymerization, per 100 parts of the monomer(s) used in each stage.", "In multi-stage polymerization, the radical polymerization initiator and the amount thereof each may be the same or different in each stage.", "When the amount of the radical polymerization initiator is too small, the rate of reaction is low and, accordingly, the production efficiency tends to worsen.", "When it is excessive, the generation of heat during reaction tends to become intensive, making it difficult to produce the desired graft copolymer.", "A chain transfer agent may also be used where necessary in carrying out the radical polymerization.", "The chain transfer agent may be any of those generally used in emulsion polymerization, without any particular limitation.", "As specific examples of the chain transfer agent, there may be mentioned tert-dodecylmercaptan, n-octylmercaptan, n-tetradecylmercaptan, and n-hexylmercaptan, among others.", "Although it is an optional component, the chain transfer agent is used preferably in an amount of 0.01 to 5 parts per 100 parts of the sum of the vinyl monomer (B) and/or vinyl monomer (C) employed.", "In multi-stage polymerization, it is used preferably in an amount of 0.01 to 5 parts per 100 parts of the monomer(s) used in each stage.", "In multi-stage polymerization, the chain transfer agents and the addition levels thereof in the respective steps may be the same or different.", "When the amount of the chain transfer agent is smaller than 0.01 parts, no significant effect can be obtained and, when it exceeds 5 parts, the rate of polymerization slows down, hence the production efficiency tends to decrease.", "Generally, it is preferred that the reaction temperature be 30 to 120° C. When, in the above polymerization, the polyorganosiloxane particles (A1) contain vinylic polymerizable groups, the vinyl monomer (B), on the occasion of polymerization thereof by means of the radical polymerization initiator, reacts with the vinylic polymerizable groups of the polyorganosiloxane particles (A1) to form grafts.", "When the polyorganosiloxane particles (A1) have no vinylic polymerizable group, a specific radical initiator, for example tert-butyl peroxylaurate, is used to extract hydrogen atoms from organic groups such as methyl groups each bonded to a silicon atom.", "Then, the vinyl monomer (B) is polymerized by the resulting radicals to form grafts.", "Furthermore, when the vinyl monomer (C) is polymerized by a radical polymerization initiator, it reacts not only with the polyorganosiloxane particles (A1), like the vinyl monomer (B), but also with the unsaturated bonds occurring in the polymer molecules formed by the vinyl monomer (B) to give grafts resulting from the vinyl monomer (C).", "The graft copolymer produced by emulsion polymerization may be separated from the latex for use thereof, or may be used in the latex form.", "The method of recovering the polymer from the latex may be any of conventional methods.", "For example, mention may be made of the method comprising adding a metal salt, such as calcium chloride, magnesium chloride or magnesium sulfate, to the latex to cause the latex to coagulate, followed by separation, washing with water, dehydrating and drying.", "The spray drying method may also be used.", "The thus-obtained graft copolymer is incorporated in various thermoplastic resins to give flame-retardant resin compositions excellent in flame retardancy and impact resistance.", "Preferred as the thermoplastic resins are polycarbonate resins whose polycarbonate content is not less than 50%, more preferably not less than 70%, since good flame retardancy can be obtained with them.", "Specific examples of the thermoplastic resins, which are preferred from the economical viewpoint and in view of good balance between flame retardancy and impact resistance, are polycarbonates (in particular aromatic polycarbonates), polycarbonate/polyester blend resins such as polycarbonate/polyethylene terephthalate blend resins and polycarbonate/polybutylene terephthalate blend resins, polycarbonate/acrylonitrile-styrene copolymer blend resins, polycarbonate/butadiene-styrene copolymer (HIPS resin) blend resins, polycarbonate/acrylonitrile-butadiene rubber-styrene copolymer (ABS resin) blend resins, polycarbonate/acrylonitrile-butadiene rubber-α-methylstyrene copolymer blend resins, polycarbonate/styrene-butadiene rubber-acrylonitrile-N-phenylmaleimide copolymer blend resins, and polycarbonate/acrylonitrile-acrylic rubber-styrene copolymer (AAS resin) blend resins.", "Mixtures of two or more blend resins may also be used.", "The level of addition of the above-mentioned polyorganosiloxane-containing graft copolymer to such a thermoplastic resin is preferably 0.1 to 30 parts per 100 parts of the thermoplastic resin since the flame retardancy, impact resistance and economical efficacy can obtained are good.", "More preferably, the addition level is not less than 0.5 parts, still more preferably not less than 1 part.", "It is more preferably not more than 15 parts, still more preferably not more than 5 parts.", "The powder-form flame retardant comprising the polyorganosiloxane-containing graft copolymer separated from the latex can be admixed with the thermoplastic resins by mixing using a Henschel mixer or ribbon blender, for instance, followed by melting and kneading using a roll, extruder or kneader, for instance.", "On that occasion, one or more of additives in general use, namely antioxidants, dripping-preventing agents, polymer processing auxiliaries, flame retardants, impact resistance improver, plasticizers, lubricants, ultraviolet absorbers, pigments, glass fibers, fillers, polymer lubricants and so forth, may be incorporated in the resin compositions.", "As specific examples of the antioxidants, there may be mentioned, among others, phenolic antioxidants such as tris[N-(3,5-di-tert-butyl-4-hydroxybenzyl)]isocyanurate (e.g.", "ADEKA STAB AO-20, product of ASAHI DENKA), tetrakis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionyloxy-methyl]methane (e.g.", "IRGANOX 1010, product of Ciba Specialty Chemicals), butylidene-1,1-bis(2-methyl-4-hydroxy-5-tert-butylphenyl) (e.g.", "ADEKA STAB AO-40, product of ASAHI DENKA) and 1,1,3-tris(2-methyl-4-hydroxy-5-tert-butylphenyl)butane (e.g.", "Yoshinox 930, product of Yoshitomi Fine Chemicals), phosphorus-containing antioxidants such as bis(2,6-di-tert-butyl-4-methylphenyl) pentaerythritol phosphite (e.g.", "ADEKA STAB PEP-36, product of ASAHI DENKA), tris(2,4-di-tert-butylphenyl) phosphite (e.g.", "ADEKA STAB 2112, product of ASAHI DENKA) and 2,2-methylenebis(4,6-di-tert-butylphenyl) octyl phosphite (e.g.", "ADEKA STAB HP-10, product of ASAHI DENKA), and sulfur-containing antioxidants such as dilauryl 3,3′-thio-dipropionate (Yoshinox DLTP, product of Yoshitomi Fine Chemicals) and dimyristyl 3,3′-thio-dipropionate (Yoshinox DMTP, product of Yoshitomi Fine Chemicals).", "Among these, phosphorus-containing antioxidants are particularly preferred since they provide improved flame retardancy.", "As examples of the dripping-preventing agents, which are preferred in view of their high dripping-preventing effect, there may be mentioned fluorinated polyolefin resins such as polymonofluoroethylene, polydifluoroethylene, polytrifluoroethylene, polytetrafluoroethylene and tetrafluoroethylene/hexafluoroethylene copolymers, and poly(vinylidene fluoride) resins.", "As specific examples of the polymer processing auxiliaries, there may be mentioned, among others, methyl methacrylate-butyl acrylate copolymers and like methacrylate (co)polymers.", "As specific examples of the impact resistance improvers, there may be mentioned, among others, butadiene rubber type impact resistance improvers (MBS resins), butyl acrylate rubber type impact resistance improvers, and butyl acrylate rubber/silicone rubber and like composite rubber type impact resistance improvers.", "One or more of other flame retardants may also be used in combination.", "As specific examples of the flame retardants to be used in combination, which are preferred from the fact that they are halogen-free and phosphorus-free, there may be mentioned, among others, silicone compounds such as aromatic group-containing polyorganosiloxanes, triazine compounds such as cyanuric acid and melamine cyanurate, and boron compounds such as boron oxide and zinc borate.", "The combined use with a phosphorus-containing compound such as triphenyl phosphate, condensed phosphate esters or stabilized red phosphorus is also possible.", "In this case, the use of the polyorganosiloxane-containing graft copolymer according to the first aspect of the invention in compositions containing a phosphorus-containing flame retardant is advantageous in that the amount of the phosphorus-containing flame retardant can be reduced thereby.", "From the effect-cost balance viewpoint, the level of addition of these additives is preferably 0.1 to 20 parts, more preferably 0.2 to 10 parts, and most preferably 0.3 to 5 parts, per 100 parts of each thermoplastic resin.", "The flame retardant resin composition obtained can be molded by applying those molding methods used in molding conventional thermoplastic resin compositions, namely injection molding, extrusion molding, blow molding, calender molding and so forth.", "The fields of application of the moldings obtained from the flame retardant resin composition according to the first aspect of the invention are not particularly restricted but include those fields where flame retardancy is required, for example housings and chassis parts of various OA/information/household electric/electronic appliances such as desktop computers, notebook computers, tower type computers, server computers, printers, copiers, fax machines, cellular phones, PHS phones, televisions and video recorders, various building parts and various automotive parts.", "The moldings obtained show excellent impact resistance and flame retardancy.", "Second Aspect of the Invention The polyorganosiloxane-containing graft copolymer according to the second aspect of the invention is obtainable by polymerizing 0 to 10 parts of a vinyl monomer (B) comprising 100 to 50% of a polyfunctional monomer (b-1) containing two or more polymerizable unsaturated bonds and 0 to 50% of another copolymerizable monomer (b-2), in the presence of 30 to 95 parts of a polyorganosiloxane (A2) in a latex form obtainable by seed polymerization using, as a seed polymer, a hydrophilic polymer capable of swelling in the corresponding organosiloxane (such polyorganosiloxane is hereinafter referred to also as “polyorganosiloxane (A2)”), followed by further polymerization of 5 to 70 parts of a vinyl monomer (C) [the sum of (A2), (B) and (C) being 100 parts].", "The seed polymer to be used in accordance with the second aspect of invention can be obtained by ordinary emulsion polymerization but the method of synthesis is not particularly restricted.", "The seed polymer is not limited to such a rubber component as butyl acrylate rubber or butadiene rubber.", "Thus, a hard polymer such as a butyl acrylate-styrene copolymer, butyl acrylate-butadiene copolymer, butyl acrylate-acrylonitrile copolymer, butyl acrylate-styrene-acrylonitrile copolymer or styrene-acrylonitrile copolymer may also be used without any substantial trouble.", "However, the seed polymer is required to be capable of sufficiently swelling in the monomer(s) forming a rubber in the next stage and have strong hydrophilicity so that it may take up water into particles thereof.", "As a method for improving the hydrophilicity of seed polymer, lowering the glass transition point of the seed polymer may be mentioned.", "The glass transition point is preferably not higher than −10° C. The glass transition point can be determined, by measuring temperature variation of specific gravity of a polymer, as the temperature at which the specific gravity is drastically varied.", "For improving the ability of the seed polymer to swell in the organosiloxane, it is first important that the polarity or the like property of the seed polymer is adapted to the organosiloxane.", "Secondly, it is effective to markedly reduce the molecular weight of the seed polymer by selecting the use of a chain transfer agent, applying a high polymerization temperature and/or use of the initiator in a large amount.", "A number average molecular weight of the seed polymer is preferably not more than 10,000, more preferably 7,000.The number average molecular weight may be determined by GPC analysis (relative to polystyrene standard).", "The “organosiloxane”, associated with the swelling capacity of the seed polymer, is an organosiloxane which is a monomer component of a polyorganosiloxane (A2).", "For example, when a polyorganosiloxane (A2) is produced from octamethylcyclotetrasiloxane, the above “organosiloxane” corresponds to octamethylcyclotetrasiloxane.", "The hydrophilicity of the seed polymer can be determined by adding water, in an amount of 20 times (by weight) that of the seed polymer in a dry state, to the dry seed polymer, stirring the mixture at 23° C. for 1 hour, and measuring the extraction rate of the polymer into water.", "When the value is not less than 1%, the hydrophilicity is sufficient.", "Preferably, however, the value is not less than 10%, more preferably not less than 50%.", "The upper limit may be at a level not more than 100%.", "The swelling capacity of the seed polymer to the organosiloxane can be determined by adding the organosiloxane, in an amount 50 times (by weight) that of the dry seed polymer, to the seed polymer latex, stirring the mixture at 23° C. for 1 hour, and determining the rate of swelling by volume from the ratio between the latex particle diameter after stirring and the latex particle diameter before stirring.", "A value not less than 1.5 times is sufficient.", "Preferably, however, it is not less than 3.The upper limit is preferably set at a level not higher than 50 times, more preferably not higher than 15 times.", "Marked flame retardancy and impact resistance improving effects can be obtained within the above-mentioned ranges.", "The volume average particle diameter of the polyorganosiloxane (A2) in a latex form can be determined by light scattering method or electron microscopic observation, and a preferred range is 0.008 to 0.6 μm.", "More preferably, it is not less than 0.01 μm.", "As for the upper limit, the diameter is more preferably not greater than 0.2 μm.", "Those particles smaller than 0.008 μm in volume average particle diameter tend to become difficult to produce.", "When the particles are greater than 0.6 μm, the flame retardancy and impact resistance tend to decrease.", "In accordance with the second aspect of the invention, the polyorganosiloxane (A2) includes, within the meaning thereof, not only simple polyorganosiloxanes but also modified polyorganosiloxanes containing not more than 5% of another or other (co)polymers.", "Thus, the polyorganosiloxane (A2) may contain up to 5% of poly(butyl acrylate) and/or a butyl acrylate-styrene copolymer, for instance.", "As specific examples of the polyorganosiloxane (A2), there may be mentioned polydimethylsiloxane particles, polymethylphenylsiloxane particles, dimethylsiloxane-diphenylsiloxane copolymer particles, and the like.", "These may be used singly or two or more of them may be used in combination.", "The above polyorganosiloxane (A2) can be prepared, for example, by polymerizing (1) an organosiloxane, (2) a bifunctional silane compound, (3) an organosiloxane and a bifunctional silane compound, (4) an organosiloxane and a vinylic polymerizable group-containing silane compound, (5) a bifunctional silane compound and a vinylic polymerizable group-containing silane compound, or (6) an organosiloxane, a bifunctional silane compound and a vinylic polymerizable group-containing silane compound, optionally further together with an at least trifunctional silane compound.", "As these compounds, those specific examples mentioned hereinabove referring to the first aspect of the invention may be mentioned.", "The proportions thereof are also the same as mentioned hereinabove referring to the first aspect of the invention.", "The polyorganosiloxane (A2) is preferably produced by subjecting, to emulsion polymerization in the presence of the above-mentioned seed polymer, a polyorganosiloxane-forming composition comprising, for example, the organosiloxane, bifunctional silane compound, vinylic polymerizable group-containing silane compound and/or the like, optionally together with the at least trifunctional silane compound.", "The method of production is the same as mentioned above referring to the first aspect of the invention.", "The above-mentioned vinyl monomer (B) is used for enhancing the flame retardant and impact resistance-improving effects.", "It comprises 100 to 50%, preferably 100 to 80%, of a polyfunctional monomer (b-1) containing two or more polymerizable unsaturated bonds and 0 to 50%, preferably 0 to 20%, of another copolymerizable monomer (b-2).", "When the proportion of the polyfunctional monomer (b-1) is too small, or when the amount of the copolymerizable monomer (b-2) is excessive, the graft copolymer finally obtained tends to become less effective in improving the impact resistance.", "As examples of the polyfunctional monomer (b-1) and of the copolymerizable monomer (b-2), there maybe mentioned the same ones as mentioned hereinabove referring to the first aspect of the invention.", "The above-mentioned vinyl monomer (C) is a component to be used for obtaining the polyorganosiloxane-containing graft copolymer.", "It is also a component to be used for securing compatibility between the graft copolymer and a thermoplastic resin to thereby disperse the graft copolymer in the thermoplastic resin uniformly for the improvements in flame retardancy and impact resistance by incorporating the graft copolymer in the thermoplastic resin.", "As specific monomers, there may be mentioned the same ones as mentioned above as the other copolymerizable monomers (b-2) included in the vinyl monomer (B).", "The polyorganosiloxane-containing graft copolymer according to the second aspect of the invention can be obtained by polymerizing 0 to 10 parts (preferably not less than 1 part, more preferably not less than 2 parts, but preferably not more than 5 parts, more preferably not more than 4 parts) of the vinyl monomer (B) in the presence of 30 to 95 parts (as solid content) (preferably not less than 60 parts, but preferably not more than 80 parts, more preferably not more than 75 parts) of the above-mentioned polyorganosiloxane (A2) and further polymerizing 5 to 70 parts (preferably not less than 15 parts, more preferably not less than 21 parts, but preferably not more than 39 parts, more preferably not more than 38 parts) of the vinyl monomer (C) so that the total amount may become 100 parts.", "When the amount of the polyorganosiloxane (A2) is too small or too large, the flame retardant effect tends to decrease in either case.", "When the amount of the vinyl monomer (B) is too small, the flame retardant and impact resistance improving effects tend to decrease and, when it is excessive, the impact resistance improving effect tends to decrease.", "When the amount of the vinyl monomer (C) is too small or too large, the flame retardant effect tends to lower in either case.", "The graft copolymerization can be carried out in the same manner as described hereinabove referring to the first aspect of the invention.", "As the method of recovering the polymer from the graft copolymer latex obtained by emulsion polymerization, there may be mentioned, for example, the method comprising adding a metal salt, such as calcium chloride, magnesium chloride or magnesium sulfate, to the latex to cause the latex to coagulate, followed by separation, washing with water, dehydrating and drying.", "The spray drying method may also be used.", "The thus-obtained graft copolymer is incorporated in various thermoplastic resins to give flame-retardant resin compositions excellent in flame retardancy and impact resistance.", "As examples of the thermoplastic resins, there may be mentioned the same ones as mentioned hereinabove referring to the first aspect of the invention.", "The level of addition of the above-mentioned polyorganosiloxane-containing graft copolymer to such a thermoplastic resin is preferably 0.1 to 30 parts per 100 parts of the thermoplastic resin from the good flame retardancy and impact resistance viewpoint.", "More preferably, the addition level is not less than 0.5 parts, still more preferably not less than 1 part.", "It is more preferably not more than 15 parts, still more preferably not more than 5 parts.", "The powder-form flame retardant comprising the polyorganosiloxane-containing graft copolymer separated from the latex can be admixed with the thermoplastic resins by mixing using a Henschel mixer or ribbon blender, for instance, followed by melting and kneading using a roll, extruder or kneader, for instance.", "On that occasion, one or more of additives in general use, namely antioxidants, dripping-preventing agents, polymer processing auxiliaries, flame retardants, impact resistance improver, plasticizers, lubricants, ultraviolet absorbers, pigments, glass fibers, fillers, polymer lubricants and so forth, may be incorporated in the resin compositions.", "Specifically, those examples mentioned hereinabove referring to the first aspect of the invention may be mentioned.", "The flame retardant resin composition obtained can be molded by applying those molding methods used in molding conventional thermoplastic resin compositions, namely injection molding, extrusion molding, blow molding, calender molding and so forth.", "The fields of application of the moldings obtained from the flame retardant resin composition according to the second aspect of the invention are not particularly restricted but include those fields mentioned hereinabove referring to the first aspect of the invention.", "The moldings obtained show excellent impact resistance and flame retardancy.", "BEST MODES FOR CARRYING OUT THE INVENTION The following examples illustrate the invention more specifically.", "They are, however, by no means limitative of the scope of the invention.", "In the following examples and comparative example, the measurements and tests were carried out as follows.", "[Degree of Conversion in Polymerization] The latex was dried in a hot air drying chamber at 120° C. for 1 hour, the remaining solid matter was weighed, and the degree of conversion was calculated according to the formula: 100× solid matter weight/charged monomer weight (%).", "[Toluene-Insoluble Matter Content] A 0.5-g portion of the polyorganosiloxane particles recovered from the latex by drying was immersed in 80 ml of toluene at 23° C. for 24 hours and, after 60 minutes of centrifugation at 12,000 rpm, the weight fraction (%) of the toluene-insoluble matter in the polyorganosiloxane particles was determined.", "[Acetone-Insoluble Matter Content] One gram of the graft copolymer was immersed in 80 ml of acetone at 23° C. for 48 hours.", "Then, after 10 minutes of centrifugation at 18,000 rpm, the sediment was weighed, and the acetone-insoluble matter content (%) was calculated.", "[Volume Average Particle Diameter] The volume average particle diameters of the seed polymer, polyorganosiloxane particles and graft copolymer were determined each in a latex form.", "Using LEED & NORTHRUP INSTRUMENTS' MICROTRAC UPA as the measuring apparatus, the volume average particle diameter (μm) and the coefficient of variation in particle diameter distribution (standard deviation/volume average particle diameter (%)) were measured by the light scattering method.", "[Impact Resistance] The evaluation was made by carrying out the Izod type test at −10° C. or 23° C. using notched {fraction (1/8)} inch bars according to ASTM D 256.", "[Flame Retardancy] The evaluation was made by carrying out the test V according to UL 94.", "[Hydrophilicity] Latex of seed polymer was weighed out in a beaker in such an amount that solid content of the seed polymer was about 5 g. The latex was completely dried in a drier at 120° C., and was weighed precisely.", "A 100 g portion of water was added to the dry seed polymer, stirred, with a stirrer, at 23° C. for 1 hour, and then filtered through filter paper.", "The filtrate was dried in a drier at 120° C. to recover water-soluble matters, and then the water-soluble matters were weighed precisely.", "The extraction rate of the water-soluble matters in dry seed polymer was calculated.", "[Swelling Capacity] The particle diameter of the seed polymer in a latex form was measured by MICROTRAC UPA.", "A emulsified liquid obtained by mixing organosiloxane (octamethylcyclotetrasiloxane), in an amount of 50 times (by weight) that of the seed polymer in a dry state, and 0.1% (by weight) aqueous solution of Emal 2F (product of Kao) was incorporated into 5% (by weight) latex of the seed polymer and mixed.", "After one-hour stirring at 23° C., particle diameters were measured by MICROTRAC UPA.", "The rate of swelling by volume was calculated by the following equation; (The rate of swelling by volume) = { ( Particle ⁢ ⁢ ⁢ diameter ⁢ ⁢ after ⁢ ⁢ swelling ⁢ ⁢ measured ⁢ ⁢ in ⁢ ⁢ latex ) ( Particle ⁢ ⁢ diameter ⁢ ⁢ before ⁢ ⁢ swelling ⁢ ⁢ measured ⁢ ⁢ in ⁢ ⁢ latex ) } 3 - 1 First Aspect of the Invention REFERENCE EXAMPLE 1 Production of Polyorganosiloxane Particles (S-1) An emulsion was prepared by stirring an aqueous solution composed of the following components at 10,000 rpm for 5 minutes using a Homomixer.", "Component Amount (parts) Pure water 251 Sodium dodecylbenzenesulfonate (SDBS) 1.0 Octamethylcyclotetrasiloxane (D4) 95 Mercaptopropyldimethoxymethylsilane (MPDS) 5 A 5-necked flask equipped with a stirrer, reflux condenser, nitrogen inlet, inlet for adding monomer and thermometer was charged with the above emulsion all at once.", "With stirring the system, a 10% aqueous solution of dodecylbenzenesulfonic acid (DBSA) (1 part as solid content) was added, and the temperature was raised to 80° C. over about 40 minutes, and the reaction was allowed to proceed at 80° C. for 6 hours.", "Then, the mixture was cooled to 25° C. and, after 20 hours of standing, the pH of the system was returned to 6.5 with sodium hydroxide to terminate the polymerization.", "Latex containing polyorganosiloxane particles (S-1) was thus obtained.", "The degree of conversion, and the volume average particle diameter and toluene-insoluble matter content of the polyorganosiloxane particle latex were determined.", "The results are shown in Table 1.REFERENCE EXAMPLE 2 Production of Polyorganosiloxane Particles (S-2) A 5-necked flask equipped with a stirrer, reflux condenser, nitrogen inlet, inlet for adding monomer and thermometer was charged with the following components.", "Component Amount (parts) Pure water 189 SDBS 1.2 Then, while substituting the system atmosphere with nitrogen, the temperature was raised to 70° C. and an aqueous solution composed of 1 part of pure water and 0.02 parts of potassium persulfate (KPS) was added.", "Then, a mixture composed of Component Amount (parts) Styrene (St) 0.7 Butyl methacrylate (BMA) 1.3 was added all at once, and the polymerization was driven to completion by stirring for 1 hour to give an St-BMA copolymer latex.", "The degree of conversion in polymerization was 99%.", "The solid content of the latex obtained was 1.0%, and the volume average particle diameter was 0.01 μm.", "This time, the coefficient of variation was 38%.", "The solvent-insoluble matter content of the St-BMA copolymer was 0%.", "Separately, a mixture composed of the following components was stirred with Homomixer at 10,000 rpm for 5 minutes to give an emulsion of the polyorganosiloxane-forming components.", "Component Amount (parts) Pure water 70 SDBS 0.5 D4 95 γ-Methacryloyloxypropyldimethoxymethylsilane 3 Then, the St-BMA copolymer-containing latex was maintained at 80° C., a 10% aqueous solution of DBSA (2 parts as solid content) was added to the system, the emulsion of the polyorganosiloxane-forming components mentioned above was then added all at once and, after 6 hours of continued stirring, the system was cooled to 25° C. and allowed to stand for 20 hours.", "Thereafter, the pH was adjusted to 6.4 with sodium hydroxide to terminate the polymerization.", "A latex containing polyorganosiloxane particles (S-2) was obtained.", "The degree of conversion in polymerization, and the volume average particle diameter and toluene-insoluble matter content of the polyorganosiloxane particle latex were determined.", "The results are shown in Table 1.As is estimable from the charge amounts and degree of conversion, the polyorganosiloxane particles in the polyorganosiloxane particle latex is composed of 98% of the polyorganosiloxane component and 2% of the St-BMA copolymer component.", "TABLE 1 Reference Reference Example 1 Example 2 Polyorganosiloxane particles S-1 S-2 Degree of conversion of 87 87 polyorganosiloxane component (%) Average particle diameter (μm) 0.14 0.04 Coefficient of variation (%) 35 35 Toluene-insoluble matter content (%) 0 0 EXAMPLES 1 TO 5 AND COMPARATIVE EXAMPLES 1 TO 4 A 5-necked flask equipped with a stirrer, reflux condenser, nitrogen inlet, inlet for adding monomer and thermometer was charged with 300 parts of pure water, 0.2 parts of sodium formaldehyde sulfoxylate (SFS), 0.01 parts of ethylenediaminetetraacetic acid disodium salts (EDTA), 0.0025 parts of iron(II) sulfate, and an amount specified in Table 2 of the polyorganosiloxane particles (A1).", "While stirring the system, the temperature was raised to 60° C. in a nitrogen atmosphere.", "After arrival at 60° C., a mixture of the monomer(s) (B) and radical polymerization initiator each specified in Table 2 each in an amount specified in Table 2 was added all at once, and the system was stirred at 60° C. for 1 hour.", "Then, the monomer (C) specified in Table 2 was added dropwise over 3 hours and, after completion of the addition, the whole mixture was further stirred for 1 hour to give a graft copolymer in a latex form.", "The latex was then diluted with pure water to a solid concentration of 15%, a 10% aqueous solution of calcium chloride (2 parts as solid content) was added, whereby coagulated slurry was obtained.", "The coagulated slurry was heated to 80° C., then cooled to 50° C., and dehydrated and dried to give a polyorganosiloxane-based graft copolymer (any of SG-1 to SG-5 and SG′-1 to SG′-4) in a powder form.", "The degree of conversion in polymerization, volume average particle diameter and acetone-insoluble matter content for each copolymer are shown in Table 2.In Table 2, AIMA stands for allyl methacrylate, BA for butyl acrylate, MMA for methyl methacrylate, AN for acrylonitrile (they are all monomers), CHP for cumene hydroperoxide (radical polymerization initiator), and Polymer SP for solubility parameter of polymer of vinyl monomer(s) (C) (as determined by the method described herein).", "TABLE 2 Examples Comparative Examples 1 2 3 4 5 1 2 3 4 Polyorganosiloxane S-1 70 70 70 70 — 70 70 70 — particles (A1) (parts) (solid content) S-2 — — — — 60 — — — 60 Vinyl monomer (B) AIMA 3 2 1 3 3 — 1 15 — (parts) BA — — — 0.5 — — 2 — — CHP 0.01 0.01 0.01 0.01 0.01 — 0.01 0.03 — Vinyl monomer (C) MMA 27 28 29 27 — 30 27 15 — (parts) St — — — — 27.75 — — — 30 AN — — — — 9.25 — — — 10 CHP 0.06 0.06 0.06 0.06 0.08 0.06 0.06 0.06 0.08 Polymer SP (C) 9.25 9.25 9.25 9.25 9.95 9.25 9.25 9.25 9.95 ((cal/cm3)1/2) Degree of conversion (B) 99 99 99 99 99 — 99 99 — (%) (C) 99 99 99 99 99 99 99 99 99 Acetone-insoluble matter 88 85 80 88 85 45 80 92 40 content (%) Graft polymer No.", "SG-1 SG-2 SG-3 SG-4 SG-5 SG′-1 SG′-2 SG′-3 SG′-4 EXAMPLES 6 TO 11 AND COMPARATIVE EXAMPLES 5 TO 11 Rendering Polycarbonate Resins Flame-Retardant A polycarbonate resin (PC-1: Toughlon FN 2200A, product of IDEMITSU PETROCHEMICAL; or PC-2: Toughlon FN 1900A, product of IDEMITSU PETROCHEMICAL) and the polyorganosiloxane-based graft copolymer obtained in any of Examples 1 to 5 (SG-1 to SG-5) or the polyorganosiloxane-based graft copolymer obtained in any of Comparative Examples 1 to 4 (SG′-1 to SG′-4) were blended together according to the formulation shown in Table 3.The abbreviation PEP36 stands for a phosphorus-containing antioxidant (ADEKA STAB PEP36, product of ASAHI DENKA), and PTFE for polytetrafluoroethylene (Polyflon FA-500, product of DAIKIN Industries).", "Pellets were produced by melting and kneading each compound at 270° C. on a twin-screw extruder (TEX 44 SS, product of Japan Steel Works).", "The pellets obtained were molded into {fraction (1/8)} inch test specimens for impact resistance evaluation and {fraction (1/16)} inch specimens for flame retardancy evaluation using FANUC's FAS 100 B injection molding machine set at a cylinder temperature of 280° C. The test specimens obtained were evaluated by the evaluation methods described hereinabove.", "The results are shown in Table 3.TABLE 3 Examples Comparative Examples 6 7 8 9 10 11 5 6 7 8 9 10 11 Thermoplastic PC-1 100 100 100 100 100 — 100 100 100 100 — 100 — resin PC-2 — — — — — 100 — — — — 100 — 100 Graft SG-1 3 — — — — 3 — — — — — — — copolymer SG-2 — 3 — — — — — — — — — — — SG-3 — — 3 — — — — — — — — — — SG-4 — — — 3 — — — — — — — — — SG-5 — — — — 3 — — — — — — — — SG′-1 — — — — — — 3 — — — — — — SG′-2 — — — — — — — 3 — — — — — SG′-3 — — — — — — — — 3 — — — — SG′-4 — — — — — — — — — 3 3 — — Antioxidant PEP36 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Dripping- PTFE 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 preventing agent Flame Total 20 30 40 25 20 25 55 60 40 55 60 140 160 retardancy combustion time (sec) Dripping No No No No No No No No No No No Yes Yes Impact −10° C. 55 60 65 60 55 35 45 60 20 40 20 20 15 resistance (kJ/m2) From Table 3, it is seen that the graft copolymer according to the first aspect of the invention can highly improve the flame retardancy-impact resistance balance of the polycarbonate resins.", "EXAMPLE 12 AND COMPARATIVE EXAMPLES 12 AND 13 Rendering a Polycarbonate/Polyethylene Terephthalate Mixed Resin Flame-Retardant PC-1, a polyethylene terephthalate resin (PET: BELLPET EFG-70, product of Kanebo Gohsen) and the polyorganosiloxane-based graft copolymer (SG-1) obtained in Example 1 or the polyorganosiloxane-based graft copolymer (SG′-1) obtained in Comparative Example 1 were blended together according to the formulation shown in Table 4.Pellets were produced by melting and kneading each compound at 270° C. on a twin-screw extruder (TEX 44 SS, product of Japan Steel Works).", "The pellets obtained were molded into {fraction (1/8)} inch test specimens for impact resistance evaluation and {fraction (1/12)} inch specimens for flame retardancy evaluation using FANUC's FAS 100 B injection molding machine set at a cylinder temperature of 260° C. The test specimens obtained were evaluated by the evaluation methods described hereinabove.", "The results are shown in Table 4.TABLE 4 Exam- Comparative Comparative ple 12 Example 12 Example 13 Thermoplastic PC-1 90 90 90 resin PET 10 10 10 Graft SG-1 4 — — copolymer SG′-1 — 4 — Antioxidant PEP36 0.3 0.3 0.3 Dripping- PTFE 0.5 0.5 0.5 preventing agent Flame Total combustion 50 65 190 retardancy time (sec) Dripping No No Yes Impact 23° C. (kJ/m2) 75 60 41 resistance From Table 4, it is seen that the graft copolymer according to the invention can highly improve the flame retardancy-impact resistance balance of the polycarbonate/polyethylene terephthalate resin.", "Second Aspect of the Invention EXAMPLES 13 TO 18 Water (400 parts) and an amount (as solid content) given in Table 5 of sodium dodecylbenzenesulfonate (SDBS) were mixed up in a 5-necked flask equipped with a stirrer, reflux condenser, nitrogen inlet, inlet for adding monomer and thermometer, then the temperature was raised to 50° C. and, after arrival of the liquid temperature at 50° C., nitrogen substitution was effected.", "Then, a mixture of 10 parts of butyl acrylate and 3 parts of tert-dodecylmercaptan was added.", "After 30 minutes, 0.01 parts (as solid content) of paramenthane hydroperoxide was added, and the polymerization was allowed to proceed for 1 hour.", "Thereafter, a mixture of 90 parts of butyl acrylate and 27 parts of tert-dodecylmercaptan was added continuously over 3 hours.", "The subsequent 2 hours of post-polymerization gave seed latex (seed 1 to seed 4).", "The weight average particle diameter, hydrophilicity and degree of swelling after synthesis were determined.", "The results are shown in Table 5.A 5-necked flask equipped with a stirrer, reflux condenser, nitrogen inlet, inlet for adding monomer and thermometer was charged with a seed polymer specified in Table 6 in an amount (as solid content) specified in Table 6.Then, an emulsion of polyorganosiloxane-forming components as separately prepared by stirring a mixture composed of 300 parts of water, 0.5 parts (as solid content) of SDBS, 95 parts of octamethylcyclotetrasiloxane and 5 parts of dimethylmethylsilylpropyl methacrylate (DSMA) at 7,000 rpm for 5 minutes using a Homomixer was added all at once.", "Then, a 10% aqueous solution of dodecylbenzenesulfonic acid (1 part as solid content) was added and, with stirring the system, the temperature was raised to 80° C. in a nitrogen atmosphere.", "After arrival at 80° C., stirring was continued at 80° C. for 6 hours and, then, the mixture was cooled to 25° C. and allowed to stand for 20 hours.", "Thereafter, the pH was adjusted to 6.4 with sodium hydroxide to stop the polymerization.", "Latex containing polyorganosiloxane particles was thus obtained.", "Then, a 5-necked flask equipped with a stirrer, reflux condenser, nitrogen inlet, inlet for adding monomer and thermometer was charged with 240 parts of pure water and 70 parts (as solid content) of the above polyorganosiloxane particles.", "With stirring the system, the temperature was raised to 40° C. in a nitrogen atmosphere.", "After arrival at 40° C., 0.2 parts of sodium formaldehyde sulfoxylate (SFS), 0.01 parts of ethylenediaminetetraacetic acid disodium salt (EDTA) and 0.0025 parts of iron (II) sulfate were added and, then, a mixture of 30 parts of methyl methacrylate (MMA) and 0.06 parts (as solid content) of cumene hydroperoxide was added dropwise over 1.5 hours.", "After completion of the addition, stirring was continued for 1 hour.", "A graft copolymer latex was thus obtained.", "The volume average particle diameter is shown in Table 6.The latex was then diluted with pure water to a solid concentration of 15%, a 25% aqueous solution of calcium chloride (4 parts as solid content) was added.", "The thus-obtained coagulated slurry was heated to 95° C., then cooled to 50° C., and dehydrated and dried to give a polyorganosiloxane-based graft copolymer in a powder form.", "Then, a polycarbonate resin (Toughlon FN 2200A, product of IDEMITSU PETROCHEMICAL) and the above polyorganosiloxane-based graft copolymer in powder form were blended together according to the formulation shown in Table 6.The dripping-preventing agent used was polytetrafluoroethylene (Polyflon FA-500, product of DAIKIN Industries), and the stabilizer used was a mixture of a phosphorus-containing antioxidant (ADEKA STAB PEP36, product of ASAHI DENKA) and a phenolic antioxidant (Topanol CA, product of ICI Japan).", "Pellets were produced by melting and kneading the thus-obtained compound at 270° C. on a twin-screw extruder (TEX 44 SS, product of Japan Steel Works).", "The pellets obtained were molded into {fraction (1/8)} inch test specimens for impact resistance evaluation and {fraction (1/16)} inch specimens for flame retardancy evaluation using FANUC's FAS 100 B injection molding machine set at a cylinder temperature of 280° C. The test specimens obtained were evaluated by the evaluation methods described hereinabove.", "The results of the impact resistance and flame retardancy evaluations of the moldings are shown in Table 6.EXAMPLES 19 AND 20 A 5-necked flask equipped with a stirrer, reflux condenser, nitrogen inlet, inlet for adding monomer and thermometer was charged with a seed polymer specified in Table 6 in an amount (as solid content) specified in Table 6.Then, an emulsion of polyorganosiloxane-forming components as separately prepared by stirring a mixture composed of 300 parts of water, 0.5 parts (as solid content) of SDBS, 95 parts of octamethylcyclotetrasiloxane and 5 parts of mercaptopropylmethyldimethoxysilane (MPrDMS) at 7,000 rpm for 5 minutes using a Homomixer was added all at once.", "Then, a 10% aqueous solution of dodecylbenzenesulfonic acid (1 part as solid content) was added and, with stirring the system, the temperature was raised to 80° C. in a nitrogen atmosphere.", "After arrival at 80° C., stirring was continued at 80° C. for 6 hours and, then, the mixture was cooled to 25° C. and allowed to stand for 20 hours.", "Thereafter, the pH was adjusted to 6.4 with sodium hydroxide to terminate the polymerization.", "Latex containing polyorganosiloxane particles was thus obtained.", "Then, a 5-necked flask equipped with a stirrer, reflux condenser, nitrogen inlet, inlet for adding monomer and thermometer was charged with 240 parts of pure water and 70 parts (as solid content) of the above polyorganosiloxane particles.", "With stirring the system, the temperature was raised to 40° C. in a nitrogen atmosphere.", "After arrival at 40° C., 0.2 parts of sodium formaldehyde sulfoxylate (SFS), 0.01 parts of ethylenediaminetetraacetic acid disodium salt (EDTA) and 0.0025 parts of iron(II) sulfate were added and, then, a mixture of 3 parts of allyl methacrylate (ALMA) and 0.01 parts (as solid content) of cumene hydroperoxide was added all at once, and stirring was continued at 40° C. for 1 hour.", "Then, a mixture of 30 parts of methyl methacrylate (MMA) and 0.06 parts (as solid content) of cumene hydroperoxide was added dropwise over 1.5 hours.", "After completion of the addition, stirring was continued for 1 hour.", "A graft copolymer latex was thus obtained.", "The volume average particle diameter is shown in Table 6.The latex was then diluted with pure water to a solid concentration of 15%, a 25% aqueous solution of calcium chloride (4 parts as solid content) was added.", "The thus-obtained coagulated slurry was heated to 85° C., then cooled to 50° C., and dehydrated and dried to give a polyorganosiloxane-based graft copolymer in a powder form.", "Then, a polycarbonate resin (Toughlon FN 1900A, product of IDEMITSU PETROCHEMICAL) and the above polyorganosiloxane-based graft copolymer in powder form were blended together according to the formulation shown in Table 6.The dripping-preventing agent used was polytetrafluoroethylene (Polyflon FA-500, product of DAIKIN Industries).", "Pellets were produced by melting and kneading the thus-obtained compound at 270° C. on a twin-screw extruder (TEX 44 SS, product of Japan Steel Works).", "The pellets obtained were molded into {fraction (1/8)} inch test specimens for impact resistance evaluation and {fraction (1/16)} inch specimens for flame retardancy evaluation using FANUC's FAS 100 B injection molding machine set at a cylinder temperature of 280° C. The test specimens obtained were evaluated by the evaluation methods described hereinabove.", "The results of the impact resistance and flame retardancy evaluations of the moldings are shown in Table 6.COMPARATIVE EXAMPLE 14 Formulation, molding and evaluations were carried out in the same manner as in Examples 13 to 18 except that the polyorganosiloxane-based graft copolymer was not added in formulating the polycarbonate resin composition.", "The results are shown in Table 6.COMPARATIVE EXAMPLE 15 Synthesis, coagulation, heat treatment, dehydration/drying/powder formation, formulation, molding and evaluations were carried out in the same manner as in Examples 13 to 18 except that no seed polymer was added in polymerizing the latex containing polyorganosiloxane particles.", "The results are shown in Table 6.COMPARATIVE EXAMPLE 16 Formulation, molding and evaluations were carried out in the same manner as in Examples 19 and 20 except that the polyorganosiloxane-based graft copolymer was not added in formulating the polycarbonate resin compositions.", "The results are shown in Table 6.COMPARATIVE EXAMPLE 17 Synthesis, coagulation, heat treatment, dehydration/drying/powder formation, formulation, molding and evaluations were carried out in the same manner as in Examples 19 to 20 except that no seed polymer was added in polymerizing the latex containing polyorganosiloxane particles.", "The results are shown in Table 6.TABLE 5 Seed 1 Seed 2 Seed 3 Seed 4 Sodium dodecylbenzenesulfonate 8 4 2 1 (phr) Butyl acrylate (phr) 100 100 100 100 tert-Dodecylmercaptan (phr) 30 30 30 30 Average particle diameter (μm) 0.04 0.06 0.08 0.09 Hydrophilicity (%) 80 80 80 80 Swelling capacity (times) 10 8 6 5 TABLE 6 Examples Comparative Examples 13 14 15 16 17 18 19 20 14 15 16 17 Formu- Polycarbonate 100 100 100 100 100 100 100 100 100 100 100 100 lation (parts) Dripping- 0.5 0.5 0.5 0.5 0.5 0.5 0.3 0.3 0.5 0.5 0.3 0.3 preventing agent (parts) Stabilizer 0.6 0.6 0.6 0.6 0.6 0.6 — — 0.6 0.6 — — (parts) Flame 3 3 3 3 3 3 3 3 — 3 — 3 retardant addition level (parts) Flame Seed polymer Seed 1 Seed 2 Seed 1 Seed 2 Seed 3 Seed 4 Seed 1 Seed 2 — — — — retardant Seed amount (parts) 1 1 2 2 2 2 1 1 — — — — Graft DSMA DSMA DSMA DSMA DSMA DSMA MPrDMS MPrDMS — DSMA — MPrDMS crosslinking agent One-stage graft — — — — — — ALMA ALMA — — — ALMA Two-stage graft MMA MMA MMA MMA MMA MMA MMA MMA — MMA — MMA Average 0.23 0.25 0.2 0.2 0.21 0.22 0.18 0.15 — 0.2 — 0.2 particle diameter (μm) Charac- Impact −10° 50 50 50 50 50 50 50 50 10 30 20 35 teristics resistance C. (kJ/m2) Flame Total 40 40 40 40 40 40 50 45 180 40 130 50 retardancy com- bus- tion time (sec) UL 94 V-0 V-0 V-0 V-0 V-0 V-0 V-0 V-0 Not- V-0 Not- V-0 V V V grading INDUSTRIAL APPLICABILITY The invention can provide a flame retardant capable of giving thermoplastic resin compositions excellent in flame retardancy-impact resistance balance when added to thermoplastic resins.", "Furthermore, thermoplastic resin compositions excellent in flame retardancy-impact resistance can be provided when the flame retardant is incorporated in thermoplastic resins." ] ]	Patent_10466130
[ [ "Stens with drug-containing amphiphilic polymer coating", "A vascular stent having an amphiphilic polymer coating is loaded with a restenosis inhibiting agent which is sparingly soluble in water, whereby delayed release of the agent takes place after implantation of the stent." ], [ "1.Intravascular stent comprising a a metal body having a coating comprising polymer and at least 20 μg per stent of a restenosis inhibiting agent in which the restenosis inhibiting agent is a sparingly water soluble drug and the polymer in the coating is a cross-linked amphiphilic polymer which, when swollen with water containing pyrene, has hydrophobic domains observable by pyrene fluorescence having an intensity ratio I3:I1of at least 0.8.2.Stent according to claim 1 in which on at least the outer wall of the stent the coating comprises a layer of the said amphiphilic polymer in which the drug is absorbed.", "3.Stent according to claim 1 or claim 2 in which the polymer in the coating when swollen with water containing pyrene has hydrophobic domains observable by I3:I1ratio of about 1.4.Stent according to any preceding claim in which the amphiphilic polymer comprises, as the groups conferring hydrophilicity, zwitterionic groups.", "5.Stent according to any preceding claim in which the amphiphilic polymer comprises, as the groups conferring hydrophobicity, pendant hydrophobic groups selected from C4-23-alkyl, -alkenyl and -alkynyl groups any of which may be substituted by one or more fluorine atoms, aryl, C7-24 aralkyl, oligo (C3-4 alkoxy) alkyl and siloxane groups.", "6.Stent according to claim 4 and claim 5 in which the polymer is formed from ethylenically unsaturated monomers including a zwitterionic monomer and a hydrophobic monomer having the said hydrophobic group.", "7.Stent according to claim 6 in which the ethylenically unsaturated monomers include one or more reactive monomers having a pendant reactive group capable of forming intermolecular crosslinks.", "8.Stent according to claim 6 or claim 7 in which the zwitterionic monomer has the general formula 1: YBX I wherein B is a straight or branched alkylene (alkanediyl), alkyleneoxaalkylene or alkylene oligo-oxaalkylene chain optionally containing one or more fluorine atoms up to and including perfluorinated chains or, if X or Y contains a terminal carbon atom bonded to B, a valence bond; X is a zwitterionic group; and Y is an ethylenically unsaturated polymerisable group selected from CH2═C(R)CH2O—, CH2═C(R)CH2OC(O)—, CH2═C(R)OC(O)—, CH2═C(R)O—, CH2═C(R)CH2OC(O)N(R1)—, R2OOCCR═CRC(O)O—, RCH═CHC(O)O—, RCH═C(COOR2)CH2C(O)O—, wherein: R is hydrogen or a C1-C4 alkyl group; R1 is hydrogen or a C1-C4 alkyl group or R1 is —B—X where B and X are as defined above; and R2 is hydrogen or a C1-4 alkyl group; A is —O— or —NR1—; K is a group —(CH2)pOC(O)—, —(CH2)pC(O)O—, —(CH2)pOC(O)O—, —(CH2)pNR3—, —(CH2)pNR3C(O)—, —(CH2)pC(O)NR3—, —(CH2)pNR3C(O)O—, —(CH2)pOC(O)NR3—, —(CH2)pNR3C(O)NR3—(in which the groups R3 are the same or different), —(CH2)pO—, —(CH2)pSO3—, or, optionally in combination with B, a valence bond p is from 1 to 12; and R3 is hydrogen or a C1-C4 alkyl group.", "9.Stent according to claim 8 in which the cationic group in X is an amine.", "10.Stent according to claim 9 in which the cationic group is a quaternary ammonium group.", "11.Stent according to any of claims 8 to 10 in which the anionic group in X is selected from sulphate, sulphonate, phosphate, phosphonate and carboxylate, and is preferably a phosphate diester.", "12.Stent according to claim 11 in which X is selected from groups of the general formula V: in which the moieties X3 and X4, which are the same or different, are —O—, —S—, —NH— or a valence bond, preferably —O—, and W+ is a group comprising an ammonium, phosphonium or sulphonium cationic group and a group linking the anionic and cationic moieties which is preferably a C1-12-alkanediyl group.", "13.Stent according to any of claims 7 to 12 in which the hydrophobic monomer has the general formula VII Y1R13 VII wherein Y1 is selected from CH2═C(R14)CH2O—, CH2═C(R14)CH2OC(O)—, CH2═C(R14)OC(O)—, CH2═C(R14)O—, CH2═C(R14)CH2OC(O)N(R15)—, R16OOCCR14═CR14C(O)O—, R14CH═CHC(O)O—, R14CH═C(COOR16)CH2C(O)—O—, wherein: R14 is hydrogen or a C1-C4 alkyl group; R15 is hydrogen or a C1-C4 alkyl group or R15 is R13; R16 is hydrogen or a C1-4 alkyl group; A1 is —O— or —NR15—; and K1 is a group —(CH2)qOC(O)—, —(CH2)qC(O)O—, (CH2)qOC(O)O—, —(CH2)qNR17—, —(CH2)qNR17C(O)—, —(CH2)qC(O)NR17—, —(CH2)qNR17C(O)O—, —(CH2)qOC(O)NR17—, —(CH2)qNR17C(O)NR17—(in which the groups R17 are the same or different), —(CH2)qO—, —(CH2)qSO3—, or a valence bond p is from 1 to 12; and R17 is hydrogen or a C1-C4 alkyl group; and R13 is the hydrophobic group.", "14.Stent according to claim 13 in which R13 is selected from a) C4-8 alkyl groups; b) aryl and aralkyl; and c) a siloxane group —(CR182)qq(SiR192)(OSiR192)ppR19 in which each group R18 is the same or different and is hydrogen or alkyl of 1 to 4 carbon atoms, or aralkyl, for example benzyl or phenethyl, each group R19 is alkyl of 1 to 4 carbon atoms, qq is from 1 to 6 and pp is from 0 to 49.15.Stent according to claim 12 in which the or each reactive monomer has the general formula VIII Y2B2R20 VIII wherein B2 is a straight or branched alkylene, oxaalkylene or oligo-oxaalkylene chain optionally containing one or more fluorine atoms up to and including perfluorinated chains, or B2 is a valence bond; Y2 is an ethylenically unsaturated polymerisable group selected from CH2═C(R21)CH2—O—, CH2═C(R21)CH2OC(O)—, CH2═C(R21)OC(O)—, CH2═C(R21)O—, CH2═C(R21)CH2OC(O)N(R22)—, R23OOCCR21═CR21C(O)O—, R21H═CHC(O)O—, R21H═C(COOR23)CH2C(O)O— where R21 is hydrogen or C1-C4 alkyl; R23 is hydrogen, or a C1-4-alkyl group; A2 is —O— or —NR22—; R22 is hydrogen or a C1-C4 alkyl group or R22 is a group B2R20; K2 is a group —(CH2)kOC(O)—, —(CH)kC(O)O—, —(CH2)kOC(O)O—, —(CH2)kNR22—, —(CH2)kNR22C(O)—, —(CH2)kOC(O)O—, —(CH2)kNR22—, —(CH2)kNR22C(O)—, —(CH2)kC(O)NR22—, —(CH2)kNR22C(O)O—, —(CH2)kOC(O)NR22—, —(CH2)kNR22C(O)NR22—(in which the groups R22 are the same or different), —(CH2)kO—, —CH2)kSO3—, a valence bond and k is from 1 to 12; and R20 is a cross-linkable group.", "16.Stent according claim 15 in which R20 is selected from the group consisting of ethylenically and acetylenically unsaturated group containing radicals; aldehyde groups; silane and siloxane groups containing one or more substituents selected from halogen atoms and C1-4-alkoxy groups; hydroxyl; amino; carboxyl; epoxy; —CHOHCH2Hal (in which Hal is selected from chlorine, bromine and iodine atoms); succinimido; tosylate; triflate; imidazole carbonyl amino; optionally substituted triazine groups; acetoxy; mesylate; carbonyl di(cyclo)alkyl carbodiimidoyl; isocyanate, acetoacetoxy; and oximino, preferably R20 comprises a silane group containing at least one, preferably three substituents selected from halogen atoms and C1-4-alkoxy groups, more preferably containing three methoxy groups.", "17.Stent according to any preceding claim in which the restenosis inhibiting agent has a logP (where P is the partition coefficient between octanol and water) in the range 1.2-4.5, preferably in the range 1.4-4.18.Stent according to any preceding claim in which the resenosis inhibiting agent is a steroid, preferably an estrogen, for instance 17β-estradiol, or a corticosteroid, such as 6α-methylprednisolone or dexamethasone.", "19.Stent according to any preceding claim in which the restenosis inhibiting agent is present in an amount in the range of 20 to 1000 μg preferably 50 to 500 μg, more preferably more than 100 μg, per stent.", "20.Method for producing a drug coated intravascular stent comprising the steps: a) a metallic stent body is coated on its inner and outer walls with a cross-linkable amphiphilic polymer; b) the cross-linkable polymer is subjected to conditions under which cross-linking takes place to produce a stent coated with polymer which, when swollen with water containing pyrene, has hydrophobic domains observable by pyrene fluorescence having an intensity ratio I3:I1of at least 0.8.; c) at least the outer coated wall of the polymer coated stent is contacted with liquid drug composition comprising a sparingly water-soluble restenosis inhibiting agent as the drug and an organic solvent in which the drug is at least partially dissolved and which is capable of swelling the cross- linked polymer of the coating, for a time sufficient to swell the polymer coating on the outer wall, to produce a wet drug-coated stent in which drug is present in an amount of at least 20 μg per stent; d) organic solvent is evaporated from the wet stent to produce a dry drug-coated stent.", "21.Method according to claim 20 in which, in step c), the drug is both absorbed into the polymer and adsorbed at the surface of the polymer coating, whereby, upon evaporation of the solvent in step d) crystals of drug are formed which are adherent to the surface of the dry-drug coated stent.", "22.Method according to claim 20 or 21 in which, in step c) the contact is by dipping the polymer coated stent in the liquid composition, optionally repeatedly.", "23.Method according to any of claims 20 to 22 in which in step c) the contact includes flowing, spraying or dripping the liquid composition onto the stent and immediately allowing evaporation of solvent from the wet stent.", "24.Method according to any of claims 20 to 23 in which the stent is, between steps b) and c), mounted onto the stent delivery section of a is delivery catheter, whereby the stent delivery section is also contacted with the said liquid drug composition.", "25.Method according to claim 24 in which the delivery catheter is a balloon catheter in which the balloon is formed of polyamide, and in which the organic solvent in the liquid drug composition is selected from the group consisting of dichloromethane, dimethylsulphoxide, C1-4 alcohol and admixtures thereof.", "26.Method according to any of claims 20 to 25 having the further feature(s) of any of claims 4 to 19." ], [ "The present invention relates to the delivery of drugs from stents coated with polymer.", "In particular the invention relates to delivery of for inhibition of restenosis following stent implantation in the treatment of cardiovascular disease.", "A leading cause of mortality within the developed world is cardiovascular disease.", "Coronary disease is of most concern.", "Patients having such disease usually have narrowing in one or more coronary arteries.", "One treatment is coronary stenting, which involves the placement of a stent at the site of acute artery closure.", "This type of surgery has proved effective in restoring vessel patency and decreasing myocardial ischemia.", "However the exposure of currently used metallic stents to flowing blood can result in thrombus formation, smooth muscle cell proliferation and acute thrombotic occlusion of the stent.", "Non-thrombogenic and anti-thrombogenic coatings for stents have been developed.", "One type of balloon expandable stent has been coated with polymers having pendant zwitterionic groups, specifically phosphorylcholine (PC) groups, generally described in WO-A-93/01221.A particularly successful embodiment of those polymers suitable for use on balloon expandable stents has been described in WO-A-98/30615.The polymers coated onto the stent have pendant crosslinkable groups which are subsequently crosslinked by exposure to suitable conditions, generally heat and/or moisture.", "Specifically a trialkoxysilylalkyl group reacts with pendant groups of the same type and/or with hydroxyalkyl groups to generate intermolecular crosslinks.", "The coatings lead to reduced thrombogenicity.", "Fischell, T. A. in Circulation (1996) 94: 1494-1495 describes tests carried out on various polymer coated stents.", "A thinner uniform polyurethane coating, having a thickness of 23 μm was observed to have a better performance than a relatively non uniform thicker layer having a thickness in the range 75 to 125 μm.", "The thicker coatings are further described by Van der Giessen, W. J. et al., in Circulation: 1996:94:1690-1997.It has been suggested to utilise coatings on stents as reservoirs for pharmaceutically active agents desired for local delivery.", "In U.S. Pat.", "No.", "5,380,299 a stent is provided with a coating of a thrombolytic compound and optionally an outer layer of an anti-thrombotic compound.", "The stent may be precoated with a “primer” such as a cellulose ester or nitrate.", "Other drug containing stents and stent coatings are described by Topol and Serruys in Circulation (1998) 98:1802-1820.McNair et al., in Proceedings of the International Symposium on Controlled Release Bioactive Materials (1995) 338-339 describe in vitro investigations of release of three model drugs, caffeine, dicloxacillin and vitamin B12, from hydrogel polymers having pendant phosphorylcholine groups.", "Alteration of the hydrophilic/hydrophobic ratio of the (hydrophilic) phosphorylcholine monomer 2-methacryloyloxyethyl phosphorylcholine, (HEMA-PC) and a hydrophobic comonomer and crosslinking of the polymer allows preparation of polymers having water contents when swollen in the range 45 to 70 wt %.", "Crosslinking is achieved by incorporating a reactive monomer 3-chloro-2-hydroxypropylmethacrylate.", "The tests are carried out on membranes swollen in aqueous drug solutions at 37° C. The release rates of the model drugs are influenced by the molecular size, solute partitioning and degree of swelling of the polymer.", "Dicloxacillin is found to have a higher half life for release than its molecular size would indicate, and the release profile did not appear to be Fickian.", "McNair et al, in Medical Device Technology, December 1996, 16-22, describe three series of experiments.", "In one, polymers formed of HEMA-PC and lauryl methacrylate crosslinked after coating by unspecified means are cocoated with drugs onto stents.", "Release rates of dexamethasone from the stent, apparently into an aqueous surrounding environment, was determined.", "Drug release from cast membranes, as model coatings, showed that the release rate obeyed Fickian diffusion principles, for hydrophilic solutes.", "In the third series of tests, a non-crosslinked polymer coating, free of drug, coated on a stent, had a significant decrease in platelet adhesion when coated on a stent used in an ex-vivo arteriovenous shunt experiment The stent coating method was not described in detail.", "Stratford et al.", "in “Novel phosphorylcholine based hydrogel polymers:developments in medical device coatings” describe polymers formed from 2-methacryloyloxyethyl phosphorylcholine, a higher alkyl methacrylate, hydroxypropylmethacrylate and a methacrylate ester comonomer having a reactive pendant group.", "These PC polymers were investigated to determine the feasibility of delivering drugs and model drugs.", "Results are shown for caffeine, dicloxacillin, vitamin B12, rhodamine and dipyridamole.", "The device on which the drug is coated is a guidewire that is, it is not an implant.", "In EP-A-0623354, solutions of drug and polymer in a solvent were used to coat Wiktor type tantalum wire stents expanded on a 3.5 mm angioplasty balloon.", "The coating weights per stent were in the range 0.6 to 1.5 mg.", "Coating was either by dipping the stent in the solution, or by spraying the stent from an airbrush.", "In each case coating involved multiple coating steps.", "The drug was for delivery to the vessel wall.", "The drugs suggested as being useful for delivery from stents were glucocorticoids, antiplatelet agents, anticoagulants, antimitotic agents, antioxidants, antimetabolite agents and antiinflammatory agents.", "The worked examples all use dexamethasone delivered from a bioabsorbable polymer.", "In U.S. Pat.", "No.", "5,900,246 drugs are delivered from a polyurethane coated substrate such as a stent.", "The polyurethanes may be modified to control its compatibility with lipophilic or hydrophilic drugs.", "Suitable drugs are antithrombotic agents, antiinflammatory agents such as steroids, antioxidants, antiproliferative compounds and vasodilators.", "Particularly preferred drugs are lipophilic compounds.", "A polyurethane coated stent is contacted with a drug in a solvent which swells the polyurethane, whereby drug is absorbed into the polyurethane.", "Selection of a suitable solvent took into account the swellability of the polyurethane and the solubility of the drug in the solvent.", "It was observed that lipophilic drugs loaded in this way released more slowly from hydrophobic polymer than more hydrophilic drugs, by virtue of interaction of the lipophilic drug with hydrophobic polymer.", "In EP-A-0923953 coatings for implantable devices, generally stents, comprise an undercoat comprising particulate drug and polymer matrix, and an overlying topcoat which partially covers the undercoat.", "The top coat must be discontinuous in situ, in order to allow release of the drug from the undercoat.", "Examples of drugs include antiproliferatives, steroidal and non steroidal antiinflammatories, agents that inhibit hyperplasia, in particular restenosis, smooth muscle cell inhibitors, growth factor inhibitors and cell adhesion promoters.", "The worked examples use heparin and dexamethasone.", "The polymer of the undercoat is, for example, hydrophobic biostable elastomeric material such as silicones, polyurethanes, ethylene vinyl acetate copolymers, polyolefin elastomers, polyamide elastomers and EPDM rubbers.", "The top layer is suitably formed of non-porous polymer such as fluorosilicones, polyethylene glycols, polysaccharides and phospholipids.", "In the examples, the undercoat comprised silicone polymer, and coating with the polymer/drug mixture was carried out by spraying a suspension in which both drug and polymer were dispersed, followed by curing of the polymer.", "In our earlier specification WO-A-0101957, unpublished at the priority date hereof, we describe methods for loading drugs into polymer coated stents.", "The polymer coating preferably comprised a crosslinked copolymer of an ethylenically unsaturated zwitterionic monomer with a hydrophobic comonomer.", "The drug was intended to be delivered into the wall of the vessel in which the stent was implanted and the thickness of the coating on the stent was adapted so as to provide higher drug dosage on the outer surface of the stent.", "The drugs were selected from antiproliferatives, anticoagulants, vasodilators, antiinflammatories, cytotoxic agents and antiangiogenic compounds.", "It is well known to those who work in the area of surfactant chemistry that it is possible to determine critical micelle concentrations by use of hydrophobic probes, which seek out the hydrophobic interior of micelles in preference to remaining in an aqueous environment.", "Pyrene is one such molecule .", "Moreover, the fluorescence intensities of various vibronic fine structures in the pyrene molecules' fluorescence spectrum shows strong environmental effects based upon the polarity of the solvent in which it is present (Kalyanasundaram, K et al; JACC, 99(7), 2039, 1977).", "The ratio of the intensity of a pair of characteristic bands (I3:I1) is relevant to the environment.", "A value for I3:I1of about 0.63 is indicative of an aqueous environment whilst a value of about 1 is indicative of a hydrophobic environment.", "In WO-A-95/03036 it is suggested that stents are coated with antiangiogenic drugs to inhibit tumour invasion.", "Many of the drugs are sparingly water soluble.", "The antiangiogenic agent is delivered from a polymeric carrier, such as a bioerodable or biodegradable polymer.", "In WO-A-00/56283, polymers having metal chelating activities are said to have matrix metalloproteinase (MMP) inhibitory activity.", "The polymers may be coated onto a stent.", "It is suggested that MMP's contribute to the development of atherosclerotic plaques and post angioplasty restenotic plaques.", "The MMP inhibiting activity of the polymers is believed to be useful in inhibiting restenosis.", "The polymers may be coated onto a stent and may have additional pharmaceutically active agents dispersed therein, some of which may be sparingly soluble in water.", "Polymers having matrix metalloproteinase inhibitory (MMPI) activity are capable of chelating divalent metals, and are generally polymers of unsaturated carboxylic acids although sulphonated anionic hydrogels may be used.", "One example of a monomer for forming a sulphonated anionic hydrogel is N,N-dimethyl-N-methacryloyloxyethyl-N-(3-sulphopropyl) ammonium betaine.", "Other examples of polymers are acrylic acid based polymers modified with C10-30-alkyl acrylates crosslinked with di- or higher- functional ethylenically unsaturated crosslinking agents.", "There is no specific suggestion of how to provide a coating on a stent comprising both MMPI active polymer and additional therapeutic agent.", "In WO-A-99/01118, antioxidants are combined with antineoplastic drugs to improve their cytotoxicity.", "One utility of the antineoplastic combination is in the treatment of vascular disease.", "The drug combination may be administered from a controlled release system.", "The crosslinkable polymer of 2-methacryloyloxyethyl-2′-trimethyl ammoniumethylphosphate inner salt and dodecyl methacrylate with s crosslinking monomer, coated onto a stent and cured, has been shown to reduce restenosis following stent delivery for the treatment of atherosclerotic conditions.", "In PCT/GB00/02087 mentioned above, we show that a range of drugs may be loaded onto the polymer coated stents such that delivery of the drug into adjacent tissue takes place.", "The present invention relates to a stent having a polymer coating, and comprising a sparingly water soluble drug which may be delivered over an extended period of time from the stents after placement.", "A new intravascular stent comprises a metal body having a coating comprising polymer and at least 20 μg per stent of a restenosis inhibiting agent in which the restenosis inhibiting agent is a sparingly water soluble drug and the polymer in the coating is a cross-linked amphiphilic polymer which, when swollen with water containing pyrene, has hydrophobic domains observable by pyrene fluorescence having an intensity ratio I3:I1of at least 0.8.The agent should be present on the external wall of the stent at a concentration of at least about 0.05 μg/mm2, the area being based on the surface area of metal.", "The ratio of I3:I1is preferably about 1.The drug should preferably have a water solubility of less than 1 mg/mg at room temperature and a logP, where P is the partition coefficient between octanol and water, of at least 1.5, preferably at least 2 or more.", "Preferably, on at least the outer wall of the stent the, coating comprises a layer of the said amphiphilic polymer in which the drug is absorbed.", "Additionally there may be drug absorbed into polymer in the coating on the inner wall.", "It may be possible to provide a sufficiently high does of drug on the stent in form of absorbed material.", "However, sometimes it may be desirable to provide higher doses than may be loaded into the amphiphilic polymer matrix.", "For instance, it may be undesirable to increase the level of polymer on the stent so as to be able to support a higher loading of drug.", "In a preferred stent, the coating on the outer wall of the stent comprises an inner layer of the said amphiphilic polymer, and adhered to said inner layer, crystalline drug.", "Provision of crystalline drug may also confer useful release characteristics on the stent.", "The crystalline material may be controlled of a particle size, for instance, to confer desired release characteristics which complement the release of absorbed drug from the polymer coating.", "In a preferred embodiment of the invention, the coating on at least the outer wall of the stent comprises an inner layer of the said amphiphilic polymer and the top coat comprising a non-biodegradable, biocompatible semipermeable polymer.", "The semipermeable polymer is selected so as to allow permeation of drug through the top layer when the stent is in an aqueous environment.", "In such an environment, the semipermeable polymer may, for instance, be swollen, and it is in this form that it should allow permeation of the active drug.", "A topcoat may confer desirable controlled release characteristics.", "Its use of particular value for the preferred embodiment where coating comprises crystalline drug adhered to an inner layer of amphiphilic polymer.", "The topcoat in such an embodiment has several functions.", "It provides a smooth outer profile, minimises loss of drug during delivery, provides a biocompatible interface with the blood vessel after implantation and controls release of drug from the stent into the surrounding tissue in use.", "A topcoat is preferably substantially free of drug prior to implantation of the stent.", "A topcoat is preferably formed of a cross-linked amphiphilic polymer.", "The coating may be cross-linked or linear.", "Preferably it is the same as the first amphiphilic polymer.", "In the present invention, an amphiphilic polymer comprises groups conferring hydrophilicity and groups conferring hydrophobicity.", "Preferably the groups conferring hydrophilicity comprise zwitterionic groups.", "Preferably the groups conferring hydrophobicity comprise pendant hydrophobic groups selected from C4-24-alkyl, -alkenyl and -alkynyl groups any of which may be substituted by one or more fluorine atoms, aryl, C7-24 aralkyl, oligo (C3-4 alkoxy) alkyl and siloxane groups.", "Most preferably the polymer is formed from ethylenically unsaturated monomers including a zwitterionic monomer and a hydrophobic comonomer.", "For forming a crosslinkable polymer, the ethylenically unsaturated monomers preferably include one or more reactive monomer having a pendant reactive group(s) capable of forming intermolecular crosslinks.", "Preferably the zwitterionic monomer has the general formula I: YBX I wherein B is a straight or branched alkylene (alkanediyl), alkyleneoxaalkylene or alkylene oligo-oxaalkylene chain optionally containing one or more fluorine atoms up to and including perfluorinated chains or, if X or Y contains a terminal carbon atom bonded to B, a valence bond; X is a zwitterionic group; and Y is an ethylenically unsaturated polymerisable group selected from CH2═C(R)CH2O—, CH2═C(R)CH2OC(O)—, CH2═C(R)OC(O)—, CH2═C(R)O—, CH2═C(R)CH2OC(O)N(R1)—, R2OOCCR═CRC(O)O—, RCH═CHC(O)O—, RCH═C(COOR2)CH2C(O)O—, wherein: R is hydrogen or a C1-C4 alkyl group; R1 is hydrogen or a C1-C4 alkyl group or R1 is —B—X where B and X are as defined above; and R2 is hydrogen or a C1-4 alkyl group; A is —O— or —NR1—; K is a group —(CH2)pOC(O)—, —(CH2)pC(O)O—, —(CH2)pOC(O)O—, —(CH2)pNR3—, —(CH2)pNR3C(O)—, —(CH2)pC(O)NR3—, —(CH2)pNR3C(O)O—, —(CH2)pOC(O)NR3—, —(CH2)pNR3C(O)NR3—, (in which the groups R3 are the same or different), —(CH2)pO—, —(CH2)pSO3—, or, optionally in combination with B, a valence bond p is from 1 to 12; and R3 is hydrogen or a C1-C4 alkyl group.", "In group X, the atom bearing the cationic charge and the atom bearing the anionic charge are generally separated by 2 to 12 atoms, preferably 2 to 8 atoms, more preferably 3 to 6 atoms, generally including at least 2 carbon atoms.", "Preferably the cationic group in zwitterionic group X is an amine group, preferably a tertiary amine or, more preferably, a quaternary ammonium group.", "The anionic group in X may be a carboxylate, sulphate, sulphonate, phosphonate, or more preferably, phosphate group.", "Preferably the zwitterionic group has a single monovalently charged anionic moiety and a single monovalently charged cationic moiety.", "A phosphate group is preferably in the form of a diester.", "Preferably, in a pendant group X, the anion is closer to the polymer backbone than the cation.", "Alternatively group X may be a betaine group (ie in which the cation is closer to the backbone), for instance a sulpho-, carboxy- or phospho-betaine.", "A betaine group should have no overall charge and is preferably therefore a carboxy- or sulpho-betaine.", "If it is a phosphobetaine the phosphate terminal group must be a diester, i.e., be esterified with an alcohol.", "Such groups may be represented by the general formula II —X1—R4—N+(R5)2—R6—V II in which X1 is a valence bond, —O—, —S— or —NH—, preferably —O—; V is a carboxylate, sulphonate or phosphate (diester-monovalently charged) anion; R4 is a valence bond (together with X1) or alkylene —C(O)alkylene-or —C(O)NHalkylene preferably alkylene and preferably containing from 1 to 6 carbon atoms in the alkylene chain; the groups R5 are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms or the groups R5 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 atoms; and R6 is alkylene of 1 to 20, preferably 1 to 10, more preferably 1 to 6 carbon atoms.", "One preferred sulphobetaine monomer has the formula II where the groups R7 are the same or different and each is hydrogen or C1-4 alkyl and d is from 2 to 4.Preferably the groups R7 are the same.", "It is also preferable that at least one of the groups R7 is methyl, and more preferable that the groups R7 are both methyl.", "Preferably d is 2 or 3, more preferably 3.Alternatively the group X may be an amino acid moiety in which the alpha carbon atom (to which an amine group and the carboxylic acid group are attached) is joined through a linker group to the backbone of polymer A.", "Such groups may be represented by the general formula IV in which X2 is a valence bond, —O—, —S— or —NH—, preferably —O—, R9 is a valence bond (optionally together with X2) or alkylene, —C(O)alkylene- or —C(O)NHalkylene, preferably alkylene and preferably containing from 1 to 6 carbon atoms; and the groups R8 are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or two of the groups R8, together with the nitrogen to which they are attached, form a heterocyclic ring of from 5 to 7 atoms, or the three group R8 together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring.", "X is preferably of formula V in which the moieties X3 and X4, which are the same or different, are —O—, —S—, —NH— or a valence bond, preferably —O—, and W+ is a group comprising an ammonium, phosphonium or sulphonium cationic group and a group linking the anionic and cationic moieties which is preferably a C1-12-alkanediyl group.", "Preferably W contains as cationic group an ammonium group, more preferably a quaternary ammonium group.", "The group W+ may for example be a group of formula —W1—N+R103, —W1—P+R113, —W1—S+R112 or —W1—Het+ in which: W1 is alkanediyl of 1 or more, preferably 2-6 carbon atoms optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl, alkylene aryl, aryl alkylene, or alkylene aryl alkylene, disubstituted cycloalkyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene cycloalkyl alkylene, which group W1 optionally contains one or more fluorine substituents and/or one or more functional groups; and either the groups R10 are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or aryl, such as phenyl or two of the groups R10 together with the nitrogen atom to which they are attached form a heterocyclic ring containing from 5 to 7 atoms or the three groups R10 together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring, and optionally one or more of the groups R10 is substituted by a hydrophilic functional group, and the groups R11 are the same or different and each is R10 or a group OR10, where R10 is as defined above; or Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-, containing ring, for example pyridine.", "Preferably W1 is a straight-chain alkanediyl group, most preferably ethane-1,2diyl.", "Preferred groups X of the formula V are groups of formula VI: where the groups R12 are the same or different and each is hydrogen or C1-4 alkyl, and e is from 1 to 4.Preferably the groups R12 are the same.", "It is also preferable that at least one of the groups R12 is methyl, and more preferable that the groups R12 are all methyl.", "Preferably e is 2 or 3, more preferably 2.Alternatively the ammonium phosphate ester group VIII may be replaced by a glycerol derivative of the formula VB, VC or VD defined in our earlier publication No.", "WO-A-93/01221.Preferably the hydrophobic comonomer has the general formula VII Y1R13 VII wherein Y1 is selected from CH2═C(R14)CH2O—, CH2═C(R14)CH2OC(O)—, CH2═C(R14)OC(O)—, CH2═C(R14)O—, CH2═C(R14)CH2OC(O)N(R15)—, R16OOCCR14═CR14C(O)O—, R14CH═CHC(O)O—, R14CH═C(COOR16)CH2C(O)—O—, wherein: R14 is hydrogen or a C1-C4 alkyl group; R15 is hydrogen or a C1-C4 alkyl group or R15 is R13; R16 is hydrogen or a C1-4 alkyl group; A1 is —O— or —NR15—; and K1 is a group —(CH2)qOC(O)—, —(CH2)qC(O)O—, (CH2)qOC(O)O—, —(CH2)qNR17—, —(CH2)qNR17C(O)—, —(CH2)qC(O)NR17—, —CH2)qNR17C(O)O—, —(CH2)qOC(O)NR17—, —(CH2)qNR17C(O)NR17—(in which the groups R17 are the same or different), —(CH2)qO—, —(CH2)qSO3—, or a valence bond p is from 1 to 12; and R17 is hydrogen or a C1-C4 alkyl group; and R13 is the hydrophobic group.", "In the comonomer of the general formula VII, the group R13 is preferably a hydrophobic group, preferably: a) a straight or branched alkyl, alkoxyalkyl or oligoalkoxyalkyl chain containing 4 or more, preferably 6 to 24 carbon atoms, unsubstituted or substituted by one or more fluorine atoms optionally containing one or more carbon double or triple bonds; b) an aryl or aralkyl, preferably phenyl, phenethyl or benzyl; or c) a siloxane group —(CR182)qq(SiR192)(OSiR192)ppR19 in which each group R18 is the same or different and is hydrogen or alkyl of 1 to 4 carbon atoms, or aralkyl, for example benzyl or phenethyl, each group R19 is alkyl of 1 to 4 carbon atoms, qq is from 1 to 6 and pp is from 0 to 49 Most preferably R13 is a straight alkyl having 4 to 18, preferably 12 to 16 carbon atoms.", "The reactive monomer to which provides crosslinkability preferably has the general formula VIII Y2B2R20 VIII wherein B2 is a straight or branched alkylene, oxaalkylene or oligo-oxaalkylene chain optionally containing one or more fluorine atoms up to and including perfluorinated chains, or B2 is a valence bond; Y2 is an ethylenically unsaturated polymerisable group selected from CH2═C(R21)CH2—O—, CH2═C(R21)CH2OC(O)—, CH2═C(R21)OC(O)—, CH2═C(R21)O—, CH2═C(R21)CH2OC(O)N(R22)—, R23OOCCR21═CR21C(O)O—, R21H ═CHC(O)O—, R21H ═C(COOR)CH2C(O)O— where R21 is hydrogen or C1-C4 alkyl; R23 is hydrogen, or a C1-4-alkyl group; A2 is —O— or —NR22—; R22 is hydrogen or a C1-C4 alkyl group or R22 is a group B2R20; K2 is a group —(CH2)kOC(O)—, —(CH)kC(O)O—, —(CH2)kOC(O)O—, —CH2)kNR22—, —(CH2)kNR22C(O)—, —(CH2)kOC(O)O—, —CH2)kNR22—, —(CH2)kNR22C(O)—, —(CH2)kC(O)NR22—, —(CH2)kNR22C(O)O—, —(CH2)kOC(O)NR22—, —(CH2)kNR22C(O)NR22—(in which the groups R22 are the same or different), —(CH2)kO—, —(CH2)kSO3—, a valence bond and k is from 1 to.", "12; and R20 is a cross-linkable group.", "Group R20 is selected so as to be reactive with itself or with a functional group in the polymer (eg in group R13) or at a surface to be coated.", "The group R20 is preferably a reactive group selected from the group consisting of ethylenically and acetylenically unsaturated group containing radicals; aldehyde groups; silane and siloxane groups containing one or more substituents selected from halogen atoms and C1-4-alkoxy groups; hydroxyl; amino; carboxyl; epoxy; —CHOHCH2Hal (in which Hal is selected from chlorine, bromine and iodine atoms); succinimido; tosylate; triflate; imidazole carbonyl amino; optionally substituted triazine groups; acetoxy; mesylate; carbonyl di(cyclo)alkyl carbodiimidoyl; isocyanate, acetoacetoxy; and oximino.", "Most preferably R20 comprises a silane group containing at least one, preferably three substituents selected from halogen atoms and C1-4-alkoxy groups, preferably containing three methoxy groups.", "Preferably each of the groups Y to Y2 is represented by the same type of group, most preferably each being an acrylic type group, of the formula H2C═C(R)C(O)—A, H2C═C(R14)C(O)A1 or H2C═C(R21)C(O)—A2, respectively.", "Preferably the groups R, R14 and R21 are all the same and are preferably H or, more preferably, CH3.Preferably A, A1 and A2 are the same and are most preferably —O—.", "B and B2 are preferably straight chain C2-6-alkanediyl.", "Preferably the ethylenically unsaturated comonomers comprise diluent comonomers which may be used to give the polymer desired physical and mechanical properties.", "Particular examples of diluent comonomers include alkyl(alk)acrylate preferably containing 1 to 24 carbon atoms in the alkyl group of the ester moiety, such as methyl (alk)acrylate or dodecyl methacrylate; a dialkylamino alkyl(alk)acrylate, preferably containing 1 to 4 carbon atoms in each alkyl moiety of the amine and 1 to 4 carbon atoms in the alkylene chain, e.g.", "2-(dimethylamino)ethyl (alk)acrylate; an alkyl (alk)acrylamide preferably containing 1 to 4 carbon atoms in the alkyl group of the amide moiety; a hydroxyalkyl (alk)acrylate preferably containing from 1 to 4 carbon atoms in the hydroxyalkyl moiety, e.g.", "a 2-hydroxyethyl (alk)acrylate glycerylmonomethacrylate or polyethyleneglycol monomethacrylate; or a vinyl monomer such as an N-vinyl lactam, preferably containing from 5 to 7 atoms in the lactam ring, for instance vinyl pyrrolidone; styrene or a styrene derivative which for example is substituted on the phenyl ring by one or more alkyl groups containing from 1 to 6, preferably 1 to 4, carbon atoms, and/or by one or more halogen, such as fluorine atoms, e.g.", "(pentafluorophenyl)styrene.", "Other suitable diluent comonomers include polyhydroxyl, for example sugar, (alk)acrylates and (alk)acrylamides in which the alkyl group contains is from 1 to 4 carbon atoms, e.g.", "sugar acrylates, methacrylates, ethacrylates, acrylamides, methacrylamides and ethacrylamides.", "Suitable sugars include glucose and sorbitol.", "Diluent comonomers include methacryloyl glucose and sorbitol methacrylate.", "Further diluents which may be mentioned specifically include polymerisable alkenes, preferably of 2-4 carbon atoms, eg.", "ethylene, dienes such as butadiene, ethylenically unsaturated dibasic acid anhydrides such as maleic anhydride and cyano-substituted alkenes, such as acrylonitrile.", "Particularly preferred diluent monomers are nonionic monomers, most preferably alkyl(alk)acrylates or hydroxyalkyl(alk)acrylates.", "It is particularly desirable to include hydroxyalkyl(alk)acrylates in combination with reactive comonomers which contain reactive silyl moieties including one or more halogen or alkoxy substituent.", "The hydroxyalkyl group containing monomer may be considered a reactive monomer although it also acts as a diluent.", "Such reactive silyl groups are reactive with hydroxy groups to provide crosslinking of the polymer after coating, for instance.", "A particularly preferred combination of reactive monomers is ω(trialkoxysilyl)alkyl(meth)acrylate and an ω-hydroxyalkyl(meth)acrylate.", "The monomers may, in some embodiments, comprise an ionic comonomer.", "Suitable comonomers are disclosed in our earlier publication WO-A-9301221.Preferably the zwitterionic monomer is used in the monomer mixture in a molar proportion of at least 1%, preferably less than 75%, more preferably in the range 5 to 50%, most preferably 10-33%.", "The hydrophobic comonomer is generally used in molar proportion of at least 2%, preferably at least 5% or at least 10%, more preferably in the range 15 to 99%, especially 50 to 95%, more especially 60 to 90%.", "The cross-linkable monomer is preferably used in a molar amount in the range 2 to 33%, preferably 3 to 20%, more preferably 5 to 10% by mole.", "The zwitterionic polymer can be represented by the general formula IX: in which I is 1 to 75, m is 0 to 99, n is 0 to 33and m+n is 25 to 99,Y3 to Y5 are the groups derived from Y to Y2, respectively, of the radical initiated addition polymerisation of the ethylenic group in Y to Y2, and B and X are as defined for the general formula I, R13 is as defined for the general formula VII, and B2 and R20 are as defined for the general formula VIII.", "In the preferred zwitterionic polymer in which Y, Y1 and Y2 are each acrylic groups the polymer has the general formula X in which B, X, R and A are as defined for the compound of the general formula I, R14, A1 and R13 are as defined for the general formula VII, R21, D2, B2 and R20 are as defined for the general formula VII and I, m and n are as defined for the general formula IX The polymerisation is carried out using suitable conditions as known in the art.", "Thus the polymerisation involves radical initiation, using thermal or redox initiators which generate free radicals and/or actinic (e.g.", "u.v or gamma) radiation, optionally in combination with photoinitiators and/or catalysts.", "The initiator is preferably used in an amount in the range 0.05 to 5% by weight based on the weight of monomer preferably an amount in the range 0.1 to 3%, most preferably in the range 0.5 to 2%.", "The level of initiator is generally higher where the monomer includes reactive monomer and the polymer is cross-linkable, e.g.", "1 to 20%.", "The molecular weight of the polymer (as coated, where the polymer is cross-linkable) is in the range 1×104 to 106, preferably in the range 5×104 to 5×105 D. The monomer mixture and the monomer mixture may include a non-polymerisable diluent, for instance a polymerisation solvent.", "Such a solvent may provide solubility and miscibility of the monomers.", "The solvent may be aqueous or non-aqueous.", "The polymer may be recovered by precipitation from the polymerisation mixture using a precipitating solvent, or recovery may involve removal of any non polymerisable diluent by evaporation, for instance.", "In the present invention the term sparingly water soluble means that at room temperature the solubility of the compound in water is less than 1 ml.", "The restenosis inhibiting agent (drug) is preferably a compound having a log P, where P is the octanol/water partition coefficient, of at least 1.5 for instance more than 2.The drug should have a logP of at least 1.5, preferably at least 2.When such drugs are absorbed into polymers having the hydrophobic domains, observed using the pyrene fluorescence test, interaction apparently takes place, affecting the release rate of the drug.", "It is believed that the hydrophobic drug will preferentially partition into the hydrophobic domains in a similar fashion to pyrene.", "Preferred drugs are steroids, especially estrogens, especially estradiol (E2) and corticosteroids, especially dexamethasone (Dex) and 6α-methylprednisolone (MP).", "Estradiol has a logP of 4.3 and a water solubility of 0.003 mg/ml; MP has a logP of 1.42; Dex has a logP of 2.55 and a water solubility of 0.01 mg/ml.", "Other examples of sparingly water-soluble drugs which may be used in the inevntion include statins, such as simvastatin (logP 2.06), and, though less preferably lovastatin (logP 1.7) and atorvastatin (logP 1.6).", "Preferably the drug should be present in an amount in the range 20 to 1000 μg, preferably at least 50 μg, more preferably in the range 100 to 400 μg, per stent.", "The stent may be made of a shape memory metal, or may be elastically self-expanding, for instance, be a braided stent.", "However, preferably it is a balloon expandable stent.", "In the embodiment of the invention, in which a topcoat is provided, the topcoat may be part of a coherent coating formed over both a stent and a stent delivery device, for instance a balloon of a balloon catheter from which a balloon expandable stent is delivered.", "In this case, the balloon may additionally be provided with a coating comprising drug, for instance adsorbed onto parts of its exterior surface between stent struts.", "Such a device may be produced by loading the stent with drug after the stent has been mounted onto the delivery catheter.", "According to a further aspect of the invention there is provided a new method for producing a drug coated intravascular stent comprising the steps: a) a metallic stent body is coated on its inner and outer walls with a cross-linkable amphiphilic polymer; b) the cross-linkable polymer is subjected to conditions under which cross-linking takes place to produce a stent coated with polymer which, when swollen with water containing pyrene, has hydrophobic domains observable by pyrene fluorescence having an intensity ratio I3:I1of at least 0.8.; c) at least the outer coated wall of the polymer coated stent is contacted with liquid drug composition comprising a sparingly water-soluble restenosis inhibiting drug and an organic solvent in which the drug is at least partially dissolved and which is capable of swelling the cross-linked polymer of the coating, for a time sufficient to swell the polymer coating on the outer wall, to produce a wet drug-coated stent in which drug is present in an amount of at least 20 μg per stent; d) organic solvent is evaporated from the wet stent to produce a dry drug-coated stent.", "In the method of the invention, in step c), the drug may be both absorbed into the polymer and adsorbed at the process of the polymer coating whereby, upon evaporation of the solvent in step d) crystals of drug are formed which are adherent to the surface of the dry drug coated stent.", "In the method of the invention, contact of the polymer coated stent with the liquid drug composition may be by dipping the stent into a body of the stent, and/or by flowing, spraying or dripping liquid composition onto the stent with immediate evaporation of solvent from the wet stent.", "Such steps allow good control of drug loading onto the stent, and are particularly useful for forming the crystals of drug at the surface of polymer.", "Whilst the stent may be provided with drug coating in the invention prior to being mounted onto its delivery device, it is preferred, and most convenient, for the stent to be premounted onto its delivery device prior to carrying out step c).", "By this means, it is primarily the outer wall of the stent (as opposed to the inner wall of the stent) which becomes coated with drug.", "Whilst this method will generally result in drug being coated onto the stent delivery section of the delivery catheter, this is, in general, not disadvantageous.", "In some circumstances it may be useful for the outer surface of the delivery catheter to be provided with a coating of drug, which may be delivered to adjacent tissue upon placement of the stent in use.", "Generally the delivery catheter is in contact with such tissue for a short period, whereby contact is not maintained for a prolonged period, and limited level of transfer of drug from the balloon take place.", "The method of the invention may include a step of applying a topcoat.", "In such a method a further step e) is carried out: e) to at least the outer wall of the dry drug coated stent a polymer is applied, to form a non-biodegradable biocompatible semi-permeable polymer-containing top-coat.", "In this preferred embodiment, in step e) it is preferred that a liquid top-coating composition comprising polymer is coated onto at least the outer wall and is cured after coating to form the top-coat.", "It is desirable for the liquid coating to be sprayed onto the outer wall of the stent, as this method has been found to minimise removal of previously applied drug.", "The top-coating composition, and consequently the top coat in the product, should generally be substantially free of drug.", "Preferably it is substantially free of other pharmaceutical actives although in certain circumstances it may be useful to cocoat a mixture of polymer and another pharmaceutically active agent.", "For the embodiment of the invention where the liquid top-coating composition comprises a cross-linkable polymer of the type preferred for use to form the first amphiphilic polymer, the liquid top-coating composition comprises cross-linkable polymer and the curing step in the preferred method involves exposure of the top-coat to cross-linking conditions.", "Curing of cross-linkable polymer may involve exposure to irradiation, chemical curing agents, catalysts or, more usually raised temperature and/or reduced pressure to acceptable condensation based cross-linking reactions.", "Drying the liquid during composition usually involves raised temperature and/or reduced pressure for a time sufficient to reduce the amount of solvent remaining on the stent to undetectable levels or levels at which it will not interfere with subsequent processing steps, or with release of the drug in use, or be toxic to a patient in whom the stent is implanted.", "Where in the preferred method, the stent is preloaded onto its delivery device before being coated with drug, the top-coat is provided over both the stent and the stent delivery section of the delivery catheter.", "Preferably the top-coat forms a coherent film covering the entire stent delivery section.", "It is preferred for the device subsequently to be sterilised and to be packaged into a sterile package for storage prior to use.", "Sterilisation may involve Γ irradiation, or application of heat, but preferably involves contact with ethylene oxide.", "Where, in the preferred method, a stent is contacted with liquid drug composition whilst mounted on a delivery device, it is important to ensure that the said contact does not adversely effect the properties of the delivery catheter.", "For a balloon catheter, the contact must not significantly reduce the burst strength of the balloon.", "A preferred balloon catheter used for delivering a stent is formed of polyamide.", "We have established that the use of ethanol, methanol or dimethylsulfoxide (DMSO) do not damage the balloon such that burst strength are reduced to an unacceptable level.", "The examples of solvents and solvent mixtures include dichloromethane (DCM), mixtures of isopropanol and water and DCM/ethanol mixtures.", "The solvent must be selected to allow adequate dissolution of drug, and swelling of the cross-linked polymer coating to allow absorption of drug into the body of the polymer.", "Drug which is absorbed into the polymer will be released over a period of time after implantation of the stent.", "The liquid drug composition may comprise other components, such as crystal modifiers, polymers, salts, acids, bases etc.", "It may be convenient to include dissolved amphiphilic, optionally crosslinkable polymer, to confer compatibility with the polymer on the stent surface.", "Such a polymer may be identical to that described above used in the first aspect of the invention.", "According to a further aspect of the invention there is also provided an intravascular stent comprising a metal body and a coating on the metal body comprising an amphiphilic polymer and 17β-estradiol.", "Preferably the amphiphilic polymer has a hydrophobic domain observable by pyrene fluorescence having an intensity ratio I3:I1of at least 0.8, preferably about 1.0.Preferably at least 20 μg estradiol is incorporated per stent.", "According to a further aspect of the invention there is also provided an intravascular stent comprising a metal body and a coating on the metal body comprising an amphiphilic polymer and 6α-methylprednisolone.", "Preferably the amphiphilic polymer has a hydrophobic domain observable by pyrene fluorescence having an intensity ratio I3:I1of at least 0.8, preferably about 1.0.Preferably at least 20 μg MP is incorporated per stent.", "Preferrred embodiments of these further aspects of the invention are preferably also covered by the main aspects of the invention and comprise the preferred aspects thereof and are produced by the inventive method.", "The present inventors have established that stents according to the invention confer improved quantative coronary angioplasty results when used in animals, reduced intimal hyperplasia, increased lumen diameter and excellent clinical results as compared to control polymer coated stents.", "The drawings relate to the following: FIG.", "1 compares the I3:I1ratio from the fluorescence spectra of pyrene in different environments (Ref Ex.", "1); FIG.", "2 shows the amount of pyrene retained in a variety of polymer coatings as determined in Reference Example 1; FIG.", "3A compares the fluorescence spectra of pyrene in lauryl methacrylate and water; FIG.", "3B compares the fluorescence spectra of pyrene in water and in two amphiphilic polymers (Ref Ex 1) FIGS.", "4a and 4b show the actual and theoretical release rates of dexamethasone and 17β-estradiol from polymers (Ref Ex 2) FIG.", "5 shows the estradiol elution profile for Example 2; FIG.", "6 shows the estradiol elution profile for Example 3; FIG.", "7 shows the estradiol elution profile for Example 4; and FIG.", "8 shows the 6α-methyl prednisolone elution profile for Example 7.The present invention is illustrated in the accompanying examples: REFERENCE EXAMPLE 1 Zwitterionic polymer coatings were investigated by allowing pyrene to diffuse into the polymer and studying the degree to which it is taken up, and the effects on the ratio of the fluorescence band intensities to see if there is any significant indication of the type of environment present.", "Polymer coatings of interest were dissolved in an appropriate solvent (usually ethanol) at 20 mgml−1.The solution was used to coat polymethylmethacrylate (PMMA) fluorescence cuvettes by simply pouring into the cuvette, draining, following by an oven curing at 70° C. overnight.", "Polymers studied were: a) a copolymer of 2-methacryloyloxy ethyl-2′-trimethyl-ammoniumethyl phosphate inner salt (MPC): n-butylmethacrylate: hydroxypropyl methacrylate (HPM): trimethoxysilylpropylmethacylate (TSM) 29:51:15:5 (by weight) b) a copolymer of MPC: benzylacrylate: HPM: TSM 29:51:15:5 c) a copolymer of MPC: dodecylmethacrylate (DM): HPM: TSM: 45:35:15:5 d) a copolymer of MPC: DM: HPM: TSM: 29:51:15:5 e) a copolymer of MPC: DM: HPM: TSM: 15:65:15:5 f) poly(2-hydroxyethylmethacrylate).", "The copolymers a-e were synthesised as disclosed in WO-A-9830615.Analytical grade pyrene was used in high purity water (8.32×10−4 M).", "The fluorescence spectrum was recorded using an excitation wavelength of 335 nm and scanned from 350-440 nm on a PE LS 50B Luminescence Spectrophotometer.", "Subtraction of the spectrum of each coating in water was necessary to remove the interference of a small band at 380 nm present in all methacrylate systems.", "Environment information could be obtained by comparing the ratio of the intensity of the peaks at 373 nm (I1) and 383 nm (I3) (I3/I1).", "Where I3/I1 was similar for polymer systems, the comparative amount of pyrene present could be estimated by the maximum intensity of I1; alternatively, the entire peak area may offer an alternative measure of the comparative amount of pyrene present in different coatings.", "It was important to mark the side of the cuvette to ensure the same orientations was achieved each time it was replaced in the spectrophotometer.", "FIG.", "3A compares the fluorescence spectra of pyrene in lauryl methacrylate (dodecyl methacrylate) (8.32×10−4 M) and water (8.32×10−5 M).", "For water the I3/I1 ratio is 0.633 (literature value1 0.63) and the I3/I1 ratio for lauryl methacrylate is 1.029.This indicates the very different environments than might be expected to be seen within the polymer coating.", "Pyrene solution added to the coated cuvettes was allowed to stand for 16 h, the cuvette emptied and washed throughly with ultrapure water, refilled with ultrapure water and the fluorescence spectrum recorded.", "The comparative maximum height of I1 was used to estimate the relative amounts of pyrene in the coatings.", "This was repeated for three cuvettes of each polymer and the average taken.", "Despite some variations between cuvettes, the trends were the same, indicating that the polymer formulations with more hydrophobic content seemed to contain more pyrene (FIG.", "2).", "This is in contradiction to the water contents of these materials which vary in the opposite order.", "Hence for the varying systems, although water contents vary in the order c>d>f>e (88:40:38:27), the final fluorescence intensity (loading of pyrene achieved in the coating) varies according to e>d>c≧f.", "20 This indicates that the pyrene is preferentially associating itself with hydrophobic areas within the coating.", "The ratio of I3/I1 was also studied (FIG.", "1) and again, those polymer with formal hydrophobic chains showed a greater ratio (indicating more hydrophobic environment for the pyrene).", "This polymer containing the benzyl side chain has a lower than expected I3/I1, initially indicating poor interaction with the pyrene.", "However, measurement of the I3/I1 for pyrene in the pure benzyl acrylate monomer showed that the maximum I3/I1 that could be expected would be 0.75 (i.e.", "less of a shift in fluorescent intensity is produced in this aromatic monomer compared to the lauryl monomer).", "PHEMA coating showed I3/I1 characteristic of pyrene in an aqueous environment (FIG.", "3A), suggesting no formal hydrophobic domain exists.", "REFERENCE EXAMPLE 2 Drug-Polymer Interaction Versus Drug Solubility There are examples of stent-based release of therapeutics that rely upon the poor solubility of the active agent in water to achieve a slow release rate, i.e.", "by relying for extended release of drug on poor solubility of the drug in water.", "When a graph of solubility versus release time (T90%) is plotted however, the relationship is extremely poor (R2=0.006) indicating the solubility on its own does not account for the observed release characteristics.", "This can be modelled further by comparing the theoretical release of drug into a known volume of water based purely upon its solubility and comparing this with its actual release profile from the polymer system into the same elution volume.", "Assuming that 100 μg of the drug is place on a surface, and that the drug is eluted off into 5 ml of solution, and then at various arbitrary points, 1 ml removed, and I ml of fresh solution added, the dissolution profiles for various drugs could be calculated and compared to experimental data obtained the same way.", "The variation between calculated and observed could be attributed to the interaction with the polymer matrix.", "This is clearly illustrated by FIGS.", "4a and b.", "Here, the theoretical release of dexamethasone (which has a log P where P is the partition coefficient between octanol and water of 2.55) and estradiol have been calculated and plotted on the graph (diamonds/circles) based on the solubility of the compound and the volume of water into which it is being eluted (Details of further loading and elution studies from polymer-coated stents are given below).", "The difference between this line and the observed data (squares) is the degree of interaction of the compound with the hydrophobic domains within the polymer coating which in this case is polymer d) from Ref.", "Example 1.It is the interaction that prolongs the release of the compound and offers some capability to control the delivery of the drug to its surrounding environment.", "EXAMPLE 1 Estradiol Uptake Studies 1.1 Normal (Low) Loading Level 15 mm BiodivYsio DD stents provided with a cross-linked coating on both inner and outer walls of copolymer d) used in Reference Example 1 were provided with a coating of drug by immersing them in a 20 mg/ml solution of estradiol for 30 minutes, removing the stents from the solution and wick drying them on tissue then allowing them to dry for 2 hours at room temperature.", "The drug total loading was measured by HPLC, and found to be in the range 45-65 μg per stent.", "1.2 High Loading Level 18 mm BiodivYsio stents premounted on their balloon delivery catheter were coated by dipping the balloon and stent in a volume of a 20 mg/ml solution of estradiol in ethanol for 5 minutes, removing and drying for 5 minutes, then pipetting 10 μl of the drug solution on to the stent/balloon and allowing to dry for 1 minute, this pipetting and drying step being repeated once, with a final 10 minute drying period.", "The balloons were inflated, deflated and the stents removed.", "The level of drug on each stent and balloon was determined using HPLC.", "The level of drug on the stents was in the range 225-250 μg per stent, and the amount on each balloon was about 190-220 μg.", "1.3 High Loading Level Repeat The method of example 1.2 was repeated with the stents premounted on either a 3 mm or 4 mm diameter balloon.", "For the 4 mm balloon system the mean level of drug was 240 with a range for 3 samples of 229-254 μg, giving an area distribution of 2.4 μg/mm2 (range 2.3 to 2.6).", "For the 3 mm balloon system the mean loading per stent was 2.59 μg with a range for 5 samples of 243-276 μg.", "The area distribution is 2.6 μg/mm2 (range 2.4-2.8).", "EXAMPLE 2 Estradiol Elution Studies at 25° C.—Non-Flow System Elution studies were carried out at 25° C. for upto 1 hour in gently agitated PBS.", "This was done by placing 15 mm DD stents loaded with estradiol, from Example 1.1 individually in vials containing 5 ml phosphate buffered saline (PBS) on rollers.", "At various time intervals up to 7 hours a 1 ml aliquot was removed and replaced with 1 ml fresh PBS.", "The stents and water aliquots were measured, to give the amount of drug eluted and the amount of drug remaining on the stents.", "The results are shown in FIG.", "5.It may be seen that during the first hour estradiol was eluted relatively more quickly than the rest of the time and at 4 hours there was still 62% estradiol still remaining on the stent.", "EXAMPLE 3 Release of 17β-Estradiol The elution profile for 17β-estradiol, FIG.", "6 from the stents produced in example 1.1, was assessed using an in-vitro test where five stents were vigorously stirred in a large volume (1000 ml) of PBS saline solution at 37° C. This test demonstrates that the stent is capable of a sustained release of 17β-estradiol over a duration of at least several hours.", "Aliquots of buffer were removed at various time points over a 24 hour period and analysed for 17β-estradiol content.", "The results are shown in FIG.", "6.When translated into in-vivo conditions, the release profile is predicted to be over a prolonged period of time.", "EXAMPLE 4 Estradiol Elution Studies in Flow-System The elution of estradiol was examined in a flow system at 37° C. and evaluated over an 8 hour period.", "PBS was maintained at 37° C. in six stirred reservoirs (500 ml each) within a water bath.", "A length of silicone tubing (3 mm internal diameter) was attached from each reservoir to one of six stent chambers (4 mm internal diameter 80 mm long) and back to the respective reservoir via a peristaltic pump.", "The system was pumped using a flow rate of 100 ml/min to reach equilibrium temperature of 37° C. The flow was stopped and two estradiol loaded 15 mm stents loaded as in Example 1 were placed in each of the six stent chambers, and flow recommenced.", "A stent was then removed at various time periods and wick dried.", "These were used to measure the amount of estradiol remaining on the stent.", "The results are shown in FIG.", "7.This shows that in this model the estradiol was eluted relatively more quickly than in the stirred 5 ml PBS of Example 2.Since the total volume of PBS passing over the stents inthe flow model is 500 ml, it is likely that throughout the period the rate of desorption of drug from the stent was higher than the rate of absorption from the environment.", "This condition may not apply to the non-flow method.", "EXAMPLE 5 In Vivo Test on Estradiol Loaded Stents This study investigated the acute and short term effects of deploying estradiol (17β) loaded 18 mm stents produced generally as described in Example 1 above (ie loaded with drug whilst mounted on the balloon using either a single dipping step or the multi-step loading method) in porcine arteries.", "There were 3 arms to the study: 1) Control, using the non-drug-coated 18 mm BiodivYsio stent with the polymer coating d from the reference example 1, 2) low estradiol dose (about 45-65 μg per stent) using the dip only loading method of Example 1.1, and 3) high dose (about 225-250 μg per stent) by using the multi-step loading method (Ex.", "1.2).", "A total of 6 animals were each implanted with three stents, one each of the control, low and high dose, one stent in each of three coronary arteries.", "A balloon:artery ratio of about 1.25:1 (in the range (1.2-1.3):1) was used, the oversizing designed to cause an injury to the artery wall resulting in neointimal formation resembling that occurring in stented human coronary arteries.", "One month after implantation observations were made by quantitative coronary angiogram (QCA) of the mean lumen diameter (MLD).", "Subsequently the results were evaluated by histomorphometric analysis of intimal hyperplasia formation and vessel luminareas, as well as for the extent of re-endothelialisation.", "The results are shown in Table 1.TABLE 1 MLD mm Intimal Area mm2 (S.D) Luminal area mm2 (S.D) Control 2.26 4.31 (1.1) 3.49 (1.41) Low 2.31 3.60 (0.79) 4.20 (1.74) High 2.53 2.54 (1.0) 5.40 (1.70) The study showed a 40% reduction in intimal area in the ‘High Dose’ 17β-estradiol loaded stents compared with control stents (p<0.05), see FIG.", "4.There was also a reduction in the Intimal Area/injury score ratio in the ‘High Dose’ 17β-estradiol group compared with the ‘Control’ stents (1.32 ±0.40 mm2 vs 1.96±0.32 mm2, for 17β-estradiol vs control respectively, P<0.01).", "There was no significant difference in the injury score for all three study arms.", "A trend was noted for the Luminal Area where there was an increase in Luminal Area with an increase in dosage.", "Re-endothelialization scores were high for all three study arms, suggesting that 17b-estradiol does not inhibit the healing process.", "EXAMPLE 6 α-Methylprednisolone Uptake Studies 6.1 Low Loading Level 15 mm BiodivYsio DD stents provided with a cross-linked coating on is both inner and outer walls of copolymer d) used in Reference Example 1 were provided with a coating of drug by immersing them in a 12.35 mg/ml solution of 6α-methyl prednisolone (MP) for 5 minutes, removing the stents from the solution and wick drying them on tissue then allowing them to dry for at least 1 hour at room temperature.", "The drug total loading was measured by placing the stent in ethanol (9.0 ml) and sonicated for 30 minutes.", "The concentration in the ethanol was determined by UV at 246.9 nm compared to standards.", "The loading was found to be in the range 30-40 μg per stent.", "6.2 High Loading Level 18 mm BiodivYsio stents coated with the cross-linked polymer d) on both walls premounted on their balloon delivery catheter were coated by dipping the balloon and stent in a volume of a 12.0 mg/ml solution of MP in ethanol for 5 minutes, removing and drying for 5 minutes, then pipetting 10 μl of the drug solution on to the stent/balloon and allowing to dry for 1 minute, this pipetting and drying step being repeated thrice, with a final 10 minute drying period.", "The balloons were inflated, deflated and the stents removed.", "The level of drug on each stent was determined using the technique described above.", "The level of drug on the stents was in the range 250-300 μg per stent.", "EXAMPLE 7 —MP Elution Studies at 25° C. —Non Flow System Elution studies were carried out at 25° C. for upto 1 hour in gently agitated PBS.", "This was done by placing 15 mm DD stents loaded with MP from Examples 6.1 and 6.2 individually in vials containing 5 ml phosphate buffered saline (PBS) on rollers.", "At various time intervals a 1 ml aliquot was removed and replaced with 1 ml fresh PBS.", "The stents and water aliquots were measured, to give the amount of drug eluted and the amount of drug remaining on the stents.", "The results are shown in FIG.", "8.EXAMPLE 8 In Vivo Test on MP and Dexamethasone Loaded Stents This study investigated the acute and short term effects of deploying MP and dexamethasone loaded 18 mm stents produced generally as described in Example 1 above (i.e.", "loaded with drug whilst mounted on the balloon using either the single dipping step of example 1.1) or the multi-step loading method of example 1.2) in porcine arteries.", "There were 4 arms to the study: 1) Control, using the non-drug-coated 18 mm BiodivYsio DD stent (which is coated with polymer d), 2) low Dex dose (about 95 μg per stent) using the dip only loading method, 3) high Dex dose (about 265 μg per stent) by using the multi-step loading method, and 4) high dose MP produced as described in Example 6.2 (about 270 μg per stent).", "Stents were implanted into porcine arteries for 5 days, explanted, then assessed for inflammation by H&E staining and the results scored histopathologically and morphometrically on an arbitrary scale.", "From nine measurements for each data point the following results were obtained: (p values in parentheses in table), 1=no difference, 0.05=95% confidence of a difference), see table 2.TABLE 2 Histopathological findings Inflammation Injury Thrombus Perivasculitis Control 0.73 0.56 0.74 0.48 + Dex 0.73 0.53 0.77 0.51 Low dose + Dex 0.57 0.40 0.54* 0.45 High dose + MP 0.51* 0.42 0.50* 0.39 Morphometric Results IEL Dia- EEL Dia- Dia Sten (%) Area Sten (%) Lum.", "Dia Lum.", "Dia Control 5 9 0.16 0.47 + Dex 4 (0.02) 8 (0.14) 0.14 (0.18) 0.47 (1) Low dose + Dex 4 (0.02) 7 (0.005) 0.13 (0.009) 0.43 (0.24) High close + MP 3 (0) 6 (0.001) 0.11 (0) 0.35 (0) *indicates statistically significant difference from control (p = 0.05) The results of Examples 6 and 8 show that the high dose stents show a trend towards improved results.", "EXAMPLE 9 Re-Endothelialisation of Dexamethasone-Loaded Stents The dexamethasone-loaded stents (Low Dex) described in example 8 were implanted into porcine coronary arteries for 30 days.", "After this time the animals were sacrificed and the stented sections of the arteries removed and fixed.", "The vessel was cut longitudinally and opened out to expose the inner surface which was sputter coated and viewed under by SEM.", "SEM revealed that the inner surface of the vessel had completely re-endothelialised over the stent struts.", "EXAMPLE 10 Clinical Trial Assessment—30 Day Data for 71 Patients Study of anti-restenosis with the BiodivYsio Dexamethasone eluting stent (STRIDE) which is a multi-centre prospective study performed at 7 centres in Belgium with 71 patients.", "The primary objective of this study was to evaluate the proportion of patients with binary restenosis 6 months after receiving a BiodivYsio stent loaded with dexamethasone i.e.", "produced by the same technique as the LowDex stent described in example 8.The secondary objectives were to evaluate the incidence of sub(acute) thrombosis to 30 days post procedure and the occurrence of MACE (death, recurrent myocardial infarction or clinically driven target lesion revascularisation) at 30 days and 6 months post procedure.", "11, 15, 18 and 28 mm by 3.0 to 4.0 mm diameter BiodivYsio stents loaded with dexamethasone were under investigation.", "30 day data for 71 patients (safety analysis set) are reported in this example.", "Other endpoints have not yet been reached and therefore will not be described.", "71 patients (79% male) with an average height of 170 cm and weight of 79 Kg were enrolled into the study.", "63% of patients had a history of hypercholesterolaemia and 69% had smoked or were current smokers.", "47% of patients had multi-vessel disease and 44% had a history of previous MI.", "The vessels/lesions treated were in the following categories: Vessel Treated Lesion Classification RCA 31% A 21% LAD 41% B1 48% Cx 19% B2 27% Other 9% C 4% The mean lesion length treated was 9 mm.", "The majority of patients had either a 15 mm (34%) or an 18 mm (39%) stent implanted.", "At 30 day follow-up two patients had a MACE (1 patient died one day post procedure following coronary embolism and 1 patient had a non Q-wave MI) (Table 3).", "Three patients had serious adverse events that were unrelated to the study treatment Technical device success defined as intended stent successfully implanted as the first stent was 95%.", "Clinical device success defined as technical device success in the absence of MACE to discharge was achieved in 94% of patients.", "The data presented in this initial interim analysis suggest that the presence of dexamethasone in the coating is not associated with an increased occurrence of MACE or serious adverse events and that the BiodivYsio Dexamethasone stent is safe in the short term for use in patients.", "REFERENCE EXAMPLE 3 Assessment of Changing Solvent on DD Stent Delivery System In order to load the pre-mounted (on a balloon delivery catheter (balloon formed from a nylon blend)) DD stent with non-water soluble drug, the stent/delivery system combination must be immersed in the drug solution.", "The aim of this experiment was to check if the solvent had a detrimental effect on the balloons.", "Pre-mounted BioDivYsio stents were placed in solvent for minutes then allowed to air dry to 5 minutes.", "The mechanical properties of the balloon were then assessed by a burst pressure test.", "The samples were connected to a pressure pump and gauge and a positive pressure of 1 atm.", "(105 Pa) applied and left for 30 seconds.", "The pressure was increased by 1 atm (105 Pa) every 30 seconds until the stent was fully deployed i.e.", "there were no creases or folds in the balloon.", "The pressure was then increased to 16 atm.", "which is the rated burst pressure for the balloon system, and held for 30 seconds.", "The pressure was then increased in 1 atm.", "steps and held for 30 seconds at each step, until the balloon burst.", "The results are in Table 2.TABLE 3 Effect of Drug Loading Solvent on Balloon Burst Pressure Solvent Deployment Pressure/atm.", "Burst Pressure/atm.", "None 3 >16 Ethanol 3 >16 Methanol 3 23 ± 1 DMSO 3 24 ± 1 None of the solvents cause detrimental effects on the balloon The choice of drug loading solvent is therefore related to drying rate and solvent toxicity, drug solubility, and swellability of the polymer." ] ]	Patent_10466144
[ [ "Modulating insulin receptor signaling", "Human ISM genes are identified as modulators of INR signaling and thus are therapeutic targets for disorders associated with defective INR signaling.", "Methods for identifying modulators of ISM, comprising screening for agents that modulate the activity of ISM are provided." ], [ "1.A method of identifying a candidate INR signaling modulating agent, said method comprising the steps of: (a) providing an assay system comprising an ISM polypeptide or nucleic acid; (b) contacting the assay system with a test agent under conditions whereby, but for the presence of the test agent, the system provides a reference activity; and (c) detecting a test agent-biased activity of the assay system, wherein a difference between the test agent-biased activity and the reference activity identifies the test agent as a candidate INR signaling modulating agent.", "2.The method of claim 1 wherein the assay system includes a screening assay comprising an ISM polypeptide, and the candidate test agent is a small molecule modulator.", "3.The method of claim 2 wherein the screening assay detect an activity selected from the group consisting of kinase activity, fatty acid elongation, fatty acid desaturation, mitochondrial transport, upbiquinone biosynthesis, protein binding, and transcription.", "4.The method of claim 1 wherein the assay system includes a binding assay comprising an ISM polypeptide and the candidate test agent is an antibody.", "5.The method of claim 1 wherein the assay system includes an expression assay comprising an ISM nucleic acid and the candidate test agent is a nucleic acid modulator.", "6.The method of claim 5 wherein the nucleic acid modulator is an antisense oligomer.", "7.The method of claim 6 wherein the nucleic acid modulator is a PMO.", "8.The method of claim 1 wherein the assay system comprises cultured cells or a non-human animal expressing an ISM, and wherein the assay system includes an assay that detects an agent-biased change in INR signaling or an output of INR signaling.", "9.The method of claim 8 wherein the assay system comprises cultured cells.", "10.The method of claim 9 wherein the assay detects an event selected from the group consisting of expression of insulin-responsive genes, phosphorylation of an INR signaling pathway component, kinase activity of an INR signaling pathway component, glycogen synthesis, glucose uptake, GLUT4 translocation, and insulin secretion.", "11.The method of claim 8 wherein the assay system comprises a non-human animal.", "12.The method of Claim 11 wherein the non-human animal is a mouse providing a model of diabetes and/or insulin resistance.", "13.The method of claim 12 wherein the assay system includes an assay that detects an event selected from the group consisting of hepatic lipid accumulation, plasma lipid accumulation, adipose lipid accumulation, plasma glucose level, plasma insulin level, and insulin sensitivity.", "14.The method of claim 1, comprising the additional steps of: (d) providing a second assay system comprising cultured cells or a non-human animal expressing an ISM, (e) contacting the second assay system with the test agent of (b) or an agent derived therefrom under conditions whereby, but for the presence of the test agent or agent derived therefrom, the system provides a reference activity; and (f) detecting an agent-biased activity of the second assay system, wherein a difference between the agent-biased activity and the reference activity of the second assay system confirms the test agent or agent derived therefrom as a candidate INR signaling modulating agent, and wherein the second assay system includes a second assay that detects an agent-biased change in an activity associated with INR signaling or an output of INR signaling.", "15.The method of claim 14 wherein the second assay system comprises cultured cells.", "16.The method of claim 15 wherein the second assay detects an event selected from the group consisting of expression of insulin-responsive genes, phosphorylation of an INR signaling pathway component, kinase activity of an INR signaling pathway component, glycogen synthesis, glucose uptake, GLUT4 translocation, and insulin secretion.", "17.The method of claim 17 wherein the second assay system comprises a non-human animal.", "18.The method of claim 18 wherein the non-human animal is a mouse providing a model of diabetes and/or insulin resistance.", "19.The method of claim 18 wherein the second assay system includes an assay that detects an event selected from the group consisting of hepatic lipid accumulation, plasma lipid accumulation, adipose lipid accumulation, plasma glucose level, plasma insulin level, and insulin sensitivity.", "20.A method of modulating INR signaling in a mammalian cell comprising contacting the cell with an agent that specifically binds an ISM polypeptide or nucleic acid.", "21.The method of claim 20 wherein the agent is administered to a mammalian animal predetermined to have a pathology associated with INR signaling.", "22.The method of claim 20 wherein the agent is a small molecule modulator, a nucleic acid modulator, or an antibody." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Insulin is the central hormone governing metabolism in vertebrates (reviewed in Steiner et al., 1989, In Endocrinology, DeGroot, eds.", "Philadelphia, Saunders: 1263-1289).", "In humans, insulin is secreted by the beta cells of the pancreas in response to elevated blood glucose levels, which normally occur following a meal.", "The immediate effect of insulin secretion is to induce the uptake of glucose by muscle, adipose tissue, and the liver.", "A longer-term effect of insulin is to increase the activity of enzymes that synthesize glycogen in the liver and triglycerides in adipose tissue.", "Insulin can exert other actions beyond these “classic” metabolic activities, including increasing potassium transport in muscle, promoting cellular differentiation of adipocytes, increasing renal retention of sodium, and promoting production of androgens by the ovary.", "Defects in the secretion and/or response to insulin are responsible for the disease diabetes mellitus, which is of enormous economic significance.", "Within the United States, diabetes mellitus is the fourth most common reason for physician visits by patients; it is the leading cause of end-stage renal disease, non-traumatic limb amputations, and blindness in individuals of working age (Warram et al., 1995, In Joslin's Diabetes Mellitus, Kahn and Weir, eds., Philadelphia, Lea & Febiger, pp.", "201-215; Kahn et al., 1996, Annu.", "Rev.", "Med.", "47:509-531; Kahn, 1998, Cell 92:593-596).", "Beyond its role in diabetes mellitus, the phenomenon of insulin resistance has been linked to other pathogenic disorders including obesity, ovarian hyperandrogenism, and hypertension.", "Within the pharmaceutical industry, there is interest in understanding the molecular mechanisms that connect lipid defects and insulin resistance.", "Hyperlipidemia and elevation of free fatty acid levels correlate with “Metabolic Syndrome,” defined as the linkage between several diseases, including obesity and insulin resistance, which often occur in the same patients and which are major risk factors for development of Type 2 diabetes and cardiovascular disease.", "Current research suggests that the control of lipid levels, in addition to glucose levels, may be required to treat Type 2 Diabetes, heart disease, and other manifestations of Metabolic Syndrome (Santomauro A T et al., Diabetes (1999) 48:1836-1841).", "The ability to manipulate and screen the genomes of model organisms such as Drosophila and C. elegans provides a powerful means to analyze biochemical processes that due to significant evolutionary conservation of genes, pathways, and cellular processes, have direct relevance to more complex vertebrate organisms.", "Identification of novel functions of genes involved in particular pathways in such model organisms can directly contribute to the understanding of the correlative pathways in mammals and of methods of modulating them (see e.g., Miklos G L and Rubin G M, Cell 1996, 86:521-529).", "While Drosophila and C. elegans are not susceptible to human pathologies, various experimental models can mimic the pathological states.", "A correlation between the pathology model and the modified expression of a Drosophila or C. elegans gene can identify the association of the human ortholog with the human disease.", "In one example, a genetic screen is performed in an invertebrate model organism displaying a mutant (generally visible or selectable) phenotype due to mis-expression—generally reduced, enhanced or ectopic expression—of a known gene (the “genetic entry point”).", "Additional genes are mutated in a random or targeted manner.", "When an additional gene mutation changes the original mutant phenotype, this gene is identified as a “modifier” that directly or indirectly interacts with the genetic entry point and its associated pathway.", "If the genetic entry point is an ortholog of a human gene associated with a human pathology, such as lipid metabolic disorders, the screen can identify modifier genes that are candidate targets for novel therapeutics.", "Genetic screens may utilize RNA interference (RNAi) techniques, whereby introduction of exogenous double stranded (ds) RNA disrupts the activity of genes containing homologous sequences and induce specific loss-of-function phenotypes (Fire et al., 1998, Nature 391:806-811).", "Suitable methods for introduction of dsRNA include injection, feeding, and bathing (Tabara et al, 1998, Science 282:430-431).", "Genetic screens may also utilize mis-expression techniques to identify genes that, when over- or misexpressed in a pattern of interest, give a specific phenotype or modulate an existing mutant phenotype.", "An exemplary mis-expression screen is the Drosophila “EP” screen, which uses Drosophila lines carrying essentially random insertion of a P-element containing gene regulatory sequences oriented to act on flanking genomic sequences (Rorth P, 1996, Proc Natl Acad Sci U S A 93:12418-22; Rorth P et al., Development (1998) 125:1049-1057; WO0015843).", "The insulin receptor (INR) signaling pathway has been extensively studied in C. elegans and Drosophila.", "For instance, it has been found that signaling through daf-2, the C. elegans INR ortholog, mediates various events, including reproductive growth and normal adult life span (see, e.g., U.S. Pat.", "No.", "6,225,120; Tissenbaum H A and Ruvkun G, 1998, Genetics 148:703-17; Ogg S and Ruvkun G, 1998, Mol Cell 2:887-93; Lin K et al, 2001, Nat Genet 28:139-45).", "Modifiers of INR signaling were identified in genetic screens using Drosophila or C. elegans." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides a method of identifying candidate INR signaling modulating agents using an assay system comprising an ISM polypeptide or nucleic acid; contacting the assay system with a test agent under conditions whereby, but for the presence of the test agent, the system provides a reference activity; and detecting a test agent-biased activity of the assay system, wherein a difference between the test agent-biased activity and the reference activity identifies the test agent as a candidate INR signaling modulating agent.", "Candidate test agents include small molecule modulators, antibodies, and nucleic acid modulators such as antisense oligomers and PMOs, among others.", "In one embodiment of the invention, the assay system comprises cultured cells or a non-human animal expressing ISM, and the assay system detects an agent-biased change in INR signaling.", "In certain embodiments, candidate INR signaling modulating agents are identified in cell-free or cell-based assays, and a second assay system that detects an agent-biased change in an activity associated with INR signaling is used to confirm the INR signaling modulating activity of the candidate agent.", "In a preferred embodiment, the second assay detects an agent-biased change in an activity associated with INR signaling.", "Preferred second assay systems are carried out in cultured cells.", "The invention further provides a method of modulating INR signaling in a mammalian cell comprising contacting the cell with an agent that specifically binds an ISM polypeptide or nucleic acid.", "In a preferred embodiment, the agent is administered to a mammalian animal predetermined to have a pathology associated with INR signaling.", "Preferred agents include small molecule modulators, nucleic acid modulators, or antibodies.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "This application claims priority to U.S.", "Provisional Application Nos.", ": 60/261,335, 60/261,532, 60/261,590, 60/261,361, 60/261,531, 60/261,457, 60/261,694, 60/261,226, 60/261,304, 60/261,459, 60/261,456, 60/261,589, 60/261,461, 60/261,697, 60/261,458, 60/261,695, 60/261,336, 60/261,518, and 60/261,303, filed on Jan. 12, 2001, which are incorporated by reference in their entireties.", "BACKGROUND OF THE INVENTION Insulin is the central hormone governing metabolism in vertebrates (reviewed in Steiner et al., 1989, In Endocrinology, DeGroot, eds.", "Philadelphia, Saunders: 1263-1289).", "In humans, insulin is secreted by the beta cells of the pancreas in response to elevated blood glucose levels, which normally occur following a meal.", "The immediate effect of insulin secretion is to induce the uptake of glucose by muscle, adipose tissue, and the liver.", "A longer-term effect of insulin is to increase the activity of enzymes that synthesize glycogen in the liver and triglycerides in adipose tissue.", "Insulin can exert other actions beyond these “classic” metabolic activities, including increasing potassium transport in muscle, promoting cellular differentiation of adipocytes, increasing renal retention of sodium, and promoting production of androgens by the ovary.", "Defects in the secretion and/or response to insulin are responsible for the disease diabetes mellitus, which is of enormous economic significance.", "Within the United States, diabetes mellitus is the fourth most common reason for physician visits by patients; it is the leading cause of end-stage renal disease, non-traumatic limb amputations, and blindness in individuals of working age (Warram et al., 1995, In Joslin's Diabetes Mellitus, Kahn and Weir, eds., Philadelphia, Lea & Febiger, pp.", "201-215; Kahn et al., 1996, Annu.", "Rev.", "Med.", "47:509-531; Kahn, 1998, Cell 92:593-596).", "Beyond its role in diabetes mellitus, the phenomenon of insulin resistance has been linked to other pathogenic disorders including obesity, ovarian hyperandrogenism, and hypertension.", "Within the pharmaceutical industry, there is interest in understanding the molecular mechanisms that connect lipid defects and insulin resistance.", "Hyperlipidemia and elevation of free fatty acid levels correlate with “Metabolic Syndrome,” defined as the linkage between several diseases, including obesity and insulin resistance, which often occur in the same patients and which are major risk factors for development of Type 2 diabetes and cardiovascular disease.", "Current research suggests that the control of lipid levels, in addition to glucose levels, may be required to treat Type 2 Diabetes, heart disease, and other manifestations of Metabolic Syndrome (Santomauro A T et al., Diabetes (1999) 48:1836-1841).", "The ability to manipulate and screen the genomes of model organisms such as Drosophila and C. elegans provides a powerful means to analyze biochemical processes that due to significant evolutionary conservation of genes, pathways, and cellular processes, have direct relevance to more complex vertebrate organisms.", "Identification of novel functions of genes involved in particular pathways in such model organisms can directly contribute to the understanding of the correlative pathways in mammals and of methods of modulating them (see e.g., Miklos G L and Rubin G M, Cell 1996, 86:521-529).", "While Drosophila and C. elegans are not susceptible to human pathologies, various experimental models can mimic the pathological states.", "A correlation between the pathology model and the modified expression of a Drosophila or C. elegans gene can identify the association of the human ortholog with the human disease.", "In one example, a genetic screen is performed in an invertebrate model organism displaying a mutant (generally visible or selectable) phenotype due to mis-expression—generally reduced, enhanced or ectopic expression—of a known gene (the “genetic entry point”).", "Additional genes are mutated in a random or targeted manner.", "When an additional gene mutation changes the original mutant phenotype, this gene is identified as a “modifier” that directly or indirectly interacts with the genetic entry point and its associated pathway.", "If the genetic entry point is an ortholog of a human gene associated with a human pathology, such as lipid metabolic disorders, the screen can identify modifier genes that are candidate targets for novel therapeutics.", "Genetic screens may utilize RNA interference (RNAi) techniques, whereby introduction of exogenous double stranded (ds) RNA disrupts the activity of genes containing homologous sequences and induce specific loss-of-function phenotypes (Fire et al., 1998, Nature 391:806-811).", "Suitable methods for introduction of dsRNA include injection, feeding, and bathing (Tabara et al, 1998, Science 282:430-431).", "Genetic screens may also utilize mis-expression techniques to identify genes that, when over- or misexpressed in a pattern of interest, give a specific phenotype or modulate an existing mutant phenotype.", "An exemplary mis-expression screen is the Drosophila “EP” screen, which uses Drosophila lines carrying essentially random insertion of a P-element containing gene regulatory sequences oriented to act on flanking genomic sequences (Rorth P, 1996, Proc Natl Acad Sci U S A 93:12418-22; Rorth P et al., Development (1998) 125:1049-1057; WO0015843).", "The insulin receptor (INR) signaling pathway has been extensively studied in C. elegans and Drosophila.", "For instance, it has been found that signaling through daf-2, the C. elegans INR ortholog, mediates various events, including reproductive growth and normal adult life span (see, e.g., U.S. Pat.", "No.", "6,225,120; Tissenbaum H A and Ruvkun G, 1998, Genetics 148:703-17; Ogg S and Ruvkun G, 1998, Mol Cell 2:887-93; Lin K et al, 2001, Nat Genet 28:139-45).", "Modifiers of INR signaling were identified in genetic screens using Drosophila or C. elegans.", "SUMMARY OF THE INVENTION The invention provides a method of identifying candidate INR signaling modulating agents using an assay system comprising an ISM polypeptide or nucleic acid; contacting the assay system with a test agent under conditions whereby, but for the presence of the test agent, the system provides a reference activity; and detecting a test agent-biased activity of the assay system, wherein a difference between the test agent-biased activity and the reference activity identifies the test agent as a candidate INR signaling modulating agent.", "Candidate test agents include small molecule modulators, antibodies, and nucleic acid modulators such as antisense oligomers and PMOs, among others.", "In one embodiment of the invention, the assay system comprises cultured cells or a non-human animal expressing ISM, and the assay system detects an agent-biased change in INR signaling.", "In certain embodiments, candidate INR signaling modulating agents are identified in cell-free or cell-based assays, and a second assay system that detects an agent-biased change in an activity associated with INR signaling is used to confirm the INR signaling modulating activity of the candidate agent.", "In a preferred embodiment, the second assay detects an agent-biased change in an activity associated with INR signaling.", "Preferred second assay systems are carried out in cultured cells.", "The invention further provides a method of modulating INR signaling in a mammalian cell comprising contacting the cell with an agent that specifically binds an ISM polypeptide or nucleic acid.", "In a preferred embodiment, the agent is administered to a mammalian animal predetermined to have a pathology associated with INR signaling.", "Preferred agents include small molecule modulators, nucleic acid modulators, or antibodies.", "DETAILED DESCRIPTION OF THE INVENTION We identified novel invertebrate modifiers of insulin receptor (INR) signaling and their human orthologs.", "The human orthologs of these genes (nucleic acids and polypeptides) are collectively referred to as ISMs (Insulin receptor Signaling Modifiers).", "A reverse genetics screen was performed using a C. elegans model for defective INR function, with the goal of identifying genes that when inactivated result in restoration of INR signaling.", "The C. elegans INR signaling pathway negatively regulates entry into a dauer state, an alternate developmental fate that normally occurs only in adverse conditions (Riddle D L and P S Albert.", "1997.In C. elegans II, D L Riddle, T. Blumenthal, B J Meyer, and J R Priess, eds., p. 739-768).", "When daf-2, the C. elegans insulin receptor, is inactivated by mutation, animals enter the dauer state regardless of the environmental conditions (Kimura K D, et al,.", "1997, Science 277:942).", "We used an RNAi-based screen to identify modifiers (suppressors) of the dauer-formation phenotype of daf-2 loss-of-function mutations.", "The screen used two worm strains, each containing a missense mutation in the ligand-binding domain of daf-2, and involved RNAi treatment of these strains with dsRNA derived from cDNA or exon-rich genomic fragments of worm genes in order to cause reduction-of-function of these genes.", "Potential suppressors were identified as those genes that, when knocked down by RNAi treatment, allowed growth of the insulin-receptor mutant strains rather than larval arrest.", "Standard sequence analysis was used to determine the human (or mouse) orthologs of these genes, which are candidate targets for therapeutics designed to treat pathologies related to insulin signaling.", "Since many of the symptoms of diabetes are a result of decreased insulin signaling, candidate targets for antagonist therapies are those genes that, when mutated, restore INR signaling.", "The C. elegans screen identified the genes whose amino acid sequences are provided in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22.An overexpression screen was performed in Drosophila.", "Signaling through the Drosophila insulin receptor, designated Dinr (GI 1362614), has been implicated in controlling cell size and cell number (Fernandez R, et al, 1995, EMBO J 14:3373-3384; Brogiolo W et al., Stocker H and Hafen E, 2000, Curr Opin Genet Dev 10:529-35; Chen C et al, 1996, Endocrinology 137:846-56).", "Mis-expression of a dominant negative form of Dinr in the Drosophila eye results in small eye phenotype.", "The overexpression screen used a collection of Drosophila lines carrying insertions of a P-element vector (“EP element”) comprising a GAL4-regulated promoter oriented to transcribe flanking genomic sequences (Rorth P, 1996, Proc Natl Acad Sci U S A 93:12418-22; Rorth P et al., 1998, Development 125:1049-57).", "Each EP line was crossed to lines expressing the dominant negative Dinr, as well as GAL4, in the eye.", "Resulting progeny were examined for a change in the small eye phenotype.", "Sequence information surrounding the P insertion site was used to identify the overexpressed genes.", "The Drosophila screen identified the genes whose amino acid sequences are provided in SEQ ID NOS: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76 The invertebrate screens identified novel associations of ISM proteins and INR signaling.", "To applicants' knowledge, associations between diabetes (i.e., type II diabetes), glucose metabolism and/or INR signaling and the ISM proteins presented in SEQ ID NOs: 18, 30, 32, 34, 36, 38, 40, 50, 52, 54, 56, 58, 64, 66, 68, 70, 72, 74 and 76 have not previously been disclosed.", "For other ISM proteins, including those presented in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 42, 44, 46, 48, 60, and 62, there have been previous reports correlating activity of these proteins to diabetes, glucose metabolism, or another output of INR signaling.", "However, to applicants' knowledge, the methods described herein present the first evidence that these proteins may directly influence and/or interact with INR signaling to regulate the metabolic effects of insulin.", "ISM genes (i.e., nucleic acids and polypeptides) are attractive drug targets for the treatment of disorders related to INR signaling.", "In a preferred example, treatment involves increasing signaling through INR in order to treat pathologies related to diabetes and/or metabolic syndrome.", "The invention provides in vitro and in vivo methods of assessing ISM function, and methods of modulating (generally inhibiting or agonizing) ISM activity, which are useful for further elucidating INR signaling and for developing diagnostic and therapeutic modalities for pathologies associated with INR signaling.", "As used herein, pathologies associated with INR signaling encompass pathologies where INR signaling contributes to maintaining the healthy state, as well as pathologies whose course may be altered by modulation of the INR signaling.", "ISM Nucleic Acids and Polypeptides Human ISM nucleic acid (cDNA) sequences are provided in SEQ ID NOs: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75.Corresponding protein sequences are provided in SEQ ID NOs:.", "2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,-72, 74 and 76.ISM proteins of this invention include several categories of proteins with particular function domains.", "ISMs are identified as enzymes belonging to kinase (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 34, 36, 38, 40, 44, 46, 50, 60, and 62), protease (SEQ ID NOs: 30, 32, 74, and 76), desaturase (SEQ ID NO:48), and biosynthetic enzyme (SEQ ID NOs:42 and 52) classes.", "Other classes include mitochondrial transport proteins (SEQ ID NOs:54, 56 and 58), specific binding proteins (SEQ ID NOs:64 and 66), and transcriptional proteins (SEQ ID NOs: 68, 70, 72).", "The term “ISM polypeptide” refers to a full-length ISM protein or a fragment or derivative thereof that is “functionally active,” meaning that the ISM protein derivative or fragment exhibits one or more functional activities associated with a full-length, wild-type ISM protein.", "As one example, a fragment or derivative may have antigenicity such that it can be used in immunoassays, for immunization, for generation of inhibitory antibodies, etc, as discussed further below.", "Preferably, a functionally active ISM fragment or derivative displays one or more biological activities associated with ISM proteins such as enzymatic activity, signaling activity, ability to bind natural cellular substrates, etc.", "For those ISM proteins that are identified as enzymes, preferred ISM polypeptides display enzymatic (kinase, protease, desaturase, biosynthetic, etc.)", "activity.", "Other preferred ISM polypeptides display transport, binding, or transcriptional activity, according to the particular protein class.", "In one embodiment, a functionally active ISM polypeptide is an ISM derivative capable of rescuing defective endogenous ISM activity, such as in cell based or animal assays; the rescuing derivative may be from the same or a different species.", "If ISM fragments are used in assays to identify modulating agents, the fragments preferably comprise a ISM domain, such as a C- or N-terminal or catalytic domain, among others, and preferably comprise at least 10, preferably at least 20, more preferably at least 25, and most preferably at least 50 contiguous amino acids of a ISM protein.", "A preferred ISM fragment comprises a catalytic domain, such as a kinase domain, for those ISMs identified as kinases.", "Other preferred ISM fragments comprise specific binding domains.", "Functional domains can be identified using the PFAM program (Bateman A et al., 1999 Nucleic Acids Res 27:260-262; website at pfam.wustl.edu).", "The term “ISM nucleic acid” refers to a DNA or RNA molecule that encodes a ISM polypeptide.", "Preferably, the ISM polypeptide or nucleic acid or fragment thereof is from a human, but it can be an ortholog or derivative thereof with at least 70%, preferably with at least 80%, preferably 85%, still more preferably 90%, and most preferably at least 95% sequence identity with a human ISM.", "As used herein, “percent (%) sequence identity” with respect to a specified subject sequence, or a specified portion thereof, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0a19 (Altschul et al., J. Mol.", "Biol.", "(1997) 215:403-410; http://blast.wustl.edu/blast/README.html) with search parameters set to default values.", "The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched.", "A “% identity value” is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported.", "“Percent (%) amino acid sequence similarity” is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation.", "A conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected.", "Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.", "Alternatively, an alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman (Smith and Waterman, 1981, Advances in Applied Mathematics 2:482-489; database: European Bioinformatics Institute wwwz.ebi.ac.uk/bic.sub.—sw/; Smith and Waterman, 1981, J. of Molec.", "Biol., 147:195-197; Nicholas et al., 1998, “A Tutorial on Searching Sequence Databases and Sequence Scoring Methods” (www.psc.edu) and references cited therein.", "; W. R. Pearson, 1991, Genomics 11:635-650).", "This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.", "3:353-358, National Biomedical Research Foundation, Wash., D.C., USA), and normalized by Gribskov (Gribskov 1986 Nucl.", "Acids Res.", "14(6):6745-6763).", "The Smith-Waterman algorithm is used to search databases for sequences similar to a query sequence.", "Smith-Waterman uses dynamic programming to determine how an optimal alignment between the query sequence and a database sequence can be produced.", "This alignment is obtained by determining what transformations the query sequence would need to undergo to match the database sequence.", "Transformations include substituting one character for another and inserting or deleting a string of characters.", "A score is assigned for each character-to-character comparison—positive scores for exact matches and some substitutions, negative scores for other substitutions and insertions/deletions.", "The first character in an insertion or deletion gap is scored with a gap open penalty and subsequent characters are scored with a gap extension penalty.", "Scores are obtained from statistically-derived scoring matrices.", "The combination of transformations that results in the highest score is used to generate an alignment between the query sequence and database sequence.", "Smith-Waterman algorithm may be employed where default parameters are used for scoring (for example, gap open penalty of 12, gap extension penalty of two).", "From the data generated the “Match” value reflects “sequence identity.” Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to an ISM nucleic acid sequence.", "The stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing.", "Conditions routinely used are set out in readily available procedure texts (e.g., Current Protocol in Molecular Biology, Vol.", "1, Chap.", "2.10, John Wiley & Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)).", "In some embodiments, a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the an ISM nucleotide under stringent hybridization conditions that comprise: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C. in a solution comprising 6× single strength citrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5× Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C. in a solution containing 6×SSC, 1× Denhardt's solution, 100 μg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C. for 1 h in a solution containing 0.2×SSC and 0.1% SDS (sodium dodecyl sulfate).", "In other embodiments, moderately stringent hybridization conditions are used that comprise: pretreatment of filters containing nucleic acid for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C. in a solution containing 2×SSC and 0.1% SDS.", "Alternatively, low stringency conditions can be used that comprise: incubation for 8 hours to overnight at 37° C. in a solution comprising 20% formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1×SSC at about 37° C. for 1 hour.", "In some embodiments, the ISM is an ortholog of human ISM.", "Methods of identifying the human orthologs of these genes are known in the art.", "Normally, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3-dimensional structures.", "Orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences.", "Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen M A et al., Genome Research (2000) 10:1204-1210).", "Programs for multiple sequence alignment, such as CLUSTAL (Thompson J D et al, 1994, Nucleic Acids Res 22:4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees.", "In a phylogenetic tree representing multiple homologous sequences from diverse species (e.g., retrieved through BLAST analysis), orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species.", "Structural threading or other analysis of protein folding (e.g., using software by ProCeryon, Biosciences, Salzburg, Austria) may also identify potential orthologs.", "In evolution, when a gene duplication event follows speciation, a single gene in one species, such as Drosophila, may correspond to multiple genes (paralogs) in another, such as human.", "As used herein, the term “orthologs” encompasses paralogs.", "Isolation, Production, Expression, and Mis-expression of ISM Nucleic Acids and Polypeptides ISM nucleic acids and polypeptides are useful for identifying and testing agents that modulate ISM function and for other applications related to the involvement of ISM in INR signaling.", "ISM nucleic acids may be obtained using any available method.", "For instance, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or by using polymerase chain reaction (PCR) are well known in the art.", "A wide variety of methods are available for obtaining ISM polypeptides.", "In general, the intended use for the polypeptide will dictate the particulars of expression, production, and purification methods.", "For instance, production of polypeptides for use in screening for modulating agents may require methods that preserve specific biological activities of these proteins, whereas production of polypeptides for antibody generation may require structural integrity of particular epitopes.", "Expression of polypeptides to be purified for screening or antibody production may require the addition of specific tags (i.e., generation of fusion proteins).", "Overexpression of a ISM polypeptide for cell-based assays used to assess ISM function, such as involvement in tubulogenesis, may require expression in eukaryotic cell lines capable of these cellular activities.", "Techniques for the expression, production, and purification of proteins are well known in the art; any suitable means therefor may be used (e.g., Higgins S J and Hames B D (eds.)", "Protein Expression: A Practical Approach, Oxford University Press Inc., New York 1999; Stanbury P F et al., Principles of Fermentation Technology, 2nd edition, Elsevier Science, New York, 1995; Doonan S (ed.)", "Protein Purification Protocols, Humana Press, New Jersey, 1996; Coligan J E et al, Current Protocols in Protein Science (eds.", "), 1999, John Wiley & Sons, New York; U.S. Pat.", "No.", "6,165,992).", "The nucleotide sequence encoding an ISM polypeptide can be inserted into any appropriate vector for expression of the inserted protein-coding sequence.", "The necessary transcriptional and translational signals, including promoter/enhancer element, can derive from the native ISM gene and/or its flanking regions or can be heterologous.", "A variety of host-vector expression systems may be utilized, such as mammalian cell systems infected with virus (e.g.", "vaccinia virus, adenovirus, etc.", "); insect cell systems infected with virus (e.g.", "baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, plasmid, or cosmid DNA.", "A host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used.", "The ISM polypeptide may be optionally expressed as a fusion or chimeric product, joined via a peptide bond to a heterologous protein sequence.", "In one application the heterologous sequence encodes a transcriptional reporter gene (e.g., GFP or other fluorescent proteins, luciferase, beta-galactosidase, etc.).", "A chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other in the proper coding frame using standard methods and expressing the chimeric product.", "A chimeric product may also be made by protein synthetic techniques, e.g.", "by use of a peptide synthesizer (Hunkapiller et al., Nature (1984) 310:105-111).", "An ISM polypeptide can be isolated and purified using standard methods (e.g.", "ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis).", "Alternatively, native ISM proteins can be purified from natural sources, by standard methods (e.g.", "immunoaffinity purification).", "Once a protein is obtained, it may be quantified and its activity measured by appropriate methods, such as immunoassay, bioassay, or other measurements of physical properties, such as crystallography.", "The methods of this invention may also use cells that have been engineered for altered expression (mis-expression) of ISM or other genes associated with INR signaling.", "As used herein, mis-expression encompasses ectopic expression, over-expression, under-expression, and non-expression (e.g.", "by gene knock-out or blocking expression that would otherwise normally occur).", "Genetically Modified Animals The methods of this invention may use non-human animals that have been genetically modified to alter expression of ISM and/or other genes known to be involved in INR signaling.", "Preferred genetically modified animals are mammals, particularly mice or rats.", "Preferred non-mammalian species include Zebrafish, C. elegans, and Drosophila.", "Preferably, the altered ISM or other gene expression results in a detectable phenotype, such as modified levels of INR signaling, modified levels of plasma glucose or insulin, or modified lipid profile as compared to control animals having normal expression of the altered gene.", "The genetically modified animals can be used to further elucidate INR signaling, in animal models of pathologies associated with INR signaling, and for in vivo testing of candidate therapeutic agents, as described below.", "Preferred genetically modified animals are transgenic, at least a portion of their cells harboring non-native nucleic acid that is present either as a stable genomic insertion or as an extra-chromosomal element, which is typically mosaic.", "Preferred transgenic animals have germ-line insertions that are stably transmitted to all cells of progeny animals.", "Non-native nucleic acid is introduced into host animals by any expedient method.", "Methods of making transgenic non-human animals are well-known in the art (for mice see Brinster et al., Proc.", "Nat.", "Acad.", "Sci.", "USA 1985, 82:4438-42; U.S. Pat.", "Nos.", "4,736,866, 4,870,009, 4,873,191, 6,127,598; Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); for homologous recombination see Capecchi, Science 1989, 244:1288-1292; Joyner et al., Nature 1989, 338:153-156; for particle bombardment see U.S. Pat.", "No., 4,945,050; for Drosophila see Rubin and Spradling, Science (1982) 218:348-53, U.S. Pat.", "No.", "4,670,388; for transgenic insects see Berghammer A. J. et al., Nature 1999, 402:370-371; for Zebrafish see Lin S. Methods Mol Biol.", "(2000); 136:375-3830; for fish, amphibians and birds see Houdebine and Chourrout, Experientia (1991) 47:897-905; for rats see Hammer et al., Cell (1990) 63:1099-1112; for embryonic stem (ES) cells see Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed., IRL Press (1987); for livestock see Pursel et al., Science (1989) 244:1281-1288; for nonhuman animal clones see Wilmut, I. et al.", "(1997) Nature 385:810-813, PCT Publication Nos.", "WO 97/07668 and WO 97/07669; for recombinase systems for regulated transgene expression see, Lakso et al., PNAS (1992) 89:6232-6236; U.S. Pat.", "No.", "4,959,317 [for cre.loxP] and O'Gorman et al.", "(1991) Science 251:1351-1355; U.S. Pat.", "No.", "5,654,182 [for FLP/FRT]).", "Homozygous or heterozygous alterations in the genomes of transgenic animals may result in mis-expression of native genes, including ectopic expression, over-expression (e.g.", "by multiple gene copies), under-expression, and non-expression (e.g.", "by gene knock-out or blocking expression that would otherwise normally occur).", "In one application, a “knock-out” animal is generated, typically using homologous recombination, in which an alteration in an endogenous gene causes a decrease in that gene's function, preferably such that gene expression is undetectable or insignificant.", "ISM Modulating Agents The invention provides methods to identify agents that interact with and/or modulate the function of ISM and/or INR signaling.", "Such agents are useful in a variety of diagnostic and therapeutic applications associated with INR signaling, as well as in further analysis of the ISM protein and its contribution to INR signaling.", "Accordingly, the invention also provides methods for modulating INR signaling comprising the step of specifically modulating ISM activity by administering an ISM-interacting or -modulating agent.", "In a preferred embodiment, ISM-modulating agents inhibit or enhance ISM activity or otherwise affect normal ISM function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity.", "In a further preferred embodiment, the candidate INR signaling- modulating agent specifically modulates the function of the ISM.", "The phrases “specific modulating agent”, “specifically modulates”, etc., are used herein to refer to modulating agents that directly bind to the ISM polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter the function of the ISM.", "The term also encompasses modulating agents that alter the interaction of ISM with a binding partner or substrate (e.g.", "by binding to a binding partner of an ISM, or to a protein/binding partner complex, and inhibiting function).", "Preferred ISM-modulating agents include small molecule chemical agents, ISM-interacting proteins, including antibodies and other biotherapeutics, and nucleic acid modulators, including antisense oligomers and RNA.", "The modulating agents may be formulated in pharmaceutical compositions, for example, as compositions that may comprise other active ingredients, as in combination therapy, and/or suitable carriers or excipients.", "Techniques for formulation and administration of the compounds may be found in “Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa., 19th edition.", "Small Molecule Modulators Chemical agents, referred to in the art as “small molecule” compounds are typically organic, non-peptide molecules, having a molecular weight less than 10,000, preferably less than 5,000, more preferably less than 1,000, and most preferably less than 500.This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries.", "Synthetic compounds may be rationally designed or identified based on known or inferred properties of the ISM protein or may be identified by screening compound libraries.", "Alternative appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for ISM-modulating activity.", "Methods for generating and obtaining compounds are well known in the art (Schreiber S L, Science (2000) 151: 1964-1969; Radmann J and Gunther J, Science (2000) 151:1947-1948).", "Small molecule modulators identified from screening assays, as described below, can be used as lead compounds from which candidate clinical compounds may be designed, optimized, and synthesized.", "Such clinical compounds may have utility in treating pathologies associated with INR signaling.", "The activity of candidate small molecule modulating agents may be improved several-fold through iterative secondary functional validation, as further described below, structure determination, and candidate modulator modification and testing.", "Additionally, candidate clinical compounds are generated with specific regard to clinical and pharmacological properties.", "For example, the reagents may be derivatized and re-screened using in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.", "Protein Modulators An ISM-interacting protein may be endogenous, i.e.", "one that normally interacts genetically or biochemically with an ISM, such as a member of the ISM pathway that modulates ISM expression, localization, and/or activity.", "ISM-modulators include dominant negative forms of ISM-interacting proteins and of ISM proteins themselves.", "Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous ISM-interacting (Finley, R. L. et al.", "(1996) in DNA Cloning-Expression Systems: A Practical Approach, eds.", "Glover D. & Hames B.", "D (Oxford University Press, Oxford, England), pp.", "169-203; Fashema S F et al., Gene (2000) 250:1-14; Drees BL Curr Opin Chem Biol (1999) 3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29; and U.S. Pat.", "No.", "5,928,868).", "Mass spectrometry offers alternative preferred methods for the elucidation of protein complexes (reviewed in, e.g., Pandley A and Mann M, Nature (2000) 405:837-846; Yates J R 3rd, Trends Genet (2000) 16:5-8).", "Specific Biotherapeutics An ISM-interacting protein may have biotherapeutic applications.", "Biotherapeutic agents formulated in pharmaceutically acceptable carriers and dosages may be used to activate or inhibit signal transduction pathways.", "This modulation may be accomplished by binding a ligand, thus inhibiting the activity of the pathway; or by binding a receptor, either to inhibit activation of, or to activate, the receptor.", "Alternatively, the biotherapeutic may itself be a ligand capable of activating or inhibiting a receptor.", "Biotherapeutic agents and methods of producing them are described in detail in U.S. Pat.", "No.", "6,146,628.Antibody Modulators In a preferred embodiment, the ISM-interacting protein is an antibody.", "In a further preferred embodiment, antibody agonists or antagonists are produced that have therapeutic utility.", "Antibodies that specifically bind ISM polypeptides can be generated using known methods.", "Preferably the antibody is specific to a mammalian ISM polypeptide, and more preferably, a human ISM.", "Antibodies may be polyclonal, monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′).sub.2 fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.", "Monoclonal antibodies with affinities of 108 M−1 preferably 109 M−1 to 1010 M−1, or stronger can be made by standard procedures as described (Harlow E and Lane D, 1988, Antibodies: A Laboratory Manual, CSH Laboratory Press; (Harlow E and Lane D, 1999 Using Antibodies: A Laboratory Manual, CSH Laboratory Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and U.S. Pat.", "Nos.", "4,381,292; 4,451,570; and 4,618,577).", "Antibodies may be generated against crude cell extracts of ISM or substantially purified fragments thereof.", "If ISM fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of an ISM protein.", "In a particular embodiment, ISM-specific antigens and/or immunogens are coupled to carrier proteins that stimulate the immune response.", "For example, the subject polypeptides are covalently coupled to the keyhole limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in Freund's complete adjuvant, which enhances the immune response.", "An appropriate immune system such as a laboratory rabbit or mouse is immunized according to conventional protocols.", "Chimeric antibodies specific to ISM polypeptides can be made that contain different portions from different animal species.", "For instance, a human immunoglobulin constant region may be linked to a variable region of a murine mAb, such that the antibody derives its biological activity from the human antibody, and its binding specificity from the murine fragment.", "Chimeric antibodies are produced by splicing together genes that encode the appropriate regions from each species (Morrison et al., Proc.", "Natl.", "Acad.", "Sci.", "(1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604-608; Takeda et al., Nature (1985) 31:452-454).", "Humanized antibodies, which are a form of chimeric antibodies, can be generated by grafting complementary-determining regions (CDRs) (Carlos, T. M., J. M. Harlan.", "1994.Blood 84:2068-2101) of mouse antibodies into a background of human framework regions and constant regions by recombinant DNA technology (Riechmann L M, et al., 1988 Nature 323: 323-327).", "Humanized antibodies contain ˜10% murine sequences and ˜90% human sequences, and thus further reduce or eliminate immunogenicity, while retaining the antibody specificities (Co MS, and Queen C. 1991 Nature 351: 501-501; Morrison S L. 1992 Ann.", "Rev.", "Immun.", "10:239-265).", "Humanized antibodies and methods of their production are well-known in the art (U.S. Pat.", "No.", "5,530,101; U.S. Pat.", "No.", "5,585,089; U.S. Pat.", "No.", "5,693,762, and U.S. Pat.", "No.", "6,180,370).", "ISM-specific single chain antibodies, which are recombinant, single chain polypeptides formed by linking the heavy and light chain fragments of the Fv regions via an amino acid bridge, can be produced (U.S. Pat.", "No.", "4,946,778; Bird, Science (1988) 242:423-426; Huston et al., Proc.", "Natl.", "Acad.", "Sci.", "USA (1988) 85:5879-5883; and Ward et al., Nature (1989) 334:544-546).", "Other suitable techniques for antibody production involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors (Huse et al., Science (1989) 246:1275-1281).", "As used herein, T-cell antigen receptors are included within the scope of antibody modulators (Harlow and Lane, 1988, supra).", "The polypeptides and antibodies of the present invention may be used with or without modification.", "Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance that provides for a detectable signal, or that is toxic to cells that express the targeted protein (Menard S, et al., Int J. Biol Markers (1989) 4:131-134).", "A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature.", "Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, fluorescent emitting lanthanide metals, chemiluminescent moieties, bioluminescent moieties, magnetic particles, and the like (U.S. Pat.", "Nos.", "3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241).", "Also, recombinant immunoglobulins may be produced (U.S. Pat.", "No.", "4,816,567).", "Antibodies to cytoplasmic proteins may be delivered and reach their targets by conjugation with membrane-penetrating toxin proteins (U.S. Pat.", "No.", "6,086,900).", "When used therapeutically in a patient, the antibodies of the subject invention are typically administered parenterally, when possible at the target site, or intravenously.", "The therapeutically effective dose and dosage regimen is determined by clinical studies.", "Typically, the amount of antibody administered is in the range of about 0.1 mg/kg-to about 10 mg/kg of patient weight.", "For parenteral administration, the antibodies are formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) in association with a pharmaceutically acceptable vehicle.", "Such vehicles are inherently nontoxic and non-therapeutic.", "Examples are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.", "Nonaqueous vehicles such as fixed oils, ethyl oleate, or liposome carriers may also be used.", "The vehicle may contain minor amounts of additives, such as buffers and preservatives, which enhance isotonicity and chemical stability or otherwise enhance therapeutic potential.", "The antibodies' concentrations in such vehicles are typically in the range of about 1 mg/ml-to about 10 mg/ml.", "Immunotherapeutic methods are further described in the literature (U.S. Pat.", "No.", "5,859,206; WO0073469).", "Nucleic Acid Modulators Other preferred ISM-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA), which generally inhibit ISM activity.", "Preferred antisense oligomers interfere with the function of ISM nucleic acids, such as DNA replication, transcription, ISM RNA translocation, translation of protein from the ISM RNA, RNA splicing, and any catalytic activity in which the ISM RNA participates.", "In one embodiment, the antisense oligomer is an oligonucleotide that is sufficiently complementary to an ISM mRNA to bind to and prevent translation from the ISM mRNA, preferably by binding to the 5′ untranslated region.", "ISM-specific antisense oligonucleotides preferably range from at least 6 to about 200 nucleotides.", "In some embodiments the oligonucleotide is preferably at least 10, 15, or 20 nucleotides in length.", "In other embodiments, the oligonucleotide is preferably less than 50, 40, or 30 nucleotides in length.", "The oligonucleotide can be DNA or RNA, a chimeric mixture of DNA and RNA, derivatives or modified versions thereof, single-stranded or double-stranded.", "The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone.", "The oligonucleotide may include other appending groups such as peptides, agents that facilitate transport across the cell membrane, hybridization-triggered cleavage agents, and intercalating agents.", "In another embodiment, the antisense oligomer is a phosphorothioate morpholino oligomer (PMO).", "PMOs are assembled from four different morpholino subunits, each of which containing one of four genetic bases (A, C, G, or T) linked to a six-membered morpholine ring.", "Polymers of these subunits are joined by non-ionic phosphodiamidate inter-subunit linkages.", "Methods of producing and using PMOs and other antisense oligonucleotides are well known in the art (e.g.", "see WO99/18193; Summerton J, and Weller D, Antisense Nucleic Acid Drug Dev 1997, 7:187-95; Probst J C, Methods 2000, 22:271-281; U.S. Pat.", "No.", "5,325,033; U.S. Pat.", "No.", "5,378,841).", "Antisense oligomers are commonly used as research reagents, diagnostics, and therapeutics.", "For example, antisense oligonucleotides, which are able to specifically inhibit gene expression, are often used to elucidate the function of particular genes (see, e.g., U.S. Pat.", "No.", "6,165,790).", "Antisense oligomers are also used, for example, to distinguish between functions of various members of a biological pathway.", "Antisense oligomers have been employed as therapeutic moieties in the treatment of disease states in animals and humans and have been demonstrated in numerous clinical trials to be safe and effective (Milligan J F et al, 1993, J Med Chem 36:1923-1937; Tonkinson J L et al., 1996, Cancer Invest 14:54-65).", "Accordingly, in one aspect of the invention, an ISM-specific antisense oligomer is used in an assay to further elucidate the function of ISM in INR signaling.", "Zebrafish is a particularly useful model for the study of INR signaling using antisense oligomers.", "For example, PMOs are used to selectively inactive one or more genes in vivo in the Zebrafish embryo.", "By injecting PMOs into Zebrafish at the 1-16 cell stage candidate targets emerging from the Drosophila screens are validated in this vertebrate model system.", "In another aspect of the invention, PMOs are used to screen the Zebrafish genome for identification of other therapeutic modulators of INR signaling.", "In a further aspect of the invention, an ISM-specific antisense oligomer is used as a therapeutic agent for treatment of metabolic pathologies.", "Alternative preferred ISM-modulating agents are double-stranded RNA species mediating RNA interference (RNAi).", "RNAi is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene.", "Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and mammals are known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A.", "Trends Genet.", "15, 358-363 (1999); Sharp, P. A. RNA interference 2001.Genes Dev.", "15, 485-490 (2001); Hammond, S. M. et al., Nature Rev Genet 2, 110-1119 (2001); Tuschl, T. Chem Biochem.", "2, 239-245 (2001); Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000); Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev.", "15, 188-200 (2001); WO0129058; WO9932619, and Elbashir S M, et al., 2001, Nature 411:494-498).", "Assay Systems The invention provides assay systems for identifying specific modulators of ISM activity.", "As used herein, an “assay system” encompasses all the components required for performing and analyzing results of an assay that detects and/or measures a particular event or events.", "In general, primary assays are used to identify or confirm a modulator's specific biochemical or molecular effect with respect to the ISM nucleic acid or protein.", "In general, secondary assays further assess the activity of an ISM-modulating agent identified by a primary assay and may confirm that the modulating agent affects ISM in a manner relevant to INR signaling.", "In some cases, ISM-modulators will be directly tested in a “secondary assay,” without having been identified or confirmed in a “primary assay.” In a preferred embodiment, the assay system comprises contacting a suitable assay system comprising an ISM polypeptide or nucleic acid with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity, which is based on the particular molecular event the assay system detects.", "The method further comprises detecting the same type of activity in the presence of a candidate agent (“the agent-biased activity of the system”).", "A difference between the agent-biased activity and the reference activity indicates that the candidate agent modulates ISM activity, and hence INR signaling.", "A difference, as used herein, is statistically significant.", "The assay systems generally include positive and/or negative controls, as are well known in the art.", "Primary Assays The type of modulator tested generally determines the type of primary assay.", "Primary Assays for Small Molecule Modulators For small molecule modulators, screening assays are used to identify candidate modulators.", "Screening assays may be cell-based or may use a cell-free system that recreates or retains the relevant biochemical reaction of the target protein (reviewed in Sittampalam G S et al., Curr Opin Chem Biol (1997) 1:384-91 and accompanying references).", "As used herein the term “cell-based” refers to assays using live cells, dead cells, or a particular cellular fraction, such as a membrane, endoplasmic reticulum, or mitochondrial fraction.", "The term “cell free” encompasses assays using substantially purified protein (either endogenous or recombinantly produced), partially purified cellular extracts, or crude cellular extracts.", "Screening assays may detect a variety of molecular events, including protein-DNA interactions, protein-protein interactions (e.g., receptor-ligand binding), transcriptional activity (e.g., using a reporter gene), enzymatic activity (e.g., via a property of the substrate), activity of second messengers, immunogenicty and changes in cellular morphology or other cellular characteristics.", "Appropriate screening assays may use a wide range of detection methods including fluorescent, radioactive, calorimetric, spectrophotometric, and amperometric methods, to provide a read-out for the particular molecular event detected.", "In a preferred embodiment, screening assays use fluorescence technologies, including fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer.", "These systems offer means to monitor protein-protein or DNA-protein interactions in which the intensity of the signal emitted from dye-labeled molecules depends upon their interactions with partner molecules (e.g., Selvin P R, Nat Struct Biol (2000) 7:730-4; Fernandes P B, Curr Opin Chem Biol (1998) 2:597-603; Hertzberg R P and Pope A J, Curr Opin Chem Biol (2000) 4:445-451).", "Suitable assay formats that may be adapted to screen for ISM modulators are known in the art.", "Examples of assays useful for the various ISM protein classes are provided below.", "Protein kinases, key signal transduction proteins that may be either membrane-associated or intracellular, catalyze the transfer of gamma phosphate from adenosine triphosphate (ATP) to a serine, threonine or tyrosine residue in a protein substrate.", "Radioassays, which monitor the transfer from [gamma-32P or -33P]ATP, are frequently used to assay kinase activity.", "Separation of the phospho-labeled product from the remaining radio-labeled ATP can be accomplished by various methods including SDS-polyacrylamide gel electrophoresis, filtration using glass fiber filters or other matrices which bind peptides or proteins, and adsorption/binding of peptide or protein substrates to solid-phase matrices allowing removal of remaining radiolabeled ATP by washing.", "In one example, a scintillation assay monitors the transfer of the gamma phosphate from [gamma-33P] ATP to a biotinylated peptide substrate.", "The substrate is captured on a streptavidin coated bead that transmits the signal (Beveridge M et al., J Biomol Screen (2000) 5:205-212).", "This assay uses the scintillation proximity assay (SPA), in which only radio-ligand bound to receptors tethered to the surface of an SPA bead are detected by the scintillant immobilized within it, allowing binding to be measured without separation of bound from free ligand.", "Other assays for protein kinase activity may use antibodies that specifically recognize phosphorylated substrates.", "For instance, the kinase receptor activation (KIRA) assay measures receptor tyrosine kinase activity by ligand stimulating the intact receptor in cultured cells, then capturing solubilized receptor with specific antibodies and quantifying phosphorylation via phosphotyrosine ELISA (Sadick M D, Dev Biol Stand (1999) 97:121-133).", "Another example of antibody based assays for protein kinase activity is TRF (time-resolved fluorometry).", "This method utilizes europium chelate-labeled anti-phosphotyrosine antibodies to detect phosphate transfer to a polymeric substrate coated onto microtiter plate wells.", "The amount of phosphorylation is then detected using time-resolved, dissociation-enhanced fluorescence (Braunwalder A F, et al., Anal Biochem 1996 July 1;238(2):159-64).", "Generic assays may be established for protein kinases that rely upon the phosphorylation of substrates such as myelein basic protein, casein, histone, or synthetic peptides such as polyGlutamate/Tyrosine and radiolabeled ATP.", "Phosphoinositide kinases catalyze the phosphorylation of phosphatidylinositol substrates.", "Assays for lipid kinase activity may use labeled, such as radio-labeled substrates to detect transfer of a phosphate to lipid substrates.", "In one example, assays may use chromatography techniques to detect phosphorylation (Sbrissa D et al.", ", 1999, J Biol Chem 274:21589-21597).", "In another example, an assay uses “FlashPlate” technology (U.S. Pat.", "No.", "5,972,595), in which the hydrophobic substrate is immobilized on a solid support in each well of a multi-well plate.", "Phosphorylation of the substrate with a radio-labeled phosphate is measured as an increase in bound radioactivity, which is detected by the close proximity of the scintillant.", "Fatty acid CoA ligases catalyze the elongation of fatty acid chains.", "Assays may detect elongation of fatty acid substrates, for instance using chromatographic (e.g., HPLC) analysis of labeled elongation products.", "Long chain fatty acids may be labeled with 14-C (Moon Y A et al., 2001, J Biol Chem 276:45358-66).", "Fatty acid desaturases catalyze the insertion of double bonds into saturated fatty acid molecules.", "In one application, radioassays for inhibitors of fatty acid desaturase activity use thin layer chromatography to detect conversion of fatty acid substrates (Obukowicz et al., Biochem Pharmacol (1998) 55:1045-1058).", "Mitochondrial adenine transporters mediate the passage of adenosine from the mitochondrial compartment to the cytoplasm.", "The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle.", "Mitochondrial transport assays may directly measure transported molecules (e.g., Ruck A et al., 1998, FEBS Lett 426:97-101) and may detect transport activity in intact mitochondria or reconstituted vesicles (e.g., Haucke V and Schatz G, 1997, EMBO J 16:4560-7; Genchi G et al., 1996 Plant Physiol 112:845-51).", "In one example, vesicles are reconstituted in the presence of the labeled substrate (Palmieri F et al., 1995, Methods Enzymol 260:349-69).", "Assays for mitochondrial transport may alternatively detect electrogenic or fluorogenic properties of the mitochondrial proteins (e.g., Burstovetsky N et al., 1996, Proc Natl Acad Sci USA 93:664-8; Streicher-Scott J, 1994, Arch Biochem Biophys 15:548-54; U.S. Pat.", "No.", "6,183,948).", "p-Hydroxybenzoate polyprenyl transferase (COQ2) is involved in the biosynthesis of ubiquinone, an essential component of the electron transfer system (Ashby M N et al., 1992, J Biol Chem 267:4128-4136).", "Assays for COQ2 activity may use chromatographic methods to detect ubiquinone biosynthesis (e.g., Uchida N et al., 2000 J Bacteriol 182:6933-6939).", "Alternatively, assays may directly detect the COQ2 polyprenyltransferase activity (condensation of p-hydroxybenzoate and polyprenyl diphosphate).", "A variety of assays are available to detect the activity of proteins that have specific binding activity.", "Exemplary assays use fluorescence polarization, fluorescence polarization, and laser scanning techniques to measure binding of fluorescently labeled proteins, peptides, or other molecules (Lynch B A et al., 1999, Anal Biochem 275:62-73; Li H Y, 2001, J Cell Biochem 80:293-303; Zuck P et al., Proc Natl Acad Sci USA 1999, 96: 11122-11127).", "In another example, binding activity is detected using the scintillation proximity assay (SPA), which uses a biotinylated peptide probe captured on a streptavidin coated SPA bead and a radio-labeled partner molecule.", "The assay specifically detects the radio-labeled protein bound to the peptide probe via scintillant immobilized within the SPA bead (Sonatore L M et al., 1996, Anal Biochem 240:289-297).", "Proteins that are part of a transcriptional complex may be assayed for binding activity, as described above.", "Other suitable assays may measure transcriptional activity.", "In one example, transcriptional activity is detected using quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems).", "In another example, a transcriptional reporter (e.g., luciferase, GFP, beta-galactosidase, etc.)", "operably linked to a responsive gene regulatory sequence is used (e.g., Berg M et al, 2000, J Biomol Screen, 5:71-76).", "Preferred screening assays are high throughput or ultra high throughput and thus provide automated, cost-effective means of screening compound libraries for lead compounds (Fernandes P B, 1998, supra; Sundberg S A, Curr Opin Biotechnol 2000, 11:47-53).", "Cell-based screening assays usually require systems for recombinant expression of ISM and any auxiliary proteins demanded by the particular assay.", "Cell-free assays often use recombinantly produced purified or substantially purified proteins.", "Appropriate methods for generating recombinant proteins produce sufficient quantities of proteins that retain their relevant biological activities and are of sufficient purity to optimize activity and assure assay reproducibility.", "Yeast two-hybrid and variant screens, and mass spectrometry provide preferred methods for determining protein-protein interactions and elucidation of protein complexes.", "In certain applications when ISM-interacting proteins are used in screening assays, the binding specificity of the interacting protein to the ISM protein may be assayed by various known methods, including binding equilibrium constants (usually at least about 107 M−1, preferably at least about 108 M−1, more preferably at least about 109 M−1), and immunogenic properties.", "For enzymes and receptors, binding may be assayed by, respectively, substrate and ligand processing.", "The screening assay may measure a candidate agent's ability to specifically bind to or modulate activity of an ISM polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein.", "The ISM polypeptide can be full length or a fragment thereof that retains functional ISM activity.", "The ISM polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag.", "The ISM polypeptide is preferably human ISM, or is an ortholog or derivative thereof as described above.", "In a preferred embodiment, the screening assay detects candidate agent-based modulation of ISM interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has ISM-specific binding activity, and can be used to assess normal ISM gene function.", "Certain screening assays may also be used to test antibody and nucleic acid modulators; for nucleic acid modulators, appropriate assay systems involve ISM mRNA expression.", "Primary Assays for Antibody Modulators For antibody modulators, appropriate primary assays are binding assays that test the antibody's affinity to and specificity for the ISM protein.", "Methods for testing antibody affinity and specificity are well known in the art (Harlow and Lane, 1988, 1999, supra).", "The enzyme-linked immunosorbant assay (ELISA) is a preferred method for detecting ISM-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.", "Primary Assays for Nucleic Acid Modulators For nucleic acid modulators, primary assays may test the ability of the nucleic acid modulator to inhibit ISM gene expression, preferably mRNA expression.", "In general, expression analysis comprises comparing ISM expression in like populations of cells (e.g., two pools of cells that endogenously or recombinantly express ISM) in the presence and absence of the nucleic acid modulator.", "Methods for analyzing mRNA and protein expression are well known in the art.", "For instance, Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems), or microarray analysis may be used to confirm that ISM mRNA expression is reduced in cells treated with the nucleic acid modulator (e.g., Current Protocols in Molecular Biology (1994) Ausubel F M et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm D H and Guiseppi-Elie, A Curr Opin Biotechnol 2001, 12:41-47).", "Protein expression may also be monitored.", "Proteins are most commonly detected with specific antibodies or antisera directed against either the ISM protein or specific peptides.", "A variety of means including Western blotting, ELISA, or in situ detection, are available (Harlow E and Lane D, 1988 and 1999, supra).", "Secondary Assays Secondary assays may be used to further assess the activity of an ISM-modulating agent identified by any of the above methods to confirm that the modulating agent affects ISM in a manner relevant to INR signaling.", "As used herein, ISM-modulating agents encompass candidate clinical compounds or other agents derived from previously identified modulating agent.", "Secondary assays can also be used to test the activity of a modulator on a particular genetic or biochemical pathway or to test the specificity of the modulator's interaction with ISM.", "Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express ISM) in the presence and absence of the candidate modulator.", "In general, such assays test whether treatment of cells or animals with a candidate ISM-modulating agent results in changes in INR signaling, in comparison to untreated (or mock- or placebo-treated) cells or animals.", "Changes in INR signaling may be detected as modifications to INR pathway components, or changes in their expression or activity.", "Assays may also detect an output of normal or defective INR signaling, used herein to encompass immediate outputs, such as glucose uptake, or longer-term effects, such as changes in glycogen and triglycerides metabolism, adipocyte differentiation, or development of diabetes or other INR-related pathologies.", "Certain assays use sensitized genetic backgrounds, used herein to describe cells or animals engineered for altered expression of genes in the INR or interacting pathways, or pathways associated with INR signaling or an output of INR signaling.", "Cell-based assays Cell-based assays may use a variety of insulin-sensitive mammalian cells and may detect endogenous INR signaling or may rely on recombinant expression of INR and/or other INR pathway components.", "Exemplary insulin-sensitive cells include adipocytes, hepatocytes, and pancreatic beta cells.", "Suitable adipocytes include 3T3 L1 cells, which are most commonly used for insulin sensitivity assays, as well as primary cells from mice or human biopsy.", "Suitable hepatocytes include the rat hepatoma H4-II-E cell line.", "Suitable beta cells include rat INS-1 cells with optimized glucose-sensitive insulin secretion (such as clone 823-13, Hohmeier et al., 2000, Diabetes 49:424).", "Other suitable cells include muscle cells, such as L6 myotubes, and CHO cells engineered to over-express INR.", "For certain assay systems it may be useful to treat cells with factors such as glucosamine, free fatty acids or TNF alpha, which induce an insulin resistant state.", "Candidate modulators are typically added to the cell media but may also be injected into cells or delivered by any other efficacious means.", "Cell based assays generally test whether treatment of insulin responsive cells with the ISM—modulating agent alters INR signaling in response to insulin stimulation (“insulin sensitivity”); such assays are well-known in the art (see, e.g., Sweeney et al., 1999, J Biol Chem 274:10071).", "In a preferred embodiment, assays are performed to determine whether inhibition of ISM function increases insulin sensitivity.", "In one example, INR signaling is assessed by measuring expression of insulin-responsive genes.", "Hepatocytes are preferred for these assays.", "Many insulin responsive genes are known (e.g., p85 P13 kinase, hexokinase II, glycogen synthetase, lipoprotein lipase, etc; PEPCK is specifically down-regulated in response to INR signaling).", "Any available means for expression analysis, as previously described, may be used.", "Typically, mRNA expression is detected.", "In a preferred application, Taqman analysis is used to directly measure mRNA expression.", "Alternatively, expression is indirectly monitored from a transgenic reporter construct comprising sequences encoding a reporter gene (such as luciferase, GFP or other fluorescent proteins, beta-galactosidase, etc.)", "under control of regulatory sequences (e.g., enhancer/promoter regions) of an insulin responsive gene.", "Methods for making and using reporter constructs are well known.", "INR signaling may also be detected by measuring the activity of components of the INR-signaling pathway, which are well-known in the art (see, e.g., Kahn and Weir, Eds., Joslin's Diabetes Mellitus, Williams & Wilkins, Baltimore, Md., 1994).", "Suitable assays may detect phosphorylation of pathway members, including IRS, PI3K, Akt, GSK3 etc., for instance, using an antibody that specifically recognizes a phosphorylated protein.", "Assays may also detect a change in the specific signaling activity of pathway components (e.g., kinase activity of PI3K, GSK3, Akt, etc.).", "Kinase assays, as well as methods for detecting phosphorylated protein substrates, are well known in the art (see, e.g., Ueki K et al, 2000, Mol Cell Biol;20:8035-46).", "In another example, assays measure glycogen synthesis in response to insulin stimulation, preferably using hepatocytes.", "Glycogen synthesis may be assayed by various means, including measurement of glycogen content, and determination of glycogen synthase activity using labeled, such as radio-labeled, glucose (see, e.g., Aiston S and Agius L, 1999, Diabetes 48:15-20; Rother K I et al., 1998, J Biol Chem 273:17491-7).", "Other suitable assays measure cellular uptake of glucose (typically labeled glucose) in response to insulin stimulation.", "Adipocytes are preferred for these assays.", "Assays also measure translocation of glucose transporter (GLUT) 4, which is a primary mediator of insulin-induced glucose uptake, primarily in muscle and adipocytes, and which specifically translocates to the cell surface following insulin stimulation.", "Such assays may detect endogenous GLUT4 translocation using GLUT4-specific antibodies or may detect exogenously introduced, epitope-tagged GLUT4 using an antibody specific to the particular epitope (see, e.g., Sweeney, 1999, supra; Quon M J et al., 1994, Proc Natl Acad Sci U S A 91:5587-91).", "Other preferred assays detect insulin secretion from beta cells in response to glucose.", "Such assays typically use ELISA (see, e.g., Bergsten and Hellman, 1993, Diabetes 42:670-4) or radioimmunoassay (RIA; see, e.g., Hohmeier et al., 2000, supra).", "Animal Assays A variety of non-human animal models of metabolic disorders may be used to test candidate ISM modulators.", "Such models typically use genetically modified animals that have been engineered to mis-express (e.g., over-express or lack expression in) genes involved in lipid metabolism, adipogenesis, and/or the INR signaling pathway.", "Additionally, particular feeding conditions, and/or administration or certain biologically active compounds, may contribute to or create animal models of lipid and/or metabolic disorders.", "Assays generally required systemic delivery of the candidate modulators, such as by oral administration, injection (intravenous, subcutaneous, intraperitoneous), bolus administration, etc.", "In one embodiment, assays use mouse models of diabetes and/or insulin resistance.", "Mice carrying knockouts of genes in the leptin pathway, such as ob (leptin) or db (leptin receptor), or the INR signaling pathway, such as INR or the insulin receptor substrate (IRS), develop symptoms of diabetes, and show hepatic lipid accumulation (fatty liver) and, frequently, increased plasma lipid levels (Nishina et al., 1994, Metabolism 43:549-553; Michael et al., 2000, Mol Cell 6:87-97; Bruning J C et al., 1998, Mol Cell 2:559-569).", "Certain susceptible wild type mice, such as C57BU6, exhibit similar symptoms when fed a high fat diet (Linton and Fazio, 2001, Current Opinion in Lipidology 12:489-495).", "Accordingly, appropriate assays using these models test whether administration of a candidate modulator alters, preferably decreases lipid accumulation in the liver.", "Lipid levels in plasma and adipose tissue may also be tested.", "Methods for assaying lipid content, typically by FPLC or calorimetric assays (Shimano H et al., 1996, J Clin Invest 98:1575-1584; Hasty et al., 2001, J Biol Chem 276:37402-37408), and lipid synthesis, such as by scintillation measurement of incorporation of radio-labeled substrates (Horton J D et al., 1999, J Clin Invest 103:1067-1076), are well known in the art.", "Other useful assays test blood glucose levels, insulin levels, and insulin sensitivity (e.g., Michael M D, 2000, Molecular Cell 6: 87).", "Insulin sensitivity is routinely tested by a glucose tolerance test or an insulin tolerance test.", "In another embodiment, assays use mouse models of lipoprotein biology and cardiovascular disease.", "For instance, mouse knockouts of apolipoprotein E (apoE) display elevated plasma cholesterol and spontaneous arterial lesions (Zhang S H, 1992, Science 258:468-471).", "Transgenic mice over-expressing cholesterol ester transfer protein (CETP) also display increased plasma lipid levels (specifically, very-low-density lipoprotein [VLDL] and low-density lipoprotein [LDL] cholesterol levels) and plaque formation in arteries (Marotti K R et al., 1993, Nature 364:73-75).", "Assays using these models may test whether administration of candidate modulators alters plasma lipid levels, such as by decreasing levels of the pro-atherogenic LDL and VLDL, increasing HDL, or by decreasing overall lipid (including trigyceride) levels.", "Additionally histological analysis of arterial morphology and lesion formation (i.e., lesion number and size) may indicate whether a candidate modulator can reduce progression and/or severity of atherosclerosis.", "Numerous other mouse models for atherosclerosis are available, including knockouts of Apo-A1, PPARgamma, and scavenger receptor (SR)-B1 in LDLR- or ApoE-null background (reviewed in, e.g., Glass C K and Witztum J L, 2001, Cell 104:503-516).", "In another embodiment, the ability of candidate modulators to alter plasma lipid levels and artherosclerotic progression are tested in mouse models for multiple lipid disorders.", "For instance, mice with knockouts in both leptin and LDL receptor genes display hypercholesterolemia, hypertriglyceridemia and arterial lesions and provide a model for the relationship between impaired fuel metabolism, increased plasma remnant lipoproteins, diabetes, and atherosclerosis (Hasty AH et al, 2001, supra.).", "Diagnostic Methods The discovery that ISM is implicated in INR signaling provides for a variety of methods that can be employed for the diagnostic and prognostic evaluation of diseases and disorders associated with INR signaling and for the identification of subjects having a predisposition to such diseases and disorders.", "Any method for assessing ISM expression in a sample, as previously described, may be used.", "Such methods may, for example, utilize reagents such as the ISM oligonucleotides and antibodies directed against ISM, as described above for: (1) the detection of the presence of ISM gene mutations, or the detection of either over- or under-expression of ISM mRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of ISM gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in a biological pathway mediated by ISM.", "All references cited herein, including patents, patent applications, publications, and gene and sequence data accessible through the Genbank identifier numbers and websites provided, are incorporated in their entireties." ] ]	Patent_10466162
[ [ "Power converter control for automatic maximum power point tracking", "The invention concerns a method and a circuit for maximum power point tracking of a variable power source from a comparison of an image of the power (P) supplied by the power source, the circuit comprising two elements (14, 31) providing different propagating delays to a physical quantity proportional to the power image, a comparator (16) of the outputs of the delaying elements to control a trigger (17) supplying a signal (Q) with two automatic control states to a static power converter, and means (33) for detecting a transitory operating condition from variations in oscillations of an established operating condition and means (32) for modifying the delay input by the slower delaying element (31)." ], [ "1.A circuit for tracking the maximum power point of a variable power source (1) based on a comparison of an image of the power (P) provided by the power source, the circuit comprising: two elements (14, 31) introducing different propagation delays in a quantity proportional to the power image; a comparator (16) of the outputs of the delay elements to control a flip-flop (17) providing a two-state signal (Q) for controlling a static power converter; characterized in that it further comprises means (33) for detecting a transient state from variations of oscillations of a steady state; and means (32) for modifying the delay introduced by the slowest delay element (31).", "2.The circuit of claim 1, wherein said means (32) for modifying the delay are formed of a switching element (321) capable of, in transient state, inhibiting the operation of the slower delay element (31).", "3.The circuit of claim 1 or 2, wherein said detection means (33) compare the duration of an active state on each output signal (Q, {overscore (Q)}) of the flip-flop (17) with a predetermined threshold (TH1, TH2).", "4.The circuit of claim 3, wherein the detection means (33) compare, independently from each other, the forward (Q) and reverse ({overscore (Q)}) outputs of the flip-flop (17) and combine (36) the result of these comparisons to provide a control pulse (DEM) to the means (32) for making the delay variable.", "5.The circuit of any of claims 1 to 4, wherein the duration of the transient state is selected according to the desired oscillation amplitude around a nominal power reference value.", "6.The circuit of any of claims 1 to 5, wherein the different voltage, current, and time measurement elements are analog.", "7.The circuit of any of claims 1 to 6, comprising means for resetting the flip-flop (17) upon occurrence of a transient state.", "8.The circuit of any of claims 1 to 7, comprising means for, upon occurrence of a transient state, resetting a ramp generator (13′) conditioning the duty cycle of a pulse-width modulation control signal of the power converter.", "9.A method for controlling a circuit for tracking the maximum power point of a variable power source (1) of the type applying two delays of different value to an image of the power (P) provided by the power source, consisting of inhibiting or shortening the shortest delay during a transient state.", "10.The method of claim 9, consisting of determining the existence of a transient state from a measurement of the frequency of oscillations around a nominal operating point of the maximum power point detector." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to the field of power converters and, more specifically, power converters equipped with a maximum power point tracking control circuit.", "Such converters are generally applied to the conversion of power provided by an irregular source.", "In the context of the present invention, “irregular” power source means a power source providing a power likely to undergo abrupt variations, as opposed to power sources providing a stable or slowly-varying power, as is the case for a battery or for the A.C. supply network.", "Such sources are, for example, photovoltaic panels providing a power varying according to the lighting, wind engines providing a power varying according to the wind speed, elements of tidal power exploitation providing a power varying according to the wave intensity, etc.", "The present invention will be described hereafter in relation with photovoltaic element panels.", "However, the present invention more generally applies to different power sources for which an automated tracking of the maximum power point is needed to optimize the output in case of a power generation.", "2.Discussion of the Related Art A power converter of the type to which the present invention applies is of static converter type, with its component operating in switched mode (on/off).", "The input and output voltages may indifferently be D.C., A.C. voltages, or others (for example, pulse voltages).", "The converter can then be a D.C./D.C., D.C./A.C., A.C./D.C.", "converter, etc.", "A currently-used control technique for the switching of the converter semiconductor component(s) is the control by pulse width modulation (PWM) at the turning-off and at the turning on of a power transistor.", "The width of the pulses for controlling the turning-on of the power transistor is regulated according to the load and to the power required by said load.", "In the applications of the present invention, the pulse width is further regulated according to the power provided by the power source by tracking, for yield reasons, the maximum power point.", "FIG.", "1 very schematically shows in the form of blocks a conventional example of a power converter of the type to which the present invention applies.", "In this example, the converter is a voltage step-up D.C./D.C.", "converter.", "Assume a power source 1 formed of photovoltaic elements PV providing a voltage V which is applied across an inductive element L in series with a PWM controlled power switch 2 .", "In the example shown, power switch 2 is formed of a MOS transistor having a gate receiving a signal CTRL formed of a train of pulses of variable width according to the control orders.", "The junction 3 of inductive element L and switch 2 is connected to the anode of a free wheel diode D having a cathode connected to a first (positive) electrode 4 of a storage capacitor C. Capacitor C provides, between its electrodes 4 and 5 , a regulated voltage Vout or current Iout of D.C., A.C. or other type according to the nature of the load connected between electrodes 4 and 5 .", "Electrode 5 of capacitor C corresponds to a reference voltage, for example the ground, for voltage V of panel 1 , for power switch 2 , and for the output voltage.", "When switch 2 is on (for a MOS transistor, this corresponds to an operation in ohmic mode), diode D is reverse biased.", "Capacitor C supplies the load connected across terminals 4 and 5 .", "Power is accumulated in inductive element L across which is applied voltage V provided by the photovoltaic panel 1 .", "When transistor 2 is off, the power stored in inductance L is transferred to capacitor C by diode D. The operation of a pulse-width modulated power converter is well known and will not be detailed any further.", "Various types of power switch assemblies are known, according to whether the converter is a step-down, step-up, or step-up/step-down converter.", "When the voltage source providing voltage V is irregular, a maximum power point tracking control circuit (MPPT) 10 is generally used.", "Such a circuit has the function of modifying the width of the pulses for turning on switch 2 according to the variations of the power provided by power source 1 .", "At its input, circuit 10 thus receives a signal (for example, a voltage) proportional to power P provided by source 1 .", "In the example of FIG.", "1 , power P is obtained by means of a multiplier 7 of a current measurement I in the photovoltaic elements by a measurement of voltage V across panel 1 .", "Circuit 10 provides a two-state signal Q intended to increase, respectively, decrease, the width of the control pulses of switch 2 .", "Signal CTRL of control of switch 2 is provided by a comparator 11 (COMP) of the converter controlled by circuit 10 .", "This comparator receives, on a first input, a periodic signal provided by a generator 12 , for example, a sawtooth of constant high frequency.", "A second input of comparator 11 receives the output of a ramp generator 13 (RAMP) having its direction inversion (ascending ramp, descending ramp) conditioned by the state of signal Q.", "The frequency of the sawtooth conditions the frequency, generally constant, of the pulse train of signal CTRL.", "The instantaneous level provided by generator 13 , formed for example of an RC circuit, sets the comparison reference, and thus the pulse duty cycle.", "To generate signal Q, circuit 10 comprises two resistive and capacitive circuits 14 , 15 (RCF and RCS) forming delay lines of power signal P with different time constants.", "Circuit 14 is, for example, a high speed circuit as compared to circuit 15 , which has a longer time constant.", "The respective outputs of circuits 14 and 15 are connected to the inputs of a comparator 16 (COMP), the output of which controls a flip-flop 17 (T) providing signal Q. Hereafter, Q will indifferently be used to designate the forward (non inverted) output terminal of flip-flop 17 or the signal present on this terminal.", "Flip-flop 17 is a flip-flop with no clock signal.", "It is, for example, a JK-type flip-flop assembled as a so-called T-type flip-flop.", "The structure and operation of a circuit such as shown in FIG.", "1 is perfectly well known.", "An example of such a circuit is described in article “Step-Up Maximum Power Point Tracker for Photovoltaic Arrays” by Ziyad Salameh, published in the proceedings of the American Solar Energy Society Conference of Jun.", "20 to 24, 1988, pages 409-414.Its operation will briefly be reminded hereafter.", "The examination of the slow and fast variations of power P provides an image of the derivative of this power.", "Due to the time constant difference of RC circuits 14 and 15 , the output of comparator 16 oscillates.", "The frequency and amplitude of these oscillations depend on the time constants of the RC circuits.", "In fact, comparator 16 indicates, according to its output state (high or low) the sign of the derivative of the power.", "As long as the output of comparator 16 remains in a same state, the output of flip-flop 17 does not switch state.", "Assuming a state 1 at the input and at the output of flip-flop 17 , the resistive and capacitive circuit of ramp generator 13 builds up power.", "This increases the corresponding input level of comparator 11 and increases the duty cycle of signal CTRL.", "Assuming that the load receiving voltage Vout is constant, power P will increase to a maximum, then start decreasing along with the increase in voltage V. When the power starts decreasing, the output of comparator 16 switches, which causes a switching of output signal Q of flip-flop 17 .", "Said signal then switches low, which causes the discharge of the RC circuit of ramp generator 13 and a decrease in the duty cycle.", "The output voltage then starts increasing again.", "At constant load, the circuit converges towards a maximum power point and oscillates around this point.", "This operation is illustrated in FIG.", "2 , which shows two examples of the course of power P according to voltage V for two lighting quantities received by panel 1 .", "A first curve 21 illustrates, for example, the case of a maximum lighting.", "As just described, at constant load, the system will oscillate around maximum power point PMM 1 .", "If the lighting of panel 1 changes (for example, by the coming of shadow), characteristic P=f(V) of panel 1 becomes a curve 22 of lower level.", "This curve also exhibits a maximum power point PMM 2 .", "However, the control system shown in FIG.", "1 cannot make out a lighting change from an abrupt variation of the load connected at the converter output or from a mere variation around the maximum power point of curve P=f(V) on which stands its operating point.", "The control system is then lost and may even find itself in a steady state no longer corresponding to the maximum power point.", "In fact, the circuit diverges towards a minimum load or maximum load state according to the flip-flop state preceding the change of curve P=f (V).", "The same problem is posed in the case of an abrupt variation in the supplied load.", "A first solution consists of choosing very different time constants of delay elements 14 and 15 .", "However, this adversely affects the output because of the significant generated oscillations.", "Another known solution consists of forcing the system to start back from the origin of curves P=f (V).", "It is then started from a very small duty cycle, which is increased to converge back towards the maximum power point of the current lighting curve.", "A disadvantage of such a solution is that it considerably slows down the control by the lighting of the photovoltaic panel or by the abrupt variations of any power source connected upstream of the system.", "Further, the differentiation between a maximum power point change (curve change) and a normal variation also poses a problem in terms of detection duration and reliability.", "FIG.", "3 illustrates an example of characteristic of current I provided by the photovoltaic panel along time at a power curve change of the panel.", "It is assumed to initially be (times t0 to t1) on a maximum lighting curve ( 21 , FIG.", "2 ).", "Current I then slightly oscillates around a value Imax, assuming a constant load.", "A lighting change at time t1 causes a reference loss for the control system.", "In the example shown in FIG.", "3 , it is assumed that the system is then restarted at a time t2 subsequent to time t1 after having discovered the system reference loss.", "It is then converged until a time t3 towards a new maximum power point corresponding to a current Iomb around which the system then starts slightly oscillating.", "The amplitude of the oscillations around values Imax and Iomb of course depends on the time constants of RC circuits 14 and 15 .", "The greater the difference between time constants, the larger the amplitude of the oscillations at the output of comparator 16 .", "The faster it is converged towards the maximum power point (duration between times t2 and t3), the greater the oscillation amplitude.", "However, the greater the oscillations, the more adversely this affects the system output.", "A compromise must thus be made between output, speed, and stability.", "Problems of system convergence after maximum power point changes are posed especially in case of a variable power source.", "However, even if the input power source is stable a priori as should be the case, for example, for photovoltaic panels used in space (without clouds), such convergence problems may be encountered.", "Indeed, space infrastructures having more and more complex geometries, shadow areas due to the very structure of satellites may appear.", "Further, sensors may be partially damaged by dust impact, which leads to the same result.", "Another known solution to overcome the disadvantages linked to abrupt variations of the power source is to use a digital circuit.", "The different operating points are successively memorized to recognize a drift.", "A digital system however remains slow to isolate a drift from a normal operating variation.", "On this regard, the larger the amplitude of the accepted oscillations in steady state, the slower the system will be in recognizing a state change due to a change in the power source.", "Another disadvantage of a digital circuit is that it is in practice limited to frequencies of pulse trains for controlling switch 2 of some hundred kHz.", "On this regard, an analog control circuit such as that illustrated in FIG.", "1 has the advantage of being able to operate at higher cut-off frequencies (on the order of one MHz).", "This eases the converter integration." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention aims at overcoming the disadvantages of known circuits for tracking the maximum power point of a static converter of switched-mode power supply type.", "The present invention more specifically aims at optimizing the converter output without adversely affecting its response speed.", "The present invention also aims at enabling the control circuit to converge back towards a new maximum power point in case of a variation in the power source, due to a simple circuit of analog type.", "The present invention also aims at preserving the control performed by the circuit in case of a variation in the load connected at its output.", "The present invention further aims at providing an integrable solution compatible with a high-frequency operation of the switched-mode power supply.", "To achieve these objects, the present invention provides a circuit for tracking the maximum power point of a variable power source based on a comparison of an image of the power provided by the power source, the circuit comprising: two elements introducing different propagation delays in a quantity proportional to the power image; a comparator of the outputs of the delay elements to control a flip-flop providing a two-state signal for controlling a static power converter; means for detecting a transient state from variations of oscillations of a steady state; and means for modifying the delay introduced by the slowest delay element.", "According to an embodiment of the present invention, said means for modifying the delay are formed of a switching element capable of, in transient state, inhibiting the operation of the slower delay element.", "According to an embodiment of the present invention, said detection means compare the duration of an active state on each output signal of the flip-flop with a predetermined threshold.", "According to an embodiment of the present invention, the detection means compare, independently from each other, the forward and reverse outputs of the flip-flop and combine the result of these comparisons to provide a control pulse to the means for making the delay variable.", "According to an embodiment of the present invention, the duration of the transient state is selected according to the desired oscillation amplitude around a nominal power reference value.", "According to an embodiment of the present invention, the different voltage, current, and time measurement elements are analog.", "According to an embodiment of the present invention, the circuit comprises means for resetting the flip-flop upon occurrence of a transient state.", "According to an embodiment of the present invention, the circuit comprises means for, upon occurrence of a transient state, resetting a ramp generator conditioning the duty cycle of a pulse-width modulation control signal of the power converter.", "The present invention also provides a method for controlling a circuit for tracking the maximum power point of a variable power source of the type applying two delays of different value to an image of the power provided by the power source, which consists of inhibiting or shortening the shortest delay during a transient state.", "According to an embodiment of the present invention, the existence of a transient state is determined from a measurement of the frequency of oscillations around a nominal operating point of the maximum power point detector.", "The foregoing objects, features, and advantages of the present invention will be discussed in detail in the following non-limiting description of specific embodiments in connection with the accompanying drawings." ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to the field of power converters and, more specifically, power converters equipped with a maximum power point tracking control circuit.", "Such converters are generally applied to the conversion of power provided by an irregular source.", "In the context of the present invention, “irregular” power source means a power source providing a power likely to undergo abrupt variations, as opposed to power sources providing a stable or slowly-varying power, as is the case for a battery or for the A.C. supply network.", "Such sources are, for example, photovoltaic panels providing a power varying according to the lighting, wind engines providing a power varying according to the wind speed, elements of tidal power exploitation providing a power varying according to the wave intensity, etc.", "The present invention will be described hereafter in relation with photovoltaic element panels.", "However, the present invention more generally applies to different power sources for which an automated tracking of the maximum power point is needed to optimize the output in case of a power generation.", "2.Discussion of the Related Art A power converter of the type to which the present invention applies is of static converter type, with its component operating in switched mode (on/off).", "The input and output voltages may indifferently be D.C., A.C. voltages, or others (for example, pulse voltages).", "The converter can then be a D.C./D.C., D.C./A.C., A.C./D.C.", "converter, etc.", "A currently-used control technique for the switching of the converter semiconductor component(s) is the control by pulse width modulation (PWM) at the turning-off and at the turning on of a power transistor.", "The width of the pulses for controlling the turning-on of the power transistor is regulated according to the load and to the power required by said load.", "In the applications of the present invention, the pulse width is further regulated according to the power provided by the power source by tracking, for yield reasons, the maximum power point.", "FIG.", "1 very schematically shows in the form of blocks a conventional example of a power converter of the type to which the present invention applies.", "In this example, the converter is a voltage step-up D.C./D.C.", "converter.", "Assume a power source 1 formed of photovoltaic elements PV providing a voltage V which is applied across an inductive element L in series with a PWM controlled power switch 2.In the example shown, power switch 2 is formed of a MOS transistor having a gate receiving a signal CTRL formed of a train of pulses of variable width according to the control orders.", "The junction 3 of inductive element L and switch 2 is connected to the anode of a free wheel diode D having a cathode connected to a first (positive) electrode 4 of a storage capacitor C. Capacitor C provides, between its electrodes 4 and 5, a regulated voltage Vout or current Iout of D.C., A.C. or other type according to the nature of the load connected between electrodes 4 and 5.Electrode 5 of capacitor C corresponds to a reference voltage, for example the ground, for voltage V of panel 1, for power switch 2, and for the output voltage.", "When switch 2 is on (for a MOS transistor, this corresponds to an operation in ohmic mode), diode D is reverse biased.", "Capacitor C supplies the load connected across terminals 4 and 5.Power is accumulated in inductive element L across which is applied voltage V provided by the photovoltaic panel 1.When transistor 2 is off, the power stored in inductance L is transferred to capacitor C by diode D. The operation of a pulse-width modulated power converter is well known and will not be detailed any further.", "Various types of power switch assemblies are known, according to whether the converter is a step-down, step-up, or step-up/step-down converter.", "When the voltage source providing voltage V is irregular, a maximum power point tracking control circuit (MPPT) 10 is generally used.", "Such a circuit has the function of modifying the width of the pulses for turning on switch 2 according to the variations of the power provided by power source 1.At its input, circuit 10 thus receives a signal (for example, a voltage) proportional to power P provided by source 1.In the example of FIG.", "1, power P is obtained by means of a multiplier 7 of a current measurement I in the photovoltaic elements by a measurement of voltage V across panel 1.Circuit 10 provides a two-state signal Q intended to increase, respectively, decrease, the width of the control pulses of switch 2.Signal CTRL of control of switch 2 is provided by a comparator 11 (COMP) of the converter controlled by circuit 10.This comparator receives, on a first input, a periodic signal provided by a generator 12, for example, a sawtooth of constant high frequency.", "A second input of comparator 11 receives the output of a ramp generator 13 (RAMP) having its direction inversion (ascending ramp, descending ramp) conditioned by the state of signal Q.", "The frequency of the sawtooth conditions the frequency, generally constant, of the pulse train of signal CTRL.", "The instantaneous level provided by generator 13, formed for example of an RC circuit, sets the comparison reference, and thus the pulse duty cycle.", "To generate signal Q, circuit 10 comprises two resistive and capacitive circuits 14, 15 (RCF and RCS) forming delay lines of power signal P with different time constants.", "Circuit 14 is, for example, a high speed circuit as compared to circuit 15, which has a longer time constant.", "The respective outputs of circuits 14 and 15 are connected to the inputs of a comparator 16 (COMP), the output of which controls a flip-flop 17 (T) providing signal Q. Hereafter, Q will indifferently be used to designate the forward (non inverted) output terminal of flip-flop 17 or the signal present on this terminal.", "Flip-flop 17 is a flip-flop with no clock signal.", "It is, for example, a JK-type flip-flop assembled as a so-called T-type flip-flop.", "The structure and operation of a circuit such as shown in FIG.", "1 is perfectly well known.", "An example of such a circuit is described in article “Step-Up Maximum Power Point Tracker for Photovoltaic Arrays” by Ziyad Salameh, published in the proceedings of the American Solar Energy Society Conference of Jun.", "20 to 24, 1988, pages 409-414.Its operation will briefly be reminded hereafter.", "The examination of the slow and fast variations of power P provides an image of the derivative of this power.", "Due to the time constant difference of RC circuits 14 and 15, the output of comparator 16 oscillates.", "The frequency and amplitude of these oscillations depend on the time constants of the RC circuits.", "In fact, comparator 16 indicates, according to its output state (high or low) the sign of the derivative of the power.", "As long as the output of comparator 16 remains in a same state, the output of flip-flop 17 does not switch state.", "Assuming a state 1 at the input and at the output of flip-flop 17, the resistive and capacitive circuit of ramp generator 13 builds up power.", "This increases the corresponding input level of comparator 11 and increases the duty cycle of signal CTRL.", "Assuming that the load receiving voltage Vout is constant, power P will increase to a maximum, then start decreasing along with the increase in voltage V. When the power starts decreasing, the output of comparator 16 switches, which causes a switching of output signal Q of flip-flop 17.Said signal then switches low, which causes the discharge of the RC circuit of ramp generator 13 and a decrease in the duty cycle.", "The output voltage then starts increasing again.", "At constant load, the circuit converges towards a maximum power point and oscillates around this point.", "This operation is illustrated in FIG.", "2, which shows two examples of the course of power P according to voltage V for two lighting quantities received by panel 1.A first curve 21 illustrates, for example, the case of a maximum lighting.", "As just described, at constant load, the system will oscillate around maximum power point PMM1.If the lighting of panel 1 changes (for example, by the coming of shadow), characteristic P=f(V) of panel 1 becomes a curve 22 of lower level.", "This curve also exhibits a maximum power point PMM2.However, the control system shown in FIG.", "1 cannot make out a lighting change from an abrupt variation of the load connected at the converter output or from a mere variation around the maximum power point of curve P=f(V) on which stands its operating point.", "The control system is then lost and may even find itself in a steady state no longer corresponding to the maximum power point.", "In fact, the circuit diverges towards a minimum load or maximum load state according to the flip-flop state preceding the change of curve P=f (V).", "The same problem is posed in the case of an abrupt variation in the supplied load.", "A first solution consists of choosing very different time constants of delay elements 14 and 15.However, this adversely affects the output because of the significant generated oscillations.", "Another known solution consists of forcing the system to start back from the origin of curves P=f (V).", "It is then started from a very small duty cycle, which is increased to converge back towards the maximum power point of the current lighting curve.", "A disadvantage of such a solution is that it considerably slows down the control by the lighting of the photovoltaic panel or by the abrupt variations of any power source connected upstream of the system.", "Further, the differentiation between a maximum power point change (curve change) and a normal variation also poses a problem in terms of detection duration and reliability.", "FIG.", "3 illustrates an example of characteristic of current I provided by the photovoltaic panel along time at a power curve change of the panel.", "It is assumed to initially be (times t0 to t1) on a maximum lighting curve (21, FIG.", "2).", "Current I then slightly oscillates around a value Imax, assuming a constant load.", "A lighting change at time t1 causes a reference loss for the control system.", "In the example shown in FIG.", "3, it is assumed that the system is then restarted at a time t2 subsequent to time t1 after having discovered the system reference loss.", "It is then converged until a time t3 towards a new maximum power point corresponding to a current Iomb around which the system then starts slightly oscillating.", "The amplitude of the oscillations around values Imax and Iomb of course depends on the time constants of RC circuits 14 and 15.The greater the difference between time constants, the larger the amplitude of the oscillations at the output of comparator 16.The faster it is converged towards the maximum power point (duration between times t2 and t3), the greater the oscillation amplitude.", "However, the greater the oscillations, the more adversely this affects the system output.", "A compromise must thus be made between output, speed, and stability.", "Problems of system convergence after maximum power point changes are posed especially in case of a variable power source.", "However, even if the input power source is stable a priori as should be the case, for example, for photovoltaic panels used in space (without clouds), such convergence problems may be encountered.", "Indeed, space infrastructures having more and more complex geometries, shadow areas due to the very structure of satellites may appear.", "Further, sensors may be partially damaged by dust impact, which leads to the same result.", "Another known solution to overcome the disadvantages linked to abrupt variations of the power source is to use a digital circuit.", "The different operating points are successively memorized to recognize a drift.", "A digital system however remains slow to isolate a drift from a normal operating variation.", "On this regard, the larger the amplitude of the accepted oscillations in steady state, the slower the system will be in recognizing a state change due to a change in the power source.", "Another disadvantage of a digital circuit is that it is in practice limited to frequencies of pulse trains for controlling switch 2 of some hundred kHz.", "On this regard, an analog control circuit such as that illustrated in FIG.", "1 has the advantage of being able to operate at higher cut-off frequencies (on the order of one MHz).", "This eases the converter integration.", "SUMMARY OF THE INVENTION The present invention aims at overcoming the disadvantages of known circuits for tracking the maximum power point of a static converter of switched-mode power supply type.", "The present invention more specifically aims at optimizing the converter output without adversely affecting its response speed.", "The present invention also aims at enabling the control circuit to converge back towards a new maximum power point in case of a variation in the power source, due to a simple circuit of analog type.", "The present invention also aims at preserving the control performed by the circuit in case of a variation in the load connected at its output.", "The present invention further aims at providing an integrable solution compatible with a high-frequency operation of the switched-mode power supply.", "To achieve these objects, the present invention provides a circuit for tracking the maximum power point of a variable power source based on a comparison of an image of the power provided by the power source, the circuit comprising: two elements introducing different propagation delays in a quantity proportional to the power image; a comparator of the outputs of the delay elements to control a flip-flop providing a two-state signal for controlling a static power converter; means for detecting a transient state from variations of oscillations of a steady state; and means for modifying the delay introduced by the slowest delay element.", "According to an embodiment of the present invention, said means for modifying the delay are formed of a switching element capable of, in transient state, inhibiting the operation of the slower delay element.", "According to an embodiment of the present invention, said detection means compare the duration of an active state on each output signal of the flip-flop with a predetermined threshold.", "According to an embodiment of the present invention, the detection means compare, independently from each other, the forward and reverse outputs of the flip-flop and combine the result of these comparisons to provide a control pulse to the means for making the delay variable.", "According to an embodiment of the present invention, the duration of the transient state is selected according to the desired oscillation amplitude around a nominal power reference value.", "According to an embodiment of the present invention, the different voltage, current, and time measurement elements are analog.", "According to an embodiment of the present invention, the circuit comprises means for resetting the flip-flop upon occurrence of a transient state.", "According to an embodiment of the present invention, the circuit comprises means for, upon occurrence of a transient state, resetting a ramp generator conditioning the duty cycle of a pulse-width modulation control signal of the power converter.", "The present invention also provides a method for controlling a circuit for tracking the maximum power point of a variable power source of the type applying two delays of different value to an image of the power provided by the power source, which consists of inhibiting or shortening the shortest delay during a transient state.", "According to an embodiment of the present invention, the existence of a transient state is determined from a measurement of the frequency of oscillations around a nominal operating point of the maximum power point detector.", "The foregoing objects, features, and advantages of the present invention will be discussed in detail in the following non-limiting description of specific embodiments in connection with the accompanying drawings.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1, previously described, shows a conventional example of a power converter of the type to which the present invention applies; FIG.", "2 shows two examples of the course of the power according to the voltage in a photovoltaic panel forming a power source of a converter according to the present invention; FIG.", "3 shows the current variation along time in case of a change of lighting of a photovoltaic panel of the converter of FIG.", "1; FIG.", "4 very schematically shows in the form of blocks an embodiment of a maximum power point tracking circuit according to the present invention; FIG.", "5 shows a functional block diagram of a transient state detector of the circuit of FIG.", "4; FIG.", "6 is a more detailed electric diagram of a control circuit according to the present invention; and FIG.", "7 shows another example of a converter controllable by a circuit according to the present invention.", "DETAILED DESCRIPTION Same elements have been designated with same reference numerals in the different drawings.", "For clarity, only those elements which are necessary to the understanding of the present invention have been shown in the drawings and will be described hereafter.", "In particular, the forming of a power source exploited by a converter of the present invention has not been detailed and is no object of the present invention.", "A feature of the present invention is to make one of the two delay elements exploiting the power information provided by the power source controllable.", "Advantage is then fully taken of the different functions of the respective time constants of the delay elements.", "Indeed, the slower delay element brings stability to the system while the faster delay element accelerates the convergence towards the maximum power point in case of a drift.", "Accordingly, by making the slower delay element faster or, preferably, by inhibiting it during a transient state corresponding to starting or disturbance periods linked to a state change, the system convergence towards the maximum power point is accelerated.", "According to the present invention, when this point is reached, the slower delay element is put back into service or its time constant is lengthened.", "Thus, steady-state oscillations are minimized.", "The minimum duration of a transient state according to the present invention depends on the transient state of the converter and on the power source.", "More precisely, the duration range for a transient state depends on the converter, on the charge curve of its input impedance, as well as on the permitted charge excess, that is, the oscillation amplitude which is allowed in steady state, etc.", "In the application to photovoltaic panels, the transient state duration provided by the present invention essentially depends on the time constant set by the equivalent resistance of the panel and by an input capacitance of the converter.", "This input capacitance is generally provided across the panel to prevent propagation of the switch's switching noises.", "As a specific example of embodiment in a system where the slower delay element has a time constant on the order of one ms while the faster has a time constant on the order of some ten μs, it is provided to transiently pass onto a time constant on the order of 10 μs for a convergence phase lasting for from 10 to 50 ms. Another feature of the present invention is to provide a detection of transient states, that is, of the need for switching to an operation with an accelerated time constant, from the steady state oscillation frequency.", "Indeed, a state switching of the system, for example, a change in the maximum power point of the power source, translates as a change in the steady-state oscillation frequency, or even as a disappearing of the oscillations.", "Thus, according to the present invention, an oscillation frequency range corresponding to a steady state is defined, and a switching of the system to a transient operating mode is caused when it is detected that it is moving away from this frequency range.", "The minimum and maximum oscillation frequencies are determined based on the oscillation rates that one is ready to accept for the system.", "In practice, a maximum oscillation rate corresponds to a minimum frequency of these oscillations and corresponds to the state of maximum power provided by the power source.", "Conversely, a minimum oscillation rate corresponds to a maximum frequency and to a state of minimum power of the power source (for example, an operation in the shade of a photovoltaic panel).", "Preferably, for stability reasons, the time constant of the ramp generator controlled by the maximum power point tracking circuit is greater than the maximum system input time constant.", "This maximum time constant corresponds, for a photovoltaic panel, to the time constant under minimum lighting.", "FIG.", "4 shows, in a very simplified view in the form of blocks, an embodiment of a maximum power point tracking circuit according to the present invention.", "In FIG.", "4, only circuit 30 receiving power information P and providing a signal Q for controlling a ramp generator of the type illustrated in FIG.", "1 has been shown.", "The other elements of the power converter and of the power source, be they means for obtaining the power information or for exploiting control signal Q, are conventional, and a circuit such as illustrated in FIG.", "1 may for example be used.", "As previously, control circuit 30 uses a comparator 16 (COMP) for controlling a flip-flop 17 (T) having its forward output Q providing the control signal of the ramp generator (13, FIG.", "1).", "Conventionally still, the two inputs of comparator 16 receive a signal representative of the power information provided by the power source with a time shift provided by two respective delay elements 14 and 31.Delay element 14 is, as previously, relatively fast (RCF).", "According to the present invention, delay element 31 has a relatively slow nominal state (RCS) and is controllable, either for a decrease in its time constant during a transient state, or to be inhibited during this transient state.", "A signal CT31 for controlling delay element 31 is a pulse signal exhibiting a pulse of inhibition or acceleration each time a transient state is detected.", "Signal CT31 is, for example, provided by a circuit 32 (TIMER) functionally forming a generator of isolated pulses, of predetermined durations.", "Circuit 32 is controlled by a signal DEM causing the occurrence of a pulse.", "Signal DEM is provided by a circuit 33 (OSC-DET) for detecting an oscillation variation in output signal Q provided by flip-flop 17.Circuit 33 samples the signal at the output of flip-flop 17 to detect a variation in the oscillation frequency such that this frequency moves away from a range of predetermined nominal operation values.", "As an alternative, the oscillation detector of the present invention may sample a signal at any other location of control circuit 30 exhibiting steady-state oscillations, that is, where the signal shape reflects the steady-state regulation.", "For example, the output signal of comparator 16 may be sampled.", "According to another alternative embodiment, the detection of an instability loss may be obtained from images of the current, of the voltage, or of the power provided by the power source, all these signals having the same frequency.", "A frequency loss or variation with respect to a predetermined range of the state-state oscillation frequency is however still detected according to the present invention.", "Reference is made to an instability loss since the disappearing of oscillations translating as an undesired stability of the power converter is detected.", "FIG.", "5 schematically shows in the form of blocks an embodiment of an instability detection circuit 33 according to the present invention.", "According to this embodiment, circuit 33 exploits the two forward and reverse outputs Q and {overscore (Q)} of flip-flop 17 to detect a variation in both directions of the system stability.", "Outputs Q and {overscore (Q)} are respectively connected to the inputs of two time comparators 34 and 35 (CPT) having their second respective inputs receiving time thresholds TH1 and TH2.In other words, each comparator 34 or 35 compares the duration for which signal Q and {overscore (Q)} associated therewith remains in a stable active state with respect to a predetermined duration.", "As soon as this duration is exceeded, the comparator output switches to trigger a transient state pulse due to signal DEM.", "The respective outputs of comparators 34 and 35 are combined by an XNOR gate 36 having the function of suppressing states which are non-significant for the detection.", "Thresholds TH1 and TH2 are chosen according to the largest oscillation period of the system in steady state.", "This period is a function, among others, of the maximum and minimum lightings that the panel can receive, of the converter, and of the load for which the system is provided, and depends on the stability which is desired to be given to the system.", "For example, thresholds TH1 and TH2 are set so that they generate a pulse when, for a given time ranging between 2 and 5 times the maximum oscillation period, there has been no oscillation, that is, no state switching of outputs Q and {overscore (Q)}.", "The two time comparators 34 and 35 enable detecting a stable state of the oscillation of the signal provided by the power source (for example, the current of FIG.", "3), whether this stable state is at the low or at the high allowed oscillation level.", "The discussion of FIG.", "5 corresponds to a functional description of the instability detector of the present invention.", "In practice, it will be ascertained that the output of comparators 34 and 35 remains stable for a duration corresponding, preferably, to from two to five times the largest oscillation period that the control can generate, according to the desired sensitivity to variations.", "An untimely starting of the system is thus avoided when in steady state.", "An advantage of the present invention is that it enables detection of a loss of the maximum power point steady state on which is set the system without knowing the origin of this loss.", "In particular, it is not necessary to provide other sensors than sensors currently used for the maximum power point determination.", "Another advantage of the present invention is that it enables fast new convergence of the system in case of a maximum power point change.", "Another advantage of the present invention is that it forms a particularly reliable system due to the means used.", "FIG.", "6 shows a more detailed embodiment of a circuit 30 according to present invention.", "The example of FIG.", "6 aims at illustrating, in particular, the integrable character of the present invention.", "In the representation of FIG.", "6, a conventional example of exploitation of signals I and V (FIG.", "1) of the power source has further been illustrated.", "A measurement of voltage V, applied on a terminal 41 of circuit 30, is applied on a first input of multiplier 7, the output of which provides power signal P exploited by the control circuit.", "As for the detection of current I, its measurement is applied on a terminal 42 and crosses a scaling circuit 43 before reaching the second input of multiplier 7.The optional use of a scaling circuit 43 depends on the amplitude of the measured variations at the power source level.", "The scaling factor circuit is conventional.", "It is, for example, formed of an operational amplifier 431 having its inverting input connected, by a resistor R432, to terminal 42 and, by a resistor R433, to an output terminal 44 corresponding to the second input of multiplier 7.The non-inverting input of amplifier 431 is connected to ground by a resistor R434.The sizing of the resistors of a scale factor conversion circuit is within the abilities of those skilled in the art and is no object of the present invention.", "The output of multiplier 7 providing signal P is connected to the respective inputs of the two delay elements 14 and 31.In the example shown, these delay elements have the simplest possible form, that is, that of a resistive and capacitive circuit.", "Thus, the output of multiplier 7 is connected to a first input of a resistor R14 of element 14, a second input of which is connected to the inverting input of comparator 16 and, by a capacitor C14, to ground.", "The output of multiplier 7 is also connected to a first terminal of a resistor R31 of delay element 31, the second terminal of resistor R31 being connected to the positive input of comparator 16 and, by a capacitor C31, to ground.", "The components of RC circuits 14 and 31 are of course different to introduce the time constant difference necessary to the operation of the present invention.", "For example, resistors of same value may be used, the time constants of the two delay elements being differentiated by means of capacitors C14 and C31 of different values.", "The output of comparator 16 crosses an inverter 45 having its output connected, in the example shown, to clock input CLK of flip-flop 17 formed from a JK flip-flop.", "Inputs J and K of the flip-flop are connected to a terminal of application of the positive supply voltage Vcc, as well as flip-flop reset terminal R. The forward output Q of the flip-flop is connected to the input of a ramp generator 13′ intended to set the duty cycle of the pulses for switching the power supply (signal CTRL).", "The output of generator 13′ is connected to a first input of comparator 11, the second input of which receives a periodic signal provided by generator 12.This signal, for example, a sawtooth signal preferably is a signal of high frequency set by a clock HCLK.", "All that has first been described approximately corresponds to a conventional circuit.", "According to the present invention, the non-inverting input of comparator 16, that is, the output of delay element 31, is grounded by a switch 321 of circuit 32.Functionally, this corresponds to control signal CT31 discussed in relation with FIG.", "4.When switch 321 is off, a normal operation corresponding to a steady state and to that of a conventional circuit is reproduced.", "When switch 321 is on, the corresponding input of comparator 16 is, according to the present invention, grounded, which inhibits the operation of slow delay element 31.Circuit 32 providing a delayed control pulse to element 31 comprises, for example, a timing circuit 322, for example a monostable circuit (MONOST), the output of which controls switch 321 (for example, a MOS transistor).", "The control input of circuit 322 is connected to the midpoint of a series association of two resistors R323 and R324 across which is applied supply voltage Vcc.", "The control input of circuit 322 is further grounded by means of a capacitor C325.Circuit 322 aims at shaping a control pulse having its duration set by the resistive and capacitive elements placed at its input.", "In particular, the values of resistors R323 and R324 condition the charge time of capacitor C325 and, accordingly, the pulse duration.", "The input of circuit 322 is, preferably, grounded by means of a switch 326 controlled by signal DEM detecting a transient state.", "In steady state, switch 326 is off, the input of circuit 322 thus is high (substantially at voltage Vcc, neglecting the voltage drop in resistor 323 of relatively small value).", "Switch 321 is accordingly off.", "When signal DEM causes the turning-on of switch 326, this discharges capacitor 325 and switches the input of circuit 322 low.", "This thus causes a switching of the output of monostable circuit 322, which turns on switch 321.Since signal DEM is of pulse form, the ground connection of the input of circuit 322 rapidly disappears by the turning-off of switch 326.Capacitor C325 can then again be charged by resistive dividing bridge R323-R324, which conditions the pulse duration.", "As soon as the threshold of circuit 322 is reached, its output switches back and switch 321 turns off to place the system back in steady state.", "According to a preferred embodiment of the present invention, signal DEM is also used to reset ramp generator 13′ formed, in this example, of an RC cell (resistor R13 and capacitor C13).", "A first terminal of resistor R13 is connected to terminal Q of flip-flop 17.The other terminal of resistor R13 is connected to a first input of comparator 11 and, by capacitor C13, to ground.", "According to the preferred embodiment of the present invention, a switch 131 short-circuits capacitor C13 to force its discharge when signal DEM is active.", "A restarting of the ramp conditioning the duty cycle at a value always identical at each transient period is thus generated.", "The example shown considers a restarting from zero.", "As an alternative, a predetermined precharge level may be provided for this restarting.", "Preferably, for stability reasons, the slow time constant, here, R31C31, is chosen to range between {fraction (1/20)} and ½ of the time constant of ramp generator 13′, here, R13C13.As for fast element 14, its time constant, here, R14C14, is chosen according to the dynamic range desired for the system.", "For example, a constant R14C14 ranging between {fraction (1/10)} and ½ of the slow time constant (R31*C31) may be provided.", "Still according to this preferred embodiment, signal DEM is also used according to the present invention to reset flip-flop 17.For this purpose, input S of flip-flop 17 is connected to a circuit 46 applying a calibrated reset pulse.", "Circuit 46 is, for example, formed of a resistive dividing bridge R461, R462, supplied by voltage Vcc and used to charge a capacitor C463 connected between the midpoint and the ground.", "Terminal S of flip-flop 17 is connected to this midpoint.", "A switch 464 controllable by signal DEM is used to force the discharge of capacitor C463.In steady state, switch 464 is off, capacitor C463 is charged, and input S of flip-flop 17 is high.", "A turning-on of switch 464 by signal DEM after a detection of a transient state causes the discharge of capacitor C463 and the switching of input S to zero, and thus the resetting of flip-flop 17.As soon as signal DEM disappears, switch 464 turns off, which enables progressive charge of capacitor C463 by dividing bridge R461, R462.The function of circuit 46 is to provide a pulse of sufficient duration for the reset of flip-flop 17.By resetting flip-flop 17 at each transient state, the system reliability is optimized by setting the initial state of any transient state (starting or lighting change).", "Outputs Q and {overscore (Q)} of flip-flop 17 are also sent as an input to both instability loss detection circuits 34 and 35 according to the present invention.", "Each circuit 34, 35 is, in the example shown, based on a timing circuit, respectively, 341, 351, of a type known under trade name LM555, assembled as a monostable circuit.", "Each circuit 341 or 351 has its output connected to one of the inputs of XNOR-type gate 36.In the example shown, the output of gate 36 crosses a monostable circuit 37 to shape pulse DEM.", "This circuit is optional.", "Circuits 341 and 351 have their supply terminals Vcc and GND respectively connected to the terminals of application of the circuit supply voltage.", "Control voltage terminals CTR of circuits LM555 are left in the air.", "Their reset terminals RST are brought to voltage Vcc.", "Their respective triggering terminals TRIG are connected to outputs Q and {overscore (Q)} of flip-flop 17.Outputs Q and {overscore (Q)} are also respectively connected to first terminals of resistors R342 and R352 having their second respective terminal connected to the base of PNP-type transistors T343 and T353.The collectors of transistors T343 and T353 are grounded.", "Their respective emitters are connected to the threshold (THR) and discharge (DSCH) terminals of the corresponding circuits 341 and 351.Further, each terminal THR is connected to the junction point of a resistor R344, respectively, R354, with a capacitor C345, respectively C355.Functionally, the disappearing or the decrease of the low-state oscillations may be related to the case where the system stabilizes in open circuit.", "Conversely, the disappearing of the oscillations in the high state may be related to the case of a short-circuited system.", "Stage 34 corresponds to the open circuit detection while stage 35 corresponds to the short-circuit detection.", "Thresholds THR of timing circuits LM555 correspond to a high state (voltage Vcc minus the voltage drop in resistors R344 and 354, respectively) when the corresponding transistor T343 or T353 is off.", "In other words, when terminal Q, respectively, {overscore (Q)}, is low, transistor T343, respectively T353, is on, the corresponding capacitor C345 or C355 is short-circuited and threshold input THR of the corresponding circuit LM555 is low.", "Since, meanwhile, triggering input TRIG of the circuit is also low, its output OUT remains low.", "When one of terminals Q or {overscore (Q)} is high, transistor T343 or T353 associated therewith turns off.", "The corresponding capacitor C345 or C355 is charged via resistor R344 or R354.As a result, after a predetermined time which is a function of the sizing of resistor R344 (or R354) and of capacitor C345 (or C355), threshold THR of circuit 341 (or 351) becomes approximately equal to voltage Vcc (neglecting the voltage drop in resistor R344 or R354).", "Since triggering input TRIG is then high, output OUT of circuit 341 (or 351) is likely to switch if threshold THR switches high before terminal Q (or {overscore (Q)}) switches back low.", "In the opposite case, the output of circuit 341 (or 351) will remain low.", "It can thus be seen that when one of the outputs of flip-flop 17 remains in a stable active state, a switching of one input of logic gate 36 is caused to trigger a control pulse of signal DEM.", "An advantage is that the present invention is particularly well adapted to a high-frequency operation of the switched-mode power supply.", "In particular, conversely to a digital circuit, no calculation or processing time is required to implement the present invention.", "Another advantage of the present invention is that this particularly economical solution enables providing one circuit per panel in the case of a multiple-panel system.", "Problems of lighting inhomogeneity (for example, upon occurrence of shadow at the scale of a panel or of a cell) are then solved at lesser cost.", "FIG.", "7 shows another embodiment of the present invention to illustrate an assembly on a step-down D.C./D.C.", "converter.", "A photovoltaic panel 1 having its two terminals respectively connected to a first terminal of power switch 2 and, by a resistor R of very small value, to ground 5, is considered.", "The other terminal of switch 2 is connected to a first terminal of an inductive element L having its second terminal forming terminal 4 providing the output voltage to a power storage element, for example, a battery not shown.", "A free wheel diode D connects the first terminal of inductance L to ground 5.Generally, a capacitor Ce connects the positive output terminal of the photovoltaic panel to ground, to stabilize the voltage across panel 1 and make it insensitive to the switching noise of switch 2.Most often, voltage measurement terminal 41 is connected to the midpoint of a resistive dividing bridge formed of a resistor R411 in series with a resistor R412 between the positive terminal of panel 1 and the ground.", "The measurement of the current applied on terminal 42 (FIG.", "6) is taken on the negative terminal of the photovoltaic panel.", "Resistor R takes part in the current measurement.", "As appears from the foregoing, the present invention applies to any type of converter, be it a step-down, step-up, or step-down/step-up converter.", "Similarly, the power source may be of any type, provided that information relative to its power can be extracted therefrom.", "Of course, the present invention is likely to have various alterations, modifications, and improvements which will readily occur to those skilled in the art.", "In particular, other analog assemblies than that illustrated in FIG.", "6 may be envisaged, provided to respect the described functionalities.", "For example, delay element 31 may be formed of a capacitor, the capacitance of which varies according to the voltage applied thereacross, of a network of switchable resistors and capacitors, etc.", "Further, the sizing of the different time constants and resistive and capacitive elements is within the abilities of those skilled in the art based on the functional indications given hereabove and on the application.", "Moreover, although the foregoing description refers to a measurement of the power as being the product of a voltage by a current, the image of the power may originate from other quantities such as, for example, an impedance measurement, a quantity proportional to the current, assuming that the voltage is constant, a measurement proportional to the voltage, assuming that the current is constant, etc.", "Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and the scope of the present invention.", "Accordingly, the foregoing description is by way of example only and is not intended to be limiting.", "The present invention is limited only as defined in the following claims and the equivalents thereto." ] ]	Patent_10466303
[ [ "High shrink polyethylene films", "A homogeneous blend of a low density polyethylene with a metallocene-catalysed polyethylene having a density of from 0.906 g/cm3 and a Dow Rheology Index of at least 5/MI2, MI2 being the melt index measured according to ASTMD-1238 condition 190° C./2.16 kg and the Dow Rheology Index being determined by a dynamic rheological analysis performed at 190°." ], [ "1-8.", "(Cancelled) 9.A polyethylene composition comprising a homogenous blend of a low density polyethylene with a metallocene-catalysed polyethylene having a density of at least 0.906 g/cm3 and a Dow Rheology Index of at least 5/MI2, wherein MI2 is the melt index measured according to ASTMD-1238 condition 190° C./2.16 kg and the Dow Rheology Index being determined by a dynamic rheological analysis performed at 190°, said metallocene-catalysed polyethylene being catalysed with a catalyst system comprising a metallocene component and an activating agent selected from the group consisting of an alumoxane or an aluminum alkyl providing a mole ratio of aluminum to the transition metal of said metallocene component within the range of 50:1-300:1.10.The composition of claim 9 wherein the metallocene-catalysed polyethylene has a Dow Rheology Index of at least 10/MI2.11.The composition of claim 9 wherein the metallocene-catalysed polyethylene has a Dow Rheology Index of at least 20/MI2.12.The composition of claim 9 wherein the metallocene-catalysed polyethylene has a density of from 0.925 g/cm3 to less than 0.965 g/cm3.13.The composition of claim 12 wherein the metallocene-catalysed polyethylene has a Dow Rheology Index of at least 10/MI2 14.The composition of claim 12 wherein the metallocene-catalysed polyethylene has a Dow Rheology Index of at least 20/MI2.15.The composition of claim 9 wherein the metallocene-catalysed polyethylene has a density of from 0.906 g/cm3 to less than 0.925 g/cm3.16.The composition of claim 15 wherein the metallocene-catalysed polyethylene has a Dow Rheology Index of at least 10/MI2.17.The composition of claim 15 wherein the metallocene-catalysed polyethylene has a Dow Rheology Index of at least 20/M2.18.A blown film comprising at least one layer formed of a polyethylene composition comprising a homogenous blend of a low density polyethylene with a metallocene-catalysed polyethylene having a density of at least 0.906 g/cm3 and a Dow Rheology Index of at least 5/MI2, wherein MI2 is the melt index measured according to ASTMD-1238 condition 190° C./2.16 kg and the Dow Rheology Index being determined by a dynamic rheological analysis performed at 190°, said metallocene-catalysed polyethylene being catalysed with a catalyst system comprising a metallocene component and an activating agent selected from the group consisting of an alumoxane or an aluminum alkyl providing a mole ratio of aluminum to the transition metal of said metallocene component within the range of 50:1-300:1.19.The film of claim 18 wherein said layer is a monolayer blown film.", "20.The film of claim 18 wherein said layer comprises at least one layer of a multilayer blown film.", "21.A process for the preparation of a blown film, forming said film in a blown film line from a polyethylene composition comprising a homogenous blend of a low density polyethylene with a metallocene-catalysed polyethylene having a density of at least 0.906 g/cm3 and a Dow Rheology Index of at least 5/MI2, wherein MI2 is the melt index measured according to ASTMD-1238 condition 190° C./2.16 kg and the Dow Rheology Index being determined by a dynamic rheological analysis performed at 190°, said metallocene-catalysed polyethylene being catalysed with a catalyst system comprising a metallocene component and an activating agent selected from the group consisting of an alumoxane or an aluminum alkyl providing a mole ratio of aluminum to the transition metal of said metallocene component within the range of 50:1-300:1, said film being characterized by: a cohesion force in the transverse direction at room temperature of at least 5% greater than the cohesion force in the transverse direction of a corresponding biaxially-oriented film formed of said low density polyethylene in a pure form; and a gloss at an angle of 45° of at least 60 and a haze of less than 10% while keeping a good rigidity.", "22.A biaxially-oriented film which has been oriented in the machine direction and the transverse direction, formed from a homogenous blend of a polyethylene composition comprising a homogenous blend of a low density polyethylene with a metallocene-catalysed polyethylene having a density of at least 0.906 g/cm3 and a Dow Rheology Index of at least 5/MI2, wherein MI2 is the melt index measured according to ASTMD-1238 condition 190° C./2.16 kg and the Dow Rheology Index being determined by a dynamic rheological analysis performed at 190°, said metallocene-catalysed polyethylene being catalysed with a catalyst system comprising a metallocene component and an activating agent selected from the group consisting of an alumoxane or an aluminum alkyl providing a mole ratio of aluminum to the transition metal of said metallocene component within the range of 50:1-300:1, said film having a cohesion force in the transverse direction which is greater than the cohesion force in the transverse direction of a corresponding biaxially-oriented film formed of said low density polyethylene in a pure form.", "23.The biaxially-oriented film of claim 22, wherein said film has a cohesion force in the machine direction which is greater than the cohesion force in the machine direction of the corresponding film formed of said low density polyethylene in a pure form.", "24.The film of claim 23, having a gloss at an angle of 45° of at least 60, and a haze which is less than 10%." ], [ "The present invention relates to polyethylene compositions and high performance shrink films thereof combining excellent mechanical properties such as stiffness and toughness with good processability and good optical properties.", "These polyethylene compositions can therefore be used for film applications requiring this unique combination of properties, such as but not exclusively, packaging.", "Shrink film has been used for years in the packaging industry to wrap articles.", "The process comprises packing the article and submitting it to heating in an oven, whereby the film is retracted so as to render the packing tight and suitable to its end use.", "It is well known to use linear low density polyethylene (LLDPE) in blend with low density polyethylene (LDPE) in shrink film compositions.", "Compositions comprising of from 20 to 40% by weight of LLDPE with 80 to 60% by weight of LDPE are commonly used.", "Indeed the addition of LLDPE to LDPE in shrink film compositions is well known in order to avoid the formation of holes that could occur during the retraction of shrink film made from pure LDPE.", "Nevertheless the currently available polyethylene resins suffer from major drawbacks.", "The low density polyethylene (LDPE) resins exhibit excellent optical and processing properties but they have poor mechanical properties and poor rigidity.", "Linear low density polyethylene (LLDPE) resins have excellent mechanical properties but have mediocre optical properties and poor processability.", "Indeed LLDPE leads to bubble instability and its extrusion is difficult.", "If mixed with LDPE they have improved processability properties but their mechanical properties are reduced.", "Metallocene-catalysed linear low density polyethylene (mLLDPE) resins have excellent mechanical properties but poor optical properties and processability requiring extrusion equipment specially designed for mLLDPE with wide die gap.", "If mixed with LDPE they have very good optical and good sealing properties, but the mechanical properties are reduced.", "Wherever high rigidity is needed, LDPE and LLDPE compositions will require overly thick structures.", "Especially for LLDPE, where excellent impact and tear properties render its down-gauging capability useful, the lack of rigidity is a main drawback because high rigidity is a requirement for product packaging.", "WO 95/27005 discloses mixtures of LDPE with LLDPE or mLLDPE.", "The rigidity of the mixtures is insufficient.", "EP-A-0844277 discloses metallocene-catalysed medium density polyethylene with LDPE and/or LLDPE compositions for blown films that claim a good balance between the good optical properties of LDPE and the good mechanical and processing properties of medium density polyethylene (MDPE).", "However this specification does not address the problem of the production of shrinkable polyethylene films EP-A-1108749 relates to shrink films of blended LDPE and MDPE resins.", "However those resins can still be further improved.", "It is an object of the present invention to provide polyethylene compositions for mono or multilayers films, that achieve good balanced shrink properties in machine direction (MD) and transverse direction (TD) with fast shrink speed and high cohesion force at room temperature while keeping a good rigidity, excellent optical properties and an easy processing in film blowing process.", "In the present invention, a film is defined as an extremely thin continuous sheet: the upper limit for thickness is of about 250 microns (Hawley's Condensed Chemical Dictionary, Twelfth Edition, Rev.", "by R. J. Lewis, Van Nostrand Reinhold Co., New York) In the present invention, good balanced shrink properties in machine and transverse directions of the polyethylene resin produced according to the invention is defined as a resin having a shrinkage value in transverse direction of at least 5%, preferably of at least 10% compared to known LDPE/LLDPE film while keeping shrinkage value in machine direction similar to LDPE/LLDPE film, LDPE/LLDPE films and films according to the blend of the invention being extruded in the same conditions.", "In the present context, high cohesion force at room temperature of the polyethylene resin produced according to the invention is defined as a resin having a cohesion force in transverse direction greater than 5%, preferably greater than 10% compared to LDPE/LLDPE films.", "Fast shrink speed of the polyethylene resin produced according to the invention is defined as a resin that shrinks of at least 10%, more preferably at least 20% faster than LDPE/LLDPE films.", "Good optical properties of the polyethylene film produced according to the invention is here defined as a film having a gloss at an angle of 45° of at least 60 and a haze of less than 10%.", "This invention relates to a homogeneous blend of a low density polyethylene (LDPE) with a metallocene-catalysed polyethylene (mPE) having a density of from 0.906 g/cm3 and a Dow Rheology Index (DRI) of at least 5/MI2, MI2 being the melt index measured according to ASTMD-1238 condition 190° C./2.16 kg and the Dow Rheology Index being determined by a dynamic rheological analysis performed at 190°, this blend consisting of from 0.5% to 99.5% by weight of mPE and of from 99.5% to 0.5% by weight of LDPE, based on the total weight of the blend.", "In this specification, the density of the polyethylene is measured at 23° C. using procedures of ASTM D-1505 and the melt index is measured according to ASTM D-1238 condition 190° C./2.16 kg.", "Other polymers compatible with said blend can be added to the blend to a total amount not to exceed 33% by weight based on the total weight of the polymers.", "The densities of the mPE used in the present invention are regulated by the amount of comonomer injected in the reactor; they will range from 0.906 g/cm3 to less than 0.965 g/cm3.Examples of comonomer which can be used include 1-olefins such as propylene, butene, hexene, octene, 4-methyl-pentene, and the like, as well as mixtures thereof up to C12 1-olefins, the most preferred being hexene.", "Ethylene can also be used as such without any addition of comonomers.", "Homopolymers of ethylene are then produced.", "According to one embodiment, the density of the mPE will range from 0.906 g/cm3 to less than 0.925 g/cm3.According to another embodiment, the density of the mPE will range from 0.925 g/cm3 to less than 0.965 g/cm3 preferably from 0.925 g/cm3 to less than 0.950 g/cm3.The melt index of the mPE used in the present invention can be regulated by the amount of hydrogen injected in the reactor; it will range from 0.1 g/10′ to 15 g/10′, preferably from 0.2 g/10′ to 4 g/10′ The molecular weight distribution (D) defined as the ratio between the average molecular weight by weight (Mw) and the average molecular weight by number (Mn) of the mPE used in the present invention is of from 2 to 8, preferably of from 2 to 4 and even more preferably of from 2 to 3.5.The melt flow ratio of the mPE used in the present invention is of from 25 to 100.The melt flow ratio being the ratio HLMI/MI2, the HLMI being measured according to ASTM D-1238, condition 190° C./21.6 kg and the MI2 being measured according to ASTM D-1238, condition 190° C./2.16 kg.", "The mPE resin used in the present invention has a high Dow Rheological Index (DRI).", "To characterize the rheological behavior of substantially linear ethylene polymers, S Lai and G. W. Knight introduced a new rheological measurement, the Dow Rheology Index (DRI) which expresses a polymer's “normalized relaxation time as the result of long chain branching” (ANTEC '93 Proceedings, Insite™ Technology Polyolefins (ITP)—New Rules in the Structure/Rheology Relationship of Ethylene &-Olefin Copolymers, New Orleans, La., May 1993).", "S. Lai et al defined the DRI as the extent that the rheology of ethylene-octene copolymers known as ITP (Dow's Insite Technology Polyolefins) incorporating long chain branching into the polymer backbone deviates from the rheology of the conventional linear homogeneous polyolefins that are reported to have no long chain branching by the following normalized equation: DRI=(365000(t0/η0)−1)/10 wherein t0 is the characteristic relaxation time of the material and η0 is the zero shear viscosity of the material (Antec '94, Dow Rheology Index (DRI) for Insite™ Technology Polyolefins (ITP): Unique structure-Processing Relationships, pp.", "1814-1815).", "The DRI is calculated from the best fitting by least squares analysis of the rheological curve (complex viscosity versus frequency) as described in U.S. Pat.", "No.", "6,114,486 with the following generalized Cross equation, i.e.", "η=η0/(1+(γt0)n) wherein n is the power law index of the material, η and γ are the measured viscosity and shear rate data respectively.", "The dynamic rheological analysis is performed at 190° C. and the strain amplitude is 10%.", "Results are reported according to ASTM D 4440.The DRI of the mPE used in the present invention is at least 5/MI2, preferably at least 10/MI2, more preferably at least 20/MI2.It has been observed that when the dynamic rheological analysis is performed at a temperature lower than 190° C., higher DRI values can be obtained compared to those obtained when the dynamic rheological analysis is performed at a temperature of 190° C. and vice versa.", "Attention should thus be paid to the temperature at which the dynamic rheological analysis is performed when DRI values are compared.", "DRI values ranging from zero for polymers which do not have measurable long chain branching to about 15 are known and have been described in several U.S. patents such as for example U.S. Pat.", "Nos.", "6,114,486, 5,674,342, 5,631,069.The manufacture of the low density polyethylenes used in the present invention is known in the art and is described for example in “Encyclopedia of Polymer Science and Engineering”, second edition, Volume 6, on pages 404 to 410 (LDPE) and pages 436 to 444 (LLDPE).", "The catalyst system used to produce the polyethylene required by the present invention comprises a metallocene component.", "The metallocene component can be any metallocene component known in the art of the general formulas: (Cp)mMRnXq I. wherein Cp is a cyclopentadienyl ring, M is a group 4b, 5b or 6b transition metal, R is a hydrocarbyl group or hydrocarboxy having from 1 to 20 carbon atoms, X is a halogen, and m=1-3, n=0-3, q=0-3 and the sum m+n+q is equal to the oxidation state of the metal.", "(C5R′k)gR″s(C5R′k)MQ3−g II.", "and R″s(C5R′k)2MQ′ III.", "wherein (C5R′k) is a cyclopentadienyl or substituted cyclopentadienyl, each R′ is the same or different and is hydrogen or a hydrocarbyl radical such as alkyl, alkenyl, aryl, alkylaryl, or arylalkyl radical containing from 1 to 20 carbon atoms or two carbon atoms are joined together to form a C4-C6 ring, R″ is a C1-C4 alkylene radical, a dialkyl germanium or silicon or siloxane, or a alkyl phosphine or amine radical bridging two (C5R′k) rings, Q is a hydrocarbyl radical such as aryl, alkyl, alkenyl, alkylaryl, or aryl alkyl radical having from 1-20 carbon atoms, hydrocarboxy radical having 1-20 carbon atoms or halogen and can be the same or different from each other, Q′ is an alkylidene radical having from 1 to about 20 carbon atoms, s is 0 or 1, g is 0, 1 or 2, s is 0 when g is 0, k is 4 when s is 1 and k is 5 when s is 0, and M is as defined above.", "Among the preferred metallocenes used one can cite among others bis tetrahydro-indenyl compounds and bis indenyl compounds as disclosed for example in WO 96/35729.The most preferred metallocene catalyst is ethylene bis (4,5,6,7-tetrahydro-1-indenyl) zirconium dichloride.", "The metallocene may be supported according to any method known in the art.", "In the event it is supported, the support used in the present invention can be any organic or inorganic solids, particularly porous supports such as talc, inorganic oxides, and resinous support material such as polyolefin.", "Preferably, the support material is an inorganic oxide in its finely divided form and has a surface area comprised between 100 and 1200 m2/g.", "An active side must be created by adding a cocatalyst having an ionizing action.", "While alumoxane can be used as cocatalyst, it is not necessary to use alumoxane as cocatalyst during the polymerization procedure for preparing the mPE resin.", "When alumoxane is used as a cocatalyst, any alumoxane known in the art can be used.", "The preferred alumoxanes comprise oligomeric linear and/or cyclic alkyl alumoxanes represented by the formula: for oligomeric, linear alumoxanes and for oligomeric, cyclic alumoxanes, wherein n is 1-40, preferably 10-20, m is 3-40, preferably 3-20 and R is a C1-C8 alkyl group and preferably methyl.", "Methylalumoxane is preferably used.", "The amount of alumoxane and metallocene usefully employed in the preparation of the solid support catalyst can vary over a wide range.", "Preferably the aluminium to transition metal mole ratio is in the range between 20:1 and 500:1, preferably in the range 50:1 and 300:1.When alumoxane is not used as a cocatalyst, one or more aluminium alkyl represented by the formula AlRx are used wherein each R is the same or different and is selected from halides or from alkoxy or alkyl groups having from 1 to 12 carbon atoms and x is from 1 to 3.Especially suitable aluminiumalkyl are trialkylaluminium, the most preferred being triisobutylaluminium (TIBAL).", "In the present invention, the mPE can be monomodal, bimodal or multimodal.", "The metallocene catalyst utilized to produce the polyethylene required by the present invention can be used in gas, solution or slurry polymerizations.", "Preferably the polymerization process is conducted under slurry phase polymerization conditions.", "It is preferred that the slurry phase polymerization conditions comprise a temperature of from 20 to 125° C., preferably from 60 to 110° C. and a pressure of from 0.1 to 8 MPa, preferably from 2 to 5 MPa for a time between 10 minutes and 4 hours, preferably between 0.4 and 2.5 hours.", "High pressure range like 100 to 2000 bars can be used for polymerization in high pressure tubular or autoclave reactors.", "It is preferred that the polymerization reaction be run in a diluent at a temperature at which the polymer remains as a suspended solid in the diluent.", "Diluents include, for examples, propane, isobutane, n-hexane, n-heptane, methylcyclohexane, n-pentane, n-butane, n-decane, cyclohexane and the like as well as mixtures thereof.", "The preferred diluent is isobutane.", "The diluent can be under liquid or super critical state.", "The polymerization of the mPE used in the present invention can be conducted in a continuous reactor.", "The continuous reactor is preferably a loop reactor.", "During the polymerization process, at least one monomer, the catalytic system and a diluent are flowed in admixture through the reactor.", "Alternatively for a bimodal production of mPE, two reactors in series can be used.", "In the present invention average molecular weights can be further controlled by the introduction of some amount of hydrogen or by changing the temperature during polymerization.", "When hydrogen is used it is preferred that the relative amounts of hydrogen and olefin introduced into the polymerization reactor be within the range of about 0.001 to 15 mole percent hydrogen and 99.999 to 85 mole percent olefin based on total hydrogen and olefin present, preferably about 0.02 to 3 mole percent hydrogen and 99.98 to 97 mole percent olefin.", "Standard additives such as antioxidants may be used for both long term and processing stabilization and if desired, one or more pigments and/or dyes and/or processing aids like fluoro elastomers can also be added.", "Antistatic, antifog, antiblocking or slip additives may also be added.", "According to embodiments of the present invention, compositions of LDPE and mPE are obtained either by preliminary dry blend or extrusion or by direct blend in the hopper or via the extruder.", "The present invention further provides the use of the homogeneous blend according to the present invention to produce a monolayer blown film or one or more layers of a multilayer blown film wherein said layers are arranged in any order.", "The present invention still further provides films, prepared with the blend of the present invention, characterised by: a shrinkage value in transverse direction of more than at least 5%, preferably of more than at least 10% compared to LLDPE/LDPE films extruded in the same conditions as those us for the blend of the present invention.", "a cohesion force in transverse direction at room temperature greater than of at least 5%, preferably greater than 10% compared to LDPE/LLDPE films.", "a shrink speed value of at least 10% more preferably at least 20% faster than LDPE/LLDPE films.", "A gloss at an angle of 45° of at least 60 and a haze of less than 10%.", "while keeping a good rigidity and being easily processable.", "In the present invention, a good rigidity means e.g.", "that for a mPE/LDPE blend according to the invention, a secant modulus at 1% of deformation according to ASTM D-882 is: at least of 200 mega Pascal for a mPE/LDPE blend having a density of 0.920 g/cm3.at least of 230 mega Pascal for a mPE/LDPE blend having a density of 0.923 g/cm3.at least of 260 mega Pascal for a mPE/LDPE blend having a density of 0.926 g/cm3.at least of 320 mega Pascal for a mPE/LDPE blend having a density of 0.930 g/cm3, the secant modulus at 1% of deformation according to ASTM D-882 for the pure LDPE being of 200 mega Pascal.", "As a consequence of a higher shrinkage in transverse direction and a higher cohesion force of the film produced according to the invention and caused by the high DRI mPE, it will be possible to make thinner shrink film leading to a significant cost reduction.", "Increasing the shrink speed of the film produced according to the invention is particularly interesting because it results either in higher shrink-wrapping packaging rates or in decreasing the temperature of the oven during the packaging wrapping process, both results leading to a significant cost reduction.", "The blend as described here above may also be used in the production of lamination films, barrier films and reticulation applications.", "These films may also be metallized, corona treated, printed and laminated.", "EXAMPLES 1.Polymerization Procedure and Product Composition.", "The polymerization of the mPE (R1) used in the blend of the present invention was carried out in a liquid-full slurry loop reactor.", "Ethylene was injected with 1-hexene together with the catalyst.", "Isobutane was used as diluent.", "The polymerization conditions are indicated in Table I TABLE I Resin R1 C2 feed (kg/h) 3900 C6 feed (g/kg C2) 22 H2 feed (g/t) 42 Iso C4 feed (kg/h) 1940 Tibal conc (ppm) 100-200 T. pol (° C.) 90 C2 = ethylene C6 = 1-hexene Iso C4 = isobutane Tibal = triisobutylaluminium The bridged metallocene catalyst used was ethylene bis (tetrahydro-indenyl) zirconium dichloride R1 produced according to the above polymerization conditions is a high DRI mPE.", "The data concerning R1 in comparison with a purely linear metallocene medium density from Phillips identified as Marlex mPact D350® are summarised in Table II.", "TABLE II R1 Marlex mPact D350 ® Density g/cm3 0.934 0.933 MI2 g/10 min 0.9 0.9 DRI 36 0 SR2 = HLMI/MI2 30 16 D (Mw/Mn) 3.28 2.88 The DRI of R1 and Marlex mPact D350® were determined by fitting the generalized Cross equation on the complexe viscosity measured according to ASTM D4440 by using a RDA 700 from Rheometrics, a diameter plate-plate of 25 mm and a gap between plates of 2 mm +/−0.2 mm.", "The apparatus was callibrated according to ARES Instrument normal 902-30004 The rheological measurements were performed at 190° C. under nitrogen and at 10% of strain.", "2.Blends Preparation.", "A blend B1 according to the invention was prepared by mixing 30% by weight of mPE (R1) and 70% by weight of LDPE A blend B2 according to the invention was prepared with the same ingredients in different proportions: 70% by weight of R1 and 30% by weight of LDPE.", "A comparison blend B3 was prepared by mixing 30% by weight of LLDPE and 70% by weight of LDPE A comparison blend B4 was prepared by mixing 30% by weight of the Marlex mPact D350® from Phillips and 70% by weight of LDPE.", "The LDPE used in all the blends is characterised by a density of 0.924 g/cm3 and a MI2 of 0.8 g/10 min The LLDPE used in comparison blend B3 is a Ziegler-Natta LLDPE characterised by a density of 0.918 g/cm3, a DRI of 0.6 and a molecular weight distribution (Mw/Mn) of 4.1.3.Films Preparation.", "Six films were blown on a Macchi blown film line equipment using a low density configuration characterised by a die of 120 mm, a blow up ratio of 2.5:1 and a die gap of 0.8 mm.", "All the films were down-gauged to a thickness of 40 microns.", "Films F1 and F2 were prepared from the blend B1 and B2 according to the invention.", "Films F3 and F4 were prepared from the comparative blends B3 and B4.Film F5 was prepared from pure LDPE Film F6 was prepared from pure resin R1 4.Films Properties.", "The shrinkage in machine direction (MD) and transverse direction (TD) was measured by immersion of the film in a bath of hot oil at 140° C. during 2 minutes according ASTM D2732.The percentage of shrinkage for each direction (MD and TD) of the six films are given in table III TABLE III Shrinkage Shrinkage TD Films Component % weight MD (%) (%) F1 R1/LDPE 30/70 71 39 F2 R1/LDPE 70/30 69 37 F3 LLDPE/LDPE 30/70 70 36 F4 mPact D350 ®/LDPE 30/70 70 33 F5 LDPE 100 70 42 F6 R1 100 63 25 Pure LLDPE and Marlex mPact D350® could not be extruded by using a narrow die gap of 0.8 mm as desribed here above for the films F1 to F6.This is why no shrink results are given for those pure resins.", "In contrast pure high DRI mPE (R1) could be extruded under those conditions.", "Moreover even pure it exibits good shrinkage in TD direction.", "It can also be observed from table III that the addition of high DRI mPE to LDPE increases the shrinkage of the film (F1) in transverse direction compared to films (F3 and F4) produced from LLDPE/LDPE and mPact D350®/LDPE blends.", "Table III shows clearly that the good shrink property in transverse direction is brought by the LDPE (film F5).", "For the shrink in machine direction, all the blends studied (F1 to F4) exhibited the same behaviour.", "It is surprising that when the high DRI mPE is used even in high concentration with LDPE the film produced (F2) still exibits better shrinkage in transverse direction compared to the films using much more LDPE (F3 and F4).", "The cohesion force exerted by the film after shrink and cooling the film at room temperature was measured according to ISO 14616 method by setting the films in an oven at 180° C. during 17 seconds.", "The cohesion force was then measured at room temperature for each film in the machine direction (MD) and transverse direction (TD).", "Those forces are expressed in Newton (N).", "The measurement results are shown in table IV.", "TABLE IV % MD Cohesion TD Cohesion Films Components weight F. (N) F. (N) F1 R1/LDPE 30/70 1.102 0.835 F2 R1/LDPE 70/30 1.21 1.01 F3 LLDPE/LDPE 30/70 0.902 0.622 F4 mPact D350 ®/LDPE 30/70 1.1 0.62 F5 LDPE 100 0.85 0.722 F6 R1 100 1.354 not applicable When added to LDPE it is seen that LLDPE and mPact D350® increase the cohesion force of the film in machine direction (MD) but decrease it in transverse direction (TD) compared to pure LDPE (F3 and F4 versus F5).", "Contrary the high DRI mPE/LDPE blend not only increases substantially the cohesion force of the film in machine direction but also increases the cohesion force in transverse direction (TD) when compared to pure LDPE (F1 versus F5).", "Both cohesion forces are further increased when increasing amount of high DRI mPE with LDPE (F2 versus F1).", "The cohesion force of the film issued from the LLDPE/LDPE blend is partly related to the molecular weight distribution (D) defined as the ratio between the average molecular weight by weight (Mw) and the average molecular weight by number (Mn) of the LLDPE.", "Higher is the molecular weight distribution of the LLDPE, better will be the cohesion force of the film.", "It is remarkable that the blend according to the invention while using a mPE having a narrower molecular weight distribution compared to the LLDPE (3.28 versus 4.1) allows to get films with a better cohesion force than that produced from LLDPE/LDPE blend (F1 and F 2 versus F3).", "It has also been unexpectetly found that the shrink speed in the machine direction of the film produced according to the blend of the invention is higher compared to those of the films produced from the LDPE/LLDPE and from the mPact D350® blends.", "Table V illustrates this result.", "TABLE V MD MD Elmendorff Secant shrink Tear Gloss Haze modulus Films time (s) (N/mm) 45° (%) (mPa) F2 (example) 15 33.5 60.7 7.3 280 F3 (comparative) 20 25 57.4 8.3 189 F4 (comparative) 20 — 57.4 8.3 189 Besides a shorter shrink time, a higher MD Elmendorf tear resistance, a better rigidity, a better gloss at 45° C. and less haze are also observed for the film produced according to the invention.", "It is unexpected and remarkable that it is the film according to the invention that give higher tear resistance and better optical properties even if the density of the blend produced according to the invention is higher than that of the comparative blend The Elmendorf tear was measured using the method of ASTM D-1922.The gloss was measured at an angle of 45° with the Byk-Gardner micro-gloss reflectometer according to ASTM D2457, the haze was measured with the Byk-Gardner Hazegard® system according to ASTM D-1003.The MD shrink time was measured using the method ISO 14616.The Secant modulus at 1% of deformation was measured according to ASTM D-882." ] ]	Patent_10466307
[ [ "Extraction bedplate with laser or water jet cut apertures", "The present invention relates to extraction bedplates (10), (110), (210), (310), (410), (510), (610) for use in apparatus (5) for defiberizing paper making stock and methods for making such bedplates.", "Preferred methods for making such bedplates (10), (110), (210), (310), (410), (510), (610) include the step of cutting a disc shaped blank from a metal plate and the step of forming holes (45), (145), (245), (345), (445), (545), (645), (646) either the metal plate or the disc shaped blank.", "The holes (45), (145), (245), (345), (445), (545), (645), (646) preferably are formed using a cutting stream, most preferably either a laser or a water jet.", "Use of a cutting stream to form the holes facilitates the cutting of holes (45), (145), (245), (345), (445), (545), (645), (646) having non-circular, and preferably tesselatory, cross sections as well as holes (45), (145), (245), (345), (445), (545), (645), (646) extending at acute angles with respect to an axis (20) of the bedplate." ], [ "1.An extraction bedplate for use in defiberizing stock for making paper, comprising: a plate defining first and second surfaces; and a plurality of holes lacking substantially circular cross-sections extending from said first surface to said second planar surface.", "2.The extraction bedplate as recited in claim 1, wherein said plate defines an axis normal to said first and second surfaces and wherein said holes extend at an acute angle with respect to said axis.", "3.The extraction bedplate as recited in claim 1, wherein said plate defines an axis normal to said first and second surface; said holes are arranged along arcs coincident with anticipated stock flow lines immediately above the upstream surface of said bedplate; and said holes are extend through said bedplate at an acute angle along said anticipated stock flow lines so as to define relatively sharp downstream side edges facing said anticipated flow lines.", "4.The extraction bedplate as recited in claim 1, wherein said holes have cross sections which tesselate a plane.", "5.The extraction bedplate as recited in claim 1, wherein said holes have substantially rhombic cross sections.", "6.The extraction bedplate as recited in claim 1, wherein said holes have substantially square cross sections.", "7.The extraction bedplate as recited in claim 1, wherein said holes have substantially rectangular cross sections.", "8.The extraction bedplate as recited in claim 1, wherein said holes have substantially chevronic cross sections.", "9.The extraction bedplate as recited in claim 1, wherein said holes have substantially triangular cross sections.", "10.The extraction bedplate as recited in claim 1, wherein said holes have substantially crescentic cross sections.", "11.The extraction bedplate as recited in claim 1, wherein said holes have substantially semi-circular cross-sections.", "12.An extraction bedplate for use in defiberizing stock for making paper, comprising: a plate defining a first surface, a second surface and an axis normal to said first and second surfaces; and a plurality of holes extending through said plate symmetrically at an acute angle with respect to said axis from said first surface to said second planar surface.", "13.The extraction bedplate as recited in claim 12, wherein said holes have cross sections in the shape of polygons which tesselate a plane.", "14.The extraction bedplate as recited in claim 12, wherein said holes have substantially circular cross sections.", "15.The extraction bedplate as recited in claim 12, wherein said holes have substantially rhombic cross sections.", "16.The extraction bedplate as recited in claim 12, wherein said holes have substantially square cross sections.", "17.The extraction bedplate as recited in claim 12, wherein said holes have substantially rectangular cross sections.", "18.The extraction bedplate as recited in claim 12, wherein said holes have substantially chevronic cross sections.", "19.The extraction bedplate as recited in claim 12, wherein said holes have substantially triangular cross sections.", "20.The extraction bedplate as recited in claim 12, wherein said holes have substantially crescentic cross sections.", "21.The extraction bedplate as recited in claim 12, wherein said holes have substantially semi-circular cross sections.", "22.A method for fabricating an extraction bedplate from a metal plate defining a first surface and a second surface, said method comprising the steps of: (a) cutting a disc-shaped blank from said metal plate; and (b) forming a plurality of holes lacking substantially circular cross-sections through one of said metal plate and said disc-shaped blank from said first surface to said second surface.", "23.The method as recited in claim 22, wherein said step (b) includes directing a stream of energy against said one of said metal plate and said disc-shaped blank to ablate said plurality of holes.", "24.The method as recited in claim 23, wherein said step (b) includes directing a stream of laser energy against said one of said metal plate and said disc-shaped blank to ablate said plurality of holes.", "25.The method as recited in claim 22, wherein said step (b) includes directing a stream of pressurized fluid against said one of said metal plate and said disc-shaped blank to cut said plurality of holes.", "26.The method as recited in claim 22, wherein said step (b) includes directing a cutting stream against said one of said metal plate and said disc-shaped blank and wherein said method includes the additional step of: (c) programming a programmable electronic controller to induce said cutting stream to move across said one of said metal plate and said disc-shaped blank so as to shape said plurality of holes.", "27.A method for fabricating an extraction bedplate from a metal plate defining a first surface and a second surface, said method comprising the steps of: (a) cutting a disc-shaped blank from said metal plate; and (b) forming a plurality of holes extending symmetrically with respect to said axis at an acute angle with respect to said axis through one of said metal plate and said disc-shaped blank from said first surface to said second surface.", "28.The method as recited in claim 27, wherein said step (b) includes directing a stream of energy against said one of said metal plate and said disc-shaped blank to ablate said plurality of holes.", "29.The method as recited in claim 28, wherein said step (b) includes directing a stream of laser energy against said one of said metal plate and said disc-shaped blank to ablate said plurality of holes.", "30.The method as recited in claim 27, wherein said step (b) includes directing a stream of pressurized fluid against said one of said metal plate and said disc-shaped blank to cut said plurality of holes.", "31.The method as recited in claim 27, wherein said step (b) includes directing a cutting stream against said one of said metal plate and said disc-shaped blank and wherein said method includes the additional step of: (c) programming a programmable electronic controller to induce said cutting stream to move across said one of said metal plate and said disc-shaped blank so as to shape said plurality of holes." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to apparatus for use in defiberizing papermaking stock.", "More particularly, the invention relates to extraction bedplates with specially shaped and contoured holes cut by laser energy or a fluid jet for use in pulping apparatus.", "2.Background Art Apparatus for pulping paper making stock is shown in Chupka U.S. Pat.", "No.", "4,725,007, the disclosure of which is incorporated by reference.", "The apparatus shown in U.S. Pat.", "No.", "4,725,007 includes a tub and a rotor mounted within the tub for inducing shear forces which serve to defiberize the stock An extraction bedplate is positioned at the bottom of this tub, surrounded by a frusto-conical wall which serves as a funnel to direct the stock toward the bedplate.", "The preferred bedplate is disc-shaped, defining an upstream surface facing into the tub; a downstream surface facing oppositely from the upstream surface; and holes or apertures extending through the bedplate from the upstream surface to the downstream surface.", "The rotor is mounted near the center of the perforated bedplate and coupled to a motor for rotation about an axis normal to the upstream surface of the bedplate.", "The holes extending through the extraction bedplate allow accepted fiber, that is, pulp which has been defiberized to a degree which is acceptable for further processing to flow out from the apparatus, while retaining larger, undefiberized particles and other solids in the tub.", "Conventional bedplates typically range from 24 inches (61 cm) to 96 inches (2.4 m) in diameter and are typically approximately ⅝ inch (1.6 cm) thick.", "Typically there are 4,000 to 5,000 holes in a 96 inch diameter plate with ⅝ inch holes.", "Since such holes are formed by conventional drilling processes, they have in the past been formed parallel to the axis of the bedplate with circular cross sections.", "The holes generally range from {fraction (1/8)} inch (3.2 mm) to 1 inch (25 mm) in diameter.", "Known extraction bedplates tend to be high maintenance items because of wear.", "Bedplates are exposed to harsh treatment from sand, metal objects and other debris contained within the stock.", "The typical clearance between the rotor and the bedplate is approximately 0.060 inch (1.5 mm) to 0.120 inch (3.0 mm).", "The stock is constantly pushed against, and drug along, the upper surface of the bedplate by the mechanical and hydraulic action of the associated rotor.", "The accepted fiber along with small contaminates which flow through the bedplate contribute to wear within the holes, particularly near the upper perimeters of the downstream edge portions of the holes.", "Bedplates typically are manufactured from steel alloys resistant to wear and corrosion.", "Various stainless steels and 410 hard chrome steel have been used in forming bedplates.", "The 410 hard chrome steel is preferred because it is more wear resistant than the stainless steels.", "On the other hand, the 410 hard chrome steel requires heat treatment to harden the material to restore acceptable wear resistance after known machining and hole-drilling steps are performed.", "Once the heat treatment is performed, further machining is possible only with special tools in a slow and costly procedure.", "The heat treatment itself tends to warp the steel, so that additional manufacturing steps are required to straighten the bedplate.", "The defibering characteristics of a given bedplate are dependent to a large degree on the surface indentations defined by the upper edges of the individual holes.", "More particularly, the paper making stock flows over the upstream surface of the bedplate during operation of the pulping apparatus.", "Hydraulic shear is generated near downstream side edges (that is, edges facing the oncoming stock now) formed at the intersections of the holes with the upstream surface of the bedplate.", "This hydraulic shear acts to break up relatively large, undefiberized particles.", "Increasing the number of such downstream side edges increases the amount of the hydraulic shear, thus improving the efficiency of the pulping apparatus.", "Therefore, there remains a need in the art for extraction bedplates providing improved efficiency and wear resistance.", "Additionally, there remains a need for improved methods for making such bedplates." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Preferred extraction bedplates in accordance with the present invention have specially shaped and configured holes which provide increased densities of downstream side edges along the upstream surfaces of the bedplates.", "In accordance with one preferred embodiment of the invention, the holes have non-circular cross sections.", "Most preferably, the holes have cross sections with shapes which tesselate a plane, that is, which when laid side-to-side will fill a plane without intervening gaps.", "Individual holes having tesselatory cross sections can be arranged closely to one another, thereby improving the density of the downstream side edges on the upstream surface of the bedplate and increasing the amount of hydraulic shear acting on the unfiberized stock.", "Especially preferred hole cross sectional shapes include rhombi (that is, “diamond shapes”), squares, rectangles, triangles and chevrons.", "Other preferred shapes include crescents and semi-circles which, though not tesselatory, can be closely arranged on the bedplate surface so as to improve the density of the downstream side edges.", "In accordance with another preferred embodiment, the holes extend from one of the upstream and downstream surfaces to the other at an acute angle relative to an axis normal to the upstream and downstream surfaces.", "Preferably, the holes extend in a pattern combining a helical arrangement with a radial splay so as to present relatively sharp side edges facing into the stock flow immediately above the upstream surface of the bedplate.", "Most preferably, the holes are arranged along arcs or curves coincident with anticipated stock flow lines immediately above the upstream surface of the bedplate and are oriented such that the holes extend into the bedplate and in the anticipated flow direction of the stock so as to present the sharpest possible downstream side edges to the flow.", "This arrangement serves to reduces the drag on the flow of accepts fiber through the bedplate and improve the generation of hydraulic shear near the upstream surface.", "In accordance with yet another preferred embodiment of the invention, the bedplate is fabricated by forming a disc-shaped blank from a metal plate and then forming the holes, preferably by means of a cutting stream.", "One preferred cutting stream is an energy stream, such as a stream of laser or other electromagnetic energy.", "Another preferred stream is a pressurized fluid stream such as a water jet.", "The use of such cutting streams to form the holes simplifies the manufacture of the bedplates and reduces the both time and cost of manufacture.", "The method also facilitates the cutting of the specially shaped and configured holes to improve the density and sharpness of the downstream side edges facing the stock flow.", "The method can be practiced on highly wear resistance materials without the heat treatments or special tools required by prior art methods.", "Since the method is adapted for use with stronger, more wear resistant steels than those typically used in the prior art, it provides for the fabrication of thinner bedplates and of bedplates having useful lives longer than those typical in the prior art.", "Further advantages, objects and features of the present invention will become apparent in the following detail description when considered together with the drawing figures and appended claims." ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to apparatus for use in defiberizing papermaking stock.", "More particularly, the invention relates to extraction bedplates with specially shaped and contoured holes cut by laser energy or a fluid jet for use in pulping apparatus.", "2.Background Art Apparatus for pulping paper making stock is shown in Chupka U.S. Pat.", "No.", "4,725,007, the disclosure of which is incorporated by reference.", "The apparatus shown in U.S. Pat.", "No.", "4,725,007 includes a tub and a rotor mounted within the tub for inducing shear forces which serve to defiberize the stock An extraction bedplate is positioned at the bottom of this tub, surrounded by a frusto-conical wall which serves as a funnel to direct the stock toward the bedplate.", "The preferred bedplate is disc-shaped, defining an upstream surface facing into the tub; a downstream surface facing oppositely from the upstream surface; and holes or apertures extending through the bedplate from the upstream surface to the downstream surface.", "The rotor is mounted near the center of the perforated bedplate and coupled to a motor for rotation about an axis normal to the upstream surface of the bedplate.", "The holes extending through the extraction bedplate allow accepted fiber, that is, pulp which has been defiberized to a degree which is acceptable for further processing to flow out from the apparatus, while retaining larger, undefiberized particles and other solids in the tub.", "Conventional bedplates typically range from 24 inches (61 cm) to 96 inches (2.4 m) in diameter and are typically approximately ⅝ inch (1.6 cm) thick.", "Typically there are 4,000 to 5,000 holes in a 96 inch diameter plate with ⅝ inch holes.", "Since such holes are formed by conventional drilling processes, they have in the past been formed parallel to the axis of the bedplate with circular cross sections.", "The holes generally range from {fraction (1/8)} inch (3.2 mm) to 1 inch (25 mm) in diameter.", "Known extraction bedplates tend to be high maintenance items because of wear.", "Bedplates are exposed to harsh treatment from sand, metal objects and other debris contained within the stock.", "The typical clearance between the rotor and the bedplate is approximately 0.060 inch (1.5 mm) to 0.120 inch (3.0 mm).", "The stock is constantly pushed against, and drug along, the upper surface of the bedplate by the mechanical and hydraulic action of the associated rotor.", "The accepted fiber along with small contaminates which flow through the bedplate contribute to wear within the holes, particularly near the upper perimeters of the downstream edge portions of the holes.", "Bedplates typically are manufactured from steel alloys resistant to wear and corrosion.", "Various stainless steels and 410 hard chrome steel have been used in forming bedplates.", "The 410 hard chrome steel is preferred because it is more wear resistant than the stainless steels.", "On the other hand, the 410 hard chrome steel requires heat treatment to harden the material to restore acceptable wear resistance after known machining and hole-drilling steps are performed.", "Once the heat treatment is performed, further machining is possible only with special tools in a slow and costly procedure.", "The heat treatment itself tends to warp the steel, so that additional manufacturing steps are required to straighten the bedplate.", "The defibering characteristics of a given bedplate are dependent to a large degree on the surface indentations defined by the upper edges of the individual holes.", "More particularly, the paper making stock flows over the upstream surface of the bedplate during operation of the pulping apparatus.", "Hydraulic shear is generated near downstream side edges (that is, edges facing the oncoming stock now) formed at the intersections of the holes with the upstream surface of the bedplate.", "This hydraulic shear acts to break up relatively large, undefiberized particles.", "Increasing the number of such downstream side edges increases the amount of the hydraulic shear, thus improving the efficiency of the pulping apparatus.", "Therefore, there remains a need in the art for extraction bedplates providing improved efficiency and wear resistance.", "Additionally, there remains a need for improved methods for making such bedplates.", "SUMMARY OF THE INVENTION Preferred extraction bedplates in accordance with the present invention have specially shaped and configured holes which provide increased densities of downstream side edges along the upstream surfaces of the bedplates.", "In accordance with one preferred embodiment of the invention, the holes have non-circular cross sections.", "Most preferably, the holes have cross sections with shapes which tesselate a plane, that is, which when laid side-to-side will fill a plane without intervening gaps.", "Individual holes having tesselatory cross sections can be arranged closely to one another, thereby improving the density of the downstream side edges on the upstream surface of the bedplate and increasing the amount of hydraulic shear acting on the unfiberized stock.", "Especially preferred hole cross sectional shapes include rhombi (that is, “diamond shapes”), squares, rectangles, triangles and chevrons.", "Other preferred shapes include crescents and semi-circles which, though not tesselatory, can be closely arranged on the bedplate surface so as to improve the density of the downstream side edges.", "In accordance with another preferred embodiment, the holes extend from one of the upstream and downstream surfaces to the other at an acute angle relative to an axis normal to the upstream and downstream surfaces.", "Preferably, the holes extend in a pattern combining a helical arrangement with a radial splay so as to present relatively sharp side edges facing into the stock flow immediately above the upstream surface of the bedplate.", "Most preferably, the holes are arranged along arcs or curves coincident with anticipated stock flow lines immediately above the upstream surface of the bedplate and are oriented such that the holes extend into the bedplate and in the anticipated flow direction of the stock so as to present the sharpest possible downstream side edges to the flow.", "This arrangement serves to reduces the drag on the flow of accepts fiber through the bedplate and improve the generation of hydraulic shear near the upstream surface.", "In accordance with yet another preferred embodiment of the invention, the bedplate is fabricated by forming a disc-shaped blank from a metal plate and then forming the holes, preferably by means of a cutting stream.", "One preferred cutting stream is an energy stream, such as a stream of laser or other electromagnetic energy.", "Another preferred stream is a pressurized fluid stream such as a water jet.", "The use of such cutting streams to form the holes simplifies the manufacture of the bedplates and reduces the both time and cost of manufacture.", "The method also facilitates the cutting of the specially shaped and configured holes to improve the density and sharpness of the downstream side edges facing the stock flow.", "The method can be practiced on highly wear resistance materials without the heat treatments or special tools required by prior art methods.", "Since the method is adapted for use with stronger, more wear resistant steels than those typically used in the prior art, it provides for the fabrication of thinner bedplates and of bedplates having useful lives longer than those typical in the prior art.", "Further advantages, objects and features of the present invention will become apparent in the following detail description when considered together with the drawing figures and appended claims.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a perspective view of pulping apparatus partially cut away to show an extraction bedplate in accordance with the present invention; FIG.", "2 is a schematic view of a first preferred extraction bedplate in accordance with the present invention; FIG.", "3 is a plan view of a portion of the extraction bedplate of FIG.", "2; FIG.", "4 is a sectional view of the extraction bedplate of FIG.", "2, taken along the line 4-4 of FIG.", "3; FIG.", "5 is a sectional view of the extraction bedplate of FIG.", "2, taken along the line 5-5 of FIG.", "3; FIG.", "6 is a plan view of a portion of a second preferred extraction bedplate in accordance with the present invention with holes having circular cross sections extending at an acute angle with respect to a radius normal to the upstream and downstream surfaces thereof; FIG.", "7 is a plan view of a portion of a third preferred extraction bedplate in accordance with the present invention with holes having crescentic cross sections; FIG.", "8 is a plan view of a portion of a fourth preferred extraction bedplate in accordance with the present invention with holes having square cross sections; FIG.", "9 is a plan view of a portion of a fifth preferred extraction bedplate in accordance with the present invention with rectangular slots or holes; FIG.", "10 is a plan view of a portion of a sixth preferred extraction bedplate in accordance with the present invention with holes having chevronic cross sections; and FIG.", "11 is a schematic view of a seventh preferred extraction bedplate in accordance with the present invention with a combination of holes having rhombic cross sections and rectangular slots; and FIG.", "12 is a flow chart diagramming a preferred method for manufacturing the extraction bedplates of FIGS.", "2-11.DETAIL DESCRIPTION OF THE INVENTION Referring initially to FIG.", "1, there is shown a pulping apparatus 5 of a type used in the paper making industry to defiberize paper making stock (not shown).", "The pulping apparatus 5 includes a tub 6 defining a side wall 7; an extraction bedplate 10 located along a bottom wall 8 of the tub 6; and a rotor 15 proximate the bedplate 10.The clearance between the bedplate 10 and the rotor 15 is approximately 0.060 inch (1.5 mm) to 0.120 inch (3.0 mm).", "The rotor 15 is mounted for rotation about an axis 20.A drive motor 25 is coupled to the rotor 15 to rotate the rotor 15 about the axis 20 in a direction 26 so as to force the paper making stock (not shown) to flow over a substantially planar first or upstream surface 30 of the bedplate 10.As the rotor 15 rotates, it not only forces the paper making stock (not shown) against the upstream surface 30 of the bedplate 10 but also drags the stock along the upper surface 30 in the direction of motion of the rotor 15.As the stock (not shown) drags along the upper surface 30, hydraulic shear generated between the rotor 15 and the bedplate 10 serves to defiberize the stock.", "Defiberized stock (not shown) flows through the bedplate 10 to an accepts conduit (not shown) while larger, undefiberized stock and other solids (not shown) remain within the tub 6 for further processing.", "The pattern of the stock flow (not shown) within the preferred pulping apparatus 5 is a combination of a first circulatory component having a flow direction indicated generally by the arrow 31 and a second circulatory component flowing in the direction of the arrow 26 about the axis 20.The first circulatory component, as indicated generally by the arrow 31, moves downwardly in the region immediately surrounding the central axis 20; radially outwardly near the rotor 15 and the upstream surface 30 of the bedplate 10; upwardly along the outer perimeter of the pulping apparatus 5; and then inwardly toward the central axis 20.The resulting flow pattern (not shown) immediately above the upstream surface 30 follows flow lines symmetric about the axis 20 which lead in an arcuate or curved manner away from the axis 20 toward the side wall 7 of the tub 6.Turning to FIG.", "2, a first preferred extraction bedplate 10 in accordance with the present invention is disc shaped, comprising the first or upstream surface 30; a substantially planar second or downstream surface 35; a circumferential surface 40; and a circular central opening 41 for accommodating the rotor 15 (FIG.", "1).", "The axis 20 extends normally to the upstream and downstream surfaces 30, 35.A plurality of mounting holes 42 provide means for securing the bedplate 10 in the pulping apparatus 5 (FIG.", "1).", "A plurality of holes or apertures 45 extend through the bedplate 10 from the upstream surface 30 to the downstream surface 35.Each hole 45 defines an perimeter 50 where the hole 45 intersects the upstream surface 30.Each such perimeter 50 defines a downstream side edge 55.The bedplate 10 has wearstrips 60, 65 positioned on the upstream and downstream surfaces 30, 35, respectively.", "The wearstrips 60, 65 preferably are shaped as elongated rectangles.", "They are arranged in pairs, one each on the upstream and downstream surfaces 30, 35, extending perpendicularly or obliquely with respect to the other so as to define angles opening outwardly toward the circumferential surface 40.The wearstrips 60, 65 preferably are mounted on land areas 70 substantially free of holes 45 on the upstream and downstream surfaces 30, 35.The wearstrips 60, 65 provides several advantages.", "First, the wearstrips 60, 65 serve to protect the upstream surface 30 of the bedplate 10 from wear due to the action of the rotor 15 (FIG.", "1) and the stock flow (not shown).", "Second, the wearstrips 60, 65 provide visual indications of the relative wear of the upstream and downstream surfaces 30, 35, respectively, and of the downstream portions 55 of the holes 45.Third, the wearstrips 60, 65 are oriented so as to baffle the flow immediately above the upstream surface 30 toward a desired direction within the pulping apparatus 5.The holes 45 of the first preferred bedplate 10 have rhombic cross sections arranged such that major diagonals of the rhombi extend radially with respect to the axis 20.As shown in FIG.", "3, the holes 45 are arranged in rings extending annularly around the bedplate 10.Webs 75 defining land areas on the upstream and downstream sides 30, 35 (FIG.", "2) connect adjacent holes 10.The use of holes 45 having rhombic cross sections arranged in annularly extending rings minimizes the sizes of the land areas defined by the webs 75 and improves the density of the holes on the upstream and downstream surfaces 30, 35 (FIG.", "3) of the bedplate 10.Most preferably, the holes 45 are arranged in a series of arcs or curves 90 coincident with the anticipated direction of the stock flow (not shown) immediately above the upstream surface 30 (FIG.", "2).", "As shown in FIG.", "4, the holes 45 extend through the first preferred bedplate 10 at an obtuse angle relative to surfaces 30, 35; that is, they extend at an acute angle relative to the axis 20 (FIGS.", "1 and 2).", "Furthermore, the extensions of the holes 45 through the bedplate 10 are symmetric with respect to the axis 20 (FIGS.", "1 and 2).", "Most preferably, the holes 45 extend in a pattern combining a helical arrangement, as indicated in FIG.", "4, with a radial splay, as indicated in FIG.", "5, so that the downstream side edges 55 of the holes 45 facing into the direction 90 of the flow of stock (not shown) immediately above the upstream surface 30 are sharper or more knife-like than downstream side edges (not shown) of corresponding holes (not shown) extending perpendicularly to the upstream and downstream surfaces 30, 35 would be.", "This arrangement, wherein the downstream side edges 55 of the holes 45 facing into the anticipated direction 90 of the flow of stock (not shown) immediately above the upstream surface 30 are relatively sharp, decreases the drag on the defiberized stock (not shown) flowing through the holes 45 to the accepts conduit (not shown) while serving to generate hydraulic shear (not shown) to defiberize larger, undefiberized particles (not shown) in the stock.", "While the surfaces 30, 35 have been described as an “upstream surface” and a “downstream surface,” respectively, those skilled in the art will note that the first preferred bedplate 10 is reversible so as to face either of the two surfaces 30, 35 into the pulping apparatus 5 (FIG.", "1) during use.", "Thus, it is possible to install the bedplate 10 in the pulping apparatus 5 (FIG.", "1) such that the “upstream surface” 30 faces upstream toward the rotor 15 (FIG.", "1) and to operate the pulping apparatus 5 (FIG.", "1) until the “upstream surface” 30 undergoes a specific degree of wear.", "The, it is possible to reverse the bedplate 10 such that the formerly “downstream surface” 35 faces upstream toward the rotor 15 (FIG.", "1).", "It will be understood that the particular shapes, sizes, configurations, number and arrangement of the holes 45 shown in FIGS.", "2-5 is not critical to the invention and that other suitable shapes, sizes, configurations, numbers and arrangements of holes (not shown) will be apparent to those of ordinary skill in the art.", "Turning to FIG.", "6, a second preferred extraction bedplate 110 in accordance with the present invention includes holes 145 having circular cross sections.", "The holes 145 extend from a substantially planar first or upstream surface 130 to an opposed substantially planar second or downstream surface (not shown) at an obtuse angle with respect to a substantially planar upstream surface 130, that is, at an acute angle with respect to the axis 20 (FIG.", "1), in the manner illustrated in FIGS.", "4 and 5.Most preferably, the holes 145 extend in a pattern combining a helical arrangement with a radial splay such that downstream side edges 155 of the holes 145 facing into the anticipated direction 190 of the flow of stock (not shown) immediately above the upstream surface 130 are relatively sharp.", "The resulting bedplate 110 is reversible.", "It will be understood that the particular shapes, sizes, configurations, number and arrangement of the holes 145 shown in FIG.", "6 is not critical to the invention and that other suitable shapes, sizes, configurations, numbers and arrangements of holes (not shown) will be apparent to those of ordinary skill in the art.", "Likewise, in FIG.", "7, a third preferred extraction bedplate 210 in accordance with the present invention includes holes 245 having crescentic cross sections arranged in annular rings such that concave faces 241 of the cross sections face the anticipated direction 226 of rotation of the rotor 15 (FIG.", "1).", "Preferably, the holes 245 extend from a substantially planar first or upstream surface 230 to an opposed substantially planar second or downstream surface (not shown) in parallel, or at an acute angle, with respect to the axis 20 (FIG.", "1).", "Most preferably, the holes 245 are arranged along arcs or curves 290 coincident with anticipated stock flow lines (not shown) immediately above the upstream surface 230 of the bedplate 210 and are oriented such that the holes 245 present the sharpest possible downstream side edges 255 to the anticipated stock flow (not shown).", "Once again, it will be understood that the particular shapes, sizes, configurations, number and arrangement of the holes 245 shown in FIG.", "7 is not critical to the invention and that other suitable shapes, sizes, configurations, numbers and arrangements of holes (not shown) will be apparent to those of ordinary skill in the art.", "Likewise, in FIG.", "8, a fourth preferred extraction bedplate 310 in accordance with the present invention includes holes 345 having square cross sections.", "Preferably, the holes 345 extend from a substantially planar first or upstream surface 330 to an opposed substantially planar second or downstream surface (not shown) in parallel, or at an acute angle, with respect to the axis 20 (FIG.", "1).", "Most preferably, the holes 345 are arranged along arcs or curves 390 coincident with anticipated stock flow lines (not shown) immediately above the upstream surface 330 of the bedplate 310 and are oriented such that the holes 345 present the sharpest possible downstream side edges 355 to the anticipated stock flow (not shown).", "Once again, it will be understood that the particular shapes, sizes, configurations, number and arrangement of the holes 345 shown in FIG.", "8 is not critical to the invention and that other suitable shapes, sizes, configurations, numbers and arrangements of holes (not shown) will be apparent to those of ordinary skill in the art.", "Turning to FIG.", "9, a fifth preferred extraction bedplate 410 in accordance with the present invention includes elongated rectangular slots or holes 445 arranged in an angular ring.", "Preferably, the rectangular slots 445 are arranged such that longer side edges 455 of the slots 445 extend radially with respect to the axis 20 (FIG.", "1).", "Most preferably, the holes 445 extend helically, or in a pattern combining a helical arrangement with a radial splay, from the a substantially planar first or upstream surface 430 to a substantially planar second or downstream surface (not shown) such that the side edges 455 of the holes 445 are relatively sharp.", "Once again, it will be understood that the particular shapes, sizes, configurations, number and arrangement of the holes 445 shown in FIG.", "9 is not critical to the invention and that other suitable shapes, sizes, configurations, numbers and arrangements of holes (not shown) will be apparent to those of ordinary skill in the art.", "Likewise, in FIG.", "10, a sixth preferred extraction bedplate 510 in accordance with the present invention includes holes 545 having chevronic cross sections arranged in annular rings such that concave faces 541 of the cross sections face the anticipated direction 526 of rotation of the rotor 15 (FIG.", "1).", "Preferably, the holes 545 extend from a substantially planar first or upstream surface 530 to an opposed substantially planar second or downstream surface (not shown) in parallel, or at an acute angle, with respect to the axis 20 (FIG.", "1).", "Most preferably, the holes 545 are arranged along arcs or curves 590 coincident with anticipated stock flow lines (not shown) immediately above the upstream surface 530 of the bedplate 510 and are oriented such that the holes 545 present the sharpest possible downstream side edges 555 to the anticipated stock flow (not shown).", "Once again, it will be understood that the particular shapes, sizes, configurations, number and arrangement of the holes 545 shown in FIG.", "10 is not critical to the invention and that other suitable shapes, sizes, configurations, numbers and arrangements of holes (not shown) will be apparent to those of ordinary skill in the art.", "Turning to FIG.", "11, a seventh preferred extraction bedplate 610 in accordance with the present invention includes a plurality of holes 645 having rhombic cross sections and a plurality of elongated rectangular slots or holes 646.The holes 645 are arranged in annular rings and are oriented such that major diagonals of the rhombi extend radially with respect to the axis 20.The rectangular slots 646 are arranged in an annular ring surrounding the holes 645 and are elongated in a radial direction relative to the axis 20.Preferably, the holes 645, 646 extend from a substantially planar first or upstream surface 630 to an opposed substantially planar second or downstream surface (not shown) in parallel, or at an acute angle, with respect to the axis 20.Once again, it will be understood that the particular shapes, sizes, configurations, number and arrangement of the holes 645, 646 shown in FIG.", "11 is not critical to the invention and that other suitable shapes, sizes, configurations, numbers and arrangements of holes (not shown) will be apparent to those of ordinary skill in the art.", "From the foregoing, it will be apparent that the extraction bedplates in accordance with the present invention, including the preferred extraction bedplates 10 (FIGS.", "2-5), 110 (FIG.", "6), 210 (FIG.", "7), 310 (FIG.", "8), 410 (FIG.", "9), 510 (FIG.", "10), 610 and 610 (FIG.", "11), are adapted to provide high densities of holes 45 (FIGS.", "2-5), 145 (FIG.", "6), 245 (FIG.", "7), 345 (FIG.", "8), 445 (FIG.", "9), 545 (FIG.", "10), 645 (FIG.", "11) and 646 (FIG.", "11) so as to improve the generation of hydraulic shear near the upstream surfaces 30 (FIGS.", "2-5), 130 (FIG.", "6), 230 (FIG.", "7), 330 (FIG.", "8), 430 (FIG.", "9), 530 (FIG.", "10) and 630 (FIG.", "11) thereof during pulping operations.", "Furthermore, it will be apparent that extending the holes 45 (FIGS.", "2-5), 145 (FIG.", "6), 245 (FIG.", "7), 345 (FIG.", "8), 445 (FIG.", "9), 545 (FIG.", "10), 645 (FIG.", "11) and 646 (FIG.", "11) through the bedplates 10 (FIGS.", "2-5), 110 (FIG.", "6), 210 (FIG.", "7), 310 (FIG.", "8), 410 (FIG.", "9), 510 (FIG.", "10) and 610 (FIG.", "11) at acute angles relative to an axis 20 (FIGS.", "1, 2 and 12) thereof serves to reduce drag on the accepts flow through the holes and to improve the generation of hydraulic shear.", "Turning to FIG.", "12, a preferred method for manufacturing the extraction bedplates 10 (FIGS.", "2-5), 110 (FIG.", "6), 210 (FIG.", "7), 310 (FIG.", "8), 410 (FIG.", "9), 510 (FIG.", "10) and 610 (FIG.", "11) from a metal plate (not shown) includes the step 700 of cutting a disc shaped blank (not shown) from the metal plate and the step 702 of forming the holes 45 (FIGS.", "2-5), 145 (FIG.", "6), 245 (FIG.", "7), 345 (FIG.", "8), 445 (FIG.", "9), 545 (FIG.", "10), 645 (FIG.", "11) and 646 (FIG.", "11) in either the metal plate or the disc shaped blank.", "The order of the steps 700 and 702 is not critical to the invention.", "The step 700 of cutting the disc shaped blank (not shown) from the metal plate (not shown) may be performed by any of a number of suitable techniques well known to those of ordinary skill in the art.", "Preferably, the step 700 includes cutting a circular central opening (e.g., 40 in FIG.", "2) to accomodate the rotor 15 (FIG.", "1).", "Optionally, the step 700 includes any suitable known surface finishing or metallurgical treatment of the disc shaped blank (not shown) to secure desirable strength, wear resistance or smoothness properties.", "The manner in which step 702 is performed is not critical to the present invention and numerous options will be apparent to those of ordinary skill in the art.", "The step 702 is preferably performed using a cutting stream (not shown) such as an energy stream (not shown) or a fluid stream (not shown).", "The preferred energy stream (not shown) comprises focused laser light (not shown), although other suitable electromagnetic or thermal energy streams (not shown) including without limitation cutting torches (not shown) may be used.", "Preferred fluid streams (not shown) include jets (not shown) of water or other fluids.", "Optionally, the method includes the additional step (not shown) of securing the wearstrips (70, 71 in FIG.", "2) on the upstream and downstream surfaces 30, 35 (FIG.", "2); 110 (FIG.", "6); 210 (FIG.", "7); 310 (FIG.", "8); 410 (FIG.", "9); 510 (FIG.", "10); and 610 (FIG.", "11) of the bedplates 10 (FIGS.", "2-5), 110 (FIG.", "6), 210 (FIG.", "7), 310 (FIG.", "8), 410 (FIG.", "9), 510 (FIG.", "10) and 610 (FIG.", "11).", "The use of a laser or water jet to form the holes 45 (FIGS.", "2-5), 145 (FIG.", "6), 245 (FIG.", "7), 345 (FIG.", "8), 445 (FIG.", "9), 545 (FIG.", "10), 645 (FIG.", "11) and 646 (FIG.", "11) simplifies the manufacture of the bedplates 10 (FIGS.", "2-5), 110 (FIG.", "6), 210 (FIG.", "7), 310 (FIG.", "8), 410 (FIG.", "9), 510 (FIG.", "10) and 610 (FIG.", "11) and reduces the both time and cost of manufacture.", "The method also facilitates the cutting of the non-circular cross sections of the holes 45 (FIGS.", "2-5), 145 (FIG.", "6), 245 (FIG.", "7), 345 (FIG.", "8), 445 (FIG.", "9), 545 (FIG.", "10), 645 (FIG.", "11) and 646 (FIG.", "11) as well as the cutting of the holes at an acute angle from the axis 20 (FIGS.", "1, 2 and 11), thereby improving the performance of the bedplates 10 (FIGS.", "2-5), 110 (FIG.", "6), 210 (FIG.", "7), 310 (FIG.", "8), 410 (FIG.", "9), 510 (FIG.", "10) and 610 (FIG.", "11).", "Furthermore, the use of a laser or water jet to form the holes 45 (FIGS.", "2-5), 145 (FIG.", "6), 245 (FIG.", "7), 345 (FIG.", "8), 445 (FIG.", "9), 545 (FIG.", "10), 645 (FIG.", "11) and 646 (FIG.", "11) enables the cutting of stronger, more wear resistant metals than those typically used in the prior art, thereby permitting the fabrication of thinner bedplates 10 (FIGS.", "2-5), 110 (FIG.", "6), 210 (FIG.", "7), 310 (FIG.", "8), 410 (FIG.", "9), 510 (FIG.", "10) and 610 (FIG.", "11) and of bedplates having useful lives longer than those typical in the prior art." ] ]	Patent_10466308
[ [ "Former and headbox for said former", "A former for a machine for the production of a fibrous web, particularly a paper or cardboard web including a headbox equipped with at least one stock suspension feed, one turbulence block equipped with several channels and/or a tube generator equipped with several channels, and a headbox nozzle whose suspension jet strikes an exposed or open surface of a dewatering belt, specifically a wire.", "Turbulence generating elements are allocated to at least a section of the channels and/or the headbox nozzle, in order to create turbulent flows in the suspension substreams in the channels, or in the headbox nozzle.", "The side of the suspension stream facing away from the dewatering belt is covered at least partially by a wall.", "A danger of the suspension stream bursting open is reduced to a minimum.", "In addition, swirling motions of considerably higher intensity than occur on conventional Fourdrinier wire formers are now permissible." ], [ "1-53.", "(Canceled) 54.A former for a machine for the production of a fibrous web, comprising: a headbox having: at least one stock suspension feed; at least one turbulence block connected to said at least one stock suspension feed, at least one said turbulence block having at least one of a plurality of channels and a tube generator having said plurality of channels, said plurality of channels having a plurality of suspension sub-streams running in said channels, said plurality of channels having at least one section; a headbox nozzle connected to at least one said turbulence block, said headbox nozzle producing a suspension stream; and turbulence generating elements allocated to at least one of said at least one section of said channels and said headbox nozzle, said turbulence generating elements creating a plurality of turbulent flows in one of said plurality of suspension sub-streams and said headbox nozzle; a dewatering belt having an open surface, said suspension stream incident on said open surface; and a wall at least partially covering a side of said suspension stream facing away from said dewatering belt.", "55.The former of claim 54, wherein said former is a suction former.", "56.The former of claim 54, wherein said dewatering belt is a wire.", "57.The former of claim 54, wherein said wall is a stationary wall.", "58.The former of claim 57, wherein said stationary wall is installed on said headbox.", "59.The former of claim 58, wherein said stationary wall is adjustably installed on said headbox.", "60.The former of claim 54, wherein said wall is in motion.", "61.The former of claim 54, wherein said wall is permeable to water.", "62.The former of claim 54, wherein said wall is impermeable to water.", "63.The former of claim 54, wherein said wall is formed by a second dewatering belt.", "64.The former of claim 63, wherein said second dewatering belt is a revolving wire.", "65.The former of claim 54, wherein said wall includes a coverage area, said coverage area includes an at least partially curved progression.", "66.The former of claim 54, wherein said wall includes a coverage area, said coverage area includes an at least partially straight progression.", "67.The former of claim 54, wherein said wall includes both an upper wall with an upper wall length and a lower wall with a lower wall length, said lower wall length is not greater than 90% of said upper wall length.", "68.The former of claim 67, wherein said lower wall length is not greater than 60% of said upper wall length.", "69.The former of claim 67, wherein said lower wall length is not greater than 30% of said upper wall length.", "70.The former of claim 54, wherein at least one said section of said channels includes at least one turbulence causing insert.", "71.A former for a machine for the production of a fibrous web, comprising: a headbox having: at least one stock suspension feed; at least one turbulence block connected to said at least one stock suspension feed, at least one said turbulence block having at least one of a plurality of channels and a tube generator having said plurality of channels, said plurality of channels having a plurality of suspension sub-streams running in said channels, said plurality of channels having at least one section; a headbox nozzle connected to at least one said turbulence block; and turbulence generating elements allocated to at least one said section of said channels, said turbulence generating elements creating a plurality of turbulent flows in said plurality of suspension sub-streams, each said turbulent flow rotating in a similar direction in said plurality of suspension sub-streams.", "72.The former of claim 71, wherein said former is one of a suction former and a twin wire former.", "73.The former of claim 71, further including helix spirals installed in at least one said section of said channels for a production of said turbulent flows that rotate in said similar direction.", "74.The former of claim 71, wherein said headbox includes a tube generator with nozzles for a feeding of a stock suspension into a respective said channel, each said channel has a corresponding center plane progressing in a longitudinal direction through said channel, said feeding occurs asymmetrically relative to each said center plane.", "75.The former of claim 74, wherein each said channel has a respective channel wall, in each said channel said feeding occurs approximately tangentially to said respective channel wall.", "76.The former of claim 74, further including a plurality of jets in fluid communication with said stock suspension and corresponding said channels, said stock suspension being fed through each said jet to said corresponding channel on only one side of said corresponding center plane.", "77.The former of claim 76, wherein said stock suspension is fed through each said jet to said corresponding channel on the same side of said corresponding center plane for each said channel.", "78.The former of claim 71, wherein said headbox includes at least one inlet for a supply of at least one of dilution water, air and chemicals.", "79.The former of claim 78, wherein said supply of at least one of dilution water, air and chemicals occurs essentially in an axial direction of a respective channel.", "80.The former of claim 71, further including both a forming roll having a second radius of curvature and a wall being located in an area opposite said forming roll, said wall being both at least partially curved and having a first radius of curvature, said first radius of curvature equal to or greater than said second radius of curvature.", "81.The former of claim 80, wherein said wall is stationary.", "82.The former of claim 80, wherein said wall covers a circumferential length of said forming roll, said circumferential length approximately between 100 to 400 mm.", "83.The former of claim 81, wherein said wall is one of rigid, deflection resistant and flexible.", "84.The former of claim 80, wherein said wall is both movable and is formed by an additional dewatering belt, said wall covers a circumferential length of said forming roll, said circumferential length approximately between 100 to 1500 mm, said circumferential length approximately between 100 to 400 mm corresponds with a circumferential angle of said forming roll approximately between 25° to 120°.", "85.The former of claim 84, wherein said additional dewatering belt is a revolving wire.", "86.The former of claim 84, wherein said dewatering belt is routed around a breast roll prior to said circumferential length when viewed in a direction of belt travel, said breast roll has a third radius of curvature, said first radius of curvature in an area covering said forming roll is greater than said third radius of curvature.", "87.The former of claim 71, further including at least one lamellar plate sectioning said headbox nozzle.", "88.The former of claim 87, wherein said headbox nozzle includes at least one nozzle wall, at least one of said at least one lamellar plate and said at least one nozzle wall has a whirl producing contour.", "89.The former of claim 88, wherein said whirl producing contour is a washboard countour.", "90.The former of claim 88, wherein at least one of said at least one lamellar plate and said at least one nozzle wall include contours which generate turbulence.", "91.The former of claim 90, wherein at least one of said at least one lamellar plate and said at least one nozzle wall include at least one interference body.", "92.The former of claim 91, wherein said at least one interference body of said at least one nozzle wall is a discontinuous tapering of a cross section of said at least one nozzle wall.", "93.The former of claim 87, wherein said headbox nozzle has a headbox nozzle length, said at least one lamellar plate has a lamellar plate length not exceeding 70% of said headbox nozzle length.", "94.The former of claim 71, wherein said headbox nozzle has a headbox nozzle length less than approximately 400 mm.", "95.The former of claim 71, further including an outlet cross section of said channels being essentially round.", "96.A headbox for a machine for the production of a fibrous web, comprising: at least one stock suspension feed; at least one turbulence block connected to said at least one stock suspension feed, at least one said turbulence block having at least one of a plurality of channels and a tube generator having said plurality of channels, said plurality of channels having a plurality of suspension sub-streams running in said channels, said plurality of channels having at least one section; a headbox nozzle connected to at least one said turbulence block; and turbulence generating elements allocated to at least one said section of said channels, said turbulence generating elements creating a plurality of same direction turbulent flows in said plurality of suspension sub-streams.", "97.The headbox of claim 96, wherein said headbox is for a former.", "98.The headbox of claim 96, further including both a stationary wall connected to said headbox nozzle and a suspension stream emerging from said headbox nozzle, said stationary wall covering said suspension stream.", "99.The headbox of claim 98, wherein said stationary wall is adjustable.", "100.The headbox of claim 98, wherein said stationary wall is permeable to water.", "101.The former of claim 98, wherein said stationary wall is impermeable to water.", "102.The former of claim 98, wherein said stationary wall includes both an upper wall with an upper wall length and a lower wall with a lower wall length, said lower wall length is not greater than 90% of said upper wall length.", "103.The former of claim 102, wherein said lower wall length is not greater than 60% of said upper wall length.", "104.The former of claim 102, wherein said lower wall length is not greater than 30% of said upper wall length.", "105.The former of claim 96, wherein said at least one section of said channels includes at least one turbulence generating insert.", "106.A headbox for a machine for the production of a fibrous web, comprising: at least one stock suspension feed; at least one turbulence block connected to said at least one stock suspension feed, at least one said turbulence block having at least one of a plurality of channels and a tube generator having said plurality of channels, said plurality of channels having a plurality of suspension sub-streams running in said channels, said plurality of channels having at least one section; a headbox nozzle connected to at least one said turbulence block; and turbulence generating elements allocated to at least one said section of said channels, said turbulence generating elements creating a plurality of turbulent flows in said plurality of suspension sub-streams, each said turbulent flow rotating in a similar direction in said plurality of suspension sub-streams.", "107.The headbox of claim 106, wherein said headbox is for a former.", "108.The headbox of claim 106, further including helix spirals installed in at least one said section of said channels for a production of said turbulent flows that rotate in said similar direction.", "109.The headbox of claim 106, wherein said headbox includes a tube generator with nozzles for a feeding of a stock suspension into a respective said channel, each said channel has a corresponding center plane progressing in a longitudinal direction through said channel, said feeding occurs asymmetrically relative to each said center plane.", "110.The headbox of claim 109, wherein each said channel has a respective channel wall, in each said channel said feeding occurs approximately tangentially to said respective channel wall.", "111.The headbox of claim 109, further including a plurality of jets in fluid communication with said stock suspension and corresponding said channels, said stock suspension being fed through each said jet to said corresponding channel on only one side of said corresponding center plane.", "112.The headbox of claim 111, wherein said stock suspension is fed through each said jet to said corresponding channel on a same side of said corresponding center plane for each said channel.", "113.The headbox of claim 106, wherein said headbox includes at least one inlet for a supply of at least one of dilution water, air and chemicals.", "114.The headbox of claim 113, wherein said supply of at least one of dilution water, air and chemicals occurs essentially in an axial direction of a respective channel.", "115.The headbox of claim 106, further including at least one lamellar plate sectioning said headbox nozzle.", "116.The headbox of claim 115, wherein said headbox nozzle includes at least one nozzle wall, at least one of said at least one lamellar plate and said at least one nozzle wall has a whirl producing contour.", "117.The headbox of claim 116, wherein said whirl producing contour is a washboard countour.", "118.The headbox of claim 116, wherein at least one of said at least one lamellar plate and said at least one nozzle wall include contours which generate turbulence.", "119.The headbox of claim 118, wherein at least one of said at least one lamellar plate and said at least one nozzle wall include at least one interference body.", "120.The headbox of claim 119, wherein said at least one interference body of said at least one nozzle wall is a discontinuous tapering of a cross section of said at least one nozzle wall.", "121.The headbox of claim 115, wherein said headbox nozzle has a headbox nozzle length, said at least one lamellar plate has a lamellar plate length not exceeding 70% of said headbox nozzle length.", "122.The headbox of claim 106, wherein said headbox nozzle has a headbox nozzle length less than approximately 400 mm.", "123.The headbox of claim 106, further including an outlet cross section of said channels which is essentially round." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to a machine a machine for the production of a fibrous web, and, more particularly, to a former and former headbox.", "2.Description of the Related Art With suction formers, a longitudinal orientation of the fibers occurs at an appropriate increase in the operating speed of the paper machine in question.", "This may limit the scope of application of such suction formers to low operational speeds.", "A relatively strong transverse orientation of the fibers, as well as a low longitudinal/transverse ratio can be achieved (see for example U.S. Pat.", "No.", "5,876,364) through transverse movements that are indicated through various swirl-producing bodies in the turbulence block.", "However, a consequence of these transverse motions is the inherent danger of the suspension stream that is delivered by the headbox of the respective former bursting open when encountering an open surface, for example on the Fourdrinier wire." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides an improved former and former headbox whereby the cited problems are eliminated.", "The present invention provides a former, particularly a suction former of a machine for the production of a fibrous web, particularly a paper or cardboard web comprising a headbox equipped with at least one stock suspension feed, one turbulence block equipped with several channels and/or a tube generator equipped with several channels, and a headbox nozzle whose suspension jet strikes an exposed or open surface of a dewatering belt, specifically a wire.", "Turbulence generating elements are allocated to at least a section of the channels and/or the headbox nozzle, in order to create turbulent flows in the suspension substreams in the channels, or in the headbox nozzle.", "The side of the suspension stream facing away from the dewatering belt is covered at least partially by a wall.", "Based on this configuration, the danger of the suspension stream bursting open is reduced to a minimum.", "In addition, swirling motions of considerably higher intensity than occur on conventional Fourdrinier wire formers are now permissible.", "The wall can be stationary or in motion.", "A stationary wall can, for example, be allocated to the headbox on which it can be installed adjustably.", "The wall can in addition, be permeable to water or impermeable to water.", "A movable wall or a wall in motion can be in the embodiment of an additional dewatering belt, that can specifically be in the form of a revolving wire.", "In the coverage area the wall can possess an at least partially curved progression and/or an at least partially straight progression.", "Moreover, in a practical embodiment the lower wall has a maximum length of 90%, preferably of 60%, especially of 30% of the length of the upper wall.", "In a preferred practical embodiment of the former in accordance with the present invention, at least some of the channels are equipped with the turbulence generating inserts.", "Such turbulence generating inserts can basically be provided in the channels of a respective turbulence block and/or in the channels of a respective tube generator.", "In hitherto conventional suction formers or in twin wire formers eddies occur in pairs in opposite rotational directions, that can lead to stripes.", "These are known as the so-called “Taylor-Görtler-Eddies” in Central Europe.", "The stripes occur especially in curved dewatering surfaces.", "The present invention provides an improved former, particularly a suction former or twin wire former for a machine for the production of a fibrous web, specifically a paper or cardboard web, in which the previously cited problems are eliminated.", "In accordance with an additional aspect of the present invention a former, specifically a suction former or twin wire former of a machine for the production of a fibrous web, specifically a paper or cardboard web is provided or this purpose; comprising a headbox that is equipped with at least one stock suspension feed, one turbulence block equipped with several channels and/or a tube generator equipped with several channels and a headbox nozzle.", "Turbulence generating elements are allocated to at least a section of the channels in order to create turbulent flows that rotate in the same direction in the suspension substreams in the channels.", "Same directional turbulence movement suppresses the undesirable stripes.", "In a functional practical embodiment of the present invention helix type spirals are installed in at least a section of the channels in order to create turbulent flows that rotate in the same direction.", "Such helix type spirals can, for example, be installed in the channels of a turbulence block and/or in the channels of tube generator.", "The creation of turbulences through the means of helix type spirals can for example occur as described in U.S. Pat.", "No.", "5,876,464.Alternatively, or in addition, turbulent flows that are rotating in the same direction can be created particularly by the fact that, in a headbox comprising a tube generator, the supply of stock suspension through nozzles into a respective tube channel occurs asymmetrically and preferably at least essentially tangentially to the tube wall, relative to a center plane progressing in longitudinal direction through the tube channel.", "Advantageously, the stock suspension is fed through nozzles only on one side of the center plane respectively.", "Preferably, suspension supplied to the relevant pipe channels is fed into the various pipe channels through nozzles always on the same side of the respective center planes.", "In contrast to Fourdrinier wires, the previously discussed, deliberately produced turbulences are kept very small in the sheet formation zone in other types of formers, particularly in suction and twin wire formers, since the surface of the suspension is “covered”, resulting in improved sheet formation, even at an increased stock consistency.", "In addition the headbox can be equipped with at least one feed for dilution water, air, chemicals and/or similar substances.", "The supply of dilution water, air, chemicals and/or similar substances may essentially occur in axial direction of a respective channel.", "In accordance with an advantageous embodiment of the present invention, the wall can be located in an area opposite the forming roll and can be at least partially curved according to a radius of curvature that is preferably larger than or equal to the radius of the forming roll.", "Basically however, a smaller radius is also feasible, at least in sections.", "The wall can for example be stationary.", "Thereby it would preferably cover the circumferential surface of the forming roll, that is in a range of approximately 100 to approximately 400 mm.", "The stationary wall can particularly be rigid, deflection resistant or not deflection resistant.", "In accordance with an additional advantageous design of the present invention whereby the wall is again provided in an area opposite the forming roll and is curved, at least partially, according to a curvature radius that is preferably larger than or equal to the radius of the forming roll, the wall can be movable or in motion and can be formed by an additional dewatering belt, particularly a revolving wire.", "In this instance, the wall preferably covers a circumferential area of this forming roll that is in the range of approximately 100 mm to approximately 1500 mm and/or corresponds with a circumferential angle of the forming roll of approximately 25° to approximately 120°.", "The additional dewatering belt that forms the wall can be routed around a breast roll, prior to the area covering the forming roll, when viewed in the direction of belt travel.", "The curvature radius of the wall in the area covering the forming roll is preferably larger than the radius of the breast roll, or respectively the corresponding curvature radius of the wall in the area of this breast roll.", "The headbox nozzle can be sectioned by at least one lamellar plate.", "A configuration without lamellar plates is also feasible.", "In a functional advantageous design at least one lamellar plate and/or at least one nozzle wall exhibit contours, especially swirl-producing washboard contours.", "Alternatively, or in addition, at least one lamellar plate and/or at least one nozzle wall can have contours that serve to generate turbulent motion.", "At least one lamellar plate and/or at least one nozzle wall can for example be equipped with at least one interference body.", "Preferably the at least one interference body of the at least one nozzle wall is in the form of preferably a discontinuous tapering of the cross section.", "This formation provides a fluidic ideal turbulence chamber.", "In addition, at least one lamellar plate has a length not exceeding 70% of the length of the headbox nozzle.", "In order to attain a sufficient effect, the headbox nozzle should be as short as possible.", "The headbox nozzle should preferably be shorter than approximately 400 mm.", "It is also advantageous if the outlet cross section of the channels or pipes is at least essentially round, since a square outlet cross section would dampen the effect.", "In accordance with the present invention a headox is furthermore disclosed especially for a former of the relevant type previously described.", "Such a headbox includes at least one stock suspension feed, one turbulence block equipped with several channels and/or a tube generator equipped with several channels and a headbox nozzle.", "Turbulence generating elements are allocated to at least a section of the channels in order to create turbulent flows that rotate in the same direction in the suspension sub-streams in the channels.", "In accordance with an additional aspect of the present invention a headbox, particularly for a former of the type previously described is provided, including at least one stock suspension feed, one turbulence block equipped with several channels and/or a tube generator equipped with several channels and a headbox nozzle.", "Turbulence generating elements are allocated to at least a section of the channels in order to create turbulent flows, rotating in the same direction, in the suspension sub-streams in the channels." ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to a machine a machine for the production of a fibrous web, and, more particularly, to a former and former headbox.", "2.Description of the Related Art With suction formers, a longitudinal orientation of the fibers occurs at an appropriate increase in the operating speed of the paper machine in question.", "This may limit the scope of application of such suction formers to low operational speeds.", "A relatively strong transverse orientation of the fibers, as well as a low longitudinal/transverse ratio can be achieved (see for example U.S. Pat.", "No.", "5,876,364) through transverse movements that are indicated through various swirl-producing bodies in the turbulence block.", "However, a consequence of these transverse motions is the inherent danger of the suspension stream that is delivered by the headbox of the respective former bursting open when encountering an open surface, for example on the Fourdrinier wire.", "SUMMARY OF THE INVENTION The present invention provides an improved former and former headbox whereby the cited problems are eliminated.", "The present invention provides a former, particularly a suction former of a machine for the production of a fibrous web, particularly a paper or cardboard web comprising a headbox equipped with at least one stock suspension feed, one turbulence block equipped with several channels and/or a tube generator equipped with several channels, and a headbox nozzle whose suspension jet strikes an exposed or open surface of a dewatering belt, specifically a wire.", "Turbulence generating elements are allocated to at least a section of the channels and/or the headbox nozzle, in order to create turbulent flows in the suspension substreams in the channels, or in the headbox nozzle.", "The side of the suspension stream facing away from the dewatering belt is covered at least partially by a wall.", "Based on this configuration, the danger of the suspension stream bursting open is reduced to a minimum.", "In addition, swirling motions of considerably higher intensity than occur on conventional Fourdrinier wire formers are now permissible.", "The wall can be stationary or in motion.", "A stationary wall can, for example, be allocated to the headbox on which it can be installed adjustably.", "The wall can in addition, be permeable to water or impermeable to water.", "A movable wall or a wall in motion can be in the embodiment of an additional dewatering belt, that can specifically be in the form of a revolving wire.", "In the coverage area the wall can possess an at least partially curved progression and/or an at least partially straight progression.", "Moreover, in a practical embodiment the lower wall has a maximum length of 90%, preferably of 60%, especially of 30% of the length of the upper wall.", "In a preferred practical embodiment of the former in accordance with the present invention, at least some of the channels are equipped with the turbulence generating inserts.", "Such turbulence generating inserts can basically be provided in the channels of a respective turbulence block and/or in the channels of a respective tube generator.", "In hitherto conventional suction formers or in twin wire formers eddies occur in pairs in opposite rotational directions, that can lead to stripes.", "These are known as the so-called “Taylor-Görtler-Eddies” in Central Europe.", "The stripes occur especially in curved dewatering surfaces.", "The present invention provides an improved former, particularly a suction former or twin wire former for a machine for the production of a fibrous web, specifically a paper or cardboard web, in which the previously cited problems are eliminated.", "In accordance with an additional aspect of the present invention a former, specifically a suction former or twin wire former of a machine for the production of a fibrous web, specifically a paper or cardboard web is provided or this purpose; comprising a headbox that is equipped with at least one stock suspension feed, one turbulence block equipped with several channels and/or a tube generator equipped with several channels and a headbox nozzle.", "Turbulence generating elements are allocated to at least a section of the channels in order to create turbulent flows that rotate in the same direction in the suspension substreams in the channels.", "Same directional turbulence movement suppresses the undesirable stripes.", "In a functional practical embodiment of the present invention helix type spirals are installed in at least a section of the channels in order to create turbulent flows that rotate in the same direction.", "Such helix type spirals can, for example, be installed in the channels of a turbulence block and/or in the channels of tube generator.", "The creation of turbulences through the means of helix type spirals can for example occur as described in U.S. Pat.", "No.", "5,876,464.Alternatively, or in addition, turbulent flows that are rotating in the same direction can be created particularly by the fact that, in a headbox comprising a tube generator, the supply of stock suspension through nozzles into a respective tube channel occurs asymmetrically and preferably at least essentially tangentially to the tube wall, relative to a center plane progressing in longitudinal direction through the tube channel.", "Advantageously, the stock suspension is fed through nozzles only on one side of the center plane respectively.", "Preferably, suspension supplied to the relevant pipe channels is fed into the various pipe channels through nozzles always on the same side of the respective center planes.", "In contrast to Fourdrinier wires, the previously discussed, deliberately produced turbulences are kept very small in the sheet formation zone in other types of formers, particularly in suction and twin wire formers, since the surface of the suspension is “covered”, resulting in improved sheet formation, even at an increased stock consistency.", "In addition the headbox can be equipped with at least one feed for dilution water, air, chemicals and/or similar substances.", "The supply of dilution water, air, chemicals and/or similar substances may essentially occur in axial direction of a respective channel.", "In accordance with an advantageous embodiment of the present invention, the wall can be located in an area opposite the forming roll and can be at least partially curved according to a radius of curvature that is preferably larger than or equal to the radius of the forming roll.", "Basically however, a smaller radius is also feasible, at least in sections.", "The wall can for example be stationary.", "Thereby it would preferably cover the circumferential surface of the forming roll, that is in a range of approximately 100 to approximately 400 mm.", "The stationary wall can particularly be rigid, deflection resistant or not deflection resistant.", "In accordance with an additional advantageous design of the present invention whereby the wall is again provided in an area opposite the forming roll and is curved, at least partially, according to a curvature radius that is preferably larger than or equal to the radius of the forming roll, the wall can be movable or in motion and can be formed by an additional dewatering belt, particularly a revolving wire.", "In this instance, the wall preferably covers a circumferential area of this forming roll that is in the range of approximately 100 mm to approximately 1500 mm and/or corresponds with a circumferential angle of the forming roll of approximately 25° to approximately 120°.", "The additional dewatering belt that forms the wall can be routed around a breast roll, prior to the area covering the forming roll, when viewed in the direction of belt travel.", "The curvature radius of the wall in the area covering the forming roll is preferably larger than the radius of the breast roll, or respectively the corresponding curvature radius of the wall in the area of this breast roll.", "The headbox nozzle can be sectioned by at least one lamellar plate.", "A configuration without lamellar plates is also feasible.", "In a functional advantageous design at least one lamellar plate and/or at least one nozzle wall exhibit contours, especially swirl-producing washboard contours.", "Alternatively, or in addition, at least one lamellar plate and/or at least one nozzle wall can have contours that serve to generate turbulent motion.", "At least one lamellar plate and/or at least one nozzle wall can for example be equipped with at least one interference body.", "Preferably the at least one interference body of the at least one nozzle wall is in the form of preferably a discontinuous tapering of the cross section.", "This formation provides a fluidic ideal turbulence chamber.", "In addition, at least one lamellar plate has a length not exceeding 70% of the length of the headbox nozzle.", "In order to attain a sufficient effect, the headbox nozzle should be as short as possible.", "The headbox nozzle should preferably be shorter than approximately 400 mm.", "It is also advantageous if the outlet cross section of the channels or pipes is at least essentially round, since a square outlet cross section would dampen the effect.", "In accordance with the present invention a headox is furthermore disclosed especially for a former of the relevant type previously described.", "Such a headbox includes at least one stock suspension feed, one turbulence block equipped with several channels and/or a tube generator equipped with several channels and a headbox nozzle.", "Turbulence generating elements are allocated to at least a section of the channels in order to create turbulent flows that rotate in the same direction in the suspension sub-streams in the channels.", "In accordance with an additional aspect of the present invention a headbox, particularly for a former of the type previously described is provided, including at least one stock suspension feed, one turbulence block equipped with several channels and/or a tube generator equipped with several channels and a headbox nozzle.", "Turbulence generating elements are allocated to at least a section of the channels in order to create turbulent flows, rotating in the same direction, in the suspension sub-streams in the channels.", "BRIEF DESCRIPTION OF THE DRAWINGS The above-mentioned and other features and advantages of this invention, and the manner of attaining them, will become more apparent and the invention will be better understood by reference to the following description of embodiments of the invention taken in conjunction with the accompanying drawings, wherein: FIG.", "1 is a partially schematic side view of an embodiment of a suction former of the present invention whose headbox includes a turbulence block that is equipped with turbulence generating inserts, and that is equipped with a stationary preferably adjustable wall, that covers the suspension stream, at least partially, on one side; FIGS.", "2-2d are cross-sectional views of various designs of the turbulence generating inserts of the present invention, viewed in the direction of arrow V in FIG.", "1; FIG.", "3 is a schematic cross-sectional view of a headbox equipped with tube generator, as viewed along line I-I in FIG.", "4, whereby the feeding of the stock suspension through nozzles into a respective tube channel occurs asymmetric and at least essentially tangential to the tube wall; FIG.", "4 is a schematic longitudinal section of an embodiment of the headbox of the present invention; FIG.", "5 is a schematic longitudinal section of an embodiment of headbox of the present invention including a turbulence block or tube generator into whose channels, at least into sections, helix type spirals or helix are installed; FIG.", "6 is a schematic side view of an embodiment of a former of the present invention with a movable wall, or a wall in motion that is formed by an additional dewatering belt and is located in an area opposite the forming roll and that covers a certain circumferential length of this forming roll; FIG.", "7 is a schematic longitudinal sectional view of an embodiment of a headbox nozzle, of the present invention that is sectioned by at least one lamellar plate, whereby at least one lamellar plate is equipped with at least one interference body; FIG.", "8 is a schematic perspective view of an embodiment of a headbox nozzle of the present invention that is sectioned by at least one lamellar plate, whereby at least one lamellar plate has a washboard contour; and FIG.", "9 is a schematic side view of an embodiment of a headbox of the present invention sectioned by at least one lamellar plate, whereby the upper and lower nozzle wall respectively are contoured.", "Corresponding reference characters indicate corresponding parts throughout the several views.", "The exemplifications set out herein illustrate one preferred embodiment of the invention, in one form, and such exemplifications are not to be construed as limiting the scope of the invention in any manner.", "DETAILED DESCRIPTION OF THE INVENTION Referring now to the drawings, and more particularly to FIG.", "1, there is shown a schematic side view of a former 10, which in this example is a suction former of a machine for the production of a fibrous web that may specifically be a paper or cardboard web.", "A headbox 12 that is allocated to this former 10 includes a stock suspension feed 14, at least one turbulence block 18 that is equipped with several channels 16 and a headbox nozzle 20 whose suspension stream 22 strikes an exposed, that is an open surface 24′ of dewatering belt 24, which in this example is a wire.", "Turbulence generating elements 26, in this example turbulence generating inserts, are allocated to at least one section of channels 16, in order to create turbulent flows 28 in the suspension substreams that are guided through the channels (also see FIG.", "2).", "These turbulent flows 28 are illustrated schematically in FIGS.", "2-2d, which show front views of various designs of the turbulence generating inserts, in the direction of arrow V in FIG.", "1.The side of suspension stream 22 facing away from the dewatering belt 24 is covered at least partially by wall 30.In the present example wall 30 is stationary, installed preferably adjustably on headbox 12.As can be seen in FIG.", "1, wall 30 can, for example, be connected via link 32 with headbox nozzle 20.Wall 30 that is mounted on headbox nozzle 20 in this manner can be adjusted through pivoting by adjustment elements 34.Headbox nozzle 20 can, for example, be sectioned by at least one lamellar plate 36.As can be seen in FIG.", "1, wall 30 is curved at least slightly in the area of coverage.", "Wall 30 can, for example, be located in an area opposite forming roll 38.In the present example wall 30 is curved in accordance with a radius of curvature R1, that is larger than the radius R2 of the forming roll 38.Wall 30 can include an upper wall shown at 30 and lower wall 30′.", "Lower wall 30′ has a lower wall length not greater than 90%, preferably 60%, especially 30% of an upper wall length.", "Stationary wall 30 can for example cover a circumferential length L of forming roll 38, that is in a range of approximately 100 to approximately 400 mm.", "In FIG.", "1 the circumferential angle of forming roll 38 resulting from a respective circumferential length L is indicated as “α”.", "The stationary wall may be rigid, deflection resistant or not deflection resistant.", "Curvature radius R1 of wall 30 can, if necessary, be larger than or equal to radius R2 of forming roll 38.In certain instances a smaller curvature radius R1 is also feasible.", "In the example illustrated in FIG.", "1 an additional dewatering belt 40, especially a wire is provided that is brought together with dewatering belt 24 at a location A.", "Furthermore, at least one disturbance body 60 of at least one nozzle wall 30 is designed as preferably a discontinuous tapering of cross section 64 (shown as broken lines).", "The tapering of the cross section may however also occur continuously and/or randomly.", "In the present example wall 30 is impermeable to water.", "As can be seen especially from the following design examples, this type of wall may basically also be water permeable, especially if a movable wall or a wall in motion is provided in the embodiment of a dewatering belt, such as especially a wire.", "FIGS.", "3 and 4 are schematic illustrations of headbox 12 including tube generator 42 that is equipped with several channels 16 whereby turbulence generating elements are allocated to at least a section of channels 16, in order to create turbulent flows 28 that rotate in the same directions in the suspension sub-streams in the relating channels 16.The turbulence generating elements in the present example include nozzles 44 through which the supply of stock suspension into a respective tube channel 16 occurs asymmetrically and at least essentially tangentially to tube wall 46.Supply of the stock suspension through the nozzles occurs asymmetrically, relative to center plane E progressing in longitudinal direction through tube channel 16.In the present example this is always only on one side of this center plane E. As can be seen in FIG.", "3, feeding into the various tube channels 16 through the nozzles always occurs on the same side of the center plane E, for example the right side in FIG.", "3, resulting in same directional rotation of the various turbulent flows 28.As can be seen in FIGS.", "3 and 4, in addition to stock suspension feed 14, headbox 12 can additionally be equipped with at least one inlet 48 for dilution water, air, chemicals and/or other substances.", "As seen in FIG.", "4 the supply of dilution water, air, chemicals and/or similar substances can essentially occur in an axial direction of a respective channel.", "Again, headbox nozzle 20 can be provided with or without lamellar plates.", "A turbulence block 18 can also be provided alternatively or in addition to the tube generator 42.As illustrated in particular in FIG.", "4, an intermediate chamber 50 can be provided, for example, following tube generator 42 which is equipped with rotation channels 16.In the available design example the rotational flow is produced by the asymmetric or tangential inflow of suspension.", "FIG.", "5 is a schematic longitudinal sectional view of another design form of a headbox 12, including tube generator 42.A turbulence block can again be provided alternatively or in addition.", "In the present example helix-type spiral or helix 52 are installed in at least a section of channels 16 of tube generator 42, or in the turbulence block, for the purpose of producing turbulent flows rotating in the same direction.", "As can be seen in FIG.", "5, headbox 12 also includes stock suspension feed 14, a headbox nozzle 20 and a stationary, preferably adjustable wall 30.Headbox nozzle 20 can be sectioned by at least one lamellar plate 36, or can also be configured withouth lamellar plates.", "Helix-type spirals 52 can be configured and installed specifically so that same-directionally rotating turbulent flows again occur.", "Moreover, a respective rotational flow initiated by a relating helix-type spiral 52 can be produced, for example, as described in U.S. Pat.", "No.", "5,876,564.In the design form illustrated in FIG.", "5 an acceleration segment a of the headbox is followed by a section b that shows helix-type spirals 52.This is followed by a section c, for example constant deceleration or acceleration, further followed by an additional acceleration section d. A deceleration occurs in a subsequent section e. FIG.", "6 is a schematic partial illustration of former 10 that is equipped with a movable wall 30, or a wall in motion, that is formed by an additional dewatering belt 54 and that is located in area opposite forming roll 38 and that covers a certain circumferential length L of forming roll 38.Former 10 also includes dewatering belt 24 that is routed around forming roll 38 and is formed particularly by a wire, and has headbox 12 whose suspension stream 22 is directed into the area between forming roll 38 and breast roll 56, around which dewatering belt 54 that forms wall 30 is routed prior to the area that covers forming roll 38, viewed in direction of belt travel 1.Dewatering belt 54 can also specifically be a wire.", "After forming roll 38, two dewatering belts 24, 54 are run over an additional forming element 58.In an area opposite forming roll 38 movable wall 30 that is formed by dewatering belt 54 is curved, at least partially according to a curvature radius R1 that is preferably larger than or equal to radius R2 of forming roll 38.Movable wall 30 can, for example, cover a circumferential length L of the forming roll 38 that is in a range of approximately 100 to approximately 1500 mm and/or corresponds with a circumferential angle a of forming roll 38 of approximately 25° to approximately 120°.", "As can be seen in FIG.", "6, the additional dewatering belt 54 that forms wall 30 can be routed around breast roll 56, prior to the area covering forming roll 38, when viewed in direction of travel 1.Curvature radius R1 of wall 30 in the area covering forming roll 38 is preferably larger than radius R3 of the breast roll, or respectively the corresponding curvature radius R3 of wall 30 in the area of this breast roll 56.FIG.", "7 is a schematic longitudinal sectional view of headbox nozzle 20 that is sectioned by at least one lamellar plate 36, whereby at least one lamellar plate 36 is equipped with at least one interference body 60 in order to provide a turbulence generating profile.", "Moreover, at least one lamellar plate 36 has a length not exceeding 70% of the length of the headbox nozzle 20.At least one lamellar plate 36 and/or at least one nozzle wall 20′, 20″ can have contours that serve to generate turbulent motion.", "FIG.", "8 is a schematic, perspective partial view of headbox nozzle 20 that is sectioned by at least one lamellar plate 36, whereby at least one lamellar plate 36 has a swirl-producing contour, in this example a washboard contour.", "At least one lamellar plate 36 and/or at least one nozzle wall 20′, 20″ can possess this type of swirl-producing contour, for example in the form of a washboard contour.", "FIG.", "9 is a schematic longitudinal view of headbox 20, that is sectioned by at least one lamellar plate 36, where the upper and lower nozzle wall 20′ or 20″ respectively are contoured so that turbulence generating edges 62 are created in the flow area.", "In order to achieve an appropriate effect a respective headbox nozzle 20 should be as short as possible, whereby headbox nozzle 20 is preferably shorter than approximately 400 mm.", "The outlet cross section of channels 16 or pipes is preferably at least essentially round, since a square cross section would dampen the effect.", "Other desired combinations of the various former variations, as well as of the various headbox variations are feasible.", "While this invention has been described as having a preferred design, the present invention can be further modified within the spirit and scope of this disclosure.", "This application is therefore intended to cover any variations, uses, or adaptations of the invention using its general principles.", "Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which fall within the limits of the appended claims." ] ]	Patent_10466354
[ [ "Gmg-3 gmg-4 and gmg-6 polynucleotides and polypeptides and uses thereof", "The present invention relates to the field of metabolic research.", "Metabolic disorders, such as obesity, are a public health problem that is serious and widespread.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides have been identified that are beneficial in the treatment of metabolic disorders.", "These compounds should be effective for reducing body mass and for treating metabolic-related diseases and disorders.", "These metabolic-related diseases and disorders include hyperlipidemias, atherosclerosis, diabetes, and hypertension." ], [ "1-11.", "(canceled) 12.A method of treating or preventing a metabolic-related disease or disorder comprising the step of administering to an individual a composition comprising a polypeptide or biologically active fragment thereof, wherein said polypeptide or fragment thereof comprises all or part of a C-terminal globular C1q homology domain and wherein said polypeptide is selected from the group consisting of: (a) SEQ ID NO: 2; and (b) SEQ ID NO: 4.13.The method of claim 12, wherein said metabolic-related disease or disorder is selected from the group consisting of: (a) obesity; (b) impaired glucose tolerance; (c) insulin resistance; (d) Syndrome X; and (e) Type II diabetes.", "14.The method of claim 12, wherein said polypeptide fragment is selected from the group consisting of: (a) amino acids 20-333 of SEQ ID NO: 2; (b) amino acids 188-333 of SEQ ID NO: 2; (c) amino acids 191-333 of SEQ ID NO: 2; (d) amino acids 193-333 of SEQ ID NO: 2; (e) amino acids 252-317 of SEQ ID NO: 2; (f) amino acids 20-333 of SEQ ID NO: 4; (g) amino acids 188-333 of SEQ ID NO: 4; (h) amino acids 191-333 of SEQ ID NO: 4; (i) amino acids 193-333 of SEQ ID NO: 4; and (O) amino acids 252-317 of SEQ ID NO: 4.15.The method of claim 13, wherein said polypeptide fragment is selected from the group consisting of: (a) amino acids 20-333 of SEQ ID NO: 2; (b) amino acids 188-333 of SEQ ID NO: 2; (c) amino acids 191-333 of SEQ ID NO: 2; (d) amino acids 193-333 of SEQ ID NO: 2; (e) amino acids 252-317 of SEQ ID NO: 2; (f) amino acids 20-333 of SEQ ID NO: 4; (g) amino acids 188-333 of SEQ ID NO: 4; (h) amino acids 191-333 of SEQ ID NO: 4; (i) amino acids 193-333 of SEQ ID NO: 4; and (j) amino acids 252-317 of SEQ ID NO: 4.16.An isolated polypeptide fragment selected from the group consisting of: (a) amino acids 20-333 of SEQ ID NO: 2; (b) amino acids 188-333 of SEQ ID NO: 2; (c) amino acids 191-333 of SEQ ID NO: 2; (d) amino acids 193-333 of SEQ ID NO: 2; (e) amino acids 252-317 of SEQ ID NO: 2; (f) amino acids 20-333 of SEQ ID NO: 4; (g) amino acids 188-333 of SEQ ID NO: 4; (h) amino acids 191-333 of SEQ ID NO: 4; (i) amino acids 193-333 of SEQ ID NO: 4; (j) amino acids 252-317 of SEQ ID NO:4; (k) amino acids 20-330 of SEQ ID NO: 8; (l) amino acids 185-330 of SEQ ID NO: 8; (m) amino acids 188-330 of SEQ ID NO: 8; (n) amino acids 190-330 of SEQ ID NO: 8; (o) amino acids 249-314 of SEQ ID NO: 8; (p) amino acids 20-323 of SEQ ID NO: 10; (q) amino acids 178-323 of SEQ ID NO: 10; (r) amino acids 181-323 of SEQ ID NO: 10; (s) amino acids 183-323 of SEQ ID NO: 10; and (t) amino acids 242-307 of SEQ ID NO: 10.17.A composition comprising a carrier and one or more of the polypeptide fragments of claim 16.18.An isolated polynucleotide, or complement thereof, encoding any one of the polypeptide fragments of claim 16.19.The polynucleotide of claim 18 selected from the group consisting of: (a) DNA; (b) RNA; (c) DNA/RNA hybrid; (d) single-stranded; and (e) double-stranded.", "20.A composition comprising a carrier and an isolated polynucleotide of claim 19.21.A vector comprising an isolated polynucleotide of claim 19.22.A composition comprising a carrier and a vector of claim 21.23.A transformed host cell comprising a vector of claim 21." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.", "Obesity is a public health problem that is serious, widespread, and increasing.", "In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634).", "Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643).", "Even modest weight loss ameliorates these associated conditions.", "While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Barsh et al (2000) Nature 404:644-651).", "In agreement with these studies, is the fact that an increasing number of metabolic-related genes are being identified.", "Some of the more extensively studied genes include those encoding leptin (ob) and its receptor (db), pro-opiomelanocortin (Pomc), melanocortin-4-receptor (Mc4r), agouti protein (A y ), carboxypeptidase E (fat), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loop-helix 2 (Nhlh2), prohormone convertase 1 (PCSK1), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651)." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The instant invention is based on Genset Metabolic Genes-3, 4, and 6 (GMG-3), (GMG-4), and (GMG-6).", "GMG-3 and GMG-4 are of human origin.", "Cluster 1 full-length polypeptide can be considered to be a C-terminal fragment of GMG-4 full-length polypeptide.", "GMG-6 is the mouse orthologue of GMG-3 and GMG-4.GMG-6A and GMG-6B correspond to splice variants of GMG-6.GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B full-length polypeptides are similar at the amino acid level to APM1, a human protein that has been implicated in obesity and diabetes and which structurally resembles TNFα.", "GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B full-length polypeptides are comprised of a C-terminal globular C1q homology domain preceded by a collagen-like region.", "By analogy to TNFα, globular head polypeptide fragments of GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B comprising TVFSRNVQVSLV or the extended loop QVTGGERFNGLFAD contact receptor and to have agonist activity.", "Results from Northern blot analysis and RT-PCR indicates expression of GMG-3 and/or GMG-4 in liver, heart, and skeletal muscle, but not in adipose tissue or brain.", "The invention includes polypeptides encoded by GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B, which include both the full-length polypeptide and fragments thereof, preferably said polypeptide fragments comprising all or part of the C-terminal globular C1q homology domain.", "The GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptide fragments containing all or part of the C-terminal globular C1q homology domain have in vitro and in vivo biological activity as described herein, including utility for weight reduction, prevention of weight gain and control of blood glucose levels in humans and other mammals.", "More specifically, the biological activities of the GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides, including fragments, include reduction of elevated free fatty acid levels caused by administration of epinephrine, i.v.", "injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, reduction in glucose levels, modulation of energy expenditure, resistance to insulin and weight reduction in mammals consuming a high fat/high sucrose diet.", "Thus, the invention is drawn to GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides, polynucleotides encoding said GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides, methods of using GMG-6 genomic sequence, vectors comprising said GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polynucleotides, and cells recombinant for said GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polynucleotides, as well as to pharmaceutical and physiologically acceptable compositions comprising said GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides and methods of administering said GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B pharmaceutical and physiologically acceptable compositions in order to reduce body weight or to treat metabolic-related diseases and disorders.", "Assays for identifying agonists and antagonists of metabolic-related activity are also part of the invention.", "In a first aspect, the invention features purified, isolated, or recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides that have lipid partitioning, lipid metabolism, and insulin-like activities.", "Preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptide fragments are said polypeptide fragments having activity, wherein said activity is also selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity.", "In preferred embodiments, said polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids and not more than 333 consecutive amino acids of SEQ ID NO: 2 or 4, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 245, 247, 248, 249, 250, 251, 252, or 253, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 256-267 of SEQ ID NO: 2 or 4) or QVTGGERFNGLFAD (amino acids 304-317 of SEQ ID NO: 2 or 4); or at least 6 and not more than 225 consecutive amino acids of SEQ ID NO: 6, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 148-159 of SEQ ID NO: 6) or QVTGGERFNGLFAD (amino acids 196-209 of SEQ ID NO: 6); at least 6 consecutive amino acids and not more than 330 consecutive amino acids of SEQ ID NO: 8, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 253-264 of SEQ ID NO: 8) or QVTGGERFNGLFAD (amino acids 301-314 of SEQ ID NO: 8); or at least 6 and not more than 323 consecutive amino acids of SEQ ID NO: 10, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, or 243, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 246-257 of SEQ ID NO: 10) or QVTGGERFNGLFAD (amino acids 294-307 of SEQ ID NO: 10).", "In other preferred embodiments, GMG-3 or GMG-4 polypeptide fragments having activity are selected from amino acids 20-333, 43-333, 44-333, 45-333, 46-333, 47-333, 48-333, 49-333, 50-333, 51-333, 52-333, 53-333, 54-333, 55-333, 56-333, 57-333, 58-333, 59-333, 60-333, 61-333, 62-333, 63-333, 64-333, 65-333, 66-333, 67-333, 68-333, 69-333, 70-333, 71-333, 72-333, 73-333, 74-333, 75-333, 76-333, 77-333, 78-333, 79-333, 80-333, 81-333, 82-333, 83-333, 84-333, 85-333, 86-333, 87-333, 88-333, 89-333, 90-333, 91-333, 92-333, 93-333, 94-333, 95-333, 96-333, 97-333, 98-333, 99-333, 100-333, 101-333, 102-333, 103-333, 104-333, 105-333, 106-333, 107-333, 108-333, 109-333, 110-333, 111-333, 112-333, 113-333, 114-333, 115-333, 116-333, 117-333, 118-333, 119-333, 120-333, 121-333, 122-333, 123-333, 124-333, 125-333, 126-333, 127-333, 128-333, 129-333, 130-333, 131-333, 132-333, 133-333, 134-333, 135-333, 136-333, 137-333, 138-333, 139-333, 140-333, 141-333, 142-333, 143-333, 144-333, 145-333, 146-333, 147-333, 148-333, 149-333, 150-333, 151-333, 152-333, 153-333, 154-333, 155-333, 156-333, 157-333, 158-333, 159-333, 160-333, 161-333, 162-333, 163-333, 164-333, 165-333, 166-333, 167-333, 168-333, 169-333, 170-333, 171-333, 172-333, 173-333, 174-333, 175-333, 176-333, 177-333, 178-333, 179-333, 180-333, 181-333, 182-333, 183-333, 184-333, 185-333, 186-333, 187-333, 188-333, 189-333, 190-333, 191-333, 192-333, 193-333, 194-333, 195-333, 196-333, 197-333, 198-333, 199-333, 200-333, 201-333 or 202-333 of SEQ ID NO: 2 or 4.In other preferred embodiments, Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 2-225, 3-225, 4-225, 5-225, 6-225, 7-225, 8-225, 9-225, 10-225, 11-225, 12-225, 13-225, 14-225, 15-225, 16-225, 17-225, 18-225, 19-225, 20-225, 21-225, 22-225, 23-225, 24-225, 25-225, 26-225, 27-225, 28-225, 29-225, 30-225, 31-225, 32-225, 33-225, 34-225, 35-225, 36-225, 37-225, 38-225, 39-225, 40-225, 41-225, 42-225, 43-225, 44-225, 45-225, 46-225, 47-225, 48-225, 49-225, 50-225, 51-225, 52-225, 53-225, 54-225, 55-225, 56-225, 57-225, 58-225, 59-225, 60-225, 61-225, 62-225, 63-225, 64-225, 65-225, 66-225, 67-225, 68-225, 69-225, 70-225, 71-225, 72-225, 73-225, 74-225, 75-225, 76-225, 77-225, 78-225, 79-225, 80-225, 81-225, 82-225, 83-225, 84-225, 85-225, 86-225, 87-225, 88-225, 89-225, 90-225, 91-225, 92-225, 93-225 or 94-225 of SEQ ID NO: 6.In other preferred embodiments, GMG-6A polypeptide fragments having activity are selected from 20-330, 43-330, 44-330, 45-330, 46-330, 47-330, 48-330, 49-330, 50-330, 51-330, 52-330, 53-330, 54-330, 55-330, 56-330, 57-330, 58-330, 59-330, 60-330, 61-330, 62-330, 63-330, 64-330, 65-330, 66-330, 67-330, 68-330, 69-330, 70-330, 71-330, 72-330, 73-330, 74-330, 75-330, 76-330, 77-330, 78-330, 79-330, 80-330, 81-330, 82-330, 83-330, 84-330, 85-330, 86-330, 87-330, 88-330, 89-330, 90-330, 91-330, 92-330, 93-330, 94-330, 95-330, 96-330, 97-330, 98-330, 99-330, 100-330, 101-330, 102-330, 103-330, 104-330, 105-330, 106-330, 107-330, 108-330, 109-330, 110-330, 111-330, 112-330, 113-330, 114-330, 115-330, 116-330, 117-330, 118-330, 119-330, 120-330, 121-330, 122-330, 123-330, 124-330, 125-330, 126-330, 127-330, 128-330, 129-330, 130-330, 131-330, 132-330, 133-330, 134-330, 135-330, 136-330, 137-330, 138-330, 139-330, 140-330, 141-330, 142-330, 143-330, 144-330, 145-330, 146-330, 147-330, 148-330, 149-330, 150-330, 151-330, 152-330, 153-330, 154-330, 155-330, 156-330, 157-330, 158-330, 159-330, 160-330, 161-330, 162-330, 163-330, 164-330, 165-330, 166-330, 167-330, 168-330, 169-330, 170-330, 171-330, 172-330, 173-330, 174-330, 175-330, 176-330, 177-330, 178-330, 179-330, 180-330, 181-330, 182-330, 183-330, 184-330, 185-330, 186-330, 187-330, 188-330, 189-330, 190-330, 191-330, 192-330, 193-330, 194-330, 195-330, 196-330, 197-330, 198-330 or 199-330 of SEQ ID NO; 8.In other preferred embodiments, GMG-6B polypeptide fragments having activity are selected from 20-323, 43-323, 44-323, 45-323, 46-323, 47-323, 48-323, 49-323, 50-323, 51-323, 52-323, 53-323, 54-323, 55-323, 56-323, 57-323, 58-323, 59-323, 60-323, 61-323, 62-323, 63-323, 64-323, 65-323, 66-323, 67-323, 68-323, 69-323, 70-323, 71-323, 72-323, 73-323, 74-323, 75-323, 76-323, 77-323, 78-323, 79-323, 80-323, 81-323, 82-323, 83-323, 84-323, 85-323, 86-323, 87-323, 88-323, 89-323, 90-323, 91-323, 92-323, 93-323, 94-323, 95-323, 96-323, 97-323, 98-323, 99-323, 100-323, 101-323, 102-323, 103-323, 104-323, 105-323, 106-323, 107-323, 108-323, 109-323, 110-323, 111-323, 112-323, 113-323, 114-323, 115-323, 116-323, 117-323, 118-323, 119-323, 120-323, 121-323, 122-323, 123-323, 124-323, 125-323, 126-323, 127-323, 128-323, 129-323, 130-323, 131-323, 132-323, 133-323, 134-323, 135-323, 136-323, 137-323, 138-323, 139-323, 140-323, 141-323, 142-323, 143-323, 144-323, 145-323, 146-323, 147-323, 148-323, 149-323, 150-323, 151-323, 152-323, 153-323, 154-323, 155-323, 156-323, 157-323, 158-323, 159-323, 160-323, 161-323, 162-323, 163-323, 164-323, 165-323, 166-323, 167-323, 168-323, 169-323, 170-323, 171-323, 172-323, 173-323, 174-323, 175-323, 176-323, 177-323, 178-323, 179-323, 180-323, 181-323, 182-323, 183-323, 184-323, 185-323, 186-323, 187-323, 188-323, 189-323, 190-323, 191-323 or 192-323 of SEQ ID NO: 10.In more preferred embodiments, GMG-3 or GMG-4 polypeptide fragments having activity are selected from amino acids 20-333, 43-333, 45-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 75-333, 77-333, 81-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 109-333, 113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333, 252-333, 252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2 or 4.In other more preferred embodiments, Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 5-225, 8-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225, 80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159, 144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6.In other more preferred embodiments, GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 43-330, 45-330, 46-330, 50-333, 53-330, 64-330, 68-330, 71-330, 72-330, 75-330, 78-330, 79-330, 83-330, 86-330, 92-330, 97-330, 101-330, 122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330, 198-330, 199-330, 224-330, 249-330, 249-264,249-314, 253-264, 253-314 or 301-314 of SEQ ID NO: 8.In other more preferred embodiments, GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 43-323, 46-323, 57-323, 61-323, 64-323, 65-323, 68-323, 71-323, 72-323, 76-323, 79-323, 85-323, 90-323, 94-323, 115-323, 118-323, 139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQ ID NO: 10.In yet more preferred embodiments, GMG-3 or GMG-4 polypeptide fragments having activity are selected from amino acids 20-333, 109-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333, 252-333, 252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2 or 4.In other yet more preferred embodiments, Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225, 80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159, 144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6.In other yet more preferred embodiments, GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 75-330, 122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330, 198-330, 199-330, 224-330, 249-330, 249-264, 249-314, 253-264, 253-314 or 301-314 of SEQ ID NO: 8.In other yet more preferred embodiments, GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 68-323, 115-323, 118-323, 139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQ ID NO: 10.In further preferred embodiments, said polypeptide fragment comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of the polypeptide sequences identified in SEQ ID NO: 2, 4, 6, 8, or 10.The invention further provides a purified or isolated polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of: (a) a full-length at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids polypeptide of SEQ ID NOs: 2, 4, 6, 8, or 10; (b) a full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of SEQ ID NOs: 2, 4, 6, 8, or 10 absent the N-terminal Met; (c) a mature GMG-3, GMG-4, GMG-6A, or GMG-6B polypeptide of SEQ ID NOs: 2, 4, 8, or 10 lacking signal peptide; (d) a GMG-3 or GMG-4 polypeptide of SEQ ID NO: 2 or 4 wherein said GMG-3 or GMG-4 polypeptide is of any one integer in length between 6 amino acids and 333 amino acids (full-length) inclusive of SEQ ID NO: 2 or 4, a Cluster 1 polypeptide of SEQ ID NO: 6 wherein said Cluster 1 polypeptide is of any one integer in length between 6 amino acids and 225 amino acids (full-length) inclusive of SEQ ID NO: 6, a GMG-6A polypeptide of SEQ ID NO: 8 wherein said GMG-6A polypeptide is of any one integer in length between 6 amino acids and 330 amino acids (full-length) inclusive of SEQ ID NO: 8, or a GMG-6B polypeptide of SEQ ID NO: 10 wherein said GMG-6B polypeptide is of any one integer in length between 6 amino acids and 323 amino acids (full-length) inclusive of SEQ ID NO: 10; (e) the epitope-bearing fragments of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of SEQ ID NO: 2, 4, 6, 8, or 10; (f) a fragment of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of SEQ ID NOs: 2, 4, 6, 8, or 10 comprising the globular head sequence TVFSRNVQVSLV and having agonist activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity; (g) a fragment of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of SEQ ID NOs: 2, 4, 6, 8, or 10 comprising the globular head sequence QVTGGERFNGLFAD and having agonist activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity; (h) the allelic variant polypeptides of any of the polypeptides of (a)-(g).", "The invention further provides for fragments of the polypeptides of (a)-(h) above, such as those having biological activity or comprising biologically functional domain(s).", "In other highly preferred embodiments, GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides comprise, consist essentially of, or consist of, a purified, isolated, or a recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment comprised of all or part of the C-terminal globular C1q homology domain.", "Preferably, said GMG-3 or GMG-4 polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 20-333 of SEQ ID NO: 2 or 4.Preferably, said Cluster 1 polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 1-225 of SEQ ID NO: 6.Preferably, said GMG-6A polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 20-330 of SEQ ID NO: 8.Preferably, said GMG-6B polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 20-323 of SEQ ID NO: 10.In other preferred embodiments, said GMG-3 or GMG-4 polypeptide fragments having activity are selected from amino acids 20-333, 43-333, 44-333, 45-333, 46-333, 47-333, 48-333, 49-333, 50-333, 51-333, 52-333, 53-333, 54-333, 55-333, 56-333, 57-333, 58-333, 59-333, 60-333, 61-333, 62-333, 63-333, 64-333, 65-333, 66-333, 67-333, 68-333, 69-333, 70-333, 71-333, 72-333, 73-333, 74-333, 75-333, 76-333, 77-333, 78-333, 79-333, 80-333, 81-333, 82-333, 83-333, 84-333, 85-333, 86-333, 87-333, 88-333, 89-333, 90-333, 91-333, 92-333, 93-333, 94-333, 95-333, 96-333, 97-333, 98-333, 99-333, 100-333, 101-333, 102-333, 103-333, 104-333, 105-333, 106-333, 107-333, 108-333, 109-333, 110-333, 111-333, 112-333, 113-333, 114-333, 115-333, 116-333, 117-333, 118-333, 119-333, 120-333, 121-333, 122-333, 123-333, 124-333, 125-333, 126-333, 127-333, 128-333, 129-333, 130-333, 131-333, 132-333, 133-333, 134-333, 135-333, 136-333, 137-333, 138-333, 139-333, 140-333, 141-333, 142-333, 143-333, 144-333, 145-333, 146-333, 147-333, 148-333, 149-333, 150-333, 151-333, 152-333, 153-333, 154-333, 155-333, 156-333, 157-333, 158-333, 159-333, 160-333, 161-333, 162-333, 163-333, 164-333, 165-333, 166-333, 167-333, 168-333, 169-333, 170-333, 171-333, 172-333, 173-333, 174-333, 175-333, 176-333, 177-333, 178-333, 179-333, 180-333, 181-333, 182-333, 183-333, 184-333, 185-333, 186-333, 187-333, 188-333, 189-333, 190-333, 191-333, 192-333, 193-333, 194-333, 195-333, 196-333, 197-333, 198-333, 199-333, 200-333, 201-333 or 202-333 of SEQ ID NO: 2 or 4.In other preferred embodiments, said Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 2-225, 3-225, 4-225, 5-225, 6-225, 7-225, 8-225, 9-225, 10-225, 11-225, 12-225, 13-225, 14-225, 15-225, 16-225, 17-225, 18-225, 19-225, 20-225, 21-225, 22-225, 23-225, 24-225, 25-225, 26-225, 27-225, 28-225, 29-225, 30-225, 31-225, 32-225, 33-225, 34-225, 35-225, 36-225, 37-225, 38-225, 39-225, 40-225, 41-225, 42-225, 43-225, 44-225, 45-225, 46-225, 47-225, 48-225, 49-225, 50-225, 51-225, 52-225, 53-225, 54-225, 55-225, 56-225, 57-225, 58-225, 59-225, 60-225, 61-225, 62-225, 63-225, 64-225, 65-225, 66-225, 67-225, 68-225, 69-225, 70-225, 71-225, 72-225, 73-225, 74-225, 75-225, 76-225, 77-225, 78-225, 79-225, 80-225, 81-225, 82-225, 83-225, 84-225, 85-225, 86-225, 87-225, 88-225, 89-225, 90-225, 91-225, 92-225, 93-225 or 94-225 of SEQ ID NO: 6.In other preferred embodiments, said GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 43-330, 44-330, 45-330, 46-330, 47-330, 48-330, 49-330, 50-330, 51-330, 52-330, 53-330, 54-330, 55-330, 56-330, 57-330, 58-330, 59-330, 60-330, 61-330, 62-330, 63-330, 64-330, 65-330, 66-330, 67-330, 68-330, 69-330, 70-330, 71-330, 72-330, 73-330, 74-330, 75-330, 76-330, 77-330, 78-330, 79-330, 80-330, 81-330, 82-330, 83-330, 84-330, 85-330, 86-330, 87-330, 88-330, 89-330, 90-330, 91-330, 92-330, 93-330, 94-330, 95-330, 96-330, 97-330, 98-330, 99-330, 100-330, 101-330, 102-330, 103-330, 104-330, 105-330, 106-330, 107-330, 108-330, 109-330, 110-330, 111-330, 112-330, 113-330, 114-330, 115-330, 116-330, 117-330, 118-330, 119-330, 120-330, 121-330, 122-330, 123-330, 124-330, 125-330, 126-330, 127-330, 128-330, 129-330, 130-330, 131-330, 132-330, 133-330, 134-330, 135-330, 136-330, 137-330, 138-330, 139-330, 140-330, 141-330, 142-330, 143-330, 144-330, 145-330, 146-330, 147-330, 148-330, 149-330, 150-330, 151-330, 152-330, 153-330, 154-330, 155-330, 156-330, 157-330, 158-330, 159-330, 160-330, 161-330, 162-330, 163-330, 164-330, 165-330, 166-330, 167-330, 168-330, 169-330, 170-330, 171-330, 172-330, 173-330, 174-330, 175-330, 176-330, 177-330, 178-330, 179-330, 180-330, 181-330, 182-330, 183-330, 184-330, 185-330, 186-330, 187-330, 188-330, 189-330, 190-330, 191-330, 192-330, 193-330, 194-330, 195-330, 196-330, 197-330, 198-330 or 199-330 of SEQ ID NO: 8.In other preferred embodiments, said GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 43-323, 44-323, 45-323, 46-323, 47-323, 48-323, 49-323, 50-323, 51-323, 52-323, 53-323, 54-323, 55-323, 56-323, 57-323, 58-323, 59-323, 60-323, 61-323, 62-323, 63-323, 64-323, 65-323, 66-323, 67-323, 68-323, 69-323, 70-323, 71-323, 72-323, 73-323, 74-323, 75-323, 76-323, 77-323, 78-323, 79-323, 80-323, 81-323, 82-323, 83-323, 84-323, 85-323, 86-323, 87-323, 88-323, 89-323, 90-323, 91-323, 92-323, 93-323, 94-323, 95-323, 96-323, 97-323, 98-323, 99-323, 100-323, 101-323, 102-323, 103-323, 104-323, 105-323, 106-323, 107-323, 108-323, 109-323, 110-323, 111-323, 112-323, 113-323, 114-323, 115-323, 116-323, 117-323, 118-323, 119-323, 120-323, 121-323, 122-323, 123-323, 124-323, 125-323, 126-323, 127-323, 128-323, 129-323, 130-323, 131-323, 132-323, 133-323, 134-323, 135-323, 136-323, 137-323, 138-323, 139-323, 140-323, 141-323, 142-323, 143-323, 144-323, 145-323, 146-323, 147-323, 148-323, 149-323, 150-323, 151-323, 152-323, 153-323, 154-323, 155-323, 156-323, 157-323, 158-323, 159-323, 160-323, 161-323, 162-323, 163-323, 164-323, 165-323, 166-323, 167-323, 168-323, 169-323, 170-323, 171-323, 172-323, 173-323, 174-323, 175-323, 176-323, 177-323, 178-323, 179-323, 180-323, 181-323, 182-323, 183-323, 184-323, 185-323, 186-323, 187-323, 188-323, 189-323, 190-323, 191-323 or 192-323 of SEQ ID NO: 10.In more preferred embodiments, said GMG-3 or GMG-4 polypeptide fragments comprised of all or part of the C-terminal globular C1q homology domain and having activity are selected from amino acids 20-333, 43-333, 45-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 75-333, 77-333, 81-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 109-333, 113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333, 252-333, 252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2 or 4.In other more preferred embodiments, said Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 5-225, 8-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225, 80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159, 144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6.In other more preferred embodiments, said GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 43-330, 45-330, 46-330, 50-333, 53-330, 64-330, 68-330, 71-330, 72-330, 75-330, 78-330, 79-330, 83-330, 86-330, 92-330, 97-330, 101-330, 122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330, 198-330, 199-330, 224-330, 249-330, 249-264, 249-314, 253-264, 253-314 or 301-314 of SEQ ID NO: 8.In other more preferred embodiments, said GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 43-323, 46-323, 57-323, 61-323, 64-323, 65-323, 68-323, 71-323, 72-323, 76-323, 79-323, 85-323, 90-323, 94-323, 115-323, 118-323, 139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQ ID NO: 10.In yet more preferred embodiments, said GMG-3 or GMG-4 polypeptide fragments comprised of all or part of the C-terminal globular C1q homology domain and having activity are selected from amino acids 20-333, 109-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333, 252-333, 252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2 or 4.In other yet more preferred embodiments, said Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225, 80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159, 144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6.In other yet more preferred embodiments, said GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 75-330, 122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330, 198-330, 199-330, 224-330, 249-330, 249-264, 249-314, 253-264, 253-314 or 301-314 of SEQ ID NO: 8.In other yet more preferred embodiments, said GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 68-323, 115-323, 118-323, 139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQ ID NO: 10.Alternatively, said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment comprises, consists essentially of, or consists of, an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 197-333 of SEQ ID NO: 2 or 4, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 88-225 of SEQ ID NO: 6, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 197-330 of SEQ ID NO: 8, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 190-323 of SEQ ID NO: 10.In a further preferred embodiment, GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are able to lower circulating (either in blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.", "Further preferred polypeptides of the invention demonstrating free fatty acid level lowering activity, glucose level lowering activity, and/or triglyceride level lowering activity, have an activity that is the same or greater than full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides at the same molar concentration, have the same or greater than transient activity and/or have a sustained activity.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that cause C2C 12 cells differentiated in the presence of said polypeptides to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase leptin uptake in a liver cell line (preferably BPRCL mouse liver cells (ATCC CRL-2217)).", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that significantly reduce the postprandial increase in plasma free fatty acids due to a high fat meal.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that significantly reduce or eliminate ketone body production as the result of a high fat meal.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase glucose uptake in skeletal muscle cells.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase glucose uptake in adipose cells.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase glucose uptake in neuronal cells.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase glucose uptake in red blood cells.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase glucose uptake in the brain.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase insulin sensitivity.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that inhibit the progression from impaired glucose tolerance to insulin resistance.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that form multimers (e.g., heteromultimers or homomultimers) in vitro and/or in vivo.", "Preferred multimers are homodimers or homotrimers.", "Other preferred multimers are homomultimers comprising at least 4, 6, 8, 9, 10 or 12 GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide subunits.", "Other preferred mulimers are hetero multimers comprising a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of the invention.", "Further preferred embodiments include heterologous polypeptides comprising one of the GMG 3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention.", "In a second aspect, the invention features purified, isolated, or recombinant polynucleotides encoding said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides described in the first aspect, or the complement thereof.", "A further preferred embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, or fragments thereof.", "In one aspect the sequence is derived from a human, mouse or other mammal.", "In a preferred aspect, the genomic sequence includes isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 2000, 5000, 10000 or 50000 nucleotides of any one of the polynucleotide sequences described in SEQ ID NOs: 1, 3, 5, 7, or 9, or the complements thereof, wherein said contiguous span comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding nucleotide sequence of the C-terminal globular C1q homology domains of SEQ ID NOs: 1, 3, 5, 7, or 9.In further embodiments the polynucleotides are DNA, RNA, DNA/RNA hybrids, single-stranded, and double-stranded.", "In a third aspect, the invention features a recombinant vector comprising, consisting essentially of, or consisting of, said polynucleotide described in the second aspect.", "In a fourth aspect, the invention features a recombinant cell comprising, consisting essentially of, or consisting of, said recombinant vector described in the third aspect.", "A further embodiment includes a host cell recombinant for a polynucleotide of the invention.", "In a fifth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides described in the first aspect and, alternatively, a pharmaceutical or physiologically acceptable diluent.", "In a sixth aspect, the invention features a method of reducing body mass comprising providing or administering to individuals in need of reducing body mass said pharmaceutical or physiologically acceptable composition described in the fifth aspect.", "In preferred embodiments, the identification of said individuals in need of reducing body mass to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B single nucleotide polymorphisms (SNPs) or measuring metabolic polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of plasma, urine, and saliva.", "Preferably, a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment of the present invention is administered to an individual with at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in blood, serum or plasma levels of full-length any one or all of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides or the naturally proteolytically cleaved GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragments as compared to healthy, non-obese patients.", "In a seventh aspect, the invention features a method of preventing or treating an metabolic-related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect.", "In preferred embodiments, the identification of said individuals in need of such treatment to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B single nucleotide polymorphisms (SNPs) or measuring GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide, wherein said biological response is selected from the group consisting of: (a) modulating circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids, wherein said modulating is preferably lowering; (b) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose, wherein said modulating is preferably lowering; (c) modulating circulating (either blood, serum or plasma) levels (concentration) of triglycerides, wherein said modulating is preferably lowering; (d) stimulating muscle lipid or free fatty acid oxidation; (c) modulating leptin uptake in the liver or liver cells, wherein said modulating is preferably increasing; (e) modulating the postprandial increase in plasma free fatty acids due to a high fat meal, wherein said modulating is preferably reducing; (f) modulating ketone body production as the result of a high fat meal, wherein said modulating is preferably reducing or eliminating; (g) increasing cell or tissue sensitivity to insulin, particularly muscle, adipose, liver or brain; and (h) inhibiting the progression from impaired glucose tolerance to insulin resistance; and further wherein said biological response is significantly greater than, or at least 10%, 20%, 30%, 35%, 40%, 50% 75% 100% or 500% greater than, the biological response caused or induced by insulin alone at the same molar concentration.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy.", "In a further preferred embodiment, the present invention may be used in complementary therapy of NIDDM patients to improve their weight or glucose control in combination with an insulin secretagogue (preferably oral form) or an insulin sensitising (preferably oral form) agent.", "Preferably, the oral insulin secretagogue is 1, 1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.", "Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.", "The present invention further provides a method of improving the body weight or glucose control of NIDDM patients alone, without an insulin secretagogue or an insulin sensitising agent.", "In a further preferred embodiment, the present invention may be used in complementary therapy of IDDM patients to improve their weight or glucose control in combination with an insulin secretagogue (preferably oral form) or an insulin sensitising (preferably oral form) agent.", "Preferably, the insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.", "Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.", "The present invention further provides a method of improving the body weight or glucose control of IDDM patients alone, without an insulin secretagogue or an insulin sensitising agent.", "In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with the insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent).", "Accordingly, the present invention further provides for a composition of pharmaceutical or physiologically acceptable composition and an insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of body weight or glucose control in NIDDM or IDDM patients.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin sensitiser.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) without insulin therapy.", "In an eighth aspect, the invention features a method of making the GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides described in the first aspect, wherein said method is selected from the group consisting of: proteolytic cleavage, recombinant methodology and artificial synthesis.", "In a ninth aspect, the present invention provides a method of making a recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment or a full length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide, the method comprising providing a transgenic, non-human mammal whose milk contains said recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment or full-length protein, and purifying said recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment or said full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide from the milk of said non-human mammal.", "In one embodiment, said non-human mammal is a cow, goat, sheep, rabbit, or mouse.", "In another embodiment, the method comprises purifying a recombinant full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide from said milk, and further comprises cleaving said protein in vitro to obtain a desired GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment.", "In a tenth aspect, the invention features a purified or isolated antibody capable of specifically binding to a polypeptide of the present invention.", "In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 6 consecutive amino acids, at least 8 consecutive amino acids, or at least 10 consecutive amino acids of the sequence of one of the polypeptide sequences described in SEQ ID NOs: 2, 4, 6, 8, or 10.In an eleventh aspect, the invention features a use of the polypeptide described in the first aspect for treatment of metabolic-related diseases and disorders and/or reducing or increasing body mass.", "Preferably, said metabolic-related diseases and disorders are selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In a twelth aspect, the invention provides a polypeptide of the first aspect of the invention, or a composition of the fifth aspect of the invention, for use in a method of treatment of the human or animal body.", "In a thirteenth aspect, the invention features methods of reducing body weight for cosmetic purposes comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect, or a polypeptide described in the first aspect.", "Preferably, for said reducing body weight said individual has a BMI of at least 20 and no more than 25.Alternatively, for said increasing body weight said individual preferably has a BMI of at least 15 and no more than 20.In a fourteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect for reducing body mass and/or for treatment or prevention of metabolic-related diseases or disorders.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, ADS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B single nucleotide polymorphisms (SNPs) or measuring GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "In a fifteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect for reducing body weight for cosmetic reasons.", "In a sixteenth aspect, the invention features methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect, or a polypeptide described in the first aspect.", "In a seventeenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with normal glucose tolerance (NGT) who are obese or who have fasting hyperinsulinemia, or who have both.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with gestational diabetes.", "Gestational diabetes refers to the development of diabetes in an individual during pregnancy, usually during the second or third trimester of pregnancy.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with impaired fasting glucose (IFG).", "Impaired fasting glucose (IFG) is that condition in which fasting plasma glucose levels in an individual are elevated but not diagnostic of overt diabetes, i.e.", "plasma glucose levels of less than 126 mg/dl and less than or equal to 110 mg/dl.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating and preventing impaired glucose tolerance (IGT) in an individual.", "By providing therapeutics and methods for reducing or preventing IGT, i.e., for normalizing insulin resistance, the progression to NIDDM can be delayed or prevented.", "Furthermore, by providing therapeutics and methods for reducing or preventing insulin resistance, the invention provides methods for reducing and/or preventing the appearance of Insulin-Resistance Syndrome.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating a subject having polycystic ovary syndrome (PCOS).", "PCOS is among the most common disorders of premenopausal women, affecting 5-10% of this population.", "Insulin-sensitizing agents, e.g., troglitazone, have been shown to be effective in PCOS and that, in particular, the defects in insulin action, insulin secretion, ovarian steroidogenosis and fibrinolysis are improved (Ehrman et al.", "(1997) J Clin Invest 100:1230), such as in insulin-resistant humans.", "Accordingly, the invention provides methods for reducing insulin resistance, normalizing blood glucose thus treating and/or preventing PCOS.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating a subject having insulin resistance.", "In further preferred embodiments, a subject having insulin resistance is treated according to the methods of the invention to reduce or cure the insulin-resistance.", "As insulin resistance is also often associated with infections and cancer, prevention or reducing insulin resistance according to the methods of the invention may prevent or reduce infections and cancer.", "In further preferred embodiment, the methods of the invention are used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.", "Thus, any of the above-described tests or other tests known in the art can be used to determine that a subject is insulin-resistant, which patient can then be treated according to the methods of the invention to reduce or cure the insulin-resistance.", "Alternatively, the methods of the invention can also be used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.", "In an eighteenth aspect, the invention features a method of preventing or treating an metabolic-related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect.", "In preferred embodiments, the identification of said individuals in need of such treatment to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B single nucleotide polymorphisms (SNPs) or measuring GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, FIV-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably non-human, preferably a cat or a dog.", "In a nineteenth aspect, the invention features a method of using a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptide fragment to screen compounds for one or more antagonists of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptide fragment activity, wherein said activity is selected from but not restricted to lipid partitioning, lipid metabolism, and insulin-like activity.", "In preferred embodiment, said compound is selected from but is not restricted to small molecular weight organic or inorganic compound, protein, peptide, carbohydrate, or lipid.", "In a twentieth aspect, the invention features a method of using a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide to identify one or more cell types expressing a cell surface receptor for said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide, preferably wherein said polypeptide comprises all or part of the C-terminal globular C1q homology domain and has lipid partitioning, lipid metabolism, or insulin-like activities.", "In a twenty-first aspect, the invention features a method of using a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide to clone cDNA encoding a cell surface receptor for said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide, preferably wherein said polypeptide comprises all or part of the C-terminal globular C1q homology domain and has lipid partitioning, lipid metabolism, or insulin-like activities.", "In a twenty-second aspect, the invention features a method of using a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide to generate transgenic non-human mammals expressing GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, preferably wherein said non-human mammal is mouse, cow, sheep, goat, pig, or rabbit.", "In a twenty-third aspect, the invention features a method of using a genomic polynucleotide or fragment thereof of SEQ ID NO: 11, 12, or 13 to generate a transgenic mouse in which expression of the gene encoding GMG-6A and GMG-6B is knocked-out either globally or in a tissue-specific manner.", "In a preferred aspect of the methods above and disclosed herein, the amount of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polynucleotide administered to an individual is sufficient to bring circulating (blood, serum, or plasma) levels (concentration) of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides to their normal levels (levels in non-obese individuals).", "“Normal levels” may be specified as the total concentration of all circulating GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides (full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B proteins and fragments thereof) or the concentration of all circulating proteolytically cleaved GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides only.", "In a further preferred aspect of the methods above and disclosed herein, weight loss is due in part or in whole to a decrease in mass of either a) subcutaneous adipose tissue and/or b) visceral (omental) adipose tissue.", "Full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides and polynucleotides encoding the same may be specifically substituted for a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment or polynucleotide encoding the same in any embodiment of the present invention.", "It is further understood that by GMG-3 polypeptide is meant the amino acid sequence of SEQ ID NO: 2 as well as any related polypeptide incorporating one or more of the amino acid polymorphisms indicated in the sequence listing, namely the related polypeptide with Val at position 219, the related polypeptide with Met at position 301, and the related polypeptide with Val at position 219 and Met at position 301.It is further understood that by GMG-4 polypeptide is meant the amino acid sequence of SEQ ID NO: 4 as well as any related polypeptide incorporating one or more of the amino acid polymorphisms indicated in the sequence listing, namely the related polypeptide with Ala at position 238.It is further understood that by Cluster 1 polypeptide is meant the amino acid sequence of SEQ ID NO: 6 as well as any related polypeptide incorporating one or more of the amino acid polymorphisms indicated in the sequence listing, namely the related polypeptide with Ala at position 130." ], [ "FIELD OF THE INVENTION The present invention relates to the field of metabolic research, in particular the discovery of compounds effective for reducing body mass and useful for treating metabolic-related diseases and disorders.", "The metabolic-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to, hyperlipidemia, atherosclerosis, diabetes, and hypertension.", "BACKGROUND OF THE INVENTION The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.", "Obesity is a public health problem that is serious, widespread, and increasing.", "In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634).", "Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643).", "Even modest weight loss ameliorates these associated conditions.", "While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Barsh et al (2000) Nature 404:644-651).", "In agreement with these studies, is the fact that an increasing number of metabolic-related genes are being identified.", "Some of the more extensively studied genes include those encoding leptin (ob) and its receptor (db), pro-opiomelanocortin (Pomc), melanocortin-4-receptor (Mc4r), agouti protein (Ay), carboxypeptidase E (fat), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loop-helix 2 (Nhlh2), prohormone convertase 1 (PCSK1), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651).", "SUMMARY OF THE INVENTION The instant invention is based on Genset Metabolic Genes-3, 4, and 6 (GMG-3), (GMG-4), and (GMG-6).", "GMG-3 and GMG-4 are of human origin.", "Cluster 1 full-length polypeptide can be considered to be a C-terminal fragment of GMG-4 full-length polypeptide.", "GMG-6 is the mouse orthologue of GMG-3 and GMG-4.GMG-6A and GMG-6B correspond to splice variants of GMG-6.GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B full-length polypeptides are similar at the amino acid level to APM1, a human protein that has been implicated in obesity and diabetes and which structurally resembles TNFα.", "GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B full-length polypeptides are comprised of a C-terminal globular C1q homology domain preceded by a collagen-like region.", "By analogy to TNFα, globular head polypeptide fragments of GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B comprising TVFSRNVQVSLV or the extended loop QVTGGERFNGLFAD contact receptor and to have agonist activity.", "Results from Northern blot analysis and RT-PCR indicates expression of GMG-3 and/or GMG-4 in liver, heart, and skeletal muscle, but not in adipose tissue or brain.", "The invention includes polypeptides encoded by GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B, which include both the full-length polypeptide and fragments thereof, preferably said polypeptide fragments comprising all or part of the C-terminal globular C1q homology domain.", "The GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptide fragments containing all or part of the C-terminal globular C1q homology domain have in vitro and in vivo biological activity as described herein, including utility for weight reduction, prevention of weight gain and control of blood glucose levels in humans and other mammals.", "More specifically, the biological activities of the GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides, including fragments, include reduction of elevated free fatty acid levels caused by administration of epinephrine, i.v.", "injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, reduction in glucose levels, modulation of energy expenditure, resistance to insulin and weight reduction in mammals consuming a high fat/high sucrose diet.", "Thus, the invention is drawn to GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides, polynucleotides encoding said GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides, methods of using GMG-6 genomic sequence, vectors comprising said GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polynucleotides, and cells recombinant for said GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polynucleotides, as well as to pharmaceutical and physiologically acceptable compositions comprising said GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides and methods of administering said GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B pharmaceutical and physiologically acceptable compositions in order to reduce body weight or to treat metabolic-related diseases and disorders.", "Assays for identifying agonists and antagonists of metabolic-related activity are also part of the invention.", "In a first aspect, the invention features purified, isolated, or recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides that have lipid partitioning, lipid metabolism, and insulin-like activities.", "Preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptide fragments are said polypeptide fragments having activity, wherein said activity is also selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity.", "In preferred embodiments, said polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids and not more than 333 consecutive amino acids of SEQ ID NO: 2 or 4, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 245, 247, 248, 249, 250, 251, 252, or 253, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 256-267 of SEQ ID NO: 2 or 4) or QVTGGERFNGLFAD (amino acids 304-317 of SEQ ID NO: 2 or 4); or at least 6 and not more than 225 consecutive amino acids of SEQ ID NO: 6, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 148-159 of SEQ ID NO: 6) or QVTGGERFNGLFAD (amino acids 196-209 of SEQ ID NO: 6); at least 6 consecutive amino acids and not more than 330 consecutive amino acids of SEQ ID NO: 8, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 253-264 of SEQ ID NO: 8) or QVTGGERFNGLFAD (amino acids 301-314 of SEQ ID NO: 8); or at least 6 and not more than 323 consecutive amino acids of SEQ ID NO: 10, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, or 243, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 246-257 of SEQ ID NO: 10) or QVTGGERFNGLFAD (amino acids 294-307 of SEQ ID NO: 10).", "In other preferred embodiments, GMG-3 or GMG-4 polypeptide fragments having activity are selected from amino acids 20-333, 43-333, 44-333, 45-333, 46-333, 47-333, 48-333, 49-333, 50-333, 51-333, 52-333, 53-333, 54-333, 55-333, 56-333, 57-333, 58-333, 59-333, 60-333, 61-333, 62-333, 63-333, 64-333, 65-333, 66-333, 67-333, 68-333, 69-333, 70-333, 71-333, 72-333, 73-333, 74-333, 75-333, 76-333, 77-333, 78-333, 79-333, 80-333, 81-333, 82-333, 83-333, 84-333, 85-333, 86-333, 87-333, 88-333, 89-333, 90-333, 91-333, 92-333, 93-333, 94-333, 95-333, 96-333, 97-333, 98-333, 99-333, 100-333, 101-333, 102-333, 103-333, 104-333, 105-333, 106-333, 107-333, 108-333, 109-333, 110-333, 111-333, 112-333, 113-333, 114-333, 115-333, 116-333, 117-333, 118-333, 119-333, 120-333, 121-333, 122-333, 123-333, 124-333, 125-333, 126-333, 127-333, 128-333, 129-333, 130-333, 131-333, 132-333, 133-333, 134-333, 135-333, 136-333, 137-333, 138-333, 139-333, 140-333, 141-333, 142-333, 143-333, 144-333, 145-333, 146-333, 147-333, 148-333, 149-333, 150-333, 151-333, 152-333, 153-333, 154-333, 155-333, 156-333, 157-333, 158-333, 159-333, 160-333, 161-333, 162-333, 163-333, 164-333, 165-333, 166-333, 167-333, 168-333, 169-333, 170-333, 171-333, 172-333, 173-333, 174-333, 175-333, 176-333, 177-333, 178-333, 179-333, 180-333, 181-333, 182-333, 183-333, 184-333, 185-333, 186-333, 187-333, 188-333, 189-333, 190-333, 191-333, 192-333, 193-333, 194-333, 195-333, 196-333, 197-333, 198-333, 199-333, 200-333, 201-333 or 202-333 of SEQ ID NO: 2 or 4.In other preferred embodiments, Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 2-225, 3-225, 4-225, 5-225, 6-225, 7-225, 8-225, 9-225, 10-225, 11-225, 12-225, 13-225, 14-225, 15-225, 16-225, 17-225, 18-225, 19-225, 20-225, 21-225, 22-225, 23-225, 24-225, 25-225, 26-225, 27-225, 28-225, 29-225, 30-225, 31-225, 32-225, 33-225, 34-225, 35-225, 36-225, 37-225, 38-225, 39-225, 40-225, 41-225, 42-225, 43-225, 44-225, 45-225, 46-225, 47-225, 48-225, 49-225, 50-225, 51-225, 52-225, 53-225, 54-225, 55-225, 56-225, 57-225, 58-225, 59-225, 60-225, 61-225, 62-225, 63-225, 64-225, 65-225, 66-225, 67-225, 68-225, 69-225, 70-225, 71-225, 72-225, 73-225, 74-225, 75-225, 76-225, 77-225, 78-225, 79-225, 80-225, 81-225, 82-225, 83-225, 84-225, 85-225, 86-225, 87-225, 88-225, 89-225, 90-225, 91-225, 92-225, 93-225 or 94-225 of SEQ ID NO: 6.In other preferred embodiments, GMG-6A polypeptide fragments having activity are selected from 20-330, 43-330, 44-330, 45-330, 46-330, 47-330, 48-330, 49-330, 50-330, 51-330, 52-330, 53-330, 54-330, 55-330, 56-330, 57-330, 58-330, 59-330, 60-330, 61-330, 62-330, 63-330, 64-330, 65-330, 66-330, 67-330, 68-330, 69-330, 70-330, 71-330, 72-330, 73-330, 74-330, 75-330, 76-330, 77-330, 78-330, 79-330, 80-330, 81-330, 82-330, 83-330, 84-330, 85-330, 86-330, 87-330, 88-330, 89-330, 90-330, 91-330, 92-330, 93-330, 94-330, 95-330, 96-330, 97-330, 98-330, 99-330, 100-330, 101-330, 102-330, 103-330, 104-330, 105-330, 106-330, 107-330, 108-330, 109-330, 110-330, 111-330, 112-330, 113-330, 114-330, 115-330, 116-330, 117-330, 118-330, 119-330, 120-330, 121-330, 122-330, 123-330, 124-330, 125-330, 126-330, 127-330, 128-330, 129-330, 130-330, 131-330, 132-330, 133-330, 134-330, 135-330, 136-330, 137-330, 138-330, 139-330, 140-330, 141-330, 142-330, 143-330, 144-330, 145-330, 146-330, 147-330, 148-330, 149-330, 150-330, 151-330, 152-330, 153-330, 154-330, 155-330, 156-330, 157-330, 158-330, 159-330, 160-330, 161-330, 162-330, 163-330, 164-330, 165-330, 166-330, 167-330, 168-330, 169-330, 170-330, 171-330, 172-330, 173-330, 174-330, 175-330, 176-330, 177-330, 178-330, 179-330, 180-330, 181-330, 182-330, 183-330, 184-330, 185-330, 186-330, 187-330, 188-330, 189-330, 190-330, 191-330, 192-330, 193-330, 194-330, 195-330, 196-330, 197-330, 198-330 or 199-330 of SEQ ID NO; 8.In other preferred embodiments, GMG-6B polypeptide fragments having activity are selected from 20-323, 43-323, 44-323, 45-323, 46-323, 47-323, 48-323, 49-323, 50-323, 51-323, 52-323, 53-323, 54-323, 55-323, 56-323, 57-323, 58-323, 59-323, 60-323, 61-323, 62-323, 63-323, 64-323, 65-323, 66-323, 67-323, 68-323, 69-323, 70-323, 71-323, 72-323, 73-323, 74-323, 75-323, 76-323, 77-323, 78-323, 79-323, 80-323, 81-323, 82-323, 83-323, 84-323, 85-323, 86-323, 87-323, 88-323, 89-323, 90-323, 91-323, 92-323, 93-323, 94-323, 95-323, 96-323, 97-323, 98-323, 99-323, 100-323, 101-323, 102-323, 103-323, 104-323, 105-323, 106-323, 107-323, 108-323, 109-323, 110-323, 111-323, 112-323, 113-323, 114-323, 115-323, 116-323, 117-323, 118-323, 119-323, 120-323, 121-323, 122-323, 123-323, 124-323, 125-323, 126-323, 127-323, 128-323, 129-323, 130-323, 131-323, 132-323, 133-323, 134-323, 135-323, 136-323, 137-323, 138-323, 139-323, 140-323, 141-323, 142-323, 143-323, 144-323, 145-323, 146-323, 147-323, 148-323, 149-323, 150-323, 151-323, 152-323, 153-323, 154-323, 155-323, 156-323, 157-323, 158-323, 159-323, 160-323, 161-323, 162-323, 163-323, 164-323, 165-323, 166-323, 167-323, 168-323, 169-323, 170-323, 171-323, 172-323, 173-323, 174-323, 175-323, 176-323, 177-323, 178-323, 179-323, 180-323, 181-323, 182-323, 183-323, 184-323, 185-323, 186-323, 187-323, 188-323, 189-323, 190-323, 191-323 or 192-323 of SEQ ID NO: 10.In more preferred embodiments, GMG-3 or GMG-4 polypeptide fragments having activity are selected from amino acids 20-333, 43-333, 45-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 75-333, 77-333, 81-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 109-333, 113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333, 252-333, 252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2 or 4.In other more preferred embodiments, Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 5-225, 8-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225, 80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159, 144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6.In other more preferred embodiments, GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 43-330, 45-330, 46-330, 50-333, 53-330, 64-330, 68-330, 71-330, 72-330, 75-330, 78-330, 79-330, 83-330, 86-330, 92-330, 97-330, 101-330, 122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330, 198-330, 199-330, 224-330, 249-330, 249-264,249-314, 253-264, 253-314 or 301-314 of SEQ ID NO: 8.In other more preferred embodiments, GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 43-323, 46-323, 57-323, 61-323, 64-323, 65-323, 68-323, 71-323, 72-323, 76-323, 79-323, 85-323, 90-323, 94-323, 115-323, 118-323, 139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQ ID NO: 10.In yet more preferred embodiments, GMG-3 or GMG-4 polypeptide fragments having activity are selected from amino acids 20-333, 109-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333, 252-333, 252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2 or 4.In other yet more preferred embodiments, Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225, 80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159, 144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6.In other yet more preferred embodiments, GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 75-330, 122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330, 198-330, 199-330, 224-330, 249-330, 249-264, 249-314, 253-264, 253-314 or 301-314 of SEQ ID NO: 8.In other yet more preferred embodiments, GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 68-323, 115-323, 118-323, 139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQ ID NO: 10.In further preferred embodiments, said polypeptide fragment comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of the polypeptide sequences identified in SEQ ID NO: 2, 4, 6, 8, or 10.The invention further provides a purified or isolated polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of: (a) a full-length at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids polypeptide of SEQ ID NOs: 2, 4, 6, 8, or 10; (b) a full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of SEQ ID NOs: 2, 4, 6, 8, or 10 absent the N-terminal Met; (c) a mature GMG-3, GMG-4, GMG-6A, or GMG-6B polypeptide of SEQ ID NOs: 2, 4, 8, or 10 lacking signal peptide; (d) a GMG-3 or GMG-4 polypeptide of SEQ ID NO: 2 or 4 wherein said GMG-3 or GMG-4 polypeptide is of any one integer in length between 6 amino acids and 333 amino acids (full-length) inclusive of SEQ ID NO: 2 or 4, a Cluster 1 polypeptide of SEQ ID NO: 6 wherein said Cluster 1 polypeptide is of any one integer in length between 6 amino acids and 225 amino acids (full-length) inclusive of SEQ ID NO: 6, a GMG-6A polypeptide of SEQ ID NO: 8 wherein said GMG-6A polypeptide is of any one integer in length between 6 amino acids and 330 amino acids (full-length) inclusive of SEQ ID NO: 8, or a GMG-6B polypeptide of SEQ ID NO: 10 wherein said GMG-6B polypeptide is of any one integer in length between 6 amino acids and 323 amino acids (full-length) inclusive of SEQ ID NO: 10; (e) the epitope-bearing fragments of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of SEQ ID NO: 2, 4, 6, 8, or 10; (f) a fragment of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of SEQ ID NOs: 2, 4, 6, 8, or 10 comprising the globular head sequence TVFSRNVQVSLV and having agonist activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity; (g) a fragment of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of SEQ ID NOs: 2, 4, 6, 8, or 10 comprising the globular head sequence QVTGGERFNGLFAD and having agonist activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity; (h) the allelic variant polypeptides of any of the polypeptides of (a)-(g).", "The invention further provides for fragments of the polypeptides of (a)-(h) above, such as those having biological activity or comprising biologically functional domain(s).", "In other highly preferred embodiments, GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides comprise, consist essentially of, or consist of, a purified, isolated, or a recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment comprised of all or part of the C-terminal globular C1q homology domain.", "Preferably, said GMG-3 or GMG-4 polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 20-333 of SEQ ID NO: 2 or 4.Preferably, said Cluster 1 polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 1-225 of SEQ ID NO: 6.Preferably, said GMG-6A polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 20-330 of SEQ ID NO: 8.Preferably, said GMG-6B polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 20-323 of SEQ ID NO: 10.In other preferred embodiments, said GMG-3 or GMG-4 polypeptide fragments having activity are selected from amino acids 20-333, 43-333, 44-333, 45-333, 46-333, 47-333, 48-333, 49-333, 50-333, 51-333, 52-333, 53-333, 54-333, 55-333, 56-333, 57-333, 58-333, 59-333, 60-333, 61-333, 62-333, 63-333, 64-333, 65-333, 66-333, 67-333, 68-333, 69-333, 70-333, 71-333, 72-333, 73-333, 74-333, 75-333, 76-333, 77-333, 78-333, 79-333, 80-333, 81-333, 82-333, 83-333, 84-333, 85-333, 86-333, 87-333, 88-333, 89-333, 90-333, 91-333, 92-333, 93-333, 94-333, 95-333, 96-333, 97-333, 98-333, 99-333, 100-333, 101-333, 102-333, 103-333, 104-333, 105-333, 106-333, 107-333, 108-333, 109-333, 110-333, 111-333, 112-333, 113-333, 114-333, 115-333, 116-333, 117-333, 118-333, 119-333, 120-333, 121-333, 122-333, 123-333, 124-333, 125-333, 126-333, 127-333, 128-333, 129-333, 130-333, 131-333, 132-333, 133-333, 134-333, 135-333, 136-333, 137-333, 138-333, 139-333, 140-333, 141-333, 142-333, 143-333, 144-333, 145-333, 146-333, 147-333, 148-333, 149-333, 150-333, 151-333, 152-333, 153-333, 154-333, 155-333, 156-333, 157-333, 158-333, 159-333, 160-333, 161-333, 162-333, 163-333, 164-333, 165-333, 166-333, 167-333, 168-333, 169-333, 170-333, 171-333, 172-333, 173-333, 174-333, 175-333, 176-333, 177-333, 178-333, 179-333, 180-333, 181-333, 182-333, 183-333, 184-333, 185-333, 186-333, 187-333, 188-333, 189-333, 190-333, 191-333, 192-333, 193-333, 194-333, 195-333, 196-333, 197-333, 198-333, 199-333, 200-333, 201-333 or 202-333 of SEQ ID NO: 2 or 4.In other preferred embodiments, said Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 2-225, 3-225, 4-225, 5-225, 6-225, 7-225, 8-225, 9-225, 10-225, 11-225, 12-225, 13-225, 14-225, 15-225, 16-225, 17-225, 18-225, 19-225, 20-225, 21-225, 22-225, 23-225, 24-225, 25-225, 26-225, 27-225, 28-225, 29-225, 30-225, 31-225, 32-225, 33-225, 34-225, 35-225, 36-225, 37-225, 38-225, 39-225, 40-225, 41-225, 42-225, 43-225, 44-225, 45-225, 46-225, 47-225, 48-225, 49-225, 50-225, 51-225, 52-225, 53-225, 54-225, 55-225, 56-225, 57-225, 58-225, 59-225, 60-225, 61-225, 62-225, 63-225, 64-225, 65-225, 66-225, 67-225, 68-225, 69-225, 70-225, 71-225, 72-225, 73-225, 74-225, 75-225, 76-225, 77-225, 78-225, 79-225, 80-225, 81-225, 82-225, 83-225, 84-225, 85-225, 86-225, 87-225, 88-225, 89-225, 90-225, 91-225, 92-225, 93-225 or 94-225 of SEQ ID NO: 6.In other preferred embodiments, said GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 43-330, 44-330, 45-330, 46-330, 47-330, 48-330, 49-330, 50-330, 51-330, 52-330, 53-330, 54-330, 55-330, 56-330, 57-330, 58-330, 59-330, 60-330, 61-330, 62-330, 63-330, 64-330, 65-330, 66-330, 67-330, 68-330, 69-330, 70-330, 71-330, 72-330, 73-330, 74-330, 75-330, 76-330, 77-330, 78-330, 79-330, 80-330, 81-330, 82-330, 83-330, 84-330, 85-330, 86-330, 87-330, 88-330, 89-330, 90-330, 91-330, 92-330, 93-330, 94-330, 95-330, 96-330, 97-330, 98-330, 99-330, 100-330, 101-330, 102-330, 103-330, 104-330, 105-330, 106-330, 107-330, 108-330, 109-330, 110-330, 111-330, 112-330, 113-330, 114-330, 115-330, 116-330, 117-330, 118-330, 119-330, 120-330, 121-330, 122-330, 123-330, 124-330, 125-330, 126-330, 127-330, 128-330, 129-330, 130-330, 131-330, 132-330, 133-330, 134-330, 135-330, 136-330, 137-330, 138-330, 139-330, 140-330, 141-330, 142-330, 143-330, 144-330, 145-330, 146-330, 147-330, 148-330, 149-330, 150-330, 151-330, 152-330, 153-330, 154-330, 155-330, 156-330, 157-330, 158-330, 159-330, 160-330, 161-330, 162-330, 163-330, 164-330, 165-330, 166-330, 167-330, 168-330, 169-330, 170-330, 171-330, 172-330, 173-330, 174-330, 175-330, 176-330, 177-330, 178-330, 179-330, 180-330, 181-330, 182-330, 183-330, 184-330, 185-330, 186-330, 187-330, 188-330, 189-330, 190-330, 191-330, 192-330, 193-330, 194-330, 195-330, 196-330, 197-330, 198-330 or 199-330 of SEQ ID NO: 8.In other preferred embodiments, said GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 43-323, 44-323, 45-323, 46-323, 47-323, 48-323, 49-323, 50-323, 51-323, 52-323, 53-323, 54-323, 55-323, 56-323, 57-323, 58-323, 59-323, 60-323, 61-323, 62-323, 63-323, 64-323, 65-323, 66-323, 67-323, 68-323, 69-323, 70-323, 71-323, 72-323, 73-323, 74-323, 75-323, 76-323, 77-323, 78-323, 79-323, 80-323, 81-323, 82-323, 83-323, 84-323, 85-323, 86-323, 87-323, 88-323, 89-323, 90-323, 91-323, 92-323, 93-323, 94-323, 95-323, 96-323, 97-323, 98-323, 99-323, 100-323, 101-323, 102-323, 103-323, 104-323, 105-323, 106-323, 107-323, 108-323, 109-323, 110-323, 111-323, 112-323, 113-323, 114-323, 115-323, 116-323, 117-323, 118-323, 119-323, 120-323, 121-323, 122-323, 123-323, 124-323, 125-323, 126-323, 127-323, 128-323, 129-323, 130-323, 131-323, 132-323, 133-323, 134-323, 135-323, 136-323, 137-323, 138-323, 139-323, 140-323, 141-323, 142-323, 143-323, 144-323, 145-323, 146-323, 147-323, 148-323, 149-323, 150-323, 151-323, 152-323, 153-323, 154-323, 155-323, 156-323, 157-323, 158-323, 159-323, 160-323, 161-323, 162-323, 163-323, 164-323, 165-323, 166-323, 167-323, 168-323, 169-323, 170-323, 171-323, 172-323, 173-323, 174-323, 175-323, 176-323, 177-323, 178-323, 179-323, 180-323, 181-323, 182-323, 183-323, 184-323, 185-323, 186-323, 187-323, 188-323, 189-323, 190-323, 191-323 or 192-323 of SEQ ID NO: 10.In more preferred embodiments, said GMG-3 or GMG-4 polypeptide fragments comprised of all or part of the C-terminal globular C1q homology domain and having activity are selected from amino acids 20-333, 43-333, 45-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 75-333, 77-333, 81-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 109-333, 113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333, 252-333, 252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2 or 4.In other more preferred embodiments, said Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 5-225, 8-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225, 80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159, 144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6.In other more preferred embodiments, said GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 43-330, 45-330, 46-330, 50-333, 53-330, 64-330, 68-330, 71-330, 72-330, 75-330, 78-330, 79-330, 83-330, 86-330, 92-330, 97-330, 101-330, 122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330, 198-330, 199-330, 224-330, 249-330, 249-264, 249-314, 253-264, 253-314 or 301-314 of SEQ ID NO: 8.In other more preferred embodiments, said GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 43-323, 46-323, 57-323, 61-323, 64-323, 65-323, 68-323, 71-323, 72-323, 76-323, 79-323, 85-323, 90-323, 94-323, 115-323, 118-323, 139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQ ID NO: 10.In yet more preferred embodiments, said GMG-3 or GMG-4 polypeptide fragments comprised of all or part of the C-terminal globular C1q homology domain and having activity are selected from amino acids 20-333, 109-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333, 252-333, 252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2 or 4.In other yet more preferred embodiments, said Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225, 80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159, 144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6.In other yet more preferred embodiments, said GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 75-330, 122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330, 198-330, 199-330, 224-330, 249-330, 249-264, 249-314, 253-264, 253-314 or 301-314 of SEQ ID NO: 8.In other yet more preferred embodiments, said GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 68-323, 115-323, 118-323, 139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQ ID NO: 10.Alternatively, said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment comprises, consists essentially of, or consists of, an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 197-333 of SEQ ID NO: 2 or 4, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 88-225 of SEQ ID NO: 6, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 197-330 of SEQ ID NO: 8, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 190-323 of SEQ ID NO: 10.In a further preferred embodiment, GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are able to lower circulating (either in blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.", "Further preferred polypeptides of the invention demonstrating free fatty acid level lowering activity, glucose level lowering activity, and/or triglyceride level lowering activity, have an activity that is the same or greater than full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides at the same molar concentration, have the same or greater than transient activity and/or have a sustained activity.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that cause C2C 12 cells differentiated in the presence of said polypeptides to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase leptin uptake in a liver cell line (preferably BPRCL mouse liver cells (ATCC CRL-2217)).", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that significantly reduce the postprandial increase in plasma free fatty acids due to a high fat meal.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that significantly reduce or eliminate ketone body production as the result of a high fat meal.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase glucose uptake in skeletal muscle cells.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase glucose uptake in adipose cells.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase glucose uptake in neuronal cells.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase glucose uptake in red blood cells.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase glucose uptake in the brain.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that increase insulin sensitivity.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that inhibit the progression from impaired glucose tolerance to insulin resistance.", "Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides are those that form multimers (e.g., heteromultimers or homomultimers) in vitro and/or in vivo.", "Preferred multimers are homodimers or homotrimers.", "Other preferred multimers are homomultimers comprising at least 4, 6, 8, 9, 10 or 12 GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide subunits.", "Other preferred mulimers are hetero multimers comprising a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of the invention.", "Further preferred embodiments include heterologous polypeptides comprising one of the GMG 3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention.", "In a second aspect, the invention features purified, isolated, or recombinant polynucleotides encoding said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides described in the first aspect, or the complement thereof.", "A further preferred embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, or fragments thereof.", "In one aspect the sequence is derived from a human, mouse or other mammal.", "In a preferred aspect, the genomic sequence includes isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 2000, 5000, 10000 or 50000 nucleotides of any one of the polynucleotide sequences described in SEQ ID NOs: 1, 3, 5, 7, or 9, or the complements thereof, wherein said contiguous span comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding nucleotide sequence of the C-terminal globular C1q homology domains of SEQ ID NOs: 1, 3, 5, 7, or 9.In further embodiments the polynucleotides are DNA, RNA, DNA/RNA hybrids, single-stranded, and double-stranded.", "In a third aspect, the invention features a recombinant vector comprising, consisting essentially of, or consisting of, said polynucleotide described in the second aspect.", "In a fourth aspect, the invention features a recombinant cell comprising, consisting essentially of, or consisting of, said recombinant vector described in the third aspect.", "A further embodiment includes a host cell recombinant for a polynucleotide of the invention.", "In a fifth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides described in the first aspect and, alternatively, a pharmaceutical or physiologically acceptable diluent.", "In a sixth aspect, the invention features a method of reducing body mass comprising providing or administering to individuals in need of reducing body mass said pharmaceutical or physiologically acceptable composition described in the fifth aspect.", "In preferred embodiments, the identification of said individuals in need of reducing body mass to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B single nucleotide polymorphisms (SNPs) or measuring metabolic polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of plasma, urine, and saliva.", "Preferably, a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment of the present invention is administered to an individual with at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in blood, serum or plasma levels of full-length any one or all of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides or the naturally proteolytically cleaved GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragments as compared to healthy, non-obese patients.", "In a seventh aspect, the invention features a method of preventing or treating an metabolic-related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect.", "In preferred embodiments, the identification of said individuals in need of such treatment to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B single nucleotide polymorphisms (SNPs) or measuring GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide, wherein said biological response is selected from the group consisting of: (a) modulating circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids, wherein said modulating is preferably lowering; (b) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose, wherein said modulating is preferably lowering; (c) modulating circulating (either blood, serum or plasma) levels (concentration) of triglycerides, wherein said modulating is preferably lowering; (d) stimulating muscle lipid or free fatty acid oxidation; (c) modulating leptin uptake in the liver or liver cells, wherein said modulating is preferably increasing; (e) modulating the postprandial increase in plasma free fatty acids due to a high fat meal, wherein said modulating is preferably reducing; (f) modulating ketone body production as the result of a high fat meal, wherein said modulating is preferably reducing or eliminating; (g) increasing cell or tissue sensitivity to insulin, particularly muscle, adipose, liver or brain; and (h) inhibiting the progression from impaired glucose tolerance to insulin resistance; and further wherein said biological response is significantly greater than, or at least 10%, 20%, 30%, 35%, 40%, 50% 75% 100% or 500% greater than, the biological response caused or induced by insulin alone at the same molar concentration.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy.", "In a further preferred embodiment, the present invention may be used in complementary therapy of NIDDM patients to improve their weight or glucose control in combination with an insulin secretagogue (preferably oral form) or an insulin sensitising (preferably oral form) agent.", "Preferably, the oral insulin secretagogue is 1, 1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.", "Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.", "The present invention further provides a method of improving the body weight or glucose control of NIDDM patients alone, without an insulin secretagogue or an insulin sensitising agent.", "In a further preferred embodiment, the present invention may be used in complementary therapy of IDDM patients to improve their weight or glucose control in combination with an insulin secretagogue (preferably oral form) or an insulin sensitising (preferably oral form) agent.", "Preferably, the insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.", "Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.", "The present invention further provides a method of improving the body weight or glucose control of IDDM patients alone, without an insulin secretagogue or an insulin sensitising agent.", "In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with the insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent).", "Accordingly, the present invention further provides for a composition of pharmaceutical or physiologically acceptable composition and an insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of body weight or glucose control in NIDDM or IDDM patients.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin sensitiser.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) without insulin therapy.", "In an eighth aspect, the invention features a method of making the GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides described in the first aspect, wherein said method is selected from the group consisting of: proteolytic cleavage, recombinant methodology and artificial synthesis.", "In a ninth aspect, the present invention provides a method of making a recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment or a full length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide, the method comprising providing a transgenic, non-human mammal whose milk contains said recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment or full-length protein, and purifying said recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment or said full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide from the milk of said non-human mammal.", "In one embodiment, said non-human mammal is a cow, goat, sheep, rabbit, or mouse.", "In another embodiment, the method comprises purifying a recombinant full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide from said milk, and further comprises cleaving said protein in vitro to obtain a desired GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment.", "In a tenth aspect, the invention features a purified or isolated antibody capable of specifically binding to a polypeptide of the present invention.", "In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 6 consecutive amino acids, at least 8 consecutive amino acids, or at least 10 consecutive amino acids of the sequence of one of the polypeptide sequences described in SEQ ID NOs: 2, 4, 6, 8, or 10.In an eleventh aspect, the invention features a use of the polypeptide described in the first aspect for treatment of metabolic-related diseases and disorders and/or reducing or increasing body mass.", "Preferably, said metabolic-related diseases and disorders are selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In a twelth aspect, the invention provides a polypeptide of the first aspect of the invention, or a composition of the fifth aspect of the invention, for use in a method of treatment of the human or animal body.", "In a thirteenth aspect, the invention features methods of reducing body weight for cosmetic purposes comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect, or a polypeptide described in the first aspect.", "Preferably, for said reducing body weight said individual has a BMI of at least 20 and no more than 25.Alternatively, for said increasing body weight said individual preferably has a BMI of at least 15 and no more than 20.In a fourteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect for reducing body mass and/or for treatment or prevention of metabolic-related diseases or disorders.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, ADS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B single nucleotide polymorphisms (SNPs) or measuring GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "In a fifteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect for reducing body weight for cosmetic reasons.", "In a sixteenth aspect, the invention features methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect, or a polypeptide described in the first aspect.", "In a seventeenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with normal glucose tolerance (NGT) who are obese or who have fasting hyperinsulinemia, or who have both.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with gestational diabetes.", "Gestational diabetes refers to the development of diabetes in an individual during pregnancy, usually during the second or third trimester of pregnancy.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with impaired fasting glucose (IFG).", "Impaired fasting glucose (IFG) is that condition in which fasting plasma glucose levels in an individual are elevated but not diagnostic of overt diabetes, i.e.", "plasma glucose levels of less than 126 mg/dl and less than or equal to 110 mg/dl.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating and preventing impaired glucose tolerance (IGT) in an individual.", "By providing therapeutics and methods for reducing or preventing IGT, i.e., for normalizing insulin resistance, the progression to NIDDM can be delayed or prevented.", "Furthermore, by providing therapeutics and methods for reducing or preventing insulin resistance, the invention provides methods for reducing and/or preventing the appearance of Insulin-Resistance Syndrome.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating a subject having polycystic ovary syndrome (PCOS).", "PCOS is among the most common disorders of premenopausal women, affecting 5-10% of this population.", "Insulin-sensitizing agents, e.g., troglitazone, have been shown to be effective in PCOS and that, in particular, the defects in insulin action, insulin secretion, ovarian steroidogenosis and fibrinolysis are improved (Ehrman et al.", "(1997) J Clin Invest 100:1230), such as in insulin-resistant humans.", "Accordingly, the invention provides methods for reducing insulin resistance, normalizing blood glucose thus treating and/or preventing PCOS.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating a subject having insulin resistance.", "In further preferred embodiments, a subject having insulin resistance is treated according to the methods of the invention to reduce or cure the insulin-resistance.", "As insulin resistance is also often associated with infections and cancer, prevention or reducing insulin resistance according to the methods of the invention may prevent or reduce infections and cancer.", "In further preferred embodiment, the methods of the invention are used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.", "Thus, any of the above-described tests or other tests known in the art can be used to determine that a subject is insulin-resistant, which patient can then be treated according to the methods of the invention to reduce or cure the insulin-resistance.", "Alternatively, the methods of the invention can also be used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.", "In an eighteenth aspect, the invention features a method of preventing or treating an metabolic-related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect.", "In preferred embodiments, the identification of said individuals in need of such treatment to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B single nucleotide polymorphisms (SNPs) or measuring GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, FIV-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably non-human, preferably a cat or a dog.", "In a nineteenth aspect, the invention features a method of using a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptide fragment to screen compounds for one or more antagonists of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptide fragment activity, wherein said activity is selected from but not restricted to lipid partitioning, lipid metabolism, and insulin-like activity.", "In preferred embodiment, said compound is selected from but is not restricted to small molecular weight organic or inorganic compound, protein, peptide, carbohydrate, or lipid.", "In a twentieth aspect, the invention features a method of using a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide to identify one or more cell types expressing a cell surface receptor for said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide, preferably wherein said polypeptide comprises all or part of the C-terminal globular C1q homology domain and has lipid partitioning, lipid metabolism, or insulin-like activities.", "In a twenty-first aspect, the invention features a method of using a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide to clone cDNA encoding a cell surface receptor for said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide, preferably wherein said polypeptide comprises all or part of the C-terminal globular C1q homology domain and has lipid partitioning, lipid metabolism, or insulin-like activities.", "In a twenty-second aspect, the invention features a method of using a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide to generate transgenic non-human mammals expressing GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, preferably wherein said non-human mammal is mouse, cow, sheep, goat, pig, or rabbit.", "In a twenty-third aspect, the invention features a method of using a genomic polynucleotide or fragment thereof of SEQ ID NO: 11, 12, or 13 to generate a transgenic mouse in which expression of the gene encoding GMG-6A and GMG-6B is knocked-out either globally or in a tissue-specific manner.", "In a preferred aspect of the methods above and disclosed herein, the amount of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polynucleotide administered to an individual is sufficient to bring circulating (blood, serum, or plasma) levels (concentration) of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides to their normal levels (levels in non-obese individuals).", "“Normal levels” may be specified as the total concentration of all circulating GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides (full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B proteins and fragments thereof) or the concentration of all circulating proteolytically cleaved GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides only.", "In a further preferred aspect of the methods above and disclosed herein, weight loss is due in part or in whole to a decrease in mass of either a) subcutaneous adipose tissue and/or b) visceral (omental) adipose tissue.", "Full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides and polynucleotides encoding the same may be specifically substituted for a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment or polynucleotide encoding the same in any embodiment of the present invention.", "It is further understood that by GMG-3 polypeptide is meant the amino acid sequence of SEQ ID NO: 2 as well as any related polypeptide incorporating one or more of the amino acid polymorphisms indicated in the sequence listing, namely the related polypeptide with Val at position 219, the related polypeptide with Met at position 301, and the related polypeptide with Val at position 219 and Met at position 301.It is further understood that by GMG-4 polypeptide is meant the amino acid sequence of SEQ ID NO: 4 as well as any related polypeptide incorporating one or more of the amino acid polymorphisms indicated in the sequence listing, namely the related polypeptide with Ala at position 238.It is further understood that by Cluster 1 polypeptide is meant the amino acid sequence of SEQ ID NO: 6 as well as any related polypeptide incorporating one or more of the amino acid polymorphisms indicated in the sequence listing, namely the related polypeptide with Ala at position 130.DETAILED DESCRIPTION OF THE SEQUENCE LISTING SEQ ID NO: 1 represents the cDNA sequence of GMG-3.SEQ ID NO:2 represents the amino acid sequence encoded by the cDNA of SEQ ID NO: 1.SEQ ID NO:3 represents the cDNA sequence of GMG-4.SEQ ID NO:4 represents the amino acid sequence encoded by the cDNA of SEQ ID NO:3.SEQ ID NO:5 represents the polynucleotide sequence of Cluster 1.SEQ ID NO:6 represents the amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO:5.SEQ ID NO:7 represents the cDNA sequence of GMG-6A.", "SEQ ID NO:8 represents the amino acid sequence encoded by the cDNA of SEQ ID NO:7.SEQ ID NO:9 represents the cDNA sequence of GMG-6B.", "SEQ ID NO: 10 represents the amino acid sequence encoded by the cDNA of SEQ ID NO:9.SEQ ID NO: 11 represents GMG-6 genomic sequence comprising the coding region of the first coding exon for GMG-6A and GMG-6B.", "SEQ ID NO:12 represents GMG-6 genomic sequence comprising the coding region of the second coding exon for GMG-6A and GMG-6B.", "SEQ ID NO: 13 represents GMG-6 genomic sequence comprising the coding region of the third coding exon for GMG-6A and GMG-6B.", "DETAILED DESCRIPTION OF THE INVENTION Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein.", "As used interchangeably herein, the terms “oligonucleotides”, and “polynucleotides” and nucleic acid include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.", "The terms encompass “modified nucleotides” which comprise at least one modification, including by way of example and not limitation: (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar.", "For examples of analogous linking groups, purines, pyrimidines, and sugars see for example PCT publication No.", "WO 95/04064.The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.", "The terms polynucleotide construct, recombinant polynucleotide and recombinant polypeptide are used herein consistently with their use in the art.", "The terms “upstream” and “downstream” are also used herein consistently with their use in the art.", "The terms “base paired” and “Watson & Crick base paired” are used interchangeably herein and consistently with their use in the art.", "Similarly, the terms “complementary”, “complement thereof”, “complement”, “complementary polynucleotide”, “complementary nucleic acid” and “complementary nucleotide sequence” are used interchangeably herein and consistently with their use in the art.", "The term “purified” is used herein to describe a polynucleotide or polynucleotide vector of the invention that has been separated from other compounds including, but not limited to, other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide).", "Purified can also refer to the separation of covalently closed polynucleotides from linear polynucleotides, or vice versa, for example.", "A polynucleotide is substantially pure when at least about 50%, 60%, 75%, or 90% of a sample contains a single polynucleotide sequence.", "In some cases this involves a determination between conformations (linear versus covalently closed).", "A substantially pure polynucleotide typically comprises about 50, 60, 70, 80, 90, 95, 99% weight/weight of a nucleic acid sample.", "Polynucleotide purity or homogeneity may be indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel.", "For certain purposes, higher resolution can be achieved by using HPLC or other means well known in the art.", "Similarly, the term “purified” is used herein to describe a polypeptide of the invention that has been separated from other compounds including, but not limited to, nucleic acids, lipids, carbohydrates and other proteins.", "In some preferred embodiments, a polypeptide is substantially pure when at least about 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the polypeptide molecules of a sample have a single amino acid sequence.", "In some preferred embodiments, a substantially pure polypeptide typically comprises about 50%, 60%, 70%, 80%, 90% 95%, 96%, 97%, 98%, 99% or 99.5% weight/weight of a protein sample.", "Polypeptide purity or homogeneity is indicated by a number of methods well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel.", "For certain purposes, higher resolution can be achieved by using HPLC or other methods well known in the art.", "Further, as used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition.", "Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.", "Alternatively, purification may be expressed as “at least” a percent purity relative to heterologous polynucleotides (DNA, RNA or both) or polypeptides.", "As a preferred embodiment, the polynucleotides or polypeptides of the present invention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 98%, 99%, 99.5% or 100% pure relative to heterologous polynucleotides or polypeptides.", "As a further preferred embodiment the polynucleotides or polypeptides have an “at least” purity ranging from any number, to the thousandth position, between 90% and 100% (e.g., at least 99.995% pure) relative to heterologous polynucleotides or polypeptides.", "Additionally, purity of the polynucleotides or polypeptides may be expressed as a percentage (as described above) relative to all materials and compounds other than the carrier solution.", "Each number, to the thousandth position, may be claimed as individual species of purity.", "The term “isolated” requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).", "For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.", "Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.", "Specifically excluded from the definition of “isolated” are: naturally occurring chromosomes (e.g., chromosome spreads), artificial chromosome libraries, genomic libraries, and cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies.", "Also specifically excluded are the above libraries wherein a 5′ EST makes up less than 5% (or alternatively 1%, 2%, 3%, 4%, 10%, 25%, 50%, 75%, or 90%, 95%, or 99%) of the number of nucleic acid inserts in the vector molecules.", "Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including said whole cell preparations which are mechanically sheared or enzymatically digested).", "Further specifically excluded are the above whole cell preparations as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis (including blot transfers of the same) wherein the polynucleotide of the invention have not been further separated from the heterologous polynucleotides in the electrophoresis medium (e.g., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).", "The term “primer” denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.", "A primer serves as an initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase, or reverse transcriptase.", "The term “probe” denotes a defined nucleic acid segment that can be used to identify a specific polynucleotide sequence present in a sample, said nucleic acid segment comprising a nucleotide sequence complementary to the specific polynucleotide sequence to be identified.", "The term “polypeptide” refers to a polymer of amino acids without regard to the length of the polymer.", "Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.", "This term also does not specify or exclude post-expression modifications of polypeptides.", "For example, polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.", "Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.", "), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.", "Without being limited by theory, the compounds/polypeptides of the invention are capable of modulating the partitioning of dietary lipids between the liver and peripheral tissues, and thus of treating “diseases involving the partitioning of dietary lipids between the liver and peripheral tissues.” The term “peripheral tissues” is meant to include muscle and adipose tissue.", "In preferred embodiments, the compounds/polypeptides of the invention partition the dietary lipids toward the muscle.", "In alternative preferred embodiments, the dietary lipids are partitioned toward the adipose tissue.", "In other preferred embodiments, the dietary lipids are partitioned toward the liver.", "In yet other preferred embodiments, the compounds/polypeptides of the invention increase or decrease the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle.", "Dietary lipids include, but are not limited to triglycerides and free fatty acids.", "Preferred diseases believed to involve the partitioning of dietary lipids include obesity and obesity-related diseases and disorders such as obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Non-Insulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).", "Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "The term “heterologous”, when used herein, is intended to designate any polypeptide or polynucleotide other than a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or a polynucleotide encoding a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of the present invention.", "The terms “comprising”, “consisting of” and “consisting essentially of” are defined according to their standard meaning.", "A defined meaning set forth in the M.P.E.P.", "controls over a defined meaning in the art and a defined meaning set forth in controlling Federal Circuit case law controls over a meaning set forth in the M.P.E.P.", "With this in mind, the terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.", "The term “host cell recombinant for” a particular polynucleotide of the present invention, means a host cell that has been altered by the hands of man to contain said polynucleotide in a way not naturally found in said cell.", "For example, said host cell may be transiently or stably transfected or transduced with said polynucleotide of the present invention.", "The term “obesity” as used herein is defined in the WHO classifications of weight (Kopelman (2000) Nature 404:635-643).", "Underweight is less than 18.5 (thin); Healthy is 18.5-24.9 (normal); grade 1 overweight is 25.0-29.9 (overweight); grade 2 overweight is 30.0-39.0 (obesity); grade 3 overweight is greater than or equal to 40.0 BMI.", "BMI is body mass index (morbid obesity) and is kg/m2.Waist circumference can also be used to indicate a risk of metabolic complications where in men a circumference of greater than or equal to 94 cm indicates an increased risk, and greater than or equal to 102 cm indicates a substantially increased risk.", "Similarly for women, greater than or equal to 88 cm indicates an increased risk, and greater than or equal to 88 cm indicates a substantially increased risk.", "The waist circumference is measured in cm at midpoint between lower border of ribs and upper border of the pelvis.", "Other measures of obesity include, but are not limited to, skinfold thickness which is a measurement in cm of skinfold thickness using calipers, and bioimpedance, which is based on the principle that lean mass conducts current better than fat mass because it is primarily an electrolyte solution; measurement of resistance to a weak current (impedance) applied across extremities provides an estimate of body fat using an empirically derived equation.", "The term “diabetes” as used herein is intended to encompass the usual diagnosis of diabetes made from any of the methods included, but not limited to, the following list: symptoms of diabetes (eg.", "polyuria, polydipsia, polyphagia) plus casual plasma glucose levels of greater than or equal to 200 mg/dl, wherein casual plasma glucose is defined any time of the day regardless of the timing of meal or drink consumption; 8 hour fasting plasma glucose levels of less than or equal to 126 mg/dl; and plasma glucose levels of greater than or equal to 200 mg/dl 2 hours following oral administration of 75 g anhydrous glucose dissolved in water.", "The term “impaired glucose tolerance (IGT)” as used herein is intended to indicate that condition associated with insulin-resistance that is intermediate between frank, NIDDM and normal glucose tolerance (NGT).", "A high percentage of the IGT population is known to progress to NIDDM relative to persons with normal glucose tolerance (Sad et al., New Engl J Med 1988; 319:1500-6).", "Thus, by providing therapeutics and methods for reducing or preventing IGT, i.e., for normalizing insulin resistance, the progression to NIDDM can be delayed or prevented.", "IGT is diagnosed by a procedure wherein an affected person's postprandial glucose response is determined to be abnormal as assessed by 2-hour postprandial plasma glucose levels.", "In this test, a measured amount of glucose is given to the patient and blood glucose levels measured regular intervals, usually every half hour for the first two hours and every hour thereafter.", "In a “normal” or non-IGT individual, glucose levels rise during the first two hours to a level less than 140 mg/dl and then drop rapidly.", "In an IGT individual, the blood glucose levels are higher and the drop-off level is at a slower rate.", "The term “Insulin-Resistance Syndrome” as used herein is intended to encompass the cluster of abnormalities resulting from an attempt to compensate for insulin resistance that sets in motion a series of events that play an important role in the development of both hypertension and coronary artery disease (CAD), such as premature atherosclerotic vascular disease.", "Increased plasma triglyceride and decreased HDL-cholesterol concentrations, conditions that are known to be associated with CAD, have also been reported to be associated with insulin resistance.", "Thus, by providing therapeutics and methods for reducing or preventing insulin resistance, the invention provides methods for reducing and/or preventing the appearance of insulin-resistance syndrome.", "The term “polycystic ovary syndrome (PCOS)” as used herein is intended to designate that etiologically unassigned disorder of premenopausal women, affecting 5-10% of this population, characterized by hyperandrogenism, chronic anovulation, defects in insulin action, insulin secretion, ovarian steroidogenesis and fibrinolysis.", "Women with PCOS frequently are insulin resistant and at increased risk to develop glucose intolerance or NIDDM in the third and fourth decades of life (Dunaif et al.", "(1996) J Clin Endocrinol Metab 81:3299).", "Hyperandrogenism also is a feature of a variety of diverse insulin-resistant states, from the type A syndrome, through leprechaunism and lipoatrophic diabetes, to the type B syndrome, when these conditions occur in premenopausal women.", "It has been suggested that hyperinsulinemia per se causes hyperandrogenism.", "Insulin-sensitizing agents, e.g., troglitazone, have been shown to be effective in PCOS and that, in particular, the defects in insulin action, insulin secretion, ovarian steroidogenosis and fibrinolysis are improved (Ehrman et al.", "(1997) J Clin Invest 100:1230), such as in insulin-resistant humans.", "The term “insulin resistance” as used herein is intended to encompass the usual diagnosis of insulin resistance made by any of a number of methods, including but not restricted to: the intravenous glucose tolerance test or measurement of the fasting insulin level.", "It is well known that there is an excellent correlation between the height of the fasting insulin level and the degree of insulin resistance.", "Therefore, one could use elevated fasting insulin levels as a surrogate marker for insulin resistance for the purpose of identifying which normal glucose tolerance (NGT) individuals have insulin resistance.", "Another way to do this is to follow the approach as disclosed in The New England Journal of Medicine, No.", "3, pp.", "1188 (1995), i.e.", "to select obesity as an initial criterion for entry into the treatment group.", "Some obese subjects have impaired glucose tolerance (IGT) while others have normal glucose tolerance (NGT).", "Since essentially all obese subjects are insulin resistant, i.e.", "even the NGT obese subjects are insulin resistant and have fasting hyperinsulinemia.", "Therefore, the target of the treatment according to the present invention can be defined as NGT individuals who are obese or who have fasting hyperinsulinemia, or who have both.", "A diagnosis of insulin resistance can also be made using the euglycemic glucose clamp test.", "This test involves the simultaneous administration of a constant insulin infusion and a variable rate glucose infusion.", "During the test, which lasts 3-4 hours, the plasma glucose concentration is kept constant at euglycemic levels by measuring the glucose level every 5-10 minutes and then adjusting the variable rate glucose infusion to keep the plasma glucose level unchanged.", "Under these circumstances, the rate of glucose entry into the bloodstream is equal to the overall rate of glucose disposal in the body.", "The difference between the rate of glucose disposal in the basal state (no insulin infusion) and the insulin infused state, represents insulin mediated glucose uptake.", "In normal individuals, insulin causes brisk and large increase in overall body glucose disposal, whereas in NIDDM subjects, this effect of insulin is greatly blunted, and is only 20-30% of normal.", "In insulin resistant subjects with either IGT or NGT, the rate of insulin stimulated glucose disposal is about half way between normal and NIDDM.", "For example, at a steady state plasma insulin concentration of about 100 uU/ml (a physiologic level) the glucose disposal rate in normal subjects is about 7 mg/kg/min.", "In NIDDM subjects, it is about 2.5 mg/kg/min., and in patients with IGT (or insulin resistant subjects with NGT) it is about 4-5 mg/kg/min.", "This is a highly reproducible and precise test, and can distinguish patients within these categories.", "It is also known that as subjects become more insulin resistant, the fasting insulin level rises.", "There is an excellent positive correlation between the height of the fasting insulin level and the magnitude of the insulin resistance as measured by euglycemic glucose clamp tests and, therefore, this provides the rationale for using fasting insulin levels as a surrogate measure of insulin resistance.", "The term “agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the partitioning of dietary lipids between the liver and the peripheral tissues as previously described.", "Preferably, the agent increases or decreases the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle.", "Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an obesity-related disease or disorder such as obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Non-Insulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).", "Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "The terms “response to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.", "The terms “side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.", "“Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.", "The term “GMG-3-, GMG-4-, Cluster 1-, GMG-6A-, or GMG-6B-related diseases and disorders” as used herein refers to any disease or disorder comprising an aberrant functioning of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B, or which could be treated or prevented by modulating GMG-3, GMG-1, Cluster 1, GMG-6A, or GMG-6B levels or activity.", "“Aberrant functioning of GMG-3, GMG-1, Cluster 1, GMG-6A, or GMG-6B” includes, but is not limited to, aberrant levels of expression of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B (either increased or decreased, but preferably decreased), aberrant activity of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B (either increased or decreased), and aberrant interactions with ligands or binding partners (either increased or decreased).", "By “aberrant” is meant a change from the type, or level of activity seen in normal cells, tissues, or patients, or seen previously in the cell, tissue, or patient prior to the onset of the illness.", "In preferred embodiments, these GMG-3-, GMG-4-, Cluster 1-, GMG-6A-, or GMG-6B-related diseases and disorders include obesity and the metabolic-related diseases and disorders described previously.", "The term “cosmetic treatments” is meant to include treatments with compounds or polypeptides of the invention that increase or decrease the body mass of an individual where the individual is not clinically obese or clinically thin.", "Thus, these individuals have a body mass index (BMI) below the cut-off for clinical obesity (e.g.", "below 25 kg/m2) and above the cut-off for clinical thinness (e.g.", "above 18.5 kg/m2).", "In addition, these individuals are preferably healthy (e.g.", "do not have an metabolic-related disease or disorder of the invention).", "“Cosmetic treatments” are also meant to encompass, in some circumstances, more localized increases in adipose tissue, for example, gains or losses specifically around the waist or hips, or around the hips and thighs, for example.", "These localized gains or losses of adipose tissue can be identified by increases or decreases in waist or hip size, for example.", "The term “preventing” as used herein refers to administering a compound prior to the onset of clinical symptoms of a disease or condition so as to prevent a physical manifestation of aberrations associated with obesity or GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B.", "The term “treating” as used herein refers to administering a compound after the onset of clinical symptoms.", "The term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g.", "physician, nurse, nurse practitioner, etc in the case of humans; veterinarian in the case of animals, including non-human mammals) that an individual or animal requires or will benefit from treatment.", "This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that include the knowledge that the individual or animal is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention.", "The term “perceives a need for treatment” refers to a sub-clinical determination that an individual desires to reduce weight for cosmetic reasons as discussed under “cosmetic treatment” above.", "The term “perceives a need for treatment” in other embodiments can refer to the decision that an owner of an animal makes for cosmetic treatment of the animal.", "The term “individual” or “patient” as used herein refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.", "The term may specify male or female or both, or exclude male or female.", "The term “non-human animal” refers to any non-human vertebrate, including birds and more usually mammals, preferably primates, animals such as swine, goats, sheep, donkeys, horses, cats, dogs, rabbits or rodents, more preferably rats or mice.", "Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term “non-human”.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are able to significantly reduce the postprandial response of plasma free fatty acids, glucose, and triglycerides in mammals fed a high fat/sucrose meal, while not affecting levels of leptin, insulin or glucagon.", "In addition, GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides modulate muscle free fatty acid oxidation in vitro and ex vivo, preferably increase oxidation.", "Further, GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention modulate weight gain in mammals that are fed a high fat/sucrose diet.", "The instant invention encompasses the use of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides in the partitioning of free fatty acid (FFA) and as an important new tool to control energy homeostasis.", "Of the tissues that can significantly remove lipids from circulation and cause FFA oxidation, muscle is believed to be quantitatively the most important.", "PREFERRED EMBODIMENTS OF THE INVENTION I. GMG-3.GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptides of the Invention GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides have been identified that have measurable activity in vitro and in vivo.", "These activities include, but are not limited to, modulation, preferably reduction, of the postprandial response of plasma free fatty acids, glucose, and triglycerides in mammals fed a high fat/sucrose meal (Example 6), change, preferably an increase, in muscle free fatty acid oxidation in vitro and ex vivo (Example 10), and sustained weight loss in mammals on a high fat/sucrose diet.", "Other assays for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide activity in vitro and in vivo are also provided (Examples 2, 5, 7, 9, 11, for example), and equivalent assays can be designed by those with ordinary skill in the art.", "The term “GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides” includes both the “full-length” polypeptide and fragments of the “full-length” GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides (although each of the above species may be particularly specified).", "By “intact” or “full-length” GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides as used herein is meant the full-length polypeptide sequence of any GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide, from the N-terminal methionine to the C-terminal stop codon.", "Examples of intact or full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are found in the sequence listing.", "The term “metabolic-related activity” as used herein refers to at least one, and preferably all, of the activities described herein for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides.", "Assays for the determination of these activities are provided herein (e.g.", "Examples 2, 5-7, 9-11), and equivalent assays can be designed by those with ordinary skill in the art.", "Optionally, “metabolic-related activity” can be selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity, or an activity within one of these categories.", "By “lipid partitioning” activity is meant the ability to effect the location of dietary lipids among the major tissue groups including, adipose tissue, liver, and muscle.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention play a role in the partitioning of lipids to the muscle, liver or adipose tissue.", "By “lipid metabolism” activity is meant the ability to influence the metabolism of lipids.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention have the ability to affect the level of free fatty acids in the plasma as well as to modulate, preferably increase, the metabolism of lipids in the muscle through free fatty acid oxidation experiments (Examples 2, 6, 8, 9, 10) and to transiently affect the levels of triglycerides in the plasma and the muscle (Examples 6, 8, 11).", "By “insulin-like” activity is meant the ability of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides to modulate the levels of glucose in the plasma.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides do not significantly impact insulin levels but do impact glucose levels similarly to the effects of insulin (examples 7 & 8).", "These effects may vary in the presence of the intact (full-length) GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides or are significantly greater in the presence of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments compared with the full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides.", "The term “significantly greater” as used herein refers to a comparison of the activity of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide in an metabolic-related assay compared with untreated cells in the same assay.", "By “significantly” as used herein is meant statistically significant as it is typically determined by those with ordinary skill in the art.", "For example, data are typically calculated as a mean±SEM, and a p-value≦0.05 is considered statistically significant.", "Statistical analysis is typically done using either the unpaired Student's t test or the paired Student's t test, as appropriate in each study.", "Examples of a significant change in activity as a result of the presence of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of the invention compared to untreated cells include an increase or a decrease in a given parameter of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.", "One or more, but not necessarily all, of the measurable parameters will change significantly in the presence of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide as compared to untreated cells.", "Representative “metabolic-related assays” are provided in the Examples.", "These assays include, but are not limited to, methods of measuring the postprandial response, methods of measuring free fatty acid oxidation, and methods of measuring weight modulation.", "In preferred embodiments, the post-prandial response is measured in non-human animals, preferably mice.", "In preferred embodiments changes in dietary lipids are measured, preferably free fatty acids and/or triglycerides.", "In other embodiments, other physiologic parameters are measured including, but not limited to, levels of glucose, insulin, and leptin.", "In other preferred embodiments, free fatty acid oxidation is measured in cells in vitro or ex vivo, preferably in muscle cells or tissue of non-human animals, preferably mice.", "In yet other preferred embodiments weight modulation is measured in human or non-human animals, preferably rodents (rats or mice), primates, canines, felines or procines, on a high fat/sucrose diet.", "Optionally, “metabolic-related activity” includes other activities not specifically identified herein.", "In general, “measurable parameters” relating to obesity and the field of metabolic research can be selected from the group consisting of free fatty acid levels, free fatty acid oxidation, triglyceride levels, glucose levels, insulin levels, leptin levels, food intake, weight, leptin and lipoprotein binding, uptake and degradation and lipolysis stimulated receptor (LSR) expression.", "In these metabolic-related assays, preferred GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides would cause a significant change in at least one of the measurable parameters selected from the group consisting of post-prandial lipemia, free fatty acid levels, triglyceride levels, glucose levels, free fatty acid oxidation, and weight.", "Alternatively, preferred GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides would have a significant change in at least one of the measurable parameters selected from the group consisting of an increase in LSR activity, an increase in leptin activity and an increase in lipoprotein activity.", "By “LSR” activity is meant expression of LSR on the surface of the cell, or in a particular conformation, as well as its ability to bind, uptake, and degrade leptin and lipoprotein.", "By “leptin” activity is meant its binding, uptake and degradation by LSR, as well as its transport across a blood brain barrier, and potentially these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.", "Similarly, by “lipoprotein” activity is meant its binding, uptake and degradation by LSR, as well as these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.", "The invention is drawn, inter alia, to isolated, purified or recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention are useful for reducing or, using antagonists of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, increasing body weight either as a cosmetic treatment or for treatment or prevention of metabolic-related diseases and disorders.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are also useful inter alia in screening assays for agonists or antagonists of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide activity; for raising GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide-specific antibodies; and in diagnostic assays.", "When used for cosmetic treatments, or for the treatment or prevention of metabolic-related diseases, disorders or conditions, one or more GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B or GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments can be provided to a subject.", "Thus, various fragments of the full-length protein can be combined into a “cocktail” for use in the various treatment regimens.", "The full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG B polypeptide is comprised of about four distinct regions including: 1.an N-terminal putative signal peptide sequence about from amino acids 1-19 of SEQ ID NO: 2, 4, 8, or 10; 2.a unique region about from amino acids 20-25 of SEQ ID NO: 2, 4, 8, or 10; 3.a collagen-like region about from amino acids 26-196 of SEQ ID NO: 2 or 4, from about amino acids 1-87 of SEQ ID NO: 6, from about amino acids 26-193 of SEQ ID NO: 8, or from about amino acids 26-186 of SEQ ID NO: 10; and 4.a C-terminal globular C1q homology domain about from amino acids 200-333 of SEQ ID NO: 2 or 4, about from amino acids 88-225 of SEQ ID NO: 6, about from amino acids 197-330 of SEQ ID NO: 8, or about from amino acids 190-323 of SEQ ID NO: 10.GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention include variants, fragments, analogs and derivatives of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides described above, including modified GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides.", "The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the present invention are preferably provided in an isolated form, and may be partially or substantially purified.", "A recombinantly produced version of any one of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides can be substantially purified by the one-step method described by Smith et al.", "((1988) Gene 67:31-40) or by the methods described herein or known in the art Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies directed against the polypeptides of the invention by methods known in the art of protein purification.", "Preparations of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention involving a partial purification of or selection for the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are also specifically contemplated.", "These crude preparations are envisioned to be the result of the concentration of cells expressing GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides with perhaps a few additional purification steps, but prior to complete purification of the fragment.", "The cells expressing GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are present in a pellet, they are lysed, or the crude polypeptide is lyophilized, for example.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments can be any integer in length from at least 6 consecutive amino acids to one amino acid less than a full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide.", "Thus, for the polypeptides of SEQ ID NO: 2 or 4, a GMG-3 or GMG-4 polypeptide fragment can be any integer of consecutive amino acids from 6 to 332, for example.", "The term “integer” is used herein in its mathematical sense and thus representative integers include, but are not limited to: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 and 332.Each GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment as described above can be further specified in terms of its N-terminal and C-terminal positions.", "For example, every combination of a N-terminal and C-terminal position that fragments of from 6 contiguous amino acids to one amino acid less than the full-length polypeptide of SEQ ID NO: 2 or 4 could occupy, on any given intact and contiguous full-length polypeptide sequence of SEQ ID NO: 2 or 4 are included in the present invention.", "Thus, a 6 consecutive amino acid fragment could occupy positions selected from the group consisting of 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20, 16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30, 26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40, 36-41, 3742, 38-43, 39-44, 40-45, 41-46, 4247, 43-48, 44-49, 45-50, 46-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-60, 56-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70, 66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, 75-80, 76-81, 77-82, 78-83, 79-84, 80-85, 81-86, 82-87, 83-88, 84-89, 85-90, 86-91, 87-92, 88-93, 89-94, 90-95, 91-96, 92-97, 93-98, 94-99, 95-100, 96-101, 97-102, 98-103, 99-104, 100-105, 101-106, 102-107, 103-108, 104-109, 105-110, 106-111, 107-112, 108-113, 109-114, 110-115, 111-116, 112-117, 113-118, 114-119, 115-120, 116-121, 117-122, 118-123, 119-124, 120-125, 121-126, 122-127, 123-128, 124-129, 125-130, 126-131, 127-132, 128-133, 129-134, 130-135, 131-136, 132-137, 133-138, 134-139, 135-140, 136-141, 137-142, 138-143, 139-144, 140-145, 141-146, 142-147, 143-148, 144-149, 145-150, 146-151, 147-152, 148-153, 149-154, 150-155, 151-156, 152-157, 153-158, 154-159, 155-160, 156-161, 157-162, 158-163, 159-164, 160-165, 161-166, 162-167, 163-168, 164-169, 165-170, 166-171, 167-172, 168-173, 169-174, 170-175, 171-176, 172-177, 173-178, 174-179, 175-180, 176-181, 177-182, 178-183, 179-184, 180-185, 181-186, 182-187, 183-188, 184-189, 185-190, 186-191, 187-192, 188-193, 189-194, 190-195, 191-196, 192-197, 193-198, 194-199, 195-200, 196-201, 197-202, 198-203, 199-204, 200-205, 201-206, 202-207, 203-208, 204-209, 205-210, 206-211, 207-212, 208-213, 209-214, 210-215, 211-216, 212-217, 213-218, 214-219, 215-220,216-221, 217-222, 218-223, 219-224,220-225, 221-226, 222-227, 223-228, 224-229, 225-230, 226-231, 227-232, 228-233, 229-234, 230-235, 231-236, 232-237, 233-238, 234-239, 235-240, 236-241, 237-242, 238-243, 240-245, 241-246, 242-247, 243-248, 244-249, 245-250, 246-251, 247-252, 248-253, 249-254, 250-255, 251-256, 252-257, 253-258, 254-259, 255-260, 256-261, 257-262, 258-263, 259-264, 260-265, 261-266, 262-267, 263-268, 264-269, 265-270, 266-271, 267-272, 268-273, 269-274, 270-275, 271-276, 272-277, 273-278, 274-279, 275-280, 276-281, 277-282, 278-283, 279-284, 280-285, 281-286, 282-287, 283-288, 284-289, 285-290, 286-291, 287-292, 288-293, 289-294, 290-295, 291-296, 292-297, 293-298, 294-299, 295-300, 296-301, 297-302, 298-303, 299-304, 300-305, 301-306, 302-307, 303-308, 304-309, 305-310, 306-311, 307-312, 308-313, 309-314, 310-315, 311-316, 312-317, 313-318, 314-319, 315-320, 316-321, 317-322, 318-323, 319-324, 320-325, 321-326, 322-327, 323-328, 324-329, 325-330, 326-331, 327-332 and 328-333 of a 333 consecutive amino acid fragment.", "A 327 consecutive amino acid fragment could occupy positions selected from the group consisting of 1-327, 2-328, 3-329, 4-330, 5-331, 6-332 and 7-333.Similarly, the positions occupied by all the other fragments of sizes between 6 amino acids and 332 amino acids in SEQ ID NO: 2 or 4, by all the other fragments of sizes between 6 amino acids and 224 amino acids in SEQ ID NO: 6, by all the other fragments of sizes between 6 amino acids and 329 amino acids in SEQ ID NO: 8, and by all the other fragments of sizes between 6 amino acids and 322 amino acids in SEQ ID NO: 10 are included in the present invention and can also be immediately envisaged based on these two examples and therefore, are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "Furthermore, the positions occupied by fragments of 6 to 332 consecutive amino acids in SEQ ID NO: 2 or 4, by fragments of 6 to 224 consecutive amino acids in SEQ ID NO: 6, by fragments of 6 to 329 amino acids in SEQ ID NO: 8, and by fragments of 6 to 322 amino acids in SEQ ID NO: 10 are included in the present invention and can also be immediately envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "In addition, the positions occupied by fragments of 6 consecutive amino acids to 1 amino acid less than any other full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide can also be envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the present invention may alternatively be described by the formula “n to c” (inclusive); where “n” equals the N-terminal most amino acid position (as defined by the sequence listing) and “c” equals the C-terminal most amino acid position (as defined by the sequence listing) of the polypeptide; and further where “n” equals an integer between 1 and the number of amino acids of the full-length polypeptide sequence of the present invention minus 6; and where “c” equals an integer between 7 and the number of amino acids of the full-length polypeptide sequence; and where “n” is an integer smaller then “c” by at least 6.Therefore, for the sequences provided in SEQ ID NO: 2 or 4, “n” is any integer selected from the list consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326 or 327 and “c” is any integer selected from the group consisting of: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332 or 333.Every combination of “n” and “c” positions are included as specific embodiments of the invention.", "Moreover, the formula “n” to “c” may be modified as “n1-n2” to “c1-c2”, wherein “n1-n2” and “c1-c2” represent positional ranges selected from any two integers above which represent amino acid positions of the sequence listing.", "Alternative formulas include “n1-n2” to “c” and “n” to “c1-c2”.", "In a preferred embodiment, GMG-3 or GMG-4 polypeptide fragments of the invention may be described by the formula where n1=20, n2=202, and c=333 of SEQ ID NO: 2 or 4; Cluster 1 polypeptide fragments of the invention may be described by the formula n1=1, n2=94, and c=225 of SEQ ID NO: 6; GMG-6A polypeptide fragments of the invention may be described by the formula n1=20, n2=199, and c=330 of SEQ ID NO: 8; or GMG-6B polypeptide fragments of the invention may be described by the formula where n1=20, n2=192, and c=323 of SEQ ID NO: 10.Furthermore, the positions occupied by polypeptides of 6 to 225 consecutive amino acids on SEQ ID NO: 6, by polypeptides of 6 to 330 consecutive amino acids on SEQ ID NO:8, or by polypeptides of 6 to 323 consecutive amino acids on SEQ ID NO: 10, are included in the present invention and can also be immediately envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "In addition, the positions occupied by fragments of 6 consecutive amino acids to 1 amino acid less than full-length Cluster 1, GMG-6A, or GMG-6B polypeptide can also be envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "In preferred embodiments, GMG-3 or GMG-4 polypeptide fragments having activity are selected from amino acids 20-333, 43-333, 44-333, 45-333, 46-333, 47-333, 48-333, 49-333, 50-333, 51-333, 52-333, 53-333, 54-333, 55-333, 56-333, 57-333, 58-333, 59-333, 60-333, 61-333, 62-333, 63-333, 64-333, 65-333, 66-333, 67-333, 68-333, 69-333, 70-333, 71-333, 72-333, 73-333, 74-333, 75-333, 76-333, 77-333, 78-333, 79-333, 80-333, 81-333, 82-333, 83-333, 84-333, 85-333, 86-333, 87-333, 88-333, 89-333, 90-333, 91-333, 92-333, 93-333, 94-333, 95-333, 96-333, 97-333, 98-333, 99-333, 100-333, 101-333, 102-333, 103-333, 104-333, 105-333, 106-333, 107-333, 108-333, 109-333, 110-333, 111-333, 112-333, 113-333, 114-333, 115-333, 116-333, 117-333, 118-333, 119-333, 120-333, 121-333, 122-333, 123-333, 124-333, 125-333, 126-333, 127-333, 128-333, 129-333, 130-333, 131-333, 132-333, 133-333, 134-333, 135-333, 136-333, 137-333, 138-333, 139-333, 140-333, 141-333, 142-333, 143-333, 144-333, 145-333, 146-333, 147-333, 148-333, 149-333, 150-333, 151-333, 152-333, 153-333, 154-333, 155-333, 156-333, 157-333, 158-333, 159-333, 160-333, 161-333, 162-333, 163-333, 164-333, 165-333, 166-333, 167-333, 168-333, 169-333, 170-333, 171-333, 172-333, 173-333, 174-333, 175-333, 176-333, 177-333, 178-333, 179-333, 180-333, 181-333, 182-333, 183-333, 184-333, 185-333, 186-333, 187-333, 188-333, 189-333, 190-333, 191-333, 192-333, 193-333, 194-333, 195-333, 196-333, 197-333, 198-333, 199-333, 200-333, 201-333 or 202-333 of SEQ ID NO: 2 or 4.In other preferred embodiments, Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 2-225, 3-225, 4-225, 5-225, 6-225, 7-225, 8-225, 9-225, 10-225, 11-225, 12-225, 13-225, 14-225, 15-225, 16-225, 17-225, 18-225, 19-225, 20-225, 21-225, 22-225, 23-225, 24-225, 25-225, 26-225, 27-225, 28-225, 29-225, 30-225, 31-225, 32-225, 33-225, 34-225, 35-225, 36-225, 37-225, 38-225, 39-225, 40-225, 41-225, 42-225, 43-225, 44-225, 45-225, 46-225, 47-225, 48-225, 49-225, 50-225, 51-225, 52-225, 53-225, 54-225, 55-225, 56-225, 57-225, 58-225, 59-225, 60-225, 61-225, 62-225, 63-225, 64-225, 65-225, 66-225, 67-225, 68-225, 69-225, 70-225, 71-225, 72-225, 73-225, 74-225, 75-225, 76-225, 77-225, 78-225, 79-225, 80-225, 81-225, 82-225, 83-225, 84-225, 85-225, 86-225, 87-225, 88-225, 89-225, 90-225, 91-225, 92-225, 93-225 or 94-225 of SEQ ID NO: 6.In other preferred embodiments, GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 43-330, 44-330, 45-330, 46-330, 47-330, 48-330, 49-330, 50-330, 51-330, 52-330, 53-330, 54-330, 55-330, 56-330, 57-330, 58-330, 59-330, 60-330, 61-330, 62-330, 63-330, 64-330, 65-330, 66-330, 67-330, 68-330, 69-330, 70-330, 71-330, 72-330, 73-330, 74-330, 75-330, 76-330, 77-330, 78-330, 79-330, 80-330, 81-330, 82-330, 83-330, 84-330, 85-330, 86-330, 87-330, 88-330, 89-330, 90-330, 91-330, 92-330, 93-330, 94-330, 95-330, 96-330, 97-330, 98-330, 99-330, 100-330, 101-330, 102-330, 103-330, 104-330, 105-330, 106-330, 107-330, 108-330, 109-330, 110-330, 111-330, 112-330, 113-330, 114-330, 115-330, 116-330, 117-330, 118-330, 119-330, 120-330, 121-330, 122-330, 123-330, 124-330, 125-330, 126-330, 127-330, 128-330, 129-330, 130-330, 131-330, 132-330, 133-330, 134-330, 135-330, 136-330, 137-330, 138-330, 139-330, 140-330, 141-330, 142-330, 143-330, 144-330, 145-330, 146-330, 147-330, 148-330, 149-330, 150-330, 151-330, 152-330, 153-330, 154-330, 155-330, 156-330, 157-330, 158-330, 159-330, 160-330, 161-330, 162-330, 163-330, 164-330, 165-330, 166-330, 167-330, 168-330, 169-330, 170-330, 171-330, 172-330, 173-330, 174-330, 175-330, 176-330, 177-330, 178-330, 179-330, 180-330, 181-330, 182-330, 183-330, 184-330, 185-330, 186-330, 187-330, 188-330, 189-330, 190-330, 191-330, 192-330, 193-330, 194-330, 195-330, 196-330, 197-330, 198-330 or 199-330 of SEQ ID NO: 8.In other preferred embodiments, GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 43-323, 44-323, 45-323, 46-323, 47-323, 48-323, 49-323, 50-323, 51-323, 52-323, 53-323, 54-323, 55-323, 56-323, 57-323, 58-323, 59-323, 60-323, 61-323, 62-323, 63-323, 64-323, 65-323, 66-323, 67-323, 68-323, 69-323, 70-323, 71-323, 72-323, 73-323, 74-323, 75-323, 76-323, 77-323, 78-323, 79-323, 80-323, 81-323, 82-323, 83-323, 84-323, 85-323, 86-323, 87-323, 88-323, 89-323, 90-323, 91-323, 92-323, 93-323, 94-323, 95-323, 96-323, 97-323, 98-323, 99-323, 100-323, 101-323, 102-323, 103-323, 104-323, 105-323, 106-323, 107-323, 108-323, 109-323, 110-323, 111-323, 112-323, 113-323, 114-323, 115-323, 116-323, 117-323, 118-323, 119-323, 120-323, 121-323, 122-323, 123-323, 124-323, 125-323, 126-323, 127-323, 128-323, 129-323, 130-323, 131-323, 132-323, 133-323, 134-323, 135-323, 136-323, 137-323, 138-323, 139-323, 140-323, 141-323, 142-323, 143-323, 144-323, 145-323, 146-323, 147-323, 148-323, 149-323, 150-323, 151-323, 152-323, 153-323, 154-323, 155-323, 156-323, 157-323, 158-323, 159-323, 160-323, 161-323, 162-323, 163-323, 164-323, 165-323, 166-323, 167-323, 168-323, 169-323, 170-323, 171-323, 172-323, 173-323, 174-323, 175-323, 176-323, 177-323, 178-323, 179-323, 180-323, 181-323, 182-323, 183-323, 184-323, 185-323, 186-323, 187-323, 188-323, 189-323, 190-323, 191-323 or 192-323 of SEQ ID NO: 10.These specific embodiments, and other polypeptide and polynucleotide fragment embodiments described herein may be modified as being “at least”, “equal to”, “equal to or less than”, “less than”, “at least ______ but not greater than ______” or “from ______ to ______” a specified size or specified N-terminal and/or C-terminal positions.", "It is noted that all ranges used to describe any embodiment of the present invention are inclusive unless specifically set forth otherwise.", "The present invention also provides for the exclusion of any individual fragment specified by N-terminal and C-terminal positions or of any fragment specified by size in amino acid residues as described above.", "In addition, any number of fragments specified by N-terminal and C-terminal positions or by size in amino acid residues as described above may be excluded as individual species.", "Further, any number of fragments specified by N-terminal and C-terminal positions or by size in amino acid residues as described above may make up a polypeptide fragment in any combination and may optionally include non-GMG-3, -GMG-4, -Cluster 1, -GMG-6A, or -GMG-6B polypeptide sequences as well.", "In preferred embodiments, GMG-3 or GMG-4 polypeptide fragments having activity are selected from amino acids 20-333, 43-333, 44-333, 45-333, 46-333, 47-333, 48-333, 49-333, 50-333, 51-333, 52-333, 53-333, 54-333, 55-333, 56-333, 57-333, 58-333, 59-333, 60-333, 61-333, 62-333, 63-333, 64-333, 65-333, 66-333, 67-333, 68-333, 69-333, 70-333, 71-333, 72-333, 73-333, 74-333, 75-333, 76-333, 77-333, 78-333, 79-333, 80-333, 81-333, 82-333, 83-333, 84-333, 85-333, 86-333, 87-333, 88-333, 89-333, 90-333, 91-333, 92-333, 93-333, 94-333, 95-333, 96-333, 97-333, 98-333, 99-333, 100-333, 101-333, 102-333, 103-333, 104-333, 105-333, 106-333, 107-333, 108-333, 109-333, 110-333, 111-333, 112-333, 113-333, 114-333, 115-333, 116-333, 117-333, 118-333, 119-333, 120-333, 121-333, 122-333, 123-333, 124-333, 125-333, 126-333, 127-333, 128-333, 129-333, 130-333, 131-333, 132-333, 133-333, 134-333, 135-333, 136-333, 137-333, 138-333, 139-333, 140-333, 141-333, 142-333, 143-333, 144-333, 145-333, 146-333, 147-333, 148-333, 149-333, 150-333, 151-333, 152-333, 153-333, 154-333, 155-333, 156-333, 157-333, 158-333, 159-333, 160-333, 161-333, 162-333, 163-333, 164-333, 165-333, 166-333, 167-333, 168-333, 169-333, 170-333, 171-333, 172-333, 173-333, 174-333, 175-333, 176-333, 177-333, 178-333, 179-333, 180-333, 181-333, 182-333, 183-333, 184-333, 185-333, 186-333, 187-333, 188-333, 189-333, 190-333, 191-333, 192-333, 193-333, 194-333, 195-333, 196-333, 197-333, 198-333, 199-333, 200-333, 201-333 or 202-333 of SEQ ID NO: 2 or 4.In other preferred embodiments, Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 2-225, 3-225, 4-225, 5-225, 6-225, 7-225, 8-225, 9-225, 10-225, 11-225, 12-225, 13-225, 14-225, 15-225, 16-225, 17-225, 18-225, 19-225, 20-225, 21-225, 22-225, 23-225, 24-225, 25-225, 26-225, 27-225, 28-225, 29-225, 30-225, 31-225, 32-225, 33-225, 34-225, 35-225, 36-225, 37-225, 38-225, 39-225, 40-225, 41-225, 42-225, 43-225, 44-225, 45-225, 46-225, 47-225, 48-225, 49-225, 50-225, 51-225, 52-225, 53-225, 54-225, 55-225, 56-225, 57-225, 58-225, 59-225, 60-225, 61-225, 62-225, 63-225, 64-225, 65-225, 66-225, 67-225, 68-225, 69-225, 70-225, 71-225, 72-225, 73-225, 74-225, 75-225, 76-225, 77-225, 78-225, 79-225, 80-225, 81-225, 82-225, 83-225, 84-225, 85-225, 86-225, 87-225, 88-225, 89-225, 90-225, 91-225, 92-225, 93-225 or 94-225 of SEQ ID NO: 6.In other preferred embodiments, GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 43-330, 44-330, 45-330, 46-330, 47-330, 48-330, 49-330, 50-330, 51-330, 52-330, 53-330, 54-330, 55-330, 56-330, 57-330, 58-330, 59-330, 60-330, 61-330, 62-330, 63-330, 64-330, 65-330, 66-330, 67-330, 68-330, 69-330, 70-330, 71-330, 72-330, 73-330, 74-330, 75-330, 76-330, 77-330, 78-330, 79-330, 80-330, 81-330, 82-330, 83-330, 84-330, 85-330, 86-330, 87-330, 88-330, 89-330, 90-330, 91-330, 92-330, 93-330, 94-330; 95-330, 96-330, 97-0.330, 98-330, 99-330, 100-330, 101-330, 102-330, 103-330, 104-330, 105-330, 106-330, 107-330, 108-330, 109-330, 110-330, 111-330, 112-330, 113-330, 114-330, 115-330, 116-330, 117-330, 118-330, 119-330, 120-330, 121-330, 122-330, 123-330, 124-330, 125-330, 126-330, 127-330, 128-330, 129-330, 130-330, 131-330, 132-330, 133-330, 134-330, 135-330, 136-330, 137-330, 138-330, 139-330, 140-330, 141-330, 142-330, 143-330, 144-330, 145-330, 146-330, 147-330, 148-330, 149-330, 150-330, 151-330, 152-330, 153-330, 154-330, 155-330, 156-330, 157-330, 158-330, 159-330, 160-330, 161-330, 162-330, 163-330, 164-330, 165-330, 166-330, 167-330, 168-330, 169-330, 170-330, 171-330, 172-330, 173-330, 174-330, 175-330, 176-330, 177-330, 178-330, 179-330, 180-330, 181-330, 182-330, 183-330, 184-330, 185-330, 186-330, 187-330, 188-330, 189-330, 190-330, 191-330, 192-330, 193-330, 194-330, 195-330, 196-330, 197-330, 198-330 or 199-330 of SEQ ID NO: 8.In other preferred embodiments, GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 43-323, 44-323, 45-323, 46-323, 47-323, 48-323, 49-323, 50-323, 51-323, 52-323, 53-323, 54-323, 55-323, 56-323, 57-323, 58-323, 59-323, 60-323, 61-323, 62-323, 63-323, 64-323, 65-323, 66-323, 67-323, 68-323, 69-323, 70-323, 71-323, 72-323, 73-323, 74-323, 75-323, 76-323, 77-323, 78-323, 79-323, 80-323, 81-323, 82-323, 83-323, 84-323, 85-323, 86-323, 87-323, 88-323, 89-323, 90-323, 91-323, 92-323, 93-323, 94-323, 95-323, 96-323, 97-323, 98-323, 99-323, 100-323, 101-323, 102-323, 103-323, 104-323, 105-323, 106-323, 107-323, 108-323, 109-323, 110-323, 111-323, 112-323, 113-323, 114-323, 115-323, 116-323, 117-323, 118-323, 119-323, 120-323, 121-323, 122-323, 123-323, 124-323, 125-323, 126-323, 127-323, 128-323, 129-323, 130-323, 131-323, 132-323, 133-323, 134-323, 135-323, 136-323, 137-323, 138-323, 139-323, 140-323, 141-323, 142-323, 143-323, 144-323, 145-323, 146-323, 147-323, 148-323, 149-323, 150-323, 151-323, 152-323, 153-323, 154-323, 155-323, 156-323, 157-323, 158-323, 159-323, 160-323, 161-323, 162-323, 163-323, 164-323, 165-323, 166-323, 167-323, 168-323, 169-323, 170-323, 171-323, 172-323, 173-323, 174-323, 175-323, 176-323, 177-323, 178-323, 179-323, 180-323, 181-323, 182-323, 183-323, 184-323, 185-323, 186-323, 187-323, 188-323, 189-323, 190-323, 191-323 or 192-323 of SEQ ID NO: 10.In more preferred embodiments, said GMG-3 or GMG-4 polypeptide fragments comprised of all or part of the C-terminal globular C1q homology domain and having activity are selected from amino acids 20-333, 43-333, 45-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 75-333, 77-333, 81-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 109-333, 113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333, 252-333, 252-267,252-317, 256-267,256-317, or 304-317 of SEQ ID NO: 2 or 4.In other more preferred embodiments, said Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 5-225, 8-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225, 80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159, 144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6.In other more preferred embodiments, said GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 43-330, 45-330, 46-330, 50-333, 53-330, 64-330, 68-330, 71-330, 72-330, 75-330, 78-330, 79-330, 83-330, 86-330, 92-330, 97-330, 101-330, 122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330, 198-330, 199-330,224-330, 249-330, 249-264, 249-314, 253-264,253-314 or 301-314 of SEQ ID NO: 8.In other more preferred embodiments, said GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 43-323, 46-323, 57-323, 61-323, 64-323, 65-323, 68-323, 71-323, 72-323, 76-323, 79-323, 85-323, 90-323, 94-323, 115-323, 118-323, 139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQ ID NO: 10.In yet more preferred embodiments, said GMG-3 or GMG-4 polypeptide fragments comprised of all or part of the C-terminal globular C1q homology domain and having activity are selected from amino acids 20-333, 109-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333, 252-333, 252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2 or 4.In other yet more preferred embodiments, said Cluster 1 polypeptide fragments having activity are selected from amino acids 1-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225, 80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159, 144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6.In other yet more preferred embodiments, said GMG-6A polypeptide fragments having activity are selected from amino acids 20-330, 75-330, 122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330, 198-330, 199-330, 224-330, 249-330, 249-264, 249-314, 253-264, 253-314 or 301-314 of SEQ ID NO: 8.In other yet more preferred embodiments, said GMG-6B polypeptide fragments having activity are selected from amino acids 20-323, 68-323, 115-323, 118-323, 139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQ ID NO: 10.In yet other preferred embodiments, the invention features a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment comprising at least 115, but not more than 175 contiguous amino acids of any one of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment sequences set forth in the sequence listing, wherein no more than 24 of said at least 115 and no more than 175 contiguous amino acids are present in the collagen-like region of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B.", "Preferably, the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment comprises at least 125, but not more than 165, or at least 140, but not more than 165 amino acids, and no more than 24 amino acids are in the collagen-like region; more preferably at least 125 but not more than 165, or at least 140 but not more than 165 amino acids, and no more than 12 amino acids are in the collagen-like region; or at least 140 and not more than 150 amino acids, and no more than 8 amino acids are present in the collagen-like region.", "Preferably the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment is mammalian, preferably human or mouse, but most preferably human.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments of the invention include variants, fragments, analogs and derivatives of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments described above, including modified GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments.", "Proteolytic cleavage of full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention in vivo is believed to be subject to regulation that facilitates the appropriate and effective generation of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments of the invention comprised of all or part of the C-terminal globular C1q homology domain and having lipid partitioning, lipid metabolism, and insulin-like activity.", "Said proteolytic cleavage at least in part is regulated at the level of the protease, for example at the level of tissue distribution of the protease and at the level of amount of the protease, which itself can be regulated by physiological signals such as those associated with inflammation.", "Particularly preferred GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments of the invention comprised of all or part of the C-terminal globular C1q homology domain and having lipid partitioning, lipid metabolism, and insulin-like activity are said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments of SEQ ID NO: 2, 4, 6, 8, or 10 believed to be generated proteolytically in vivo.", "Said particularly preferred are GMG-3 or GMG-4 polypeptide fragments of amino acids 45-333, 75-333, or 81-333 of SEQ ID NO: 2 or 4 generated by collagenase cleavage.", "Also said particularly preferred are GMG-6A polypeptide fragments of amino acids 45-330, 72-330, or 78-330 of SEQ ID NO: 8 generated by collagenase cleavage.", "Also said particularly preferred are GMG-6B polypeptide fragments of amino acids 65-323 or 71-323 of SEQ ID NO: 10 generated by collagenase cleavage.", "Also said particularly preferred are GMG-3 or GMG-4 polypeptide fragments of amino acids 45-333, 75-333, or 81-333 of SEQ ID NO: 2 or 4 generated by matrix metalloproteinase-1 (MMP-1) cleavage.", "Also said particularly preferred are GMG-6A polypeptide fragments of amino acids 45-330, 72-330, or 78-330 of SEQ ID NO: 8 generated by matrix metalloproteinase-1 (MMP-1) cleavage.", "Also said particularly preferred are GMG-6B polypeptide fragments of amino acids 65-323 or 71-323 of SEQ ID NO: 10 generated by matrix metalloproteinase-1 (MMP-1) cleavage.", "Also said particularly preferred are GMG-3 polypeptide fragments of amino acids 43-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 77-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 109-333, 113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, or 202-333 of SEQ ID NO:2 generated by plasmin cleavage.", "Also said particularly preferred are GMG-4 polypeptide fragments of amino acids 43-333, 46-333, 50-333, 53-333, 67-333, 74-333, 77-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 109-333, 113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, or 202-333 of SEQ ID NO:4 generated by plasmin cleavage.", "Also said particularly preferred are Cluster 1 polypeptide fragments of amino acids 5-225, 8-225, 17-225, 20-225, 32-225, 52-15-225, 56-225, 71-225, 74-225, 77-225, 80-225, 83-225, 85-225 or 94-225 of SEQ ID NO: 6 generated by plamsin cleavage.", "Also particularly preferred are GMG-6A polypeptide fragments of amino acids 43-330, 46-330, 50-333, 53-330, 64-330, 68-330, 71-330, 79-330, 83-330, 86-330, 92-330, 97-330, 101-330, 122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330 or 199-330 of SEQ ID NO: 8 generated by plasmin cleavage.", "Also said particularly preferred are GMG-6B polypeptide fragments of amino acids 43-323, 46-323, 57-323, 61-323, 64-323, 72-323, 76-323, 79-323, 85-323, 90-323, 94-323, 115-323, 118-323, 139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323, 183-323 or 192-323 of SEQ ID NO: 10 generated by plasmin cleavage.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention include variants, fragments, analogs and derivatives of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides described above, including modified GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides.", "Variants It will be recognized by one of ordinary skill in the art that some amino acids of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide sequences of the present invention can be varied without significant effect on the structure or function of the proteins; there will be critical amino acids in the sequence that determine activity.", "Thus, the invention further includes variants of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides that have metabolic-related activity as described above.", "Such variants include GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide sequences with one or more amino acid deletions, insertions, inversions, repeats, and substitutions either from natural mutations or human manipulation selected according to general rules known in the art so as to have little effect on activity.", "Guidance concerning how to make phenotypically silent amino acid substitutions is provided below.", "There are two main approaches for studying the tolerance of an amino acid sequence to change (see, Bowie, et al.", "(1990) Science, 247, 1306-10).", "The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection.", "The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality.", "These studies have revealed that proteins are surprisingly tolerant of amino acid substitutions and indicate which amino acid changes are likely to be permissive at a certain position of the protein.", "For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.", "Other such phenotypically silent substitutions are described by Bowie et al.", "(supra) and the references cited therein.", "In the case of an amino acid substitution in the amino acid sequence of a polypeptide according to the invention, one or several amino acids can be replaced by “equivalent” amino acids.", "The expression “equivalent” amino acid is used herein to designate any amino acid that may be substituted for one of the amino acids having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.", "In particular embodiments, conservative substitutions of interest are shown in Table 1 under the heading of preferred substitutions.", "If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 1, or as further described below in reference to amino acid classes, are introduced and the products screened.", "TABLE 1 Original Residue Exemplary Substitutions Preferred Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu Substantial modifications in function or immunological identity of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.", "Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.", "Non-conservative substitutions will entail exchanging a member of one of these classes for another class.", "Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.", "The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.", "Site-directed mutagenesis [Carter et al., Nucl Acids Res, 13:4331 (1986); Zoller et al., Nucl Acids Res, 10:6487 (1987)], cassette mutagenesis [Wells et al.", "Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos.", "Trans.", "R. Soc.", "London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B variant DNA.", "Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence.", "Among the preferred scanning amino acids are relatively small, neutral amino acids.", "Such amino acids include alanine, glycine, serine, and cysteine.", "Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)].", "Alanine is also typically preferred because it is the most common amino acid.", "Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W.H.", "Freeman & Co., N.Y.); Chothia, J. Mol.", "Biol., 150:1 (1976)].", "If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.", "Amino acids in the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide sequences of the invention that are essential for function can also be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham, et al.", "(1989) Science 244:1081-5).", "The latter procedure introduces single alanine mutations at every residue in the molecule.", "The resulting mutant molecules are then tested for metabolic-related activity using assays as described above.", "Of special interest are substitutions of charged amino acids with other charged or neutral amino acids that may produce proteins with highly desirable improved characteristics, such as less aggregation.", "Aggregation may not only reduce activity but also be problematic when preparing pharmaceutical or physiologically acceptable formulations, because aggregates can be immunogenic (see, e.g., Pinckard, et al., (1967) Clin Exp Immunol 2:331-340; Robbins, et al., (1987) Diabetes 36:838-41; and Cleland, et al., (1993) Crit Rev Ther Drug Carrier Syst 10:307-77).", "Thus, the fragment, derivative, analog, or homolog of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the present invention may be, for example: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code (i.e.", "may be a non-naturally occurring amino acid); or (ii) one in which one or more of the amino acid residues includes a substituent group; or (iii) one in which the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are fused with another compound, such as a compound to increase the half-life of the fragment (for example, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the above form of the fragment, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the fragment or a pro-protein sequence.", "Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.", "A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, not more than 40 conservative amino acid substitutions, not more than 30 conservative amino acid substitutions, and not more than 20 conservative amino acid substitutions.", "Also provided are polypeptides which comprise the amino acid sequence of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment, having at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.", "In addition, amino acids have chirality within the body of either L or D. In some embodiments it is preferable to alter the chirality of the amino acids in the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments of the invention in order to extend half-life within the body.", "Thus, in some embodiments, one or more of the amino acids are preferably in the L configuration.", "In other embodiments, one or more of the amino acids are preferably in the D configuration.", "Percent Identity The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 50% identical, at least 60% identical, or 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide as described above.", "By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide amino acid sequence is meant that the amino acid sequence is identical to the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide sequence except that it may include up to five amino acid alterations per each 100 amino acids of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide amino acid sequence.", "The reference sequence is the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide with a sequence corresponding to the sequences provided in SEQ ID NOs: 2, 4, 6, 8, or 10.Thus, to obtain a polypeptide having an amino acid sequence at least 95% identical to a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the sequence may be inserted, deleted, or substituted with another amino acid compared with the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide sequence.", "These alterations may occur at the amino or carboxy termini or anywhere between those terminal positions, interspersed either individually among residues in the sequence or in one or more contiguous groups within the sequence.", "As a practical matter, whether any particular polypeptide is a percentage identical to a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide can be determined conventionally using known computer programs.", "Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, (1988) Proc Natl Acad Sci USA 85:2444-8; Altschul et al., (1990) J Mol Biol 215:403-410; Thompson et al., (1994) Nucleic Acids Res 22(2):4673-4680; Higgins et al, (1996) Meth Enzymol 266:383-402; Altschul et al, (1997) Nucleic Acids Res 25:3389-3402; Altschul et al., (1993) Nature Genetics 3:266-272).", "In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”), which is well known in the art (See, e.g., Karlin and Altschul (1990) Proc Natl Acad Sci USA 87:2264-8; Altschul et al., 1990, 1993, 1997, all supra).", "In particular, five specific BLAST programs are used to perform the following tasks: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.", "The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.", "High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.", "Preferably, the scoring matrix used is the BLOSUM62 matrix (see, Gonnet et al., (1992) Science 256:1443-5; Henikoff and Henikoff (1993) Proteins 17:49-61).", "Less preferably, the PAM or PAM250 matrices may also be used (See, e.g., Schwartz and Dayhoff, eds, (1978) Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).", "The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology.", "Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin.", "(See, e.g., Karlin and Altschul, (1990) Proc Natl Acad Sci USA 87:2264-8).", "The BLAST programs may be used with the default parameters or with modified parameters provided by the user.", "Preferably, the parameters are default parameters.", "A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al.", "(1990) Comp App Biosci 6:237-245.In a sequence alignment the query and subject sequences are both amino acid sequences.", "The result of said global sequence alignment is in percent identity.", "Preferred parameters used in a FASTDB amino acid alignment are: Matrix-PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty-20, Randomization Group=25 Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=247 or the length of the subject amino acid sequence, whichever is shorter.", "If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, the results, in percent identity, must be manually corrected because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity.", "For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, that are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.", "Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.", "This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.", "This final percent identity score is what is used for the purposes of the present invention.", "Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score.", "That is, only query amino acid residues outside the farthest N- and C-terminal residues of the subject sequence.", "For example, a 90 amino acid residue subject sequence is aligned with a 100-residue query sequence to determine percent identity.", "The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not match/align with the first residues at the N-terminus.", "The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program.", "If the remaining 90 residues were perfectly matched the final percent identity would be 90%.", "In another example, a 90-residue subject sequence is compared with a 100-residue query sequence.", "This time the deletions are internal so there are no residues at the N- or C-termini of the subject sequence, which are not matched/aligned with the query.", "In this case, the percent identity calculated by FASTDB is not manually corrected.", "Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected.", "No other manual corrections are made for the purposes of the present invention.", "Production Note, throughout the disclosure, wherever GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are discussed, GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragments, variants and derivatives are specifically intended to be included as a preferred subset of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are preferably isolated from human or mammalian tissue samples or expressed from human or mammalian genes in human or mammalian cells.", "The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention can be made using routine expression methods known in the art.", "The polynucleotide encoding the desired polypeptide is ligated into an expression vector suitable for any convenient host.", "Both eukaryotic and prokaryotic host systems are used in forming recombinant polypeptides.", "The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use.", "Purification is by any technique known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like.", "See, for example, Methods in Enzymology for a variety of methods for purifying proteins.", "In a alternative embodiment, the polypeptides of the invention are isolated from milk.", "The polypeptides can be purified as full length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, which can then be cleaved, if appropriate, in vitro to generate a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment, or, alternatively, GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragments themselves can be purified from the milk.", "Any of a large number of methods can be used to purify the present polypeptides from milk including those taught in Protein Purification Applications, A Practical Approach (New Edition), Edited by Simon Roe, AEA Technology Products and Systems, Biosciences, Harwell; Clark (1998) J Mammary Gland Biol Neoplasia 3:337-50; Wilkins and Velander (1992) 49:333-8; U.S. Pat.", "Nos.", "6,140,552; 6,025,540; Hennighausen, Protein Expression and Purification, vol.", "1, pp.", "3-8 (1990); Harris et al.", "(1997) Bioseparation 7:31-7; Degener et al.", "(1998) J Chromatog 799:125-37; Wilkins (1993) J Cell Biochem.", "Suppl.", "0 (17 part A):39; the entire disclosures of each of which are herein incorporated by reference.", "In a typical embodiment, milk is centrifuged, e.g.", "at a relatively low speed, to separate the lipid fraction, and the aqueous supernatant is then centrifuged at a higher speed to separate the casein in the milk from the remaining, “whey” fraction.", "Often, biomedical proteins are found in this whey fraction, and can be isolated from this fraction using standard chromatographic or other procedures commonly used for protein purification, e.g.", "as described elsewhere in the present application.", "In one preferred embodiment, GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are purified using antibodies specific to GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, e.g.", "using affinity chromatography.", "In addition, methods can be used to isolate particular GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragments, e.g.", "electrophoretic or other methods for isolating proteins of a particular size.", "The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides isolating using these methods can be naturally occurring, as GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides have been discovered to be naturally present in the milk of mammals, or can be the result of the recombinant production of the protein in the mammary glands of a non-human mammal, as described infra.", "In one such embodiment, the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B is produced as a fusion protein with a heterologous, antigenic polypeptide sequence, which antigenic sequence can be used to purify the protein, e.g., using standard immuno-affinity methodology.", "In addition, shorter protein fragments may be produced by chemical synthesis.", "Alternatively, the proteins of the invention are extracted from cells or tissues of humans or non-human animals.", "Methods for purifying proteins are known in the art, and include the use of detergents or chaotropic agents to disrupt particles followed by differential extraction and separation of the polypeptides by ion exchange chromatography, affinity chromatography, sedimentation according to density, and gel electrophoresis.", "Any GMG-3, GMG-4, GMG-6A, or GMG-6B cDNA or Cluster 1 polynucleotide, including those in SEQ ID NO: 1, 3, 5, 7, or 9, can be used to express GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides.", "The nucleic acid encoding the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology.", "The GMG-3, GMG-4, GMG-6A, or GMG-6B cDNA or Cluster 1 polynucleotide insert in the expression vector may comprise the coding sequence for: the full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide (to be later modified); from 6 amino acids to 6 amino acids any integer less than the full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide; a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment; or variants and % similar polypeptides.", "The expression vector is any of the mammalian, yeast, insect or bacterial expression systems known in the art, some of which are described herein.", "Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, Mass.", "), Stratagene (La Jolla, Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego, Calif.).", "If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence can be optimized for the particular expression organism into which the expression vector is introduced, as explained by Hatfield, et al., U.S. Pat.", "No.", "5,082,767, the disclosures of which are incorporated by reference herein in their entirety.", "If the nucleic acid encoding any one of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques.", "Similarly, if the insert from the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide cDNA lacks a poly A signal, this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using BglI and SalI restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene).", "pXT1 contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus.", "The position of the LTRs in the construct allows efficient stable transfection.", "The vector includes the Herpes Simplex Thymidine Kinase promoter and the selectable neomycin gene.", "The nucleic acid encoding GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B can be obtained by PCR from a vector containing the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B nucleotide sequence using oligonucleotide primers complementary to the desired GMG-3, GMG-4, GMG-6A, or GMG-6B cDNA or Cluster 1 polynucleotide and containing restriction endonuclease sequences for Pst I incorporated into the 5′ primer and BglII at the 5′ end of the corresponding cDNA 3′ primer, taking care to ensure that the sequence encoding the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B is positioned properly with respect to the poly A signal.", "The purified polynucleotide obtained from the resulting PCR reaction is digested with PstI, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXT1, now containing a poly A signal and digested with BglII.", "Transfection of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B expressing vector into mouse NIH 3T3 cells is one embodiment of introducing polynucleotides into host cells.", "Introduction of a polynucleotide encoding a polypeptide into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods.", "Such methods are described in many standard laboratory manuals, such as Davis et al.", "((1986) Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., Amsterdam).", "It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.", "A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.", "Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.", "Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.", "Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.", "Preferably the polypeptides of the invention are non-glycosylated.", "In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.", "Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells.", "While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.", "In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides.", "For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, see, e.g.", "U.S. Pat.", "No.", "5,641,670, issued Jun.", "24, 1997; International Publication No.", "WO 96/29411, published Sep. 26, 1996; International Publication No.", "WO 94/12650, published Aug. 4, 1994; Koller et al., (1989) Proc Natl Acad Sci USA 86:8932-5; Koller et al., (1989) Proc Natl Acad Sci USA 86:8927-31; and Zijlstra et al.", "(1989) Nature 342:435-8; the disclosures of each of which are incorporated by reference in their entireties).", "Modifications In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (See, e.g., Creighton, 1983 Proteins.", "New York, N.Y.: W.H.", "Freeman and Company; and Hunkapiller et al., (1984) Nature 310:105-11).", "For example, a relative short fragment of the invention can be synthesized by use of a peptide synthesizer.", "Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the fragment sequence.", "Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.", "Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).", "The invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.", "Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.", "Additional post-translational modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.", "The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.", "Also provided by the invention are chemically modified derivatives of the polypeptides of the invention that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity.", "See U.S. Pat.", "No.", "4,179,337.The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.", "The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.", "The polymer may be of any molecular weight, and may be branched or unbranched.", "For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.", "Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).", "The polyethylene glycol molecules (or other chemical moieties) should be attached to the polypeptide with consideration of effects on functional or antigenic domains of the polypeptide.", "There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al.", "(1992) Exp Hematol (8):1028-35, reporting pegylation of GM-CSF using tresyl chloride).", "For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.", "Reactive groups are those to which an activated polyethylene glycol molecule may be bound.", "The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue.", "Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules.", "Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.", "One may specifically desire proteins chemically modified at the N-terminus.", "Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.", "), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.", "The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules.", "Selective proteins chemically modified at the N-terminus may be accomplished by reductive alkylation, which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein.", "Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.", "Multimers The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers).", "Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, pharmaceutical or physiologically acceptable compositions) containing them.", "In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers.", "In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.", "Multimers encompassed by the invention may be homomers or heteromers.", "As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention (including polypeptide fragments, variants, splice variants, and fusion proteins corresponding to these polypeptide fragments as described herein).", "These homomers may contain polypeptide fragments having identical or different amino acid sequences.", "In a specific embodiment, a homomer of the invention is a multimer containing only polypeptide fragments having an identical amino acid sequence.", "In another specific embodiment, a homomer of the invention is a multimer containing polypeptide fragments having different amino acid sequences.", "In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences).", "In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.", "As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., corresponding to different proteins or polypeptides thereof) in addition to the polypeptides of the invention.", "In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.", "In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.", "Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.", "Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution.", "In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.", "In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention.", "Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone).", "In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences, which interact in the native (i.e., naturally occurring) polypeptide.", "In another instance, the covalent associations are the consequence of chemical or recombinant manipulation.", "Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.", "In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat.", "No.", "5,478,925).", "In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein).", "In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety).", "In another embodiment, two or more polypeptides of the invention are joined through peptide linkers.", "Examples include those peptide linkers described in U.S. Pat.", "No.", "5,073,627 (hereby incorporated by reference).", "Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.", "Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence.", "Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found.", "Leucine zippers were originally identified in several DNA-binding proteins, and have since been found in a variety of different proteins (Landschulz et al., (1988) Genes Dev 2:786-800).", "Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.", "Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference.", "Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.", "Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity.", "Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers.", "One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al.", "FEBS Letters (1994) 344:191-5 and in U.S. patent application Ser.", "No.", "08/446,922, hereby incorporated by reference.", "Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.", "In another example, proteins of the invention are associated by interactions between Flag® & polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence.", "In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti Flag® antibody.", "The multimers of the invention may be generated using chemical techniques known in the art.", "For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "Additionally, at least 30 techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art.", "In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hyrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (See, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "II.", "GMG-3, GMG-4, Cluster 1, GMG-6A or GMG-6B Polynucleotides of the Invention Preferred polynucleotides are those that encode GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention.", "The recombinant polynucleotides encoding GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides can be used in a variety of ways, including, but not limited to, expressing the polypeptides in recombinant cells for use in screening assays for antagonists and agonists of its activity as well as to facilitate its purification for use in a variety of ways including, but not limited to screening assays for agonists and antagonists of its activity, diagnostic screens, and raising antibodies, as well as treatment and/or prevention of metabolic-related diseases and disorders and/or to reduce body mass.", "The invention relates to the polynucleotides encoding GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides and variant polypeptides thereof as described herein.", "These polynucleotides may be purified, isolated, and/or recombinant.", "In all cases, the desired GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotides of the invention are those that encode GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention having metabolic-related activity as described and discussed herein.", "Fragments A polynucleotide fragment is a polynucleotide having a sequence that entirely is the same as part, but not all, of the full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or a specified GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide nucleotide sequence.", "Such fragments may be “free-standing”, i.e.", "not part of or fused to other polynucleotides, or they may be comprised within another non-GMG-3, -GMG-4, -Cluster 1, -GMG-6A, or -GMG-6B (heterologous) polynucleotide of which they form a part or region.", "However, several GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide fragments may be comprised within a single polynucleotide.", "The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotides of the invention comprise from 18 consecutive bases to the full-length polynucleotide sequences encoding the intact GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, for example the full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide polynucleotide sequences in SEQ ID NO: 1, 3, 5, 7, or 9.In one aspect of this embodiment, the polynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 740, 770, 800, 850, 879, 900, 950, 972, 993, 1000 or 1002 consecutive nucleotides of a polynucleotide of the present invention.", "In addition to the above preferred nucleic acid sizes, further preferred nucleic acids comprise at least 18 nucleotides, wherein “at least 18” is defined as any integer between 18 and the integer representing the 3′ most nucleotide position of the intact GMG-3, GMG-4, GMG-6A, or GMG-6B polypeptides cDNA or Cluster 1 polynucleotide as set forth in SEQ ID NOs: 1, 3, 5, 7, or 9 or elsewhere herein.", "Further included as preferred polynucleotides of the present invention are nucleic acid fragments at least 18 nucleotides in length, as described above, that are further specified in terms of their 5′ and 3′ position.", "The 5′ and 3′ positions are represented by the position numbers set forth in the sequence listing below.", "For allelic and degenerate and other variants, position I is defined as the 5′ most nucleotide of the ORF, i.e., the nucleotide “A” of the start codon (ATG) with the remaining nucleotides numbered consecutively.", "Therefore, every combination of a 5′ and 3′ nucleotide position that a polynucleotide fragment, at least 18 contiguous nucleotides in length, could occupy on an intact GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide encoding a polynucleotide of the present invention is included in the invention as an individual species.", "The polynucleotide fragments specified by 5′ and 3′ positions can be immediately envisaged and are therefore not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "It is noted that the above species of polynucleotide fragments of the present invention may alternatively be described by the formula “x to y”; where “x” equals the 5′ most nucleotide position and “y” equals the 3′ most nucleotide position of the polynucleotide; and further where “x” equals an integer between 1 and the number of nucleotides of the polynucleotide sequence of the present invention minus 18, and where “y” equals an integer between 19 and the number of nucleotides of the polynucleotide sequence of the present invention minus 18 nucleotides; and where “x” is an integer less than “y” by at least 18.The present invention also provides for the exclusion of any species of polynucleotide fragments of the present invention specified by 5′ and 3′ positions or polynucleotides specified by size in nucleotides as described above.", "Any number of fragments specified by 5′ and 3′ positions or by size in nucleotides, as described above, may be excluded.", "The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide fragments of the invention comprise from 18 consecutive bases to the full-length polynucleotide sequence encoding the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragments described in Section II of the Preferred Embodiments of the Invention.", "In one aspect of this embodiment, the polynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 740, 770, 800, 850, 879, 900, 950, 972, 993, 1000 or 1002 consecutive nucleotides of a polynucleotide of the present invention.", "In addition to the above preferred nucleic acid sizes, further preferred nucleic acids comprise at least 18 nucleotides, wherein “at least 18” is defined as any integer between 18 and the integer corresponding to the 3′ most nucleotide position of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment cDNA herein.", "Further included as preferred polynucleotides of the present invention are nucleic acid fragments at least 18 nucleotides in length, as described above, that are further specified in terms of their 5′ and 3′ position.", "The 5′ and 3′ positions are represented by the position numbers set forth in the sequence listing below.", "For allelic and degenerate and other variants, position 1 is defined as the 5′ most nucleotide of the open reading frame (ORF), i.e., the nucleotide “A” of the start codon (ATG) with the remaining nucleotides numbered consecutively.", "Therefore, every combination of a 5′ and 3′ nucleotide position that a polynucleotide fragment invention, at least 18 contiguous nucleotides in length, could occupy on a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment polynucleotide of the present invention is included in the invention as an individual species.", "The polynucleotide fragments specified by 5′ and 3′ positions can be immediately envisaged and are therefore not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "It is noted that the above species of polynucleotide fragments of the present invention may alternatively be described by the formula “x to y”; where “x” equals the 5′ most nucleotide position and “y” equals the 3′ most nucleotide position of the polynucleotide; and further where “x” equals an integer between 1 and the number of nucleotides of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide sequences of the present invention minus 18, and where “y” equals an integer between 9 and the number of nucleotides of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide sequences of the present invention; and where “x” is an integer smaller than “y” by at least 18.Every combination of “x” and “y” positions are included as specific embodiments of the invention.", "Moreover, the formula “x” to “y” may be modified as “x1-x2” to “y1-y2”, wherein “x1-x2” and “y1-y2” represent positional ranges selected from any two nucleotide positions of the sequence listing.", "Alternative formulas include “x1-x2” to “y” and “x” to “y1-y2”.", "These specific embodiments, and other polynucleotide fragment embodiments described herein may be modified as being “at least”, “equal to”, “equal to or less than”, “less than”, “at least ______ but not greater than ______” or “from ______ to ______” a specified size or specified 5′ and/or 3′ positions.", "The present invention also provides for the exclusion of any species of polynucleotide fragments of the present invention specified by 5′ and 3′ positions or polynucleotides specified by size in nucleotides as described above.", "Any number of fragments specified by 5′ and 3′ positions or by size in nucleotides, as described above, may be excluded.", "Variants In other preferred embodiments, variants of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotides encoding GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are envisioned.", "Variants of polynucleotides, as the term is used herein, are polynucleotides whose sequence differs from a reference polynucleotide.", "A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.", "Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.", "Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.", "Polynucleotide variants that comprise a sequence substantially different from those described above but that, due to the degeneracy of the genetic code, still encode GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the present invention are also specifically envisioned.", "It would also be routine for one skilled in the art to generate the degenerate variants described above, for instance, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by other mammalian or bacterial host cells).", "As stated above, variant polynucleotides may occur naturally, such as a natural allelic variant, or by recombinant methods.", "By an “allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (See, e.g., B. Lewin, (1990) Genes IV, Oxford University Press, New York).", "Non-naturally occurring variants may be produced using art-known mutagenesis techniques.", "Such nucleic acid variants include those produced by nucleotide substitutions, deletions, or additions.", "The substitutions, deletions, or additions may involve one or more nucleotides.", "Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.", "Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention.", "Also preferred in this regard are conservative substitutions.", "Nucleotide changes present in a variant polynucleotide are preferably silent, which means that they do not alter the amino acids encoded by the polynucleotide.", "However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence.", "In cases where the nucleotide substitutions result in one or more amino acid changes, preferred GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides include those that retain one or more metabolic-related activity as described in Section I of the Preferred Embodiments of the Invention.", "By “retain the same activities” is meant that the activity measured using the polypeptide encoded by the variant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide in assays is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, and not more than 101%, 102%, 103%, 104%, 105%, 110%, 115%, 120% or 125% of the activity measured using a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide described in the Examples Section herein.", "By the activity being “increased” is meant that the activity measured using the polypeptide encoded by the variant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide in assays is at least 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 450%, or 500% of the activity measured using a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide described in the Examples Section herein.", "By the activity being “decreased” is meant that the activity measured using the polypeptide encoded by the variant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide in assays is decreased by at least 25%, 30%, 35%, 40%, 45%, 50%, 75%, 80%, 90% or 95% of the activity measured using a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide described in the Examples Section herein Percent Identity The present invention is further directed to nucleic acid molecules having sequences at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, or 9 or fragments thereof that encode a polypeptide having metabolic-related activity as described in Section I of the Preferred Embodiments of the Invention.", "Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences shown in SEQ ID NOs: 1, 3, 5, 7, or 9 or fragments thereof will encode a polypeptide having biological activity.", "In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.", "It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having biological activity.", "This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described previously in Section I of the Preferred Embodiments of the Invention.", "By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide.", "In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted, inserted, or substituted with another nucleotide.", "The query sequence may be an entire sequence or any fragment specified as described herein.", "The methods of determining and defining whether any particular nucleic acid molecule or polypeptide is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be done by using known computer programs.", "A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., ((1990) Comput Appl Biosci.", "July; 6(3):237-45).", "In a sequence alignment the query and subject sequences are both DNA sequences.", "An RNA sequence can be compared by first converting U's to T's.", "The result of said global sequence alignment is in percent identity.", "Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.", "If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results.", "This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity.", "For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence.", "Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.", "This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.", "This corrected score is what is used for the purposes of the present invention.", "Only nucleotides outside the 5′ and 3′ nucleotides of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.", "For example, a 90-nucleotide subject sequence is aligned to a 100-nucleotide query sequence to determine percent identity.", "The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 nucleotides at 5′ end.", "The 10 unpaired nucleotides represent 10% of the sequence (number of nucleotides at the 5′ and 3′ ends not matched/total number of nucleotides in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program.", "If the remaining 90 nucleotides were perfectly matched the final percent identity would be 90%.", "In another example, a 90 nucleotide subject sequence is compared with a 100 nucleotide query sequence.", "This time the deletions are internal deletions so that there are no nucleotides on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query.", "In this case the percent identity calculated by FASTDB is not manually corrected.", "Once again, only nucleotides 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for.", "No other manual corrections are made for the purposes of the present invention.", "Fusions Further included in the present invention are polynucleotides encoding the polypeptides of the present invention that are fused in frame to the coding sequences for additional heterologous amino acid sequences.", "Also included in the present invention are nucleic acids encoding polypeptides of the present invention together with additional, non-coding sequences, including for example, but not limited to non-coding 5′ and 3′ sequences, vector sequence, sequences used for purification, probing, or priming.", "For example, heterologous sequences include transcribed, nontranslated sequences that may play a role in transcription, and mRNA processing, for example, ribosome binding and stability of mRNA.", "The heterologous sequences may alternatively comprise additional coding sequences that provide additional functionalities.", "Thus, a nucleotide sequence encoding a polypeptide may be fused to a tag sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide.", "In certain preferred embodiments of this aspect of the invention, the tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Chatsworth, Calif.), among others, many of which are commercially available.", "For instance, hexa-histidine provides for convenient purification of the fusion protein (See, Gentz et al., (1989) Proc Natl Acad Sci USA 86: 821-4).", "The “HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein (See, Wilson et al., (1984) Cell 37:767-78).", "As discussed above, other such fusion proteins include GMG-3, GMG-4, GMG-6A, or GMG-6B cDNA or Cluster 1 polynucleotide fused to Fc at the N- or C-terminus.", "III.", "Recombinant Vectors of the Invention The term “vector” is used herein to designate either a circular or a linear DNA or RNA molecule, that is either double-stranded or single-stranded, and that comprises at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism.", "The present invention relates to recombinant vectors comprising any one of the polynucleotides described herein.", "The present invention encompasses a family of recombinant vectors that comprise polynucleotides encoding GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention.", "In a first preferred embodiment, a recombinant vector of the invention is used to amplify the inserted polynucleotide in a suitable cell host, this polynucleotide being amplified every time that the recombinant vector replicates.", "The inserted polynucleotide can be one that encodes GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention.", "A second preferred embodiment of the recombinant vectors according to the invention consists of expression vectors comprising polynucleotides encoding GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention.", "Within certain embodiments, expression vectors are employed to express a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of the invention, preferably a modified GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B described in the present invention, which can be then purified and, for example, be used as a treatment for metabolic-related diseases, or simply to reduce body mass of individuals.", "Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources, that drive expression of the genes of interest in host cells.", "Dominant drug selection markers for establishing permanent, stable, cell clones expressing the products are generally included in the expression vectors of the invention, as they are elements that link expression of the drug selection markers to expression of the polypeptide.", "More particularly, the present invention relates to expression vectors which include nucleic acids encoding a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of the invention, or a modified GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B as described herein, or variants or fragments thereof, under the control of a regulatory sequence selected among GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, or alternatively under the control of an exogenous regulatory sequence.", "Consequently, preferred expression vectors of the invention are selected from the group consisting of: (a) a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B regulatory sequence and driving the expression of a coding polynucleotide operably linked thereto; and (b) a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B coding sequence of the invention, operably linked to regulatory sequences allowing its expression in a suitable cell host and/or host organism.", "Some of the elements that can be found in the vectors of the present invention are described in further detail in the following sections.", "1) General Features of the Expression Vectors of the Invention A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid, or even a linear DNA molecule which may consist of a chromosomal, non-chromosomal, semi-synthetic or synthetic DNA.", "Such a recombinant vector can comprise a transcriptional unit comprising an assembly of: (1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers.", "Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription; (2) a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in (1); and (3) appropriate transcription initiation and termination sequences.", "Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.", "Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include a N-terminal residue.", "This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.", "Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.", "The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium.", "In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5′-flanking non-transcribed sequences.", "DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.", "2) Regulatory Elements Promoters The suitable promoter regions used in the expression vectors of the present invention are chosen taking into account the cell host in which the heterologous gene is expressed.", "The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.", "Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter.", "A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed.", "Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.", "Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.", "Preferred bacterial promoters are the LacI, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trp promoters (EP 0036776), the polyhedrin promoter, or the p10 protein promoter from baculovirus (Kit Novagen) (Smith et al., (1983) Mol Cell Biol 3:2156-65; O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.", "Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L.", "In addition, promoters specific for a particular cell type may be chosen, such as those facilitating expression in adipose tissue, muscle tissue, or liver.", "Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.", "The choice of a promoter is well within the ability of a person skilled in the field of genetic engineering.", "For example, one may refer to Sambrook et al.", "(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, Vol.", "1, 2, 3 (1989), or also to the procedures described by Fuller et al.", "(1996) Immunology in Current Protocols in Molecular Biology.", "Other Regulatory Elements Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.", "The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.", "Also contemplated as an element of the expression cassette is a terminator.", "These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.", "Vectors containing the appropriate DNA sequence as described above can be utilized to transform an appropriate host to allow the expression of the desired polypeptide or polynucleotide.", "3) Selectable Markers Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.", "The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRP 1 for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli, or levan saccharase for mycobacteria, this latter marker being a negative selection marker.", "4) Preferred Vectors Bacterial Vectors As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017).", "Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and pGEM1 (Promega Biotec, Madison, Wis. USA).", "Large numbers of other suitable vectors are known to those of skill in the art, and are commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).", "Baculovirus Vectors A suitable vector for the expression of polypeptides of the invention is a baculovirus vector that can be propagated in insect cells and in insect cell lines.", "A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC No.", "CRL 1711) which is derived from Spodoptera frugiperda.", "Other suitable vectors for the expression of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide comprising the C-terminal globular C1q homology domain in a baculovirus expression system include those described by Chai et al.", "(1993; Biotechnol Appl Biochem 18 (Pt 3):259-73); Vlasak et al.", "(1983; Eur J Biochem 135:123-6); and Lenhard et al.", "(1996; Gene 169:187-90).", "Plasmid Vectors A suitable vector for the expression of polypeptides of the invention is a plasmid vector that contains an SV40-derived origin of replication and that can be used for transient transfection of COS cells (ATCC No.", "CRL1650; No.", "CRL1651).", "Plasmid vectors suitable for transient transfection of COS cells include but are not limited to CDM8 (Invitrogen).", "Viral Vectors In one specific embodiment, the vector is derived from an adenovirus.", "Preferred adenovirus vectors according to the invention are those described by Feldman and Steg (1996; Semin Interv Cardiol 1:203-8) or Ohno et al.", "(1994; Science 265:781-4).", "Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin (French patent application No.", "FR-93.05954).", "Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans.", "These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.", "Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vivo gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus.", "Particularly preferred Murine Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298).", "Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728).", "Other preferred retroviral vectors are those described in Roth et al.", "(1996), PCT Application No WO 93/25234, PCT Application No WO 94/06920, Roux et al., ((1989) Proc Natl Acad Sci USA 86:9079-83), Julan et al., (1992) J. Gen. Virol 3:3251-3255 and Neda et al., ((1991) J Biol Chem 266:14143-6).", "Yet another viral vector system that is contemplated by the invention consists of the adeno-associated virus (AAV).", "The adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., (1992) Curr Top Microbiol Immunol 158:97-129).", "It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al., (1992) Am J Respir Cell Mol Biol 7:349-56; Samulski et al., (1989) J Virol 63:3822-8; McLaughlin et al., (1989) Am J Hum Genet 59:561-569).", "One advantageous feature of AAV derives from its reduced efficacy for transducing primary cells relative to transformed cells.", "5) Delivery of the Recombinant Vectors In order to effect expression of the polynucleotides of the invention, these constructs must be delivered into a cell.", "This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain disease states.", "One mechanism is viral infection where the expression construct is encapsulated in an infectious viral particle.", "Several non-viral methods for the transfer of polynucleotides into cultured mammalian cells are also contemplated by the present invention, and include, without being limited to, calcium phosphate precipitation (Graham et al., (1973) Virology 54:536-9; Chen et al., (1987) Mol Cell Biol 7:2745-52), DEAE-dextran (Gopal, (1985) Mol Cell Biol, 5:1188-90), electroporation (Tur-Kaspa et al., (1986) Mol Cell Biol 6:716-8; Potter et al., (1984) Proc Natl Acad Sci USA 81:7161-5.", "), direct microinjection (Harland et al., (1985) J Cell Biol 101:1094-9), DNA-loaded liposomes (Nicolau et al., (1982) Biochim Biophys Acta 721:185-90; Fraley et al., (1979) Proc Natl Acad Sci USA 76:3348-52), and receptor-mediated transfection (Wu and Wu, (1987) J Biol Chem 262:4429-32; Wu and Wu (1988) Biochemistry 27:887-92).", "Some of these techniques may be successfully adapted for in vivo or ex vivo use.", "Once the expression polynucleotide has been delivered into the cell, it may be stably integrated into the genome of the recipient cell.", "This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).", "In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.", "Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.", "One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect.", "This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.", "Compositions for use in vitro and in vivo comprising a “naked” polynucleotide are described in PCT application No.", "WO 90/11092 (Vical Inc.) and also in PCT application No.", "WO 95/11307 (Institut Pasteur, INSERM, Université d'Ottawa) as well as in the articles of Tascon et al.", "(1996) Nature Medicine 2:888-892 and of Huygen et al.", "((1996) Nat Med 2:893-8).", "In still another embodiment of the invention, the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al.", "((1990) Curr Genet 17:97-103).", "In a further embodiment, the polynucleotide of the invention may be entrapped in a liposome (Ghosh and Bacchawat, (1991) Targeted Diagn Ther 4:87-103; Wong et al., (1980) Gene 10:87-94; Nicolau et al., (1987) Methods Enzymol.", "149:157-76).", "These liposomes may further be targeted to cells expressing LSR by incorporating leptin, triglycerides, or other known LSR ligands into the liposome membrane.", "In a specific embodiment, the invention provides a composition for the in vivo production of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B globular head polypeptide described herein.", "It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells of the tissue to express the said polypeptide.", "The amount of vector to be injected to the desired host organism varies according to the site of injection.", "As an indicative dose, it will be injected between 0.1 and 100 μg of the vector in an animal body, preferably a mammal body, for example a mouse body.", "In another embodiment of the vector according to the invention, it may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell.", "In a subsequent step, the cell that has been transformed with the vector coding for the desired GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B globular head polypeptide or the desired fragment thereof is reintroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.", "IV.", "Recombinant Cells of the Invention Another object of the invention consists of host cells recombinant for, i.e., that have been transformed or transfected with one of the polynucleotides described herein, and more precisely a polynucleotide comprising a polynucleotide encoding a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of the invention such as any one of those described in “Polynucleotides of the Invention”.", "These polynucleotides can be present in cells as a result of transient or stable transfection.", "The invention includes host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as any one of those described in “Recombinant Vectors of the Invention”.", "Generally, a recombinant host cell of the invention comprises at least one of the polynucleotides or the recombinant vectors of the invention that are described herein.", "Preferred host cells used as recipients for the recombinant vectors of the invention are the following: a) Prokaryotic host cells: Escherichia coli strains (I.E.", "DH5-α strain), Bacillus subtilis, Salmonella typhimurium, and strains from species like Pseudomonas, Streptomyces and Staphylococcus, and b) Eukaryotic host cells: HeLa cells (ATCC No.", "CCL2; No.", "CCL2.1; No.", "CCL2.2), Cv 1 cells (ATCC No.", "CCL70), COS cells (ATCC No.", "CRL1650; No.", "CRL1651), Sf-9 cells (ATCC No.", "CRL1711), C127 cells (ATCC No.", "CRL-1804), 3T3 (ATCC No.", "CRL-6361), CHO (ATCC No.", "CCL-61), human kidney 293 (ATCC No.", "45504; No.", "CRL-1573), BHK (ECACC No.", "84100501; No.", "84111301), PLC cells, HepG2, and Hep3B.", "The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.", "Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.", "In a preferred embodiment, recombinant protein expressed by cells that have been stably or transiently transfected with a recombinant vector such as any one of those described in “recombinant Vectors of the Invention” is recovered from culture supernatant.", "Alternatively, cells may be harvested (typically by centrifugation), disrupted by physical or chemical means, and the resulting crude extract retained for further purification.", "Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.", "Such methods are well known by the skilled artisan.", "Further, according to the invention, these recombinant cells can be created in vitro or in vivo in an animal, preferably a mammal, most preferably selected from the group consisting of mice, rats, dogs, pigs, sheep, cattle, and primates, not to include humans.", "Recombinant cells created in vitro can also be later surgically implanted in an animal, for example.", "Methods to create recombinant cells in vivo in animals are well-known in the art.", "The present invention also encompasses primary, secondary, and immortalized homologously recombinant host cells of vertebrate origin, preferably mammalian origin and particularly human origin, that have been engineered to: a) insert exogenous (heterologous) polynucleotides into the endogenous chromosomal DNA of a targeted gene, b) delete endogenous chromosomal DNA, and/or c) replace endogenous chromosomal DNA with exogenous polynucleotides.", "Insertions, deletions, and/or replacements of polynucleotide sequences may be to the coding sequences of the targeted gene and/or to regulatory regions, such as promoter and enhancer sequences, operably associated with the targeted gene.", "The present invention further relates to a method of making a homologously recombinant host cell in vitro or in vivo, wherein the expression of a targeted gene not normally expressed in the cell is altered.", "Preferably the alteration causes expression of the targeted gene under normal growth conditions or under conditions suitable for producing the polypeptide encoded by the targeted gene.", "The method comprises the steps of: (a) transfecting the cell in vitro or in vivo with a polynucleotide construct, the polynucleotide construct comprising; (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; and (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination.", "The present invention further relates to a method of altering the expression of a targeted gene in a cell in vitro or in vivo wherein the gene is not normally expressed in the cell, comprising the steps of: (a) transfecting the cell in vitro or in vivo with a polynucleotide construct, the polynucleotide construct comprising: (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; and (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell; and (c) maintaining the homologously recombinant cell in vitro or in vivo under conditions appropriate for expression of the gene.", "The present invention further relates to a method of making a polypeptide of the present invention by altering the expression of a targeted endogenous gene in a cell in vitro or in vivo wherein the gene is not normally expressed in the cell, comprising the steps of: a) transfecting the cell in vitro with a polynucleotide construct, the polynucleotide construct comprising: (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell; and c) maintaining the homologously recombinant cell in vitro or in vivo under conditions appropriate for expression of the gene thereby making the polypeptide.", "The present invention further relates to a polynucleotide construct that alters the expression of a targeted gene in a cell type in which the gene is not normally expressed.", "This occurs when a polynucleotide construct is inserted into the chromosomal DNA of the target cell, wherein the polynucleotide construct comprises: a) a targeting sequence; b) a regulatory sequence and/or coding sequence; and c) an unpaired splice-donor site, if necessary.", "Further included are polynucleotide constructs, as described above, wherein the construct further comprises a polynucleotide that encodes a polypeptide and is in-frame with the targeted endogenous gene after homologous recombination with chromosomal DNA.", "The compositions may be produced, and methods performed, by techniques known in the art, such as those described in U.S. Pat.", "Nos.", "6,054,288; 6,048,729; 6,048,724; 6,048,524; 5,994,127; 5,968,502; 5,965,125; 5,869,239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580,734; International Publication Nos: WO96/29411, WO 94/12650; and scientific articles described by Koller et al., (1994) Annu Rev Immunol 10:705-730; the disclosures of each of which are incorporated by reference in their entireties).", "The expression of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B in mammalian, and typically human, cells may be rendered defective, or alternatively it may be enhanced, with the insertion of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B genomic or cDNA sequence with the replacement of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B gene counterpart in the genome of an animal cell by a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide according to the invention.", "These genetic alterations may be generated by homologous recombination events using specific DNA constructs that have been previously described.", "One kind of host cell that may be used are mammalian zygotes, such as murine zygotes.", "For example, murine zygotes may undergo microinjection with a purified DNA molecule of interest, for example a purified DNA molecule that has previously been adjusted to a concentration range from 1 ng/ml—for BAC inserts—3 ng/μl—for P1 bacteriophage inserts—in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine, and 70 μM spermidine.", "When the DNA to be microinjected has a large size, polyamines and high salt concentrations can be used in order to avoid mechanical breakage of this DNA, as described by Schedl et al ((1993) Nature 362:258-61).", "Any one of the polynucleotides of the invention, including the DNA constructs described herein, may be introduced in an embryonic stem (ES) cell line, preferably a mouse ES cell line.", "ES cell lines are derived from pluripoient, uncommitted cells of the inner cell mass of pre-implantation blastocysts.", "Preferred ES cell lines are the following: ES-E14TG2a (ATCC No.", "CRL-1821), ES-D3 (ATCC No.", "CRL1934 and No.", "CRL-11632), YS001 (ATCC No.", "CRL-11776), 36.5 (ATCC No.", "CRL-11116).", "To maintain ES cells in an uncommitted state, they are cultured in the presence of growth inhibited feeder cells which provide the appropriate signals to preserve this embryonic phenotype and serve as a matrix for ES cell adherence.", "Preferred feeder cells are primary embryonic fibroblasts that are established from tissue of day 13-day 14 embryos of virtually any mouse strain, that are maintained in culture, such as described by Abbondanzo et al.", "(1993; Methods Enzymol 225:803-23) and are inhibited in growth by irradiation, such as described by Robertson ((1987) Embryo-derived stem cell lines.", "In: E. J. Robertson Ed.", "Teratocarcinomas and embrionic stem cells: a practical approach.", "IRL Press, Oxford), or by the presence of an inhibitory concentration of LIF, such as described by Pease and Williams (1990; Exp Cell Res 190:209-11).", "The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.", "Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.", "Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.", "Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.", "Such methods are well known by the skilled artisan.", "V. DNA Construct that Enables Directing Temporal and Spatial GMG-3.GMG-4, Cluster 1, GMG-6A, or GMG-6B Gene Expression in Transgenic Animals DNA Constructs Allowing Homologous Recombination: Replacement Vectors A preferred DNA construct will comprise, from 5′-end to 3′-end: (a) a first nucleotide sequence that is comprised in the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B genomic sequence; (b) a nucleotide sequence comprising a positive selection marker, such as the marker for neomycine resistance (neo); and (c) a second nucleotide sequence that is comprised in the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B genomic sequence, and is located on the genome downstream the first GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B nucleotide sequence (a).", "In a preferred embodiment, this DNA construct also comprises a negative selection marker located upstream the nucleotide sequence (a) or downstream the nucleotide sequence (c).", "Preferably, the negative selection marker comprises the thymidine kinase (tk) gene (Thomas et al., 1986), the hygromycine beta gene (Te Riele et al., 1990), the hprt gene (Van der Lugt et al., 1991; Reid et al., 1990) or the Diphteria toxin A fragment (Dt-A) gene (Nada et al., 1993; Yagi et al.", "1990), which disclosures are hereby incorporated by reference in their entireties.", "Preferably, the positive selection marker is located within a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B exon sequence so as to interrupt the sequence encoding a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein.", "These replacement vectors are described, for example, by Thomas et al.", "(1986; 1987), Mansour et al.", "(1988) and Koller et al.", "(1992).", "The first and second nucleotide sequences (a) and (c) may be indifferently located within a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B regulatory sequence, an intronic sequence, an exon sequence or a sequence containing both regulatory and/or intronic and/or exon sequences.", "The size of the nucleotide sequences (a) and (c) ranges from 1 to 50 kb, preferably from 1 to 10 kb, more preferably from 2 to 6 kb and most preferably from 2 to 4 kb.", "Preferably the nucleotide sequences (a) and (c) comprise SEQ ID NO: 11, 12, or 13.DNA Constructs Allowing Homologous Recombination: Cre-LoxP System These new DNA constructs make use of the site specific recombination system of the P1 phage.", "The P1 phage possesses a recombinase called Cre that interacts specifically with a 34 base pairs loxP site.", "The loxP site is composed of two palindromic sequences of 13 bp separated by a 8 bp conserved sequence (Hoess et al., 1986), which disclosure is hereby incorporated by reference in its entirety.", "The recombination by the Cre enzyme between two loxP sites having an identical orientation leads to the deletion of the DNA fragment.", "The Cre-loxP system used in combination with a homologous recombination technique has been first described by Gu et al.", "(1993, 1994), which disclosures are hereby incorporated by reference in their entireties.", "Briefly, a nucleotide sequence of interest to be inserted in a targeted location of the genome harbors at least two loxP sites in the same orientation and located at the respective ends of a nucleotide sequence to be excised from the recombinant genome.", "The excision event requires the presence of the recombinase (Cre) enzyme within the nucleus of the recombinant cell host.", "The recombinase enzyme may be brought at the desired time either by (a) incubating the recombinant cell hosts in a culture medium containing this enzyme, by injecting the Cre enzyme directly into the desired cell, such as described by Araki et al (1995), which disclosure is hereby incorporated by reference in its entirety, or by lipofection of the enzyme into the cells, such as described by Baubonis et al (1993), which disclosure is hereby incorporated by reference in its entirety; (b) transfecting the cell host with a vector comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter being optionally inducible, said vector being introduced in the recombinant cell host, such as described by Gu et al.", "(1993) and Sauer et al (1988), which disclosures are hereby incorporated by reference in their entireties; (c) introducing in the genome of the cell host a polynucleotide comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter is optionally inducible, and said polynucleotide being inserted in the genome of the cell host either by a random insertion event or an homologous recombination event, such as described by Gu et at.", "(1994).", "In a specific embodiment, the vector containing the sequence to be inserted in the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B gene by homologous recombination is constructed in such a way that selectable markers are flanked by loxP sites of the same orientation, it is possible, by treatment by the Cre enzyme, to eliminate the selectable markers while leaving the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B sequences of interest that have been inserted by an homologous recombination event.", "Again, two selectable markers are needed: a positive selection marker to select for the recombination event and a negative selection marker to select for the homologous recombination event.", "Vectors and methods using the Cre-loxP system are described by Zou et al.", "(1994), which disclosure is hereby incorporated by reference in its entirety.", "Thus, a second preferred DNA construct of the invention comprises, from 5′-end to 3′-end: (a) a first nucleotide sequence that is comprised in the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B genomic sequence; (b) a nucleotide sequence comprising a polynucleotide encoding a positive selection marker, said nucleotide sequence comprising additionally two sequences defining a site recognized by a recombinase, such as a loxP site, the two sites being placed in the same orientation; and (c) a second nucleotide sequence that is comprised in the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B genomic sequence, and is located on the genome downstream of the first GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B nucleotide sequence (a).", "Preferably the nucleotide sequences (a) and (c) comprise SEQ ID NO: 11, 12, or 13.The sequences defining a site recognized by a recombinase, such as a loxP site, are preferably located within the nucleotide sequence (b) at suitable locations bordering the nucleotide sequence for which the conditional excision is sought.", "In one specific embodiment, two loxP sites are located at each side of the positive selection marker sequence, in order to allow its excision at a desired time after the occurrence of the homologous recombination event.", "In a preferred embodiment of a method using the third DNA construct described above, the excision of the polynucleotide fragment bordered by the two sites recognized by a recombinase, preferably two loxP sites, is performed at a desired time, due to the presence within the genome of the recombinant host cell of a sequence encoding the Cre enzyme operably linked to a promoter sequence, preferably an inducible promoter, more preferably a tissue-specific promoter sequence and most preferably a promoter sequence which is both inducible and tissue-specific, such as described by Gu et al (1994).", "The presence of the Cre enzyme within the genome of the recombinant cell host may result from the breeding of two transgenic animals, the first transgenic animal bearing the GMG-3-, GMG-4-, Cluster 1-, GMG-6A-, or GMG-6B-derived sequence of interest containing the loxP sites as described above and the second transgenic animal bearing the Cre coding sequence operably linked to a suitable promoter sequence, such as described by Gu et al.", "(1994).", "Spatio-temporal control of the Cre enzyme expression may also be achieved with an adenovirus based vector that contains the Cre gene thus allowing infection of cells, or in vivo infection of organs, for delivery of the Cre enzyme, such as described by Anton and Graham (1995) and Kanegae et al (1995), which disclosures are hereby incorporated by reference in their entireties.", "The DNA constructs described above may be used to introduce a desired nucleotide sequence of the invention, preferably a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B genomic sequence or a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B cDNA sequence, and most preferably an altered copy of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B genomic or cDNA sequence, within a predetermined location of the targeted genome, leading either to the generation of an altered copy of a targeted gene (knock-out homologous recombination) or to the replacement of a copy of the targeted gene by another copy sufficiently homologous to allow an homologous recombination event to occur (knock-in homologous recombination).", "VI.", "Transgenic Animals The present invention also provides methods and compositions for the generation of non-human animals and plants that express the recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, of the present invention.", "The animals or plants can be transgenic, i.e.", "each of their cells contains a gene encoding a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide, or, alternatively, a polynucleotide encoding a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide can be introduced into somatic cells of the animal or plant, e.g.", "into mammary secretory epithelial cells of a mammal.", "In preferred embodiments, the non-human animal is a mammal such as a cow, sheep, goat, pig, or rabbit.", "Methods of making transgenic animals such as mammals are well known to those of skill in the art, and any such method can be used in the present invention.", "Briefly, transgenic mammals can be produced, e.g., by transfecting a pluripotential stem cell such as an ES cell with a polynucleotide encoding a polypeptide of interest.", "Successfully transformed ES cells can then be introduced into an early stage embryo that is then implanted into the uterus of a mammal of the same species.", "In certain cases, the transformed (“transgenic”) cells will comprise part of the germ line of the resulting animal, and adult animals comprising the transgenic cells in the germ line can then be mated to other animals, thereby eventually producing a population of transgenic animals that have the transgene in each of their cells, and which can stably transmit the transgene to each of their offspring.", "Other methods of introducing the polynucleotide can be used, for example introducing the polynucleotide encoding the polypeptide of interest into a fertilized egg or early stage embryo via microinjection.", "Alternatively, the transgene may be introduced into an animal by infection of zygotes with a retrovirus containing the transgene (Jaenisch, R. (1976) Proc.", "Natl.", "Acad.", "Sci.", "USA 73, 1260-1264).", "Methods of making transgenic mammals are described, e.g., in Wall et al.", "(1992) J Cell Biochem 1992 49:113-20; Hogan, et al.", "(1986) in Manipulating the mouse embryo.", "A Laboratory Manual.", "Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; in WO 91/08216, or in U.S. Pat.", "No.", "4,736,866.In a preferred method, the polynucleotides are microinjected into the fertilized oocyte.", "Typically, fertilized oocytes are microinjected using standard techniques, and then cultured in vitrountil a “pre-implantation embryo” is obtained.", "Such pre-implantation embryos preferably contain approximately 16 to 150 cells.", "Methods for culturing fertilized oocytes to the pre-implantation stage are described, e.g., by Gordon et al.", "((1984) Methods in Enzymology, 101, 414); Hogan et al.", "((1986) in Manipulating the mouse embryo.", "A Laboratory Manual.", "Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) (for the mouse embryo); Hammer et al.", "((1985) Nature, 315, 680) (for rabbit and porcine embryos); Gandolfi et al.", "((1987) J. Reprod.", "Fert.", "81, 23-28); Rexroad et al.", "((1988) J. Anim.", "Sci.", "66, 947-953) (for ovine embryos); and Eyestone et al.", "((1989) J. Reprod.", "Fert.", "85, 715-720); Camous et al.", "((1984) J. Reprod.", "Pert.", "72, 779-785); and Heyman et al.", "((1987) Theriogenology 27, 5968) (for bovine embryos); the disclosures of each of which are incorporated herein in their entireties.", "Pre-implantation embryos are then transferred to an appropriate female by standard methods to permit the birth of a transgenic or chimeric animal, depending upon the stage of development when the transgene is introduced.", "As the frequency of transgene incorporation is often low, the detection of transgene integration in pre-implantation embryos is often desirable using any of the herein-described methods.", "Any of a number of methods can be used to detect the presence of a transgene in a pre-implantation embryo.", "For example, one or more cells may be removed from the pre-implantation embryo, and the presence or absence of the transgene in the removed cell or cells can be detected using any standard method e.g.", "PCR.", "Alternatively, the presence of a transgene can be detected in utero or post partum using standard methods.", "In a particularly preferred embodiment of the present invention, transgenic mammals are generated that secrete recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides in their milk.", "As the mammary gland is a highly efficient protein-producing organ, such methods can be used to produce protein concentrations in the gram per liter range, and often significantly more.", "Preferably, expression in the mammary gland is accomplished by operably linking the polynucleotide encoding the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide to a mammary gland specific promoter and, optionally, other regulatory elements.", "Suitable promoters and other elements include, but are not limited to, those derived from mammalian short and long WAP, alpha, beta, and kappa, casein, alpha and beta lactoglobulin, beta-CN 5′ genes, as well as the the mouse mammary tumor virus (MMTV) promoter.", "Such promoters and other elements may be derived from any mammal, including, but not limited to, cows, goats, sheep, pigs, mice, rabbits, and guinea pigs.", "Promoter and other regulatory sequences, vectors, and other relevant teachings are provided, e.g., by Clark (1998) J Mammary Gland Biol Neoplasia 3:337-50; Jost et al.", "(1999) Nat Biotechnol 17:160-4; U.S. Pat.", "Nos.", "5,994,616; 6,140,552; 6,013,857; Sohn et al.", "(1999) DNA Cell Biol.", "18:845-52; Kim et al.", "(1999) J Biochem (Japan) 126:320-5; Soulier et al.", "(1999) Euro J Biochem 260:533-9; Zhang et al.", "(1997) Chin J Biotech 13:271-6; Rijnkels et al.", "(1998) Transgen Res 7:5-14; Korhonen et al.", "(1997) Euro J Biochem 245:482-9; Uusi-Oukari et al.", "(1997) Transgen Res 6:75-84; Hitchin et al.", "(1996) Prot Expr Purif 7:247-52; Platenburg et al.", "(1994) Transgen Res 3:99-108; Heng-Cherl et al.", "(1993) Animal Biotech 4:89-107; and Christa et al.", "(2000) Euro J Biochem 267:1665-71; the entire disclosures of each of which is herein incorporated by reference.", "In another embodiment, the polypeptides of the invention can be produced in milk by introducing polynucleotides encoding the polypeptides into somatic cells of the mammary gland in vivo, e.g.", "mammary secreting epithelial cells.", "For example, plasmid DNA can be infused through the nipple canal, e.g.", "in association with DEAE-dextran (see, e.g., Hens et al.", "(2000) Biochim.", "Biophys.", "Acta 1523:161-171), in association with a ligand that can lead to receptor-mediated endocytosis of the construct (see, e.g., Sobolev et al.", "(1998) 273:7928-33), or in a viral vector such as a retroviral vector, e.g.", "the Gibbon ape leukemia virus (see, e.g., Archer et al.", "(1994) PNAS 91:6840-6844).", "In any of these embodiments, the polynucleotide may be operably linked to a mammary gland specific promoter, as described above, or, alternatively, any strongly expressing promoter such as CMV or MoMLV LTR.", "The suitability of any vector, promoter, regulatory element, etc.", "for use in the present invention can be assessed beforehand by transfecting cells such as mammary epithelial cells, e.g.", "MacT cells (bovine mammary epithelial cells) or GME cells (goat mammary epithelial cells), in vitro and assessing the efficiency of transfection and expression of the transgene in the cells.", "For in vivo administration, the polynucleotides can be administered in any suitable formulation, at any of a range of concentrations (e.g.", "1-500 μg/ml, preferably 50-100 μg/ml), at any volume (e.g.", "1-100 ml, preferably 1 to 20 ml), and can be administered any number of times (e.g.", "1, 2, 3, 5, or 10 times), at any frequency (e.g.", "every 1, 2, 3, 5, 10, or any number of days).", "Suitable concentrations, frequencies, modes of administration, etc.", "will depend upon the particular polynucleotide, vector, animal, etc., and can readily be determined by one of skill in the art.", "In a preferred embodiment, a retroviral vector such as as Gibbon ape leukemia viral vector is used, as described in Archer et al.", "((1994) PNAS 91:6840-6844).", "As retroviral infection typically requires cell division, cell division in the mammary glands can be stimulated in conjunction with the administration of the vector, e.g.", "using a factor such as estrodiol benzoate, progesterone, reserpine, or dexamethasone.", "Further, retroviral and other methods of infection can be facilitated using accessory compounds such as polybrene.", "In any of the herein-described methods for obtaining GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides from milk, the quantity of milk obtained, and thus the quantity of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides produced, can be enhanced using any standard method of lactation induction, e.g.", "using hexestrol, estrogen, and/or progesterone.", "The polynucleotides used in such embodiments can either encode a full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment.", "Typically, the encoded polypeptide will include a signal sequence to ensure the secretion of the protein into the milk.", "Where a full length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B sequence is used, the full length protein can, e.g., be isolated from milk and cleaved in vitro using a suitable protease.", "Alternatively, a second, protease-encoding polynucleotide can be introduced into the animal or into the mammary gland cells, whereby expression of the protease results in the cleavage of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide in vivo, thereby allowing the direct isolation of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragments from milk.", "VII Pharmaceutical or Physiologically Acceptable Compositions of the Invention The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention can be administered to non-human animals and/or humans, alone or in pharmaceutical or physiologically acceptable compositions where they are mixed with suitable carriers or excipient(s).", "The pharmaceutical or physiologically acceptable composition is then provided at a therapeutically effective dose.", "A therapeutically effective dose refers to that amount of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG 6B polypeptide sufficient to result in prevention or amelioration of symptoms or physiological status of metabolic-related diseases or disorders as determined by the methods described herein.", "A therapeutically effective dose can also refer to the amount of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide necessary for a reduction in weight or a prevention of an increase in weight or prevention of an increase in the rate of weight gain in persons desiring this affect for cosmetic reasons.", "A therapeutically effective dosage of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of the invention is that dosage that is adequate to promote weight loss or weight gain with continued periodic use or administration.", "Techniques for formulation and administration of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.", "Other diseases or disorders that GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention could be used to treat or prevent include, but are not limited to, obesity and obesity-related diseases and disorders such as obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Non-Insulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).", "Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides may also be used to enhance physical performance during work or exercise or enhance a feeling of general well-being.", "Physical performance activities include walking, running, jumping, lifting and/or climbing.", "The GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptides or antagonists thereof may also be used to treat dyslexia, attention-deficit disorder (ADD), attention-deficit/hyperactivity disorder (ADHD), and psychiatric disorders such as schizophrenia by modulating fatty acid metabolism, more specifically, the production of certain long-chain polyunsaturated fatty acids.", "It is expressly considered that the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention may be provided alone or in combination with other pharmaceutically or physiologically acceptable compounds.", "Other compounds useful for the treatment of obesity and other diseases and disorders are currently well-known in the art.", "In a preferred embodiment, the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are useful for, and used in, the treatment of insulin resistance and diabetes using methods described herein and known in the art.", "More particularly, a preferred embodiments relates to process for the therapeutic modification and regulation of glucose metabolism in an animal or human subject, which comprises administering to a subject in need of treatment (alternatively on a timed daily basis) GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.", "Further preferred embodiments relate to methods for the prophylaxis or treatment of diabetes comprising administering to a subject in need of treatment (alternatively on a timed daily basis) a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.", "Routes of Administration.", "Suitable routes of administration include oral, nasal, rectal, transmucosal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocular injections using methods known in the art.", "A particularly useful method of administering compounds for promoting weight loss involves surgical implantation, for example into the abdominal cavity of the recipient, of a device for delivering GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides over an extended period of time.", "Other particularly preferred routes of administration are aerosol and depot formulation.", "Sustained release formulations, particularly depot, of the invented medicaments are expressly contemplated.", "Composition/Formulation Pharmaceutical or physiologically acceptable compositions and medicaments for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries.", "Proper formulation is dependent upon the route of administration chosen.", "Certain of the medicaments described herein will include a pharmaceutically or physiologically acceptable acceptable carrier and at least one polypeptide that is a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of the invention.", "For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer such as a phosphate or bicarbonate buffer.", "For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.", "Such penetrants are generally known in the art.", "Pharmaceutical or physiologically acceptable preparations that can be taken orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.", "The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.", "In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.", "In addition, stabilizers may be added.", "All formulations for oral administration should be in dosages suitable for such administration.", "For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.", "For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable gaseous propellant, e.g., carbon dioxide.", "In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.", "Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator, may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.", "The compounds may be formulated for parenteral administration by injection, e.g.", "by bolus injection or continuous infusion.", "Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.", "The compositions may take such forms as suspensions, solutions or emulsions in aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.", "Pharmaceutical or physiologically acceptable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.", "Aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.", "Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.", "Alternatively, the active ingredient may be in powder or lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.", "In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.", "Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.", "Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.", "Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.", "Various sustained release materials have been established and are well known by those skilled in the art.", "Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.", "Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.", "The pharmaceutical or physiologically acceptable compositions also may comprise suitable solid or gel phase carriers or excipients.", "Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.", "Effective Dosage.", "Pharmaceutical or physiologically acceptable compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose.", "More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.", "Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.", "For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays.", "For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range shown to increase leptin or lipoprotein uptake or binding in an in vitro system.", "Such information can be used to more accurately determine useful doses in humans.", "A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient.", "Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50, (the dose lethal to 50% of the test population) and the ED50 (the dose therapeutically effective in 50% of the population).", "The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD5O and ED5O.", "Compounds that exhibit high therapeutic indices are preferred.", "The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.", "The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50, with little or no toxicity.", "The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.", "The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.", "(See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.", "1).", "Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain or prevent weight loss or gain, depending on the particular situation.", "Dosages necessary to achieve these effects will depend on individual characteristics and route of administration.", "Dosage intervals can also be determined using the value for the minimum effective concentration.", "Compounds should be administered using a regimen that maintains plasma levels above the minimum effective concentration for 10-90% of the time, preferably between 30-90%; and most preferably between 50-90%.", "In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.", "The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.", "A preferred dosage range for the amount of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of the invention, which can be administered on a daily or regular basis to achieve desired results, including a reduction in levels of circulating plasma triglyceride-rich lipoproteins, range from 0.05-1.0 mg/kg body mass.", "A more preferred dosage range is from 0.1-5 mg/kg.", "A more preferred dose is 0.25-2.5 mg/kg.", "Of course, these daily dosages can be delivered or administered in small amounts periodically during the course of a day.", "It is noted that these dosage ranges are only preferred ranges and are not meant to be limiting to the invention.", "VIII.", "Methods of Treatment The invention is drawn inter alia to methods of preventing or treating metabolic-related diseases and disorders comprising providing an individual in need of such treatment with a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of the invention.", "Preferably, the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide has metabolic-related activity either in vitro or in vivo.", "Preferably the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide is provided to the individual in a pharmaceutical composition that is preferably taken orally.", "Preferably the individual is a mammal, and most preferably a human.", "In preferred embodiments, the metabolic-related disease or disorder is selected from the group consisting of atherosclerosis, cardiovascular disease, impaired glucose tolerance, insulin resistance, hypertension, stroke, Syndrome X, Type I diabetes, Type II diabetes and lipoatrophic diabetes.", "Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia, hypertriglyceridemia, and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, neoplasia-related weight loss, anorexia, and bulimia.", "In preferred embodiments, GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides in pharmaceutical compositions are used to modulate body weight in healthy individuals for cosmetic reasons.", "The invention also features a method of preventing or treating metabolic-related diseases and disorders comprising providing an individual in need of such treatment with a compound identified by assays of the invention (described in Section VI of the Preferred Embodiments of the Invention and in the Examples).", "Preferably these compounds antagonize or agonize effects of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides in cells in vitro, muscles ex vivo, or in animal models.", "Alternatively, these compounds agonize or antagonize the effects of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on leptin and/or lipoprotein uptake and/or binding.", "Optionally, these compounds prevent the interaction, binding, or uptake of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides with LSR in vitro or in vivo.", "Preferably, the compound is provided to the individual in a pharmaceutical composition that is preferably taken orally.", "Preferably the individual is a mammal, and most preferably a human.", "In preferred embodiments, the metabolic-related disease or disorder is selected from the group consisting of obesity and metabolic-related diseases and disorders such as atherosclerosis, heart disease, insulin resistance, hypertension, stroke, Syndrome X, Type I diabetes, Type II diabetes, and lipoatrophic diabetes.", "Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia, hypertriglyceridemia, and hyperuricemia.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some individuals, particularly those with Type II diabetes or insulin resistance, alone, without combination of insulin therapy.", "In still a further preferred embodiment, the control of body weight is due in part or in whole to a decrease in mass of 1) subcutaneous adipose tissue and/or 2) viseral (omental) adipose tissue.", "In a further preferred embodiment, the present invention may be used in complementary therapy, particularly in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, to improve their weight or glucose control in combination with an insulin secretagogue or an insulin sensitising agent.", "Preferably, the insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide, and glidazide.", "Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.", "The present invention further provides a method of improving the body weight or glucose control of some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, alone, without an insulin secretagogue or an insulin sensitising agent.", "In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with the insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent).", "Accordingly, the present invention further provides for a composition of pharmaceutical or physiologically acceptable composition and an oral insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of body weight or glucose control in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin sensitiser.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some individuals, particularly those with Type II diabetes or insulin resistance, without insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an inhibitor of the progression from impaired glucose tolerance to insulin resistance.", "IX.", "Ligands Interacting with GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptides.", "For the purpose of the present invention, a Ligand means a molecule, such as a protein, a peptide, an antibody or any synthetic chemical compound capable of binding to a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein or one of its fragments or variants or to modulate the expression of the polynucleotide coding for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B or a fragment or variant thereof.", "In the Ligand screening method according to the present invention, a biological sample or a defined molecule to be tested as a putative Ligand of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein is brought into contact with the corresponding purified GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, for example the corresponding purified recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein produced by a recombinant cell host as described herein, in order to form a complex between this protein and the putative Ligand molecule to be tested.", "As an illustrative example, to study the interaction of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selected from the group consisting of of SEQ ID NO: 2, 4, 6, 8, and 10, with drugs or small molecules, such as molecules generated through combinatorial chemistry approaches, the microdialysis coupled to HPLC method described by Wang et al.", "(1997) or the affinity capillary electrophoresis method described by Bush et al.", "(1997), the disclosures of which are incorporated by reference, can be used.", "In further methods, peptides, drugs, fatty acids, lipoproteins, or small molecules which interact with a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selected from the group consisting of sequences of SEQ ID NO: 2, 4, 6, 8, or 10 may be identified using assays such as the following.", "The molecule to be tested for binding is labelled with a detectable label, such as a fluorescent, radioactive, or enzymatic tag and placed in contact with immobilized GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment thereof under conditions that permit specific binding to occur.", "After removal of non-specifically bound molecules, bound molecules are detected using appropriate means.", "Various candidate substances or molecules can be assayed for interaction with a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide.", "These substances or molecules include, without being limited to, natural or synthetic organic compounds or molecules of biological origin such as polypeptides.", "When the candidate substance or molecule comprises a polypeptide, this polypeptide may be the resulting expression product of a phage clone belonging to a phage-based random peptide library, or alternatively the polypeptide may be the resulting expression product of a cDNA library cloned in a vector suitable for performing a two-hybrid screening assay.", "A.", "Candidate Ligands Obtained by Affinity Chromatography.", "Proteins or other molecules interacting with a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment thereof comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selected from the group consisting of sequences of SEQ ID NO: 2, 4, 6, 8, and 10, can also be found using affinity columns which contain the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment thereof.", "The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment thereof, may be attached to the column using conventional techniques including chemical coupling to a suitable column matrix such as agarose, Affi Gel®, or other matrices familiar to those of skill in art.", "In some embodiments of this method, the affinity column contains chimeric proteins in which the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment thereof, is fused to glutathion S transferase (GST).", "A mixture of cellular proteins or pool of expressed proteins as described above is applied to the affinity column.", "Proteins or other molecules interacting with the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment thereof, attached to the column can then be isolated and analyzed on 2-D electrophoresis gel as described in Ramunsen et al.", "(1997), the disclosure of which is incorporated by reference.", "Alternatively, the proteins retained on the affinity column can be purified by electrophoresis-based methods and sequenced.", "The same method can be used to isolate antibodies, to screen phage display products, or to screen phage display human antibodies.", "B.", "Candidate Ligands Obtained by Optical Biosensor Methods Proteins interacting with a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selected from the group consisting of sequences of SEQ ID NO: 2, 4, 6, 8, or 10, can also be screened by using an Optical Biosensor as described in Edwards and Leatherbarrow (1997) and also in Szabo et al.", "(1995), the disclosures of which are incorporated by reference.", "This technique permits the detection of interactions between molecules in real time, without the need of labelled molecules.", "This technique is based on the surface plasmon resonance (SPR) phenomenon.", "Briefly, the candidate Ligand molecule to be tested is attached to a surface (such as a carboxymethyl dextran matrix).", "A light beam is directed towards the side of the surface that does not contain the sample to be tested and is reflected by said surface.", "The SPR phenomenon causes a decrease in the intensity of the reflected light with a specific association of angle and wavelength.", "The binding of candidate Ligand molecules cause a change in the refraction index on the surface, which change is detected as a change in the SPR signal.", "For screening of candidate Ligand molecules or substances that are able to interact with the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment thereof, the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment thereof, is immobilized onto a surface.", "This surface comprises one side of a cell through which flows the candidate molecule to be assayed.", "The binding of the candidate molecule on the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment thereof, is detected as a change of the SPR signal.", "The candidate molecules tested may be proteins, peptides, carbohydrates, lipids, or small molecules generated by combinatorial chemistry.", "This technique may also be performed by immobilizing eukaryotic or prokaryotic cells or lipid vesicles exhibiting an endogenous or a recombinantly expressed GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein at their surface.", "The main advantage of the method is that it allows the determination of the association rate between the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein and molecules interacting with the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein.", "It is thus possible to select specifically Ligand molecules interacting with the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, or a fragment thereof, through strong or conversely weak association constants.", "C. Candidate Ligands Obtained Through a Two-Hybrid Screening Assay.", "The yeast two-hybrid system is designed to study protein-protein interactions in vivo (Fields and Song, 1989), which disclosure is hereby incorporated by reference in its entirety, and relies upon the fusion of a bait protein to the DNA binding domain of the yeast Gal4 protein.", "This technique is also described in the U.S. Pat.", "No.", "5,667,973 and the U.S. Pat.", "No.", "5,283,173, the technical teachings of both patents being herein incorporated by reference.", "The general procedure of library screening by the two-hybrid assay may be performed as described by Harper et al.", "(1993) or as described by Cho et al.", "(1998) or also Fromont-Racine et al.", "(1997), which disclosures are hereby incorporated by reference in their entireties.", "The bait protein or polypeptide comprises, consists essentially of, or consists of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or a fragment thereof comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selected from the group consisting of sequences of SEQ ID NO: 2, 4, 6, 8, and 10.More precisely, the nucleotide sequence encoding the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or a fragment or variant thereof is fused to a polynucleotide encoding the DNA binding domain of the GAL4 protein, the fused nucleotide sequence being inserted in a suitable expression vector, for example pAS2 or pM3.Then, a human cDNA library is constructed in a specially designed vector, such that the human cDNA insert is fused to a nucleotide sequence in the vector that encodes the transcriptional domain of the GAL4 protein.", "Preferably, the vector used is the pACT vector.", "The polypeptides encoded by the nucleotide inserts of the human cDNA library are termed “prey” polypeptides.", "A third vector contains a detectable marker gene, such as beta galactosidase gene or CAT gene that is placed under the control of a regulation sequence that is responsive to the binding of a complete Gal4 protein containing both the transcriptional activation domain and the DNA binding domain.", "For example, the vector pG5EC may be used.", "Two different yeast strains are also used.", "As an illustrative but non-limiting example the two different yeast strains may be the following: Y190, the phenotype of which is (MATa, Leu2-3, 112 ura3-12, trp1-901, his3-D200, ade2-101, gal4Dgal180D URA3 GAL-LacZ, LYS GAL-HIS3, cyhr); Y187, the phenotype of which is (MATa gal4 gal80his3 trp1-901 ade2-101 ura3-52 leu2-3, -112 URA3 GAL-lacZmet−), which is the opposite mating type of Y190.Briefly, 20 μg of pAS2/GMG-3, pAS2/GMG-4, pAS2/Cluster 1, pAS2/GMG-6A, or pAS2/GMG-6B and 20 μg of pACT-cDNA library are co-transformed into yeast strain Y190.The transformants are selected for growth on minimal media lacking histidine, leucine and tryptophan, but containing the histidine synthesis inhibitor 3-AT (50 mM).", "Positive colonies are screened for beta galactosidase by filter lift assay.", "The double positive colonies (His+, beta-gal+) are then grown on plates lacking histidine, leucine, but containing tryptophan and cycloheximide (10 mg/ml) to select for loss of pAS2/GMG-3, pAS2/GMG-4, pAS2/Cluster 1, pAS2/GMG-6A, or pAS2/GMG-6B plasmids but retention of pACT-cDNA library plasmids.", "The resulting Y190 strains are mated with Y187 strains expressing GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B or non-related control proteins; such as cyclophilin B, lamin, or SNF1, as Gal4 fusions as described by Harper et al.", "(1993) and by Bram et al.", "(1993), which disclosures are hereby incorporated by reference in their entireties, and screened for beta galactosidase by filter lift assay.", "Yeast clones that are beta gal- after mating with the control Gal4 fusions are considered false positives.", "In another embodiment of the two-hybrid method according to the invention, interaction between the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B or a fragment or variant thereof with cellular proteins may be assessed using the Matchmaker Two Hybrid System 2 (Catalog No.", "K1604-1, Clontech).", "As described in the manual accompanying the kit, the disclosure of which is incorporated herein by reference, nucleic acids encoding the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein or a portion thereof, are inserted into an expression vector such that they are in frame with DNA encoding the DNA binding domain of the yeast transcriptional activator GAL4.A desired cDNA, preferably human cDNA, is inserted into a second expression vector such that they are in frame with DNA encoding the activation domain of GAL4.The two expression plasmids are transformed into yeast and the yeast are plated on selection medium which selects for expression of selectable markers on each of the expression vectors as well as GALA dependent expression of the HIS3 gene.", "Transformants capable of growing on medium lacking histidine are screened for GAL4 dependent lacZ expression.", "Those cells that are positive in both the histidine selection and the lacZ assay contain interaction between GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B and the protein or peptide encoded by the initially selected cDNA insert.", "X. Assays for Identifying Modulators of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptide Activity The invention features methods of screening for one or more compounds that modulate the activity of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B in cells, which includes providing potential compounds to be tested to the cells.", "Exemplary assays that may be used are described in the Examples section.", "To these assays would be added compounds to be tested for their inhibitory or stimulatory activity as compared to the effects of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides alone.", "Other assays in which an effect is observed based on the addition of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides can also be used to screen for modulators of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide activity or effects of the presence of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on cells.", "The essential step is to apply an unknown compound and then to monitor an assay for a change from what is seen when only GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are applied to the cell.", "A change is defined as something that is significantly different in the presence of the compound plus GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide compared to GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide alone.", "In this case, significantly different would be an “increase” or a “decrease” in a measurable effect of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.", "The term “modulation” as used herein refers to a measurable change in an activity.", "Examples include, but are not limited to, lipolysis stimulated receptor (LSR) modulation, leptin modulation, lipoprotein modulation, plasma FFA levels, FFA oxidation, TG levels, glucose levels, and weight.", "These effects can be in vitro or preferably in vivo.", "Modulation of an activity can be either an increase or a decrease in the activity.", "Thus, LSR activity can be increased or decreased, leptin activity can be increased or decreased, and lipoprotein activity can be increased or decreased.", "Similarly, FFA, TG, glucose levels and weight can be increased or decreased in vivo Free Fatty Acid oxidation can be increased or decreased in vivo or ex vivo.", "By “LSR” activity is meant expression of LSR on the surface of the cell, or in a particular conformation, as well as its ability to bind, uptake, and degrade leptin and lipoprotein.", "By “leptin” activity is meant its binding, uptake and degradation by LSR, as well as its transport across a blood brain barrier, and potentially these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.", "Similarly, by “lipoprotein” activity is meant its binding, uptake and degradation by LSR, as well as these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.", "Exemplary assays are provided in the Examples.", "These assay and other comparable assays can be used to determine/identify compounds that modulate GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide activity.", "In some cases it may be important to identify compounds that modulate some but not all of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide activities, although preferably all activities are modified.", "The term “increasing” as used herein refers to the ability of a compound to increase the activity of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides in some measurable way compared to the effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides in its absence.", "As a result of the presence of the compound leptin binding and/or uptake might increase, for example, as compared to controls in the presence of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide alone.", "Preferably, an increase in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% compared to the level of activity in the presence of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide.", "Preferably said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide is GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment comprising all or part of the C-terminal globular C1q homology domain.", "Similarly, the term “decreasing” as used herein refers to the ability of a compound to decrease an activity in some measurable way compared to the effect of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide in its absence.", "For example, the presence of the compound decreases the plasma concentrations of FFA, TG, and glucose in mice.", "Also as a result of the presence of a compound leptin binding and/or uptake might decrease, for example, as compared to controls in the presence of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides alone.", "Preferably, an decrease in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% as compared to the level of activity in the presence of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides alone.", "Preferably said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide is GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment comprising all or part of the C-terminal globular C1q homology domain.", "The invention features a method for identifying a potential compound to decrease body mass in individuals in need of decreasing body mass comprising: a) contacting a cell with a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide and a candidate compound; b) detecting a result selected from the group consisting of LSR modulation, leptin modulation, increase in glucose uptake or oxidation, decrease in blood lipid or triglyceride levels, increase in lipoprotein binding, uptake or degradation; FFA oxidation increase; and c) wherein said result identifies said potential compound if said result differs from said result when said cell is contacted with the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide alone.", "Alternatively, the invention features a method for identifying a potential compound to increase body mass in individuals in need of increasing body mass comprising: a) contacting a cell with a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide and a candidate compound; b) detecting a result selected from the group consisting of LSR modulation, leptin modulation, decrease in glucose uptake or oxidation, increase in blood lipid or triglyceride levels, decrease in lipoprotein binding, uptake or degradation; FFA oxidation decrease; and c) wherein said result identifies said potential compound if said result differs from said result when said cell is contacted with the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide alone.", "In still other preferred embodiments, said potential compound is selected from the group consisting of peptides, peptide libraries, non-peptide libraries, peptoids, fatty acids, lipoproteins, medicaments, antibodies, small molecules, proteases and protease inhibitors.", "XI.", "Epitopes and Antibody Fusions A preferred embodiment of the present invention is directed to epitope-bearing polypeptides and epitope-bearing polypeptide fragments.", "These epitopes may be “antigenic epitopes” or both an “antigenic epitope” and an “immunogenic epitope”.", "An “immunogenic epitope” is defined as a part of a protein that elicits an antibody response in vivo when the polypeptide is the immunogen.", "On the other hand, a region of polypeptide to which an antibody binds is defined as an “antigenic determinant” or “antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes.", "See, e.g., Geysen, et al.", "(1983) Proc Natl Acad Sci USA 81:3998-4002.It is particularly noted that although a particular epitope may not be immunogenic, it is nonetheless useful since antibodies can be made in vitro to any epitope.", "An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope.", "Generally an epitope consists of at least 6 such amino acids, and more often at least 8-10 such amino acids.", "In preferred embodiment, antigenic epitopes comprise a number of amino acids that is any integer between 3 and 50.Fragments which function as epitopes may be produced by any conventional means.", "See, e.g., Houghten, R. A., Proc Natl Acad Sci USA 82:5131-5135 (1985), further described in U.S. Pat.", "No.", "4,631,211.Methods for determining the amino acids which make up an immunogenic epitope include x-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping, e.g., the Pepscan method described by H. Mario Geysen et al.", "(1984); Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 81:3998-4002; PCT Publication No.", "WO 84/03564; and PCT Publication No.", "WO 84/03506.Another example is the algorithm of Jameson and Wolf, Comp.", "Appl.", "Biosci.", "4:181-186 (1988) (said references incorporated by reference in their entireties).", "The Jameson-Wolf antigenic analysis, for example, may be performed using the computer program PROTEAN, using default parameters (Version 4.0 Windows, DNASTAR, Inc., 1228 South Park Street Madison, Wis.).", "The epitope-bearing fragments of the present invention preferably comprise 6 to 50 amino acids (i.e.", "any integer between 6 and 50, inclusive) of a polypeptide of the present invention.", "Also, included in the present invention are antigenic fragments between the integers of 6 and the full-length sequence of the sequence listing.", "All combinations of sequences between the integers of 6 and the full-length sequence of a polypeptide of the present invention are included.", "The epitope-bearing fragments may be specified by either the number of contiguous amino acid residues (as a sub-genus) or by specific N-terminal and C-terminal positions (as species) as described above for the polypeptide fragments of the present invention.", "Any number of epitope-bearing fragments of the present invention may also be excluded in the same manner.", "Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies that specifically bind the epitope (See, Wilson et al., 1984; and Sutcliffe, J. G. et al., 1983).", "The antibodies are then used in various techniques such as diagnostic and tissue/cell identification techniques, as described herein, and in purification methods.", "Similarly, immunogenic epitopes can be used to induce antibodies according to methods well known in the art (See, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al.", "; (1985) and Bittle, F. J. et al., (1985).", "A preferred immunogenic epitope includes the polypeptides of the sequence listing.", "The immunogenic epitopes may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) if necessary.", "Immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.).", "Epitope-bearing polypeptides of the present invention are used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods (See, e.g., Sutcliffe, et al., supra; Wilson, et al., supra, and Bittle, et al., 1985).", "If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.", "For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as -maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.", "Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μgs of peptide or carrier protein and Freund's adjuvant.", "Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody, which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.", "The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.", "As one of skill in the art will appreciate, and discussed above, the polypeptides of the present invention including, but not limited to, polypeptides comprising an immunogenic or antigenic epitope can be fused to heterologous polypeptide sequences.", "For example, the polypeptides of the present invention may be fused with the constant region comprising portions of immunoglobulins (IgA, IgE, IgG, IgM), or portions of the constant region (CH1, CH2, CH3, any combination thereof including both entire domains and portions thereof) resulting in chimeric polypeptides.", "These fusion proteins facilitate purification, and show an increased half-life in vivo.", "This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (See, e.g., EPA 0,394,827; and Traunecker et al., 1988).", "Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion can also be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone (See, e.g., Fountoulakis et al., 1995).", "Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag to aid in detection and purification of the expressed polypeptide.", "Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, or codon-shuffling (collectively referred to as “DNA shuffling”).", "DNA shuffling may be employed to modulate the activities of polypeptides of the present invention thereby effectively generating agonists and antagonists of the polypeptides.", "See, for example, U.S. Pat.", "Nos.", "5,605,793; 5,811,238; 5,834,252; 5,837,458; and Patten, P. A., et al., (1997); Harayama, S., (1998); Hansson, L. O., et al (1999); and Lorenzo, M. M. and Blasco, R., (1998).", "(Each of these documents are hereby incorporated by reference).", "In one embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of coding polynucleotides of the invention, or the polypeptides encoded thereby may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc.", "of one or more heterologous molecules.", "Antibodies The present invention further relates to antibodies and T-cell antigen receptors (TCR) that specifically bind the polypeptides, and more specifically, the epitopes of the polypeptides of the present invention.", "The antibodies of the present invention include IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY.", "As used herein, the term “antibody” (Ab) is meant to include whole antibodies, including single-chain whole antibodies, and antigen binding fragments thereof.", "In a preferred embodiment the antibodies are human antigen binding antibody fragments of the present invention include, but are not limited to, Fab, Fab′ F(ab)2 and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.", "The antibodies may be from any animal origin including birds and mammals.", "Preferably, the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken.", "Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire or partial of the following: hinge region, CH1, CH2, and CH3 domains.", "Also included in the invention are any combinations of variable region(s) and hinge region, CH1, CH2, and CH3 domains.", "The present invention further includes chimeric, humanized, and human monoclonal and polyclonal antibodies, which specifically bind the polypeptides of the present invention.", "The present invention further includes antibodies that are anti-idiotypic to the antibodies of the present invention.", "The antibodies of the present invention may be monospecific, bispecific, and trispecific or have greater multispecificity.", "Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or solid support material.", "See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al.", "(1991); U.S. Pat.", "Nos.", "5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; Kostelny, S. A. et al.", "(1992).", "Antibodies of the present invention may be described or specified in terms of the epitope(s) or epitope-bearing portion(s) of a polypeptide of the present invention, which are recognized or specifically bound by the antibody.", "In the case of proteins of the present invention secreted proteins, the antibodies may specifically bind a full-length protein encoded by a nucleic acid of the present invention, a mature protein (i.e., the protein generated by cleavage of the signal peptide) encoded by a nucleic acid of the present invention, a signal peptide encoded by a nucleic acid of the present invention, or any other polypeptide of the present invention.", "Therefore, the epitope(s) or epitope bearing polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or otherwise described herein.", "Antibodies that specifically bind any epitope or polypeptide of the present invention may also be excluded as individual species.", "Therefore, the present invention includes antibodies that specifically bind specified polypeptides of the present invention, and allows for the exclusion of the same.", "Antibodies of the present invention may also be described or specified in terms of their cross-reactivity.", "Antibodies that do not specifically bind any other analog, ortholog, or homolog of the polypeptides of the present invention are included.", "Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein, eg., using FASTDB and the parameters set forth herein) to a polypeptide of the present invention are also included in the present invention.", "Further included in the present invention are antibodies, which only bind polypeptides encoded by polynucleotides, which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein).", "Antibodies of the present invention may also be described or specified in terms of their binding affinity.", "Preferred binding affinities include those with a dissociation constant or Kd value less than 5×10−6 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, and 10−15 M. Antibodies of the present invention have uses that include, but are not limited to, methods known in the art to purify, detect, and target the polypeptides of the present invention including both in vitro and in vivo diagnostic and therapeutic methods.", "For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples (See, e.g., Harlow et al., 1988).", "The antibodies of the present invention may be used either alone or in combination with other compositions.", "The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.", "For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins.", "See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.", "No.", "5,314,995; and EP 0 396 387.The antibodies of the present invention may be prepared by any suitable method known in the art.", "For example, a polypeptide of the present invention or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.", "The term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.", "The term “antibody” refers to a polypeptide or group of polypeptides which are comprised of at least one binding domain, where a binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen, which allows an immunological reaction with the antigen.", "The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.", "Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technology.", "Hybridoma techniques include those known in the art (See, e.g., Harlow et al.", "1988); Hammerling, et al, 1981) (said references incorporated by reference in their entireties).", "Fab and F(ab′)2 fragments may be produced, for example, from hybridoma-produced antibodies by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments).", "Alternatively, antibodies of the present invention can be produced through the application of recombinant DNA technology or through synthetic chemistry using methods known in the art.", "For example, the antibodies of the present invention can be prepared using various phage display methods known in the art.", "In phage display methods, functional antibody domains are displayed on the surface of a phage particle, which carries polynucleotide sequences encoding them.", "Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g.", "human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead.", "Phage used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.", "Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman U. et al.", "(1995); Ames, R. S. et al.", "(1995); Kettleborough, C. A. et al.", "(1994); Persic, L. et al.", "(1997); Burton, D. R. et al.", "(1994); PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat.", "Nos.", "5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743.As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria.", "For example, techniques to recombinantly produce Fab, Fab° F(ab)2 and F(ab)2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax, R. L. et al.", "(1992); and Sawai, H. et al.", "(1995); and Better, M. et al.", "(1988).", "Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat.", "Nos.", "4,946,778 and 5,258,498; Huston et al.", "(1991); Shu, L. et al.", "(1993); and Skerra, A. et al.", "(1988).", "For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies.", "Methods for producing chimeric antibodies are known in the art.", "See e.g., Morrison, (1985); Oi et al., (1986); Gillies, S. D. et al.", "(1989); and U.S. Pat.", "No.", "5,807,715.Antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat.", "Nos.", "5,530,101; and 5,585,089), veneering or resurfacing, (EP 0 592 106; EP 0 519 596; Padlan E. A., 1991; Studnicka G. M. et al., 1994; Roguska M. A. et al., 1994), and chain shuffling (U.S. Pat.", "No.", "5,565,332).", "Human antibodies can be made by a variety of methods known in the art including phage display methods described above.", "See also, U.S. Pat.", "Nos.", "4,444,887, 4,716,111, 5,545,806, and 5,814,318; WO 98/46645; WO 98/50433; WO 98/24893; WO 96/34096; WO 96/33735; and WO 91/10741.Further included in the present invention are antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide of the present invention.", "The antibodies may be specific for antigens other than polypeptides of the present invention.", "For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.", "Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art (See e.g., Harbor et al.", "supra; WO 93/21232; EP 0 439 095; Naramura, M. et al.", "0.1994; U.S. Pat.", "No.", "5,474,981; Gillies, S. O. et al., 1992; Fell, H. P. et al., 1991).", "The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.", "For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.", "The antibody portion fused to a polypeptide of the present invention may comprise the hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.", "The polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half-life of the polypeptides or for use in immunoassays using methods known in the art.", "The polypeptides may also be fused or conjugated to the above antibody portions to form multimers.", "For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.", "Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.", "Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art.", "See e.g., U.S. Pat.", "Nos.", "5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. et al.", "(1991); Zheng, X. X. et al.", "(1995); and Vil, H. et al.", "(1992).", "The invention further relates to antibodies that act as agonists or antagonists of the polypeptides of the present invention.", "For example, the present invention includes antibodies that disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.", "Included are both receptor-specific antibodies and ligand-specific antibodies.", "Included are receptor-specific antibodies, which do not prevent ligand binding but prevent receptor activation.", "Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art.", "Also include are receptor-specific antibodies which both prevent ligand binding and receptor activation.", "Likewise, included are neutralizing antibodies that bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies that bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.", "Further included are antibodies that activate the receptor.", "These antibodies may act as agonists for either all or less than all of the biological activities affected by ligand-mediated receptor activation.", "The antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein.", "The above antibody agonists can be made using methods known in the art.", "See e.g., WO 96/40281; U.S. Pat.", "No.", "5,811,097; Deng, B. et al.", "(1998); Chen, Z. et al.", "(1998); Harrop, J.", "A. et al.", "(1998); Zhu, Z. et al.", "(1998); Yoon, D. Y. et al.", "(1998); Prat, M. et al.", "(1998) J.; Pitard, V. et al.", "(1997); Liautard, J. et al.", "(1997); Carlson, N. G. et al.", "(1997) J.; Taryman, R. E. et al.", "(1995); Muller, Y.", "A. et al.", "(1998); Bartunek, P. et al.", "(1996).", "As discussed above, antibodies of the polypeptides of the invention can, in turn, be utilized to generate anti-idiotypic antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art (See, e.g.", "Greenspan and Bona (1989); and Nissinoff (1991).", "For example, antibodies which bind to and competitively inhibit polypeptide multimerization or binding of a polypeptide of the invention to ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization or binding domain and, as a consequence, bind to and neutralize polypeptide or its ligand.", "Such neutralization anti-idiotypic antibodies can be used to bind a polypeptide of the invention or to bind its ligands/receptors, and therby block its biological activity, The invention also concerns a purified or isolated antibody capable of specifically binding to a mutated full length or mature polypeptide of the present invention or to a fragment or variant thereof comprising an epitope of the mutated polypeptide.", "In another preferred embodiment, the present invention concerns an antibody capable of binding to a polypeptide comprising at least 10 consecutive amino acids of a polypeptide of the present invention and including at least one of the amino acids which can be encoded by the trait causing mutations.", "Non-human animals or mammals, whether wild-type or transgenic, which express a different species of a polypeptide of the present invention than the one to which antibody binding is desired, and animals which do not express a polypeptide of the present invention (i.e.", "a knockout animal) are particularly useful for preparing antibodies.", "Gene knock out animals will recognize all or most of the exposed regions of a polypeptide of the present invention as foreign antigens, and therefore produce antibodies with a wider array of epitopes.", "Moreover, smaller polypeptides with only 10 to 30 amino acids may be useful in obtaining specific binding to any one of the polypeptides of the present invention.", "In addition, the humoral immune system of animals that produce a species of a polypeptide of the present invention that resembles the antigenic sequence will preferentially recognize the differences between the animal's native polypeptide species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence.", "Such a technique will be particularly useful in obtaining antibodies that specifically bind to any one of the polypeptides of the present invention.", "Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.", "The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.", "The antibodies of the invention may be labelled by any one of the radioactive, fluorescent or enzymatic labels known in the art.", "Consequently, the invention is also directed to a method for detecting specifically the presence of a polypeptide of the present invention according to the invention in a biological sample, said method comprising the following steps: a) obtaining a biological sample suspected of containing a polypeptide of the present invention; b) contacting the biological sample with a polyclonal or monoclonal antibody that specifically binds a polypeptide of the present invention under conditions suitable for antigen-antibody binding; and c) detecting the antigen-antibody complex formed.", "The invention also concerns a diagnostic kit for detecting in vitro the presence of a polypeptide of the present invention in a biological sample, wherein said kit comprises: a) a polyclonal or monoclonal antibody that specifically binds a polypeptide of the present invention, optionally labelled; b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labelled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labelled by itself.", "A.", "Monoclonal Antibody Production by Hybridoma Fusion Monoclonal antibody to epitopes of any of the peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler, G. and Milstein, C., Nature 256:495 (1975) or derivative methods thereof.", "Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom over a period of a few weeks.", "The mouse is then sacrificed, and the antibody producing cells of the spleen isolated.", "The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media).", "The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.", "Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as Elisa, as originally described by Engvall, E., Meth Enzymol 70:419 (1980), and derivative methods thereof.", "Selected positive clones can be expanded and their monoclonal antibody product harvested for use.", "Detailed procedures for monoclonal antibody production are described in Davis, L. et al.", "Basic Methods in Molecular Biology Elsevier, New York.", "Section 21-2.In a preferred embodiment, said monoclonal antibody is specific for a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptide fragment comprising all or part of the C-terminal globular C1q homology domain.", "In preferred embodiments, said polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids and not more than 333 consecutive amino acids of SEQ ID NO: 2 or 4, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 245, 247, 248, 249, 250, 251, 252, or 253, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 256-267 of SEQ ID NO: 2 or 4) or QVTGGERFNGLFAD (amino acids 304-317 of SEQ ID NO: 2 or 4); or at least 6 and not more than 225 consecutive amino acids of SEQ ID NO: 6, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 148-159 of SEQ ID NO: 6) or QVTGGERFNGLFAD (amino acids 196-209 of SEQ ID NO: 6); at least 6 consecutive amino acids and not more than 330 consecutive amino acids of SEQ ID NO: 8, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 253-264 of SEQ ID NO: 8) or QVTGGERFNGLFAD (amino acids 301-314 of SEQ ID NO: 8); or at least 6 and not more than 323 consecutive amino acids of SEQ ID NO: 10, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, or 243, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 246-257 of SEQ ID NO: 10) or QVTGGERFNGLFAD (amino acids 294-307 of SEQ ID NO: 10).", "B. Polyclonal Antibody Production by Immunization Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein or peptides derived therefrom described above, which can be unmodified or modified to enhance immunogenicity.", "Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species.", "For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant.", "Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera.", "Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable.", "An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al.", "J. Clin.", "Endocrinol.", "Metab.", "33:988-991 (1971).", "Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall.", "See, for example, Ouchterlony, O. et al., Chap.", "19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973).", "Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 □M).", "Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap.", "42 in: Manual of Clinical Immunology, 2d Ed.", "(Rose and Friedman, Eds.)", "Amer.", "Soc.", "For Microbiol., Washington, D.C. (1980).", "In a preferred embodiment, said polyclonal antibody is specific for a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptide fragment comprising all or part of the C-terminal globular C1q homology domain.", "In preferred embodiments, said polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids and not more than 333 consecutive amino acids of SEQ ID NO: 2 or 4, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 245, 247, 248, 249, 250, 251, 252, or 253, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 256-267 of SEQ ID NO: 2 or 4) or QVTGGERFNGLFAD (amino acids 304-317 of SEQ ID NO: 2 or 4); or at least 6 and not more than 225 consecutive amino acids of SEQ ID NO: 6, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 148-159 of SEQ ID NO: 6) or QVTGGERFNGLFAD (amino acids 196-209 of SEQ ID NO: 6); at least 6 consecutive amino acids and not more than 330 consecutive amino acids of SEQ ID NO: 8, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 253-264 of SEQ ID NO: 8) or QVTGGERFNGLFAD (amino acids 301-314 of SEQ ID NO: 8); or at least 6 and not more than 323 consecutive amino acids of SEQ ID NO: 10, preferably wherein said polypeptide fragment is comprised of one or more of amino acids 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, or 243, and more preferably wherein said polypeptide fragment is comprised of the sequence TVFSRNVQVSLV (amino acids 246-257 of SEQ ID NO: 10) or QVTGGERFNGLFAD (amino acids 294-307 of SEQ ID NO: 10).", "Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.", "The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.", "XII.", "Identifying One or More Cell Types Expressing a Cell Surface Receptor for GMG-3.GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptide The invention features methods of identifying one or more cell types expressing a cell surface receptor for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide comprised of contacting said cell type with labelled GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide and measuring the amount of said polypeptide bound.", "Preferably said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide comprises all or part of the C-terminal globular C1q homology domain and has lipid partitioning, lipid metabolism, or insulin-like activities.", "Preferably said labelled GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide is selected from but not restricted to fluorescein-coupled GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B or biotin coupled GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B.", "Bound fluorescein-coupled GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B is detected directly by FACS.", "Bound biotin-coupled GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B is detected by FACS after secondary binding of phycoerythrin-coupled streptavidin or by radioassay after secondary binding of 125I-streptavidin.", "Alternatively, GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide is tagged with an antibody epitope at the N- or C-terminus as described supra with regard to polynucleotides encoding polypeptides of the invention that are fused in frame to the coding sequences for additional heterologous amino acid sequences.", "Binding of said epitope-tagged GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide is detected with antibody specific for the epitope.", "XIII.", "Cloning cDNA Encoding Cell Surface Receptor for GMG-3.GMG-4, Cluster 1, GMG-6A, or GMG-6B GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptide The invention features methods of using GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide to clone cDNA encoding a cell surface receptor for said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide.", "Preferably said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide comprises all or part of the C-terminal globular C1q homology domain and has lipid partitioning, lipid metabolism, or insulin-like activities.", "In a preferred embodiment, said method of cloning a cell surface receptor for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide comprises: isolating mRNA from a cell type expressing said cell surface receptor for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide; converting said mRNA to cDNA; ligating said cDNA into a eukaryotic expression vector containing the origin for SV40 replication; transiently transfecting pools of said ligated cDNA into COS cells using dextran sulfate; culturing the transfected COS cells for about 48 h; detecting cell surface expression of said receptor for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide by contacting said transfected COS cells with biotinylated or epitope-tagged GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-4B polypeptide; contacting said biotinylated or eptiope-tagged GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide bound to said transfected COS cells directly (biotinylated said polypeptide) or indirectly (epitope-tagged said polypeptide) with 125I-streptavidin; identifying said transfected COS cells labelled with 125I-streptavidin; recovering cDNA from said labelled COS cells; and repeating said transient transfection with smaller pools of said recovered cDNA until transfection with a single clone of cDNA leads to cell surface expression of said receptor for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide.", "Said method of cloning cDNA encoding a cell surface receptor for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide by transient transfection of COS cells is well known to those skilled in the art.", "Other characteristics and advantages of the invention are described in the Examples.", "These are meant to be exemplary only, and not to limit the invention in any way.", "Throughout this application, various publications, patents and published patent applications are cited.", "The disclosures of these publications, patents and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure.", "EXAMPLES The following Examples are provided for illustrative purposes and not as a means of limitation.", "One of ordinary skill in the art would be able to design equivalent assays and methods based on the disclosure herein all of which form part of the instant invention.", "Example 1 Northern Analysis of GMG-3, GMG-4 Cluster 1, GMG-6A, or GMG-6B DNA Analysis of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B expression in different human tissues (adult and fetal) and cell lines, as well as mouse embryos in different stages of development, is accomplished by using poly A+ RNA blots purchased from Clontech (e.g.", "#7780-1, 7757-1, 7756-1, 7768-1 and 7763-1).", "Labeling of RNA probes is performed using the RNA Strip-EZ kit from Ambion as per manufacture's instructions.", "Hybridization of RNA probes to RNA blots is performed Ultrahyb hybridization solution (Ambion).", "Briefly, blots are prehybridized for 30 min at 58° C. (low-strigency) or 65° C. (high stringency).", "After adding the labelled probe (2×106 cpm/ml), blots are hybridized overnight (14-24 hrs), and washed 2×20 min at 50° C. with 2×SSC/0.1% SDS (low stringency), 2×20 min at 58° C. with 1×SSC/0.1% SDS (medium stringency) and 2×20 min at 65° C. with 1×SSC/0.1% SDS (high stringency).", "After washings are completed blots are exposed on the phosphoimager (Molecular Dynamics) for 1-3 days.", "Example 2 In Vitro Tests of Metabolic-Related Activity The activity of various preparations and various sequence variants of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are assessed using various in vitro assays including those provided below.", "These assays are also exemplary of those that can be used to develop GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide antagonists and agonists.", "To do that, the effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides in the above assays, e.g.", "on leptin and/or LSR activity, in the presence of the candidate molecules would be compared with the effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides in the assays in the absence of the candidate molecules.", "Since GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides reduce body weight in mice on a high-cafeteria diet (Example 5), these assays also serve to identify candidate treatments for reducing (or increasing) body weight.", "Liver Cell Line: Tests of efficacy of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on LSR can be performed using liver cell lines, including for example, PLC, HepG2, Hep3B (human), Hepa 1-6, BPRCL (mouse), or MCA-RH777, MCA-RH8994 (rat).", "BPRCL mouse liver cells (ATCC Repository) are plated at a density of 300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose) containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992).", "Media is changed on day 2.On day 3, the confluent monolayers are washed once with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well).", "Cells are incubated at 37° C. for 30 min with increasing concentrations of recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment comprising the C-terminal globular C1q homology domain in DMEM containing 0.2% (w/v) BSA, 5 mM Hepes, 2 mM CaCl2, 3.7 g/L sodium bicarbonate, pH 7.5.Incubations are continued for 3 h at 37° C. after addition of 10 ng/mL 125I-mouse leptin (specific activity, 22100 cpm/ng).", "Monolayers are washed 2 times consecutively with PBS containing 0.2% BSA, followed by 1 wash with PBS/BSA, and then 2 times consecutively with PBS.", "Cells are lysed with 0.1 N NaOH containing 0.24 mM EDTA.", "Lysates are collected into tubes, and counted in a gamma-counter.", "Blood Brain Barrier Model: The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on leptin transport in the brain can be determined using brain-derived cells.", "One method that is envisioned is to use the blood/brain barrier model described by Dehouck et al (J Neurochem 54:1798-801, 1990; hereby incorporated herein by reference in its entirety including any figures, tables, or drawings) that uses a co-culture of brain capillary endothelial cells and astrocytes to test the effects of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on leptin (or other molecules) transport via LSR or other receptors.", "This assay would be an indicator of the potential effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on leptin transport to the brain and could be used to screen GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide variants for their ability to modulate leptin transport through LSR or other receptors in the brain.", "In addition, putative agonists and antagonists of the effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on leptin transport through LSR or other receptors could also be screened using this assay.", "Increased transport of leptin across the blood/brain barrier would presumably increase its action as a satiety factor.", "FACS Analysis of LSR Expression The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on LSR can also be determined by measuring the level of LSR expression at the cell surface by flow surface cytometry, using anti-LSR antibodies and fluorescent secondary antibodies.", "Flow cytometry is a laser-based technology that is used to measure characteristics of biological particles.", "The underlying principle of flow cytometry is that light is scattered and fluorescence is emitted as light from the excitation source strikes the moving particles.", "This is a high throughput assay that could be easily adapted to screen GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides and variants as well as putative agonists or antagonists of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides.", "Two assays are provided below.", "The antibody, cell-line and GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide analogs would vary depending on the experiment, but a human cell-line, human anti-LSR antibody and GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment comprising the C-terminal globular C1q homology domain could be used to screen for variants, agonists, and antagonists to be used to treat humans.", "Assay 1: Cells are pretreated with either intact GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment comprising the C-terminal globular C1q homology domain (or untreated) before harvesting and analysis by FACS.", "Cells are harvested using non-enzymatic dissociation solution (Sigma), and then are incubated for 1 h at 4° C. with a 1:200 dilution of anti-LSR 81B or an irrelevant anti-serum in PBS containing 1% (w/v) BSA.", "After washing twice with the same buffer, goat anti-rabbit FITC-conjugated antibody (Rockland, Gilbertsville, Pa.) is added to the cells, followed by a further incubation for 30 min at 4° C. After washing, the cells are fixed in 2% formalin.", "Flow cytometry analysis is done on a FACSCalibur cytometer (Becton-Dickinson, Franklin Lakes, N.J.).", "Assay 2: Cells are cultured in T175 flasks according to manufacturer's instructions for 48 hours prior to analysis.", "Cells are washed once with FACS buffer (1×PBS/2% FBS, filter sterilized), and manually scraped from the flask in 10 mLs of FACS buffer.", "The cell suspension is transferred to a 15 mL conical tube and centrifuged at 1200 rpm, 4° C. for 5 minutes.", "Supernatant is discarded and cells are resuspended in 10 mL FACS buffer chilled to 4° C. A cell count is performed and the cell density adjusted with FACS buffer to a concentration of 1×106 cells/mL.", "One milliliter of cell suspension was added to each well of a 48 well plate for analysis.", "Cells are centrifuged at 1200 rpm for 5 minutes at 4° C. Plates are checked to ensure that cells are pelleted, the supernatant is removed and cells resuspended by running plate over a vortex mixer.", "One milliliter of FACS buffer is added to each well, followed by centrifugation at 1200 rpm for 5 minutes at 4° C. This described cell washing was performed a total of 3 times.", "Primary antibody, titered in screening experiments to determine proper working dilutions (for example 1:25, 1:50, 1:100, 1:200, 1:400, 1:500, 1:800, 1:1000, 1:2000, 1:4000, 1:5000, or 1:10000), is added to cells in a total volume of 50 μL FACS buffer.", "Plates are incubated for 1 h at 4° C. protected from light.", "Following incubation, cells are washed 3 times as directed above.", "Appropriate secondary antibody, titered in screening experiments to determine proper working dilutions (for example 1:25, 1:50, 1:100, 1:200, 1:400, 1:500, 1:800, 1:1000, 1:2000, 1:4000, 1:5000, or 1:10000), is added to cells in a total volume of 50 μL FACS buffer.", "Plates are incubated for 1 h at 4° C. protected from light.", "Following incubation, cells are washed 3 times as directed above.", "Upon final wash, cells are resuspended in 500 μL FACS buffer and transferred to a FACS acquisition tube.", "Samples are placed on ice protected from light and analyzed within 1 hour.", "Cellular Binding and Uptake of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptides as Detected by Fluorescence Microscopy Fluorecein isothiocyanate (FITC) conjugation of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides: Purified GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B proteins at 1 mg/mL concentration are labelled with FITC using Sigma's FluoroTag FITC conjugation kit (Stock No.", "FITC-1).", "Protocol outlined in the Sigma Handbook for small-scale conjugation is followed for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein labeling.", "Cell Culture: C2C12 mouse skeletal muscle cells (ATCC, Manassas, Va. CRL-1772) and Hepa-1-6 mouse hepatocytes (ATCC, Manassas, Va. CRL-1830) are seeded into 6 well plates at a cell density of 2×105 cells per well.", "C2C12 and Hepa-1-6 cells are cultured according to repository's instructions for 24-48 hours prior to analysis.", "Assay is performed when cells were 80% confluent.", "FITC labelled GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein cellular binding and uptake using microscopy: C2C12 and Hepa 1-6 cells are incubated in the presence/absence of antibody directed against human LSR (81B: N-terminal sequence of human LSR; does not cross react with mouse LSR and 93A: c-terminal sequence, cross reacts with mouse LSR) or an antiserum directed against gC1qr (953) for 1 hour at 37° C., 5% CO2.LSR antibodies are added to the media at a concentration of 2 μg/mL.", "The anti-gC1qr antiserum is added to the media at a volume of 2.5 μL undiluted serum (high concentration) or 1:100 dilution (low concentration).", "Following incubation with specified antibody, FITC-GMG-3, -GMG-4, -Cluster 1, -GMG-6A, or -GMG-6B polypeptide (50 nM/mL) is added to each cell culture well.", "Cells are again incubated for 1 hour at 37° C., 5% CO2.Cells are washed 2× with PBS, cells are scraped from well into 1 mL of PBS.", "Cell suspension is transferred to an eppendorf tube and centrifuged at 1000 rpm for 2 minutes.", "Supernatant is removed and cells resuspended in 200 μL of PBS.", "Binding and uptake of FITC-GMG-3, -GMG-4, -Cluster 1, -GMG-6A, or -GMG-6B polypeptide is analyzed by fluorescence microscopy under 40× magnification.", "This assay may be useful for identifying agents that facilitate or prevent the uptake and/or binding of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides to cells.", "Effect on LSR as a Lipoprotein Receptor The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein on the lipoprotein binding, internalizing and degrading activity of LSR can also be tested.", "Measurement of LSR as lipoprotein receptor is described in Bihain & Yen, ((1992) Biochemistry 31:4628-36; hereby incorporated herein in its entirety including any drawings, tables, or figures).", "The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein on the lipoprotein binding, internalizing and degrading activity of LSR (or other receptors) can be compared with that of intact GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, with untreated cells as an additional control.", "This assay can also be used to screen for active and inhibitory variants of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, as well as agonists and antagonists of metabolic-related activity.", "Human liver PLC cells (ATCC Repository) are plated at a density of 300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose) containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992).", "Media is changed on day 2.On day 3, the confluent monolayers are washed once with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well).", "Cells are incubated at 37° C. for 30 min with 10 ng/mL human recombinant leptin in DMEM containing 0.2% (w/v) BSA, 5 mM Hepes, 2 mM CaCl2, 3.7 g/L sodium bicarbonate, pH 7.5, followed by another 30 min incubation at 37° C. with increasing concentrations of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide.", "Incubations are continued for 2 h at 37° C. after addition of 0.8 mM oleate and 20 μg/mL 125I-LDL.", "Monolayers are washed 2 times consecutively with PBS containing 0.2% BSA, followed by 1 wash with PBS/BSA, and then 2 times consecutively with PBS.", "The amounts of oleate-induced binding, uptake and degradation of 125I-LDL are measured as previously described (Bihain & Yen, 1992, supra).", "Results are shown as the mean of triplicate determinations.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein leads to an increased activity of LSR as a lipoprotein receptor.", "The oleate-induced binding and uptake of LDL would be more affected by GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein as compared to the degradation.", "This increased LSR activity would potentially result in an enhanced clearance of triglyceride-rich lipoproteins during the postprandial state.", "Thus, more dietary fat would be removed through the liver, rather than being deposited in the adipose tissue.", "This assay could be used to determine the efficiency of a compound (or agonists or antagonists) to increase or decrease LSR activity (or lipoprotein uptake, binding and degradation through other receptors), and thus affect the rate of clearance of triglyceride-rich lipoproteins.", "Effect on Muscle Differentiation C2C12 cells (murine skeletal muscle cell line; ATCC CRL 1772, Rockville, Md.)", "are seeded sparsely (about 15-20%) in complete DMEM (w/glutamine, pen/strep, etc)+10% FCS.", "Two days later they become 80-90% confluent.", "At this time, the media is changed to DMEM+2% horse serum to allow differentiation.", "The media is changed daily.", "Abundant myotube formation occurs after 3-4 days of being in 2% horse serum, although the exact time course of C2C12 differentiation depends on how long they have been passaged and how they have been maintained, among other things.", "To test the effect of the presence of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein on muscle differentiation, GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptide fragment comprising the C-terminal globular C1q homology domain (1 to 2.5 μg/mL) is added the day after seeding when the cells are still in DMEM w/ 10% FCS.", "Two days after plating the cells (one day after said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptide fragment was first added), at about 80-90% confluency, the media is changed to DMEM+2% horse serum plus said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptide fragment.", "Effect on Muscle Cell Fatty Acid Oxidation C2C12 cells are differentiated in the presence or absence of 2 μg/mL GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein for 4 days.", "On day 4, oleate oxidation rates are determined by measuring conversion of 1-14C-oleate (0.2 mM) to 14CO2 for 90 min.", "This experiment can be used to screen for active polypeptides and peptides as well as agonists and antagonists or activators and inhibitors of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides.", "The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptide fragment comprising the C-terminal globular C1q homology domain on the rate of oleate oxidation can be compared in differentiated C2C12 cells (murine skeletal muscle cells; ATCC, Manassas, Va. CRL-1772) and in a hepatocyte cell line (Hepa1-6; ATCC, Manassas, Va. CRL-1830).", "Cultured cells are maintained according to manufacturer's instructions.", "The oleate oxidation assay is performed as previously described (Muoio et al (1999) Biochem J 338;783-791).", "Briefly, nearly confluent myocytes are kept in low serum differentiation media (DMEM, 2.5% Horse serum) for 4 days, at which time formation of myotubes became maximal.", "Hepatocytes are kept in the same DMEM medium supplemented with 10% FCS for 2 days.", "One hour prior to the experiment the media is removed and 1 mL of preincubation media (MEM, 2.5% Horse serum, 3 mM glucose, 4 mM Glutamine, 25 mM Hepes, 1% FFA free BSA, 0.25 mM Oleate, 5 μg/mL gentamycin) is added.", "At the start of the oxidation experiment 14C-Oleic acid (1 μCi/mL, American Radiolabelled Chemical Inc., St. Louis, Mo.)", "is added and cells are incubated for 90 min at 37° C. in the absence/presence of 2.5 μg/mL GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptide fragment.", "After the incubation period 0.75 mL of the media is removed and assayed for 14C-oxidation products as described below for the muscle FFA oxidation experiment.", "Triglyceride and Protein Analysis Following Oleate Oxidation in Cultured Cells Following transfer of media for oleate oxidation assay, cells are placed on ice.", "To determine triglyceride and protein content, cells are washed with 1 mL of 1×PBS to remove residual media.", "To each well 300 μL of cell dissociation solution (Sigma) is added and incubated at 37° C. for 10 min.", "Plates are tapped to loosen cells, and 0.5 mL of 1×PBS was added.", "The cell suspension is transferred to an eppendorf tube, each well is rinsed with an additional 0.5 mL of 1×PBS, and is transferred to appropriate eppendorf tube.", "Samples are centrifuged at 1000 rpm for 10 minutes at room temperature.", "Supernatant is discarded and 750 μL of 1×PBS/2% chaps is added to cell pellet.", "Cell suspension is vortexed and placed on ice for 1 hour.", "Samples are then centrifuged at 13000 rpm for 20 min at 4° C. Supernatants are transferred to new tube and frozen at −20° C. until analyzed.", "Quantitative measure of triglyceride level in each sample is determined using Sigma Diagnostics GPO-TRINDER enzymatic kit.", "The procedure outlined in the manual is adhered to, with the following exceptions: assay is performed in 48 well plate, 350 μL of sample volume was assayed, control blank consisted of 350 μL PBS/2% chaps, and standard contained 10 μL-standard provide in kit plus 690 μL PBS/2% chaps.", "Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 mm Protein analysis is carried out on 25 μL of each supernatant sample using the BCA protein assay (Pierce) following manufacturer's instructions.", "Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm.", "In Vitro Glucose Uptake by Muscle Cells L6 Muscle cells are obtained from the European Culture Collection (Porton Down) and are used at passages 7-11.Cells are maintained in standard tissue culture medium DMEM, and glucose uptake is assessed using [3H]-2-deoxyglucose (2DG) with or without GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment in the presence or absence of insulin (10−8 M) as has been previously described (Walker, P. S. et al.", "(1990) Glucose transport activity in L6 muscle cells is regulated by the coordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription.", "JBC 265(3):1516-1523; and Kilp, A. et al.", "(1992) Stimulation of hexose transport by metformin in L6 muscle cells in culture.", "Endocrinology 130(5):2535-2544, which disclosures are hereby incorporated by reference in their entireties).", "Uptake of 2DG is expressed as the percentage change compared with control (no added insulin or GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment).", "Values are presented as mean±SEM of sets of 4 wells per experiment.", "Differences between sets of wells are evaluated by Student's t test, probability values p<0.05 are considered to be significant.", "Example 3 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptides on Mice Fed a High-Fat Diet Experiments are performed using approximately 6 week old C57B1/6 mice (8 per group).", "All mice are housed individually.", "The mice are maintained on a high fat diet throughout each experiment.", "The high fat diet (cafeteria diet; D12331 from Research Diets, Inc.) has the following composition: protein kcal % 16, sucrose kcal % 26, and fat kcal % 58.The fat is primarily composed of coconut oil, hydrogenated.", "After the mice are fed a high fat diet for 6 days, micro-osmotic pumps are inserted using isoflurane anesthesia, and are used to provide full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments, saline, and an irrelevant peptide to the mice subcutaneously (s.c.) for 18 days.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are provided at doses of 100, 50, 25, and 2.5 μg/day and the irrelevant peptide is provided at 10 μg/day.", "Body weight is measured on the first, third and fifth day of the high fat diet, and then daily after the start of treatment.", "Final blood samples are taken by cardiac puncture and are used to determine triglyceride (TG), total cholesterol (TC), glucose, leptin, and insulin levels.", "The amount of food consumed per day is also determined for each group.", "Example 4 Tests of Metabolic-Related Activity in Humans Tests of the efficacy of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides in humans are performed in accordance with a physician's recommendations and with established guidelines.", "The parameters tested in mice are also tested in humans (e.g.", "food intake, weight, TG, TC, glucose, insulin, leptin, FFA).", "It is expected that the physiological factors would show changes over the short term.", "Changes in weight gain might require a longer period of time.", "In addition, the diet would need to be carefully monitored.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, preferably GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides comprising the C-terminal globular C1q homology domain, would be given in daily doses of about 6 mg protein per 70 kg person or about 10 mg per day.", "Other doses would also be tested, for instance 1 mg or 5 mg per day up to 20 mg, 50 mg, or 100 mg per day.", "Example 5 Tests of Metabolic-Related Activity in a Murine Lipoatrophic Diabetes Model Previously, leptin was reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al.", "Nature 401:73-76 (1999); hereby incorporated herein in its entirety including any drawings, figures, or tables).", "Leptin was found to be less effective in a different lipodystrophic mouse model of lipoatrophic diabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporated herein in its entirety including any drawings, figures, or tables).", "The instant invention encompasses the use of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides for reducing the insulin resistance and hyperglycaemia in this model either alone or in combination with leptin, the leptin peptide (U.S. provisional application No.", "60/155,506), or other compounds.", "Assays include that described previously in Gavrilova et al.", "((2000) Diabetes 49:1910-6; (2000) Nature 403:850) using A-ZIP/F-1 mice, except that GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides would be administered using the methods previously described in Example 3 (or Examples 6-8).", "The glucose and insulin levels of the mice would be tested, and the food intake and liver weight monitored, as well as other factors, such as leptin, FFA, and TG levels, typically measured in our experiments (see Example 3, above, or Examples 6-8).", "Example 6 Effect of GMG-3, GMG-4Cluster 1, GMG-6A, or GMG-6B Polypeptides on Plasma Free Fatty Acid in C57 BL/6 Mice The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on postprandial lipemia (PPL) in normal C57BL6/J mice is tested.", "The mice used in this experiment are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.", "All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point).", "At time 0 (8:30 AM), a standard high fat meal (6 g butter, 6 g sunflower oil, 10 g nonfat dry milk, 10 g sucrose, 12 mL distilled water prepared fresh following Nb#6, JF, pg.", "1) is given by gavage (vol.=1% of body weight) to all animals.", "Immediately following the high fat meal, 25 μg a GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptide is injected i.p.", "in 100 μL saline.", "The same dose (25 μg/mL in 100 μL) is again injected at 45 min and at 1 hr 45 min.", "Control animals are injected with saline (3×100 μL).", "Untreated and treated animals are handled in an alternating mode.", "Blood samples are taken in hourly intervals, and are immediately put on ice; Plasma is prepared by centrifugation following each time point.", "Plasma is kept at −20° C. and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).", "Due to the limited amount of plasma available, glucose is determined in duplicate using pooled samples.", "For each time point, equal volumes of plasma from all 8 animals per treatment group are pooled.", "Example 7 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptides on Plasma Leptin and Insulin in C57 BL/6 Mice The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on plasma leptin and insulin levels during postprandial lipemia (PPL) in normal C57BL6/J mice is tested.", "The experimental procedure is the same as that described in Example 6, except that blood was drawn only at 0, 2 and 4 hours to allow for greater blood samples needed for the determination of leptin and insulin by RIA.", "Briefly, 16 mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.", "All blood samples are taken from the tail using EDTA coated capillary tubes (100 μL each time point).", "At time 0 (9:00 AM), a standard high fat meal (see Example 6) is given by gavage (vol.=1% of body weight) to all animals.", "Immediately following the high fat meal, 25 μg of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide is injected i.p.", "in 100 μL saline.", "The same dose (25 μg in 100 μL) is again injected at 45 min and at 1 hr 45 min (treated group).", "Control animals are injected with saline (3×100 μL).", "Untreated and treated animals are handled in an alternating mode.", "Blood samples are immediately put on ice and plasma is prepared by centrifugation following each time point.", "Plasma is kept at −20° C. and free fatty acids (FFA) are determined within 24 hours using a standard test kit (Wako).", "Leptin and Insulin are determined by RIA (ML-82K and SRI-13K, LINCO Research, Inc., St. Charles, Mo.)", "following the manufacturer's protocol; however; only 20 μL plasma is used.", "Each determination is done in duplicate.", "Due to the limited amount of plasma available, leptin and insulin are determined in 4 pools of 2 animals each in both treatment groups.", "Example 8 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptides on Plasma FFA, TG, and Glucose in C57 BL/6 Mice The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on plasma FFA, TG, glucose, leptin and insulin levels during postprandial lipemia (PPL) in normal C57BL6/J mice has been described.", "Weight loss resulting from GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides (2.5 μg/day) given to normal C57BL6/J mice on a high fat diet has also been shown (Example 3).", "The experimental procedure is similar to that described in Example 6.Briefly, 14 mice re fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.", "All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point).", "At time 0 (9:00 AM), a standard high fat meal (see Example 6) is given by gavage (vol.=1% of body weight) to all animals.", "Immediately following the high fat meal, 4 mice are injected 25 μg of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide i.p.", "in 100 μL saline.", "The same dose (25 μg in 100 μL) is again injected at 45 min and at 1 hr 45 min.", "A second treatment group receives 3 times 50 μg GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide at the same intervals.", "Control animals are injected with saline (3×100 μL).", "Untreated and treated animals are handled in an alternating mode.", "Blood samples are immediately put on ice.", "Plasma is prepared by centrifugation following each time point.", "Plasma is kept at −20° C. and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).", "Example 9 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptides on FFA Following Epinephrine Injection In mice, plasma free fatty acids increase after intragastric administration of a high fat/sucrose test meal.", "These free fatty acids are mostly produced by the activity of lipolytic enzymes i.e.", "lipoprotein lipase (LPL) and hepatic lipase (HL).", "In this species, these enzymes are found in significant amounts both bound to endothelium and freely circulating in plasma.", "Another source of plasma free fatty acids is hormone sensitive lipase (HSL) that releases free fatty acids from adipose tissue after β-adrenergic stimulation.", "To test whether GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides also regulate the metabolism of free fatty acid released by HSL, mice are injected with epinephrine.", "Two groups of mice are given epinephrine (5 μg) by intraperitoneal injection.", "A treated group is injected with a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide (25 μg) one hour before and again together with epinephrine, while control animals receive saline.", "Plasma is isolated and free fatty acids and glucose are measured as described above (Example 8).", "Example 10 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptides on Muscle FFA Oxidation To investigate the effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on muscle free fatty acid oxidation, intact hind limb muscles from C57BL/6J mice are isolated and FFA oxidation is measured using oleate as substrate (Clee, S. M. et al.", "Plasma and vessel wall lipoprotein lipase have different roles in atherosclerosis.", "J Lipid Res 41, 521-531 (2000); Muoio, D. M., Dohm, G. L., Tapscott, E. B.", "& Coleman, R. A. Leptin opposes insulin's effects on fatty acid partitioning in muscles isolated from obese ob/ob mice.", "Am J Physiol 276, E913-921 (1999)) Oleate oxidation in isolated muscle is measured as previously described (Cuendet et al (1976) J Clin Invest 58:1078-1088; Le Marchand-Brustel, Y., Jeanrenaud, B.", "& Freychet, P. Insulin binding and effects in isolated soleus muscle of lean and obese mice.", "Am J Physiol 234, E348-E358 (1978).", "Briefly, mice are sacrificed by cervical dislocation and soleus and EDL muscles are rapidly isolated from the hind limbs.", "The distal tendon of each muscle is tied to a piece of suture to facilitate transfer among different media.", "All incubations are carried out at 30° C. in 1.5 mL of Krebs-Henseleit bicarbonate buffer (118.6 mM NaCl, 4.76 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgSO4, 2.54 mM CaCl2, 25 mM NaHCO3, 10 mM Hepes, pH 7.4) supplemented with 4% FFA free bovine serum albumin (fraction V, RIA grade, Sigma) and 5 mM glucose (Sigma).", "The total concentration of oleate (Sigma) throughout the experiment is 0.25 mM.", "All media are oxygenated (95% O2; 5% CO2) prior to incubation.", "The gas mixture is hydrated throughout the experiment by bubbling through a gas washer (Kontes Inc., Vineland, N.J.).", "Muscles are rinsed for 30 min in incubation media with oxygenation.", "The muscles are then transferred to fresh media (1.5 mL) and incubated at 30° C. in the presence of 1 μCi/mL [1-14C] oleic acid (American Radiolabelled Chemicals).", "The incubation vials containing this media are sealed with a rubber septum from which a center well carrying a piece of Whatman paper (1.5 cm×11.5 cm) is suspended.", "After an initial incubation period of 10 min with constant oxygenation, gas circulation is removed to close the system to the outside environment and the muscles are incubated for 90 min at 30° C. At the end of this period, 0.45 mL of Solvable (Packard Instruments, Meriden, Conn.) is injected onto the Whatman paper in the center well and oleate oxidation by the muscle is stopped by transferring the vial onto ice.", "After 5 min, the muscle is removed from the medium, and an aliquot of 0.5 mL medium is also removed.", "The vials are closed again and 1 mL of 35% perchloric acid is injected with a syringe into the media by piercing through the rubber septum.", "The CO2 released from the acidified media is collected by the Solvable in the center well.", "After a 90 min collection period at 30° C., the Whatman paper is removed from the center well and placed in scintillation vials containing 15 mL of scintillation fluid (HionicFlour, Packard Instruments, Meriden, Conn.).", "The amount of 14C radioactivity is quantitated by liquid scintillation counting.", "The rate of oleate oxidation is expressed as nmol oleate produced in 90 min/g muscle.", "To test the effect of full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment comprising the C-terminal globular C1q homology domain on oleate oxidation, these proteins are added to the media at a final concentration of 2.5 μg/mL and maintained in the media throughout the procedure.", "Example 11 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptides on Triglyceride in Muscle & Liver Isolated from Mice To determine whether the increased FFA oxidation induced by GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides is also accompanied by increased FFA delivery into muscle or liver, the hindlimb muscle and liver triglyceride content is measured after the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide treatment of mice.", "Hind limb muscles as well as liver samples are removed from treated and untreated animals and the triglyceride and free fatty acid concentration is determined following a standard lipid extraction method (Shimabukuro, M. et al.", "Direct antidiabetic effect of leptin through triglyceride depletion of tissues.", "Proc Natl Acad Sci USA 94:4637-4641 (1997)) followed by TG and FFA analysis using standard test kits.", "Example 12 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A or GMG-6B Polypeptides on FFA Following Intralipid Injection Two groups of mice are intravenously (tail vein) injected with 30 μL bolus of Intralipid-20% (Clintec) to generate a sudden rise in plasma FFAs, thus by-passing intestinal absorption.", "(Intralipid is an intravenous fat emulsion used in nutritional therapy).", "A treated group (GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide-treated) is injected with a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide (25 μg) at 30 and 60 minutes before Intralipid is given, while control animals (□ control) received saline.", "Plasma is isolated and FFAs are measured as described previously.", "The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on the decay in plasma FFAs following the peak induced by Intralipid injection is then monitored.", "Example 13 In Vitro Glucose Uptake by Muscle Cells L6 Muscle cells are obtained from the European Culture Collection (Porton Down) and are used at passages 7-11.Cells are maintained in standard tissue culture medium DMEM, and glucose uptake is assessed using [3]-2-deoxyglucose (2DG) with or without GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides in the presence or absence of insulin (10−8 M) as has been previously described (Walker, P. S. et al.", "(1990) Glucose transport activity in L6 muscle cells is regulated by the coordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription.", "JBC 265:1516-1523; and Kilp, A. et al.", "(1992) Stimulation of hexose transport by metformin in L6 muscle cells in culture, Endocrinology 130:2535-2544, which disclosures are hereby incorporated by reference in their entireties).", "Uptake of 2DG is expressed as the percentage change compared with control (no added insulin or GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B).", "Values are presented as mean±SEM of sets of 4 wells per experiment.", "Differences between sets of wells are evaluated by Student's t test, probability values p<0.05 are considered to be significant.", "Example 14 In Vivo Tests for Metabolic-Related Activity in Rodent Diabetes Models As metabolic profiles differ among various animal models of obesity and diabetes, analysis of multiple models is undertaken to separate the effects GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on hyperglycemia, hyperinsulinemia, hyperlipidemia and obesity.", "Mutations within colonies of laboratory animals and different sensitivities to dietary regimens have made the development of animal models with non-insulin dependent diabetes associated with obesity and insulin resistance possible.", "Genetic models such as db/db and ob/ob (See Diabetes, (1982) 31(1): 1-6 in mice and fa/fa in zucker rats have been developed by the various laboratories for understanding the pathophysiology of disease and testing the efficacy of new antidiabetic compounds (Diabetes, (1983) 32: 830-838; Annu Rep Sankyo Res Lab (1994) 46: 1-57).", "The homozygous animals, C57 BL/KsJ-db/db mice developed by Jackson Laboratory, US, are obese, hyperglycemic, hyperinsulinemic and insulin resistant (J Clin Invest, (1990) 85: 962-967), whereas heterozygous are lean and normoglycemic.", "In db/db model, mouse progressively develops insulinopenia with age, a feature commonly observed in late stages of human type II diabetes when blood sugar levels are insufficiently controlled.", "The state of pancreas and its course vary according to the models.", "Since this model resembles that of type II diabetes mellitus, the compounds of the present invention are tested for blood sugar and triglycerides lowering activities.", "Zucker (fa/fa) rats are severely obese, hyperinsulinemic, and insulin resistant (Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds.", "(Elsevier Science Publishing Co., Inc., New York, ed.", "4, 1990), pp.", "299-340), and the fa/fa mutation may be the rat equivalent of the murine db mutation (Friedman et al., Cell 69:217-220, 1992; Truett et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 88:7806, 1991).", "Tubby (tub/tub) mice are characterized by obesity, moderate insulin resistance and hyperinsulinemia without significant hyperglycemia (Coleman et al., J. Heredity 81:424, 1990).", "Previously, leptin was reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al.", "Nature 401: 73-76 (1999).", "Leptin is found to be less effective in a different lipodystrophic mouse model of lipoatrophic diabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporated herein in its entirety including any drawings, figures, or tables).", "The streptozotocin (STZ) model for chemically-induced diabetes is tested to examine the effects of hyperglycemia in the absence of obesity.", "STZ-treated animals are deficient in insulin and severely hyperglycemic (Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds.", "(Elsevier Science Publishing Co., Inc., New York, ed.", "4, 1990), pp.", "299-340).", "The monosodium glutamate (MSG) model for chemically-induced obesity (Olney, Science 164:719, 1969; Cameron et al., Clin Exp Pharmacol Physiol 5:41, 1978), in which obesity is less severe than in the genetic models and develops without hyperphagia, hyperinsulinemia and insulin resistance, is also examined.", "Finally, a non-chemical, non-genetic model for induction of obesity includes feeding rodents a high fat/high carbohydrate (cafeteria diet) diet ad libitum.", "The instant invention encompasses the use of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides for reducing the insulin resistance and hyperglycemia in any or all of the above rodent diabetes models or in humans with Type I or Type II diabetes or other prefered metabolic diseases described previously or models based on other mammals.", "In the compositions of the present invention the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides may, if desired, be associated with other compatible pharmacologically active antidiabetic agents such as insulin, leptin (U.S. provisional application No.", "60/155,506), or troglitazone, either alone or in combination.", "Assays include that described previously in Gavrilova et al.", "((2000) Diabetes 49:1910-6; (2000) Nature 403:850) using A-ZIP/F-1 mice, except that GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are administered intraperitoneally, subcutaneously, intramuscularly or intravenously.", "The glucose and insulin levels of the mice would be tested, and the food intake and liver weight monitored, as well as other factors, such as leptin, FFA, and TG levels, typically measured in our experiments.", "In Vivo Assay for Anti-Hyperglycemic Activity of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptides Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old) are housed (7-9 mice/cage) under standard laboratory conditions at 22° C. and 50% relative humidity, and maintained on a diet of Purina rodent chow and water ad libitum.", "Prior to treatment, blood is collected from the tail vein of each animal and blood glucose concentrations are determined using One Touch Basic Glucose Monitor System (Lifescan).", "Mice that have plasma glucose levels between 250 to 500 mg/dl are used.", "Each treatment group consists of seven mice that are distributed so that the mean glucose levels are equivalent in each group at the start of the study.", "db/db mice are dosed by micro-osmotic pumps, inserted using isoflurane anesthesia, to provide GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, saline, and an irrelevant peptide to the mice subcutaneously (s.c.).", "Blood is sampled from the tail vein hourly for 4 hours and at 24, 30 h post-dosing and analyzed for blood glucose concentrations.", "Food is withdrawn from 0-4 h post dosing and reintroduced thereafter.", "Individual body weights and mean food consumption (each cage) are also measured after 24 h. Significant differences between groups (comparing GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B treated to saline-treated) are evaluated using Student t-test.", "In Vivo Insulin Sensitivity Assay In vivo insulin sensitivity is examined by utilizing two-step hyperinsulinemic-euglycemic clamps according to the following protocol.", "Rodents from any or all of the various models described in Example 2 are housed for at least a week prior to experimental procedures.", "Surgeries for the placement of jugular vein and carotid artery catheters are performed under sterile conditions using ketamine and xylazine (i.m.)", "anesthesia.", "After surgery, all rodents are allowed to regain consciousness and placed in individual cages.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides or vehicle is administered through the jugular vein after complete recovery and for the following two days.", "Sixteen hours after the last treatment, hyperinsulinemic-euglycemic clamps are performed.", "Rodents are placed in restrainers and a bolus of 4 μCi [3-3H] glucose (NEN) is administered, followed by a continuous infusion of the tracer at a dose of 0.2 μCi/min (20 μl/min).", "Two hours after the start of the tracer infusion, 3 blood samples (0.3 ml each) are collected at 10 minute intervals (−20-0 min) for basal measurements.", "An insulin infusion is then started (5 mU/kg/min), and 100 μl blood samples are taken every 10 min.", "to monitor plasma glucose.", "A 30% glucose solution is infused using a second pump based on the plasma glucose levels in order to reach and maintain euglycemia.", "Once a steady state is established at 5 mU/kg/min insulin (stable glucose infusion rate and plasma glucose), 3 additional blood samples (0.3 ml each) are obtained for measurements of glucose, [3-3H] glucose and insulin (100-120 min.).", "A higher dose of insulin (25 mU/kg/min.)", "is then administered and glucose infusion rates are adjusted for the second euglycemic clamp and blood samples are taken at min.", "220-240.Glucose specific activity is determined in deproteinized plasma and the calculations of Rd and hepatic glucose output (HGO) are made, as described (Lang et al., Endocrinology 130:43, 1992).", "Plasma insulin levels at basal period and after 5 and 25 mU/kg/min, infusions are then determined and compared between GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B treated and vehicle treated rodents.", "Insulin regulation of glucose homeostasis has two major components; stimulation of peripheral glucose uptake and suppression of hepatic glucose output.", "Using tracer studies in the glucose clamps, it is possible to determine which portion of the insulin response is affected by the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides." ] ]	Patent_10466375
[ [ "Metabolic gene polynucleotides and polypeptides and uses thereof", "The present invention relates to the field of metabolic research.", "Metabolic disorders, such as obesity, are a public health problem that is serious and widespread.", "GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides have been identified that are beneficial in the treatment of metabolic disorders.", "These compounds should be effective for reducing body mass and for treating metabolic-related diseases and disorders.", "These metabolic-related diseases and disorders include hyperlipidemias, atherosclerosis, diabetes, and hypertension." ], [ "1-11.", "(canceled) 12.A method of treating or preventing a metabolic-related disease or disorder comprising the step of administering to an individual a composition comprising a polypeptide or biologically active fragment thereof, wherein said polypeptide is selected from the group consisting of: (a) SEQ ID NO: 2; (b) SEQ ID NO: 4; (c) SEQ ID NO: 6; (d) SEQ ID NO: 8; (e) SEQ ID NO: 10; and (f) SEQ ID NO: 12.13.The method of claim 12, wherein said metabolic-related disease or disorder is selected from the group consisting of: (a) obesity; (b) impaired glucose tolerance; (c) insulin resistance; (d) Syndrome X; and (e) Type II diabetes.", "14.The method of claim 12, wherein said polypeptide fragment is selected from the group consisting of: (a) amino acids 2-710 of SEQ ID NO: 2; (b) amino acids 1-262 of SEQ ID NO: 2; (c) amino acids 1-263 of SEQ ID NO: 2; (d) amino acids 1-275 of SEQ ID NO: 2; (e) amino acids 553-710 of SEQ ID NO: 2; (f) amino acids 554-710 of SEQ ID NO: 2; (g) amino acids 568-710 of SEQ ID NO: 2; (h) amino acids 575-710 of SEQ ID NO: 2; (i) amino acids 2-471 of SEQ ID NO: 4; (j) amino acids 1-262 of SEQ ID NO: 4; (k) amino acids 1-263 of SEQ ID NO: 4; (l) amino acids 1-275 of SEQ ID NO: 4; (m) amino acids 1-442 of SEQ ID NO: 4; (n) amino acids 1-443 of SEQ ID NO: 4; (o) amino acids 28-201 of SEQ ID NO: 6; (p) amino acids 54-201 of SEQ ID NO: 6; (q) amino acids 66-201 of SEQ ID NO: 6; (r) amino acids 25-446 of SEQ ID NO: 8; (s) amino acids 228-356 of SEQ ID NO: 8; (t) amino acids 228-360 of SEQ ID NO: 8; (u) amino acids 228-431 of SEQ ID NO: 8; (v) amino acids 228-446 of SEQ ID NO: 8; (w) amino acids 231-356 of SEQ ID NO: 8; (x) amino acids 231-360 of SEQ ID NO: 8; (y) amino acids 231-431 of SEQ ID NO: 8; (z) amino acids 231-446 of SEQ ID NO: 8; (aa) amino acids 241-356 of SEQ ID NO: 8; (bb) amino acids 241-360 of SEQ ID NO: 8; (cc) amino acids 241-431 of SEQ ID NO: 8; (dd) amino acids 241-446 of SEQ ID NO: 8; (ee) amino acids 24-296 of SEQ ID NO: 10; (ff) amino acids 132-296 of SEQ ID NO: 10; (gg) amino acids 135-296 of SEQ ID NO: 10; (hh) amino acids 148-296 of SEQ ID NO: 10; (ii) amino acids 33-205 of SEQ ID NO: 12; (jj) amino acids 53-205 of SEQ ID NO: 12; (kk) amino acids 54-205 of SEQ ID NO: 12; and (ll) amino acids 59-205 of SEQ ID NO: 12.15.The method of claim 13, wherein said polypeptide fragment is selected from the group consisting of: (a) amino acids 2-710 of SEQ ID NO: 2; (b) amino acids 1-262 of SEQ ID NO: 2; (c) amino acids 1-263 of SEQ ID NO: 2; (d) amino acids 1-275 of SEQ ID NO: 2; (e) amino acids 553-710 of SEQ ID NO: 2; (f) amino acids 554-710 of SEQ ID NO: 2; (g) amino acids 568-710 of SEQ ID NO: 2; (h) amino acids 575-710 of SEQ ID NO: 2; (i) amino acids 2-471 of SEQ ID NO: 4; (j) amino acids 1-262 of SEQ ID NO: 4; (k) amino acids 1-263 of SEQ ID NO: 4; (l) amino acids 1-275 of SEQ ID NO: 4; (m) amino acids 1-442 of SEQ ID NO: 4; (n) amino acids 1-443 of SEQ ID NO: 4; (o) amino acids 28-201 of SEQ ID NO: 6; (p) amino acids 54-201 of SEQ ID NO: 6; (q) amino acids 66-201 of SEQ ID NO: 6; (r) amino acids 25-446 of SEQ ID NO: 8; (s) amino acids 228-356 of SEQ ID NO: 8; (t) amino acids 228-360 of SEQ ID NO: 8; (u) amino acids 228-431 of SEQ ID NO: 8; (v) amino acids 228-446 of SEQ ID NO: 8; (w) amino acids 231-356 of SEQ ID NO: 8; (x) amino acids 231-360 of SEQ ID NO: 8; (y) amino acids 231-431 of SEQ ID NO: 8; (z) amino acids 231-446 of SEQ ID NO: 8; (aa) amino acids 241-356 of SEQ ID NO: 8; (bb) amino acids 241-360 of SEQ ID NO: 8; (cc) amino acids 241-431 of SEQ ID NO: 8; (dd) amino acids 241-446 of SEQ ID NO: 8; (ee) amino acids 24-296 of SEQ ID NO: 10; (ff) amino acids 132-296 of SEQ ID NO: 10; (gg) amino acids 135-296 of SEQ ID NO: 10; (hh) amino acids 148-296 of SEQ ID NO: 10; (ii) amino acids 33-205 of SEQ ID NO: 12; (jj) amino acids 53-205 of SEQ ID NO: 12; (kk) amino acids 54-205 of SEQ ID NO: 12; and (ll) amino acids 59-205 of SEQ ID NO: 12.16.An isolated polypeptide fragment selected from the group consisting of: (a) amino acids 2-710 of SEQ ID NO: 2; (b) amino acids 1-262 of SEQ ID NO: 2; (c) amino acids 1-263 of SEQ ID NO: 2; (d) amino acids 1-275 of SEQ ID NO: 2; (e) amino acids 553-710 of SEQ ID NO: 2; (f) amino acids 554-710 of SEQ ID NO: 2; (g) amino acids 568-710 of SEQ ID NO: 2; (h) amino acids 575-710 of SEQ ID NO: 2; (i) amino acids 2-471 of SEQ ID NO: 4; (j) amino acids 1-262 of SEQ ID NO: 4; (k) amino acids 1-263 of SEQ ID NO: 4; (l) amino acids 1-275 of SEQ ID NO: 4; (m) amino acids 1-442 of SEQ ID NO: 4; (n) amino acids 1-443 of SEQ ID NO: 4; (o) amino acids 28-201 of SEQ ID NO: 6; (p) amino acids 54-201 of SEQ ID NO: 6; (q) amino acids 66-201 of SEQ ID NO: 6; (r) amino acids 25-446 of SEQ ID NO: 8; (s) amino acids 228-356 of SEQ ID NO: 8; (t) amino acids 228-360 of SEQ ID NO: 8; (u) amino acids 228-431 of SEQ ID NO: 8; (v) amino acids 228-446 of SEQ ID NO: 8; (w) amino acids 231-356 of SEQ ID NO: 8; (x) amino acids 231-360 of SEQ ID NO: 8; (y) amino acids 231-431 of SEQ ID NO: 8; (z) amino acids 231-446 of SEQ ID NO: 8; (aa) amino acids 241-356 of SEQ ID NO: 8; (bb) amino acids 241-360 of SEQ ID NO: 8; (cc) amino acids 241-431 of SEQ ID NO: 8; (dd) amino acids 241-446 of SEQ ID NO: 8; (ee) amino acids 24-296 of SEQ ID NO: 10; (ff) amino acids 132-296 of SEQ ID NO: 10; (gg) amino acids 135-296 of SEQ ID NO: 10; (hh) amino acids 148-296 of SEQ ID NO: 10; (ii) amino acids 33-205 of SEQ ID NO: 12; (jj) amino acids 53-205 of SEQ ID NO: 12; (kk) amino acids 54-205 of SEQ ID NO: 12; and (ll) amino acids 59-205 of SEQ ID NO: 12.17.A composition comprising a carrier and one or more of the polypeptide fragments of claim 16.18.An isolated polynucleotide, or complement thereof, encoding any one of the polypeptide fragments of claim 16.19.The polynucleotide of claim 18 selected from the group consisting of: (a) DNA; (b) RNA; (c) DNA/RNA hybrid; (d) single-stranded; and (e) double-stranded.", "20.A composition comprising a carrier and an isolated polynucleotide of claim 19.21.A vector comprising an isolated polynucleotide of claim 19.22.A composition comprising a carrier and a vector of claim 21.23.A transformed host cell comprising a vector of claim 21." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.", "Obesity is a public health problem that is serious, widespread, and increasing.", "In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634).", "Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643).", "Even modest weight loss ameliorates these associated conditions.", "While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Harsh et al (2000) Nature 404:644-651).", "In agreement with these studies, is the fact that an increasing number of metabolic-related genes are being identified.", "Some of the more extensively studied genes include those encoding leptin (ob) and its receptor (db), pro-opiomelanocortin (Poinc), melanocortin-4-receptor (Mc4r), agouti protein (A y ), carboxypeptidase E (at), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loop-helix 2 (Nhlh2), prohormone convertase 1 (PCSK1), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651)." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The instant invention is based on Genset Metabolic Genes-7, 8, 9, 10, and 11 (GMG-7), (GMG-8; previously referred to as Cluster 9), (GMG-9; previously referred to as Cluster 10), (GMG-10; previously referred to as Cluster 17(a)) and (GMG-11; previously referred to as Cluster 19) of human origin.", "GMG-7A (previously referred to as Cluster 6 (1900)) and GMG-7B (previously referred to as Cluster 6 (d)) correspond to splice variants of GMG-7.GMG-7A, GMG-8, GMG-10, and GMG-11 are comprised of a C-terminal globular C1q homology domain.", "GMG-7B lacks the C-terminal globular C1q homology domain present in GMG-7A.", "GMG-9 is comprised near its N-terminus of a truncated globular C1q homology domain.", "Analysis of the C-terminal globular C1q homology domain of APM1 has shown it to structurally resemble TNFα.", "By analogy to APM1, biological activity can also reside in polypeptide fragments exclusive of all or part of the globular C1q homology domain.", "Results from Northern blot analysis indicate expression of GMG-7 in heart and brain, expression of GMG-8 in brain, and expression of GMG-11 in brain and pancreas.", "The invention includes polypeptides encoded by GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11, which include both the full-length polypeptide and fragments thereof, preferably but not intended to be limited to said polypeptide fragments comprising all or part of the C-terminal globular C1q homology domain.", "The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptide fragments of the invention have in vitro and in vivo biological activity as described herein, including utility for weight reduction, prevention of weight gain and control of blood glucose levels in humans and other mammals.", "More specifically, the biological activities of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides, including fragments, include reduction of elevated free fatty acid levels caused by administration of epinephrine, i.v.", "injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, reduction in glucose levels, modulation of energy expenditure, resistance to insulin and weight reduction in mammals consuming a high fat/high sucrose diet.", "Polypeptide fragments of the invention have activities overlapping but distinct from that of the full-length polypeptide.", "Thus, the invention is drawn to GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides, polynucleotides encoding said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides, vectors comprising said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polynucleotides, and cells recombinant for said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polynucleotides, as well as to pharmaceutical and physiologically acceptable compositions comprising said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides and methods of administering said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 pharmaceutical and physiologically acceptable compositions in order to reduce body weight or to treat metabolic-related diseases and disorders.", "Assays for identifying agonists and antagonists of metabolic-related activity are also part of the invention.", "In a first aspect, the invention features purified, isolated, or recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides or GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptide fragments that have lipid partitioning, lipid metabolism, and insulin-like activities.", "Preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptide fragments have activity, wherein said activity is also selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity.", "In preferred embodiments, said polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids and not more than 710 consecutive amino acids of SEQ ID NO: 2; at least 6 and not more than 471 consecutive amino acids of SEQ ID NO: 4; at least 6 consecutive amino acids and not more than 201 consecutive amino acids of SEQ ID NO: 6; at least 6 and not more than 446 consecutive amino acids of SEQ ID NO: 8; at least 6 consecutive amino acids and not more than 296 consecutive amino acids of SEQ ID NO: 10; or at least 6 and not more than 205 consecutive amino acids of SEQ ID NO: 12.In preferred embodiments, GMG-7A polypeptide fragments having activity are selected from amino acids 2-710, 1-262, 263-710, 1-263, 264-710 1-552, 553-710, 554-710, 561-710, 568-710, 575-710, 580-710, 581-710, 1-275 or 276-710 of SEQ ID NO: 2.In other preferred embodiments, GMG-7B polypeptide fragments having activity are selected from amino acids 2-471, 1-442, 443-471, 1-443, 444-471, 1-262, 263-471, 1-263, 264-471, 1-275 or 276-471 of SEQ ID NO: 4.In other preferred embodiments, GMG-8 polypeptide fragments having activity are selected from amino acids 28-201, 40-201, 54-201, 66-201, 70-201, or 71-201 of SEQ ID NO: 6.In other preferred embodiments, GMG-9 polypeptide fragments having activity are selected from amino acids 25-446, 228-356, 228-360, 228-431, 228-446, 231-356, 231-360, 231-431, 231-446, 233-356, 233-360, 233-431, 233-446, 236-356, 236-360, 236-431, 236-446, 240-356, 240-360, 240-431, 240-446, 241-356, 241-360, 241-431, 241-446, 242-356, 242-360, 242-431, or 242-446 of SEQ ID NO: 8.In other preferred embodiments, GMG-10 polypeptide fragments having activity are selected from amino acids 8-296, 9-296, 24-296, 32-296, 39-296, 52-296, 65-296, 71-296, 74-296, 77-296, 78-296, 81-296, 84-296, 90-296, 92-296, 102-296, 110-296, 111-296, 120-296, 132-296, 135-296, 148-296, 154-296 or 155-296 of SEQ ID NO: 10.In other preferred embodiments, GMG-11 polypeptide fragments having activity are selected from amino acids 33-205, 53-205, 54-205, 59-205, 71-205 or 72-205 of SEQ ID NO: 12.In more preferred embodiments, GMG-7A polypeptide fragments having activity are selected from amino acids 2-710, 1-262, 1-263, 553-710, 554-710, 568-710, 575-710 or 1-275 of SEQ ID NO: 2.In other more preferred embodiments, GMG-7B polypeptide fragments having activity are selected from amino acids 2-471, 14-42, 1-443, 1-262, 1-263, or 1-275 of SEQ ID NO: 4.In other more preferred embodiments, GMG-8 polypeptide fragments having activity are selected from amino acids 28-201, 54-201 or 66-201 of SEQ ID NO: 6.In other more preferred embodiments, GMG-9 polypeptide fragments having activity are selected from amino acids 254-46, 228-356, 228-360, 228-431, 228-446, 231-356, 231-360, 231-431, 231-446, 241-356, 241-360, 241-431 or 241-446 of SEQ ID NO: 8.In other more preferred embodiments, GMG-10 polypeptide fragments having activity are selected from amino acids 8-296, 9-296, 24-296, 52-296, 71-296, 74-296, 81-296, 84-296, 110-296, 111-296, 120-296, 132-296, 135-296, 148-296, 154-296 or 155-296 of SEQ ID NO: 10.In other more preferred embodiments, GMG-11 polypeptide fragments having activity are selected from amino acids 33-205, 53-205, 54-205 or 59-205 of SEQ ID NO: 12.In further preferred embodiments, said polypeptide fragment comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of the polypeptide sequences identified in SEQ ED NO: 2, 4, 6, 8, 10, or 12.The invention further provides a purified or isolated polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of: (a) a full-length at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, or 12; (b) a full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, or 12 absent the N-terminal Met; (c) a mature GMG-8, GMG-9, GMG-10 or GMG-11 polypeptide of SEQ ID NOs: 6, 8, 10, or 12 lacking signal peptide; (d) a GMG-7A polypeptide of SEQ ID NO: 2 wherein said GMG-7A polypeptide is of any one integer in length between 6 amino acids and 710 amino acids (full-length) inclusive of SEQ ED NO: 2, a GMG-7B polypeptide of SEQ ID NO: 4 wherein said GMG-7B polypeptide is of any one integer in length between 6 amino acids and 471 amino acids (full-length) inclusive of SEQ ID NO: 4, a GMG-8 polypeptide of SEQ ID NO: 6 wherein said GMG-8 polypeptide is of any one integer in length between 6 amino acids and 201 amino acids (full-length) inclusive of SEQ ID NO: 6, a GMG-9 polypeptide of SEQ ID NO: 8 wherein said GMG-8 polypeptide is of any one integer in length between 6 amino acids and 446 amino acids (full-length) inclusive of SEQ ID NO: 8; or a GMG-10 polypeptide of SEQ ID NO: 10 wherein said GMG-10 polypeptide is of any one integer in length between 6 amino acids and 296 amino acids (full-length) inclusive of SEQ ID NO: 10; or a GMG-11 polypeptide of SEQ ID NO: 12 wherein said GMG-11 polypeptide is of any one integer in length between 6 amino acids and 205 amino acids (full-length) inclusive of SEQ ID NO: 12; (e) the epitope-bearing fragments of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of SEQ ED NO: 2, 4, 6, 8, 10, or 12; (f) the allelic variant polypeptides of any of the polypeptides of (a)-(e).", "The invention further provides for fragments of the polypeptides of (a)-(f) above, such as those having biological activity or comprising biologically functional domain(s).", "In other highly preferred embodiments, GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides comprise, consist essentially of, or consist of, a purified, isolated, or a recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment comprised of all or part of the C-terminal globular C1q homology domain.", "Preferably, said GMG-7A polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 2-710 of SEQ ID NO: 2.Preferably, said GMG-8 polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 28-201 of SEQ ID NO: 6.Preferably, said GMG-10 polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 24296 of SEQ ID NO: 10.Preferably, said GMG-11 polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 33-205 of SEQ ID NO: 12.In preferred embodiments, said GMG-7A polypeptide fragments comprised of all or part of the C-terminal globular C1q homology domain and having activity are selected from amino acids 2-710, 263-710, 264-710, 553-710, 554-710, 561-710, 568-710, 575-710, 580-710, 581-710, or 276-710 of SEQ ID NO: 2.In other preferred embodiments, said GMG-8 polypeptide fragments having activity are selected from amino acids 28-201, 40-201, 54-201, 66-201, 70-210 or 71-201 of SEQ ID NO: 6.In other preferred embodiments, said GMG-10 polypeptide fragments having activity are selected from amino acids 8-296, 9-296, 24-296, 32-296, 39-296, 52-296, 65-296, 71-296, 74-296, 77-296, 78-296, 81-296, 84-296, 90-296, 92-296, 102-296, 110-296, 111-296, 120-296, 132-296, 135-296, 148-296, 154-296 or 155-296 of SEQ ID NO: 10.In other preferred embodiments, said GMG-11 polypeptide fragments having activity are selected from amino acids 33-205, 53-205, 54-205, 59-205, 71-205 or 72-205 of SEQ ID NO: 12.In more preferred embodiments, said GMG-7A polypeptide fragments comprised of all or part of the C-terminal globular C1q homology domain and having activity are selected from amino acids 2-710, 553-710, 554-710, 568-710, or 575-710 of SEQ ID NO: 2.In other more preferred embodiments, said GMG-8 polypeptide fragments having activity are selected from amino acids 28-201, 54-201 or 66-201 of SEQ ID NO: 6.In other more preferred embodiments, said GMG-10 polypeptide fragments having activity are selected from amino acids 8-296, 9-296, 24-296, 52-296, 71-296, 74-296, 81-296, 84-296, 110-296, 111-296, 120-296, 132-296, 135-296, 148-296, 154-296 or 155-296 of SEQ ID NO: 10.In other more preferred embodiments, said GMG-11 polypeptide fragments having activity are selected from amino acids 33-205, 53-205, 54-205 or 59-205 of SEQ ID NO: 12.Alternatively, said GMG-7A, GMG-8, GMG-10, or GMG-11 polypeptide fragment comprises, consists essentially of, or consists of, an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 568-710 of SEQ ID NO: 2, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 54-201 of SEQ ID NO: 6, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 135-296 of SEQ ED NO: 10, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 53-205 of SEQ ID NO: 12.In a further preferred embodiment, GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are able to lower circulating (either in blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.", "Further preferred polypeptides of the invention demonstrating free fatty acid level lowering activity, glucose level lowering activity, and/or triglyceride level lowering activity, have an activity that is the same or greater than full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides at the same molar concentration, have the same or greater than transient activity and/or have a sustained activity.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that cause C2C12 cells differentiated in the presence of said polypeptides to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase leptin uptake in a liver cell line (preferably BPRCL mouse liver cells (ATCC CRL-2217)).", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that significantly reduce the postprandial increase in plasma free fatty acids due to a high fat meal.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that significantly reduce or eliminate ketone body production as the result of a high fat meal.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase glucose uptake in skeletal muscle cells.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase glucose uptake in adipose cells.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase glucose uptake in neuronal cells.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase glucose uptake in red blood cells.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase glucose uptake in the brain.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase insulin sensitivity.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that inhibit the progression from impaired glucose tolerance to insulin resistance.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that form multimers (e.g., heteromultimers or homomultimers) in vitro and/or in vivo.", "Preferred multimers are homodimers or homotrimers.", "Other preferred multimers are homomultimers comprising at least 4, 6, 8, 9, 10 or 12 GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide subunits.", "Other preferred mulimers are hetero multimers comprising a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of the invention.", "Further preferred embodiments include heterologous polypeptides comprising one of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention.", "In a second aspect, the invention features purified, isolated, or recombinant polynucleotides encoding said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides described in the first aspect, or the complement thereof.", "A further preferred embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, or fragments thereof.", "In one aspect the sequence is derived from a human, mouse or other mammal.", "In a preferred aspect, the genomic sequence includes isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 2000, 5000, 10000 or 50000 nucleotides of any one of the polynucleotide sequences described in SEQ ID NOs: 1, 3, 5, 7, 9, or 11 or the complements thereof, wherein said contiguous span comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding nucleotide sequence of the C-terminal globular C1q homology domains of SEQ ID NOs: 1, 3, 5, 7, 9, or 11.In further embodiments the polynucleotides are DNA, RNA, DNA/RNA hybrids, single-stranded, and double-stranded.", "In a third aspect, the invention features a recombinant vector comprising, consisting essentially of, or consisting of, said polynucleotide described in the second aspect.", "In a fourth aspect, the invention features a recombinant cell comprising, consisting essentially of, or consisting of, said recombinant vector described in the third aspect.", "A further embodiment includes a host cell recombinant for a polynucleotide of the invention.", "In a fifth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides described in the first aspect and, alternatively, a pharmaceutical or physiologically acceptable diluent.", "In a sixth aspect, the invention features a method of reducing body mass comprising providing or administering to individuals in need of reducing body mass said pharmaceutical or physiologically acceptable composition described in the fifth aspect.", "In preferred embodiments, the identification of said individuals in need of reducing body mass to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 single nucleotide polymorphisms (SNPs) or measuring metabolic polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of plasma, urine, and saliva.", "Preferably, a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment of the present invention is administered to an individual with at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in blood, serum or plasma levels of full-length any one or all of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-11, or GMG-11 polypeptides or the naturally proteolytically cleaved GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 fragments as compared to healthy, non-obese patients.", "In a seventh aspect, the invention features a method of preventing or treating an metabolic-related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect.", "In preferred embodiments, the identification of said individuals in need of such treatment to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 single nucleotide polymorphisms (SNPs) or measuring GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, wherein said biological response is selected from the group consisting of: (a) modulating circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids, wherein said modulating is preferably lowering; (b) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose, wherein said modulating is preferably lowering; (c) modulating circulating (either blood, serum or plasma) levels (concentration) of triglycerides, wherein said modulating is preferably lowering; (d) stimulating muscle lipid or free fatty acid oxidation; (c) modulating leptin uptake in the liver or liver cells, wherein said modulating is preferably increasing; (e) modulating the postprandial increase in plasma free fatty acids due to a high fat meal, wherein said modulating is preferably reducing; (f) modulating ketone body production as the result of a high fat meal, wherein said modulating is preferably reducing or eliminating; (g) increasing cell or tissue sensitivity to insulin, particularly muscle, adipose, liver or brain; and (h) inhibiting the progression from impaired glucose tolerance to insulin resistance; and further wherein said biological response is significantly greater than, or at least 10%, 20%, 30%, 35%, 40%, 50% 75% 100% or 500% greater than, the biological response caused or induced by insulin alone at the same molar concentration.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy.", "In a further preferred embodiment, the present invention may be used in complementary therapy of NIDDM patients to improve their weight or glucose control in combination with an insulin secretagogue (preferably oral form) or an insulin sensitising (preferably oral form) agent.", "Preferably, the oral insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.", "Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.", "The present invention further provides a method of improving the body weight or glucose control of NIDDM patients alone, without an insulin secretagogue or an insulin sensitising agent.", "In a further preferred embodiment, the present invention may be used in complementary therapy of IDDM patients to improve their weight or glucose control in combination with an insulin secretagogue (preferably oral form) or an insulin sensitising (preferably oral form) agent.", "Preferably, the insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.", "Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.", "The present invention further provides a method of improving the body weight or glucose control of IDDM patients alone, without an insulin secretagogue or an insulin sensitising agent.", "In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with the insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent).", "Accordingly, the present invention further provides for a composition of pharmaceutical or physiologically acceptable composition and an insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of body weight or glucose control in NIDDM or IDDM patients.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin sensitiser.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) without insulin therapy.", "In an eighth aspect, the invention features a method of making the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides described in the first aspect, wherein said method is selected from the group consisting of: proteolytic cleavage, recombinant methodology and artificial synthesis.", "In a ninth aspect, the present invention provides a method of making a recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment or a full length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, the method comprising providing a transgenic, non-human mammal whose milk contains said recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment or full length protein, and purifying said recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment or said full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide from the milk of said non-human mammal.", "In one embodiment, said non-human mammal is a cow, goat, sheep, rabbit, or mouse.", "In another embodiment, the method comprises purifying a recombinant full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide from said milk, and further comprises cleaving said protein in vitro to obtain a desired GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment.", "In a tenth aspect, the invention features a purified or isolated antibody capable of specifically binding to a polypeptide of the present invention.", "In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 6 consecutive amino acids, at least 8 consecutive amino acids, or at least 10 consecutive amino acids of the sequence of one of the polypeptide sequences described in SEQ ID NO: 2, 4, 6, 8, 10, or 12.In an eleventh aspect, the invention features a use of the polypeptide described in the first aspect for treatment of metabolic-related diseases and disorders and/or reducing or increasing body mass.", "Preferably, said metabolic-related diseases and disorders are selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In a twelth aspect, the invention provides a polypeptide of the first aspect of the invention, or a composition of the fifth aspect of the invention, for use in a method of treatment of the human or animal body.", "In a thirteenth aspect, the invention features methods of reducing body weight for cosmetic purposes comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect, or a polypeptide described in the first aspect.", "Preferably, for said reducing body weight said individual has a BMI of at least 20 and no more than 25.Alternatively, for said increasing body weight said individual preferably has a BMI of at least 15 and no more than 20.In a fourteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect for reducing body mass and/or for treatment or prevention of metabolic-related diseases or disorders.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 single nucleotide polymorphisms (SNPs) or measuring GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "In a fifteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect for reducing body weight for cosmetic reasons.", "In a sixteenth aspect, the invention features methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect, or a polypeptide described in the first aspect.", "In a seventeenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with normal glucose tolerance (NGT) who are obese or who have fasting hyperinsulinemia, or who have both.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with gestational diabetes.", "Gestational diabetes refers to the development of diabetes in an individual during pregnancy, usually during the second or third trimester of pregnancy.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with impaired fasting glucose (IFG).", "Impaired fasting glucose (IFG) is that condition in which fasting plasma glucose levels in an individual are elevated but not diagnostic of overt diabetes, i.e.", "plasma glucose levels of less than 126 mg/dl and less than or equal to 110 mg/dl.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating and preventing impaired glucose tolerance (IGT) in an individual.", "By providing therapeutics and methods for reducing or preventing IGT, i.e., for normalizing insulin resistance, the progression to NIDDM can be delayed or prevented.", "Furthermore, by providing therapeutics and methods for reducing or preventing insulin resistance, the invention provides methods for reducing and/or preventing the appearance of Insulin-Resistance Syndrome.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating a subject having polycystic ovary syndrome (PCOS).", "PCOS is among the most common disorders of premenopausal women, affecting 5-10% of this population.", "Insulin-sensitizing agents, e.g., troglitazone, have been shown to be effective in PCOS and that, in particular, the defects in insulin action, insulin secretion, ovarian steroidogenosis and fibrinolysis are improved (Ehrman et al.", "(1997) J Clin Invest 100:1230), such as in insulin-resistant humans.", "Accordingly, the invention provides methods for reducing insulin resistance, normalizing blood glucose thus treating and/or preventing PCOS.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating a subject having insulin resistance.", "In further preferred embodiments, a subject having insulin resistance is treated according to the methods of the invention to reduce or cure the insulin-resistance.", "As insulin resistance is also often associated with infections and cancer, prevention or reducing insulin resistance according to the methods of the invention may prevent or reduce infections and cancer.", "In further preferred embodiment, the methods of the invention are used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.", "Thus, any of the above-described tests or other tests known in the art can be used to determine that a subject is insulin-resistant, which patient can then be treated according to the methods of the invention to reduce or cure the insulin-resistance.", "Alternatively, the methods of the invention can also be used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.", "In an eighteenth aspect, the invention features a method of preventing or treating an metabolic-related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect.", "In preferred embodiments, the identification of said individuals in need of such treatment to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 single nucleotide polymorphisms (SNPs) or measuring GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, FIV-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably non-human, preferably a cat or a dog.", "In a nineteenth aspect, the invention features a method of using a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or polypeptide fragment to screen compounds for one or more antagonists of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or polypeptide fragment activity, wherein said activity is selected from but not restricted to lipid partitioning, lipid metabolism, and insulin-like activity.", "In preferred embodiment, said compound is selected from but is not restricted to small molecular weight organic or inorganic compound, protein, peptide, carbohydrate, or lipid.", "In a preferred aspect of the methods above and disclosed herein, the amount of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or polynucleotide administered to an individual is sufficient to bring circulating (blood, serum, or plasma) levels (concentration) of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides to their normal levels (levels in non-obese individuals).", "“Normal levels” may be specified as the total concentration of all circulating GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides (full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 proteins and fragments thereof) or the concentration of all circulating proteolytically cleaved GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides only.", "In a further preferred aspect of the methods above and disclosed herein, weight loss is due in part or in whole to a decrease in mass of either a) subcutaneous adipose tissue and/or b) visceral (omental) adipose tissue.", "Full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides and polynucleotides encoding the same may be specifically substituted for a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment or polynucleotide encoding the same in any embodiment of the present invention." ], [ "FIELD OF THE INVENTION The present invention relates to the field of metabolic research, in particular the discovery of compounds effective for reducing body mass and useful for treating metabolic-related diseases and disorders.", "The metabolic-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to, hyperlipidemia, atherosclerosis, diabetes, and hypertension.", "BACKGROUND OF THE INVENTION The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.", "Obesity is a public health problem that is serious, widespread, and increasing.", "In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634).", "Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643).", "Even modest weight loss ameliorates these associated conditions.", "While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Harsh et al (2000) Nature 404:644-651).", "In agreement with these studies, is the fact that an increasing number of metabolic-related genes are being identified.", "Some of the more extensively studied genes include those encoding leptin (ob) and its receptor (db), pro-opiomelanocortin (Poinc), melanocortin-4-receptor (Mc4r), agouti protein (Ay), carboxypeptidase E (at), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loop-helix 2 (Nhlh2), prohormone convertase 1 (PCSK1), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651).", "SUMMARY OF THE INVENTION The instant invention is based on Genset Metabolic Genes-7, 8, 9, 10, and 11 (GMG-7), (GMG-8; previously referred to as Cluster 9), (GMG-9; previously referred to as Cluster 10), (GMG-10; previously referred to as Cluster 17(a)) and (GMG-11; previously referred to as Cluster 19) of human origin.", "GMG-7A (previously referred to as Cluster 6 (1900)) and GMG-7B (previously referred to as Cluster 6 (d)) correspond to splice variants of GMG-7.GMG-7A, GMG-8, GMG-10, and GMG-11 are comprised of a C-terminal globular C1q homology domain.", "GMG-7B lacks the C-terminal globular C1q homology domain present in GMG-7A.", "GMG-9 is comprised near its N-terminus of a truncated globular C1q homology domain.", "Analysis of the C-terminal globular C1q homology domain of APM1 has shown it to structurally resemble TNFα.", "By analogy to APM1, biological activity can also reside in polypeptide fragments exclusive of all or part of the globular C1q homology domain.", "Results from Northern blot analysis indicate expression of GMG-7 in heart and brain, expression of GMG-8 in brain, and expression of GMG-11 in brain and pancreas.", "The invention includes polypeptides encoded by GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11, which include both the full-length polypeptide and fragments thereof, preferably but not intended to be limited to said polypeptide fragments comprising all or part of the C-terminal globular C1q homology domain.", "The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptide fragments of the invention have in vitro and in vivo biological activity as described herein, including utility for weight reduction, prevention of weight gain and control of blood glucose levels in humans and other mammals.", "More specifically, the biological activities of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides, including fragments, include reduction of elevated free fatty acid levels caused by administration of epinephrine, i.v.", "injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, reduction in glucose levels, modulation of energy expenditure, resistance to insulin and weight reduction in mammals consuming a high fat/high sucrose diet.", "Polypeptide fragments of the invention have activities overlapping but distinct from that of the full-length polypeptide.", "Thus, the invention is drawn to GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides, polynucleotides encoding said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides, vectors comprising said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polynucleotides, and cells recombinant for said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polynucleotides, as well as to pharmaceutical and physiologically acceptable compositions comprising said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides and methods of administering said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 pharmaceutical and physiologically acceptable compositions in order to reduce body weight or to treat metabolic-related diseases and disorders.", "Assays for identifying agonists and antagonists of metabolic-related activity are also part of the invention.", "In a first aspect, the invention features purified, isolated, or recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides or GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptide fragments that have lipid partitioning, lipid metabolism, and insulin-like activities.", "Preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptide fragments have activity, wherein said activity is also selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity.", "In preferred embodiments, said polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids and not more than 710 consecutive amino acids of SEQ ID NO: 2; at least 6 and not more than 471 consecutive amino acids of SEQ ID NO: 4; at least 6 consecutive amino acids and not more than 201 consecutive amino acids of SEQ ID NO: 6; at least 6 and not more than 446 consecutive amino acids of SEQ ID NO: 8; at least 6 consecutive amino acids and not more than 296 consecutive amino acids of SEQ ID NO: 10; or at least 6 and not more than 205 consecutive amino acids of SEQ ID NO: 12.In preferred embodiments, GMG-7A polypeptide fragments having activity are selected from amino acids 2-710, 1-262, 263-710, 1-263, 264-710 1-552, 553-710, 554-710, 561-710, 568-710, 575-710, 580-710, 581-710, 1-275 or 276-710 of SEQ ID NO: 2.In other preferred embodiments, GMG-7B polypeptide fragments having activity are selected from amino acids 2-471, 1-442, 443-471, 1-443, 444-471, 1-262, 263-471, 1-263, 264-471, 1-275 or 276-471 of SEQ ID NO: 4.In other preferred embodiments, GMG-8 polypeptide fragments having activity are selected from amino acids 28-201, 40-201, 54-201, 66-201, 70-201, or 71-201 of SEQ ID NO: 6.In other preferred embodiments, GMG-9 polypeptide fragments having activity are selected from amino acids 25-446, 228-356, 228-360, 228-431, 228-446, 231-356, 231-360, 231-431, 231-446, 233-356, 233-360, 233-431, 233-446, 236-356, 236-360, 236-431, 236-446, 240-356, 240-360, 240-431, 240-446, 241-356, 241-360, 241-431, 241-446, 242-356, 242-360, 242-431, or 242-446 of SEQ ID NO: 8.In other preferred embodiments, GMG-10 polypeptide fragments having activity are selected from amino acids 8-296, 9-296, 24-296, 32-296, 39-296, 52-296, 65-296, 71-296, 74-296, 77-296, 78-296, 81-296, 84-296, 90-296, 92-296, 102-296, 110-296, 111-296, 120-296, 132-296, 135-296, 148-296, 154-296 or 155-296 of SEQ ID NO: 10.In other preferred embodiments, GMG-11 polypeptide fragments having activity are selected from amino acids 33-205, 53-205, 54-205, 59-205, 71-205 or 72-205 of SEQ ID NO: 12.In more preferred embodiments, GMG-7A polypeptide fragments having activity are selected from amino acids 2-710, 1-262, 1-263, 553-710, 554-710, 568-710, 575-710 or 1-275 of SEQ ID NO: 2.In other more preferred embodiments, GMG-7B polypeptide fragments having activity are selected from amino acids 2-471, 14-42, 1-443, 1-262, 1-263, or 1-275 of SEQ ID NO: 4.In other more preferred embodiments, GMG-8 polypeptide fragments having activity are selected from amino acids 28-201, 54-201 or 66-201 of SEQ ID NO: 6.In other more preferred embodiments, GMG-9 polypeptide fragments having activity are selected from amino acids 254-46, 228-356, 228-360, 228-431, 228-446, 231-356, 231-360, 231-431, 231-446, 241-356, 241-360, 241-431 or 241-446 of SEQ ID NO: 8.In other more preferred embodiments, GMG-10 polypeptide fragments having activity are selected from amino acids 8-296, 9-296, 24-296, 52-296, 71-296, 74-296, 81-296, 84-296, 110-296, 111-296, 120-296, 132-296, 135-296, 148-296, 154-296 or 155-296 of SEQ ID NO: 10.In other more preferred embodiments, GMG-11 polypeptide fragments having activity are selected from amino acids 33-205, 53-205, 54-205 or 59-205 of SEQ ID NO: 12.In further preferred embodiments, said polypeptide fragment comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of the polypeptide sequences identified in SEQ ED NO: 2, 4, 6, 8, 10, or 12.The invention further provides a purified or isolated polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of: (a) a full-length at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, or 12; (b) a full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, or 12 absent the N-terminal Met; (c) a mature GMG-8, GMG-9, GMG-10 or GMG-11 polypeptide of SEQ ID NOs: 6, 8, 10, or 12 lacking signal peptide; (d) a GMG-7A polypeptide of SEQ ID NO: 2 wherein said GMG-7A polypeptide is of any one integer in length between 6 amino acids and 710 amino acids (full-length) inclusive of SEQ ED NO: 2, a GMG-7B polypeptide of SEQ ID NO: 4 wherein said GMG-7B polypeptide is of any one integer in length between 6 amino acids and 471 amino acids (full-length) inclusive of SEQ ID NO: 4, a GMG-8 polypeptide of SEQ ID NO: 6 wherein said GMG-8 polypeptide is of any one integer in length between 6 amino acids and 201 amino acids (full-length) inclusive of SEQ ID NO: 6, a GMG-9 polypeptide of SEQ ID NO: 8 wherein said GMG-8 polypeptide is of any one integer in length between 6 amino acids and 446 amino acids (full-length) inclusive of SEQ ID NO: 8; or a GMG-10 polypeptide of SEQ ID NO: 10 wherein said GMG-10 polypeptide is of any one integer in length between 6 amino acids and 296 amino acids (full-length) inclusive of SEQ ID NO: 10; or a GMG-11 polypeptide of SEQ ID NO: 12 wherein said GMG-11 polypeptide is of any one integer in length between 6 amino acids and 205 amino acids (full-length) inclusive of SEQ ID NO: 12; (e) the epitope-bearing fragments of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of SEQ ED NO: 2, 4, 6, 8, 10, or 12; (f) the allelic variant polypeptides of any of the polypeptides of (a)-(e).", "The invention further provides for fragments of the polypeptides of (a)-(f) above, such as those having biological activity or comprising biologically functional domain(s).", "In other highly preferred embodiments, GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides comprise, consist essentially of, or consist of, a purified, isolated, or a recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment comprised of all or part of the C-terminal globular C1q homology domain.", "Preferably, said GMG-7A polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 2-710 of SEQ ID NO: 2.Preferably, said GMG-8 polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 28-201 of SEQ ID NO: 6.Preferably, said GMG-10 polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 24296 of SEQ ID NO: 10.Preferably, said GMG-11 polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 33-205 of SEQ ID NO: 12.In preferred embodiments, said GMG-7A polypeptide fragments comprised of all or part of the C-terminal globular C1q homology domain and having activity are selected from amino acids 2-710, 263-710, 264-710, 553-710, 554-710, 561-710, 568-710, 575-710, 580-710, 581-710, or 276-710 of SEQ ID NO: 2.In other preferred embodiments, said GMG-8 polypeptide fragments having activity are selected from amino acids 28-201, 40-201, 54-201, 66-201, 70-210 or 71-201 of SEQ ID NO: 6.In other preferred embodiments, said GMG-10 polypeptide fragments having activity are selected from amino acids 8-296, 9-296, 24-296, 32-296, 39-296, 52-296, 65-296, 71-296, 74-296, 77-296, 78-296, 81-296, 84-296, 90-296, 92-296, 102-296, 110-296, 111-296, 120-296, 132-296, 135-296, 148-296, 154-296 or 155-296 of SEQ ID NO: 10.In other preferred embodiments, said GMG-11 polypeptide fragments having activity are selected from amino acids 33-205, 53-205, 54-205, 59-205, 71-205 or 72-205 of SEQ ID NO: 12.In more preferred embodiments, said GMG-7A polypeptide fragments comprised of all or part of the C-terminal globular C1q homology domain and having activity are selected from amino acids 2-710, 553-710, 554-710, 568-710, or 575-710 of SEQ ID NO: 2.In other more preferred embodiments, said GMG-8 polypeptide fragments having activity are selected from amino acids 28-201, 54-201 or 66-201 of SEQ ID NO: 6.In other more preferred embodiments, said GMG-10 polypeptide fragments having activity are selected from amino acids 8-296, 9-296, 24-296, 52-296, 71-296, 74-296, 81-296, 84-296, 110-296, 111-296, 120-296, 132-296, 135-296, 148-296, 154-296 or 155-296 of SEQ ID NO: 10.In other more preferred embodiments, said GMG-11 polypeptide fragments having activity are selected from amino acids 33-205, 53-205, 54-205 or 59-205 of SEQ ID NO: 12.Alternatively, said GMG-7A, GMG-8, GMG-10, or GMG-11 polypeptide fragment comprises, consists essentially of, or consists of, an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 568-710 of SEQ ID NO: 2, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 54-201 of SEQ ID NO: 6, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 135-296 of SEQ ED NO: 10, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 53-205 of SEQ ID NO: 12.In a further preferred embodiment, GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are able to lower circulating (either in blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.", "Further preferred polypeptides of the invention demonstrating free fatty acid level lowering activity, glucose level lowering activity, and/or triglyceride level lowering activity, have an activity that is the same or greater than full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides at the same molar concentration, have the same or greater than transient activity and/or have a sustained activity.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that cause C2C12 cells differentiated in the presence of said polypeptides to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase leptin uptake in a liver cell line (preferably BPRCL mouse liver cells (ATCC CRL-2217)).", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that significantly reduce the postprandial increase in plasma free fatty acids due to a high fat meal.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that significantly reduce or eliminate ketone body production as the result of a high fat meal.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase glucose uptake in skeletal muscle cells.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase glucose uptake in adipose cells.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase glucose uptake in neuronal cells.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase glucose uptake in red blood cells.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase glucose uptake in the brain.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that increase insulin sensitivity.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that inhibit the progression from impaired glucose tolerance to insulin resistance.", "Further preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides are those that form multimers (e.g., heteromultimers or homomultimers) in vitro and/or in vivo.", "Preferred multimers are homodimers or homotrimers.", "Other preferred multimers are homomultimers comprising at least 4, 6, 8, 9, 10 or 12 GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide subunits.", "Other preferred mulimers are hetero multimers comprising a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of the invention.", "Further preferred embodiments include heterologous polypeptides comprising one of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention.", "In a second aspect, the invention features purified, isolated, or recombinant polynucleotides encoding said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides described in the first aspect, or the complement thereof.", "A further preferred embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, or fragments thereof.", "In one aspect the sequence is derived from a human, mouse or other mammal.", "In a preferred aspect, the genomic sequence includes isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 2000, 5000, 10000 or 50000 nucleotides of any one of the polynucleotide sequences described in SEQ ID NOs: 1, 3, 5, 7, 9, or 11 or the complements thereof, wherein said contiguous span comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding nucleotide sequence of the C-terminal globular C1q homology domains of SEQ ID NOs: 1, 3, 5, 7, 9, or 11.In further embodiments the polynucleotides are DNA, RNA, DNA/RNA hybrids, single-stranded, and double-stranded.", "In a third aspect, the invention features a recombinant vector comprising, consisting essentially of, or consisting of, said polynucleotide described in the second aspect.", "In a fourth aspect, the invention features a recombinant cell comprising, consisting essentially of, or consisting of, said recombinant vector described in the third aspect.", "A further embodiment includes a host cell recombinant for a polynucleotide of the invention.", "In a fifth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides described in the first aspect and, alternatively, a pharmaceutical or physiologically acceptable diluent.", "In a sixth aspect, the invention features a method of reducing body mass comprising providing or administering to individuals in need of reducing body mass said pharmaceutical or physiologically acceptable composition described in the fifth aspect.", "In preferred embodiments, the identification of said individuals in need of reducing body mass to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 single nucleotide polymorphisms (SNPs) or measuring metabolic polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of plasma, urine, and saliva.", "Preferably, a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment of the present invention is administered to an individual with at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in blood, serum or plasma levels of full-length any one or all of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-11, or GMG-11 polypeptides or the naturally proteolytically cleaved GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 fragments as compared to healthy, non-obese patients.", "In a seventh aspect, the invention features a method of preventing or treating an metabolic-related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect.", "In preferred embodiments, the identification of said individuals in need of such treatment to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 single nucleotide polymorphisms (SNPs) or measuring GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, wherein said biological response is selected from the group consisting of: (a) modulating circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids, wherein said modulating is preferably lowering; (b) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose, wherein said modulating is preferably lowering; (c) modulating circulating (either blood, serum or plasma) levels (concentration) of triglycerides, wherein said modulating is preferably lowering; (d) stimulating muscle lipid or free fatty acid oxidation; (c) modulating leptin uptake in the liver or liver cells, wherein said modulating is preferably increasing; (e) modulating the postprandial increase in plasma free fatty acids due to a high fat meal, wherein said modulating is preferably reducing; (f) modulating ketone body production as the result of a high fat meal, wherein said modulating is preferably reducing or eliminating; (g) increasing cell or tissue sensitivity to insulin, particularly muscle, adipose, liver or brain; and (h) inhibiting the progression from impaired glucose tolerance to insulin resistance; and further wherein said biological response is significantly greater than, or at least 10%, 20%, 30%, 35%, 40%, 50% 75% 100% or 500% greater than, the biological response caused or induced by insulin alone at the same molar concentration.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy.", "In a further preferred embodiment, the present invention may be used in complementary therapy of NIDDM patients to improve their weight or glucose control in combination with an insulin secretagogue (preferably oral form) or an insulin sensitising (preferably oral form) agent.", "Preferably, the oral insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.", "Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.", "The present invention further provides a method of improving the body weight or glucose control of NIDDM patients alone, without an insulin secretagogue or an insulin sensitising agent.", "In a further preferred embodiment, the present invention may be used in complementary therapy of IDDM patients to improve their weight or glucose control in combination with an insulin secretagogue (preferably oral form) or an insulin sensitising (preferably oral form) agent.", "Preferably, the insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.", "Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.", "The present invention further provides a method of improving the body weight or glucose control of IDDM patients alone, without an insulin secretagogue or an insulin sensitising agent.", "In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with the insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent).", "Accordingly, the present invention further provides for a composition of pharmaceutical or physiologically acceptable composition and an insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of body weight or glucose control in NIDDM or IDDM patients.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin sensitiser.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) without insulin therapy.", "In an eighth aspect, the invention features a method of making the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, and GMG-11 polypeptides described in the first aspect, wherein said method is selected from the group consisting of: proteolytic cleavage, recombinant methodology and artificial synthesis.", "In a ninth aspect, the present invention provides a method of making a recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment or a full length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, the method comprising providing a transgenic, non-human mammal whose milk contains said recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment or full length protein, and purifying said recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment or said full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide from the milk of said non-human mammal.", "In one embodiment, said non-human mammal is a cow, goat, sheep, rabbit, or mouse.", "In another embodiment, the method comprises purifying a recombinant full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide from said milk, and further comprises cleaving said protein in vitro to obtain a desired GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment.", "In a tenth aspect, the invention features a purified or isolated antibody capable of specifically binding to a polypeptide of the present invention.", "In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 6 consecutive amino acids, at least 8 consecutive amino acids, or at least 10 consecutive amino acids of the sequence of one of the polypeptide sequences described in SEQ ID NO: 2, 4, 6, 8, 10, or 12.In an eleventh aspect, the invention features a use of the polypeptide described in the first aspect for treatment of metabolic-related diseases and disorders and/or reducing or increasing body mass.", "Preferably, said metabolic-related diseases and disorders are selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In a twelth aspect, the invention provides a polypeptide of the first aspect of the invention, or a composition of the fifth aspect of the invention, for use in a method of treatment of the human or animal body.", "In a thirteenth aspect, the invention features methods of reducing body weight for cosmetic purposes comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect, or a polypeptide described in the first aspect.", "Preferably, for said reducing body weight said individual has a BMI of at least 20 and no more than 25.Alternatively, for said increasing body weight said individual preferably has a BMI of at least 15 and no more than 20.In a fourteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect for reducing body mass and/or for treatment or prevention of metabolic-related diseases or disorders.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 single nucleotide polymorphisms (SNPs) or measuring GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "In a fifteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect for reducing body weight for cosmetic reasons.", "In a sixteenth aspect, the invention features methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect, or a polypeptide described in the first aspect.", "In a seventeenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with normal glucose tolerance (NGT) who are obese or who have fasting hyperinsulinemia, or who have both.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with gestational diabetes.", "Gestational diabetes refers to the development of diabetes in an individual during pregnancy, usually during the second or third trimester of pregnancy.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with impaired fasting glucose (IFG).", "Impaired fasting glucose (IFG) is that condition in which fasting plasma glucose levels in an individual are elevated but not diagnostic of overt diabetes, i.e.", "plasma glucose levels of less than 126 mg/dl and less than or equal to 110 mg/dl.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating and preventing impaired glucose tolerance (IGT) in an individual.", "By providing therapeutics and methods for reducing or preventing IGT, i.e., for normalizing insulin resistance, the progression to NIDDM can be delayed or prevented.", "Furthermore, by providing therapeutics and methods for reducing or preventing insulin resistance, the invention provides methods for reducing and/or preventing the appearance of Insulin-Resistance Syndrome.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating a subject having polycystic ovary syndrome (PCOS).", "PCOS is among the most common disorders of premenopausal women, affecting 5-10% of this population.", "Insulin-sensitizing agents, e.g., troglitazone, have been shown to be effective in PCOS and that, in particular, the defects in insulin action, insulin secretion, ovarian steroidogenosis and fibrinolysis are improved (Ehrman et al.", "(1997) J Clin Invest 100:1230), such as in insulin-resistant humans.", "Accordingly, the invention provides methods for reducing insulin resistance, normalizing blood glucose thus treating and/or preventing PCOS.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating a subject having insulin resistance.", "In further preferred embodiments, a subject having insulin resistance is treated according to the methods of the invention to reduce or cure the insulin-resistance.", "As insulin resistance is also often associated with infections and cancer, prevention or reducing insulin resistance according to the methods of the invention may prevent or reduce infections and cancer.", "In further preferred embodiment, the methods of the invention are used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.", "Thus, any of the above-described tests or other tests known in the art can be used to determine that a subject is insulin-resistant, which patient can then be treated according to the methods of the invention to reduce or cure the insulin-resistance.", "Alternatively, the methods of the invention can also be used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.", "In an eighteenth aspect, the invention features a method of preventing or treating an metabolic-related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect.", "In preferred embodiments, the identification of said individuals in need of such treatment to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 single nucleotide polymorphisms (SNPs) or measuring GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, FIV-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "In preferred embodiments, said individual is a mammal, preferably non-human, preferably a cat or a dog.", "In a nineteenth aspect, the invention features a method of using a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or polypeptide fragment to screen compounds for one or more antagonists of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or polypeptide fragment activity, wherein said activity is selected from but not restricted to lipid partitioning, lipid metabolism, and insulin-like activity.", "In preferred embodiment, said compound is selected from but is not restricted to small molecular weight organic or inorganic compound, protein, peptide, carbohydrate, or lipid.", "In a preferred aspect of the methods above and disclosed herein, the amount of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or polynucleotide administered to an individual is sufficient to bring circulating (blood, serum, or plasma) levels (concentration) of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides to their normal levels (levels in non-obese individuals).", "“Normal levels” may be specified as the total concentration of all circulating GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides (full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 proteins and fragments thereof) or the concentration of all circulating proteolytically cleaved GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides only.", "In a further preferred aspect of the methods above and disclosed herein, weight loss is due in part or in whole to a decrease in mass of either a) subcutaneous adipose tissue and/or b) visceral (omental) adipose tissue.", "Full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides and polynucleotides encoding the same may be specifically substituted for a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment or polynucleotide encoding the same in any embodiment of the present invention.", "DETAILED DESCRIPTION OF THE SEQUENCE LISTING SEQ ID NO:1 represents the cDNA sequence of GMG-7A.", "SEQ ID NO:2 represents the amino acid sequence encoded by the cDNA of SEQ ID NO:1.SEQ ID NO:3 represents the cDNA sequence of GMG-7B.", "SEQ ID NO:4 represents the amino acid sequence encoded by the cDNA of SEQ ID NO:3.SEQ ID NO:5 represents the polynucleotide sequence of GMG-8.SEQ ID NO:6 represents the amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO:5.SEQ ID NO:7 represents the cDNA sequence of GMG-9.SEQ ID NO:8 represents the amino acid sequence encoded by the cDNA of SEQ ID NO:7.SEQ ID NO:9 represents the cDNA sequence of GMG-10.SEQ ID NO:10 represents the amino acid sequence encoded by the cDNA of SEQ ID NO:9.SEQ ID NO:11 represents the cDNA sequence of GMG-11.SEQ ID NO:12 represents the amino acid sequence encoded by the cDNA of SEQ ID NO:11.DETAILED DESCRIPTION OF THE INVENTION Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein.", "As used interchangeably herein, the terms “oligonucleotides”, and “polynucleotides” and nucleic acid include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.", "The terms encompass “modified nucleotides” which comprise at least one modification, including by way of example and not limitation: (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar.", "For examples of analogous linking groups, purines, pyrimidines, and sugars see for example PCT publication No.", "WO 95/04064.The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.", "The terms polynucleotide construct, recombinant polynucleotide and recombinant polypeptide are used herein consistently with their use in the art.", "The terms “upstream” and “downstream” are also used herein consistently with their use in the art.", "The terms “base paired” and “Watson & Crick base paired” are used interchangeably herein and consistently with their use in the art.", "Similarly, the terms “complementary”, “complement thereof”, “complement”, “complementary polynucleotide”, “complementary nucleic acid” and “complementary nucleotide sequence” are used interchangeably herein and consistently with their use in the art.", "The term “purified” is used herein to describe a polynucleotide or polynucleotide vector of the invention that has been separated from other compounds including, but not limited to, other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide).", "Purified can also refer to the separation of covalently closed polynucleotides from linear polynucleotides, or vice versa, for example.", "A polynucleotide is substantially pure when at least about 50%, 60%, 75%, or 90% of a sample contains a single polynucleotide sequence.", "In some cases this involves a determination between conformations (linear versus covalently closed).", "A substantially pure polynucleotide typically comprises about 50, 60, 70, 80, 90, 95, 99% weight/weight of a nucleic acid sample.", "Polynucleotide purity or homogeneity may be indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel.", "For certain purposes, higher resolution can be achieved by using HPLC or other means well known in the art.", "Similarly, the term “purified” is used herein to describe a polypeptide of the invention that has been separated from other compounds including, but not limited to, nucleic acids, lipids, carbohydrates and other proteins.", "In some preferred embodiments, a polypeptide is substantially pure when at least about 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the polypeptide molecules of a sample have a single amino acid sequence.", "In some preferred embodiments, a substantially pure polypeptide typically comprises about 50%, 60%, 70%, 80%, 90% 95%, 96%, 97%, 98%, 99% or 99.5% weight/weight of a protein sample.", "Polypeptide purity or homogeneity is indicated by a number of methods well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel.", "For certain purposes, higher resolution can be achieved by using HPLC or other methods well known in the art.", "Further, as used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition.", "Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.", "Alternatively, purification may be expressed as “at least” a percent purity relative to heterologous polynucleotides (DNA, RNA or both) or polypeptides.", "As a preferred embodiment, the polynucleotides or polypeptides of the present invention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 98%, 99%, 99.5% or 100% pure relative to heterologous polynucleotides or polypeptides.", "As a further preferred embodiment the polynucleotides or polypeptides have an “at least” purity ranging from any number, to the thousandth position, between 90% and 100% (e.g., at least 99.995% pure) relative to heterologous polynucleotides or polypeptides.", "Additionally, purity of the polynucleotides or polypeptides may be expressed as a percentage (as described above) relative to all materials and compounds other than the carrier solution.", "Each number, to the thousandth position, may be claimed as individual species of purity.", "The term “isolated” requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).", "For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.", "Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.", "Specifically excluded from the definition of “isolated” are: naturally occurring chromosomes (e.g., chromosome spreads), artificial chromosome libraries, genomic libraries, and cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies.", "Also specifically excluded are the above libraries wherein a 5′ EST makes up less than 5% (or alternatively 1%, 2%, 3%, 4%, 10%, 25%, 50%, 75%, or 90%, 95%, or 99%) of the number of nucleic acid inserts in the vector molecules.", "Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including said whole cell preparations which are mechanically sheared or enzymatically digested).", "Further specifically excluded are the above whole cell preparations as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis (including blot transfers of the same) wherein the polynucleotide of the invention have not been further separated from the heterologous polynucleotides in the electrophoresis medium (e.g., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).", "The term “primer” denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.", "A primer serves as an initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase, or reverse transcriptase.", "The term “probe” denotes a defined nucleic acid segment that can be used to identify a specific polynucleotide sequence present in a sample, said nucleic acid segment comprising a nucleotide sequence complementary to the specific polynucleotide sequence to be identified.", "The term “polypeptide” refers to a polymer of amino acids without regard to the length of the polymer.", "Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.", "This term also does not specify or exclude post-expression modifications of polypeptides.", "For example, polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.", "Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.", "), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.", "Without being limited by theory, the compounds/polypeptides of the invention are capable of modulating the partitioning of dietary lipids between the liver and peripheral tissues, and thus of treating “diseases involving the partitioning of dietary lipids between the liver and peripheral tissues.” The term “peripheral tissues” is meant to include muscle and adipose tissue.", "In preferred embodiments, the compounds/polypeptides of the invention partition the dietary lipids toward the muscle.", "In alternative preferred embodiments, the dietary lipids are partitioned toward the adipose tissue.", "In other preferred embodiments, the dietary lipids are partitioned toward the liver.", "In yet other preferred embodiments, the compounds/polypeptides of the invention increase or decrease the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle.", "Dietary lipids include, but are not limited to triglycerides and free fatty acids.", "Preferred diseases believed to involve the partitioning of dietary lipids include obesity and obesity-related diseases and disorders such as obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Non-Insulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).", "Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "The term “heterologous”, when used herein, is intended to designate any polypeptide or polynucleotide other than a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or a polynucleotide encoding a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of the present invention.", "The terms “comprising”, “consisting of” and “consisting essentially of” are defined according to their standard meaning.", "A defined meaning set forth in the M.P.E.P.", "controls over a defined meaning in the art and a defined meaning set forth in controlling Federal Circuit case law controls over a meaning set forth in the M.P.E.P.", "With this in mind, the terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.", "The term “host cell recombinant for” a particular polynucleotide of the present invention, means a host cell that has been altered by the hands of man to contain said polynucleotide in a way not naturally found in said cell.", "For example, said host cell may be transiently or stably transfected or transduced with said polynucleotide of the present invention.", "The term “obesity” as used herein is defined in the WHO classifications of weight (Kopelman (2000) Nature 404:635643).", "Underweight is less than 18.5 (thin); Healthy is 18.5-24.9 (normal); grade 1 overweight is 25.0-29.9 (overweight); grade 2 overweight is 30.0-39.0 (obesity); grade 3 overweight is greater than or equal to 40.0 BMI.", "BMI is body mass index (morbid obesity) and is kg/m2.Waist circumference can also be used to indicate a risk of metabolic complications where in men a circumference of greater than or equal to 94 cm indicates an increased risk and greater than or equal to 102 cm indicates a substantially increased risk.", "Similarly for women, greater than or equal to 88 cm indicates an increased risk, and greater than or equal to 88 cm indicates a substantially increased risk.", "The waist circumference is measured in cm at midpoint between lower border of ribs and upper border of the pelvis.", "Other measures of obesity include, but are not limited to, skinfold thickness which is a measurement in cm of skinfold thickness using calipers, and bioimpedance, which is based on the principle that lean mass conducts current better than fat mass because it is primarily an electrolyte solution; measurement of resistance to a weak current (impedance) applied across extremities provides an estimate of body fat using an empirically derived equation.", "The term “diabetes” as used herein is intended to encompass the usual diagnosis of diabetes made from any of the methods included, but not limited to, the following list: symptoms of diabetes (eg.", "polyuria, polydipsia, polyphagia) plus casual plasma glucose levels of greater than or equal to 200 mg/dl, wherein casual plasma glucose is defined any time of the day regardless of the timing of meal or drink consumption; 8 hour fasting plasma glucose levels of less than or equal to 126 mg/dl; and plasma glucose levels of greater than or equal to 200 mg/dl 2 hours following oral administration of 75 g anhydrous glucose dissolved in water.", "The term “impaired glucose tolerance (IGT)” as used herein is intended to indicate that condition associated with insulin-resistance that is intermediate between frank, NIDDM and normal glucose tolerance (NGT).", "A high percentage of the IGT population is known to progress to NIDDM relative to persons with normal glucose tolerance (Sad et al., New Engl J Med 1988; 319:1500-6).", "Thus, by providing therapeutics and methods for reducing or preventing IGT, i.e., for normalizing insulin resistance, the progression to NIDDM can be delayed or prevented.", "IGT is diagnosed by a procedure wherein an affected person's postprandial glucose response is determined to be abnormal as assessed by 2-hour postprandial plasma glucose levels.", "In this test, a measured amount of glucose is given to the patient and blood glucose levels measured regular intervals, usually every half hour for the first two hours and every hour thereafter.", "In a “normal” or non-IGT individual, glucose levels rise during the first two hours to a level less than 140 mg/dl and then drop rapidly.", "In an IGT individual, the blood glucose levels are higher and the drop-off level is at a slower rate.", "The term “Insulin-Resistance Syndrome” as used herein is intended to encompass the cluster of abnormalities resulting from an attempt to compensate for insulin resistance that sets in motion a series of events that play an important role in the development of both hypertension and coronary artery disease (CAD), such as premature atherosclerotic vascular disease.", "Increased plasma triglyceride and decreased HDL-cholesterol concentrations, conditions that are known to be associated with CAD, have also been reported to be associated with insulin resistance.", "Thus, by providing therapeutics and methods for reducing or preventing insulin resistance, the invention provides methods for reducing and/or preventing the appearance of insulin-resistance syndrome.", "The term “polycystic ovary syndrome (PCOS)” as used herein is intended to designate that etiologically unassigned disorder of premenopausal women, affecting 5-10% of this population, characterized by hyperandrogenism, chronic anovulation, defects in insulin action, insulin secretion, ovarian steroidogenesis and fibrinolysis.", "Women with PCOS frequently are insulin resistant and at increased risk to develop glucose intolerance or NIDDM in the third and fourth decades of life (Dunaif et al.", "(1996) J Clin Endocrinol Metab 81:3299).", "Hyperandrogenism also is a feature of a variety of diverse insulin-resistant states, from the type A syndrome, through leprechaunism and lipoatrophic diabetes, to the type B syndrome, when these conditions occur in premenopausal women.", "It has been suggested that hyperinsulinemia per se causes hyperandrogenism.", "Insulin-sensitizing agents, e.g., troglitazone, have been shown to be effective in PCOS and that, in particular, the defects in insulin action, insulin secretion, ovarian steroidogenosis and fibrinolysis are improved (Ehrman et al.", "(1997) J Clin Invest 100:1230), such as in insulin-resistant humans.", "The term “insulin resistance” as used herein is intended to encompass the usual diagnosis of insulin resistance made by any of a number of methods, including but not restricted to: the intravenous glucose tolerance test or measurement of the fasting insulin level.", "It is well known that there is an excellent correlation between the height of the fasting insulin level and the degree of insulin resistance.", "Therefore, one could use elevated fasting insulin levels as a surrogate marker for insulin resistance for the purpose of identifying which normal glucose tolerance (NGT) individuals have insulin resistance.", "Another way to do this is to follow the approach as disclosed in The New England Journal of Medicine, No.", "3, pp.", "1188 (1995), i.e.", "to select obesity as an initial criterion for entry into the treatment group.", "Some obese subjects have impaired glucose tolerance (IGT) while others have normal glucose tolerance (NGT).", "Since essentially all obese subjects are insulin resistant, i.e.", "even the NGT obese subjects are insulin resistant and have fasting hyperinsulinemia.", "Therefore, the target of the treatment according to the present invention can be defined as NGT individuals who are obese or who have fasting hyperinsulinemia, or who have both.", "A diagnosis of insulin resistance can also be made using the euglycemic glucose clamp test.", "This test involves the simultaneous administration of a constant insulin infusion and a variable rate glucose infusion.", "During the test, which lasts 3-4 hours, the plasma glucose concentration is kept constant at euglycemic levels by measuring the glucose level every 5-10 minutes and then adjusting the variable rate glucose infusion to keep the plasma glucose level unchanged.", "Under these circumstances, the rate of glucose entry into the bloodstream is equal to the overall rate of glucose disposal in the body.", "The difference between the rate of glucose disposal in the basal state (no insulin infusion) and the insulin infused state, represents insulin mediated glucose uptake.", "In normal individuals, insulin causes brisk and large increase in overall body glucose disposal, whereas in NIDDM subjects, this effect of insulin is greatly blunted, and is only 20-30% of normal.", "In insulin resistant subjects with either IGT or NGT, the rate of insulin stimulated glucose disposal is about half way between normal and NIDDM.", "For example, at a steady state plasma insulin concentration of about 100 uU/ml (a physiologic level) the glucose disposal rate in normal subjects is about 7 mg/kg/min.", "In NIDDM subjects, it is about 2.5 mg/.kg/min., and in patients with IGT (or insulin resistant subjects with NGT) it is about 4-5 mg/kg/min.", "This is a highly reproducible and precise test, and can distinguish patients within these categories.", "It is also known that as subjects become more insulin resistant, the fasting insulin level rises.", "There is an excellent positive correlation between the height of the fasting insulin level and the magnitude of the insulin resistance as measured by euglycemic glucose clamp tests and, therefore, this provides the rationale for using fasting insulin levels as a surrogate measure of insulin resistance.", "The term “agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the partitioning of dietary lipids between the liver and the peripheral tissues as previously described.", "Preferably, the agent increases or decreases the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle.", "Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an obesity-related disease or disorder such as obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Non-Insulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).", "Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "The terms “response to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.", "The terms “side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.", "“Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.", "The term “GMG-7A-, GMG-7B-, GMG-8-, GMG-9-, GMG-10-, or GMG-11-related diseases and disorders” as used herein refers to any disease or disorder comprising an aberrant functioning of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11, or which could be treated or prevented by modulating GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 levels or activity.", "“Aberrant functioning of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11” includes, but is not limited to, aberrant levels of expression of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 (either increased or decreased, but preferably decreased), aberrant activity of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 (either increased or decreased), and aberrant interactions with ligands or binding partners (either increased or decreased).", "By “aberrant” is meant a change from the type, or level of activity seen in normal cells, tissues, or patients, or seen previously in the cell, tissue, or patient prior to the onset of the illness.", "In preferred embodiments, these GMG-7A-, GMG-7B-, GMG-8-, GMG-9-, GMG-10-, or GMG-11-related diseases and disorders include obesity and the metabolic-related diseases and disorders described previously.", "The term “cosmetic treatments” is meant to include treatments with compounds or polypeptides of the invention that increase or decrease the body mass of an individual where the individual is not clinically obese or clinically thin.", "Thus, these individuals have a body mass index (BMI) below the cut-off for clinical obesity (e.g.", "below 25 kg/m2) and above the cut-off for clinical thinness (e.g.", "above 18.5 kg/m2).", "In addition, these individuals are preferably healthy (e.g.", "do not have an metabolic-related disease or disorder of the invention).", "“Cosmetic treatments” are also meant to encompass, in some circumstances, more localized increases in adipose tissue, for example, gains or losses specifically around the waist or hips, or around the hips and thighs, for example.", "These localized gains or losses of adipose tissue can be identified by increases or decreases in waist or hip size, for example.", "The term “preventing” as used herein refers to administering a compound prior to the onset of clinical symptoms of a disease or condition so as to prevent a physical manifestation of aberrations associated with obesity or GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11.The term “treating” as used herein refers to administering a compound after the onset of clinical symptoms.", "The term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g.", "physician, nurse, nurse practitioner, etc in the case of humans; veterinarian in the case of animals, including non-human mammals) that an individual or animal requires or will benefit from treatment.", "This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that include the knowledge that the individual or animal is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention.", "The term “perceives a need for treatment” refers to a sub-clinical determination that an individual desires to reduce weight for cosmetic reasons as discussed under “cosmetic treatment” above.", "The term “perceives a need for treatment” in other embodiments can refer to the decision that an owner of an animal makes for cosmetic treatment of the animal.", "The term “individual” or “patient” as used herein refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.", "The term may specify male or female or both, or exclude male or female.", "The term “non-human animal” refers to any non-human vertebrate, including birds and more usually mammals, preferably primates, animals such as swine, goats, sheep, donkeys, horses, cats, dogs, rabbits or rodents, more preferably rats or mice.", "Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term “non-human”.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GM 10, or GMG-11 polypeptides are able to significantly reduce the postprandial response of plasma free fatty acids, glucose, and triglycerides in mammals fed a high fat/sucrose meal, while not affecting levels of leptin, insulin or glucagon.", "In addition, GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides modulate muscle free fatty acid oxidation in vitro and ex vivo, preferably increase oxidation.", "Further, GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-O, or GMG-11 polypeptides of the invention modulate weight gain in mammals that are fed a high fat/sucrose diet.", "The instant invention encompasses the use of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides in the partitioning of free fatty acid (FFA) and as an important new tool to control energy homeostasis.", "Of the tissues that can significantly remove lipids from circulation and cause FFA oxidation, muscle is believed to be quantitatively the most important.", "PREFERRED EMBODIMENTS OF THE INVENTION I. GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Polypeptides of the Invention GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides have been identified that have measurable activity in vitro and in vivo.", "These activities include, but are not limited to, modulation, preferably reduction, of the postprandial response of plasma free fatty acids, glucose, and triglycerides in mammals fed a high fat/sucrose meal (Example 6), change, preferably an increase, in muscle free fatty acid oxidation in vitro and ex vivo (Example 10), and sustained weight loss in mammals on a high fat/sucrose diet.", "Other assays for GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide activity in vitro and in vivo are also provided (Examples 2, 5, 7, 9, 11, for example), and equivalent assays can be designed by those with ordinary skill in the art.", "The term “GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides” includes both the “full-length” polypeptide and fragments of the “full-length” GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides (although each of the above species may be particularly specified).", "By “intact” or “full-length” GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides as used herein is meant the full-length polypeptide sequence of any GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, from the N-terminal methionine to the C-terminal stop codon.", "Examples of intact or full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are found in the sequence listing.", "The term “metabolic-related activity” as used herein refers to at least one, and preferably all, of the activities described herein for GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides.", "Assays for the determination of these activities are provided herein (e.g.", "Examples 2, 5-7, 9-11), and equivalent assays can be designed by those with ordinary skill in the art.", "Optionally, “metabolic-related activity” can be selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity, or an activity within one of these categories.", "By “lipid partitioning” activity is meant the ability to effect the location of dietary lipids among the major tissue groups including, adipose tissue, liver, and muscle.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention play a role in the partitioning of lipids to the muscle, liver or adipose tissue.", "By “lipid metabolism” activity is meant the ability to influence the metabolism of lipids.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention have the ability to affect the level of free fatty acids in the plasma as well as to modulate, preferably increase, the metabolism of lipids in the muscle through free fatty acid oxidation experiments (Examples 2, 6, 8, 9, 10) and to transiently affect the levels of triglycerides in the plasma and the muscle (Examples 6, 8, 11).", "By “insulin-like” activity is meant the ability of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides to modulate the levels of glucose in the plasma.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides do not significantly impact insulin levels but do impact glucose levels similarly to the effects of insulin (Examples 7 & 8).", "These effects may vary in the presence of the intact (full-length) GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides or may be significantly greater in the presence of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragments compared with the full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides.", "The term “significantly greater” as used herein refers to a comparison of the activity of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide in a metabolic-related assay compared with untreated cells in the same assay.", "By “significantly” as used herein is meant statistically significant as it is typically determined by those with ordinary skill in the art.", "For example, data are typically calculated as a mean±SEM, and a p-value ≦0.05 is considered statistically significant.", "Statistical analysis is typically done using either the unpaired Student's t test or the paired Student's t test, as appropriate in each study.", "Examples of a significant change in activity as a result of the presence of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of the invention compared to untreated cells include an increase or a decrease in a given parameter of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.", "One or more, but not necessarily all, of the measurable parameters will change significantly in the presence of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide as compared to untreated cells.", "Representative “metabolic-related assays” are provided in the Examples.", "These assays include, but are not limited to, methods of measuring the postprandial response, methods of measuring free fatty acid oxidation, and methods of measuring weight modulation.", "In preferred embodiments, the post-prandial response is measured in non-human animals, preferably mice.", "In preferred embodiments changes in dietary lipids are measured, preferably free fatty acids and/or triglycerides.", "In other embodiments, other physiologic parameters are measured including, but not limited to, levels of glucose, insulin, and leptin.", "In other preferred embodiments, free fatty acid oxidation is measured in cells in vitro or ex vivo, preferably in muscle cells or tissue of non-human animals, preferably mice.", "In yet other preferred embodiments weight modulation is measured in human or non-human animals, preferably rodents (rats or mice), primates, canines, felines or procines.", "on a high fat/sucrose diet.", "Optionally, “metabolic-related activity” includes other activities not specifically identified herein.", "In general, “measurable parameters” relating to obesity and the field of metabolic research can be selected from the group consisting of free fatty acid levels, free fatty acid oxidation, triglyceride levels, glucose levels, insulin levels, leptin levels, food intake, weight, leptin and lipoprotein binding, uptake and degradation and lipolysis stimulated receptor (LSR) expression.", "In these metabolic-related assays, preferred GMG-7A, GMG-7B, GM 8, GMG-9, GMG-10, or GMG-11 polypeptides would cause a significant change in at least one of the measurable parameters selected from the group consisting of post-prandial lipemia, free fatty acid levels, triglyceride levels, glucose levels, free fatty acid oxidation, and weight.", "Alternatively, preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides would have a significant change in at least one of the measurable parameters selected from the group consisting of an increase in LSR activity, an increase in leptin activity and an increase in lipoprotein activity.", "By “LSR” activity is meant expression of LSR on the surface of the cell, or in a particular conformation, as well as its ability to bind, uptake, and degrade leptin and lipoprotein.", "By “leptin” activity is meant its binding, uptake and degradation by LSR, as well as its transport across a blood brain barrier, and potentially these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.", "Similarly, by “lipoprotein” activity is meant its binding, uptake and degradation by LSR, as well as these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.", "The invention is drawn, inter alia, to isolated, purified or recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention are useful for reducing or, using antagonists of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides, increasing body weight either as a cosmetic treatment or for treatment or prevention of metabolic-related diseases and disorders.", "GMG-7A, GMG-7B, GMG-8, GM 9, GMG-10, or GMG-11 polypeptides are also useful inter alia in screening assays for agonists or antagonists of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide activity; for raising GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide-specific antibodies; and in diagnostic assays.", "When used for cosmetic treatments, or for the treatment or prevention of metabolic-related diseases, disorders or conditions, one or more GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 or GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragments can be provided to a subject.", "Thus, various fragments of the full-length protein can be combined into a “cocktail” for use in the various treatment regimens.", "The full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide is comprised of distinct regions including: 1.an N-terminal putative signal peptide sequence about from amino acids 1-27 of SEQ ID NO: 6, about from amino acids 1-24 of SEQ ID NO: 8, about from amino acids 1-23 of SEQ ID NO: 10, or about from amino acids 1-32 of SEQ ID NO: 12; 2.a collagen-like region about from amino acids 45-146 of SEQ ID NO: 10; 3.a C-terminal globular C1q homology domain about from amino acids 579-710 of SEQ ID NO: 2, about from amino acids 69-201 of SEQ ID NO: 6, about from amino acids 149-285 of SEQ ID NO: 10, or about from amino acids 70-205 of SEQ ID NO: 12; and 4.an N-terminally disposed truncated globular C1q homology domain about from amino acids 45-110 of SEQ ID NO: 8.GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention include variants, fragments, analogs and derivatives of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides described above, including modified GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides.", "The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the present invention are preferably provided in an isolated form, and may be partially or substantially purified.", "A recombinantly produced version of any one of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides can be substantially purified by the one-step method described by Smith et al.", "((1988) Gene 67:31-40) or by the methods described herein or known in the art.", "Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies directed against the polypeptides of the invention by methods known in the art of protein purification.", "Preparations of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention involving a partial purification of or selection for the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are also specifically contemplated.", "These crude preparations are envisioned to be the result of the concentration of cells expressing GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides with perhaps a few additional purification steps, but prior to complete purification of the fragment.", "The cells expressing GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are present in a pellet, they are lysed, or the crude polypeptide is lyophilized, for example.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragments can be any integer in length from at least 6 consecutive amino acids to one amino acid less than a full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide.", "Thus, for the polypeptide of SEQ ED NO: 10, a GMG-10 polypeptide fragment can be any integer of consecutive amino acids from 6 to 295, for example.", "The term “integer” is used herein in its mathematical sense and thus representative integers include, but are not limited to: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294 and 295.Each GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment as described above can be further specified in terms of its N-terminal and C-terminal positions.", "For example, every combination of a N-terminal and C-terminal position that fragments of from 6 contiguous amino acids to one amino acid less than the full-length polypeptide of SEQ ID NO: 10 could occupy, on any given intact and contiguous full-length polypeptide sequence of SEQ ID NO: 10 are included in the present invention.", "Thus, a 6 consecutive amino acid fragment could occupy positions selected from the group consisting of 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20, 16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30, 26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40, 36-41, 37-42, 38-43, 39-44, 40-45, 41-46, 42-47, 43-48, 44-49, 45-50, 46-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-60, 56-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70, 66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, 75-80, 76-81, 77-82, 78-83, 79-84, 80-85, 81-86, 82-87, 83-88, 84-89, 85-90, 86-91, 87-92, 88-93, 89-94, 90-95, 91-96, 92-97, 93-98, 94-99, 95-100, 96-101, 97-102, 98-103, 99-104, 100-105, 101-106, 102-107, 103-108, 104-109, 105-110, 106-111, 107-112, 108-113, 109-114, 110-115, 111-116, 112-117, 113-118, 114-119, 115-120, 116-121, 117-122, 118-123, 119-124, 120-125, 121-126, 122-127, 123-128, 124-129, 125-130, 126-131, 127-132, 128-133, 129-134, 130-135, 131-136, 132-137, 133-138, 134-139, 135-140, 136-141, 137-142, 138-143, 139-144, 140-145, 141-146, 142-147, 143-148, 144-149, 145-150, 146-151, 147-152, 148-153, 149-154, 150-155, 151-156, 152-157, 153-158, 154-159, 155-160, 156-161, 157-162, 158-163, 159-164, 160-165, 161-166, 162-167, 163-168, 164-169, 165-170, 166-171, 167-172, 168-173, 169-174, 170-175, 171-176, 172-177, 173-178, 174-179, 175-180, 176-181, 177-182, 178-183, 179-184, 180-185, 181-186, 182-187, 183-188, 184-189, 185-190, 186-191, 187-192, 188-193, 189-194, 190-195, 191-196, 192-197, 193-198, 194-199, 195-200, 196-201, 197-202, 198-203, 199-204, 200-205, 201-206, 202-207, 203-208, 204-209, 205-210, 206-211, 207-212, 208-213, 209-214, 210-215, 211-216, 212-217, 213-218, 214-219, 215-220, 216-221, 217-222, 218-223, 219-224, 220-225, 221-226, 222-227, 223-228, 224-229, 225-230, 226-231, 227-232, 228-233, 229-234, 230-235, 231-236, 232-237, 233-238, 234-239, 235-240, 236-241, 237-242, 238-243, 240-245, 241-246, 242-247, 243-248, 244-249, 245-250, 246-251, 247-252, 248-253, 249-254, 250-255, 251-256, 252-257, 253-258, 254-259, 255-260, 256-261, 257-262, 258-263, 259-264, 260-265, 261-266, 262-267, 263-268, 264-269, 265-270, 266-271, 267-272, 268-273, 269-274, 270-275, 271-276, 272-277, 273-278, 274-279, 275-280, 276-281, 277-282, 278-283, 279-284 280-285, 281-286, 282-287, 283-288, 284-289, 285-290, 286-291, 287-292, 288-293, 289-294, 290-295 and 291-296 of a 296 consecutive amino acid fragment.", "A 290 consecutive amino acid fragment could occupy positions selected from the group consisting of 1-290, 2-291, 3-292, 4-293, 5-294, 6-295 and 7-296.Similarly, the positions occupied by all the other fragments of sizes between 6 amino acids and 295 amino acids in SEQ ID NO: 10, by all the other fragments of sizes between 6 amino acids and 709 amino acids in SEQ ID NO: 2, by all the other fragments of sizes between 6 amino acids and 470 amino acids in SEQ ID NO: 4, by all the other fragments of sizes between 6 amino acids and 200 amino acids in SEQ ID NO: 6, by all the other fragments of sizes between 6 amino acids and 445 amino acids in SEQ ID NO: 8, and by all the other fragments of sizes between 6 amino acids and 204 amino acids in SEQ ID NO: 12 are included in the present invention and can also be immediately envisaged based on these two examples and therefore, are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "Furthermore, the positions occupied by fragments of 6 to 295 consecutive amino acids in SEQ ID NO: 10, by fragments of 6 to 709 consecutive amino acids in SEQ ID NO: 2, by fragments of 6 to 470 amino acids in SEQ ID NO: 4, by fragments of 6 to 200 amino acids in SEQ ID NO: 6, by fragments of 6 to 445 amino acids in SEQ ID NO: 8, and by fragments of 6 to 204 amino acids in SEQ ID NO: 12 are included in the present invention and can also be immediately envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "In addition, the positions occupied by fragments of 6 consecutive amino acids to 1 amino acid less than any other full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide can also be envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the present invention may alternatively be described by the formula “n to c” (inclusive); where “n” equals the N-terminal most amino acid position (as defined by the sequence listing) and “c” equals the C-terminal most amino acid position (as defined by the sequence listing) of the polypeptide; and further where “n” equals an integer between 1 and the number of amino acids of the full-length polypeptide sequence of the present invention minus 6; and where “c” equals an integer between 7 and the number of amino acids of the full-length polypeptide sequence; and where “n” is an integer smaller then “c” by at least 6.Therefore, for the sequences provided in SEQ ID NO: 10, “n” is any integer selected from the list consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 or 290 and “c” is any integer selected from the group consisting of: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 291, 293, 294, 295 or 296.Every combination of “n” and “c” positions are included as specific embodiments of the invention.", "Moreover, the formula “n” to “c” may be modified as “‘n1-n2” to “c1-c2’”, wherein “n1-n2” and “c1-c2” represent positional ranges selected from any two integers above which represent amino acid positions of the sequence listing.", "Alternative formulas include “‘n1-n2” to “c’” and “‘n” to “c1-c2’”.", "In a preferred embodiment, GMG-10 polypeptide fragments of the invention may be described by the n1=24, n2=155, and c=296 of SEQ ID NO: 10; GMG-8 polypeptide fragments of the invention by the formula n1=28, n2=71, and c=201 of SEQ ID NO: 6; and GMG-11 polypeptide fragments of the invention by the formula n1=33, n2=72, and c=205 of SEQ ID NO: 12.Furthermore, the positions occupied by polypeptides of 6 to 710 consecutive amino acids on SEQ ID NO: 2, by polypeptides of 6 to 471 consecutive amino acids on SEQ ID NO: 4, by polypeptides of 6 to 201 consecutive amino acids on SEQ ID NO: 6, by polypeptides of 6 to 446 consecutive amino acids on SEQ ID NO: 8, or by polypeptides of 6 to 205 consecutive amino acids on SEQ ID NO: 12, are included in the present invention and can also be immediately envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "In addition, the positions occupied by fragments of 6 consecutive amino acids to 1 amino acid less than full-length GMG-7A, GMG-7B, GMG-8, GMG 9, or GMG-11 polypeptide can also be envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "These specific embodiments, and other polypeptide and polynucleotide fragment embodiments described herein may be modified as being “at least”, “equal to”, “equal to or less than”, “less than”, “at least ______ but not greater than ______” or “from ______ to ______” a specified size or specified N-terminal and/or C-terminal positions.", "It is noted that all ranges used to describe any embodiment of the present invention are inclusive unless specifically set forth otherwise.", "The present invention also provides for the exclusion of any individual fragment specified by N-terminal and C-terminal positions or of any fragment specified by size in amino acid residues as described above.", "In addition, any number of fragments specified by N-terminal and C-terminal positions or by size in amino acid residues as described above may be excluded as individual species.", "Further, any number of fragments specified by N-terminal and C-terminal positions or by size in amino acid residues as described above may make up a polypeptide fragment in any combination and may optionally include non-GMG-7A, -GMG-7B, -GMG-8, -GMG-9, -GMG-10, or -GMG-11 polypeptide sequences as well.", "In preferred embodiments, said GMG-7A polypeptide fragments having activity are selected from amino acids acids 2-710, 1-262, 263-710, 1-263, 264-710 1-552, 553-710, 554-710, 561-710, 568-710, 575-710, 580-710, 581-710, 1-275 or 276-710 of SEQ ID NO: 2.In other preferred embodiments, said GMG-7B polypeptide fragments having activity are selected from amino acids 2-471, 1-442, 443-471, 1-443, 444-471, 1-262, 263-471, 1-263, 264-471, 1-275 or 276-471 of SEQ ID NO: 4.In other preferred embodiments, said GMG-8 polypeptide fragments having activity are selected from amino acids 28-201, 40-201, 54-201, 66-201, 70-201, or 71-201 of SEQ ID NO: 6.In other preferred embodiments, said GMG-9 polypeptide fragments having activity are selected from amino acids 25-446, 228-356, 228-360, 228-431, 228-446, 231-356, 231-360, 231-431, 231-446, 233-356, 233-360, 233-431, 233-446, 236-356, 236-360, 236-431, 236-446, 240-356, 240-360, 240-431, 240-446, 241-356, 241-360, 241-431, 241-446, 242-356, 242-360, 242-431, or 242-446 of SEQ ID NO: 8.In other preferred embodiments, GMG-10 polypeptide fragments having activity are selected from amino acids 8-296, 9-296, 24-296, 32-296, 39-296, 52-296, 65-296, 71-296, 74-296, 77-296, 78-296, 81-296, 84-296, 90-296, 92-296, 102-296, 110-296, 111-296, 120-296, 132-296, 135-296, 148-296, 154-296 or 155-296 of SEQ ID NO: 10.In other preferred embodiments, GMG-11 polypeptide fragments having activity are selected from amino acids 33-205, 53-205, 54-205, 59-205, 71-205 or 72-205 of SEQ ID NO: 12.In more preferred embodiments, said GMG-7A polypeptide fragments having activity are selected from amino acids 2-710, 1-262, 1-263, 553-710, 554-710, 568-710, 575-710 or 1-275 of SEQ ID NO: 2.In other more preferred embodiments, said GMG-7B polypeptide fragments having activity are selected from amino acids 2-471, 1-442, 1-443, 1-262, 1-263, or 1-275 of SEQ ID NO: 4.In other more preferred embodiments, said GMG-8 polypeptide fragments having activity are selected from amino acids 28-201, 54-201 or 66-201 of SEQ ID NO: 6.In other more preferred embodiments, said GMG-9 polypeptide fragments having activity are selected from amino acids 25-446, 228-356, 228-360, 228-431, 228-446, 231-356, 231-360, 231-431, 231-446, 241-356, 241-360, 241-431 or 241-446 of SEQ ID NO: 8.In other more preferred embodiments, GMG-10 polypeptide fragments having activity are selected from amino acids 8-296, 9-296, 24-296, 52-296, 71-296, 74-296, 81-296, 84-296, 110-296, 111-296, 120-296, 132-296, 135-296, 148-296, 154-296 or 155-296 of SEQ ID NO: 10.In other more preferred embodiments, GMG-11 polypeptide fragments having activity are selected from amino acids 33-205, 53-205, 54-205 or 59-205 of SEQ ID NO: 12.In yet other preferred embodiment, the invention features a GMG-10 polypeptide fragment comprising at least 115, but not more than 175 contiguous amino acids of the SEQ ID NO: 10, wherein no more than 24 of said at least 115 and no more than 175 contiguous amino acids are present in the collagen-like region of GMG-10.Preferably, the GMG-10 polypeptide fragment comprises at least 125, but not more than 165, or at least 140, but not more than 165 amino acids, and no more than 24 amino acids are in the collagen-like region; more preferably at least 125 but not more than 165, or at least 140 but not more than 165 amino acids, and no more than 12 amino acids are in the collagen-like region; or at least 140 and not more than 150 amino acids, and no more than 8 amino acids are present in the collagen-like region.", "Preferably, the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment is mammalian, preferably human or mouse, but most preferably human.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragments of the invention include variants, fragments, analogs and derivatives of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragments described above, including modified GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragments.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention include variants, fragments, analogs and derivatives of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides described above, including modified GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides.", "Variants It will be recognized by one of ordinary skill in the art that some amino acids of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide sequences of the present invention can be varied without significant effect on the structure or function of the proteins; there will be critical amino acids in the sequence that determine activity.", "Thus, the invention further includes variants of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides that have metabolic-related activity as described above.", "Such variants include GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide sequences with one or more amino acid deletions, insertions, inversions, repeats, and substitutions either from natural mutations or human manipulation selected according to general rules known in the art so as to have little effect on activity.", "Guidance concerning how to make phenotypically silent amino acid substitutions is provided below.", "There are two main approaches for studying the tolerance of an amino acid sequence to change (see, Bowie, et al.", "(1990) Science, 247, 1306-10).", "The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection.", "The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality.", "These studies have revealed that proteins are surprisingly tolerant of amino acid substitutions and indicate which amino acid changes are likely to be permissive at a certain position of the protein.", "For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.", "Other such phenotypically silent substitutions are described by Bowie et al.", "(supra) and the references cited therein.", "In the case of an amino acid substitution in the amino acid sequence of a polypeptide according to the invention, one or several amino acids can be replaced by “equivalent” amino acids.", "The expression “equivalent” amino acid is used herein to designate any amino acid that may be substituted for one of the amino acids having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.", "In particular embodiments, conservative substitutions of interest are shown in Table 1 under the heading of preferred substitutions.", "If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 1, or as further described below in reference to amino acid classes, are introduced and the products screened.", "TABLE 1 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu Substantial modifications in function or immunological identity of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.", "Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.", "Non-conservative substitutions will entail exchanging a member of one of these classes for another class.", "Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.", "The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.", "Site-directed mutagenesis [Carter et al., Nucl Acids Res, 13:4331 (1986); Zoller et al., Nucl Acids Res, 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos.", "Trans.", "R. Soc.", "London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 variant DNA.", "Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence.", "Among the preferred scanning amino acids are relatively small, neutral amino acids.", "Such amino acids include alanine, glycine, serine, and cysteine.", "Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)].", "Alanine is also typically preferred because it is the most common amino acid.", "Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W.H.", "Freeman & Co., N.Y.); Chothia, J Mol Biol, 150:1 (1976)].", "If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.", "Amino acids in the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide sequences of the invention that are essential for function can also be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham, et al.", "(1989) Science 244:1081-5).", "The latter procedure introduces single alanine mutations at every residue in the molecule.", "The resulting mutant molecules are then tested for metabolic-related activity using assays as described above.", "Of special interest are substitutions of charged amino acids with other charged or neutral amino acids that may produce proteins with highly desirable improved characteristics, such as less aggregation.", "Aggregation may not only reduce activity but also be problematic when preparing pharmaceutical or physiologically acceptable formulations, because aggregates can be immunogenic (see, e.g., Pinckard, et al., (1967) Clin Exp Immunol 2:331-340; Robbins, et al., (1987) Diabetes 36:838-41; and Cleland, et al., (1993) Crit Rev Ther Drug Carrier Syst 10:307-77).", "Thus, the fragment, derivative, analog, or homolog of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the present invention may be, for example: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code (i.e.", "may be a non-naturally occurring amino acid); or (ii) one in which one or more of the amino acid residues includes a substituent group; or (iii) one in which the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are fused with another compound, such as a compound to increase the half-life of the fragment (for example, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the above form of the fragment, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the fragment or a pro-protein sequence.", "Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.", "A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, not more than 40 conservative amino acid substitutions, not more than 30 conservative amino acid substitutions, and not more than 20 conservative amino acid substitutions.", "Also provided are polypeptides which comprise the amino acid sequence of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 fragment, having at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.", "In addition, amino acids have chirality within the body of either L or D. In some embodiments it is preferable to alter the chirality of the amino acids in the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragments of the invention in order to extend half-life within the body.", "Thus, in some embodiments, one or more of the amino acids are preferably in the L configuration.", "In other embodiments, one or more of the amino acids are preferably in the D configuration.", "Percent Identity The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 50% identical, at least 60% identical, or 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide as described above.", "By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide amino acid sequence is meant that the amino acid sequence is identical to the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide sequence except that it may include up to five amino acid alterations per each 100 amino acids of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide amino acid sequence.", "The reference sequence is the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide with a sequence corresponding to the sequences provided in SEQ ED NOs: 2, 4, 6, 8, 10, or 12.Thus, to obtain a polypeptide having an amino acid sequence at least 95% identical to a GMG-7A, GMG-713, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the sequence may be inserted, deleted, or substituted with another amino acid compared with the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide sequence.", "These alterations may occur at the amino or carboxy termini or anywhere between those terminal positions, interspersed either individually among residues in the sequence or in one or more contiguous groups within the sequence.", "As a practical matter, whether any particular polypeptide is a percentage identical to a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide can be determined conventionally using known computer programs.", "Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, (1988) Proc Natl Acad Sci USA 85:2444-8; Altschul et al., (1990) J Mol Biol 215:403-410; Thompson et al., (1994) Nucleic Acids Res 22(2):4673-4680; Higgins et al, (1996) Meth Enzymol 266:383-402; Altschul et al, (1997) Nucleic Acids Res 25:3389-3402; Altschul et al., (1993) Nature Genetics 3:266-272).", "In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”), which is well known in the art (See, e.g., Karlin and Altschul (1990) Proc Natl Acad Sci USA 87:2264-8; Altschul et al., 1990, 1993, 1997, all supra).", "In particular, five specific BLAST programs are used to perform the following tasks: (1) BLASTP and BLAST3 compare an ammo acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.", "The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.", "High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.", "Preferably, the scoring matrix used is the BLOSUM62 matrix (see, Gonnet et al., (1992) Science 256:1443-5; Henikoff and Henikoff (1993) Proteins 17:49-61).", "Less preferably, the PAM or PAM250 matrices may also be used (See, e.g., Schwartz and Dayhoff, eds, (1978) Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).", "The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology.", "Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (See, e.g., Karlin and Altschul, (1990) Proc Natl Acad Sci USA 87:2264-8).", "The BLAST programs may be used with the default parameters or with modified parameters provided by the user.", "Preferably, the parameters are default parameters.", "A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al.", "(1990) Comp App Biosci 6:237-245.In a sequence alignment the query and subject sequences are both amino acid sequences.", "The result of said global sequence alignment is in percent identity.", "Preferred parameters used in a FASTDB amino acid alignment are: Matrx=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group=25 Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=247 or the length of the subject amino acid sequence, whichever is shorter.", "If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, the results, in percent identity, must be manually corrected because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity.", "For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, that are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.", "Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.", "This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.", "This final percent identity score is what is used for the purposes of the present invention.", "Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score.", "That is, only query amino acid residues outside the farthest N- and C-terminal residues of the subject sequence.", "For example, a 90 amino acid residue subject sequence is aligned with a 100-residue query sequence to determine percent identity.", "The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not match/align with the first residues at the N-terminus.", "The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program.", "If the remaining 90 residues were perfectly matched the final percent identity would be 90%.", "In another example, a 90-residue subject sequence is compared with a 100-residue query sequence.", "This time the deletions are internal so there are no residues at the N- or C-termini of the subject sequence, which are not matched/aligned with the query.", "In this case, the percent identity calculated by FASTDB is not manually corrected.", "Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected.", "No other manual corrections are made for the purposes of the present invention.", "Production Note, throughout the disclosure, wherever GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are discussed, GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 fragments, variants and derivatives are specifically intended to be included as a preferred subset of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are preferably isolated from human or mammalian tissue samples or expressed from human or mammalian genes in human or mammalian cells.", "The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention can be made using routine expression methods known in the art.", "The polynucleotide encoding the desired polypeptide is ligated into an expression vector suitable for any convenient host.", "Both eukaryotic and prokaryotic host systems are used in forming recombinant polypeptides.", "The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use.", "Purification is by any technique known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like.", "See, for example, Methods in Enzymology for a variety of methods for purifying proteins.", "In an alternative embodiment, the polypeptides of the invention are isolated from milk.", "The polypeptides can be purified as full length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides, which can then be cleaved, if appropriate, in vitro to generate a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 fragment, or, alternatively, GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 fragments themselves can be purified from the milk.", "Any of a large number of methods can be used to purify the present polypeptides from milk, including those taught in Protein Purification Applications, A Practical Approach (New Edition), Edited by Simon Roe, ABA Technology Products and Systems, Biosciences, Harwell; Clark (1998) J Mammary Gland Biol Neoplasia 3:337-50; Wilkins and Velander (1992) 49:333-8; U.S. Pat.", "Nos.", "6,140,552; 6,025,540; Hennighausen, Protein Expression and Purification, vol.", "1, pp.", "3-8 (1990); Harris et al.", "(1997) Bioseparation 7:31-7; Degener et al.", "(1998) J Chromatog 799:125-37; Wilkins (1993) J Cell Biochem.", "Suppl.", "0 (17 part A):39; the entire disclosures of each of which are herein incorporated by reference.", "In a typical embodiment, milk is centrifuged, e.g.", "at a relatively low speed, to separate the lipid fraction, and the aqueous supernatant is then centrifuged at a higher speed to separate the casein in the milk from the remaining, “whey” fraction.", "Often, biomedical proteins are found in this whey fraction, and can be isolated from this fraction using standard chromatographic or other procedures commonly used for protein purification, e.g.", "as described elsewhere in the present application.", "In one preferred embodiment, GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are purified using antibodies specific to GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides, e.g.", "using affinity chromatography.", "In addition, methods can be used to isolate particular GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 fragments, e.g.", "electrophoretic or other methods for isolating proteins of a particular size.", "The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides isolating using these methods can be naturally occurring, as GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides have been discovered to be naturally present in the milk of mammals, or can be the result of the recombinant production of the protein in the mammary glands of a non-human mammal, as described infra.", "In one such embodiment, the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 is produced as a fusion protein with a heterologous, antigenic polypeptide sequence, which antigenic sequence can be used to purify the protein, e.g., using standard immuno-affinity methodology.", "In addition, shorter protein fragments may be produced by chemical synthesis.", "Alternatively, the proteins of the invention are extracted from cells or tissues of humans or non-human animals.", "Methods for purifying proteins are known in the art, and include the use of detergents or chaotropic agents to disrupt particles followed by differential extraction and separation of the polypeptides by ion exchange chromatography, affinity chromatography, sedimentation according to density, and gel electrophoresis.", "Any GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-DNA, including those in SEQ ED NO: 1, 3, 5, 7, 9 or 11, can be used to express GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides.", "The nucleic acid encoding the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology.", "The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 cDNA insert in the expression vector may comprise the coding sequence for: the full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide (to be later modified); from 6 amino acids to any integer less than the full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide; a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 fragment; or variants and % similar polypeptides.", "The expression vector is any of the mammalian, yeast, insect or bacterial expression systems known in the art, some of which are described herein.", "Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, Mass.", "), Stratagene (La Jolla, Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego, Calif.).", "If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence can be optimized for the particular expression organism into which the expression vector is introduced, as explained by Hatfield, et al., U.S. Pat.", "No.", "5,082,767, the disclosures of which are incorporated by reference herein in their entirety.", "If the nucleic acid encoding any one of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG 10, or GMG-11 polypeptides lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques.", "Similarly, if the insert from the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide cDNA lacks a poly A signal, this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using BglI and Sail restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene).", "pXT1 contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus.", "The position of the LTRs in the construct allows efficient stable transfection.", "The vector includes the Herpes Simplex Thyridine Kinase promoter and the selectable neomycin gene.", "The nucleic acid encoding GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 can be obtained by PCR from a vector containing the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 nucleotide sequence using oligonucleotide primers complementary to the desired GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 cDNA and containing restriction endonuclease sequences for Pst I incorporated into the 5′ primer and BglII at the 5′ end of the corresponding cDNA 3′ primer, taking care to ensure that the sequence encoding the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 is positioned properly with respect to the poly A signal.", "The purified polynucleotide obtained from the resulting PCR reaction is digested with PstI, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXT1, now containing a poly A signal and digested with BglII.", "Transfection of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 expressing vector into mouse NIH 3T3 cells is one embodiment of introducing polynucleotides into host cells.", "Introduction of a polynucleotide encoding a polypeptide into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods.", "Such methods are described in many standard laboratory manuals, such as Davis et al.", "((1986) Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., Amsterdam).", "It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.", "A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.", "Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.", "Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.", "Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.", "Preferably the polypeptides of the invention are non-glycosylated.", "In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.", "Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells.", "While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.", "In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides.", "For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, see, e.g., U.S. Pat.", "No.", "5,641,670, issued Jun.", "24, 1997; International Publication No.", "WO 96/29411, published Sep. 26, 1996; International Publication No.", "WO 94/12650, published Aug. 4, 1994; Koller et al., (1989) Proc Natl Acad Sci USA 86:8932-5; Koller et al., (1989) Proc Natl Acad Sci USA 86:8927-31; and Zijistra et al.", "(1989) Nature 342:435-8; the disclosures of each of which are incorporated by reference in their entireties).", "Modifications In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (See, e.g., Creighton, 1983 Proteins.", "New York, N.Y.: W.H.", "Freeman and Company; and Hunkapiller et al., (1984) Nature 310:105-11).", "For example, a relative short fragment of the invention can be synthesized by use of a peptide synthesizer.", "Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the fragment sequence.", "Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.", "Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).", "The invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.", "Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.", "Additional post-translational modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.", "The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.", "Also provided by the invention are chemically modified derivatives of the polypeptides of the invention that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity.", "See U.S. Pat.", "No.", "4,179,337.The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.", "The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.", "The polymer may be of any molecular weight, and may be branched or unbranched.", "For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.", "Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).", "The polyethylene glycol molecules (or other chemical moieties) should be attached to the polypeptide with consideration of effects on functional or antigenic domains of the polypeptide.", "There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al.", "(1992) Exp Hematol (8):1028-35, reporting pegylation of GMG-CSF using tresyl chloride).", "For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.", "Reactive groups are those to which an activated polyethylene glycol molecule may be bound.", "The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue.", "Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules.", "Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.", "One may specifically desire proteins chemically modified at the N-terminus.", "Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.", "), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.", "The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules.", "Selective proteins chemically modified at the N-terminus may be accomplished by reductive alkylation, which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein.", "Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.", "Multimers The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers).", "Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, pharmaceutical or physiologically acceptable compositions) containing them.", "In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers.", "In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.", "Multimers encompassed by the invention may be homomers or heteromers.", "As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention (including polypeptide fragments, variants, splice variants, and fusion proteins corresponding to these polypeptide fragments as described herein).", "These homomers may contain polypeptide fragments having identical or different amino acid sequences.", "In a specific embodiment, a homomer of the invention is a multimer containing only polypeptide fragments having an identical amino acid sequence.", "In another specific embodiment, a homomer of the invention is a multimer containing polypeptide fragments having different amino acid sequences.", "In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences).", "In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.", "As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., corresponding to different proteins or polypeptides thereof) in addition to the polypeptides of the invention.", "In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.", "In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.", "Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.", "Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution.", "In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.", "In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention.", "Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone).", "In one instance, the covalent associations are cross-inking between cysteine residues located within the polypeptide sequences, which interact in the native (i.e., naturally occurring) polypeptide.", "In another instance, the covalent associations are the consequence of chemical or recombinant manipulation.", "Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.", "In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat.", "No.", "5,478,925).", "In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein).", "In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety).", "In another embodiment, two or more polypeptides of the invention are joined through peptide linkers.", "Examples include those peptide linkers described in U.S. Pat.", "No.", "5,073,627 hereby incorporated by reference).", "Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.", "Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence.", "Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found.", "Leucine zippers were originally identified in several DNA-binding proteins, and have since been found in a variety of different proteins (Landschulz et al., (1988) Genes Dev 2:786-800).", "Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.", "Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCr application WO 94/10308, hereby incorporated by reference.", "Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.", "Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity.", "Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers.", "One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al.", "FEBS Letters (1994) 344:191-5 and in U.S. patent application Ser.", "No.", "08/446,922, hereby incorporated by reference.", "Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.", "In another example, proteins of the invention are associated by interactions between Flag® & polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence.", "In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti Flag® antibody.", "The multimers of the invention may be generated using chemical techniques known in the art.", "For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "Additionally, at least 30 techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art.", "In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hyrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (See, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "II.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Polynucleotides of the Invention Preferred polynucleotides are those that encode GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention.", "The recombinant polynucleotides encoding GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides can be used in a variety of ways, including, but not limited to, expressing the polypeptides in recombinant cells for use in screening assays for antagonists and agonists of its activity as well as to facilitate its purification for use in a variety of ways including, but not limited to screening assays for agonists and antagonists of its activity, diagnostic screens, and raising antibodies, as well as treatment and/or prevention of metabolic-related diseases and disorders and/or to reduce body mass.", "The invention relates to the polynucleotides encoding GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides and variant polypeptides thereof as described herein.", "These polynucleotides may be purified, isolated, and/or recombinant.", "In all cases, the desired GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polynucleotides of the invention are those that encode GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention having metabolic-related activity as described and discussed herein.", "Fragments A polynucleotide fragment is a polynucleotide having a sequence that entirely is the same as part, but not all, of the full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or a specified GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide nucleotide sequence.", "Such fragments may be “free-standing”, i.e.", "not part of or fused to other polynucleotides, or they may be comprised within another non-GMG-7A, -GMG-7B, -GMG-8, -GMG-9, -GMG-10, or -GMG-11 (heterologous) polynucleotide of which they form a part or region.", "However, several GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polynucleotide fragments may be comprised within a single polynucleotide.", "The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polynucleotides of the invention comprise from 18 consecutive bases to the full-length polynucleotide sequences encoding the intact GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides, for example the full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide polynucleotide sequences in SEQ ID NO: 1, 3, 5, 7, 9, or 11.In one aspect of this embodiment, the polynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 611, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 740, 770, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1242, 1250, 1300, 1338, 1350, 1400, 1450, 1500, 1550, 1600, 1630, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250 or 2257 consecutive nucleotides of a polynucleotide of the present invention.", "In addition to the above preferred nucleic acid sizes, further preferred nucleic acids comprise at least 18 nucleotides, wherein “at least 18” is defined as any integer between 18 and the integer representing the 3′ most nucleotide position of the intact GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides cDNA as set forth in SEQ ID NOs: 1, 3, 5, 7, 9, or 11 or elsewhere herein.", "Further included as preferred polynucleotides of the present invention are nucleic acid fragments at least 18 nucleotides in length, as described above, that are further specified in terms of their 5′ and 3′ position.", "The 5′ and 3′ positions are represented by the position numbers set forth in the sequence listing below.", "For allelic and degenerate and other variants, position 1 is defined as the 5′ most nucleotide of the ORF, i.e., the nucleotide “A” of the start codon (ATG) with the remaining nucleotides numbered consecutively.", "Therefore, every combination of a 5′ and 3′ nucleotide position that a polynucleotide fragment, at least 18 contiguous nucleotides in length, could occupy on an intact GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polynucleotide encoding a polypeptide of the present invention is included in the invention as an individual species.", "The polynucleotide fragments specified by 5′ and 3′ positions can be immediately envisaged and are therefore not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "It is noted that the above species of polynucleotide fragments of the present invention may alternatively be described by the formula “x to y”; where “x” equals the 5′ most nucleotide position and “y” equals the 3′ most nucleotide position of the polynucleotide; and further where “x” equals an integer between 1 and the number of nucleotides of the polynucleotide sequence of the present invention minus 18, and where “y” equals an integer between 19 and the number of nucleotides of the polynucleotide sequence of the present invention minus 18 nucleotides; and where “x” is an integer less than “y” by at least 18.The present invention also provides for the exclusion of any species of polynucleotide fragments of the present invention specified by 5′ and 3′ positions or polynucleotides specified by size in nucleotides as described above.", "Any number of fragments specified by 5′ and 3′ positions or by size in nucleotides, as described above, may be excluded.", "The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polynucleotide fragments of the invention comprise from 18 consecutive bases to the full-length polynucleotide sequence encoding the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragments described in Section II of the Preferred Embodiments of the Invention.", "In one aspect of this embodiment, the polynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 611, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 740, 770, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1242, 1250, 1300, 1338, 1350, 1400, 1450, 1500, 1550, 1600, 1630, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250 or 2257 consecutive nucleotides of a polynucleotide of the present invention.", "In addition to the above preferred nucleic acid sizes, further preferred nucleic acids comprise at least 18 nucleotides, wherein “at least 18” is defined as any integer between 18 and the integer corresponding to the 3′ most nucleotide position of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 fragment cDNA herein.", "Further included as preferred polynucleotides of the present invention are nucleic acid fragments at least 18 nucleotides in length, as described above, that are further specified in terms of their 5′ and 3′ position.", "The 5′ and 3′ positions are represented by the position numbers set forth in the sequence listing below.", "For allelic and degenerate and other variants, position 1 is defined as the 5′ most nucleotide of the open reading frame (ORF), i.e., the nucleotide “A” of the start codon (ATG) with the remaining nucleotides numbered consecutively.", "Therefore, every combination of a 5′ and 3′ nucleotide position that a polynucleotide fragment invention, at least 18 contiguous nucleotides in length, could occupy on a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 fragment polynucleotide of the present invention is included in the invention as an individual species.", "The polynucleotide fragments specified by 5′ and 3′ positions can be immediately envisaged and are therefore not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "It is noted that the above species of polynucleotide fragments of the present invention may alternatively be described by the formula “x to y”; where “x” equals the 5′ most nucleotide position and “y” equals the 3′ most nucleotide position of the polynucleotide; and further where “x” equals an integer between 1 and the number of nucleotides of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polynucleotide sequences of the present invention minus 18, and where “y” equals an integer between 9 and the number of nucleotides of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polynucleotide sequences of the present invention; and where “x” is an integer smaller than “y” by at least 18.Every combination of “x” and ‘y’ positions are included as specific embodiments of the invention.", "Moreover, the formula “x” to “y” may be modified as “‘x1-x2” to “y1-y2’”, wherein “x1-x2” and “y1-y2” represent positional ranges selected from any two nucleotide positions of the sequence listing.", "Alternative formulas include “‘x1-x2” to “y’” and “‘x” to “y1-y2’”.", "These specific embodiments, and other polynucleotide fragment embodiments described herein may be modified as being “at least”, “equal to”, “equal to or less than”, “less than”, “at least ______ but not greater than ______” or “from ______ to ______”, a specified size or specified 5′ and/or 3′ positions.", "The present invention also provides for the exclusion of any species of polynucleotide fragments of the present invention specified by 5′ and 3′ positions or polynucleotides specified by size in nucleotides as described above.", "Any number of fragments specified by 5′ and 3′ positions or by size in nucleotides, as described above, may be excluded.", "Variants In other preferred embodiments, variants of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polynucleotides encoding GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are envisioned.", "Variants of polynucleotides, as the term is used herein, are polynucleotides whose sequence differs from a reference polynucleotide.", "A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.", "Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.", "Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.", "Polynucleotide variants that comprise a sequence substantially different from those described above but that, due to the degeneracy of the genetic code, still encode GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the present invention are also specifically envisioned.", "It would also be routine for one skilled in the art to generate the degenerate variants described above, for instance, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by other mammalian or bacterial host cells).", "As stated above, variant polynucleotides may occur naturally, such as a natural allelic variant, or by recombinant methods.", "By an “allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (See, e.g., B. Lewin, (1990) Genes IV, Oxford University Press, New York).", "Non-naturally occurring variants may be produced using art-known mutagenesis techniques.", "Such nucleic acid variants include those produced by nucleotide substitutions, deletions, or additions.", "The substitutions, deletions, or additions may involve one or more nucleotides.", "Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.", "Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention.", "Also preferred in this regard are conservative substitutions.", "Nucleotide changes present in a variant polynucleotide are preferably silent, which means that they do not alter the amino acids encoded by the polynucleotide.", "However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence.", "In cases where the nucleotide substitutions result in one or more amino acid changes, preferred GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides include those that retain one or more metabolic-related activity as described in Section I of the Preferred Embodiments of the Invention.", "By “retain the same activities” is meant that the activity measured using the polypeptide encoded by the variant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polynucleotide in assays is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, and not more than 101%, 102%, 103%, 104%, 105%, 110%, 115%, 120% or 125% of the activity measured using a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide described in the Examples Section herein.", "By the activity being “increased” is meant that the activity measured using the polypeptide encoded by the variant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polynucleotide in assays is at least 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 450%, or 500% of the activity measured using a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide described in the Examples Section herein.", "By the activity being “decreased” is meant that the activity measured using the polypeptide encoded by the variant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polynucleotide in assays is decreased by at least 25%, 30%, 35%, 40%, 45%, 50%, 75%, 80%, 90% or 95% of the activity measured using a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide described in the Examples Section herein Percent Identity The present invention is further directed to nucleic acid molecules having sequences at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, or 11 or fragments thereof that encode a polypeptide having metabolic-related activity as described in Section I of the Preferred Embodiments of the Invention.", "Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, or 11 or fragments thereof will encode a polypeptide having biological activity.", "In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.", "It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having biological activity.", "This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described previously in Section I of the Preferred Embodiments of the Invention.", "By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide.", "In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted, inserted, or substituted with another nucleotide.", "The query sequence may be an entire sequence or any fragment specified as described herein.", "The methods of determining and defining whether any particular nucleic acid molecule or polypeptide is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be done by using known computer programs.", "A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., ((1990) Comput Appl Biosci.", "July; 6(3):23745).", "In a sequence alignment the query and subject sequences are both DNA sequences.", "An RNA sequence can be compared by first converting U's to T's.", "The result of said global sequence alignment is in percent identity.", "Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrx=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.", "If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results.", "This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity.", "For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence.", "Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.", "This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.", "This corrected score is what is used for the purposes of the present invention.", "Only nucleotides outside the 5′ and 3′ nucleotides of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.", "For example, a 90-nucleotide subject sequence is aligned to a 100-nucleotide query sequence to determine percent identity.", "The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 nucleotides at 5′ end.", "The 10 unpaired nucleotides represent 10% of the sequence (number of nucleotides at the 5′ and 3′ ends not matched/total number of nucleotides in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program.", "If the remaining 90 nucleotides were perfectly matched the final percent identity would be 90%.", "In another example, a 90 nucleotide subject sequence is compared with a 100 nucleotide query sequence.", "This time the deletions are internal deletions so that there are no nucleotides on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query.", "In this case the percent identity calculated by FASTDB is not manually corrected.", "Once again, only nucleotides 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for.", "No other manual corrections are made for the purposes of the present invention.", "Fusions Further included in the present invention are polynucleotides encoding the polypeptides of the present invention that are fused in frame to the coding sequences for additional heterologous amino acid sequences.", "Also included in the present invention are nucleic acids encoding polypeptides of the present invention together with additional, non-coding sequences, including for example, but not limited to non-coding 5′ and 3′ sequences, vector sequence, sequences used for purification, probing, or priming.", "For example, heterologous sequences include transcribed, non-translated sequences that may play a role in transcription, and mRNA processing, for example, ribosome binding and stability of mRNA.", "The heterologous sequences may alternatively comprise additional coding sequences that provide additional functionalities.", "Thus, a nucleotide sequence encoding a polypeptide may be fused to a tag sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide.", "In certain preferred embodiments of this aspect of the invention, the tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Chatsworth, Calif.), among others, many of which are commercially available.", "For instance, hexa-histidine provides for convenient purification of the fusion protein (See, Gentz et al., (1989) Proc Natl Acad Sci USA 86:821-4).", "The “HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein (See, Wilson et al., (1984) Cell 37:767-78).", "As discussed above, other such fusion proteins include GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-O, or GMG-11 cDNA fused to Fc at the N- or C-terminus.", "III.", "Recombinant Vectors of the Invention The term “vector” is used herein to designate either a circular or a linear DNA or RNA molecule, that is either double-stranded or single-stranded, and that comprises at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism.", "The present invention relates to recombinant vectors comprising any one of the polynucleotides described herein.", "The present invention encompasses a family of recombinant vectors that comprise polynucleotides encoding GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention.", "In a first preferred embodiment, a recombinant vector of the invention is used to amplify the inserted polynucleotide in a suitable cell host, this polynucleotide being amplified every time that the recombinant vector replicates.", "The inserted polynucleotide can be one that encodes GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention.", "A second preferred embodiment of the recombinant vectors according to the invention consists of expression vectors comprising polynucleotides encoding GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-O, or GMG-11 polypeptides of the invention.", "Within certain embodiments, expression vectors are employed to express a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of the invention, preferably a modified GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 described in the present invention, which can be then purified and, for example, be used as a treatment for metabolic-related diseases, or simply to reduce body mass of individuals.", "Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources, that drive expression of the genes of interest in host cells.", "Dominant drug selection markers for establishing permanent, stable, cell clones expressing the products are generally included in the expression vectors of the invention, as they are elements that link expression of the drug selection markers to expression of the polypeptide.", "More particularly, the present invention relates to expression vectors which include nucleic acids encoding a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of the invention, or a modified GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 as described herein, or variants or fragments thereof, under the control of a regulatory sequence selected among GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides, or alternatively under the control of an exogenous regulatory sequence.", "Consequently, preferred expression vectors of the invention are selected from the group consisting of: (a) a regulatory sequence driving the expression of a coding polynucleotide operably linked thereto; and (b) a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 coding sequence of the invention, operably linked to regulatory sequences allowing its expression in a suitable cell host and/or host organism.", "Some of the elements that can be found in the vectors of the present invention are described in further detail in the following sections.", "1) General Features of the Expression Vectors of the Invention A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid, or even a linear DNA molecule which may consist of a chromosomal, non-chromosomal, semi-synthetic or synthetic DNA.", "Such a recombinant vector can comprise a transcriptional unit comprising an assembly of: (1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers.", "Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription; (2) a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in (1); and (3) appropriate transcription initiation and termination sequences.", "Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.", "Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include a N-terminal residue.", "This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.", "Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.", "The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium.", "In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5′-flanking non-transcribed sequences.", "DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.", "2) Regulatory Elements Promoters The suitable promoter regions used in the expression vectors of the present invention are chosen taking into account the cell host in which the heterologous gene is expressed.", "The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.", "Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter.", "A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed.", "Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.", "Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.", "Preferred bacterial promoters are the LacI, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trp promoters (EP 0036776), the polyhedrin promoter, or the p10 protein promoter from baculovirus (Kit Novagen) (Smith et al., (1983) Mol Cell Biol 3:2156-65; O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.", "Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L.", "In addition, promoters specific for a particular cell type may be chosen, such as those facilitating expression in adipose tissue, muscle tissue, or liver.", "Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.", "The choice of a promoter is well within the ability of a person skilled in the field of genetic engineering.", "For example, one may refer to Sambrook et al.", "(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, Vol.", "1, 2, 3 (1989), or also to the procedures described by Fuller et al.", "(1996) Immunology in Current Protocols in Molecular Biology.", "Other Regulatory Elements Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.", "The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.", "Also contemplated as an element of the expression cassette is a terminator.", "These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.", "Vectors containing the appropriate DNA sequence as described above can be utilized to transform an appropriate host to allow the expression of the desired polypeptide or polynucleotide.", "3) Selectable Markers Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.", "The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRP1 for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli, or levan saccharase for mycobacteria, this latter marker being a negative selection marker.", "4) Preferred Vectors Bacterial Vectors As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017).", "Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and pGEM1 (Promega Biotec, Madison, Wis., USA).", "Large numbers of other suitable vectors are known to those of skill in the art, and are commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).", "Baculovirus Vectors A suitable vector for the expression of polypeptides of the invention is a baculovirus vector that can be propagated in insect cells and in insect cell lines.", "A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC No CRL 1711) which is derived from Spodoptera frugiperda.", "Other suitable vectors for the expression of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide in a baculovirus expression system include those described by Chai et al.", "(1993; Biotechnol Appl Biochem 18 (Pt 3):259-73); Vlasak et al.", "(1983; Eur J Biochem 135:123-6); and Lenhard et al.", "(1996; Gene 169:187-90).", "Plasmid Vectors A suitable vector for the expression of polypeptides of the invention is a plasmid vector that contains an SV40-derived origin of replication and that can be used for transient transfection of COS cells (ATCC No CRL1650; No CRL1651).", "Plasmid vectors suitable for transient transfection of COS cells include but are not limited to CDM8 (Invitrogen).", "Viral Vectors In one specific embodiment, the vector is derived from an adenovirus.", "Preferred adenovirus vectors according to the invention are those described by Feldman and Steg (1996; Semin Interv Cardiol 1:203-8) or Ohno et al.", "(1994; Science 265:781-4).", "Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin (French patent application No.", "FR-93.05954).", "Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans.", "These vectors provide-efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.", "Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vivo gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus.", "Particularly preferred Murine Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298).", "Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728).", "Other preferred retroviral vectors are those described in Roth et al.", "(1996), PCT Application No WO 93/25234, PCT Application No WO 94/06920, Roux et al., ((1989) Proc Natl Acad Sci USA 86:9079-83), Julan et al., (1992) J Gen. Virol 3:3251-3255 and Neda et al., ((1991) J Biol Chem 266:14143-6).", "Yet another viral vector system that is contemplated by the invention consists of the adeno-associated virus (AAV).", "The adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., (1992) Curr Top Microbiol Immunol 158:97-129).", "It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al., (1992) Am J Respir Cell Mol Biol 7:349-56; Samulski et al., (1989) J Virol 63:3822-8; McLaughlin et al., (1989) Am J Hum Genet 59:561-569).", "One advantageous feature of AAV derives from its reduced efficacy for transducing primary cells relative to transformed cells.", "5) Delivery of the Recombinant Vectors In order to effect expression of the polynucleotides of the invention, these constructs must be delivered into a cell.", "This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain disease states.", "One mechanism is viral infection where the expression construct is encapsulated in an infectious viral particle.", "Several non-viral methods for the transfer of polynucleotides into cultured mammalian cells are also contemplated by the present invention, and include, without being limited to, calcium phosphate precipitation (Graham et al., (1973) Virology 54:536-9; Chen et al., (1987) Mol Cell Biol 7:2745-52), DEAE-dextran (Gopal, (1985) Mol Cell Biol 5:1188-90), electroporation (Tur-Kaspa et al., (1986) Mol Cell Biol 6:716-8; Potter et al., (1984) Proc Natl Acad Sci USA 81:7161-5.", "), direct microinjection (Harland et al., (1985) J Cell Biol 101:1094-9), DNA-loaded liposomes (Nicolau et al., (1982) Biochim Biophys Acta 721:185-90; Fraley et al., (1979) Proc Natl Acad Sci USA 76:3348-52), and receptor-mediated transfection (Wu and Wu, (1987) J Biol Chem 262:4429-32; Wu and Wu (1988) Biochemistry 27:887-92).", "Some of these techniques may be successfully adapted for in vivo or ex vivo use.", "Once the expression polynucleotide has been delivered into the cell, it may be stably integrated into the genome of the recipient cell.", "This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).", "In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.", "Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.", "One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect.", "This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.", "Compositions for use in vitro and in vivo comprising a “naked” polynucleotide are described in PCT application No.", "WO 90/11092 (Vical Inc.) and also in PCT application No.", "WO 95/11307 (Institut Pasteur, INSERM, Université d'Ottawa) as well as in the articles of Tascon et al.", "(1996) Nature Medicine 2:888-892 and of Huygen et al.", "((1996) Nat Med 2:893-8).", "In still another embodiment of the invention, the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al.", "((1990) Curr Genet 17:97-103).", "In a further embodiment, the polynucleotide of the invention may be entrapped in a liposome (Ghosh and Bacchawat, (1991) Targeted Diagn Ther 4:87-103; Wong et al., (1980) Gene 10:87-94; Nicolau et al., (1987) Methods Enzymot.", "149:157-76).", "These liposomes may further be targeted to cells expressing LSR by incorporating leptin, triglycerides, or other known LSR ligands into the liposome membrane.", "In a specific embodiment, the invention provides a composition for the in vivo production of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide described herein.", "It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells of the tissue to express the said polypeptide.", "The amount of vector to be injected to the desired host organism varies according to the site of injection.", "As an indicative dose, it will be injected between 0.1 and 100 μg of the vector in an animal body, preferably a mammal body, for example a mouse body.", "In another embodiment of the vector according to the invention, it may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell.", "In a subsequent step, the cell that has been transformed with the vector coding for the desired GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or the desired fragment thereof is reintroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.", "IV.", "Recombinant Cells of the Invention Another object of the invention consists of host cells recombinant for, i.e., that have been transformed or transfected with one of the polynucleotides described herein, and more precisely a polynucleotide comprising a polynucleotide encoding a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of the invention such as any one of those described in “Polynucleotides of the Invention”.", "These polynucleotides can be present in cells as a result of transient or stable transfection.", "The invention includes host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as any one of those described in “Recombinant Vectors of the Invention”.", "Generally, a recombinant host cell of the invention comprises at least one of the polynucleotides or the recombinant vectors of the invention that are described herein.", "Preferred host cells used as recipients for the recombinant vectors of the invention are the following: a) Prokaryotic host cells: Escherichia coli strains (I.E.", "DH5-x strain), Bacillus subtilis, Salmonella typhimurium, and strains from species like Pseudomonas, Streptomyces and Staphylococcus, and b) Eukaryotic host cells: HeLa cells (ATCC No CCL2; No CCL2.1; No CCL2.2), Cv 1 cells (ATCC No CCL70), COS cells (ATCC No CRL1650; No CRL1651), Sf-9 cells (ATCC No CRL1711), C127 cells (ATCC No CRL-1804), 3T3 (ATCC No CRL-6361), CHO (ATCC No CCL-61), human kidney 293 (ATCC No 45504; No CRL-1573), BHK (ECACC No 84100501; No 84111301), PLC cells, HepG2, and Hep3B.", "The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.", "Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.", "In a preferred embodiment, recombinant protein expressed by cells that have been stably or transiently transfected with a recombinant vector such as any one of those described in “Recombinant Vectors of the Invention” is recovered from culture supernatant.", "Alternatively, cells may be harvested (typically by centrifugation), disrupted by physical or chemical means, and the resulting crude extract retained for further purification.", "Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.", "Such methods are well known by the skilled artisan.", "Further, according to the invention, these recombinant cells can be created in vitro or in vivo in an animal, preferably a mammal, most preferably selected from the group consisting of mice, rats, dogs, pigs, sheep, cattle, and primates, not to include humans.", "Recombinant cells created in vitro can also be later surgically implanted in an animal, for example.", "Methods to create recombinant cells in vivo in animals are well-known in the art.", "The present invention also encompasses primary, secondary, and immortalized homologously recombinant host cells of vertebrate origin, preferably mammalian origin and particularly human origin, that have been engineered to: a) insert exogenous (heterologous) polynucleotides into the endogenous chromosomal DNA of a targeted gene, b) delete endogenous chromosomal DNA, and/or c) replace endogenous chromosomal DNA with exogenous polynucleotides.", "Insertions, deletions, and/or replacements of polynucleotide sequences may be to the coding sequences of the targeted gene and/or to regulatory regions, such as promoter and enhancer sequences, operably associated with the targeted gene.", "The present invention further relates to a method of making a homologously recombinant host cell in vitro or in vivo, wherein the expression of a targeted gene not normally expressed in the cell is altered.", "Preferably the alteration causes expression of the targeted gene under normal growth conditions or under conditions suitable for producing the polypeptide encoded by the targeted gene.", "The method comprises the steps of: (a) transfecting the cell in vitro or in vivo with a polynucleotide construct, the polynucleotide construct comprising; (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; and (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination.", "The present invention further relates to a method of altering the expression of a targeted gene in a cell in vitro or in vivo wherein the gene is not normally expressed in the cell, comprising the steps of: (a) transfecting the cell in vitro or in vivo with a polynucleotide construct, the polynucleotide construct comprising: (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; and (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell; and (c) maintaining the homologously recombinant cell in vitro or in vivo under conditions appropriate for expression of the gene.", "The present invention further relates to a method of making a polypeptide of the present invention by altering the expression of a targeted endogenous gene in a cell in vitro or in vivo wherein the gene is not normally expressed in the cell, comprising the steps of: a) transfecting the cell in vitro with a polynucleotide construct, the polynucleotide construct comprising: (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; (b) maintaining the transfected cell it vitro or in vivo under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell; and c) maintaining the homologously recombinant cell in vitro or in vivo under conditions appropriate for expression of the gene thereby making the polypeptide.", "The present invention further relates to a polynucleotide construct that alters the expression of a targeted gene in a cell type in which the gene is not normally expressed.", "This occurs when a polynucleotide construct is inserted into the chromosomal DNA of the target cell, wherein the polynucleotide construct comprises: a) a targeting sequence; b) a regulatory sequence and/or coding sequence; and c) an unpaired splice-donor site, if necessary.", "Further included are polynucleotide constructs, as described above, wherein the construct further comprises a polynucleotide that encodes a polypeptide and is in-frame with the targeted endogenous gene after homologous recombination with chromosomal DNA.", "The compositions may be produced, and methods performed, by techniques known in the art, such as those described in U.S. Pat.", "Nos.", "6,054,288; 6,048,729; 6,048,724; 6,048,524; 5,994,127; 5,968,502; 5,965,125; 5,869,239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580,734; International Publication Nos: WO96/29411, WO 94/12650; and scientific articles described by Koller et al., (1994) Annu Rev Immunol 10:705-730; the disclosures of each of which are incorporated by reference in their entireties).", "The expression of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 in mammalian, and typically human, cells may be rendered defective, or alternatively it may be enhanced, with the insertion of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 genomic or cDNA sequence with the replacement of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 gene counterpart in the genome of an animal cell by a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polynucleotide according to the invention.", "These genetic alterations may be generated by homologous recombination events using specific DNA constructs that have been previously described.", "One kind of host cell that may be used are mammalian zygotes, such as murine zygotes.", "For example, murine zygotes may undergo microinjection with a purified DNA molecule of interest, for example a purified DNA molecule that has previously been adjusted to a concentration range from 1 ng/ml—for BAC inserts—3 ng/μl—for P1 bacteriophage inserts—in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine, and 70 μM spermidine.", "When the DNA to be microinjected has a large size, polyamines and high salt concentrations can be used in order to avoid mechanical breakage of this DNA, as described by Schedl et al ((1993) Nature 362:258-61).", "Any one of the polynucleotides of the invention, including the DNA constructs described herein, may be introduced in an embryonic stem (ES) cell line, preferably a mouse ES cell line.", "ES cell lines are derived from pluripotent, uncommitted cells of the inner cell mass of pre-implantation blastocysts.", "Preferred ES cell lines are the following: ES-E14TG2a (ATCC No.", "CRL-1821), ES-D3 (ATCC No.", "CRL-1934 and No.", "CRL-11632), YS001 (ATCC No.", "CRL-11776), 36.5 (ATCC No.", "CRL-11116).", "To maintain ES cells in an uncommitted state, they are cultured in the presence of growth inhibited feeder cells which provide the appropriate signals to preserve this embryonic phenotype and serve as a matrix for ES cell adherence.", "Preferred feeder cells are primary embryonic fibroblasts that are established from tissue of day 13-day 14 embryos of virtually any mouse strain, that are maintained in culture, such as described by Abbondanzo et al.", "(1993; Methods Enzymol 225:803-23) and are inhibited in growth by irradiation, such as described by Robertson ((1987) Embryo-derived stem cell lines.", "In: E. J. Robertson Ed.", "Teratocarcinomas and embrionic stem cells: a practical approach.", "IRL Press, Oxford), or by the presence of an inhibitory concentration of LIF, such as described by Pease and Williams (1990; Exp Cell Res 190:209-11).", "The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.", "Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.", "Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.", "Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.", "Such methods are well known by the skilled artisan.", "V. Transgenic Animals The present invention also provides methods and compositions for the generation of non-human animals and plants that express the recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides, of the present invention.", "The animals or plants can be transgenic, i.e.", "each of their cells contains a gene encoding a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or, alternatively, a polynucleotide encoding a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide can be introduced into somatic cells of the animal or plant, e.g.", "into mammary secretory epithelial cells of a mammal.", "In preferred embodiments, the non-human animal is a mammal such as a cow, sheep, goat, pig, or rabbit.", "Methods of making transgenic animals such as mammals are well known to those of skill in the art, and any such method can be used in the present invention.", "Briefly, transgenic mammals can be produced, e.g., by transfecting a pluripotential stem cell such as an ES cell with a polynucleotide encoding a polypeptide of interest.", "Successfully transformed ES cells can then be introduced into an early stage embryo that is then implanted into the uterus of a mammal of the same species.", "In certain cases, the transformed (“transgenic”) cells will comprise part of the germ line of the resulting animal, and adult animals comprising the transgenic cells in the germ line can then be mated to other animals, thereby eventually producing a population of transgenic animals that have the transgene in each of their cells, and which can stably transmit the transgene to each of their offspring.", "Other methods of introducing the polynucleotide can be used, for example introducing the polynucleotide encoding the polypeptide of interest into a fertilized egg or early stage embryo via microinjection.", "Alternatively, the transgene may be introduced into an animal by infection of zygotes with a retrovirus containing the transgene (Jaenisch, R. (1976) Proc.", "Natl.", "Acad.", "Sci.", "USA 73, 1260-1264).", "Methods of making transgenic mammals are described, e.g., in Wall et al.", "(1992) J Cell Biochem 1992 49:113-20; Hogan, et al.", "(1986) in Manipulating the mouse embryo.", "A Laboratory Manual.", "Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; in WO 91/08216, or in U.S. Pat.", "No.", "4,736,866.In a preferred method, the polynucleotides are microinjected into the fertilized oocyte.", "Typically, fertilized oocytes are microinjected using standard techniques, and then cultured in vitrountil a “pre-implantation embryo” is obtained.", "Such pre-implantation embryos preferably contain approximately 16 to 150 cells.", "Methods for culturing fertilized oocytes to the pre-implantation stage are described, e.g., by Gordon et al.", "((1984) Methods in Enzymology, 101, 414); Hogan et al.", "((1986) in Manipulating the mouse embryo.", "A Laboratory Manual.", "Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) (for the mouse embryo); Hammer et al.", "((1985) Nature, 315, 680) (for rabbit and porcine embryos); Gandolfi et al.", "((1987) J. Reprod.", "Fert.", "81, 23-28); Rexroad et al.", "((1988) J. Anim.", "Sci.", "66, 947-953) (for ovine embryos); and Eyestone et al.", "((1989) J. Reprod.", "Fert.", "85, 715-720); Camous et al.", "((1984) J. Reprod.", "Fert.", "72, 779-785); and Heyman et al.", "((1987) Theriogenology 27, 5968) (for bovine embryos); the disclosures of each of which are incorporated herein in their entireties.", "Pre-implantation embryos are then transferred to an appropriate female by standard methods to permit the birth of a transgenic or chimeric animal, depending upon the stage of development when the transgene is introduced.", "As the frequency of transgene incorporation is often low, the detection of transgene integration in pre-implantation embryos is often desirable using any of the herein-described methods.", "Any of a number of methods can be used to detect the presence of a transgene in a pre-implantation embryo.", "For example, one or more cells may be removed from the pre-implantation embryo, and the presence or absence of the transgene in the removed cell or cells can be detected using any standard method e.g.", "PCR.", "Alternatively, the presence of a transgene can be detected in utero or post partum using standard methods.", "In a particularly preferred embodiment of the present invention, transgenic mammals are generated that secrete recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides in their milk.", "As the mammary gland is a highly efficient protein-producing organ, such methods can be used to produce protein concentrations in the gram per liter range, and often significantly more.", "Preferably, expression in the mammary gland is accomplished by operably linking the polynucleotide encoding the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide to a mammary gland specific promoter and, optionally, other regulatory elements.", "Suitable promoters and other elements include, but are not limited to, those derived from mammalian short and long WAP, alpha, beta, and kappa, casein, alpha and beta lactoglobulin, beta-CN 5′ genes, as well as the the mouse mammary tumor virus (MMTV) promoter.", "Such promoters and other elements may be derived from any mammal, including, but not limited to, cows, goats, sheep, pigs, mice, rabbits, and guinea pigs.", "Promoter and other regulatory sequences, vectors, and other relevant teachings are provided, e.g., by Clark (1998) J Mammary Gland Biol Neoplasia 3:337-50; Jost et al.", "(1999) Nat Biotechnol 17:160-4; U.S. Pat.", "Nos.", "5,994,616; 6,140,552; 6,013,857; Sohn et al.", "(1999) DNA Cell Biol.", "18:845-52; Kim et al.", "(1999) J Biochem (Japan) 126:320-5; Soulier et al.", "(1999) Euro J Biochem 260:533-9; Zhang et al.", "(1997) Chin J Biotech 13:271-6; Rijnkels et al.", "(1998) Transgen Res 7:5-14; Korhonen et al.", "(1997) Euro J Biochem 245:482-9; Uusi-Oukari et al.", "(1997) Transgen Res 6:75-84; Hitchin et al.", "(1996) Prot Expr Purif 7:247-52; Platenburg et al.", "(1994) Transgen Res 3:99-108; Heng-Cherl et al.", "(1993) Animal Biotech.", "4:89-107; and Christa et al.", "(2000) Euro J Biochem 267:1665-71; the entire disclosures of each of which is herein incorporated by reference.", "In another embodiment, the polypeptides of the invention can be produced in milk by introducing polynucleotides encoding the polypeptides into somatic cells of the mammary gland in vivo, e.g.", "mammary secreting epithelial cells.", "For example, plasmid DNA can be infused through the nipple canal, e.g.", "in association with DEAE-dextran (see, e.g., Hens et al.", "(2000) Biochim.", "Biophys.", "Acta 1523:161-171), in association with a ligand that can lead to receptor-mediated endocytosis of the construct (see, e.g., Sobolev et al.", "(1998) 273:7928-33), or in a viral vector such as a retroviral vector, e.g.", "the Gibbon ape leukemia virus (see, e.g., Archer et al.", "(1994) PNAS 91:6840-6844).", "In any of these embodiments, the polynucleotide may be operably linked to a mammary gland specific promoter, as described above, or, alternatively, any strongly expressing promoter such as CMV or MoMLV LTR.", "The suitability of any vector, promoter, regulatory element, etc.", "for use in the present invention can be assessed beforehand by transfecting cells such as mammary epithelial cells, e.g.", "MacT cells (bovine mammary epithelial cells) or GME cells (goat mammary epithelial cells), in vitro and assessing the efficiency of transfection and expression of the transgene in the cells.", "For in vivo administration, the polynucleotides can be administered in any suitable formulation, at any of a range of concentrations (e.g.", "1-500 μg/ml, preferably 50-100 μg/ml), at any volume (e.g.", "1-100 ml, preferably 1 to 20 ml), and can be administered any number of times (e.g.", "1, 2, 3, 5, or 10 times), at any frequency (e.g.", "every 1, 2, 3, 5, 10, or any number of days).", "Suitable concentrations, frequencies, modes of administration, etc.", "will depend upon the particular polynucleotide, vector, animal, etc., and can readily be determined by one of skill in the art.", "In a preferred embodiment, a retroviral vector such as as Gibbon ape leukemia viral vector is used, as described in Archer et al.", "((1994) PNAS 91:6840-6844).", "As retroviral infection typically requires cell division, cell division in the mammary glands can be stimulated in conjunction with the administration of the vector, e.g.", "using a factor such as estrodiol benzoate, progesterone, reserpine, or dexamethasone.", "Further, retroviral and other methods of infection can be facilitated using accessory compounds such as polybrene.", "In any of the herein-described methods for obtaining GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides from milk, the quantity of milk obtained, and thus the quantity of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides produced, can be enhanced using any standard method of lactation induction, e.g.", "using hexestrol, estrogen, and/or progesterone.", "The polynucleotides used in such embodiments can either encode a full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 fragment.", "Typically, the encoded polypeptide will include a signal sequence to ensure the secretion of the protein into the milk.", "Where a full length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 sequence is used, the full length protein can, e.g., be isolated from milk and cleaved in vitro using a suitable protease.", "Alternatively, a second, protease-encoding polynucleotide can be introduced into the animal or into the mammary gland cells, whereby expression of the protease results in the cleavage of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide in vivo, thereby allowing the direct isolation of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 fragments from milk.", "VI.", "Pharmaceutical or Physiologically Acceptable Compositions of the Invention The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention can be administered to non-human animals and/or humans, alone or in pharmaceutical or physiologically acceptable compositions where they are mixed with suitable carriers or excipient(s).", "The pharmaceutical or physiologically acceptable composition is then provided at a therapeutically effective dose.", "A therapeutically effective dose refers to that amount of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide sufficient to result in prevention or amelioration of symptoms or physiological status of metabolic-related diseases or disorders as determined by the methods described herein.", "A therapeutically effective dose can also refer to the amount of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide necessary for a reduction in weight or a prevention of an increase in weight or prevention of an increase in the rate of weight gain in persons desiring this affect for cosmetic reasons.", "A therapeutically effective dosage of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of the invention is that dosage that is adequate to promote weight loss or weight gain with continued periodic use or administration.", "Techniques for formulation and administration of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.", "Other diseases or disorders that GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention could be used to treat or prevent include, but are not limited to, obesity and obesity-related diseases and disorders such as obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Non-Insulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).", "Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.", "The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides may also be used to enhance physical performance during work or exercise or enhance a feeling of general well-being.", "Physical performance activities include walking, running, jumping, lifting and/or climbing.", "The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides or antagonists thereof may also be used to treat dyslexia, attention-deficit disorder (ADD), attention-deficit/hyperactivity disorder (ADHD), and psychiatric disorders such as schizophrenia by modulating fatty acid metabolism, more specifically, the production of certain long-chain polyunsaturated fatty acids.", "It is expressly considered that the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides of the invention may be provided alone or in combination with other pharmaceutically or physiologically acceptable compounds.", "Other compounds useful for the treatment of obesity and other diseases and disorders are currently well-known in the art.", "In a preferred embodiment, the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are useful for, and used in, the treatment of insulin resistance and diabetes using methods described herein and known in the art.", "More particularly, a preferred embodiments relates to process for the therapeutic modification and regulation of glucose metabolism in an animal or human subject, which comprises administering to a subject in need of treatment (alternatively on a timed daily basis) GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.", "Further preferred embodiments relate to methods for the prophylaxis or treatment of diabetes comprising administering to a subject in need of treatment (alternatively on a timed daily basis) a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.", "Routes of Administration.", "Suitable routes of administration include oral, nasal, rectal, transmucosal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocular injections using methods known in the art.", "A particularly useful method of administering compounds for promoting weight loss involves surgical implantation, for example into the abdominal cavity of the recipient, of a device for delivering GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides over an extended period of time.", "Other particularly preferred routes of administration are aerosol and depot formulation.", "Sustained release formulations, particularly depot, of the invented medicaments are expressly contemplated.", "Composition/Formulation Pharmaceutical or physiologically acceptable compositions and medicaments for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries.", "Proper formulation is dependent upon the route of administration chosen.", "Certain of the medicaments described herein will include a pharmaceutically or physiologically acceptable acceptable carrier and at least one polypeptide that is a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of the invention.", "For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer such as a phosphate or bicarbonate buffer.", "For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.", "Such penetrants are generally known in the art.", "Pharmaceutical or physiologically acceptable preparations that can be taken orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.", "The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.", "In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.", "In addition, stabilizers may be added.", "All formulations for oral administration should be in dosages suitable for such administration.", "For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.", "For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable gaseous propellant, e.g., carbon dioxide.", "In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.", "Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator, may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.", "The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.", "Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.", "The compositions may take such forms as suspensions, solutions or emulsions in aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.", "Pharmaceutical or physiologically acceptable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.", "Aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.", "Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.", "Alternatively, the active ingredient may be in powder or lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.", "In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.", "Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.", "Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.", "Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.", "Various sustained release materials have been established and are well known by those skilled in the art.", "Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.", "Depending on the chemical nature and the biological stability of the therapeutic reagent additional strategies for protein stabilization may be employed.", "The pharmaceutical or physiologically acceptable compositions also may comprise suitable solid or gel phase carriers or excipients.", "Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.", "Effective Dosage.", "Pharmaceutical or physiologically acceptable compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose.", "More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.", "Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.", "For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays.", "For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range shown to increase leptin or lipoprotein uptake or binding in an in vitro system.", "Such information can be used to more accurately determine useful doses in humans.", "A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient.", "Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g.", "for determining the LD50, (the dose lethal to 50% of the test population) and the ED50 (the dose therapeutically effective in 50% of the population).", "The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD5O and ED5O.", "Compounds that exhibit high therapeutic indices are preferred.", "The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.", "The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50, with little or no toxicity.", "The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.", "The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.", "(See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.", "1).", "Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain or prevent weight loss or gain, depending on the particular situation.", "Dosages necessary to achieve these effects will depend on individual characteristics and route of administration.", "Dosage intervals can also be determined using the value for the minimum effective concentration.", "Compounds should be administered using a regimen that maintains plasma levels above the minimum effective concentration for 10-90% of the time, preferably between 30-90%; and most preferably between 50-90%.", "In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.", "The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.", "A preferred dosage range for the amount of a GMG-7A, GM 7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of the invention, which can be administered on a daily or regular basis to achieve desired results, including a reduction in levels of circulating plasma triglyceride-rich lipoproteins, range from 0.05-1.0 mg/kg body mass.", "A more preferred dosage range is from 0.1-5 mg/kg.", "A more preferred dose is 0.25-2.5 mg/kg.", "Of course, these daily dosages can be delivered or administered in small amounts periodically during the course of a day.", "It is noted that these dosage ranges are only preferred ranges and are not meant to be limiting to the invention.", "VII.", "Methods of Treatment The invention is drawn inter alia to methods of preventing or treating metabolic-related diseases and disorders comprising providing an individual in need of such treatment with a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide of the invention.", "Preferably, the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide has metabolic-related activity either in vitro or in vivo.", "Preferably the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide is provided to the individual in a pharmaceutical composition that is preferably taken orally.", "Preferably the individual is a mammal, and most preferably a human.", "In preferred embodiments, the metabolic-related disease or disorder is selected from the group consisting of atherosclerosis, cardiovascular disease, impaired glucose tolerance, insulin resistance, hypertension, stroke, Syndrome X, Type I diabetes, Type II diabetes and lipoatrophic diabetes.", "Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia, hypertriglyceridemia, and hyperuricemia.", "Yet other metabolic-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, neoplasia-related weight loss, anorexia, and bulimia.", "In preferred embodiments, GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides in pharmaceutical compositions are used to modulate body weight in healthy individuals for cosmetic reasons.", "The invention also features a method of preventing or treating metabolic-related diseases and disorders comprising providing an individual in need of such treatment with a compound identified by assays of the invention (described in Section VI of the Preferred Embodiments of the Invention and in the Examples).", "Preferably these compounds antagonize or agonize effects of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides in cells in vitro, muscles ex vivo, or in animal models.", "Alternatively, these compounds agonize or antagonize the effects of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on leptin and/or lipoprotein uptake and/or binding.", "Optionally, these compounds prevent the interaction, binding, or uptake of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides with LSR in vitro or in vivo.", "Preferably, the compound is provided to the individual in a pharmaceutical composition that is preferably taken orally.", "Preferably the individual is a mammal, and most preferably a human.", "In preferred embodiments, the metabolic-related disease or disorder is selected from the group consisting of obesity and metabolic-related diseases and disorders such as atherosclerosis, heart disease, insulin resistance, hypertension, stroke, Syndrome X Type I diabetes, Type II diabetes, and lipoatrophic diabetes.", "Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia, hypertriglyceridemia, and hyperuricemia.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, alone, without combination of insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some individuals, particularly those with Type II diabetes or insulin resistance, alone, without combination of insulin therapy.", "In still a further preferred embodiment, the control of body weight is due in part or in whole to a decrease in mass of 1) subcutaneous adipose tissue and/or 2) viseral (omental) adipose tissue.", "In a further preferred embodiment, the present invention may be used in complementary therapy, particularly in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, to improve their weight or glucose control in combination with an insulin secretagogue or an insulin sensitising agent.", "Preferably, the insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.", "Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.", "The present invention further provides a method of improving the body weight or glucose control of some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, alone, without an insulin secretagogue or an insulin sensitising agent.", "In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with the insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent).", "Accordingly, the present invention further provides for a composition of pharmaceutical or physiologically acceptable composition and an oral insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of body weight or glucose control in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin sensitiser.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some individuals, particularly those with Type II diabetes or insulin resistance, without insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an inhibitor of the progression from impaired glucose tolerance to insulin resistance.", "VII.", "Ligands Interacting with GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Polypeptides For the purpose of the present invention, a Ligand means a molecule, such as a protein, a peptide, an antibody or any synthetic chemical compound capable of binding to a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or one of its fragments or variants or to modulate the expression of the polynucleotide coding for GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or a fragment or variant thereof.", "In the Ligand screening method according to the present invention, a biological sample or a defined molecule to be tested as a putative Ligand of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide is brought into contact with the corresponding purified GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, for example the corresponding purified recombinant GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide produced by a recombinant cell host as described herein, in order to form a complex between this protein and the putative Ligand molecule to be tested.", "As an illustrative example, to study the interaction of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selected from the group consisting of of SEQ ID NO: 2, 4, 6, 8, 10, and 12, with drugs or small molecules, such as molecules generated through combinatorial chemistry approaches, the microdialysis coupled to HPLC method described by Wang et al.", "(1997) or the affinity capillary electrophoresis method described by Bush et al.", "(1997), the disclosures of which are incorporated by reference, can be used.", "In further methods, peptides, drugs, fatty acids, lipoproteins, or small molecules which interact with a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selected from the group consisting of sequences of SEQ ID NO: 2, 4, 6, 8, 10, or 12 may be identified using assays such as the following.", "The molecule to be tested for binding is labelled with a detectable label, such as a fluorescent, radioactive, or enzymatic tag and placed in contact with immobilized GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment thereof under conditions that permit specific binding to occur.", "After removal of non-specifically bound molecules, bound molecules are detected using appropriate means.", "Various candidate substances or molecules can be assayed for interaction with a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide.", "These substances or molecules include, without being limited to, natural or synthetic organic compounds or molecules of biological origin such as polypeptides.", "When the candidate substance or molecule comprises a polypeptide, this polypeptide may be the resulting expression product of a phage clone belonging to a phage-based random peptide library, or alternatively the polypeptide may be the resulting expression product of a cDNA library cloned in a vector suitable for performing a two-hybrid screening assay.", "A.", "Candidate Ligands Obtained by Affinity Chromatography.", "Proteins or other molecules interacting with a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment thereof comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selected from the group consisting of sequences of SEQ ID NO: 2, 4, 6, 8, 10, and 12, can also be found using affinity columns which contain the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment thereof.", "The GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment thereof, may be attached to the column using conventional techniques including chemical coupling to a suitable column matrix such as agarose, Affi Gel®, or other matrices familiar to those of skill in art.", "In some embodiments of this method, the affinity column contains chimeric proteins in which the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment thereof, is fused to glutathion S transferase (GST).", "A mixture of cellular proteins or pool of expressed proteins as described above is applied to the affinity column.", "Proteins or other molecules interacting with the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment thereof, attached to the column can then be isolated and analyzed on 2-D electrophoresis gel as described in Ramunsen et al.", "(1997), the disclosure of which is incorporated by reference.", "Alternatively, the proteins retained on the affinity column can be purified by electrophoresis-based methods and sequenced.", "The same method can be used to isolate antibodies, to screen phage display products, or to screen phage display human antibodies.", "B.", "Candidate Ligands Obtained by Optical Biosensor Methods Proteins interacting with a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selected from the group consisting of sequences of SEQ ID NO: 2, 4, 6, 8, 10, or 12, can also be screened by using an Optical Biosensor as described in Edwards and Leatherbarrow (1997) and also in Szabo et al.", "(1995), the disclosures of which are incorporated by reference.", "This technique permits the detection of interactions between molecules in real time, without the need of labelled molecules.", "This technique is based on the surface plasmon resonance (SPR) phenomenon.", "Briefly, the candidate Ligand molecule to be tested is attached to a surface (such as a carboxymethyl dextran matrix).", "A light beam is directed towards the side of the surface that does not contain the sample to be tested and is reflected by said surface.", "The SPR phenomenon causes a decrease in the intensity of the reflected light with a specific association of angle and wavelength.", "The binding of candidate Ligand molecules cause a change in the refraction index on the surface, which change is detected as a change in the SPR signal.", "For screening of candidate Ligand molecules or substances that are able to interact with the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment thereof, the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment thereof, is immobilized onto a surface.", "This surface comprises one side of a cell through which flows the candidate molecule to be assayed.", "The binding of the candidate molecule on the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment thereof, is detected as a change of the SPR signal.", "The candidate molecules tested may be proteins, peptides, carbohydrates, lipids, or small molecules generated by combinatorial chemistry.", "Screening of candidate ligands by optical biosensor methods may also be performed by immobilizing eukaryotic or prokaryotic cells or lipid vesicles exhibiting an endogenous or a recombinantly expressed GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide at their surface.", "The main advantage of the method is that it allows the determination of the association rate between the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide and molecules interacting with the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide.", "It is thus possible to select specifically Ligand molecules interacting with the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, or a fragment thereof, through strong or conversely weak association constants.", "C. Candidate Ligands Obtained Through a Two-Hybrid Screening Assay.", "The yeast two-hybrid system is designed to study protein-protein interactions in vivo (Fields and Song, 1989), which disclosure is hereby incorporated by reference in its entirety, and relies upon the fusion of a bait protein to the DNA binding domain of the yeast Gal4 protein.", "This technique is also described in the U.S. Pat.", "No.", "5,667,973 and the U.S. Pat.", "No.", "5,283,173, the technical teachings of both patents being herein incorporated by reference.", "The general procedure of library screening by the two-hybrid assay may be performed as described by Harper et al.", "(1993) or as described by Cho et al.", "(1998) or also Fromont-Racine et al.", "(1997), which disclosures are hereby incorporated by reference in their entireties.", "The bait protein or polypeptide comprises, consists essentially of, or consists of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or a fragment thereof comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selected from the group consisting of sequences of SEQ ID NO: 2, 4, 6, 8, 10, and 12.More precisely, the nucleotide sequence encoding the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or a fragment or variant thereof is fused to a polynucleotide encoding the DNA binding domain of the GAL4 protein, the fused nucleotide sequence being inserted in a suitable expression vector, for example pAS2 or pM3.Then, a human cDNA library is constructed in a specially designed vector, such that the human cDNA insert is fused to a nucleotide sequence in the vector that encodes the transcriptional domain of the GAL4 protein.", "Preferably, the vector used is the pACT vector.", "The polypeptides encoded by the nucleotide inserts of the human cDNA library are termed “prey” polypeptides.", "A third vector contains a detectable marker gene, such as beta galactosidase gene or CAT gene that is placed under the control of a regulation sequence that is responsive to the binding of a complete Gal4 protein containing both the transcriptional activation domain and the DNA binding domain.", "For example, the vector pG5EC may be used.", "Two different yeast strains are also used.", "As an illustrative but non-limiting example the two different yeast strains may be the following: Y190, the phenotype of which is (MATa, Leu2-3, 112 ura3-12, trp1-901, his3-D200, ade2-101, gal4Dgal180D URA3 GAL-LacZ, LYS GAL-HIS3, cyhr); Y187, the phenotype of which is (MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3, -112 URA3 GAL-lacZmet−), which is the opposite mating type of Y190.Briefly, 20 μg of pAS2/GMG-7A, pAS2/GMG-7B, pAS2/GMG-8, pAS2/GMG-9, pAS2/GMG-10, or pAS2/GMG-11 and 20 μg of pACT-cDNA library are co-transformed into yeast strain Y190.The transformants are selected for growth on minimal media lacking histidine, leucine and tryptophan, but containing the histidine synthesis inhibitor 3-AT (50 nM).", "Positive colonies are screened for beta galactosidase by filter lift assay.", "The double positive colonies (Mis+, beta-gal+) are then grown on plates lacking histidine, leucine, but containing tryptophan and cycloheximide (10 mg/ml) to select for loss of pAS2/GMG-7A, pAS2/GMG-7B, pAS2/GMG-8, pAS2/GMG-9, pAS2/GMG-10, or pAS2/GMG-11 plasmids but retention of pACT-cDNA library plasmids.", "The resulting Y190 strains are mated with Y187 strains expressing GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 or non-related control polypeptides such as cyclophilin B, lamin, or SNF1, as Gal4 fusions as described by Harper et al.", "(1993) and by Bram et al.", "(1993), which disclosures are hereby incorporated by reference in their entireties, and screened for beta galactosidase by filter lift assay.", "Yeast clones that are beta gal-after mating with the control Gal4 fusions are considered false positives.", "In another embodiment of the two-hybrid method according to the invention, interaction between the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or a fragment or variant thereof with cellular proteins may be assessed using the Matchmaker Two Hybrid System 2 (Catalog No.", "K1604-1, Clontech).", "As described in the manual accompanying the kit, the disclosure of which is incorporated herein by reference, nucleic acids encoding the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or a portion thereof, are inserted into an expression vector such that they are in frame with DNA encoding the DNA binding domain of the yeast transcriptional activator GAL4.A desired cDNA, preferably human cDNA, is inserted into a second expression vector such that they are in frame with DNA encoding the activation domain of GAL4.The two expression plasmids are transformed into yeast and the yeast are plated on selection medium which selects for expression of selectable markers on each of the expression vectors as well as GAL4 dependent expression of the HIS3 gene.", "Transformants capable of growing on medium lacking histidine are screened for GAL4 dependent lacZ expression.", "Those cells that are positive in both the histidine selection and the lacZ assay contain interaction between GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide and the protein or peptide encoded by the initially selected cDNA insert.", "IX.", "Assays for Identifying Modulators of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Polypeptide Activity The invention features methods of screening for one or more compounds that modulate the activity of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 in cells, which includes providing potential compounds to be tested to the cells.", "Exemplary assays that may be used are described in the Examples section.", "To these assays would be added compounds to be tested for their inhibitory or stimulatory activity as compared to the effects of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides alone.", "Other assays in which an effect is observed based on the addition of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides can also be used to screen for modulators of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide activity or effects of the presence of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on cells.", "The essential step is to apply an unknown compound and then to monitor an assay for a change from what is seen when only GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are applied to the cell.", "A change is defined as something that is significantly different in the presence of the compound plus GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide compared to GMG 7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide alone.", "In this case, significantly different would be an “increase” or a “decrease” in a measurable effect of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.", "The term “modulation” as used herein refers to a measurable change in an activity.", "Examples include, but are not limited to, lipolysis stimulated receptor (LSR) modulation, leptin modulation, lipoprotein modulation, plasma FFA levels, FFA oxidation, TG levels, glucose levels, and weight.", "These effects can be in vitro or preferably in vivo.", "Modulation of an activity can be either an increase or a decrease in the activity.", "Thus, LSR activity can be increased or decreased, leptin activity can be increased or decreased, and lipoprotein activity can be increased or decreased.", "Similarly, FFA, TG, glucose levels and weight can be increased or decreased in vivo.", "Free Fatty Acid oxidation can be increased or decreased in vivo or ex vivo.", "By “LSR” activity is meant expression of LSR on the surface of the cell, or in a particular conformation, as well as its ability to bind, uptake, and degrade leptin and lipoprotein.", "By “leptin” activity is meant its binding, uptake and degradation by LSR, as well as its transport across a blood brain barrier, and potentially these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.", "Similarly, by “lipoprotein” activity is meant its binding, uptake and degradation by LSR, as well as these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.", "Exemplary assays are provided in the Examples.", "These assay and other comparable assays can be used to determine/identify compounds that modulate GMG-7A, GMG-7, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide activity.", "In some cases it may be important to identify compounds that modulate some but not all of the GMG-7A, GMG-7B, GMG 8, GMG-9, GMG-10, or GMG-11 polypeptide activities, although preferably all activities are modified.", "The term “increasing” as used herein refers to the ability of a compound to increase the activity of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides in some measurable way compared to the effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides in its absence.", "As a result of the presence of the compound leptin binding and/or uptake might increase, for example, as compared to controls in the presence of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide alone.", "Preferably, an increase in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% compared to the level of activity in the presence of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide.", "Preferably, but not intended to be limiting, said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide is GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment comprising all or part of the C-terminal globular C1q homology domain.", "Similarly, the term “decreasing” as used herein refers to the ability of a compound to decrease an activity in some measurable way compared to the effect of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide in its absence.", "For example, the presence of the compound decreases the plasma concentrations of FFA, TG, and glucose in mice.", "Also as a result of the presence of a compound leptin binding and/or uptake might decrease, for example, as compared to controls in the presence of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides alone.", "Preferably, an decrease in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% as compared to the level of activity in the presence of the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides alone.", "Preferably, but not intended to be limiting, said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide is GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment comprising all or part of the C-terminal globular C1q homology domain.", "The invention features a method for identifying a potential compound to decrease body mass in individuals in need of decreasing body mass comprising: a) contacting a cell with a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide and a candidate compound; b) detecting a result selected from the group consisting of LSR modulation, leptin modulation, increase in glucose uptake or oxidation, decrease in blood lipid or triglyceride levels, increase in lipoprotein binding, uptake or degradation; FFA oxidation increase; and c) wherein said result identifies said potential compound if said result differs from said result when said cell is contacted with the GMG 7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide alone.", "Alternatively, the invention features a method for identifying a potential compound to increase body mass in individuals in need of increasing body mass comprising: a) contacting a cell with a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide and a candidate compound; b) detecting a result selected from the group consisting of LSR modulation, leptin modulation, decrease in glucose uptake or oxidation, increase in blood lipid or triglyceride levels, decrease in lipoprotein binding, uptake or degradation; FFA oxidation decrease; and c) wherein said result identifies said potential compound if said result differs from said result when said cell is contacted with the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide alone.", "In still other preferred embodiments, said potential compound is selected from the group consisting of peptides, peptide libraries, non-peptide libraries, peptoids, fatty acids, lipoproteins, medicaments, antibodies, small molecules, proteases and protease inhibitors.", "X. Epitopes and Antibody Fusions A preferred embodiment of the present invention is directed to eiptope-bearing polypeptides and epitope-bearing polypeptide fragments.", "These epitopes may be “antigenic epitopes” or both an “antigenic epitope” and an “immunogenic epitope”.", "An “immunogenic epitope” is defined as a part of a protein that elicits an antibody response in vivo when the polypeptide is the immunogen.", "On the other hand, a region of polypeptide to which an antibody binds is defined as an “antigenic determinant” or “antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes.", "See, e.g., Geysen, et al.", "(1983) Proc Natl Acad Sci USA 81:39984002.It is particularly noted that although a particular epitope nay not be immunogenic, it is nonetheless useful since antibodies can be made in vitro to any epitope.", "An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope.", "Generally an epitope consists of at least 6 such amino acids, and more often at least 8-10 such amino acids.", "In preferred embodiment, antigenic epitopes comprise a number of amino acids that is any integer between 3 and 50.Fragments which function as epitopes may be produced by any conventional means.", "See, e.g., Houghten, R. A., Proc Natl Acad Sci USA 82:5131-5135 (1985), further described in U.S. Pat.", "No.", "4,631,211.Methods for determining the amino acids which make up an immunogenic epitope include x-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping, e.g., the Pepscan method described by H. Mario Geysen et al.", "(1984); Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 81:3998-4002; PCT Publication No.", "WO 84/03564; and PCT Publication No.", "WO 84/03506.Another example is the algorithm of Jameson and Wolf, Comp.", "Appl.", "Biosci.", "4:181-186 (1988) (said references incorporated by reference in their entireties).", "The Jameson-Wolf antigenic analysis, for example, may be performed using the computer program PROTEAN, using default parameters (Version 4.0 Windows, DNASTAR, Inc., 1228 South Park Street Madison, Wis.).", "The epitope-bearing fragments of the present invention preferably comprise 6 to 50 amino acids (i.e.", "any integer between 6 and 50, inclusive) of a polypeptide of the present invention.", "Also, included in the present invention are antigenic fragments between the integers of 6 and the full-length sequence of the sequence listing.", "All combinations of sequences between the integers of 6 and the full-length sequence of a polypeptide of the present invention are included.", "The epitope-bearing fragments may be specified by either the number of contiguous amino acid residues (as a sub-genus) or by specific N-terminal and C-terminal positions (as species) as described above for the polypeptide fragments of the present invention.", "Any number of epitope-bearing fragments of the present invention may also be excluded in the same manner.", "Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies that specifically bind the epitope (See, Wilson et al., 1984; and Sutcliffe, J. G. et al., 1983).", "The antibodies are then used in various techniques such as diagnostic and tissue/cell identification techniques, as described herein, and in purification methods.", "Similarly, immunogenic epitopes can be used to induce antibodies according to methods well known in the art (See, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al.", "; (1985) and Bittle, F. J. et al., (1985).", "A preferred immunogenic epitope includes the polypeptides of the sequence listing.", "The immunogenic epitopes may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) if necessary.", "Immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.).", "Epitope-bearing polypeptides of the present invention are used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods (See, e.g., Sutcliffe, et al., supra; Wilson, et al., supra, and Bittle, et al., 1985).", "If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.", "For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as—maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.", "Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μgs of peptide or carrier protein and Freund's adjuvant.", "Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody, which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.", "The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.", "As one of skill in the art will appreciate, and discussed above, the polypeptides of the present invention including, but not limited to, polypeptides comprising an immunogenic or antigenic epitope can be fused to heterologous polypeptide sequences.", "For example, the polypeptides of the present invention may be fused with the constant region comprising portions of immunoglobulins (IgA, IgE, IgG, IgM), or portions of the constant region (CH1, CH2, CH3, any combination thereof including both entire domains and portions thereof) resulting in chimeric polypeptides.", "These fusion proteins facilitate purification, and show an increased half-life in vivo.", "This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (See, e.g., EPA 0,394,827; and Traunecker et al., 1988).", "Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion can also be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone (See, e.g., Fountoulakis et al., 1995).", "Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag to aid in detection and purification of the expressed polypeptide.", "Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, or codon-shuffling (collectively referred to as “DNA shuffling”).", "DNA shuffling may be employed to modulate the activities of polypeptides of the present invention thereby effectively generating agonists and antagonists of the polypeptides.", "See, for example, U.S. Pat.", "Nos.", "5,605,793; 5,811,238; 5,834,252; 5,837,458; and Patten, P. A., et al., (1997); Harayama, S., (1998); Hansson, L. O., et al (1999); and Lorenzo, M. M. and Blasco, R., (1998).", "(Each of these documents are hereby incorporated by reference).", "In one embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of coding polynucleotides of the invention, or the polypeptides encoded thereby may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc.", "of one or more heterologous molecules.", "Antibodies The present invention further relates to antibodies and T-cell antigen receptors (TCR) that specifically bind the polypeptides, and more specifically, the epitopes of the polypeptides of the present invention.", "The antibodies of the present invention include IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY.", "As used herein, the term “antibody” (Ab) is meant to include whole antibodies, including single-chain whole antibodies, and antigen binding fragments thereof.", "In a preferred embodiment the antibodies are human antigen binding antibody fragments of the present invention include, but are not limited to, Fab, Fab′ F(ab)2 and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.", "The antibodies may be from any animal origin including birds and mammals.", "Preferably, the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken.", "Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire or partial of the following: hinge region, CH1, CH2, and CH3 domains.", "Also included in the invention are any combinations of variable region(s) and hinge region, CH1, CH2, and CH3 domains.", "The present invention further includes chimeric, humanized, and human monoclonal and polyclonal antibodies, which specifically bind the polypeptides of the present invention.", "The present invention further includes antibodies that are anti-idiotypic to the antibodies of the present invention.", "The antibodies of the present invention may be monospecific, bispecific, and trispecific or have greater multispecificity.", "Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or solid support material.", "See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al.", "(1991); U.S. Pat.", "Nos.", "5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; Kostelny, S. A. et al.", "(1992).", "Antibodies of the present invention may be described or specified in terms of the epitope(s) or epitope-bearing portion(s) of a polypeptide of the present invention, which are recognized or specifically bound by the antibody.", "In the case of proteins of the present invention secreted proteins, the antibodies may specifically bind a full-length protein encoded by a nucleic acid of the present invention, a mature protein (i.e., the protein generated by cleavage of the signal peptide) encoded by a nucleic acid of the present invention, a signal peptide encoded by a nucleic acid of the present invention, or any other polypeptide of the present invention.", "Therefore, the epitope(s) or epitope bearing polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or otherwise described herein.", "Antibodies that specifically bind any epitope or polypeptide of the present invention may also be excluded as individual species.", "Therefore, the present invention includes antibodies that specifically bind specified polypeptides of the present invention, and allows for the exclusion of the same.", "Antibodies of the present invention may also be described or specified in terms of their cross-reactivity.", "Antibodies that do not specifically bind any other analog, ortholog, or homolog of the polypeptides of the present invention are included.", "Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein, eg., using FASTDB and the parameters set forth herein) to a polypeptide of the present invention are also included in the present invention.", "Further included in the present invention are antibodies, which only bind polypeptides encoded by polynucleotides, which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein).", "Antibodies of the present invention may also be described or specified in terms of their binding affinity.", "Preferred binding affinities include those with a dissociation constant or Kd value less than 5×10−6M, 10−6M, 5×10−7M, 10−7M, 5×10−8M, 10−8M, 5×10−9M, 10−9M, 5×10−10M, 10−10M, 5×10−11M, 10−11M, 5×10−12M, 10−12M, 5×10−13M, 10−13M, 5×10−14M, 10−14M, 5×10−15M, and 10−15M.", "Antibodies of the present invention have uses that include, but are not limited to, methods known in the art to purify, detect, and target the polypeptides of the present invention including both in vitro and in vivo diagnostic and therapeutic methods.", "For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples (See, e.g., Harlow et al., 1988).", "The antibodies of the present invention may be used either alone or in combination with other compositions.", "The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.", "For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins.", "See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.", "No.", "5,314,995; and EP 0 396 387.The antibodies of the present invention may be prepared by any suitable method known in the art.", "For example, a polypeptide of the present invention or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.", "The term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.", "The term “antibody” refers to a polypeptide or group of polypeptides which are comprised of at least one binding domain, where a binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen, which allows an immunological reaction with the antigen.", "The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.", "Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technology.", "Hybridoma techniques include those known in the art (See, e.g., Harlow et al.", "1988); Hammerling, et al, 1981) (said references incorporated by reference in their entireties).", "Fab and F(ab′)2 fragments may be produced, for example, from hybridoma-produced antibodies by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments).", "Alternatively, antibodies of the present invention can be produced through the application of recombinant DNA technology or through synthetic chemistry using methods known in the art.", "For example, the antibodies of the present invention can be prepared using various phage display methods known in the art.", "In phage display methods, functional antibody domains are displayed on the surface of a phage particle, which carries polynucleotide sequences encoding them.", "Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g.", "human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead.", "Phage used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.", "Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman U. et al.", "(1995); Ames, R. S. et al.", "(1995); Kettleborough, C. A. et al.", "(1994); Persic, L. et al.", "(1997); Burton, D. R. et al.", "(1994); PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat.", "Nos.", "5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743.As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria.", "For example, techniques to recombinantly produce Fab, Fab′ F(ab)2 and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax, R. L. et al.", "(1992); and Sawai, H. et al.", "(1995); and Better, M. et al.", "(1988).", "Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat.", "Nos.", "4,946,778 and 5,258,498; Huston et al.", "(1991); Shu, L. et al.", "(1993); and Skerra, A. et al.", "(1988).", "For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies.", "Methods for producing chimeric antibodies are known in the art.", "See e.g., Morrison, (1985); Oi et al., (1986); Gillies, S. D. et al.", "(1989); and U.S. Pat.", "No.", "5,807,715.Antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 23 9 400; WO 91/09967; U.S. Pat.", "Nos.", "5,530,101; and 5,585,089), veneering or resurfacing, (EP 0 592 106; EP 0 519 596; Padlan E. A., 1991; Studnicka G. M. et al., 1994; Roguska M. A. et al., 1994), and chain shuffling (U.S. Pat.", "No.", "5,565,332).", "Human antibodies can be made by a variety of methods known in the art including phage display methods described above.", "See also, U.S. Pat.", "Nos.", "4,444,887, 4,716,111, 5,545,806, and 5,814,318; WO 98/46645; WO 98/50433; WO 98/24893; WO 96/34096; WO 96/33735; and WO 91/10741.Further included in the present invention are antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide of the present invention.", "The antibodies may be specific for antigens other than polypeptides of the present invention.", "For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.", "Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art (See e.g., Harbor et al.", "supra; WO 93/21232; EP 0 439 095; Naramura, M. et al.", "1994; U.S. Pat.", "No.", "5,474,981; Gillies, S. O. et al., 1992; Fell, H. P. et al., 1991).", "The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.", "For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.", "The antibody portion fused to a polypeptide of the present invention may comprise the hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.", "The polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half-life of the polypeptides or for use in immunoassays using methods known in the art.", "The polypeptides may also be fused or conjugated to the above antibody portions to form multimers.", "For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.", "Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.", "Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art.", "See e.g., U.S. Pat.", "Nos.", "5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. et al.", "(1991); Zheng, X. X. et al.", "(1995); and Vil, H. et al.", "(1992).", "The invention further relates to antibodies that act as agonists or antagonists of the polypeptides of the present invention.", "For example, the present invention includes antibodies that disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.", "Included are both receptor-specific antibodies and ligand-specific antibodies.", "Included are receptor-specific antibodies, which do not prevent ligand binding but prevent receptor activation.", "Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art.", "Also include are receptor-specific antibodies which both prevent ligand binding and receptor activation.", "Likewise, included are neutralizing antibodies that bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies that bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.", "Further included are antibodies that activate the receptor.", "These antibodies may act as agonists for either all or less than all of the biological activities affected by ligand-mediated receptor activation.", "The antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein.", "The above antibody agonists can be made using methods known in the art.", "See e.g., WO 96/40281; U.S. Pat.", "No.", "5,811,097; Deng, B. et al.", "(1998); Chen, Z. et al.", "(1998); Harrop, J.", "A. et al.", "(1998); Zhu, Z. et al.", "(1998); Yoon, D. Y. et al.", "(1998); Prat, M. et al.", "(1998) J.; Pitard, V. et al.", "(1997); Liautard, J. et al.", "(1997); Carlson, N. G. et al.", "(1997) J.; Taryman, R. E. et al.", "(1995); Muller, Y.", "A. et al.", "(1998); Bartunek, P. et al.", "(1996).", "As discussed above, antibodies of the polypeptides of the invention can, in turn, be utilized to generate anti-idiotypic antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art (See, e.g.", "Greenspan and Bona (1989); and Nissinoff (1991).", "For example, antibodies which bind to and competitively inhibit polypeptide multimerization or binding of a polypeptide of the invention to ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization or binding domain and, as a consequence, bind to and neutralize polypeptide or its ligand.", "Such neutralization anti-idiotypic antibodies can be used to bind a polypeptide of the invention or to bind its ligands/receptors, and therby block its biological activity, The invention also concerns a purified or isolated antibody capable of specifically binding to a mutated full length or mature polypeptide of the present invention or to a fragment or variant thereof comprising an epitope of the mutated polypeptide.", "In another preferred embodiment, the present invention concerns an antibody capable of binding to a polypeptide comprising at least 10 consecutive amino acids of a polypeptide of the present invention and including at least one of the amino acids which can be encoded by the trait causing mutations.", "Non-human animals or mammals, whether wild-type or transgenic, which express a different species of a polypeptide of the present invention than the one to which antibody binding is desired, and animals which do not express a polypeptide of the present invention (i.e.", "a knockout animal) are particularly useful for preparing antibodies.", "Gene knock out animals will recognize all or most of the exposed regions of a polypeptide of the present invention as foreign antigens, and therefore produce antibodies with a wider array of epitopes.", "Moreover, smaller polypeptides with only 10 to 30 amino acids may be useful in obtaining specific binding to any one of the polypeptides of the present invention.", "In addition, the humoral immune system of animals that produce a species of a polypeptide of the present invention that resembles the antigenic sequence will preferentially recognize the differences between the animal's native polypeptide species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence.", "Such a technique will be particularly useful in obtaining antibodies that specifically bind to any one of the polypeptides of the present invention.", "Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.", "The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.", "The antibodies of the invention may be labelled by any one of the radioactive, fluorescent or enzymatic labels known in the art.", "Consequently, the invention is also directed to a method for detecting specifically the presence of a polypeptide of the present invention according to the invention in a biological sample, said method comprising the following steps: a) obtaining a biological sample suspected of containing a polypeptide of the present invention; b) contacting the biological sample with a polyclonal or monoclonal antibody that specifically binds a polypeptide of the present invention under conditions suitable for antigen-antibody binding; and c) detecting the antigen-antibody complex formed.", "The invention also concerns a diagnostic kit for detecting in vitro the presence of a polypeptide of the present invention in a biological sample, wherein said kit comprises: a) a polyclonal or monoclonal antibody that specifically binds a polypeptide of the present invention, optionally labelled; b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labelled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labelled by itself.", "A.", "Monoclonal Antibody Production by Hybridoma Fusion Monoclonal antibody to epitopes of any of the peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler, G. and Milstein, C., Nature 256:495 (1975) or derivative methods thereof.", "Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom over a period of a few weeks.", "The mouse is then sacrificed, and the antibody producing cells of the spleen isolated.", "The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media).", "The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.", "Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as Elisa, as originally described by Engvall, E., Meth Enzymol 70:419 (1980), and derivative methods thereof.", "Selected positive clones can be expanded and their monoclonal antibody product harvested for use.", "Detailed procedures for monoclonal antibody production are described in Davis, L. et al.", "Basic Methods in Molecular Biology Elsevier, New York.", "Section 21-2.In a preferred embodiment, said monoclonal antibody is specific for a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or biologically active fragment thereof, wherein said biological activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity.", "Preferably, but not intented to be limiting, said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment comprises all or part of the C-terminal globular C1q homology domain.", "In preferred embodiments, said polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids and not more than 710 consecutive amino acids of SEQ ID NO: 2; at least 6 and not more than 471 consecutive amino acids of SEQ ID NO: 4; at least 6 consecutive amino acids and not more than 201 consecutive amino acids of SEQ ID NO: 6; at least 6 and not more than 446 consecutive amino acids of SEQ ID NO: 8; at least 6 and not more than 296 consecutive amino acids of SEQ ID NO: 10; or at least 6 and not more than 205 consecutive amino acids of SEQ ID NO: 12.B.", "Polyclonal Antibody Production by Immunization Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein or peptides derived therefrom described above, which can be unmodified or modified to enhance immunogenicity.", "Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species.", "For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant.", "Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera.", "Small doses (ng level) of antigen administered at multiple intradermal sites appear to be most reliable.", "An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al.", "J. Clin.", "Endocrinol.", "Metab.", "33:988-991 (1971).", "Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall.", "See, for example, Ouchterlony, O. et al., Chap.", "19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973).", "Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 □M).", "Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap.", "42 in: Manual of Clinical Immunology, 2d Ed.", "(Rose and Friedman, Eds.)", "Amer.", "Soc.", "For Microbiol., Washington, D.C. (1980).", "In a preferred embodiment, said polyclonal antibody is specific for a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or biologically active fragment thereof, wherein said biological activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity.", "Preferably, but not intented to be limiting, said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment comprises all or part of the C-terminal globular C1q homology domain.", "In preferred embodiments, said polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids and not more than 710 consecutive amino acids of SEQ ID NO: 2; at least 6 and not more than 471 consecutive amino acids of SEQ ED NO: 4; at least 6 consecutive amino acids and not more than 201 consecutive amino acids of SEQ ID NO: 6; at least 6 and not more than 446 consecutive amino acids of SEQ ID NO: 8; at least 6 and not more than 296 consecutive amino acids of SEQ ID NO: 10; or at least 6 and not more than 205 consecutive amino acids of SEQ ID NO: 12.Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.", "The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.", "Other characteristics and advantages of the invention are described in the Examples.", "These are meant to be exemplary only, and not to limit the invention in any way.", "Throughout this application, various publications, patents and published patent applications are cited.", "The disclosures of these publications, patents and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure.", "EXAMPLES The following Examples are provided for illustrative purposes and not as a means of limitation.", "One of ordinary skill in the art would be able to design equivalent assays and methods based on the disclosure herein all of which form part of the instant invention.", "Example 1 Northern Analysis of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Expression Analysis of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 expression in different human tissues (adult and fetal) and cell lines, as well as mouse embryos in different stages of development, is accomplished by using poly A+ RNA blots purchased from Clontech (e.g.", "#7780-1, 7757-1, 7756-1, 7768-1 and 7763-1).", "Labeling of RNA probes is performed using the RNA Strip-EZ kit from Ambion as per manufacture's instructions.", "Hybridization of RNA probes to RNA blots is performed Ultrahyb hybridization solution (Ambion).", "Briefly, blots are prehybridized for 30 min at 58° C. (low-strigency) or 65° C. (high stringency).", "After adding the labelled probe (2×106 cpm/ml), blots are hybridized overnight (14-24 hrs), and washed 2×20 min at 50° C. with 2×SSC/0.1% SDS (low stringency), 2×20 min at 58° C. with 1×SSC/0.1% SDS (medium stringency) and 2×20 min at 65° C. with 1×SSC/0.1% SDS (high stringency).", "After washings are completed blots are exposed on the phosphoimager (Molecular Dynamics) for 1-3 days.", "Example 2 In Vitro Tests of Metabolic-Related Activity The activity of various preparations and various sequence variants of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are assessed using various in vitro assays including those provided below.", "These assays are also exemplary of those that can be used to develop GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide antagonists and agonists.", "To do that, the effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides in the above assays, e.g.", "on leptin and/or LSR activity, in the presence of the candidate molecules would be compared with the effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides in the assays in the absence of the candidate molecules.", "Since GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides reduce body weight in mice on a high-cafeteria diet (Example 5), these assays also serve to identify candidate treatments for reducing (or increasing) body weight.", "Liver Cell Line: Tests of efficacy of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on LSR can be performed using liver cell lines, including for example, PLC, HepG2, Hep3B (human), Hepa 1-6, BPRCL (mouse), or MCA-RH777, MCA-RH8994 (rat).", "BPRCL mouse liver cells (ATCC Repository) are plated at a density of 300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose) containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992).", "Media is changed on day 2.On day 3, the confluent monolayers are washed once with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well).", "Cells are incubated at 37° C. for 30 min with increasing concentrations of recombinant GMG-7A, GMG-7B, GMC-8, GMG-9, GMG-10, or GMG-11 polypeptide or GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment in DMEM containing 0.2% (w/v) BSA, 5 mM Hepes, 2 mM CaCl2, 3.7 g/L sodium bicarbonate, pH 7.5.Incubations are continued for 3 h at 37° C. after addition of 10 ng/mL 125I-mouse leptin (specific activity, 22100 cpm/ng).", "Monolayers are washed 2 times consecutively with PBS containing 0.2% BSA, followed by 1 wash with PBS/BSA, and then 2 times consecutively with PBS.", "Cells are lysed with 0.1 N NaOH containing 0.24 mM EDTA.", "Lysates are collected into tubes, and counted in a gamma-counter.", "Blood Brain Barrier Model: The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on leptin transport in the brain can be determined using brain-derived cells.", "One method that is envisioned is to use the blood/brain barrier model described by Dehouck, et al (J Neurochem 54:1798-801, 1990; hereby incorporated herein by reference in its entirety including any figures, tables, or drawings) that uses a co-culture of brain capillary endothelial cells and astrocytes to test the effects of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on leptin (or other molecules) transport via LSR or other receptors.", "This assay would be an indicator of the potential effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on leptin transport to the brain and could be used to screen GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide variants for their ability to modulate leptin transport through LSR or other receptors in the brain.", "In addition, putative agonists and antagonists of the effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on leptin transport through LSR or other receptors could also be screened using this assay.", "Increased transport of leptin across the blood/brain barrier would presumably increase its action as a satiety factor.", "FACS Analysis of LSR Expression The effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on LSR can also be determined by measuring the level of LSR expression at the cell surface by flow surface cytometry, using anti-LSR antibodies and fluorescent secondary antibodies.", "Flow cytometry is a laser-based technology that is used to measure characteristics of biological particles.", "The underlying principle of flow cytometry is that light is scattered and fluorescence is emitted as light from the excitation source strikes the moving particles.", "This is a high throughput assay that could be easily adapted to screen GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides and variants as well as putative agonists or antagonists of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides.", "Two assays are provided below.", "The antibody, cell-line and GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide analogs would vary depending on the experiment, but a human cell-line, human anti-LSR antibody and GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment could be used to screen for variants, agonists, and antagonists to be used to treat humans.", "Assay 1: Cells are pretreated with either intact GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment (or untreated) before harvesting and analysis by FACS.", "Cells are harvested using non-enzymatic dissociation solution (Sigma), and then are incubated for 1 h at 4° C. with a 1:200 dilution of anti-LSR 81B or an irrelevant anti-serum in PBS containing 1% (w/v) BSA.", "After washing twice with the same buffer, goat anti-rabbit FITC-conjugated antibody (Rockland, Gilbertsville, Pa.) is added to the cells, followed by a further incubation for 30 min at 4° C. After washing, the cells are fixed in 2% formalin.", "Flow cytometry analysis is done on a FACSCalibur cytometer (Becton-Dickinson, Franklin Lakes, N.J.).", "Assay 2: Cells are cultured in T175 flasks according to manufacturer's instructions for 48 hours prior to analysis.", "Cells are washed once with FACS buffer (1×PBS/2% FBS, filter sterilized), and manually scraped from the flask in 10 mLs of FACS buffer.", "The cell suspension is transferred to a 15 mL conical tube and centrifuged at 1200 rpm, 4° C. for 5 minutes.", "Supernatant is discarded and cells are resuspended in 10 mL FACS buffer chilled to 4° C. A cell count is performed and the cell density adjusted with FACS buffer to a concentration of 1×106 cells/mL.", "One milliliter of cell suspension was added to each well of a 48 well plate for analysis.", "Cells are centrifuged at 1200 rpm for 5 minutes at 4° C. Plates are checked to ensure that cells are pelleted, the supernatant is removed and cells resuspended by running plate over a vortex mixer.", "One milliliter of FACS buffer is added to each well, followed by centrifugation at 1200 rpm for 5 minutes at 4° C. This described cell washing was performed a total of 3 times.", "Primary antibody, titered in screening experiments to determine proper working dilutions (for example 1:25, 1:50, 1:100, 1:200, 1:400, 1:500, 1:800, 1:1000, 1:2000, 1:4000, 1:5000, or 1:10000), is added to cells in a total volume of 50 μL FACS buffer.", "Plates are incubated for 1 h at 4° C. protected from light.", "Following incubation, cells are washed 3 times as directed above.", "Appropriate secondary antibody, titered in screening experiments to determine proper working dilutions (for example 1:25, 1:50, 1:100, 1:200, 1:400, 1:500, 1:800, 1:1000, 1:2000, 1:4000, 1:5000, or 1:10000), is added to cells in a total volume of 50 μL FACS buffer.", "Plates are incubated for 1 h at 4° C. protected from light.", "Following incubation, cells are washed 3 times as directed above.", "Upon final wash, cells are resuspended in 500 μL FACS buffer and transferred to a FACS acquisition tube.", "Samples are placed on ice protected from light and analyzed within 1 hour.", "Cellular Binding and Uptake of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Polypeptides as Detected by Fluorescence Microscopy Fluorecein isothiocyanate (FITC) conjugation of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides: Purified GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides at 1 mg/mL concentration are labelled with FITC using Sigma's FluoroTag FITC conjugation kit (Stock No.", "FITC-1).", "Protocol outlined in the Sigma Handbook for small-scale conjugation is followed for GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide labeling.", "Cell Culture: C2C12 mouse skeletal muscle cells (ATCC, Manassas, Va. CRL-1772) and Hepa-1-6 mouse hepatocytes (ATCC, Manassas, Va. CRL-1830) are seeded into 6 well plates at a cell density of 2×105 cells per well.", "C2C12 and Hepa-1-6 cells are cultured according to repository's instructions for 24-48 hours prior to analysis.", "Assay is performed when cells were 80% confluent.", "FITC labelled GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide cellular binding and uptake using microscopy: C2C12 and Hepa 1-6 cells are incubated in the presence/absence of antibody directed against human LSR (81B: N-terminal sequence of human LSR; does not cross react with mouse LSR and 93A: c-terminal sequence, cross reacts with mouse LSR) or an antiserum directed against gC1qr (953) for 1 hour at 37° C., 5% CO2.LSR antibodies are added to the media at a concentration of 2 μg/mL.", "The anti-gC1qr antiserum is added to the media at a volume of 2.5 μL undiluted serum (high concentration) or 1:100 dilution (low concentration).", "Following incubation with specified antibody, FITC-GMG-7A, -GMG-7B, -GMG-8, -GMG-9, -GMG-10, or -GMG-11 polypeptide (50 nM/mL) is added to each cell culture well.", "Cells are again incubated for 1 hour at 37° C., 5% CO2.Cells are washed 2× with PBS, cells are scraped from well into 1 mL of PBS.", "Cell suspension is transferred to an eppendorf tube and centrifuged at 1000 rpm for 2 minutes.", "Supernatant is removed and cells resuspended in 200 μL of PBS.", "Binding and uptake of FITC-GMG-7A, -GMG-7B, -GMG-8, -GMG-9, -GMG-10, or -GMG-11 polypeptide is analyzed by fluorescence microscopy under 40× magnification.", "This assay may be useful for identifying agents that facilitate or prevent the uptake and/or binding of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides to cells.", "Effect on LSR as a Lipoprotein Receptor The effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide on the lipoprotein binding, internalizing and degrading activity of LSR can also be tested.", "Measurement of LSR as lipoprotein receptor is described in Bihain & Yen, ((1992) Biochemistry 31:4628-36; hereby incorporated herein in its entirety including any drawings, tables, or figures).", "The effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide on the lipoprotein binding, internalizing and degrading activity of LSR (or other receptors) can be compared with that of intact GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, with untreated cells as an additional control.", "This assay can also be used to screen for active and inhibitory variants of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide, as well as agonists and antagonists of metabolic-related activity.", "Human liver PLC cells (ATCC Repository) are plated at a density of 300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose) containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992).", "Media is changed on day 2.On day 3, the confluent monolayers are washed once with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well).", "Cells are incubated at 37° C. for 30 min with 10 ng/mL human recombinant leptin in DMEM containing 0.2% (w/v) BSA, 5 mM Hepes, 2 mM CaCl2, 3.7 g/L sodium bicarbonate, pH 7.5, followed by another 30 min incubation at 37° C. with increasing concentrations of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide.", "Incubations are continued for 2 h at 37° C. after addition of 0.8 mM oleate and 20 μg/mL 125I-LDL.", "Monolayers are washed 2 times consecutively with PBS containing 0.2% BSA, followed by 1 wash with PBS/BSA, and then 2 times consecutively with PBS.", "The amounts of oleate-induced binding, uptake and degradation of 125I-LDL are measured as previously described (Bihain & Yen, 1992, supra).", "Results are shown as the mean of triplicate determinations.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide leads to an increased activity of LSR as a lipoprotein receptor.", "The oleate-induced binding and uptake of LDL would be more affected by GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide as compared to the degradation.", "This increased LSR activity would potentially result in an enhanced clearance of triglyceride-rich lipoproteins during the postprandial state.", "Thus, more dietary fat would be removed through the liver, rather than being deposited in the adipose tissue.", "This assay could be used to determine the efficiency of a compound (or agonists or antagonists) to increase or decrease LSR activity (or lipoprotein uptake, binding and degradation through other receptors), and thus affect the rate of clearance of triglyceride-rich lipoproteins.", "Effect on Muscle Differentiation C2C12 cells (murine skeletal muscle cell line; ATCC CRL 1772, Rockville, Md.)", "are seeded sparsely (about 15-20%) in complete DMEM (w/glutamine, pen/strep, etc)+10% FCS.", "Two days later they become 80-90% confluent.", "At this time, the media is changed to DMEM+2% horse serum to allow differentiation.", "The media is changed daily.", "Abundant myotube formation occurs after 3-4 days of being in 2% horse serum, although the exact time course of C2C12 differentiation depends on how long they have been passaged and how they have been maintained, among other things.", "To test the effect of the presence of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide on muscle differentiation, GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or polypeptide fragment (1 to 2.5 μg/mL) is added the day after seeding when the cells are still in DMEM w/10% FCS.", "Two days after plating the cells (one day after said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or polypeptide fragment was first added), at about 80-90% confluency, the media is changed to DMEM+2% horse serum plus said GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or polypeptide fragment.", "Effect on Muscle Cell Fatty Acid Oxidation C2C12 cells are differentiated in the presence or absence of 2 μg/mL GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide for 4 days.", "On day 4, oleate oxidation rates are determined by measuring conversion of 1-14C-oleate (0.2 mM) to 14CO2 for 90 min.", "This experiment can be used to screen for active polypeptides and peptides as well as agonists and antagonists or activators and inhibitors of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides.", "The effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or polypeptide fragment on the rate of oleate oxidation can be compared in differentiated C2C12 cells (murine skeletal muscle cells; ATCC, Manassas, Va. CRL-1772) and in a hepatocyte cell line (Hepal-6; ATCC, Manassas, Va. CRL-1830).", "Cultured cells are maintained according to manufacturer's instructions.", "The oleate oxidation assay is performed as previously described Muoio et al (1999) Biochem J 338; 783-791).", "Briefly, nearly confluent myocytes are kept in low serum differentiation media (DMEM, 2.5% Horse serum) for 4 days, at which time formation of myotubes became maximal.", "Hepatocytes are kept in the same DMEM medium supplemented with 10% FCS for 2 days.", "One hour prior to the experiment the media is removed and 1 mL of preincubation media (MEM, 2.5% Horse serum, 3 mM glucose, 4 mM Glutamine, 25 mM Hepes, 1% FFA free BSA, 0.25 mM Oleate, 5 μg/mL gentamycin) is added.", "At the start of the oxidation experiment 14C-Oleic acid (1 μCi/mL, American Radiolabelled Chemical Inc., St. Louis, Mo.)", "is added and cells are incubated for 90 min at 37° C. in the absence/presence of 2.5 μg/mL GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or polypeptide fragment.", "After the incubation period 0.75 mL of the media is removed and assayed for 14C-oxidation products as described below for the muscle FFA oxidation experiment.", "Triglyceride and Protein Analysis Following Oleate Oxidation in Cultured Cells Following transfer of media for oleate oxidation assay, cells are placed on ice.", "To determine triglyceride and protein content, cells are washed with 1 mL of 1×PBS to remove residual media.", "To each well 300 μL of cell dissociation solution (Sigma) is added and incubated at 37° C. for 10 min.", "Plates are tapped to loosen cells, and 0.5 mL of 1×PBS was added.", "The cell suspension is transferred to an eppendorf tube, each well is rinsed with an additional 0.5 mL of 1×PBS, and is transferred to appropriate eppendorf tube.", "Samples are centrifuged at 1000 rpm for 10 minutes at room temperature.", "Supernatant is discarded and 750 μL of 1×PBS/2% chaps is added to cell pellet.", "Cell suspension is vortexed and placed on ice for 1 hour.", "Samples are then centrifuged at 13000 rpm for 20 min at 4° C. Supernatants are transferred to new tube and frozen at −20° C. until analyzed.", "Quantitative measure of triglyceride level in each sample is determined using Sigma Diagnostics GPO-TRIDER enzymatic kit.", "The procedure outlined in the manual is adhered to, with the following exceptions: assay is performed in 48 well plate, 350 μL of sample volume was assayed, control blank consisted of 350 μL PBS/2% chaps, and standard contained 10 μL standard provide in kit plus 690 μL PBS/2% chaps.", "Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm.", "Protein analysis is carried out on 25 μL of each supernatant sample using the BCA protein assay Pierce) following manufacturer's instructions.", "Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm.", "In Vitro Glucose Uptake by Muscle Cells L6 Muscle cells are obtained from the European Culture Collection (Porton Down) and are used at passages 7-11.Cells are maintained in standard tissue culture medium DMEM, and glucose uptake is assessed using [3H]-2-deoxyglucose (2DG) with or without GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment in the presence or absence of insulin (10−8M) as has been previously described (Walker, P. S. et al.", "(1990) Glucose transport activity in L6 muscle cells is regulated by the coordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription, JBC 265:1516-1523; and Kilp, A. et al.", "(1992) Stimulation of hexose transport by metformin in L6 muscle cells in culture, Endocrinology 130:2535-2544, which disclosures are hereby incorporated by reference in their entireties).", "Uptake of 2DG is expressed as the percentage change compared with control (no added insulin or GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment).", "Values are presented as mean±SEM of sets of 4 wells per experiment.", "Differences between sets of wells are evaluated by Student's t test, probability values p<0.05 are considered to be significant.", "Example 3 Effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Polypeptides on Mice Fed a High-Fat Diet Experiments are performed using approximately 6 week old C57Bl/6 mice (8 per group).", "All mice are housed individually.", "The mice are maintained on a high fat diet throughout each experiment.", "The high fat diet (cafeteria diet; D12331 from Research Diets, Inc.) has the following composition: protein kcal % 16, sucrose kcal % 26, and fat kcal % 58.The fat is primarily composed of coconut oil, hydrogenated.", "After the mice are fed a high fat diet for 6 days, micro-osmotic pumps are inserted using isoflurane anesthesia, and are used to provide full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides, GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragments, saline, and an irrelevant peptide to the mice subcutaneously (s.c.) for 18 days.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are provided at doses of 100, 50, 25, and 2.5 μg/day and the irrelevant peptide is provided at 10 μg/day.", "Body weight is measured on the first, third and fifth day of the high fat diet, and then daily after the start of treatment.", "Final blood samples are taken by cardiac puncture and are used to determine triglyceride (TG), total cholesterol (TC), glucose, leptin, and insulin levels.", "The amount of food consumed per day is also determined for each group.", "Example 4 Tests of Metabolic-Related Activity in Humans Tests of the efficacy of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides in humans are performed in accordance with a physician's recommendations and with established guidelines.", "The parameters tested in mice are also tested in humans (e.g.", "food intake, weight, TG, TC, glucose, insulin, leptin, FFA).", "It is expected that the physiological factors would show changes over the short term.", "Changes in weight gain might require a longer period of time.", "In addition, the diet would need to be carefully monitored.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides, preferably biologically active polypeptide fragments thereof, would be given in daily doses of about 6 mg protein per 70 kg person or about 10 mg per day.", "Other doses would also be tested, for instance 1 mg or 5 mg per day up to 20 mg, 50 mg, or 100 mg per day.", "Example 5 Tests of Metabolic-Related Activity in a Murine Lipoatrophic Diabetes Model Previously, leptin was reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al.", "Nature 401:73-76 (1999); hereby incorporated herein in its entirety including any drawings, figures, or tables).", "Leptin was found to be less effective in a different lipodystrophic mouse model of lipoatrophic diabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporated herein in its entirety including any drawings, figures, or tables).", "The instant invention encompasses the use of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides for reducing the insulin resistance and hyperglycaemia in this model either alone or in combination with leptin, the leptin peptide (U.S. provisional application No.", "60/155,506), or other compounds.", "Assays include that described previously in Gavrilova et al.", "((2000) Diabetes 49:1910-6; (2000) Nature 403:850) using A-ZIP/F-1 mice, except that GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides would be administered using the methods previously described in Example 3 (or Examples 6-8).", "The glucose and insulin levels of the mice would be tested, and the food intake and liver weight monitored, as well as other factors, such as leptin, FFA, and TG levels, typically measured in our experiments (see Example 3, above, or Examples 6-8).", "Example 6 Effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Polypeptides on Plasma Free Fatty Acid in C57 BL/6 Mice The effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on postprandial lipemia (PPL) in normal C57BL6/J mice is tested.", "The mice used in this experiment are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.", "All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point).", "At time 0 (8:30 AM), a standard high fat meal (6 g butter, 6 g sunflower oil, 10 g nonfat dry milk, 10 g sucrose, 12 mL distilled water prepared fresh following Nb#6, JF, pg.", "1) is given by gavage (vol.=1% of body weight) to all animals.", "Immediately following the high fat meal, 25 μg a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide is injected i.p.", "in 100 μL saline.", "The same dose (25 μg/mL in 100 μL) is again injected at 45 min and at 1 hr 45 min.", "Control animals are injected with saline (3×100 μL).", "Untreated and treated animals are handled in an alternating-mode.", "Blood samples are taken in hourly intervals, and are immediately put on ice.", "Plasma is prepared by centrifugation following each time point.", "Plasma is kept at −20° C. and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).", "Due to the limited amount of plasma available, glucose is determined in duplicate using pooled samples.", "For each time point, equal volumes of plasma from all 8 animals per treatment group are pooled.", "Example 7 Effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Polypeptides on Plasma Leptin and Insulin in C57 BL/6 Mice The effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on plasma leptin and insulin levels during postprandial lipemia (PPL) in normal C57BL6/J mice is tested.", "The experimental procedure is the same as that described in Example 6, except that blood was drawn only at 0, 2 and 4 hours to allow for greater blood samples needed for the determination of leptin and insulin by RIA.", "Briefly, 16 mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.", "All blood samples are taken from the tail using EDTA coated capillary tubes (100 μL each time point).", "At time 0 (9:00 AM), a standard high fat meal (see Example 6) is given by gavage (vol.=1% of body weight) to all animals.", "Immediately following the high fat meal, 25 fig of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide is injected i.p.", "in 100 μL saline.", "The same dose (25 μg in 100 μL) is again injected at 45 min and at 1 hr 45 min (treated group).", "Control animals are injected with saline (3×100 μL).", "Untreated and treated animals are handled in an alternating mode.", "Blood samples are immediately put on ice and plasma is prepared by centrifugation following each time point.", "Plasma is kept at −20° C. and free fatty acids (FFA) are determined within 24 hours using a standard test kit (Wako).", "Leptin and Insulin are determined by RIA (ML-82K and SRI-13K, LINCO Research, Inc., St. Charles, Mo.)", "following the manufacturer's protocol; however, only 20 μL plasma is used.", "Each determination is done in duplicate.", "Due to the limited amount of plasma available, leptin and insulin are determined in 4 pools of 2 animals each in both treatment groups.", "Example 8 Effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Polypeptides on Plasma FFA, TG and Glucose in C57 BL/6 Mice The effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on plasma FFA, TG, glucose, leptin and insulin levels during postprandial lipemia (PPL) in normal C57BL6/J mice has been described.", "Weight loss resulting from GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides (2.5 μg/day) given to normal C57BL6/J mice on a high fat diet has also been shown (Example 3).", "The experimental procedure is similar to that described in Example 6.Briefly, 14 mice re fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.", "All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point).", "At time 0 (9:00 AM), a standard high fat meal (see Example 6) is given by gavage (vol.=1% of body weight) to all animals.", "Immediately following the high fat meal, 4 mice are injected 25 μg of a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide i.p.", "in 100 μL saline.", "The same dose (25 μg in 100 μL) is again injected at 45 min and at 1 hr 45 min.", "A second treatment group receives 3 times 50 μg GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide at the same intervals.", "Control animals are injected with saline (3×100 μL).", "Untreated and treated animals are handled in an alternating mode.", "Blood samples are immediately put on ice.", "Plasma is prepared by centrifugation following each time point.", "Plasma is kept at −20° C. and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).", "Example 9 Effect of GMG-7A, GMG-7B, GMG-8, GM 9, GMG-10, or GMG-11 Polypeptides on FFA Following Epinephrine Injection In mice, plasma free fatty acids increase after intragastric administration of a high fat/sucrose test meal.", "These free fatty acids are mostly produced by the activity of lipolytic enzymes i.e.", "lipoprotein lipase (LPL) and hepatic lipase (HL).", "In this species, these enzymes are found in significant amounts both bound to endothelium and freely circulating in plasma.", "Another source of plasma free fatty acids is hormone sensitive lipase (HSL) that releases free fatty acids from adipose tissue after β-adrenergic stimulation.", "To test whether GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides also regulate the metabolism of free fatty acid released by HSL, mice are injected with epinephrine.", "Two groups of mice are given epinephrine (5 μg) by intraperitoneal injection.", "A treated group is injected with a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide (25 μg) one hour before and again together with epinephrine, while control animals receive saline.", "Plasma is isolated and free fatty acids and glucose are measured as described above (Example 8).", "Example 10 Effect of GMG-7A GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Polypeptides on Muscle FFA Oxidation To investigate the effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on muscle free fatty acid oxidation, intact hind limb muscles from C57BL/6J mice are isolated and FFA oxidation is measured using oleate as substrate (Clee, S. M. et al.", "Plasma and vessel wall lipoprotein lipase have different roles in atherosclerosis.", "J Lipid Res 41, 521-531 (2000); Muoio, D. M., Dohm, G. L., Tapscott, E. B.", "& Coleman, R. A. Leptin opposes insulin's effects on fatty acid partitioning in muscles isolated from obese ob/ob mice.", "Am J Physiol 276, E913-921 (1999)) Oleate oxidation in isolated muscle is measured as previously described (Cuendet et al (1976) J Clin Invest 58:1078-1088; Le Marchand-Brustel, Y., Jeanrenaud, B.", "& Freychet, P. Insulin binding and effects in isolated soleus muscle of lean and obese mice.", "Am J Physiol 234, E348-E358 (1978).", "Briefly, mice are sacrificed by cervical dislocation and soleus and EDL muscles are rapidly isolated from the hind limbs.", "The distal tendon of each muscle is tied to a piece of suture to facilitate transfer among different media.", "All incubations are carried out at 30° C. in 1.5 mL of Krebs-Henseleit bicarbonate buffer (118.6 mM NaCl, 4.76 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgSO4, 2.54 mM CaCl2, 25 mM NaHCO3, 10 mM Hepes, pH 7.4) supplemented with 4% FFA free bovine serum albumin (fraction V, RIA grade, Sigma) and 5 mM glucose (Sigma).", "The total concentration of oleate (Sigma) throughout the experiment is 0.25 mM.", "All media are oxygenated (95% O2; 5% CO2) prior to incubation.", "The gas mixture is hydrated throughout the experiment by bubbling through a gas washer (Kontes Inc., Vineland, N.J.).", "Muscles are rinsed for 30 min in incubation media with oxygenation.", "The muscles are then transferred to fresh media (1.5 mL) and incubated at 30° C. in the presence of 1 μCi/mL [1-14C] oleic acid (American Radiolabelled Chemicals).", "The incubation vials containing this media are sealed with a rubber septum from which a center well carrying a piece of Whatman paper (1.5 cm×11.5 cm) is suspended.", "After an initial incubation period of 10 min with constant oxygenation, gas circulation is removed to close the system to the outside environment and the muscles are incubated for 90 min at 30° C. At the end of this period, 0.45 mL of Solvable (Packard Instruments, Meriden, Conn.) is injected onto the Whatman paper in the center well and oleate oxidation by the muscle is stopped by transferring the vial onto ice.", "After 5 min, the muscle is removed from the medium, and an aliquot of 0.5 mL medium is also removed.", "The vials are closed again and 1 mL of 35% perchloric acid is injected with a syringe into the media by piercing through the rubber septum.", "The CO2 released from the acidified media is collected by the Solvable in the center well.", "After a 90 min collection period at 30° C., the Whatman paper is removed from the center well and placed in scintillation vials containing 15 mL of scintillation fluid (HionicFlour, Packard Instruments, Meriden, Conn.).", "The amount of 14C radioactivity is quantitated by liquid scintillation counting.", "The rate of oleate oxidation is expressed as nmol oleate produced in 90 min/g muscle.", "To test the effect of full-length GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide or GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide fragment on oleate oxidation, these proteins are added to the media at a final concentration of 2.5 μg/mL and maintained in the media throughout the procedure.", "Example 11 Effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Polypeptides on Triglyceride in Muscle & Liver Isolated from Mice To determine whether the increased FFA oxidation induced by GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides is also accompanied by increased FFA delivery into muscle or liver, the hindlimb muscle and liver triglyceride content is measured after the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide treatment of mice.", "Hind limb muscles as well as liver samples are removed from treated and untreated animals and the triglyceride and free fatty acid concentration is determined following a standard lipid extraction method (Shimabukuro, M. et al.", "Direct antidiabetic effect of leptin through triglyceride depletion of tissues.", "Proc Natl Acad Sci USA 94:4637-4641 (1997)) followed by TG and FFA analysis using standard test kits.", "Example 12 Effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 Polypeptides on FFA Following Intralipid Injection Two groups of mice are intravenously (tail vein) injected with 30 μL bolus of Intralipid-20% (Clintec) to generate a sudden rise in plasma FFAs, thus by-passing intestinal absorption.", "(Intralipid is an intravenous fat emulsion used in nutritional therapy).", "A treated group (GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide-treated) is injected with a GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptide (25 μg) at 30 and 60 minutes before Intralipid is given, while control animals (0 control) received saline.", "Plasma is isolated and FFAs are measured as described previously.", "The effect of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on the decay in plasma FFAs following the peak induced by Intralipid injection is then monitored.", "Example 13 In Vitro Glucose Uptake by Muscle Cells L6 Muscle cells are obtained from the European Culture Collection (Porton Down) and are used at passages 7-11.Cells are maintained in standard tissue culture medium DMEM, and glucose uptake is assessed using [3H]-2-deoxyglucose (2DG) with or without GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides in the presence or absence of insulin (10−8 M) as has been previously described (Walker, P. S. et al.", "(1990) Glucose transport activity in L6 muscle cells is regulated by the coordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription.", "JBC 265:1516-1523; and Kilp, A. et al.", "(1992) Stimulation of hexose transport by metformin in L6 muscle cells in culture, Endocrinology 130:2535-2544, which disclosures are hereby incorporated by reference in their entireties).", "Uptake of 2DG is expressed as the percentage change compared with control (no added insulin or GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11).", "Values are presented as mean±SEM of sets of 4 wells per experiment.", "Differences between sets of wells are evaluated by Student's t test, probability values p<0.05 are considered to be significant.", "Example 14 In Vivo Tests for Metabolic-Related Activity in Rodent Diabetes Models As metabolic profiles differ among various animal models of obesity and diabetes, analysis of multiple models is undertaken to separate the effects GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides on hyperglycemia, hyperinsulinemia, hyperlipidemia and obesity.", "Mutations within colonies of laboratory animals and different sensitivities to dietary regimens have made the development of animal models with non-insulin dependent diabetes associated with obesity and insulin resistance possible.", "Genetic models such as db/db and ob/ob (See Diabetes, (1982) 31:1-6) in mice and fa/fa in zucker rats have been developed by the various laboratories for understanding the pathophysiology of disease and testing the efficacy of new antidiabetic compounds (Diabetes, (1983) 32:830-838; Annu Rep Sankyo Res Lab (1994) 46:1-57).", "The homozygous animals, C57 BL/KsJ-db/db mice developed by Jackson Laboratory, US, are obese, hyperglycemic, hyperinsulinemic and insulin resistant (J Clin Invest, (1990) 85:962-967), whereas heterozygous are lean and normoglycemic.", "In db/db model, mouse progressively develops insulinopenia with age, a feature commonly observed in late stages of human type II diabetes when blood sugar levels are insufficiently controlled.", "The state of pancreas and its course vary according to the models.", "Since this model resembles that of type II diabetes mellitus, the compounds of the present invention are tested for blood sugar and triglycerides lowering activities.", "Zucker (fa/fa) rats are severely obese, hyperinsulinemic, and insulin resistant (Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Riflin and D. Porte, Jr. Eds.", "(Elsevier Science Publishing Co., Inc., New York, ed.", "4, 1990), pp.", "299-340), and the fa/fa mutation may be the rat equivalent of the murine db mutation (Friedman et al., Cell 69:217-220, 1992; Truett et al., Proc Natl Acad Sci USA 88:7806, 1991).", "Tubby (tub/tub) mice are characterized by obesity, moderate insulin resistance and hyperinsulinemia without significant hyperglycemia (Coleman et al., J. Heredity 81:424, 1990).", "Previously, leptin was reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al.", "Nature 401: 73-76 (1999).", "Leptin is found to be less effective in a different lipodystrophic mouse model of lipoatrophic diabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporated herein in its entirety including any drawings, figures, or tables).", "The streptozotocin (STZ) model for chemically-induced diabetes is tested to examine the effects of hyperglycemia in the absence of obesity.", "STZ-treated animals are deficient in insulin and severely hyperglycemic (Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds.", "(Elsevier Science Publishing Co., Inc., New York, ed.", "4, 1990), pp.", "299-340).", "The monosodium glutamate (MSG) model for chemically-induced obesity (Olney, Science 164:719, 1969; Cameron et al., Clin Exp Pharmacol Physiol 5:41, 1978), in which obesity is less severe than in the genetic models and develops without hyperphagia, hyperinsulinemia and insulin resistance, is also examined.", "Finally, a non-chemical, non-genetic model for induction of obesity includes feeding rodents a high fat/high carbohydrate (cafeteria diet) diet ad libitum.", "The instant invention encompasses the use of GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides for reducing the insulin resistance and hyperglycemia in any or all of the above rodent diabetes models or in humans with Type I or Type II diabetes or other prefered metabolic diseases described previously or models based on other mammals.", "In the compositions of the present invention the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides may, if desired, be associated with other compatible pharmacologically active antidiabetic agents such as insulin, leptin (U.S. provisional application No.", "60/155,506), or troglitazone, either alone or in combination.", "Assays include that described previously in Gavrilova et al.", "((2000) Diabetes 49:1910-6; (2000) Nature 403:850) using A-ZIP/F-1 mice, except that GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides are administered intraperitoneally, subcutaneously, intramuscularly or intravenously.", "The glucose and insulin levels of the mice would be tested, and the food intake and liver weight monitored, as well as other factors, such as leptin, FFA, and TG levels, typically measured in our experiments.", "In Vivo Assay for Anti-Hyperglycemic Activity of GMG-7A, GMG-7B GMG-8, GMG-9, GMG-10, or GMG-11 Polypeptides Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old) are housed (7-9 mice/cage) under standard laboratory conditions at 22° C. and 50% relative humidity, and maintained on a diet of Purina rodent chow and water ad libitum.", "Prior to treatment, blood is collected from the tail vein of each animal and blood glucose concentrations are determined using One Touch Basic Glucose Monitor System (Lifescan).", "Mice that have plasma glucose levels between 250 to 500 mg/dl are used.", "Each treatment group consists of seven mice that are distributed so that the mean glucose levels are equivalent in each group at the start of the study.", "db/db mice are dosed by micro-osmotic pumps, inserted using isoflurane anesthesia, to provide GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides, saline, and an irrelevant peptide to the mice subcutaneously (s.c.).", "Blood is sampled from the tail vein hourly for 4 hours and at 24, 30 h post-dosing and analyzed for blood glucose concentrations.", "Food is withdrawn from 0-4 h post dosing and reintroduced thereafter.", "Individual body weights and mean food consumption (each cage) are also measured after 24 h. Significant differences between groups (comparing GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 treated to saline-treated) are evaluated using Student t-test.", "In Vivo Insulin Sensitivity Assay In vivo insulin sensitivity is examined by utilizing two-step hyperinsulinemic-euglycemic clamps according to the following protocol.", "Rodents from any or all of the various models described in Example 2 are housed for at least a week prior to experimental procedures.", "Surgeries for the placement of jugular vein and carotid artery catheters are performed under sterile conditions using ketamine and xylazine (i.m.)", "anesthesia.", "After surgery, all rodents are allowed to regain consciousness and placed in individual cages.", "GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides or vehicle is administered through the jugular vein after complete recovery and for the following two days.", "Sixteen hours after the last treatment, hyperinsulinemic-euglycemic clamps are performed.", "Rodents are placed in restrainers and a bolus of 4 μCi [3-3H] glucose (NEN) is administered, followed by a continuous infusion of the tracer at a dose of 0.2 μCi/min (20 μl/min).", "Two hours after the start of the tracer infusion, 3 blood samples (0.3 ml each) are collected at 10 minute intervals (−20-0 min) for basal measurements.", "An insulin infusion is then started (5 mU/kg/min), and 100 μl blood samples are taken every 10 min.", "to monitor plasma glucose.", "A 30% glucose solution is infused using a second pump based on the plasma glucose levels in order to reach and maintain euglycemia.", "Once a steady state is established at 5 mU/kg/min insulin (stable glucose infusion rate and plasma glucose), 3 additional blood samples (0.3 ml each) are obtained for measurements of glucose, [3-3H] glucose and insulin (100-120 min.).", "A higher dose of insulin (25 mU/kg/min.)", "is then administered and glucose infusion rates are adjusted for the second euglycemic clamp and blood samples are taken at min.", "220-240.Glucose specific activity is determined in deproteinized plasma and the calculations of Rd and hepatic glucose output (HGO) are made, as described (Lang et al., Endocrinology 130:43, 1992).", "Plasma insulin levels at basal period and after 5 and 25 mU/kg/min.", "infusions are hen determined and compared between GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 treated and vehicle treated rodents.", "Insulin regulation of glucose homeostasis has two major components; stimulation of peripheral glucose uptake and suppression of hepatic glucose output.", "Using tracer studies in the glucose clamps, it is possible to determine which portion of the insulin response is affected by the GMG-7A, GMG-7B, GMG-8, GMG-9, GMG-10, or GMG-11 polypeptides." ] ]	Patent_10466376
[ [ "Watermarked paper", "Watermarked paper, preferably shadow-watermarked paper is produced in conventional manner and then size-press dyed using a pigment dyestuff composition.", "This results in unusual and attractive decorative effects as a result of preferential dye take-up in parts of the watermarked areas, resulting in enhanced contrast between watermarked and non-watermarked areas of the paper.", "The invention finds particular application in wallpaper base, but can also be used in decorative or security papers.", "The pigment dyestuff can be fluorescent for security paper use." ], [ "1.A method of producing a dyed watermarked paper, wherein paper carrying a watermark is produced on a papermachine in conventional manner, characterized in that dyeing is carried out at the size press of the papermachine using a pigment dyestuff composition.", "2.A method as claimed in claim 1, wherein the watermark carried by the paper is a shadow watermark.", "3.A method as claimed in claim 2, wherein the shadow watermark is applied by means of a dandy roll which is driven slightly faster than the papermachine wire.", "4.A method as claimed in any preceding claim, wherein the pigment dyestuff provides colour under daylight illumination.", "5.A method as claimed in any of claims 1 to 3, wherein the pigment dyestuff is fluorescent and provides colour under ultra-violet illumination.", "6.A method as claimed in any preceding claim, wherein the paper is a wallpaper base made from a blend of a major proportion of hardwood pulp to provide bulk characteristics and a minor, but still quite high, proportion of softwood pulp to provide strength and dimensional stability.", "7.A method as claimed in claim 6, wherein said blend comprises about 70% by weight of eucalyptus pulp and 30% by weight of Kraft or pine softwood pulp.", "8.A method as claimed in claim 6 or claim 7, wherein the paper is calendered to a Bendtsen roughness value of about 350-400 ml min−1.9.A method as claimed in any of claims 1 to 5, wherein the paper is a security paper or a decorative paper." ], [ "This invention relates to dyed watermarked paper suitable for conversion into wallpaper or for use for decorative, security or other purposes.", "Watermarking is a long-established paper industry technique for incorporating words, images and patterns into paper in an unobtrusive manner which does not interfere with the use to which the paper is to be put but which indicates the origin of the paper or guarantees that it is genuine.", "Thus watermarking is widely employed in the manufacture of banknotes, security papers, and high-quality branded or bespoke stationery for business or personal use.", "The watermark image is produced by inducing localized variation in the thickness of the paper.", "This in turn creates localized variation in the opacity and texture of the paper, and so creates a contrast which makes the watermark visible, particularly in transmitted light.", "The desired localized variation in paper thickness is typically effected by fibre displacement by means of a so-called dandy roll which runs on top of the wet web on the wire of a Fourdrinier papermachine and carries an array of electrotype- or intaglio-type designs corresponding to the watermark to be applied.", "Electrotype designs stand proud of the surface of the dandy roll, and so the fibre displacement they cause results in localized thinning of the paper, and therefore areas of reduced opacity/greater light transmittance.", "By contrast, intaglio designs are recessed into the dandy roll surface, usually by embossing, and so the fibre displacement they cause results in localized thickening of the paper and therefore areas of greater opacity/lesser light transmittance.", "These areas appear darker than the surrounding paper when held up to the light, and so are often referred to as “shadow” or “shade” watermarks.", "It is also common when embossing a dandy roll with an intaglio design to incorporate raised as well as recessed areas in the dandy roll surface.", "The resulting papers therefore include areas of both greater and reduced thickness and opacity, and this enables aesthetically pleasing high-contrast watermarks to be produced, with shading, half-tones and fine detail.", "In the case of a cylinder mould papermachine, the wire mesh of the mould can be adapted to carry similar electrotype- or intaglio-style designs which likewise give rise to localized variation in paper thickness and watermarking effects as already described.", "Although watermarking has hitherto generally been used to indicate the origin or genuineness of the paper, this does not preclude the simultaneous achievement of a decorative effect.", "Conventionally, however, such an effect is limited by its inherently monochrome character, i.e.", "the watermarked paper is either white or is dyed to a single uniform colour by addition of a direct dye to the pulp suspension from which the paper is made.", "We have now discovered that unusual and attractive decorative or security effects can be achieved with a watermarked paper, particularly where the watermark is a shadow watermark, if the paper is size-press dyed after it has been produced using one or more pigment dyestuffs.", "Such dyestuffs differ from the soluble direct dyestuffs commonly used for colouring paper during its production.", "The decorative effect appears to result from selective preferential dye take-up in parts of the watermarked areas of the paper, and a consequential enhanced contrast between the watermarked and non-watermarked areas of the paper.", "The preferential dye take-up is at its greatest in relatively thick areas of the paper and this is thought to be the reason why the most noticeable and attractive effects are obtained with a paper which has been shadow-watermarked.", "Accordingly, the present invention provides, in a first aspect, a method of producing a dyed watermarked paper wherein paper carrying a watermark, preferably a shadow watermark, is produced on a papermachine in conventional manner, characterized in that dyeing is carried out at the size press of the papermachine using a pigment dyestuff composition.", "In a second aspect, the present invention resides in dyed watermarked paper produced by a method according to said first aspect of the invention.", "The pigment dyestuff composition can be made up in conventional manner, for example by initial dispersion in water in a holding or mixing tank, followed by transfer to a high shear mixer and thence to the size press.", "The use of pigment dyestuffs as opposed to more commonly used and cheaper soluble dyestuffs gives a more pronounced decorative or security effect.", "It also makes it easier to control batch-to-batch colour shade variations, and leads to a more fade-resistant pattern in the final product.", "The pigment dyestuff used can provide colour under normal daylight illumination or it can be fluorescent so as to provide colour under ultra-violet illumination for security paper applications.", "A combination of fluorescent and non-fluorescent pigment dyestuffs can also be used.", "The present invention can be applied to Fourdrinier or cylinder mould papers of a wide range of basis weights (grammages), including heavyweight papers of the kind sometimes referred to as card or board.", "The size press used in the present method may be of the kind traditionally used in papermaking, or of the modified form known as a metered size press and commercialised more recently under names such as the “Speedsizer” by Voith Sulzer, the “Symsizer” by Valmet, the “Film Press” by Jagenberg, and the “Twin-HSM” by BTG Kalle.", "The present invention can advantageously be applied to the production of wallpaper base.", "For this application, it is important that the product should have a high bulk for a given grammage, and yet be strong with good dimensional stability.", "Accordingly the furnish should contain a blend of a major proportion of hardwood pulp to provide the desired bulk characteristics and a minor, but still quite high, proportion of softwood pulp to provide strength and dimensional stability.", "The hardwood pulp can be for example, eucalyptus or birch pulp.", "The softwood pulp can be, for example, Kraft or pine pulp.", "We have found a blend of about 70% by weight of eucalyptus pulp and 30% by weight of Kraft or pine softwood pulp to be particularly suitable.", "No filler, dyestuffs, or optical brightening agent (“OBA”) or broke containing these materials should be included in the furnish (the presence of filler would be likely to produce undesirable two-sidedness in the sheet, and the presence of dyestuffs or OBA would be likely to interfere with the subsequent dyeing of the paper, making it difficult to control batch-to-batch variations in shade).", "The presence of soluble dyestuffs would also be likely to result in gradual colour change, as soluble dyestuffs are prone to fading, particularly when permanently exposed to light as in the case of wallpaper.", "The furnish desirably also includes a conventional amount of a wet strength agent, say 1.5 to 1.7% by weight, based on dry fibre content, and an internal sizing agent, for example a resin/polyaluminium chloride (PAC) composition in an amount of about 2% by weight, based on dry fibre content.", "When a dandy roll is used, to form the watermark, we have found it advantageous to drive the dandy roll slightly faster than the papermachine wire.", "This “drags” more fibre into the recessed areas of the dandy roll and produces a more pronounced shadow effect whilst maximising the dimensional stability of the paper.", "The resulting increased fibre content results in higher pigment take up during the subsequent dyeing operation, and so in an enhanced colouring effect and a more prominent watermark.", "After production and dyeing of the paper, it is normally calendered in conventional manner to a desired smoothness value, although this is not critical.", "In this context, smoothness can conveniently be measured in terms of a Bendtsen roughness value.", "We have found a Bendtsen roughness value of 350-400 ml min−1 is suitable, but the invention can be applied to papers with higher Bendtsen values, i.e.", "rougher papers, to achieve particular desired aesthetic effects.", "After calendering, the paper can be reeled up for subsequent conversion into rolls of wallpaper or paper for other end uses.", "In the case of wallpaper, the rolls can be directly prepasted if desired, or the dyed watermarked paper can be laminated onto a backing paper which can itself be prepasted.", "When the present invention is employed in the manufacture of security papers, or decorative papers, for example decorative printing papers, the furnish and production methods used can be as is conventional for those types of paper.", "Although the invention is particularly suited to pigment-dyed shadow-watermarked papers, we have observed that a more modest colour—accentuation effect is obtained with embossed watermarks.", "For example, the lines on a discrete image embossed watermark can be made more visible by pigment dyeing.", "The invention will now be illustrated by the following Examples in which all parts and percentages are by weight unless otherwise stated: EXAMPLE 1 A 70% eucalyptus hardwood/30% Kraft softwood fibre stock was prepared in conventional manner in a pulper at about 5 to 6% consistency and refined in a generally conventional manner appropriate to the production of a high bulk yet strong and dimensionally stable paper suitable for use as a wallpaper base.", "The resulting stock was then pumped to a header tank, diluted, and projected on to a Fourdrinier papermaking wire from a headbox slice to produce paper in the normal way.", "The process conditions were selected with the aim of producing a final dried paper of a grammage of about 120 g m−2 (after size-press sizing and dyeing).", "No fillers, dyestuff or OBA were present, but the stock contained 1.7% wet strength agent, based on the dry weight of as-supplied pulp used, and 2% resin/PAC internal sizing agent.", "The papermachine was equipped with a shadow-watermarking dandy roll which imparted an all-over diagonal lattice pattern of which the lines of the lattice resembled a rope (a “cordage” pattern).", "The papermachine was equipped with a horizontal size press to which a beige-green pigment dyestuff size composition was supplied at approximately 40% solids content.", "The size press formulation was as follows: Parts Pigment dyestuff 10 Starch 8 Water 82 The size press pick-up was approximately 4% on a dry basis, and the average grammage of the final product was 125 g m−2.The mean Bendtsen roughness after calendering was 385 ml min−1.The product had an attractive appearance, with the cordage watermark pattern clearly visible in both reflected and transmitted light.", "The colouring in certain areas of individual plies of the “rope” was noticeably accentuated, giving an unusual and attractive effect.", "EXAMPLE 2 The procedure of Example 1 was repeated, except that a violet pigment dye formulation was used and two differently-watermarked papers were produced.", "The pattern for the first of these was as in Example 1, but the second had a continuous wavy line or “spaghetti” watermark pattern, achieved by the use of a different dandy roll.", "The size press formulation was: Parts Pigment dyestuff 5 Starch size 8 Water 87 The size press pick-up was approximately 4%, and the final product had an average grammage of 120 g m−2.The mean Bendtsen roughness value after calendering was 385 ml min−1.Both of the products had an attractive appearance, with the watermark pattern clearly visible in both reflected and transmitted light.", "The colouring in certain areas of individual plies of the “rope” and “spaghetti” watermark patterns was noticeably accentuated, giving an unusual and attractive effect." ] ]	Patent_10466416
[ [ "Device for metering a urea soulution", "A device for metering urea solutions permitting a reliable reduction of nitrogen oxides in the exhaust gas of an internal combustion engine is provided.", "This is achieved by the fact that the device for metering the urea solution includes a sensor unit for monitoring one or more physical state variables of an enzyme-free Urea solution." ], [ "1.A device for metering a urea solution, in particular for spraying the urea solution into the exhaust gas stream of an internal combustion engine, wherein a sensor unit is provided for monitoring one or more physical state variables of an enzyme-free urea solution using a physical measuring sensor (3, 6, 9).", "2.The device as recited in claim 1, wherein the measuring sensor (3, 6) is designed for detecting an electric state variable.", "3.The device as recited in one of the preceding claims, wherein the measuring sensor (3, 6, 7) is designed for detecting the pH, the dielectric constant, and/or the conductance of the enzyme-free urea solution.", "4.The device as recited in one of the preceding claims, wherein the measuring sensor (3, 6, 7) includes at least two electrodes.", "5.The device as recited in one of the preceding claims, wherein at least one electrode (3, 6, 7) has a structure for increasing the surface area.", "6.The device as recited in one of the preceding claims, wherein two electrodes (3, 6) have an intermeshing comb-like structure.", "7.The device as recited in one of the preceding claims, wherein at least one third electrode (7) is provided for detecting at least one second electric state variable.", "8.The device as recited in one of the preceding claims, wherein the measuring sensor (9) is designed for detecting a physicomechanical state variable.", "9.The device as recited in one of the preceding claims, wherein the measuring sensor (9) is designed for measuring the viscosity and/or density of the enzyme-free urea solution.", "10.The device as recited in one of the preceding claims, wherein a vibration generator (9) is provided.", "11.The device as recited in one of the preceding claims, wherein the vibration generator includes a quartz oscillator (9) and/or a piezoelectric crystal.", "12.The device as recited in one of the preceding claims, wherein a sensor unit (1) having a measuring sensor (3, 6, 7) for an electric state variable of the urea solution and having a measuring sensor (9) for a physicomechanical state variable is provided, an analyzer unit being provided for determining the urea concentration from the two measured values.", "13.The device as recited in one of the preceding claims, wherein a temperature sensor is provided.", "14.The device as recited in one of the preceding claims, wherein a filling level sensor is provided for a storage container.", "15.The device as recited in one of the preceding claims, wherein the filling level sensor is a measuring sensor according to one of the preceding claims.", "16.The device as recited in one of the preceding claims, wherein a plurality of filling level sensors is provided.", "17.An internal combustion engine having catalytic exhaust gas treatment, wherein a device for metering a urea solution according to one of the preceding claims is provided." ], [ "<SOH> BACKGROUND INFORMATION <EOH>To reduce nitrogen oxides in the exhaust gas of motor vehicles, urea solution has in the past been sprayed into the exhaust gas during catalytic reduction.", "Urea is broken down into carbon dioxide and ammonia by chemical reaction on a hydrolysis catalyst.", "Ammonia then reacts selectively with nitrogen oxides to form nitrogen and water, thus removing nitrogen oxides from the exhaust gas.", "For reliable reduction of nitrogen oxides with a urea solution, various parameters are important, in particular the urea concentration in the aqueous solution.", "Sensor applications known in the past for measuring the urea concentration in the fields of medicine and biology have used urease, which enzymatically and selectively breaks down urea to form ammonia.", "Sensors then detect the influence of the ammonia on the pH of the solution.", "Information regarding the urea concentration is obtainable in this way.", "One disadvantage of this method of measuring the concentration of a urea solution is the instability of urease, in particular in an environment where temperatures may vary greatly.", "However, such temperature variations occur during use in motor vehicles, so that previous sensors according to the related art are not suitable for such an application.", "Therefore, the object of the present invention is to propose a device for metering urea solutions which may be used reliably for reduction of nitrogen oxides, even under difficult conditions, e.g., within broad temperature intervals." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Accordingly, a device according to the present invention for metering urea is characterized in that a sensor unit is provided for monitoring a physical state variable of an enzyme-free urea solution.", "The sensor unit here preferably includes a measuring sensor.", "In this way, a measurement is possible directly on the basis of the physical properties of urea in solution without intermediate enzymatic breakdown.", "Accordingly, this measurement is not subject to the instabilities to which an enzyme such as urease is subject.", "In an exemplary embodiment of the present invention, a measuring sensor is provided for detecting one or more electric state variables.", "Such a state variable may include, for example, the pH, the dielectric constant and/or the conductance of the solution.", "By measuring these or other electric state variables, it is possible to obtain information regarding the properties of the urea solution, e.g., its concentration.", "Measurement of these state variables is comparatively unproblematical and in particular it is possible to perform these measurements in situations of extreme temperature variations.", "Two electrodes may be provided to detect the electric state variables, these electrodes protruding into the urea solution.", "By applying an electric d.c. or a.c. voltage to the electrodes, it is possible to determine directly the aforementioned electric state variables, such as the pH, the dielectric constant, and/or the conductance.", "To improve the sensitivity of the measuring sensor the electrodes may be provided with a structure which increases their surface area.", "Such a surface area enlarging structure may be achieved, e.g., by a comb-shaped design of the electrodes, which additionally has the advantage that two electrodes designed in this way may be arranged to intermesh, so that a small distance between the two electrodes is adjustable simultaneously with a comparatively large surface area.", "Due to the large surface area, in particular in combination with the small distance, the test voltage and/or test current may be reduced and therefore the control and analyzing unit for a measuring sensor according to the present invention may be designed with small dimensions.", "A separate electrode may be provided for simultaneous determination of multiple state variables, if necessary.", "For example, by using such a third electrode, it is possible to determine the pH, while another state variable, e.g., the dielectric constant, is determined using the two aforementioned electrodes.", "In an exemplary embodiment of the present invention, a measuring sensor is provided for detecting one or more physicomechanical state variables of the urea solution.", "Such a physicomechanical state variable may be the viscosity or density, for example.", "Such physicomechanical state variables may be determined in a traditional manner, e.g., by weighing the solution and/or a part of the solution or by measuring the buoyancy of a displacement body, etc.", "However, in an exemplary embodiment the physicomechanical state variable is detected by a dynamic sensor.", "Thus, a physicomechanical state variable may be measured with the help of a vibration generator, for example.", "The behavior of the urea solution when agitated with the help of mechanical vibration depends to a significant extent on the physico-mechanical state variables to be detected, e.g., the density or viscosity.", "In an exemplary embodiment, this property may be detected directly on the vibration generator itself by measurement technology, e.g., by measuring the electric current, the frequency, etc.", "A quartz oscillator may be used as the vibration generator.", "However, any other known or future means for inducing mechanical vibration is also conceivable.", "For example, a piezoelectric crystal could also be used as well as a high-speed out-of-balance motor or an electromagnetic coil in conjunction with a diaphragm based on the loudspeaker principle.", "In an exemplary embodiment, a sensor unit is provided with a measuring sensor for an electric state variable and with a measuring sensor for a physicomechanical state variable.", "The measured values of the two measuring sensors are used in an analyzer unit to determine the urea concentration in solution.", "By analyzing two independent state variables, this yields the possibility of a more accurate and more selective determination of the urea concentration.", "In addition, a device according to the present invention may be combined with a temperature sensor.", "Since the state variables to be determined may under some circumstances be dependent upon temperature, correction of errors due to temperature variations is possible through simultaneous measurement and consideration of temperature in analysis of the state variable detected, e.g., for determination of the urea concentration in solution.", "In combination with a metering device for urea solution a filling level sensor may be provided for measuring the degree of filling of a storage container for the urea solution.", "In an exemplary embodiment, such a filling level sensor is combined directly with a measuring sensor according to the present invention for detecting one or more physical state variables.", "The measuring sensor according to an exemplary embodiment of the present invention shows definite differences in the measurement in solution in comparison with the measurement in the gas phase, so a filling level may also be readily measured in this way.", "To do so, various embodiments of the measuring sensor according to the present invention are again conceivable.", "For example, a measuring sensor according to the present invention may be mounted at a certain filling level and used as a threshold value sensor as the filling level passes the threshold value.", "For a more precise filling level measurement at different filling levels, a plurality of sensors may also be mounted at different levels.", "Such a sensor system may be mounted, e.g., in a sensor housing which extends over the corresponding height or on a rod-shaped sensor mount, for example.", "A continuous filling level measurement may be achieved by designing the measuring sensor according to an exemplary embodiment of the present invention to extend over a corresponding height.", "The sensor signal here is a function of the ratio of sensor areas situated in the gas phase or in the liquid solution.", "These sensor areas in turn vary with the filling level, so that information about the filling level is obtainable from the sensor signal in this way." ], [ "FIELD OF THE INVENTION The present invention relates to a device for metering a urea solution.", "BACKGROUND INFORMATION To reduce nitrogen oxides in the exhaust gas of motor vehicles, urea solution has in the past been sprayed into the exhaust gas during catalytic reduction.", "Urea is broken down into carbon dioxide and ammonia by chemical reaction on a hydrolysis catalyst.", "Ammonia then reacts selectively with nitrogen oxides to form nitrogen and water, thus removing nitrogen oxides from the exhaust gas.", "For reliable reduction of nitrogen oxides with a urea solution, various parameters are important, in particular the urea concentration in the aqueous solution.", "Sensor applications known in the past for measuring the urea concentration in the fields of medicine and biology have used urease, which enzymatically and selectively breaks down urea to form ammonia.", "Sensors then detect the influence of the ammonia on the pH of the solution.", "Information regarding the urea concentration is obtainable in this way.", "One disadvantage of this method of measuring the concentration of a urea solution is the instability of urease, in particular in an environment where temperatures may vary greatly.", "However, such temperature variations occur during use in motor vehicles, so that previous sensors according to the related art are not suitable for such an application.", "Therefore, the object of the present invention is to propose a device for metering urea solutions which may be used reliably for reduction of nitrogen oxides, even under difficult conditions, e.g., within broad temperature intervals.", "SUMMARY OF THE INVENTION Accordingly, a device according to the present invention for metering urea is characterized in that a sensor unit is provided for monitoring a physical state variable of an enzyme-free urea solution.", "The sensor unit here preferably includes a measuring sensor.", "In this way, a measurement is possible directly on the basis of the physical properties of urea in solution without intermediate enzymatic breakdown.", "Accordingly, this measurement is not subject to the instabilities to which an enzyme such as urease is subject.", "In an exemplary embodiment of the present invention, a measuring sensor is provided for detecting one or more electric state variables.", "Such a state variable may include, for example, the pH, the dielectric constant and/or the conductance of the solution.", "By measuring these or other electric state variables, it is possible to obtain information regarding the properties of the urea solution, e.g., its concentration.", "Measurement of these state variables is comparatively unproblematical and in particular it is possible to perform these measurements in situations of extreme temperature variations.", "Two electrodes may be provided to detect the electric state variables, these electrodes protruding into the urea solution.", "By applying an electric d.c. or a.c. voltage to the electrodes, it is possible to determine directly the aforementioned electric state variables, such as the pH, the dielectric constant, and/or the conductance.", "To improve the sensitivity of the measuring sensor the electrodes may be provided with a structure which increases their surface area.", "Such a surface area enlarging structure may be achieved, e.g., by a comb-shaped design of the electrodes, which additionally has the advantage that two electrodes designed in this way may be arranged to intermesh, so that a small distance between the two electrodes is adjustable simultaneously with a comparatively large surface area.", "Due to the large surface area, in particular in combination with the small distance, the test voltage and/or test current may be reduced and therefore the control and analyzing unit for a measuring sensor according to the present invention may be designed with small dimensions.", "A separate electrode may be provided for simultaneous determination of multiple state variables, if necessary.", "For example, by using such a third electrode, it is possible to determine the pH, while another state variable, e.g., the dielectric constant, is determined using the two aforementioned electrodes.", "In an exemplary embodiment of the present invention, a measuring sensor is provided for detecting one or more physicomechanical state variables of the urea solution.", "Such a physicomechanical state variable may be the viscosity or density, for example.", "Such physicomechanical state variables may be determined in a traditional manner, e.g., by weighing the solution and/or a part of the solution or by measuring the buoyancy of a displacement body, etc.", "However, in an exemplary embodiment the physicomechanical state variable is detected by a dynamic sensor.", "Thus, a physicomechanical state variable may be measured with the help of a vibration generator, for example.", "The behavior of the urea solution when agitated with the help of mechanical vibration depends to a significant extent on the physico-mechanical state variables to be detected, e.g., the density or viscosity.", "In an exemplary embodiment, this property may be detected directly on the vibration generator itself by measurement technology, e.g., by measuring the electric current, the frequency, etc.", "A quartz oscillator may be used as the vibration generator.", "However, any other known or future means for inducing mechanical vibration is also conceivable.", "For example, a piezoelectric crystal could also be used as well as a high-speed out-of-balance motor or an electromagnetic coil in conjunction with a diaphragm based on the loudspeaker principle.", "In an exemplary embodiment, a sensor unit is provided with a measuring sensor for an electric state variable and with a measuring sensor for a physicomechanical state variable.", "The measured values of the two measuring sensors are used in an analyzer unit to determine the urea concentration in solution.", "By analyzing two independent state variables, this yields the possibility of a more accurate and more selective determination of the urea concentration.", "In addition, a device according to the present invention may be combined with a temperature sensor.", "Since the state variables to be determined may under some circumstances be dependent upon temperature, correction of errors due to temperature variations is possible through simultaneous measurement and consideration of temperature in analysis of the state variable detected, e.g., for determination of the urea concentration in solution.", "In combination with a metering device for urea solution a filling level sensor may be provided for measuring the degree of filling of a storage container for the urea solution.", "In an exemplary embodiment, such a filling level sensor is combined directly with a measuring sensor according to the present invention for detecting one or more physical state variables.", "The measuring sensor according to an exemplary embodiment of the present invention shows definite differences in the measurement in solution in comparison with the measurement in the gas phase, so a filling level may also be readily measured in this way.", "To do so, various embodiments of the measuring sensor according to the present invention are again conceivable.", "For example, a measuring sensor according to the present invention may be mounted at a certain filling level and used as a threshold value sensor as the filling level passes the threshold value.", "For a more precise filling level measurement at different filling levels, a plurality of sensors may also be mounted at different levels.", "Such a sensor system may be mounted, e.g., in a sensor housing which extends over the corresponding height or on a rod-shaped sensor mount, for example.", "A continuous filling level measurement may be achieved by designing the measuring sensor according to an exemplary embodiment of the present invention to extend over a corresponding height.", "The sensor signal here is a function of the ratio of sensor areas situated in the gas phase or in the liquid solution.", "These sensor areas in turn vary with the filling level, so that information about the filling level is obtainable from the sensor signal in this way.", "BRIEF DESCRIPTION OF THE DRAWING FIG.", "1 shows a schematic diagram of an exemplary embodiment of a measuring sensor according to the present invention.", "DETAILED DESCRIPTION Sensor unit 1 is mounted on a sensor plate 2.A comb-shaped electrode 3 is divided into two areas 4, 5.Individual teeth of the comb structure are spaced farther apart in upper area 5 than in lower area 4.In upper area 5, another electrode 6 engages with a corresponding comb structure.", "The two electrodes 5 and 6 extend over a large area of sensor plate 2 and constitute a filling level sensor.", "A third electrode 7 is situated opposite lower area 4 of electrode 3.The comb structure of electrode 7 corresponds to the finer comb structure of lower area 4 of electrode 3, i.e., the teeth are not as far apart.", "Together with lower area 4 of electrode 3, electrode 7 forms a measuring sensor according to an exemplary embodiment of the present invention for measuring an electric state variable, e.g., the conductivity, the dielectric constant, etc.", "Electric terminals 8 for electrodes 3, 6, 7 are provided in the lower area of sensor plate 2.These electric terminals 8 may be connected via a plug connector in a manner not shown in greater detail here.", "Beneath lower area 4 of electrode 3, i.e., beneath electrode 7, a quartz oscillator 9 is shown as an oscillation generator for detecting a physicomechanical state variable, e.g., viscosity or density.", "Quartz oscillator 9 is also contacted via terminals 8.In an exemplary embodiment, sensor plate 2 may be designed at least partially as a PC board on which the electrodes are implemented in the form of flat printed conductors.", "In exemplary embodiment, however, sensor plate 2 may function as a mounting plate for mountable electrodes.", "With the help of sensor unit 1 according to FIG.", "1, one or more electric state variables such as the dielectric constant, the conductivity, the pH or the like, as well as one or more physicomechanical state variables such as density or viscosity may be detected.", "At the same time, sensor unit 1 also functions as a filling level sensor because of the extent of upper area 5 of electrode 3 and opposing electrode 6.Sensor unit 1 is therefore mounted in the interior of a container for a urea solution, so that electrodes 3 and 6 are at least partially immersed in the urea solution.", "With the help of sensor unit 1 according to the exemplary embodiment of the present invention, it is possible to reliably monitor the state of a urea solution even under adverse conditions, e.g., over a wide temperature interval.", "Such a sensor unit 1 is therefore suitable for use even in the area of exhaust gas processing of motor vehicles.", "List of Reference Numbers: 1 sensor unit 2 sensor plate 3 electrode 4 area 5 area 6 electrode 7 electrode 8 terminals 9 quartz oscillator" ] ]	Patent_10466505
[ [ "Surface passivation organic polymers and elastomers", "Surface treatment of organic polymer materials and material surfaces with oligo- or polysaccharide as passivation agent for deliberate alteration of the sorption properties, the diffusion barrier function, lubricative wetting and/or biocompatibility of organic polymers, is described.", "The passivating agent is derivatized with chemical entities that allow tight adsorption and/or covalent binding of the passivating agent to organic polymers or elastomer surfaces.", "The passivating agent may be derivatized with a primary functional group to allow covalent surface passivation by photo- or thermal activation of the derivative.", "The passivating agent may comprise one or more secondary functional groups that allow covalent binding of probe molecules and receptors to the passivated surface.", "The surface treatment improves the biocompatibility of medical devices and the performance of bioanalytical systems.", "Its application for heterogeneous affinity based assays, biosensor analysis platforms, microcontact printing, and lubrication of medical devices is described.", "Devices comprising surfaces passivated PDMS organic polymer are also described." ], [ "1.Surface treatment of organic polymer materials and material surfaces with homo- or heteropolysaccharides as passivation agent for deliberate alteration of the sorption properties, the diffusion barrier function, lubricative wetting and/or biocompatibility of organic polymers, wherein the passivating agent is derivatized with chemical entities that allow tight adsorption and/or covalent binding of the passivating agent to organic polymers or elastomer surfaces.", "2.Surface treatment according to claim 1 where the organic polymer or elastomers are prepared by chemical precursor polymerization or materials of natural origin.", "3.Surface treatment according to claim 1 where the organic polymer or elastomers are microstructured.", "4.Surface treatment according to claim 1, wherein the passivating reagent is an oligo- or polysaccharide, preferably aminodextran or chitosan.", "5.Surface treatment according to claim 1 where the passivating agent is a polysaccharide containing a defined number of primary functional groups, fully or partially substituted with photoreagents such as benzophenone-4-isothiocyanate or with diazirino-aryl-isothiocyanates.", "6.Surface treatment according to claim 1, whereby the photoactivatable reagents are converted to reactive species by irradiation with light.", "7.Surface treatment according to claim 1 wherein the photoactivatable reagents are converted to reactive species by heating to 80-115° C. 8.Surface treatment according to claim 1 where the passivating agent carries one or more secondary functional groups that allow covalent binding of probe molecules and receptors to passivated material surfaces.", "9.Surface treatment according to claim 8 where the secondary functional group(s) are amino groups, carboxyl groups, maleimides, thiols, biotin, epoxides, chelating- or photoreactive entities.", "10.A device in which the organic polymer is polydimethylsiloxane forming a replicated microchannel network on glass, quartz, silicium or organic polymers wherein channel surface passivation is attained by in situ or ex situ physisorption and subsequent light or temperature induced immobilization of aryldiazirine derivatized aminodextran.", "11.A device in which the organic polymer is polydimethylsiloxane forming a replicated microchannel network on glass, quartz, silicium or organic polmer, whereby channel surface passivation is attained by in situ or ex situ physisorption of a protein-biotin conjugate and subsequent attachment of avidin analogues, the generated protein-based layer being additionally capped with biotin or biotinylated macromolecules including receptors, antibodies, nucleic acids, oligonucleotides, oligosaccharides or polysaccharides.", "12.Application of the surface treatment according to claim 1 for heterogeneous affinity-based binding assays.", "13.Application of the surface treatment according to claim 1 for molecular engineering of biosensor analysis platforms and fluidic devices to suppress physisorption and diffusion of molecules.", "14.Application of the surface treatment according to claim 1 for the passivation of structured organic polymer surfaces used for microcontact printing.", "15.Application of the surface treatment of claim 1 for the lubrication of medical devices including catheters and implants.", "16.A biosensor analysis platform, a fluidic device, a structured organic polymer surface such as for use in microcontact printing, or a medical device, fabricated using the surface treatment method of claim 1.17.A biosensor analysis platform, a fluidic device, a structured organic polymer surface such as for use in microcontact printing, or a medical device, incorporating a device as defined in claim 10.18.A biosensor analysis platform, a fluidic device, a structured organic polymer surface such as for use in microcontact printing, or a medical device, incorporating a device as defined in claim 11." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention in its various aspects is defined in the appended independent claims, to which reference should now be made.", "Preferred or advantageous features of the invention are set out in dependent subclaims.", "Thus, the invention may advantageously overcome the problems in the prior art discussed above.", "In its various aspects, the invention relates to the treatment of material surfaces, in particular organic polymers and elastomers of natural or synthetic origin that require unique or particular surface properties for adequate device function.", "Although organic polymers are numerous with respect to their chemistry, complexity, physical properties, their mode of use and fields of application, there are restricted means to render them compatible with biological systems.", "In view of engineering of material surfaces for bioanalytical purposes, surface structuring with biomolecules, and medical applications there is a need for adaptation of organic materials and composites for appropriate function when contacted with biological systems.", "This invention relates to material surface treatment that may advantageously result in a close mimic of biological systems: the generic approach of in situ generation of chemically reactive species in conjunction with the passivating properties of polysaccharide may advantageously render novel properties to materials and may thus improve the efficiency of biocompatible devices.", "The surfaces of solid organic polymers and elastomers are prone to physical adsorption and bi-directional diffusion of low molecular weight molecules.", "Diffusive sorption and release processes, as well as adsorptive binding properties of polymeric materials may advantageously be drastically suppressed by treatment of the polymer surfaces with oligo- or polysaccharides.", "Surface passivation by covalent or adsorptive thin-film coating may be attained by depositing and subsequent immobilization of biopolymers onto organic polymer or elastomer surfaces.", "Preferably, the passivating material is covalently bonded to the organic material yielding biocompatible surfaces as used in medical and analytical applications.", "With aryldiazirine or benzophenone derivatized oligo- or polysaccharides, covalent surface passivation may be attained by photo- or thermal activation of the polysaccharide derivative.", "Surface passivation is exemplified by polydimethyldioxane microchannel treatment and application of the resulting systems for the detection of immunointeractions." ], [ "PRIOR ART It is known that the properties of polymer surfaces may be modulated by integrating polar or charged components or monomeric precursor analogues in the polymerization process.", "Post-polymerization surface treatments are equally established, e.g.", "plasma treatment followed by exposure to aqueous media renders organic polymers highly wettable, and surface silylation has been applied to introduce selectively reactive functionalities on organic polymer material surfaces.", "In a recent approach, PDMS (polydimethylsiloxane) microchannels were fabricated using replica molding techniques.", "Surface passivation was achieved by multimeric protein (Immunoglobulin M) is adsorption or by theromochemical crosslinking of a capture protein (protein A) with glutaraldehyde.", "(E Eteshola, D. Leckband, Sensors and Actuators B 72 (2001) 129-133).", "The increasing use of binary or ternary composite polymers in bio-analytical and medical devices demands unifying surface modification processes that are indistinguishably applicable to different polymer chemistries.", "It is widely documented that C—H, C═C and O—H bonds—the most frequent chemical bonds in organic polymers and elastomers—are chemically reactive with generated carbenes and ketyl radicals.", "Such chemical intermediates can be generated locally by thermal or light activation.", "The reactivity of many organic polymers with e.g.", "photogenerated carbenes was shown using low molecular weight crosslinkers or photolabel derivatized macromolecules including proteins and nucleic acids.", "Several patents and scientific research articles report on the advantageous properties of oligo- and polysaccharides to adsorb and store water or aqueous media.", "When conjugated to material surfaces polysaccharides form biocompatible passivating layers leading to improved performance of bioanalytical systems.", "However, establishment of such passivating layers requires a specific substrate and a sequence of at least two surface chemical steps to attain the requested conjugate.", "SUMMARY OF THE INVENTION The invention in its various aspects is defined in the appended independent claims, to which reference should now be made.", "Preferred or advantageous features of the invention are set out in dependent subclaims.", "Thus, the invention may advantageously overcome the problems in the prior art discussed above.", "In its various aspects, the invention relates to the treatment of material surfaces, in particular organic polymers and elastomers of natural or synthetic origin that require unique or particular surface properties for adequate device function.", "Although organic polymers are numerous with respect to their chemistry, complexity, physical properties, their mode of use and fields of application, there are restricted means to render them compatible with biological systems.", "In view of engineering of material surfaces for bioanalytical purposes, surface structuring with biomolecules, and medical applications there is a need for adaptation of organic materials and composites for appropriate function when contacted with biological systems.", "This invention relates to material surface treatment that may advantageously result in a close mimic of biological systems: the generic approach of in situ generation of chemically reactive species in conjunction with the passivating properties of polysaccharide may advantageously render novel properties to materials and may thus improve the efficiency of biocompatible devices.", "The surfaces of solid organic polymers and elastomers are prone to physical adsorption and bi-directional diffusion of low molecular weight molecules.", "Diffusive sorption and release processes, as well as adsorptive binding properties of polymeric materials may advantageously be drastically suppressed by treatment of the polymer surfaces with oligo- or polysaccharides.", "Surface passivation by covalent or adsorptive thin-film coating may be attained by depositing and subsequent immobilization of biopolymers onto organic polymer or elastomer surfaces.", "Preferably, the passivating material is covalently bonded to the organic material yielding biocompatible surfaces as used in medical and analytical applications.", "With aryldiazirine or benzophenone derivatized oligo- or polysaccharides, covalent surface passivation may be attained by photo- or thermal activation of the polysaccharide derivative.", "Surface passivation is exemplified by polydimethyldioxane microchannel treatment and application of the resulting systems for the detection of immunointeractions.", "DESCRIPTION OF THE INVENTION The implementation of the invention will now be described, including description of several embodiments.", "Further details are also set out in the accompanying drawings.", "In a preferred embodiment, the invention may provide a surprisingly simple process to passivate organic polymer or elastomer surfaces.", "Thus, in tests, different types of polymer materials, individually or as composite material, were passivated by depositing carbene- or ketyl radical forming derivatives of polysaccharides in target surfaces.", "Derivatives of aminated polysaccharides such as aminodextran or chitosan were prepared: the polysaccharides were thermochemically modified with benzophenone-4-isothiocyanate or isothiocyano-aryl-diazirines, yielding benzophenone-dextran and benzophenone-chitosan, or aryldiazirine-dextran and aryldiazirine-chitosan, respectively.", "For covalent immobilization of these passivating agents, the reagents were thin-film deposited on the organic polymer surfaces and irradiated with activating light (wavelength: 350+20 nanometer, power: 10 microWatts per square centremetre, exposure time: 4 mintutes) or by heating to 80-115° C. Such surface treatment was effective for passivation of PDMS (polydimethylsiloxane) elastomers and polymers as for example, parylene, polystyrene, polyurethane, polycarbonate, polyvinylalcohol or polyvinyl difluoride.", "For analytical purposes, PDMS microchannels, forming parts of microfluidic systems, were passivated by either protein-mediated binding of dextran, or by direct immobilization of radical or carbene generating polysaccharides on PDMS.", "Immobilization was feasible ex situ and in situ, in particular after the formation of functional microchannels.", "Both passivation procedures yielded surfaces that prevented diffusion of charged or polar low molecular weight chemicals into the PDMS matrix and suppressed the adsorption of proteins e.g.", "immunoglobulins to analytically non-interfering levels.", "Furthermore, attachment of immunoreagents to passivated microfluidic channels or organic polymer surfaces via secondary functions (e.g.", "binding of antigens to photoimmobilized carboxy-dextran) generated immunoreactive polymer or elastomer surfaces.", "The described surface treatment may thus open new routes to fast and simply-implemented in situ passivation of structured organic materials, making them available for bioanalytical and biomedical applications.", "Surface Bio-Passivation of Replicated μ-Fluidic Channels Polydimethylsiloxane (PDMS) appeared recently as a material of choice for rapid and accurate replication of polymer-based microfluidic networks.", "However, due to its hydrophobicity, the surface strongly interacts with apolar analytes or species containing apolar domains, resulting in significant loss of sample to the substrate and, consequently, poor analytical performance.", "This contribution describes, with reference to the accompanying drawings and figure legends, the characterization of a native PDMS surface passivation treatment in microchannels.", "FIGS.", "1 and 2 show the behaviour of a fluorescent neutral marker in fused-silica/Pyrex; FIG.", "3 shows schematically PDMS surface bio-passivation; FIG.", "4 shows EO mobility in coated channels; FIG.", "5 shows passivation and coating stability; and FIG.", "6 shows biomolecule mobility in coated channels.", "FIGURE LEGENDS FIG.", "1 Comparative capillary zone electrophoresis (CZE) runs of TMR-dextran and/or Caffeine.", "Samples: in Na-acetate buffer 30 mM pH=4.7 Working buffer: 30 mM Na-acetate pH=4.7 Instrument: P/ACE 5510, UV detection at 214 nm Exp.", "Conditions: 6 kV, 5 sec injection time, 25° C. Capillary: 20/27 cm, ID=50 μm.", "Tetramethylrhodamine labeled dextran 70 KDa (TMR-dextran) was shown to be neutral over the pH range 4.7 to 9.3.CZE runs of both caffeine (UV neutral marker) and TMR-dextran gave identical migration times.", "FIG.", "2 Boltzman fit for EO mobility measurements in Pyrex channels using TMR-dextran.", "Pyrex channel, HF etched (20 μm deep) Separation channel: total length 6 cm, 70 μm wide All buffers ionic strength I=14 mM E=650 Vcm−1 (buffers pH 4.0 to 8.5) E=430 Vcm−1 (buffers pH 8.95 to 10) LIF detection (488 nm excitation) Pinched injection TMR-dextran was used as a fluorescent marker for EO mobility determination in Pyrex channels with a selection of acetate, phosphate and tetraborate buffers ranging from pH 4.0 to 10.Ionic strength remained constant.", "Results compare well with published data.", "FIG.", "3 Schematic diagram of the coating used for passivation.", "(1) Physisorption of biotin conjugate of IgG to PDMS; (2) Neutravidin; (3) Biotin conjugate of dextran 10 kDa.", "FIG.", "4 Migration time of TMR-dextran in coated channels and calculated EO mobility.", "Experimental conditions: running buffer (RB) 6.74 mM Tetraborate buffer pH 9.3; sample 50 mM TMR-dextran in RB; E=825 Vcm−1; LIF detection (488 nm excitation), pinched injection.", "EO mobility was measured in microchannels by monitoring TMR-dextran detection times.", "Good reproducibility was obtained (RSD (n=10)<2%).", "However, EO mobility doubled within one month; this variation requires the use of an internal standard.", "FIG.", "5 Adsorption of 20 mM BODIPY-Digoxigenin in (a) uncoated and (b) coated channels (2 minutes incubation, hydrodynamic flow).", "Channel preparation: replicas were obtained by moulding PDMS using a micromachined Si wafer as master.", "Drilled PDMS slabs were sealed against Pyrex wafers.", "The three layer coating dramatically decreased adsorption for a variety of fluorescently labeled molecules and bio-polymers, such as BODIPY-digoxigenin (MW 0.8 kDa), TMR-dextran (MN 70 kDa), FITC-human IgG (MW 150 kDa), and others.", "The three layer coating appeared to be stable upon extensive exposure to buffers of neutral to basic pH, urine and human blood plasma.", "Short exposure to weak detergents (such as Tween 20) may be considered, but strong detergents (SDS) and very basic solutions (NaOH 0.1M) damage the coating rapidly.", "FIG.", "6 Recorded peak compared with its Gaussian fit (zoom).", "Experimental conditions: working buffer 7.92 mM Na-phosphate buffer pH 7.0; sample 5 μg/ml fluorescein mouse-IgG in diluted PBS; E=453 Vcm−1; LIF detection (488 nm excitation), pinched injection.", "Full run recorded at 5 Hz, peak (zoom) at 500 Hz.", "Fluorescein-labeled mouse-IgG can be analyzed by CZE in a coated channel.", "The Gaussian peaks that are recorded indicate that no significant adsorption occurs.", "In uncoated channels, IgG adsorbs to the elastomer surface: it does not reach the detector.", "Conclusion The protein-based surface modification considerably decreases adsorption of fluorescently labelled low and high molecular weight substances.", "Moreover, the passivation layer is stable in buffers, as well as in biological fluids such as serum or urine.", "Electroosmotic pumping in modified channels is also possible, making this an attractive surface treatment approach for many analytical applications." ] ]	Patent_10466507
[ [ "Method of enhancing the efficiency of data flow in communication systems", "A method of improving the flow of data in a communication system having at least two links, comprising inserting into one of said links at least one enhancer, the operation of which is to a) communicate with a the unit upstream of the data flow such that the upstream unit perceives that it is communicating with the unit (s) downstream of the enhancer; b) obtaining a measure of the efficacy of operation of the down stream unit(s), and c) dependent upon the measure, varying the control of data flow sent to downstream units." ], [ "1.A method of improving the flow of data in a communication system having at least two links connecting units at least one link has multiple simultaneous connections, comprising splitting and inserting into one of said links at least one enhancer, the operation of which is to a) communicate with a unit upstream of the data flow such that the upstream unit perceives that it is communicating with unit(s) downstream of the enhancer; b) obtaining a measure of the efficacy of operation of the down stream unit(s), by assessing the aggregate amount of unacknowledged data set (10) on the link having multiple simultaneous connections; and c) dependent upon the measure, varying the control of data flow sent to downstream units, by not sending data down a particular connection if the amount of unacknowledged data sent down that particular connection exceeds a first predetermined amount and the amount of said aggregate data exceeds a second predetermined amount.", "2.A method as claimed in claim 1, wherein said communication with the upstream unit by the enhancer uses standard TCP and communication with downstream units uses a modified and adaptive protocol.", "3.A method as claimed in claim 1, wherein the enhancer(s) are located in positions where upstream links are generally faster than those downstream from the enhancer.", "4.A method as claimed in claim 1, wherein the upstream unit is the Internet.", "5.A method as claimed in claim 1, wherein the downstream links are wireless links.", "6.A communication system having at least two links connecting units at least one link having multiple simultaneous connections, and including at least one enhancer located by splitting and inserting it into one of said links, said enhancer including means to communicate with a unit upstream of the data flow such that the upstream unit perceives that it is communicating with the unit(s) downstream of the enhancer; means to obtain a measure of the efficacy of operation of the down stream unit(s) by assessing the aggregate amount of unacknowledged data set on the link having multiple simultaneous connections, and means to adjustably control data sent to downstream units, dependent upon the said measure, by not sending data down a particular connection if the amount of unacknowledged data sent down that particular connection exceeds a first predetermined amount and the amount of said aggregate data exceeds a second predetermined amount.", "7.A communication system as claimed in claim 6 wherein the enhancer(s) is/are located in positions where upstream links are generally faster than those downstream from the enhancer.", "8.A communication system as claimed in claim 6, wherein the upstream unit is the Internet.", "9.A communication system as claimed in claim 6, wherein downstream links are wireless links." ], [ "This invention relates to communication systems in general and has particular application to communication systems having a plurality of links where one link is considerably slower in terms of data flow than other links.", "In order to illustrate the flow of data and its control in conventional systems, FIG.", "1 shows a schematic representation of the communication system.", "Unit 1, which may be referred to as the sender (of information) represents a source of data to be transmitted and may be for example the Internet.", "This has a wire link with 2 to a wireless access base station 3.This wireless access base station communicates via radio links 4 to unit 5, A, B and C which in practice may comprise a number of mobile telephones.", "Under a conventional operation known system unit 1 will send a packet of data to the wire station under standard Transmission Control protocol.", "(TCP).", "Under the TCP system, station B will send an acknowledgement to the sender that it has received the data packet and sender (or server) will double the transmission data rate and send two data packets.", "After receiving an acknowledgement that the base station has received these two data packets it will further double the data flow rate and send four data packets.", "This will continue until a maximum data transfer rate has been reached.", "The function of the base station is to forward on the data to mobile phones.", "As this second link is a radio link, the data transmission rate is considerably lower than that of the first link i.e.", "it has limited bandwidth.", "As the wireless access base station has finite buffer storage space and has to handle incoming data and acknowledge it as well as controlling and sending forwarded-on data packets, errors will occur such as loss of data packets.", "Under TCP data packet loss will be recognised by different mechanisms such as not receiving an acknowledgement for a particular data packet within a predetermined time or by the numbered data packets being out of synchrony.", "In the latter cases the base station will for example will send duplicate (or repeated) request for data packet No.", "3, e.g., should data packets Nos.", "4 and 5 etc come after data packet No.", "2.When the server recognises data packet loss it will re-send the lost data and assume it is sending the data too fast.", "As a consequence it will half the data transmission rate.", "Further data packet losses will again further half the data transmission rate and result in meltdown.", "As consequence the server cannot do anything else and the data flow rate is severely slowed down and time is required for the data transmission rate to be ramped up again.", "In summary thereforeTCP implements a number of algorithms under the general heading of ‘congestion avoidance’.", "These are designed to make TCP ‘reactive’ to congestion in the network and to control its sending rate to avoid causing ‘congestion collapse’.", "This is a very real danger in the Internet, and so the algorithms are essential.", "The important aspects of congestion avoidance are these: slow-start—an exponential increase in the TCP sending rate.", "Slow start is performed on every new connection; after a connection has been idle for some time; and after a retransmission timeout has occurred.", "fast-retransmit/recovery—dropped packets can be detected by duplicate acknowledgements.", "When this happens the current sending rate is effectively halved and then increases linearly.", "In summary therefore, the wireless access environment and other communication scenario's present a set of challenges to achieving a high level of TCP performance such as limited bandwidth, variable bandwidth, long latency and possible lengthy disconnections It is an object of the invention to limit and prevent packet loss events and to manage the flow of data to make the optimum usage of transmission sources in a balanced way, and to reduce the possibility of overflowing available buffer resources and thus to increase the overall efficiency of the system.", "The invention comprises a method of improving the flow of data in a communication system having at least two links, comprising inserting into one of said links at least one enhancer, the operation of which is to a) communicate with a the unit upstream of the data flow such that the upstream unit perceives that it is communicating with the unit (s) downstream of the enhancer; b) obtaining a measure of the efficacy of operation of the down stream unit, and c) dependent upon the measure adjustably varying the control of data sent to downstream units.", "The enhancer, by sending out data more smoothly and controllably and at a rate at which the downstream units can handle, data packet loss can be minimised or eliminated.", "Moreover, the enhancer can make optimum use of fast links such as would be usual for the link between sender and enhancer.", "The enhancer can rapidly send out acknowledgements to the sender that it has received data or not without the sender having to wait for units downstream (with slower links) to send back acknowledgements.", "In prior art systems, the sender may have sent many data packets out during the time after which there has been overload and data loss which would be a waste of time.", "By doing this, flow control and congestion management is no longer performed end-to-end, as would happen with ordinary TCP.", "This is important, as the wireless link has very different characteristics from the other, terrestrial, hops that packets will use.", "By splitting the connection, rather than simply acting as a router, the enhancer can control access to the wireless resource without having to drop packets.", "This is a significant advantage over a bottleneck router which, if overwhelmed with data, would have to discard packets.", "An important point for the use of asymmetric enhancers according to the invention, is that once a packet has reached the interface to the wireless link, the data is transferred to the mobile station via a point-to-point link.", "This link is not shared and cannot suffer from congestion.", "It is also important to note that the network attached to the mobile is a ‘stub’ network.", "That is, there are no networks beyond it that are routed through it.", "Because of these points, it should be seen that congestion control mechanisms are not required once the wireless link has been reached.", "This, then, is the how the enhancement is achieved.", "The connection is split at the enhancer.", "A TCP flow that is regenerated back into the Internet remains a standard, responsive, TCP.", "A TCP that is regenerated towards a mobile, however, has the congestion avoidance mechanisms removed.", "In this way, the protocol used by the enhancer is fully interoperable with the host attached to the mobile station—all the datagrams are perfectly standard TCP/IP.", "The performance is enhanced because congestion avoidance is not performed over a link for which it is inappropriate.", "The air interface used to communicate with the mobile stations has a fixed maximum capacity.", "However, the actual rate at which user data can be passed over the link depends upon the link error rate and the split between up-link and down-link.", "Since the connection is split at the enhancer it is possible to adjust not only congestion avoidance but also the retransmission strategy between the enhancer and a mobile host.", "Again, the link characteristics are sufficiently different from the wired Internet case that this is a useful exercise.", "The invention will now be described by way of example only and with reference to the following figures of which: FIG.", "1 shows a simple schematic figure of a prior art system.", "FIG.", "2 shows a basic embodiment of the invention applied to the system shown in FIG.", "1 which includes an enhancer.", "FIG.", "3 show one embodiment of the system where the build up of data to units downstream is monitored by the enhancer.", "FIG.", "4 shows physical and representation of a further embodiment of the invention where there is one link with three connections.", "FIG.", "5 shows a further embodiment of the invention whereby the amount of unacknowledged data sent downstream can be monitored.", "FIG.", "6 shows a representation of an embodiment of the invention showing an example of the software architecture of the enhancer.", "FIG.", "2 shows a simple embodiment of the invention comprising essentially the same features of FIG.", "1 but with the additional insertion of an enhancer 6 located between the Internet server and the wireless Access Base Station.", "The enhancer effectively isolates or splits the communication system into two halves and operates such that perceived problems on one side of the enhancer do not affect the operation on the other side.", "Connection splitting is the key mechanism for enabling TCP performance enhancement.", "Rather than allowing the TCP connection to run end-to-end, as usual, the connection is segmented.", "The enhancer terminates a TCP connection on one interface and re-generates it on another.", "Connection Splitting is done by a technique referred to in the art as “spoofing”, whereby the enhancer forges an acknowledgement to the sender to say I am the wireless base station, so that as far as the sender is concerned the connection is terminated at the enhancer.", "The enhancer operates with downstream systems and units to send data out to these in a more controlled, aggressive and thus efficient manner in an adaptive fashion using knowledge of the how the downstream systems are functioning.", "In other words the enhancer obtains a measure of how well downstream units are working, or clogged up the downstream systems are; and sent out data controllably and such that it does not give the downstream units more than they can handle and such that data packet loss does not occur.", "One way in which this may be implemented is by the enhancer using a modified TCP to communicate with downstream units, which is compatible with the links but which is adaptive.", "The term adaptive here means that the TCP can be adapted to vary according to knowledge of the type of link and units i.e.", "systems downstream and how it is operating.", "There are many ways in which the enhancer can obtain a measure of how the downstream units are currently operating.", "In general not only can the enhancer use knowledge about the type of links and systems downstream therefrom, such as whether they are mobile telephone links, but also it can obtain feedback in various ways to get a knowledge of how these downstream system are functioning and accordingly control the data flow rate to these systems.", "The effective splitting of the system by the enhancer also means that, because it pretends to be the downstream systems, it can communicate back to the server to send early feed-back of acknowledgements because it does not have to wait for acknowledgements to come from the systems further down-stream, which conventional takes longer, due to the distance of the network and the slowness of links downstream.", "Thus the nature of the rapid link between the enhancer and server means that any dropped packets can acknowledge and retransmitted fast.", "Such quick communication without reliance on downstream acknowledgement, results in high usage and efficiency of this link, thus allowing high data transmission rates from the server to the enhancer.", "The enhancer will contain a buffer, but this does not have to be larger than may be thought initially as because the enhancer, as already mentioned can increase the data transmission rate of forwarded on data packets because of its smart efficient control there of as described above.", "The following give examples of how the enhancer is adaptive according to the nature of operation of the downstream systems.", "EXAMPLE 1 Measurement of Round Trip Time FIG.", "3 shows a schematic drawing of a communication system to illustrate this embodiment.", "Again the figure is similar to that of the previous figures but additionally shows a buffer of a wireless access station.", "The radio link to the station would have a fixed capacity i.e.", "fixed buffer capacity e.g.", "64 Kbytes.", "The radio station sends data over a radio link to one or more host e.g.", "mobile telephones.", "Data sent to the mobile host in the form of data packets 7 will cause an acknowledgement 8 to be sent back to the enhancer.", "These acknowledgements are either sent immediately on receipt of a packet, or, under certain circumstances after a short delay (typically in the order of 200 ms).", "If the rate at which the packets are sent to the host remain less than the available bandwidth then there will never be more than 1 data packet in the buffer and the round trip time will remain relatively steady.", "The round trip time is defined as the time between sending a data packet and receiving an acknowledgement.", "If the rate of sending increases above the available bandwidth then the buffer will start to fill up and which could lead to buffer overload.", "The buffer will also drain at the rate determined by the available bandwidth.", "The effect of this is that the RTT will appear to increase.", "This will continue all the time until the sending rate is too high and the buffer will fill up and packet loss will result.", "The enhancer uses measurement of RTT (assuming the buffer is of adequate size) to indicate the degree to which the link is being over-utilised, and to slow down the data transmission rate accordingly.", "Thus the enhancer adapts, in a dynamic fashion, the transmission rate according to the increase in RTT.", "EXAMPLE 2 Aggregate Monitoring Many protocols such a TCP use a “sliding window” as an effective means of flow control.", "The window is the maximum amount of data that a sender can output without receiving an acknowledgement, which is a useful technique in limiting the ability of a system to flood a downstream network.", "Thus, knowing the data packet size, the downstream buffer size and number of unacknowledged packets can slow down accordingly to ensure no buffer overflow occurs It is important that the window is large enough to support the maximum.", "Typically the window is sized according to the delay bandwidth product of the link) A problem with this is that this sizing is carried out by each link, a link being defined as between two units.", "If there are multiple simultaneous connections on one link, this has the effect of multiplying the effective window size which can prove a problem in terms of controlling buffer usage and can lead to bursty network arbitration and synchronisation effects.", "In one embodiment of the invention this problem is overcome by the use of an aggregate window where each connection uses its own window as normal but that an overall window is applied to all connections.", "This is illustrated in figure.", "Between the enhancer and the destination operate a multiple of connections, in the example three of them, that are operating simultaneously.", "If each connection allows a window size of 10 kb, for example, but the buffer at the end destination only has 20 kb, then flooding of the buffer will occur.", "In order to prevent such data loss and thus minimise re-sending the enhancer control is such that it can only output data on a particular connection of a link if that connection window is not full AND the aggregate window is not full.", "In other words, the enhancer sums the amount of unacknowledged data packets are outstanding for each connection and ensures that if the sum or aggregate is greater than a predetermined size then no further data will be sent to the unit.", "EXAMPLE 3 Aggregate Rate Monitoring Yet another way in which the enhancer can obtain quantitative measurements of how data is flowing downstream will now be described.", "Often in a communications link, a base station for example, data flow from a server will be of mixed type; there may be data packets of a varied nature.", "As an example, some data packets such as audio or video data packets are unacknowledged data packets (UPD) that is to say that once received by a unit, an acknowledgement is not sent to the originator.", "In these instances, if the UPD is lost, then there is no point in re-sending as these data packets need to be sent smoothly i.e.", "in order.", "Data packet loss is not that critical and merely causes a momentary blip in a video or audio signal.", "For such mixed data transmission, there arises a problem in that the enhancer receives no information as to the progression of UDP's because the UDP's are not acknowledged and so the enhancer does not know whether they have arrived or been lost.", "The inventors have provided an innovative way of allowing the enhancer to determine how the UDP's are fairing by making use of the fact that all data packets, both acknowledged and unacknowledged, are sent out in a contiguous order.", "By “pinning” the unacknowledged packets to acknowledged packets it is possible to determine whether the UDP's have arrived.", "This is illustrated in figure.", "This shows a flow of data between the enhancer and a downstream unit.", "The data flow comprises packets to be acknowledged under the TCP protocol (denoted TCP's) as well as unacknowledged packets (denoted UDP's).", "When data packet TCP1 has been processed and stored or forwarded without errors them an acknowledgement Ac1 is sent out.", "When data packet UDP1 arrives even if it has been stored or sent on without errors an acknowledgement is not sent.", "However when data packet TCP2 has been correctly stored or sent on without errors, the enhancer uses this to assume that the UDP1 has been stored correctly and without errors.", "Thus this gives the enhancer knowledge of the outstanding data, how much buffer space has been taken up and data rate on and thus can be used in conjunction with any of the above techniques.", "Software Architecture FIG.", "6 shows a schematic representation of the one example of software architecture of an embodiment of the enhancer.", "The link to ‘host B [mobile side]’ is not directly to the mobile.", "This link carries the packets to the wireless access point, or Control Equipment (referred to hereafter as the ‘CE’).", "Two TCP stacks, connected by the spoofer, sit above a single IP stack, which in turn sits above the ethernet layer.", "The limiting module estimates the fullness of the CE buffers and switches off the transfer of data, from the outer to the inner TCP stack, if the buffer is estimated to be too full.", "There is also a weighted fair queuing (wfq) module which controls the overall maximum data flow rate from the outer to the inner stack.", "The CE has several buffers (one per mobile).", "If data is sent to the CE at too high a rate then these buffers could be flooded causing loss of packets.", "These packets would then need to be re-transmitted which would seriously impair the enhanced TCP performance.", "The enhancer has no direct way of querying the CE about how full these buffers are.", "Therefore a limiting module has been designed to estimate the fullness of these queues and, if necessary, tell the spoofer not to transfer any more data until the buffers have drained.", "The basic principle is to keep track of the minimum and mean RTT (round trip time) for each class (buffer).", "This is the time taken for an acknowledgement of a packet sent out from the inner TCP stack to arrive back at the inner stack.", "The minimum RTT is used as an indication of the inherent latency in the link Large RTTs which are thought to be due to retransmissions are ignored.", "If the mean RTT becomes larger than a certain multiple of the minimum RTT then it is assumed that the buffer is filling up (packets must queue in the buffer before being dealt with hence the RTT increases).", "In this case the spoofer is forbidden from sending any more packets for this class until either: A timeout occurs (this is a safety net and should not happen).", "The Limiter Class The limiter estimates that the buffer has drained sufficiently for data transfer to commence again In order to best describe how the limiting module works it is necessary to describe the central data structure to the module—the limiter class.", "One limiter class is maintained per buffer in the CE and has the following elements.A flag specifying whether the class is awake or asleep.", "The spoofer is not allowed to send data for a class that is sleeping.", "A packet loop.", "This loop contains several packets of data for the class (the configuration parameter loop_length).", "These packets are not the full packets being sent, but merely contain the necessary information to identify the TCP segment i.e.", "the source and destination IP address and port number, and the sequence number.", "The time between packets being logged in this loop is defined by the configuration variable time between logs.", "This packet loop has two main purposes.", "To keep track of RTTs and therefore determine when to send a class to sleep.", "To determine when to awaken a sleeping class.", "Packets are logged every time between logs milliseconds.", "The inner TCP stack passes the relevant details to the limiter before it outputs the packet.", "Packets are only cleared from the packet loop when and ACK is received back at the inner TCP stack.", "At this point an RTT for the logged packet can be calculated.", "The time the packet loop was last updated.", "This is logged to monitor when another packet should be logged in the packet loop.", "A marker for the loop head and loop tail.", "These are cyclic values modulo the loop length.", "The packet head represents the position to log the next packet, while the packet tail represents the oldest logged packet.", "Having a cyclic buffer ensures that a maximum of loop length meaningful packets can be stored at any given time.", "The time that the class was sent to sleep (only applies if the awake flag is set to FALSE).", "If the class is sent to sleep then the time is recorded so that it can be awakened if it is not awakened by an ACK of a packet in the packet loop within a set time period.", "The mean and variance of the RTT.", "The minimum RTT and RTT count are also stored.", "If the mean of the RTT becomes larger than a certain multiple of the minimum RTT then the class is sent to sleep.", "The RTT count is initially ramped up to a maximum value.", "and used as a weighting factor when computing the new mean RTT from the old value, i.e.", "〈 R 〉 new = 〈 R 〉 old ⁢ ( R c R c + 1 ) + R new ⁡ ( 1 R c + 1 ) where <R>new is the new mean RTT, <R>old is the old mean RTT, Rnew is the newly submitted RTT and Rc is the RTT count.", "This ‘ramping up’ of the RTT count ensures that early RTTs (i.e.", "when the limiter is first started up) carry a greater weighting to the mean than later ones.", "Note that, as with all rolling mean calculations, the weighting factors (in the round brackets) add up to one.", "The variance of the RTT is used to estimate when an RTT is due to a re-transmission.", "These are ignored.", "A flag which specifies whether or not RTTs should be ignored.", "Recall, RTTs are used to estimate when a class should be sent to sleep.", "When a class is sent to sleep, RTTs arising from packets logged from that time until the time the class was awoken are ignored.", "When the class is sent to sleep the flag ignore rtts is set to TRUE and te variable ignore until packet is set to the position that first packet after the class is woken up will be logged at.", "All packets previous to this in the packet loop are ignored (for the purposes of calculating RTTs).", "Once the packet at ignore until packet has been ack'ed the ignore rtts flag is set to FALSE.", "This technique makes sure that the mean RTT is not influenced by very large RTTs that have already effectively been acted upon (by sending the class to sleep).", "If these were not ignored then a class that had just been awoken would unnecessarily be sent back to sleep again.", "Estimating the CE Buffer Fullness Under the assumption that every packet sent to the CE buffer is logged in the limiter class loop and that there is no latency in the link, the limiter class loop will mirror exactly the state of the CE buffer.", "That is, the packet at the head of the limiter class loop will be at the back of the CE buffer queue and the packet at the tail of the limiter class loop will be at the front of the limiter class loop.", "Under the further assumption that the flows both in and out of the buffer are constant rate, the round trip times for these packets exactly equals length of the queue (in ms).", "Therefore the difference in log time between the packet at the head and the packet at the tail of the packet loop will be exactly the length of the CE buffer queue in ms (i.e.", "the time needed to clear the queue if no more packets were sent).", "These principles are those used to send a class to sleep and also awaken it.", "When the RTTs for packets in the class buffer become large than some multiple of the base RTT then the CE buffer is assumed to be filling up and the class is sent to sleep.", "When the difference in log time between the packet at the head and the packet at the tail of the packet loop becomes less than some multiple of the base RTT then the class is awoken.", "Retransmission.", "The provision of an assured layer-2 service (layer-2 are assumed to make use of ARQ acknowledgement repeat request schemes) on the link to the mobile host needs to be handled.", "Rather than a link with errors, it will now appear to the TCP to be a link with a highly variable RTT (any lower layer retransmission will cause the RTT to double).", "Because of the latencies involved this could cause end-to-end TCP to unnecessarily retransmit packets, as the increase in RTT may trigger a timeout.", "The enhancer is able to manage this, as the retransmission timer can be altered to have a suitable minimum value that accounts for the possibility of layer-2 retransmissions.", "Assuming that the layer-2 is reliable means that it can be considered unlikely that this timeout will occur.", "If the timeout does trigger, then it may indicate that the link to the mobile host is temporarily disconnected.", "Normal TCP backs-off in the event of a retransmission.", "That is, if a timeout occurs the packet is retransmitted.", "The timeout for this packet is then doubled.", "This is to avoid stress on the network.", "In the case with a mobile host, there may be (relatively) long periods of disconnection.", "If this exponential back-off were performed, it could take a long time for a packet to be sent which establishes that the link is available again.", "The enhancer, therefore, does not perform this back-off in the event of a retransmission.", "Instead, it keeps the original value of the retransmission timer and re-sends the dropped packet at a constant rate.", "This effectively polls the mobile host—it will elicit an immediate response when the link becomes available.", "This improves the response of the system over end-to-end TCP.", "In the upload case (data flowing from the mobile) there is one case where the enhancer can improve matters, and this is with lengthy disconnections.", "An unmodified TCP stack at the mobile host, if it does not receive an acknowledgement (due to radio link failure), will assume packet loss and retransmit.", "As described above, the TCP will exponentially back-off its retransmissions.", "This means that it will tend to take a long time to notice that the link has become available.", "The enhancer can improve the recovery in this case by detecting that the link to a given mobile is idle.", "In this case it should repeat the last acknowledgement sent on each connection at regular but infrequent intervals1.This will have the effect of ‘kicking’ the TCP at the mobile host into a response when the link becomes available.", "The detection of the link being idle (and thus potentially unavailable) can be described quite simply: if at least one TCP connection exists to the mobile and no TCP connections to that mobile has received a packet for a timeout period, then the link can be considered idle.", "The only cases in which this will be true is when the link is unavailable or when the connections all happen to be idle (i.e.", "there is no unacknowledged data outstanding or data waiting to be delivered).", "If the connections are idle, then the sending of duplicate acknowledgements will not affect the behaviour of the TCP stack on the mobile, so it is a neutral activity.", "It does consume some bandwidth, but the sending of an ‘ack’ packet every few seconds is not a heavy load.", "In the event that the link is unavailable, the TCP window advertised by the enhancer to the Internet hosts will be allowed to close.", "This signals to the sending TCP that the packets are being correctly received, but not being processed by the application.", "The enhancer will (as with standard TCP) send a ‘window update’ packet as soon as data starts being processed again to signal to the host that it can re-start sending.", "Additionally, if the window closes completely, the sending TCP will periodically send a ‘window probe’ to discover when the window opens (as the window update is not reliably delivered).", "The combined effect of the TCP ‘kick’ and the control of the sender by the enhancer mean that enhanced connections are able to recover much faster from a lengthy disconnection than normal, end-to-end TCP/IP.", "The skilled person would realise that the enhancers according to the invention can be employed in a variety of different communication systems, and would know where the most suitable location would be.", "Of course a communication system especially a complex one with many links, may well benefit from a number of enhancers." ] ]	Patent_10466598
[ [ "Image processing method and program for processing image", "The invention relates to error distribution processing used for two-valued or multi-valued reproduction on a system recording or displaying a gradation image with several levels.", "The texture by error distribution processing is suppressed and the granularity of an image is controlled minutely.", "An accumulation error for the target pixel position is separated into first correction accumulation error and second correction accumulation error, the first correction accumulation error is added to data level of target pixel to generate correction level, multi-valuation level of correction level is determined, difference between correction level and multi-valued level is calculated, multi-valuation error is added to the second correction accumulation error to calculate correction multi-valuation error, error distribution value corresponding to unprocessed pixel adjacent to the target pixel is computed from correction multi-valuation error using a specific distribution coefficient, and the results and the accumulation error are added together to update the accumulation error." ], [ "1.An image processing method for representing tone data sampled from an original image in pixels by multi-valued data, comprising the steps of: separating an accumulation error for a position of a target pixel into a first correction accumulation error and a second correction accumulation error; generating a correction level by adding the first correction accumulation error to a data level of the target pixel; determining a multi-valued level of the correction level; computing a multi-valuation error that is a difference between the correction level and the multi-valued level; computing a correction multi-valuation error by adding the second correction accumulation error to the multi-valuation error; computing an error distribution value for an unprocessed pixel around the target pixel from the correction multi-valuation error using a predetermined distribution coefficient; and adding the error distribution value to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "2-3.", "(canceled) 4.An image processing method for representing tone data sampled from an original image in pixels by multi-valued data, comprising the steps of: determining processing conditions using a data level of a target pixel; separating an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error; generating a correction level by adding the first correction accumulation error to a data level of the target pixel; determining a multi-valued level of the correction level; computing a multi-valuation error that is a difference between the correction level and the multi-valued level; computing a correction multi-valuation error by adding the second correction accumulation error to the multi-valuation error; computing an error distribution value for an unprocessed pixel around the target pixel from the correction multi-valuation error using a predetermined distribution coefficient; adding the error distribution value to an accumulation error for a position of the unprocessed pixel to update the accumulation error; and wherein the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "5-7.", "(canceled) 8.An image processing method for representing tone data sampled from an original image in pixels by multi-valued data, comprising the steps of: determining processing conditions using a data level of a target pixel; obtaining an input level of the target pixel by adding a predetermined data level for the target pixel; separating an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error; generating a correction level by adding the first correction accumulation error to the input level; determining a multi-valued level of the correction level; computing a multi-valuation error that is a difference between the correction level and the multi-valued level; computing a correction multi-valuation error by adding the second correction accumulation error to the multi-valuation error; computing an error distribution value for an unprocessed pixel around the target pixel from the correction multi-valuation error using a predetermined distribution coefficient; adding the error distribution value to an accumulation error for a position of the unprocessed pixel to update the accumulation error; and wherein at least one of the predetermined data level and the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "9-16.", "(canceled) 17.An image processing method for representing tone data sampled from an original image in pixels by multi-valued data, comprising the steps of: determining processing conditions using a data level of a target pixel; obtaining an input level of the target pixel by adding a predetermined data level for the target pixel; generating a correction level by adding an accumulation error for a position of the target pixel to the input level; determining a multi-valued level of the correction level using a fluctuating threshold value; computing a multi-valuation error that is a difference between the correction level and the multi-valued level; computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient that changes in a specific cycle; adding the error distribution value to an accumulation error for a position of the unprocessed pixel to update the accumulation error; and wherein the threshold value is generated on the basis of the processing conditions, and at least one of the distribution coefficient and the predetermined data level is controlled using the processing conditions.", "18.An image processing method for representing tone data sampled from an original image in pixels by multi-valued data, comprising the steps of: determining processing conditions using a data level of a target pixel; obtaining an input level of the target level by adding a predetermined data level for the target pixel; separating an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error; generating a correction level by adding the first correction accumulation error to the input level; determining a multi-valued level of the correction level using a fluctuating threshold value; computing a multi-valuation error that is a difference between the correction level and the multi-valued level; computing a correction multi-valuation error by adding the second correction accumulation error to the multi-valuation error; computing an error distribution value for an unprocessed pixel around the target pixel from the correction multi-valuation error using a distribution coefficient that changes in a specific cycle; adding the error distribution value to an accumulation error for a position of the unprocessed pixel to update the accumulation error; and wherein the threshold value is generated on the basis of the processing conditions, and at least one of the distribution coefficient, the predetermined data level, and the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "19.The image processing method of any one of claims 4, 8, 17 and 18 wherein the processing conditions are determined on the basis of results for detecting an area including a highlight area or a shadow area of at least one color data level.", "20-23.", "(canceled) 24.The image processing method of claim 1, 4, 8, or 18 wherein the separation is controlled by multi-valued data for other color at the same pixel position.", "25-26.", "(canceled) 27.The image processing method of claim 17 or 18 wherein the predetermined cycle of the distribution coefficient fluctuates according to the processing conditions.", "28.The image processing method of claim 17 or 18 wherein the error distribution value of the distribution coefficient fluctuates according to the processing conditions.", "29.The image processing method of claim 17 or 18 wherein a filter size of distribution coefficients fluctuates according to the processing conditions.", "30.The image processing method of claim 18 wherein the distribution coefficient comes in two kinds, one for the second correction accumulation error and the other for the multi-valuation error.", "31.The image processing method of claim 18 wherein the data level to be added to the input level is changed according to color.", "32.The image processing method of any one of claims 8, 17 and 18 wherein the predetermined data level is added to only a specific data level of the original image on the basis of the processing conditions.", "33.The image processing method of claim 32 wherein the specific data level is a high level that becomes highlighted when the number of colors is at least one color, or a shadow level that becomes a shadow when the number of colors is least one color.", "34-37.", "(canceled) 38.The image processing method of claim 17 or 18 wherein in case a threshold value is generated on the basis of the processing conditions, a threshold value in one color is differentiated from a threshold value in another color.", "39.An image processing apparatus comprising: an error storing unit operable to store a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated; an error re-distribution determining unit operable to separate an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error; an input correction unit operable to add an input level that is the data level of the target pixel and the first correction accumulation error together; a multi-valuation unit operable to determine a multi-valued level of a correction level outputted from the input correction unit; a difference operation unit operable to find the multi-valuation error that is the difference between the correction level and the multi-valued level; and an error distribution update unit operable to update an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error and the second correction accumulation error using a distribution coefficient, and add the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "40-41.", "(canceled) 42.An image processing apparatus comprising: an error storing unit operable to store a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated; a processing conditions determining unit operable to determine processing conditions using the data level of the target pixel; an error re-distribution determining unit operable to separate an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error; an input correction unit operable to add an input level that is the data level of the target pixel and the first correction accumulation error together; a multi-valuation unit operable to determine a multi-valued level of a correction level outputted from the input correction unit; a difference operation unit operable to find the multi-valuation error that is the difference between the correction level and the multi-valued level; an error distribution update unit operable to update an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error and the second correction accumulation error using a distribution coefficient, and add the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit; and wherein the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "43-45.", "(canceled) 46.An image processing apparatus comprising: an error storing unit operable to store a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated; a processing conditions determining unit operable to determine processing conditions using the data level of the target pixel; a data addition unit operable to add a predetermined data level to the data level of the original image to give an input level of the target pixel; an error re-distribution determining unit operable to separate an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error; an input correction unit operable to add the first correction accumulation error to the input level; a multi-valuation unit operable to determine a multi-valued level of a correction level outputted from the input correction unit; a difference operation unit operable to find the multi-valuation error that is the difference between the correction level and the multi-valued level; an error distribution update unit operable to update an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error and the second correction accumulation error using a distribution coefficient, and add the error distribution value to the accumulation error for a position of the unprocessed pixel, with the error distribution value being stored in the error storing unit; and wherein at least one of the separation into the first correction accumulation error and the second correction accumulation error, and the predetermined data level to be added by the data addition unit is controlled using the processing conditions.", "47-54.", "(canceled) 55.An image processing apparatus comprising: an error storing unit operable to store a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated; a processing conditions determining unit operable to determine processing conditions using the data level of the target pixel; a data addition unit operable to add a predetermined data level to the data level of the original image to give an input level of the target pixel; an input correction unit operable to add an accumulation error for a position of the target pixel to the input level; a threshold value generating unit operable to generate a threshold value for multi-valuation using the processing conditions; a multi-valuation unit operable to determine a multi-valued level of a correction level outputted from the input correction unit using the threshold value outputted from the threshold value generating unit; a difference operation unit operable to find the multi-valuation error that is the difference between the correction level and the multi-valued level; an error distribution update unit operable to update an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient, and add the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit; a distribution coefficient generating unit operable to generate the distribution coefficient used by the error distribution update unit while changing the distribution coefficient in a predetermined cycle; and wherein at least one of the predetermined data level to be added by the data addition unit and the distribution coefficient is controlled using the processing conditions.", "56.An image processing apparatus comprising: an error storing unit operable to store a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated; a processing conditions determining unit operable to determine processing conditions using the data level of the target pixel; a data addition unit operable to add a predetermined data level to the data level of the original image to give an input level of the target pixel; an error re-distribution determining unit operable to separate an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error; an input correction unit operable to add the first correction accumulation error to the input level; a threshold value generating unit operable to generate a threshold value for multi-valuation using the processing conditions; a multi-valuation unit operable to determine a multi-valued level of a correction level outputted from the input correction unit using the threshold value outputted from the threshold value generating unit; a difference operation unit operable to find the multi-valuation error that is the difference between the correction level and the multi-valued level; an error distribution update unit operable to update an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient, and add the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit; a distribution coefficient generating unit operable to generate the distribution coefficient used by the error distribution update unit while changing the distribution coefficient in a predetermined cycle; and wherein at least one of the separation into the first correction accumulation error and the second correction accumulation error, the predetermined data level to be added by the data addition unit and the distribution coefficient is controlled using the processing conditions.", "57.The image processing apparatus of any one of claims 42, 46, and 56 wherein the processing conditions determining unit detects an area including a highlight area or a shadow area of at least one color data level, and determines the processing conditions on the basis of the detection results.", "58-61.", "(canceled) 62.The image processing apparatus of any one of claims 39, 42, 46, and 56 wherein the error re-distribution determining unit uses multi-valued data in the separation for other color at the same pixel position.", "63-64.", "(canceled) 65.The image processing apparatus of any one of claims 55 or 56 wherein the predetermined cycle of the distribution coefficient fluctuates according to the processing conditions.", "66.The image processing apparatus of any one of claims 55 or 56 wherein the error distribution value of the distribution coefficient fluctuates according to the processing conditions.", "67.The image processing apparatus of any one of claims 55 or 56 wherein a filter size of distribution coefficients fluctuates according to the processing conditions.", "68.The image processing apparatus of claim 56 wherein the distribution coefficient to be outputted from the distribution coefficient generating unit comes in two kinds, one for the second correction accumulation error and the other for the multi-valuation error.", "69.The image processing apparatus of any one of claims 46, 55 and 56 wherein the data addition unit changes the data level to be added according to color.", "70.The image processing apparatus of any one of claims 46, 55 and 56 wherein the data addition unit adds the data level to only a specific data level of the original image on the basis of the processing conditions.", "71.The image processing apparatus of claim 70 wherein the specific data level is a highlight level that indicates a highlight with regard to at least one color or a shadow level that indicates a shadow with regard to at least one color.", "72-75.", "(canceled) 76.The image processing apparatus of claim 55 or 56 wherein in case a threshold value is generated on the basis of the processing conditions, the threshold value generating unit differentiates a threshold value in one color from a threshold value in another color.", "77-152.", "(canceled)" ], [ "<SOH> BACKGROUND ART <EOH>In recent years, with the spread of personal computers, the demand for printers has increase greatly and the print quality has been improved.", "In the ink jet printer, the full colors used to be each expressed with two values but now have come to be done with three or more values, so that a higher picture quality is obtained.", "To express multi-gradation with a few recording values, the expression is generally made with quasi-gradation by digital halftone processing, and the dither method or the error diffusion method are often used.", "FIG.", "53 is a diagram to explain a common error diffusion method.", "On a density level, that is, a data level for each pixel sampled from the original image (on assumption that the image processing system is a recording system, the error diffusion method will be explained with the density level as the data level), input correction unit Z 1 generates a correction level I′xy by adding an accumulation error Sxy to a density level Ixy of the notice pixel.", "Two valuation unit Z 2 compares the correction level I′xy with a specific threshold value Th.", "If the correction level I′xy is larger than the value Th, an output level Pxy of the two valuation unit Z 2 becomes “1,” otherwise, the output becomes “0.” In the description that follows, it is to be understood that the output level “1” is equal to the density level “255,” and the output level “0” is equal to “0.” Differential operation unit Z 3 generates a two-valued error Exy, a value obtained by deducting the output level (density level) Pxy from the correction level I′xy.", "The two-valued error Exy is inputted into error distribution unit Z 4 .", "The error distribution unit Z 4 distributes the two-valued error on the basis of distribution coefficients of the error, adds the distributed errors to corresponding accumulation errors on error storing unit Z 5 and stores the results.", "FIG.", "54 A is a well-known example of distribution coefficients.", "The figures in the filter of these coefficients represent distribution ratios.", "The error diffusion method has excellent characteristics with regard to graduation property and resolving power, and with this method, the occurrence of moire pattern is very low, but the problem is that a peculiar texture is caused.", "To solve this problem, techniques are proposed in Japanese patent publicized gazettes 6-66873 and 6-81257.The block diagram of the image signal processing apparatus disclosed in Japanese patent publicized gazette 6-66873 is shown in FIG.", "55 .", "A big difference between this technique and the error diffusion method described with reference to FIG.", "53 is that in the former technique the distribution coefficients of the two-valued error are changed in a specific cycle by distribution coefficient generating unit Z 14 .", "The distribution ratios of the two-valued error corresponding to the pixels around the notice pixel are not fixed, and a plurality of distribution coefficients for the positions of the adjacent pixels are selected at random from sets of distribution coefficients along with pixel processing and is utilized, whereby texture observed in the ordinary error diffusion method can be greatly curbed.", "The block diagram of the image signal processing apparatus disclosed in Japanese patent publicized gazette 6-81257 is shown in FIG.", "56 .", "A big difference between that apparatus and the apparatus disclosed in Japanese patent publicized gazette 6-66873 ( FIG.", "55 ) is that the latter is provided with density addition unit Z 20 .", "The density addition unit Z 20 adds a density level different from the density level of the original image to the density level of each pixel in the original image.", "That reduces greatly the texture observed in the conventional error diffusion method even on images with small changes in density and on image signals of a uniform density generated by the computer.", "In the techniques disclosed in Japanese patent publicized gazettes 6-66873 and 6-81257, the texture can be kept down, while all density levels and images are processed the same way, and the granularity in an area where the processing is usually not needed is raised, and the picture quality will be degraded.", "Another problem is that that arrangement could not sufficiently curb overlapping of color dots with poor granularity.", "Still another problem is that in half-tone area, too, the granularity is different from area to area and continuity of the granularity is lacking." ], [ "<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.", "1 is a block diagram of an image processing apparatus in the first embodiment according to the present invention.", "FIG.", "2 is a block diagram of a color image processing apparatus.", "FIG.", "3 is a block diagram of a first error distribution determining circuit, an example of an error re-distribution determining unit.", "FIG.", "4 is a block diagram of a first error distribution update circuit, an example of error distribution update unit.", "FIG.", "5 is a block diagram of an image processing apparatus in the second embodiment according to the present invention.", "FIG.", "6 is a block diagram of a first distribution coefficient generating circuit, an embodiment of distribution coefficient generating unit.", "FIG.", "7 is a block diagram of an image processing apparatus in the third embodiment according to the present invention.", "FIG.", "8 is a block diagram of a first data addition circuit, an example of data addition unit.", "FIG.", "9 is a block diagram of an image processing apparatus in the fourth embodiment of the present invention.", "FIG.", "10 is diagram showing a processing condition determining circuit A, an example of processing condition determining unit.", "FIG.", "11 is processing condition determining circuit B, an example of processing condition determining unit.", "FIG.", "12 is a diagram showing a processing condition determining circuit C, an example of processing condition determining unit.", "FIG.", "13 is a diagram showing processing condition determining circuit D, an example of processing condition determining unit.", "FIG.", "14 is a diagram showing image area determining circuit, an example of image area determining unit.", "FIG.", "15 is diagram showing a second error distribution determining circuit, an example of error re-distribution determining unit.", "FIG.", "16 is a block diagram of an image processing apparatus in the fifth embodiment according to the present invention.", "FIG.", "17 is a block diagram of second distribution coefficient generating circuit, an example of distribution coefficient generating unit.", "FIG.", "18 is a block diagram of an image processing apparatus in the sixth embodiment according to the present invention.", "FIG.", "19 is a block diagram of an image processing apparatus in the seventh embodiment according to the present invention.", "FIG.", "20 is a block diagram of a second data addition circuit, an example of data addition unit.", "FIG.", "21 is a block diagram of an image processing apparatus in the eighth embodiment according to the present invention.", "FIG.", "22 is a block diagram of an image processing apparatus in the ninth embodiment according to the present invention.", "FIG.", "23 is a block diagram of an image processing apparatus in the tenth embodiment according to the present invention.", "FIG.", "24 is a block diagram of an image processing apparatus in the eleventh embodiment according to the present invention.", "FIG.", "25 is a block diagram of a threshold value generating circuit, an example of threshold value generating unit.", "FIG.", "26 is an explanatory diagram of threshold values.", "FIG.", "27 is a block diagram of an image processing apparatus in the twelfth embodiment according to the present invention.", "FIG.", "28 is a block diagram of an image processing apparatus in the thirteenth embodiment according to the present invention.", "FIG.", "29 is a block diagram of an image processing apparatus in the fourteenth embodiment according to the present invention.", "FIG.", "30 is a block diagram of an image processing apparatus in the fifteenth embodiment according to the present invention.", "FIG.", "31 is a block diagram of an image processing apparatus in the sixteenth embodiment according to the present invention.", "FIG.", "32 is a block diagram of an image processing apparatus in the seventeenth embodiment according to the present invention.", "FIG.", "33 is a block diagram of an image processing apparatus in the eighteenth embodiment according to the present invention.", "FIG.", "34 is a block diagram of an MPU system.", "FIG.", "35 is a flow chart of an image processing method in the nineteenth embodiment according to the present invention.", "FIG.", "36 is a flow chart of an image processing method in the twentieth embodiment according to the present invention.", "FIG.", "37 is a flow chart of an image processing method in the twenty-first embodiment according to the present invention.", "FIG.", "38 is a flow chart of an image processing method in the twenty-second embodiment according to the present invention.", "FIG.", "39 is a flow chart of an image processing method in the twenty-third embodiment according to the present invention.", "FIG.", "40 is a flow chart of an image processing method in the twenty-fourth embodiment according to the present invention.", "FIG.", "41 is a flow chart of an image processing method in the twenty-fifth embodiment according to the present invention.", "FIG.", "42 is a flow chart of an image processing method in the twenty-sixth embodiment according to the present invention.", "FIG.", "43 is a flow chart of an image processing method in the twenty-seventh embodiment according to the present invention.", "FIG.", "44 is a flow chart of an image processing method in the twenty-eighth embodiment according to the present invention.", "FIG.", "45 is a flow chart of an image processing method in the twenty-ninth embodiment according to the present invention.", "FIG.", "46 is a flow chart of an image processing method in the thirtieth embodiment according to the present invention.", "FIG.", "47 is a flow chart of an image processing method in the thirty-first embodiment according to the present invention.", "FIG.", "48 is a flow chart of an image processing method in the thirty-second embodiment according to the present invention.", "FIG.", "49 is a flow chart of an image processing method in the thirty-third embodiment according to the present invention.", "FIG.", "50 is a flow chart of an image processing method in thirty-fourth embodiment according to the present invention.", "FIG.", "51 is a flow chart of an image processing method in the thirty-fifth embodiment according to the present invention.", "FIG.", "52 is a flow chart of an image processing method in the thirty-sixth embodiment according to the present invention.", "FIG.", "53 is a block diagram of the prior art error diffusion processing apparatus.", "FIG.", "54 is an explanatory diagram of distribution coefficient of error.", "FIG.", "55 is a block diagram of a prior art image signal processing apparatus.", "FIG.", "56 is a block diagram of another prior art image signal processing apparatus.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to an image processing method, image processing apparatus, image processing system, and image processing program for two-valued or multi-valued reproduction in a system recording or displaying a gradation image with several levels.", "BACKGROUND ART In recent years, with the spread of personal computers, the demand for printers has increase greatly and the print quality has been improved.", "In the ink jet printer, the full colors used to be each expressed with two values but now have come to be done with three or more values, so that a higher picture quality is obtained.", "To express multi-gradation with a few recording values, the expression is generally made with quasi-gradation by digital halftone processing, and the dither method or the error diffusion method are often used.", "FIG.", "53 is a diagram to explain a common error diffusion method.", "On a density level, that is, a data level for each pixel sampled from the original image (on assumption that the image processing system is a recording system, the error diffusion method will be explained with the density level as the data level), input correction unit Z1 generates a correction level I′xy by adding an accumulation error Sxy to a density level Ixy of the notice pixel.", "Two valuation unit Z2 compares the correction level I′xy with a specific threshold value Th.", "If the correction level I′xy is larger than the value Th, an output level Pxy of the two valuation unit Z2 becomes “1,” otherwise, the output becomes “0.” In the description that follows, it is to be understood that the output level “1” is equal to the density level “255,” and the output level “0” is equal to “0.” Differential operation unit Z3 generates a two-valued error Exy, a value obtained by deducting the output level (density level) Pxy from the correction level I′xy.", "The two-valued error Exy is inputted into error distribution unit Z4.The error distribution unit Z4 distributes the two-valued error on the basis of distribution coefficients of the error, adds the distributed errors to corresponding accumulation errors on error storing unit Z5 and stores the results.", "FIG.", "54 A is a well-known example of distribution coefficients.", "The figures in the filter of these coefficients represent distribution ratios.", "The error diffusion method has excellent characteristics with regard to graduation property and resolving power, and with this method, the occurrence of moire pattern is very low, but the problem is that a peculiar texture is caused.", "To solve this problem, techniques are proposed in Japanese patent publicized gazettes 6-66873 and 6-81257.The block diagram of the image signal processing apparatus disclosed in Japanese patent publicized gazette 6-66873 is shown in FIG.", "55.A big difference between this technique and the error diffusion method described with reference to FIG.", "53 is that in the former technique the distribution coefficients of the two-valued error are changed in a specific cycle by distribution coefficient generating unit Z14.The distribution ratios of the two-valued error corresponding to the pixels around the notice pixel are not fixed, and a plurality of distribution coefficients for the positions of the adjacent pixels are selected at random from sets of distribution coefficients along with pixel processing and is utilized, whereby texture observed in the ordinary error diffusion method can be greatly curbed.", "The block diagram of the image signal processing apparatus disclosed in Japanese patent publicized gazette 6-81257 is shown in FIG.", "56.A big difference between that apparatus and the apparatus disclosed in Japanese patent publicized gazette 6-66873 (FIG.", "55) is that the latter is provided with density addition unit Z20.The density addition unit Z20 adds a density level different from the density level of the original image to the density level of each pixel in the original image.", "That reduces greatly the texture observed in the conventional error diffusion method even on images with small changes in density and on image signals of a uniform density generated by the computer.", "In the techniques disclosed in Japanese patent publicized gazettes 6-66873 and 6-81257, the texture can be kept down, while all density levels and images are processed the same way, and the granularity in an area where the processing is usually not needed is raised, and the picture quality will be degraded.", "Another problem is that that arrangement could not sufficiently curb overlapping of color dots with poor granularity.", "Still another problem is that in half-tone area, too, the granularity is different from area to area and continuity of the granularity is lacking.", "DISCLOSURE OF INVENTION This invention adopts the following configuration for solving the aforementioned problems.", "In the (first) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, an accumulation error for a position of a target pixel is separated into a first correction accumulation error and a second correction accumulation error.", "A correction level is generated by adding the first correction accumulation error to a data level of the target pixel.", "After a multi-valued level of the correction level is determined, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "A correction multi-valuation error is computed by adding the second correction accumulation error to the multi-valuation error.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a predetermined distribution coefficient.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "As set forth above, the accumulation error for the target pixel position is separated into first and second correction accumulation errors, and added the first correction accumulation error to the density level of the original image.", "Then, the data level of the target pixel can be made not larger than the original image when another color dot is presented as long as the error is not accumulated more than a specific value.", "Therefore, the overlapping of color dots can be kept down, and dots disperse, whereby the granularity will improve.", "In the (second) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, an accumulation error for a position of a target pixel is separated into a first correction accumulation error and a second correction accumulation error.", "A correction level is generated by adding the first correction accumulation error to a data level of the target pixel.", "After a multi-valued level of the correction level is determined, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "A correction multi-valuation error is computed by adding the second correction accumulation error to the multi-valuation error.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a distribution coefficient that changes in a specific cycle.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "In case the distribution coefficients fluctuating, it is possible to keep down occurrence of texture in addition to the effects of the first image processing method.", "In the (third) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, an input level of a target pixel is obtained by adding a predetermined data level for the target pixel.", "An accumulation error for a position of the target pixel is separated into a first correction accumulation error and a second correction accumulation error.", "A correction level is generated by adding the first correction accumulation error to the input level.", "After a multi-valued level of the correction level is determined, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "A correction multi-valuation error is computed by adding the second correction accumulation error to the multi-valuation error.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a distribution coefficient that changes in a specific cycle.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "As set forth above, since the data level for the target pixel and the predetermined data level are added, it is possible to substantially keep down the texture for an image with small change in density and an image with a uniform density generated by computer in addition to effects of the second image processing method.", "In the (fourth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "An accumulation error for a position of the target pixel is separated into a first correction accumulation error and a second correction accumulation error.", "A correction level is generated by adding the first correction accumulation error to a data level of the target pixel.", "After a multi-valued level of the correction level is determined, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "A correction multi-valuation error is computed by adding the second correction accumulation error to the multi-valuation error.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a predetermined distribution coefficient.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "In this case, since the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions, example detecting a edge (a character/line-drawing area), dots are overstrike in character, line drawing area even if other color dots are present, and therefore the edge sharpness of characters, line drawing increases, and the image quality in the character, line drawing area will improve.", "Also, the propagation of accumulation error can be prevented and occurrence of unnecessary noise can be kept down.", "In the (fifth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "A correction level is generated by adding an accumulation error for a position of the target pixel to a data level of the target pixel.", "After a multi-valued level of the correction level is determined, a correction multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a distribution coefficient that changes in a specific cycle.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, the distribution coefficient is controlled using the processing conditions.", "As set forth above, since the distribution coefficient is controlled using the processing conditions, the granularity of image can be controlled corresponding to an image area.", "In the (sixth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "An accumulation error for a position of the target pixel is separated into a first correction accumulation error and a second correction accumulation error.", "A correction level is generated by adding the first correction accumulation error to a data level of the target pixel.", "After a multi-valued level of the correction level is determined, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "A correction multi-valuation error is computed by adding the second correction accumulation error to the multi-valuation error.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a distribution coefficient that changes in a specific cycle.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, at least one of the separation into the first correction accumulation error and the second correction accumulation error, and the distribution coefficient is controlled using the processing conditions.", "As set forth above, the sixth image processing method is possible to obtain the effects of the fifth image processing method in addition to the effects of the fourth image processing method.", "As the two kinds of the separation of the accumulation error and the distribution coefficient are controlled together, the image quality will improve.", "In the (seventh) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "An input level of a target pixel is obtained by adding a predetermined data level for the target pixel.", "A correction level is generated by adding an accumulation error for a position of the target pixel to the input level.", "After a multi-valued level of the correction level is determined, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the multi-valuation error using a predetermined distribution coefficient.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, the predetermined data level is controlled using the processing conditions.", "As set forth above, since the data level to be added to the target pixel is controlled using the data level of the target pixel or the pixel around the target pixel, the diffusion of dots can be controlled minutely on the density level of target.", "Example, the data level can be added only the highlight area or shadow area in which the diffusion of dots is worse.", "In the (eighth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "An input level of the target pixel is obtained by adding a predetermined data level for the target pixel.", "An accumulation error for a position of the target pixel is separated into a first correction accumulation error and a second correction accumulation error.", "A correction level is generated by adding the first correction accumulation error to the input level.", "After a multi-valued level of the correction level is determined, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "A correction multi-valuation error is computed by adding the second correction accumulation error to the multi-valuation error.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a predetermined distribution coefficient.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, at least one of the separation into the first correction accumulation error and the second correction accumulation error, and the predetermined data level is controlled using the processing conditions.", "As set forth above, the eighth image processing method is possible to obtain the effects of the fourth image processing method and the seventh image processing apparatus.", "Then, as the two kinds of the separation of the accumulation error and the data level to be added are controlled together, the image quality will improve.", "In the (ninth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "An input level of a target pixel is obtained by adding a predetermined data level for the target pixel.", "A correction level is generated by adding an accumulation error for a position of the target pixel to the input level.", "After a multi-valued level of the correction level is generated, a correction multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a distribution coefficient that changes in a specific cycle.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, at least one of the distribution coefficient and the predetermined data level is controlled using the processing conditions.", "As set forth above, the ninth image processing method is possible to obtain the effects of the seventh image processing method in addition to the effects of the fifth image processing method.", "As the distribution coefficient and the data level to be added are controlled together, the image quality will improve.", "In the (tenth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "An input level of the target pixel is obtained by adding a predetermined data level for the target pixel.", "An accumulation error for a position of the target pixel is separated into a first correction accumulation error and a second correction accumulation error.", "A correction level is generated by adding the first correction accumulation error to the input level.", "After a multi-valued level of the correction level is determined, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "A correction multi-valuation error is computed by adding the second correction accumulation error to the multi-valuation error.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a distribution coefficient that changes in a specific cycle.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, at least one of the distribution coefficient, the predetermined data level, and the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "As set forth above, the tenth image processing method is possible to obtain the effects of the forth image processing method, the fifth image processing method, and the seventh image processing method at the same time.", "As the three kinds of the separation of the accumulation error, the distribution coefficient, and the data level to be added are controlled together, the image quality will improve.", "In the (eleventh) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using only a data level of a target pixel.", "A correction level is generated by adding an accumulation error for a position of the target pixel to the data level of the target pixel.", "After a multi-valued level of the correction level is determined using a fluctuating threshold value, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the multi-valuation error using a predetermined distribution coefficient.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, the threshold value is generated on the basis of the processing conditions.", "As set forth above, since the generation of threshold value using only the data level of the target pixel, processing speed is faster than detecting the image area including the pixel around the target pixel, and it can be obtained an image data in which the delay of dot can be kept down.", "In the (twelfth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "An accumulation error for a position of the target pixel is separated into a first correction accumulation error and a second correction accumulation error.", "A correction level is generated by adding the first correction accumulation error to the data level of the target pixel.", "After a multi-valued level of the correction level is determined using a fluctuating threshold value, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "A correction multi-valuation error is computed by adding the second correction accumulation error to the multi-valuation error.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a predetermined distribution coefficient.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, the threshold value is generated on the basis of the processing conditions, and the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "As set forth above, the twelfth image processing method is possible to obtain the effects of the eleventh image processing method, in addition to the effects of the fourth image processing method.", "As the two kinds of the separation of the accumulation error and the generation of the threshold value are controlled together, the image quality will improve.", "In the (thirteenth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "A correction level is generated by adding an accumulation error for a position of the target pixel to the data level of the target pixel.", "After multi-valued level of the correction level is determined using a fluctuating threshold value, a correction multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a distribution coefficient that changes in a specific cycle.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, the threshold value is generated on the basis of the processing conditions, and the distribution coefficient is controlled using the processing conditions.", "As set forth above, the thirteenth image processing method is possible to obtain the effects of the eleventh image processing method in addition to the effects of the fifth image processing method.", "As the two kinds of the distribution coefficient and the generation of the threshold value are controlled together, the image quality will improve.", "In the (fourteenth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "An accumulation error for a position of the target pixel is separated into a first correction accumulation error and a second correction accumulation error.", "A correction level is generated by adding the first correction accumulation error to the data level of the target pixel.", "After a multi-valued level of the correction level is determined using a fluctuating threshold value, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "A correction multi-valuation error is computed by adding the second correction accumulation error to the multi-valuation error.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a distribution coefficient that changes in a specific cycle.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, the threshold value is generated on the basis of the processing conditions, and at least one of the distribution coefficient and the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "As set forth above, the fourteenth image processing method is possible to obtain the effects of the eleventh image processing method in addition to the effects of the fourth image processing method and the fifth image processing method.", "As the three kinds of the separation of the accumulation error, the distribution coefficient, and the generation of the threshold value are controlled together, the image quality will improve.", "In the (fifteenth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "An input level of the target pixel is obtained by adding a predetermined data level for the target pixel.", "A correction level is generated by adding an accumulation error for a position of the target pixel to the input level.", "After a multi-valued level of the correction level is determined using a fluctuating threshold value, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the multi-valuation error using a predetermined distribution coefficient.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, the threshold value is generated on the basis of the processing conditions, and the predetermined data level is controlled using the processing conditions.", "As set forth above, the fifteenth image processing method is possible to obtain the effects of the eleventh image processing method in addition to the effects of the seventh image processing method.", "As the data level to be added to the input level and the generation of the threshold value are controlled together, the image quality will improve.", "In the (sixteenth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "An input level of the target level is obtained by adding a predetermined data level for the target pixel.", "An accumulation error for a position of the target pixel is separated into a first correction accumulation error and a second correction accumulation error.", "A correction level is generated by adding the first correction accumulation error to the input level.", "After a multi-valued level of the correction level is determined using a fluctuating threshold value, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "A correction multi-valuation error is computed by adding the second correction accumulation error to the multi-valuation error.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a predetermined distribution coefficient.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, the threshold value is generated on the basis of the processing conditions, and at least one of the predetermined data level and the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "As set forth above, the sixteenth image processing method is possible to obtain the effects of the eleventh image processing method in addition to the effects of the fourth image processing method and the seventh image processing method.", "As the three kinds of the separation of the accumulation error, the data level to be added to the input data level, and the generation of the threshold value are controlled together, the image quality will improve.", "In the (seventeenth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "An input level of the target pixel is obtained by adding a predetermined data level for the target pixel.", "A correction level is generated by adding an accumulation error for a position of the target pixel to the input level.", "After a multi-valued level of the correction level is determined using a fluctuating threshold value, a multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the multi-valuation error using a distribution coefficient that changes in a specific cycle.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, the threshold value is generated on the basis of the processing conditions, and at least one of the distribution coefficient and the predetermined data level is controlled using the processing conditions.", "As set forth above, the seventeenth image processing method is possible to obtain the effects of the eleventh image processing method in addition to the effects of the fifth image processing method and the seventh image processing method.", "As the three kinds of the distribution coefficient, the data level to be added to the input data level, and the generation of the threshold value are controlled together, the image quality will improve.", "In the (eighteenth) image processing method of this invention, for representing tone data sampled from an original image in pixels by multi-valued data, processing conditions are determined using a data level of a target pixel.", "An input level of the target level is obtained by adding a predetermined data level for the target pixel.", "An accumulation error for a position of the target pixel is separated into a first correction accumulation error and a second correction accumulation error.", "A correction level is generated by adding the first correction accumulation error to the input level.", "After a multi-valued level of the correction level is determined using a fluctuating threshold value.", "A multi-valuation error that is a difference between the correction level and the multi-valued level is computed.", "A correction multi-valuation error is computed by adding the second correction accumulation error to the multi-valuation error.", "An error distribution value for an unprocessed pixel around the target pixel is computed from the correction multi-valuation error using a distribution coefficient that changes in a specific cycle.", "The error distribution value is added to an accumulation error for a position of the unprocessed pixel to update the accumulation error.", "Then, in this method, the threshold value is generated on the basis of the processing conditions, and at least one of the distribution coefficient, the predetermined data level, and the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "As set forth above, the eighteenth image processing method is possible to obtain the effects of the eleventh image processing method in addition to the effects of the fourth image processing method, the fifth image processing apparatus, and the seventh image processing method.", "As the four kinds of the separation of the accumulation error, the distribution coefficient, the data level to be added to the input data level, and the generation of the threshold value are controlled together, the image quality will improve.", "In the image processing method of the present invention, as an example, the processing conditions are determined on the basis of results for detecting an area including a highlight area or a shadow area in at least one color data level.", "Also, the processing conditions can be determined by using only the data level of the target pixel.", "The processing conditions can be determined on the basis of results for detecting an area including at least the maximum data level or the minimum data level.", "In addition, the processing conditions can be determined on the basis of results for detecting an area where the edge quantity of the image area is not smaller than a specific value and an area where the granularity in the image area changes no smaller than a specific value.", "Also, the separation into the first correction accumulation error and the second correction accumulation error is controlled by multi-valued data for other color at the same pixel position, as an example.", "In case predetermined processing conditions are met, both the first correction accumulation error and the second correction accumulation error for a position of the target pixel may be made 0.In this case, the predetermined processing conditions are that the data level of the target pixel is the maximum data or the minimum data level as an example.", "The predetermined cycle of the distribution coefficient may fluctuate according to the processing conditions.", "The error distribution value of the distribution coefficient may fluctuate according to the processing conditions.", "Also, a filter size of the distribution coefficients may fluctuate according to the processing conditions.", "It is possible that the distribution coefficient comes in two kinds, one for the second correction accumulation error and the other for the multi-valuation error.", "Also, the data level to be added to the input level may be changed according to color.", "The predetermined data level can be added to only a specific data level of the original image on the basis of the processing conditions.", "In this case, the specific data level is a highlight level that indicates a highlight with regard to at least one color or a shadow level that indicates a shadow with regard to at least one color as an example.", "The specific data level can be a data level determined on the basis of the degree of change in granularity after multi-valuation.", "In a case that the input level is a shadow level that indicates a shadow with regard to at least one color, it is possible to decrease the threshold value of multi-valuation on the basis of the processing conditions.", "And, in a case that the input level is a highlight level that indicates a highlight with regard to at least one color, it is possible to increase the threshold value of multi-valuation on the basis of the processing conditions.", "Also, the threshold value for multi-valuation of a specific data level of the original image may be fluctuated in a specific cycle on the basis of the processing conditions.", "In case a threshold value is generated on the basis of the processing conditions, it is possible to differentiate a threshold value in one color from a threshold value in another color.", "Also, in the (first) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "An error re-distribution determining unit separates an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error.", "An input correction unit adds an input level that is the data level of the target pixel and the first correction accumulation error together.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit.", "A difference operation finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error and the second correction accumulation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "In this way, the accumulation error is separated into the first correction accumulation error and second correction accumulation error according to the error redistribution control signal, and therefore, diffusion of dots can be controlled.", "Especially when positioning information on dots of other colors is used as the error distribution control signal, overlapping of dots decreases and an image with good granularity can be obtained.", "In the (second) image processing apparatus of this invention, the error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "An error re-distribution determining unit separates an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error.", "An input correction unit adds an input level that is the data level of the target pixel and the first correction accumulation error together.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error and the second correction accumulation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "A distribution coefficient generating unit generates the distribution coefficient used by the error distribution update unit while changing the distribution coefficient in a predetermined cycle.", "In this apparatus, the error re-distribution determining unit makes it possible to obtain an image with a good granularity with less overlapping of color dots.", "In addition, provision of distribution coefficient generating unit can keep down occurrence of texture in image.", "In the (third) image processing apparatus of this invention, a data addition unit adds a predetermined data level to a data level of an original image to give an input level of a target pixel when tone data sampled from the original image by pixels is multi-valuated.", "An error storing unit stores a multi-valuation error of the target pixel by relating the multi-valuation error to pixel positions around the target pixel.", "An error re-distribution determining unit separates an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error.", "An input correction unit adds the input level and the first correction accumulation error together.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error and the second correction accumulation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "A distribution coefficient generating unit generates the distribution coefficient used by the error distribution update unit while changing the distribution coefficient in a predetermined cycle.", "In the third image processing apparatus, provision of data addition unit can substantially keep down the texture on an image with less change in density and an image with a uniform density generated by computer, in addition to the of the second image processing apparatus Also, on the (fourth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "An error re-distribution determining unit separates an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error.", "An input correction unit adds an input level that is the data level of the target pixel and the first correction accumulation error together.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error and the second correction accumulation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "Then, in this apparatus, the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "As set forth above, since the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions, example processing condition determining unit detects a edge (a character/line-drawing area), dots are overstrike in character, line drawing area even if other color dots are present, and therefore the edge sharpness of characters, line drawing increases, and the image quality in the character, line drawing area will improve.", "Also, the propagation of accumulation error can be prevented and occurrence of unnecessary noise can be kept down.", "In the (fifth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "An input correction unit adds an accumulation error for a position of the target pixel to an input level that is the data level of the target pixel.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "A distribution coefficient generating unit generates the distribution coefficient used by the error distribution update unit while changing the distribution coefficient in a predetermined cycle.", "As set forth above, since the distribution coefficient is controlled using the processing conditions, the granularity of image can be controlled corresponding to an image area.", "In the (sixth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "An error re-distribution determining unit separates an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error.", "An input correction unit adds the first correction accumulation error to an input level that is the data level of the target pixel.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error and the second correction accumulation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "A distribution coefficient generating unit generates the distribution coefficient used by the error distribution update unit while changing the distribution coefficient in a predetermined cycle.", "Then, in this apparatus, at least one of the separation into the first correction accumulation error and the second correction accumulation error, and the distribution coefficient is controlled using the processing conditions.", "As set forth above, the sixth image processing apparatus is possible to obtain the effects of the fifth image processing apparatus in addition to the effects of the fourth image processing apparatus.", "As the two kinds of the separation of the accumulation error and the distribution coefficient are controlled together, the image quality will improve.", "In the (seventh) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "A data addition unit adds the data level controlled by the processing conditions to the data level of the original image to give an input level of the target pixel.", "An input correction unit adds an accumulation error for a position of the target pixel to the input level.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "As set forth above, when the data level to be added to the target pixel is controlled using the data level of the target pixel or the pixel around the target pixel, the diffusion of dots can be controlled minutely on the density level of target.", "Example, the data level can be added only the highlight area or shadow area in which the diffusion of dots is worse.", "In the (eighth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "A data addition unit adds a predetermined data level to the data level of the original image to give an input level of the target pixel.", "An error re-distribution determining unit separates an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error.", "An input correction unit adds the first correction accumulation error to the input level.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error and the second correction accumulation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the error distribution value being stored in the error storing unit.", "Then, in this apparatus, at least one of the separation into the first correction accumulation error and the second correction accumulation error, and the predetermined data level to be added by the data addition unit is controlled using the processing conditions.", "As set forth above, the eighth image processing apparatus is possible to obtain the effects of the fourth image processing apparatus and the seventh image processing apparatus.", "Then, as the two kinds of the separation of the accumulation error and the data level to be added are controlled together, the image quality will improve.", "In the (ninth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "A data addition unit adds a predetermined data level to the data level of the original image to give an input level of the target pixel.", "An input correction unit adds the accumulation error for a position of the target pixel to the input level.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, the accumulation error being stored in the error storing unit.", "A distribution coefficient generating unit generates the distribution coefficient used by the error distribution update unit while changing the distribution coefficient in a predetermined cycle.", "Then, in this apparatus, at least one of the distribution coefficient and the predetermined data level to be added by the data addition unit is controlled using the processing conditions.", "As set forth above, the ninth image processing apparatus is possible to obtain the effects of the seventh image processing apparatus in addition to the effects of the fifth image processing apparatus.", "As the distribution coefficient and the data level to be added are controlled together, the image quality will improve.", "In the (tenth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "A data addition unit adds a predetermined data level to the data level of the original image to give an input level of the target pixel.", "An error re-distribution determining unit separates an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error.", "An input correction unit adds the first correction accumulation error to the input level.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "A distribution coefficient generating unit generates the distribution coefficient used by the error distribution update unit while changing the distribution coefficient in a predetermined cycle.", "Then, in this apparatus, at least one of the separation into the first correction accumulation error and the second correction accumulation error, the predetermined data level to be added by the data addition unit, and the distribution coefficient is controlled using the processing conditions.", "As set forth above, the tenth image processing apparatus is possible to obtain the effects of the forth image processing apparatus, the fifth image processing apparatus, and the seventh image processing apparatus at the same time.", "As the three kinds of the separation of the accumulation error, the distribution coefficient, and the data level to be added are controlled together, the image quality will improve.", "In the (eleventh) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using only the data level of the target pixel.", "An input correction unit adds an accumulation error for a position of the target pixel to an input level that is the data level of the target pixel.", "A threshold value generating unit generates a threshold value for a multi-valuation using the processing conditions.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit using the threshold value outputted from the threshold value generating unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "As set forth above, since the generation of threshold value using only the data level of the target pixel, processing speed is faster than detecting the image area including the pixel around the target pixel, and it can be obtained an image data in which the delay of dot can be kept down.", "In the (twelfth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "An error re-distribution determining unit separates an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error.", "An input correction unit adds the first correction accumulation error to an input level that is the data level of the target pixel.", "A threshold value generating unit generates a threshold value for multi-valuation using the processing conditions.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit using the threshold value outputted from the threshold value generating unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "Then, in this apparatus, the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "As set forth above, the twelfth image processing apparatus is possible to obtain the effects of the eleventh image processing apparatus, in addition to the effects of the fourth image processing apparatus.", "As the two kinds of the separation of the accumulation error and the generation of the threshold value are controlled together, the image quality will improve.", "In the (thirteenth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "An input correction unit adds an accumulation error for a position of the target pixel to an input level that is the data level of the target pixel.", "A threshold value generating unit generates a threshold value for multi-valuation using the processing conditions.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit using the threshold value outputted from the threshold value generating unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "A distribution coefficient generating unit generates the distribution coefficient used by the error distribution update unit while changing the distribution coefficient in a predetermined cycle.", "Then, in this apparatus, the distribution coefficient is controlled using the processing conditions.", "As set forth above, the thirteenth image processing apparatus is possible to obtain the effects of the eleventh image processing apparatus in addition to the effects of the fifth image processing apparatus.", "As the two kinds of the distribution coefficient and the generation of the threshold value are controlled together, the image quality will improve.", "In the (fourteenth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "An error re-distribution determining unit separates an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error.", "An input correction unit adds the first correction accumulation error to an input level that is the data level of the target pixel.", "A threshold value generating unit generates a threshold value for multi-valuation using the processing conditions.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit using the threshold value outputted from the threshold value generating unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error and the second correction accumulation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "A distribution coefficient generating unit generates the distribution coefficient used by the error distribution update unit while changing the distribution coefficient in a predetermined cycle.", "Then, in this apparatus, at least one of the separation into the first correction accumulation error and the second correction accumulation error, and the distribution coefficient is controlled using the processing conditions.", "As set forth above, the fourteenth image processing apparatus is possible to obtain the effects of the eleventh image processing apparatus in addition to the effects of the fourth image processing apparatus and the fifth image processing apparatus.", "As the three kinds of the separation of the accumulation error, the distribution coefficient, and the generation of the threshold value are controlled together, the image quality will improve.", "In the (fifteenth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "A data addition unit adds the data level controlled by the processing conditions to the data level of the original image to give an input level of the target pixel.", "An input correction unit adds an accumulation error for a position of the target pixel to the input level.", "A threshold value generating unit generates a threshold value for multi-valuation using the processing conditions.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit using the threshold value outputted from the threshold value generating unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "As set forth above, the fifteenth image processing apparatus is possible to obtain the effects of the eleventh image processing apparatus in addition to the effects of the seventh image processing apparatus.", "As the data level to be added to the input level and the generation of the threshold value are controlled together, the image quality will improve.", "In the (sixteenth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "A data addition unit adds a predetermined data level to the data level of the original image to give an input level of the target pixel.", "An error re-distribution determining unit separates an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error.", "An input correction unit adds the first correction accumulation error to the input level.", "A threshold value generating unit generates a threshold value for multi-valuation using the processing conditions.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit using the threshold value outputted from the threshold value generating unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error and the second correction accumulation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "Then, in this apparatus, at least one of the predetermined data level to be added by the data addition unit, and the separation into the first correction accumulation error and the second correction accumulation error is controlled using the processing conditions.", "As set forth above, the sixteenth image processing apparatus is possible to obtain the effects of the eleventh image processing apparatus in addition to the effects of the fourth image processing apparatus and the seventh image processing apparatus.", "As the three kinds of the separation of the accumulation error, the data level to be added to the input data level, and the generation of the threshold value are controlled together, the image quality will improve.", "In the (seventeenth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "A data addition unit adds a predetermined data level to the data level of the original image to give an input level of the target pixel.", "An input correction unit adds an accumulation error for a position of the target pixel to the input level.", "A threshold value generating unit generates a threshold value for multi-valuation using the processing conditions.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit using the threshold value outputted from the threshold value generating unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "A distribution coefficient generating unit generates the distribution coefficient used by the error distribution update unit while changing the distribution coefficient in a predetermined cycle.", "Then, in this apparatus, at least one of the predetermined data level to be added by the data addition unit and the distribution coefficient is controlled using the processing conditions.", "As set forth above, the seventeenth image processing apparatus is possible to obtain the effects of the eleventh image processing apparatus in addition to the effects of the fifth image processing apparatus and the seventh image processing apparatus.", "As the three kinds of the distribution coefficient, the data level to be added to the input data level, and the generation of the threshold value are controlled together, the image quality will improve.", "In the (eighteenth) image processing apparatus of this invention, an error storing unit stores a multi-valuation error of a target pixel by relating the multi-valuation error to pixel positions around the target pixel when tone data sampled from an original image by pixels is multi-valuated.", "A processing conditions determining unit determines processing conditions using the data level of the target pixel.", "A data addition unit adds a predetermined data level to the data level of the original image to give an input level of the target pixel.", "An error re-distribution determining unit separates an accumulation error for a position of the target pixel into a first correction accumulation error and a second correction accumulation error.", "An input correction unit adds the first correction accumulation error to the input level.", "A threshold value generating unit generates a threshold value for multi-valuation using the processing conditions.", "A multi-valuation unit determines a multi-valued level of a correction level outputted from the input correction unit using the threshold value outputted from the threshold value generating unit.", "A difference operation unit finds the multi-valuation error that is the difference between the correction level and the multi-valued level.", "An error distribution update unit updates an accumulation error by computing an error distribution value for an unprocessed pixel around the target pixel from the multi-valuation error using a distribution coefficient, and adds the error distribution value to the accumulation error for a position of the unprocessed pixel, with the accumulation error being stored in the error storing unit.", "A distribution coefficient generating unit generates the distribution coefficient used by the error distribution update unit while changing the distribution coefficient in a predetermined cycle.", "Then, in this apparatus, at least one of the separation into the first correction accumulation error and the second correction accumulation error, the predetermined data level to be added by the data addition unit and the distribution coefficient is controlled using the processing conditions.", "As set forth above, the eighteenth image processing apparatus is possible to obtain the effects of the eleventh image processing apparatus in addition to the effects of the fourth image processing apparatus, the fifth image processing apparatus, and the seventh image processing apparatus.", "As the four kinds of the separation of the accumulation error, the distribution coefficient, the data level to be added to the input data level, and the generation of the threshold value are controlled together, the image quality will improve.", "In the image processing apparatus of the present invention, the processing conditions determining unit detects, as an example, an area including a highlight area or a shadow area in at least one color data level, and determines the processing conditions on the basis of the detection results.", "The processing conditions determining unit may determine the processing conditions by using only the data level of the target pixel.", "Also, the processing conditions determining unit may detect an area including at least the maximum data level or the minimum data level and determine the processing conditions on the basis of the detection results.", "In addition, the processing conditions determining unit may detect an area where the edge quantity of the image area is not smaller than a specific value, and determine the processing conditions on the basis of the detection results.", "And the processing conditions determining unit may detect an area where the granularity in the image area changes no smaller than a specific value, and determines the processing conditions on the basis of the detection results.", "The error re-distribution determining unit uses multi-valued data in the separation for other color at the same pixel position as an example.", "In case predetermined processing conditions are met, error re-distribution determining unit may make both the first correction accumulation error and the second correction accumulation error for a position of the target pixel 0.In this case, the predetermined processing conditions are that the data level of the target pixel is the maximum data or the minimum data level.", "The predetermined cycle of the distribution coefficient may fluctuate according to the processing conditions.", "The error distribution value of the distribution coefficient may fluctuate according to the processing conditions, too.", "Also, a filter size of the distribution coefficients may fluctuate according to the processing conditions.", "It is possible that the distribution coefficient to be outputted from the distribution coefficient generating unit comes in two kinds, one for the second correction accumulation error and the other for the multi-valuation error.", "The data addition unit changes the data level to be added according to color.", "The data addition unit may add the data level to only a specific data level of the original image on the basis of the processing conditions.", "In this case, the specific data level is a highlight level that indicates a highlight with regard to at least one color or a shadow level that indicates a shadow with regard to at least one color as an example.", "Also, the specific data level can be a data level determined on the basis of the degree of change in granularity after multi-valuation.", "In a case that the input level is a shadow level that indicates a shadow with regard to at least one color, the threshold value generating unit may decrease the threshold value of multi-valuation on the basis of the processing conditions.", "And, in a case that the input level is a highlight level that indicates a highlight with regard to at least one color, the threshold value generating unit may increase the threshold value of multi-valuation on the basis of the processing conditions.", "Also, the threshold value generating unit may fluctuate the threshold value for multi-valuation of a specific data level of the original image in a specific cycle on the basis of the processing conditions.", "In case a threshold value is generated on the basis of the processing conditions, the threshold value generating unit can differentiate a threshold value in one color from a threshold value in another color.", "Also, the present invention provides not only the image processing method or the image processing apparatus but also an image processing system or an image processing program.", "In the image processing system, the same function of the image processing apparatus can be obtained in cooperation with plural units comprised in the system.", "Also, the image processing program makes a computer or a computer system operate as the image processing apparatus or the image processing system.", "It is possible to obtain the same function of the image processing apparatus or the image processing system by the image processing program cooperating with hardware of the computer or the computer system.", "BRIEF DESCRIPTION OF DRAWINGS FIG.", "1 is a block diagram of an image processing apparatus in the first embodiment according to the present invention.", "FIG.", "2 is a block diagram of a color image processing apparatus.", "FIG.", "3 is a block diagram of a first error distribution determining circuit, an example of an error re-distribution determining unit.", "FIG.", "4 is a block diagram of a first error distribution update circuit, an example of error distribution update unit.", "FIG.", "5 is a block diagram of an image processing apparatus in the second embodiment according to the present invention.", "FIG.", "6 is a block diagram of a first distribution coefficient generating circuit, an embodiment of distribution coefficient generating unit.", "FIG.", "7 is a block diagram of an image processing apparatus in the third embodiment according to the present invention.", "FIG.", "8 is a block diagram of a first data addition circuit, an example of data addition unit.", "FIG.", "9 is a block diagram of an image processing apparatus in the fourth embodiment of the present invention.", "FIG.", "10 is diagram showing a processing condition determining circuit A, an example of processing condition determining unit.", "FIG.", "11 is processing condition determining circuit B, an example of processing condition determining unit.", "FIG.", "12 is a diagram showing a processing condition determining circuit C, an example of processing condition determining unit.", "FIG.", "13 is a diagram showing processing condition determining circuit D, an example of processing condition determining unit.", "FIG.", "14 is a diagram showing image area determining circuit, an example of image area determining unit.", "FIG.", "15 is diagram showing a second error distribution determining circuit, an example of error re-distribution determining unit.", "FIG.", "16 is a block diagram of an image processing apparatus in the fifth embodiment according to the present invention.", "FIG.", "17 is a block diagram of second distribution coefficient generating circuit, an example of distribution coefficient generating unit.", "FIG.", "18 is a block diagram of an image processing apparatus in the sixth embodiment according to the present invention.", "FIG.", "19 is a block diagram of an image processing apparatus in the seventh embodiment according to the present invention.", "FIG.", "20 is a block diagram of a second data addition circuit, an example of data addition unit.", "FIG.", "21 is a block diagram of an image processing apparatus in the eighth embodiment according to the present invention.", "FIG.", "22 is a block diagram of an image processing apparatus in the ninth embodiment according to the present invention.", "FIG.", "23 is a block diagram of an image processing apparatus in the tenth embodiment according to the present invention.", "FIG.", "24 is a block diagram of an image processing apparatus in the eleventh embodiment according to the present invention.", "FIG.", "25 is a block diagram of a threshold value generating circuit, an example of threshold value generating unit.", "FIG.", "26 is an explanatory diagram of threshold values.", "FIG.", "27 is a block diagram of an image processing apparatus in the twelfth embodiment according to the present invention.", "FIG.", "28 is a block diagram of an image processing apparatus in the thirteenth embodiment according to the present invention.", "FIG.", "29 is a block diagram of an image processing apparatus in the fourteenth embodiment according to the present invention.", "FIG.", "30 is a block diagram of an image processing apparatus in the fifteenth embodiment according to the present invention.", "FIG.", "31 is a block diagram of an image processing apparatus in the sixteenth embodiment according to the present invention.", "FIG.", "32 is a block diagram of an image processing apparatus in the seventeenth embodiment according to the present invention.", "FIG.", "33 is a block diagram of an image processing apparatus in the eighteenth embodiment according to the present invention.", "FIG.", "34 is a block diagram of an MPU system.", "FIG.", "35 is a flow chart of an image processing method in the nineteenth embodiment according to the present invention.", "FIG.", "36 is a flow chart of an image processing method in the twentieth embodiment according to the present invention.", "FIG.", "37 is a flow chart of an image processing method in the twenty-first embodiment according to the present invention.", "FIG.", "38 is a flow chart of an image processing method in the twenty-second embodiment according to the present invention.", "FIG.", "39 is a flow chart of an image processing method in the twenty-third embodiment according to the present invention.", "FIG.", "40 is a flow chart of an image processing method in the twenty-fourth embodiment according to the present invention.", "FIG.", "41 is a flow chart of an image processing method in the twenty-fifth embodiment according to the present invention.", "FIG.", "42 is a flow chart of an image processing method in the twenty-sixth embodiment according to the present invention.", "FIG.", "43 is a flow chart of an image processing method in the twenty-seventh embodiment according to the present invention.", "FIG.", "44 is a flow chart of an image processing method in the twenty-eighth embodiment according to the present invention.", "FIG.", "45 is a flow chart of an image processing method in the twenty-ninth embodiment according to the present invention.", "FIG.", "46 is a flow chart of an image processing method in the thirtieth embodiment according to the present invention.", "FIG.", "47 is a flow chart of an image processing method in the thirty-first embodiment according to the present invention.", "FIG.", "48 is a flow chart of an image processing method in the thirty-second embodiment according to the present invention.", "FIG.", "49 is a flow chart of an image processing method in the thirty-third embodiment according to the present invention.", "FIG.", "50 is a flow chart of an image processing method in thirty-fourth embodiment according to the present invention.", "FIG.", "51 is a flow chart of an image processing method in the thirty-fifth embodiment according to the present invention.", "FIG.", "52 is a flow chart of an image processing method in the thirty-sixth embodiment according to the present invention.", "FIG.", "53 is a block diagram of the prior art error diffusion processing apparatus.", "FIG.", "54 is an explanatory diagram of distribution coefficient of error.", "FIG.", "55 is a block diagram of a prior art image signal processing apparatus.", "FIG.", "56 is a block diagram of another prior art image signal processing apparatus.", "BEST MODE FOR CARRYING OUT THE INVENTION Now, the embodiments of the present invention will be described with reference to the drawings.", "The embodiments will be described with the case of a recording system shown and with data level as density level.", "First Embodiment FIG.", "1 is a block diagram of an image processing apparatus in the first embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "1 comprises input correction unit 1, multi-valuation unit 2, difference operation unit 3, error re-distribution determining unit 4, error distribution update unit 5 and error storing unit 6.Error re-distribution determining unit 4 separates accumulation error 17 for the target pixel position into first correction accumulation error 12 and second correction accumulation error 16 according to error distribution control signal 19.Input correction unit 1 first adds first correction accumulation error 12 outputted from error re-distribution determining unit 4 to tone density level 10 of each pixel which is sampled from the original image and generates correction level 11.Multi-valuation unit 2 compares correction level 11 and a plurality of threshold values 13 and outputs multi-valued data 14.Difference operation unit 3 calculates multi-valuation error 15 from correction level 11 and multi-valued data 14.Error distribution update unit 5 distributes the sum of multi-valuation error 15 and second correction accumulation error 16 according to a specific distribution coefficients (distribution ratio), and adds each distributed error to the respective accumulation error 18 at the pixel position for each unprocessed pixel adjacent to a target pixel stored in error storing unit 6 (or stored in error distribution update unit 5) and updates the accumulation error.", "FIG.", "2 indicates the structure of the color image processing device in the simplified manner.", "The color signal is comprised of density level 10 of tone for respective colors of C (cyan), M (magenta), Y (yellow) and K (black).", "The color image processing device has four image processing devices 21-24 for respective colors, and density level 10 of each color is inputted to the corresponding image processing devices.", "The respective image processing devices 21-24 output multi-valued data of density level 10 as signals 19, 30, 31 and 32.In FIG.", "2, the signal 19 outputted from image processing device 21 is also inputted to the image processing device 22 as an error re-distribution control signal.", "This indicates that the present invention is applied to the image processing device 22 among the image processing devices 21-24.The present invention, however, can be applied to the other image processing devices.", "For example, in order to apply the present invention to the image processing devices 23 and 24, as shown by the broken line in FIG.", "2, the signals 30 and 31 outputted from the image processing devices 22 and 23 may be inputted to the image processing devices 23 and 24 respectively as error re-distribution control signals.", "The reason to use multi-valued data of other colors outputted from the image processing device 21 for error re-distribution control signal 19 corresponding to the image processing device 22 is that the granularity becomes finer and the image quality is more improved in case a dot of other color is not laid to the notice pixel position than other color dot is laid.", "In order to attain a high quality image, an accumulation error corresponding to the notice pixel position is not added to density level of the notice pixel, but multi-valuation is conducted only in the density level of the original image, which prevents a dot from being laid to the same position.", "Color for separating an accumulation error into the first correction accumulation error and the second correction accumulation error by using error re-distribution control signal 19 could be one color.", "Also, here, error re-distribution control signal 19 explains as a signal indicating whether other color dot exists or not, but it is not limited to the foregoing.", "FIG.", "3 is a block diagram of a first error distribution value determination circuit that is an example of error re-distribution determining unit 4.The error distribution determining circuit is made up of comparators 41, 42, logic element 43, selectors 44, 45.Accumulation error 17 for the target pixel position is first inputted into comparators 41, 42.In comparator 41, a comparison is made with specific value 46.The specific value 46 is density level “0”, for example.", "In addition, accumulation error 17 is compared with specific value 47 by comparator 42.Specific value 47 is density level “128”, for example.", "If accumulation error 17 is larger than specific threshold 46, comparator 41 makes signal line 48 a high level.", "If accumulation error 17 is smaller than specific value 47, comparator 42 makes signal line 49 a high level.", "That is, these output signals 48 and 49 judge whether accumulation error 17 is within a specific scope (larger than specific value 46 and smaller than specific value 47).", "Only when error distribution control signal 19 is at a high level (indicates that a dot of another color is stricken) and the outputs of comparators 48 and 49 are at a high level, logical element 43 sets signal line 50 at a high level.", "Selector 44 outputs specific value 51 when signal line 50 is at a high level, and outputs accumulation error 17 when signal 50 is at a low level.", "That is, when another dot is stricken at the target pixel position, and when the accumulation error for the pixel position is within a specific range, specific value 51 (“0” for example) is outputted instead of accumulation error 17.That can reduce the overlapping ratio of color dots.", "The value outputted from selector 44 becomes first correction accumulation error 12.On the other hand, selector 45 outputs accumulation error 17 when signal line 50 is at a high level and outputs specific value 52 (“0” for example) when signal line 50 is at a low level.", "The value outputted from selector 45 becomes second correction accumulation error 16.In the present example, specific values are fixed values 46, 47, but may be fluctuated depending on or density level at or around the target pixel.", "The present embodiment is so configured that either accumulation error 17 or specific value “0” is selected as first correction accumulation error 12 and second correction accumulation error.", "But it is not limited to this method, but it may be so configured that the distribution ratio is changed depending on selection signals.", "Input correction unit 1 may be formed of adders, and multi-valuation unit 2 is formed of a plurality of comparators and selectors.", "And also difference operation unit 3 can be formed of a difference device (not shown).", "A well known method may be used.", "Also as error storing unit 6, RAM (random access memory) or line buffer may be used.", "FIG.", "4 is a block diagram of a first error distribution update circuit, an example of error distribution update unit 5.The first error distribution update circuit comprises adders 61 to 64, multipliers 65 to 68, register 69-71 and divider 72.Adder 61 obtains a correction multi-valued error 76 by adding second correction accumulation error 16 outputted from error re-distribution determining unit 4 and multi-valued error 15 outputted from difference operation unit 3.The correction multi-valued error 76 is multiplied by the respective values 77a to 77d in multipliers 65 to 68.As specific values 77a to 77d, the distribution coefficients shown in FIG.", "54A may be used.", "That is, specific value 77a is a value “7”; specific value 77b is a value “1”; specific value 77c is a value “5”; specific value 77d is a value “3.” Distribution error 82 generated by multiplier 66 is stored in register 70.The register synchronizes with the sampling of the pixel and stores data.", "That is, from register 70, the multiplication result of the preceding image is outputted from register 70.Adder 63 adds distribution error 85 of the previous pixel and distribution error 83 outputted from multiplier 67 and then outputs the result to register 71.Similarly, register 71 delays data for one pixel.", "The signal outputted from adder 64 becomes accumulation error 18a, that is, an accumulation of accumulation errors distributed to the pixels located lower than the target pixel, and stored in error storing unit 6.Adder 62 adds together accumulation error 18B one line before and distribution error 81 of target pixel.", "The addition result is inputted into divider 72 and divided by a specific value.", "The specific value to be used is a value obtained by adding all distribution coefficients together.", "In the present example, the distribution coefficients shown in FIG.", "54A is used and divided by a value “16.” To simplify the circuit, the division of addition result 88 may be executed with 4 bit shift.", "Division result 89 is stored in register 69 and is outputted as final accumulation error 17 in the next pixel processing.", "In present example, second correction accumulation error 16 and multi-valuation error 15 are added together and the result is distributed according to the distribution coefficients.", "Instead, the results may be distributed according to different distribution coefficients respectively.", "As described, the accumulation error is separated into the first correction accumulation error and second correction accumulation error according to the error redistribution control signal, and therefore, an image with good granularity can be obtained.", "Especially when positioning information on dots of other colors is used as the error distribution control signal, overlapping of dots decreases and an image with good granularity can be obtained.", "Second Embodiment FIG.", "5 is a block diagram of an image processing apparatus in the second embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "5 is made up of input correction unit 91, multi-valuation mean 92, difference operation unit 93, error re-distribution determining unit 94, error distribution update unit 95, distribution coefficient generating unit 96 and error storing unit 97.Error re-distribution determining unit 94 separates accumulation error 107 for the target pixel position into first correction accumulation error 101 and second correction accumulation error 106 according to error distribution control signal 110.Input correction unit 91 adds first correction accumulation error 101 outputted from error re-distribution determining unit 94 to tone density level 100 of each pixel which is sampled from the original image, and generates correction level 102.Multi-valuation unit 92 compares correction level 102 and a plurality of specific threshold values 103, and outputs multi-valued data 104.Difference operation unit 93 works out multi-valuation error 105 from correction level 102 and multi-valued data 104.Distribution coefficient generating unit 96 generates distribution coefficient 108 at specific intervals and outputs the same to error distribution update unit 95.Error distribution update unit 95 distributes the sum of multi-valuation error 105 and second correction accumulation error 106 according to distribution coefficients 108 and adds each distributed error to the respective accumulation error 109 at the pixel position for each unprocessed pixel adjacent to a target pixel stored in error storing unit 97 (or stored in error distribution update unit 95), and updates accumulation error.", "In FIG.", "5, input correction unit 91, multi-valuation unit 92, difference operation unit 93, error re-distribution determining unit 94, error distribution update unit 95 and error storing unit 97 can be materialized in the same configuration as in the first embodiment.", "Therefore, there will be explained distribution coefficient generating unit 96.FIG.", "6 is a block diagram of a first distribution coefficient generating circuit, an example of distribution coefficient generating unit 96.The first distribution coefficient generating circuit comprises random signal generating unit 111 and selector 112.One-bit random signal 118 is outputted from random signal generating unit 111.With one-bit random value generated by computer stored in a table in advance, random signal 118 may be outputted per pixel.", "Selector 112 selects either the first distribution coefficients 113 or second distribution coefficients by random signal 118 and outputs it as distribution coefficients 108 (108A to 108D).", "The outputted distribution coefficients 108 are inputted into error distribution update unit 95.Distribution coefficients shown in FIG.", "54B may be used as first distribution coefficients 113 and distribution coefficients shown in FIG.", "54C may be used as second distribution coefficients 114.The distribution coefficients are not limited to that.", "Furthermore, the size of filter of distribution coefficients may be changed.", "The number of distribution coefficients is not limited to two.", "More than two distribution coefficients may be switched.", "(The same is applicable to the embodiments which will be described later.)", "Furthermore, distribution coefficients 108 outputted from distribution coefficient generating unit 96 may be outputted for two purposes, that is, for second correction accumulation error 106 and for multi-valuation error 105.In this case, second correction accumulation error 106 and multi-valuation error 105 are distributed according to different distribution coefficients.", "After distribution, they may be synthesized (added) and made accumulation error at each position.", "The error re-distribution determining unit 94 makes it possible to obtain an image with a good granularity with less overlapping of color dots.", "In addition, provision of distribution coefficient generating unit 96 can keep down occurrence of texture in image.", "Third Embodiment FIG.", "7 is a block diagram of an image processing apparatus in the third embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "7 comprises data addition unit 121, input correction unit 122, multi-valuation 123, difference operation unit 124, error re-distribution determining unit 125, error distribution update unit 126, distribution coefficient generating unit 127 and error storing unit 128.Data addition unit 121 adds density level (data level) fluctuating at specific intervals different from the density level of target pixel to tone density level of each pixel which is sampled from the original image, and generates input level 132.Error re-distribution determining unit 125 separates accumulation error 139 for the target pixel position into first correction accumulation error 136 and second correction accumulation error 138 in accordance with error distribution control signal 140.Input correction unit 122 adds first correction accumulation error 136 outputted from error re-distribution determining unit 125 to input level 132 outputted from data addition unit 121, and generates correction level 133.Multi-valuation unit 123 compares correction level 133 and a plurality of specific threshold values 134 and outputs multi-valued data 135.Difference operation unit 124 works out multi-valuation error 137 from correction level 133 and multi-valued data 135.Distribution coefficient generating unit 127 generates distribution coefficient 141 at specific intervals and outputs the same to error distribution update unit 126.Error distribution update unit 126 distributes the sum of multi-valuation error 137 and second correction accumulation error 136 according to distribution coefficients 141 and adds each distributed error to the respective accumulation error 142 at the pixel position for each unprocessed pixel adjacent to a target pixel stored in error storing unit 128 (or stored in error distribution update unit 126), and updates the accumulation error.", "In FIG.", "7, input correction unit 122, multi-valuation unit 123, difference operation unit 124, error re-distribution determining unit 127, error re-distribution determining unit 125, error distribution update unit 126 and error storing unit 128 can be materialized in the same configuration as in the second embodiment.", "Therefore, there will be explained data addition unit 121.FIG.", "8 is a block diagram of a first data addition circuit, an example of data addition unit 121.The first data addition circuit is made up of data generating unit 151 and adder 152.Tone density level 131 of each pixel which is sampled from the original image is added to density level 164 outputted from data generating unit 151 by adder 152 and generates input level 132.Data generating unit 151 is made up of line data generating unit 153 to 156 and selector 157.Selector 157 selects and outputs any one of addition data levels 170 to 173 outputted from line data generating unit 153 to 156 on the basis of line information 165 on the target pixel.", "In the present embodiment, selected addition data levels 170 to 173 change four lines by four lines.", "It is noted that in the present embodiment, there are provided four line data generating unit, but the number of the unit is not limited to four.", "Line data generating unit 153 is made up of a plurality of registers (or a set of flip-flops) 158 to 161.Data levels 174 to 176 outputted from registers 158 to 160 are inputted to registers 159 to 161 in the following stage respectively.", "The data level 170 outputted from the register 161 placed in the last stage is inputted to the register 158 placed in the forefront stage through the signal line 177.In line data generating unit 153, the value of register circulates by every pixel.", "As a result, the data level 170 outputted from the register 161 varies by four pixel cycles.", "An initial value of the register is set to the register 158 through the signal line 166.In the example of FIG.", "8, four registers 158 to 161 are set in line data generating unit 153, but it is not limited to four registers.", "By changing an initial value which is set in one or more registers by colors, the diffusion of dots which are different by every color is generated.", "Other line data generating units 154 to 156 are comprised in the same manner as line data generating unit 153.As an example for these, an initial value of the register of other line data generating units 154 to 156 is set through signal lines 167-169 respectively.", "For example, by using such data addition unit 121, density level 161 is added to density level 131, and input level 132 is produced.", "Provision of data addition unit 121 can substantially keep down the texture on an image with less change in density and an image with a uniform density generated by computer, in addition to the effects described in the second embodiment.", "If correction level is multi-valuated without adding data level, there is a data level for which the granularity extremely decreases.", "The continuity of impression of grain can be improved by adding data to such data level which is determined according to the degree of change of the granularity after the multi-valuation.", "Fourth Embodiment FIG.", "9 is a block diagram of an image processing apparatus in the fourth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "9 is made up of input correction unit 181, multi-valuation unit 182, difference operation unit 183, processing condition determining unit 184, error re-distribution determining unit 185, error distribution update unit 186 and error storing unit 187.Processing condition determining unit 184 determines processing conditions using the density level of the target pixel alone or density level of the target pixel and density level adjacent to the target pixel out of tone density levels 191 which are sampled from the original image by pixels, and outputs first processing condition signal 196.Error re-distribution determining unit 185 separates the accumulation error for the target pixel position into first correction accumulation error 197 and second correction accumulation error 198 on the basis of error distribution control signal 200 and first processing condition signal 196.Input correction unit 181 adds first correction accumulation error 197 to input level 191, that is, density level of the target pixel and generates correction level 192.Multi-valuation unit 182 generates multi-valued data 194 from correction level 192 and a plurality of threshold value 193.Difference operation unit 183 works out multi-valuation error 195 from correction level 192 and multi-valued data 194.Error distribution update unit 186 distributes multi-valuation error 195 according to distribution coefficient and adds each distributed error to the respective accumulation error 201 at the pixel position for each unprocessed pixels adjacent to a target pixel stored in error storing unit 187 (or stored in error distribution update unit 186), and updates the accumulation error.", "In FIG.", "9, input correction unit 181, multi-valuation unit 182, difference operation unit 183, error distribution update unit 186, and error storing unit 187 can be materialized in the same configuration as in the first embodiment.", "Therefore, there will be explained processing condition determining unit 184 and error re-distribution determining unit 185.FIG.", "10 is a block diagram of processing condition determining circuit A, a first example of processing condition determining unit 184.Processing condition determining circuit A of the present embodiment is a circuit for detecting the highlight area and shadow area.", "Processing condition determining circuit A shown in FIG.", "10 are made up of line buffers 204, 205, adders 206, 207, 213, 214, registers (a set of flip-flops) 211, 212, comparators 208, 209 and logic element 210.It is noted that blocks 231, 232, 233 are identical in configuration.", "Tone density level 191A of each pixel which is sampled from the original image is inputted into register 211.After delaying for one pixel, the output signal 228 of register 211 is inputted into register 212.After delaying by one more pixel, register 212 outputs signal 230.By these, density levels 191A, 228, 230 of three pixels can be handled at the same time.", "Adder 213 adds density level 191A and signal 228, and the addition result 229 is furthermore added to signal 230 at adder 214.That means that image data for three pixels are added.", "Line buffer 204 delays image data for one line.", "In addition, line buffer 205 delays image data by one more line.", "Blocks 232, 233 are identical with block 231 in configuration.", "These permit handling density levels of pixels on three columns, three rows.", "Image data for three lines are added by adders 206, 207.Added data 223 of a total of 9 pixels are compared with specific values 224, 225 at comparators 208, 209.If the added data 223 is smaller than specific threshold value 224, comparator 208 outputs to signal line 226 a high level signal indicating that the area is a highlight area, while if the added data 223 is larger than the threshold value 225, comparator 209 outputs to signal line 227 a high level signal indicating that the area is a shadow area.", "If either signal line 226 or 227 is a high level, logical element 210 sets (first) processing condition signal at a high level.", "In the present embodiment, the pixel position of the target pixel is the third column, third row and the image data corresponding to the target pixel is the oldest pixel data.", "Therefore, the density level 191 to be inputted into input correction unit 181 has to be delayed (no delay circuit shown).", "The delay circuit may make common use of a line buffer or register in processing condition determining circuit A shown in FIG.", "10 The target pixel position, in the above example, is not limited to data at this pixel position.", "It is also noted that the image area is detected from the density level of an area of 3×3 pixels, but the area is not limited to this area size.", "FIG.", "11 is a block diagram of processing condition determining circuit B, a second example of processing condition determining unit 184.Processing condition determining circuit B detects whether the target pixel in the original image is at the minimum level or at the maximum level.", "Processing condition determining circuit B shown in FIG.", "11 is formed of comparators 241, 242, and logic element 243.Density level 191B of the target pixel is inputted into comparator 241 and it is judged whether the density level is equal to the minimum density level 246.If density level 191B is equal to minimum density level 246, comparator 241 outputs high level to signal line 247.Furthermore, density level 191B is inputted into comparator 242 and it is judged whether the density level is equal to maximum density level 248.If density level 191B is equal to maximum density level 248, comparator 242 outputs high level to signal line 249.If one of the signal lines 247, 249 becomes a high level, logic element 243 sets (first) processing condition signal 196B at a high level.", "FIG.", "12 is a block diagram of processing condition determining circuit C, a third example of processing condition determining unit 184.Processing condition determining circuit C detects a character/line-drawing area by edge detection.", "Processing condition determining circuit C shown in FIG.", "12 is made up of line buffer 251, 252, registers 253 to 256, adders 257 to 259, multiplier 260, divider 261, comparator 262.Processing condition determining circuit C is well known as edge detection circuit.", "The line buffer delays image data by one line.", "Therefore, an image outputted from line buffer 251 delays by one line and an image outputted from line buffer 252 delays by two lines.", "Furthermore, the register delays image data by one pixel.", "The density level at the target pixel position in the present embodiment is a density level 270 outputted from register 254.Data outputted from adder 258 is a value obtained by adding all density levels 267, 269, 272, 273, adjacent to the target pixel in the vertical and horizontal directions.", "Density level 270 at the target pixel position is inputted into multiplier 260 and multiplied by a specific value (“4”, for example).", "The difference value (absolute value) between the output of multiplier 260 and the output of adder 258 is worked out by difference circuit 261.Difference value 277 is compared with specific value 278 at comparator 262, and if the difference value 277 is larger than specific value 278, the comparator sets (first) processing condition signal 196C at a high level.", "When processing condition determining circuit C of the present embodiment is used, density level 191 to be inputted in input correction unit 181 also has to be delayed (no delay circuit shown).", "FIG.", "13 is a block diagram of processing condition determining circuit D, a fourth example of processing condition determining unit 184.Processing condition determining circuit D in the present embodiment detects the average density level in a specific area of the original image.", "When an image of a specific density level is multi-valuated, there is a density level area where the granularity changes markedly (decreases), which can affect the continuity of the impression of grain in images like gradation.", "Therefore, this density area is detected.", "Processing condition determining circuit D shown in FIG.", "13 is formed of line buffer 281, registers 282, 283, adder 284, divider 285, look-up table 286.The density level at the target pixel is density level 293 outputted from register 283.Line buffer 281 delays image data by one line, and registers 282, 283 delay by one pixel.", "Therefore, adder 284 outputs to signal line 294 the results obtained by adding all density levels in an area of 2×2.At divider 285, the addition result is divided by “4” and the average density level 295 is outputted.", "In the case of value “4”, merely two-bit shift may be used.", "Lookup table 286 outputs (first) processing condition signal 196D, a signal judging whether the density level is within a specific scope from average density level 295.Lookup table 286 is used.", "As an alternative to that, it may be judged by comparator whether the density level is within a specific scope.", "It is also noted that the average density level in a 2×2 area is worked out in processing condition determining circuit D, but the present invention is not limited to this area size.", "Furthermore, when processing condition determining circuit D of the present embodiment is used, density level 191 to be inputted into input correction unit 181 also has to be delayed (no delay circuit shown).", "As set forth above, different circuits can be materialized as processing condition determining unit 184.They may be used alone, or some of them may be used in combination.", "When some are combined, image area judging unit may be provided which judges the image area from a plurality of (first) processing condition signals.", "FIG.", "14 is an image area judging circuit, an example of image area judging unit for generating first processing condition signal when the four above-mentioned processing condition determining circuits are combined.", "(First) Processing condition signal 196A outputted from processing condition determining circuit A, (first) processing condition signal 196B outputted from processing condition determining circuit B, (first) processing condition signal 196C outputted from processing condition determining circuit C and (first) processing condition signal 196D outputted from processing condition determining circuit D are inputted into lookup table 301.A control signal outputted from lookup table 301 becomes first processing condition signal 196.The way of controlling will be explained in detail later.", "The present example of processing condition determining unit 184 is so configured that image data is delayed by line buffer and at the same time density levels for a plurality of lines can be processed at the same time.", "As an alternative to that, it is may be so configured that data is read directly form the memory instead of using the line buffer.", "FIG.", "15 is a second error distribution determining circuit, an example of error re-distribution determining unit 185.The second error distribution determining circuit shown in FIG.", "15 is formed of logic elements 311 to 313, comparators 314, 315 and selectors 316, 317.Accumulation error 199 for the target pixel position is first inputted into comparators 314, 315.Comparator 314 compares the accumulation error with specific value 321.The specific value 321 is density level “0”, for example.", "Also, accumulation error 199 is compared with specific value 322 at comparator 315.Specific value 322 is density level “128,” for example.", "If accumulation error 199 is larger than specific value 321, comparator 314 sets output line 323 at a high level.", "If, on the other hand, accumulation error 199 is smaller than specific value 322, comparator 315 sets output line at a high level.", "In other words, it can be judged from these output signals 323, 324 whether accumulation error 199 is within a specific scope.", "Only when error distribution control signal 200 is high level (indicating that another color dot is stricken), output of comparators 314, 315 is high level and first processing condition signal 196C outputted from processing condition determining unit 184 is low level, logic element 311 sets signal line 325 at high level.", "As first processing condition signal 196C, detection signal of character, line drawing area shown in FIG.", "12 is desirable.", "That is, if character, line drawing areas are detected, logical element 311 outputs a low level.", "In the present embodiment, first processing condition signal 196B is input signal of the second error distribution determining circuit.", "As first processing condition signal 196B, the maximum density level or minimum density level shown in FIG.", "11 is desirable.", "When the processing condition signal 196B is high level (when maximum density level or minimum density level is detected) or the output of logical element 311 is at a high level, logical element 312 outputs high level to signal line 326.When signal line 326 is at a high level, selector 316 outputs specific value 328, and when signal line 326 is at a low level, accumulation error 199 is outputted.", "That is, when maximum density level or minimum density level is detected, or when another color dot is stricken at the target pixel position and accumulation error 199 for the target pixel position is within a specific scope, not accumulation error 199 but specific value 328 (“0”, for example) is outputted.", "That can keep down propagation of unneeded errors at the most white part or most black part on the ground etc.", "or overlapping rate of color dots.", "The value outputted from selector 316 becomes first correction accumulation error 197.When signal line 325 is at a high level and first processing condition signal 196B is at a low level (when maximum density level and maximum density level are not detected), selector 317 outputs accumulation error 199, and when signal line 325 is low level or first processing condition signal 196B is high level, specific value 329 (“0”, for example) is outputted.", "A value outputted from selector 317 becomes second correction accumulation error 198.In the present embodiment, specific values 321, 322 are fixed value.", "These values may be changed depending on the density level at or around target pixel.", "The present embodiment is so configured that either accumulation error 199 or specific value “0” is selected as first correction accumulation error 197 and second correction accumulation error 198.The present embodiment is not limited to this method, but may be so configured that the distribution ratio of accumulation error 199 is changed depending on the selection signal.", "Furthermore, first processing condition signals 196B, 196C are used.", "The present embodiment is not limited to these signals, but other first processing condition signals 196A, 196B or a combination of these signals may be used.", "Fifth Embodiment FIG.", "16 is a block diagram of an image processing apparatus in the fifth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "16 is formed of input correction unit 331, multi-valuation unit 332, difference operation unit 333, error distribution update unit 334, distribution coefficient generating unit 335, processing condition determining unit 336 and error storing unit 337.Processing condition determining unit 336 determines processing condition using the density level at or around the target pixel out of tone density levels 341 which are sampled from the original image by pixels, and outputs second processing condition signal 346.Input correction unit 331 adds accumulation error 349 for the target pixel position to tone density level 341 and generates correction level 342.Multi-valuation unit 332 generates multi-valued data 344 from correction level 342 and a plurality of threshold values 343.Difference operation unit 333 works out multi-valuation error 345 from correction level 342 and multi-valued data 344.Distribution coefficient generating unit 335 generates distribution coefficient 347 at specific intervals and outputs the same to error distribution update unit 334.Then, the specific interval of distribution coefficient generating unit 335 is controlled by second processing condition signal 346 outputted from processing condition determining unit 336.Error distribution update unit 334 distributes multi-valuation error 345 according to distribution coefficients 347, and adds each distributed error to the respective accumulation error 348 of the pixel position for each unprocessed pixels adjacent to the target pixel stored in error storing unit 337 (or stored in error distribution update unit 334), and updates accumulation error.", "In FIG.", "16, input correction unit 331, multi-valuation unit 332, difference operation unit 333, processing condition determining unit 336 and error storing unit 337 can be each materialized in the same configuration as the fourth embodiment.", "Therefore, there will be explained error distribution update unit 334 and distribution coefficient generating unit 335.A second error distribution update circuit, an example of error distribution update unit 334, can be materialized with some changes made in first error distribution update circuit shown in FIG.", "4.The second error distribution update circuit where second correction accumulation error 16 does not exist is constituted without adder 61 (not shown).", "FIG.", "17 is a block diagram of a second distribution coefficient generating circuit, an example of distribution coefficient generating unit 335.The second distribution coefficient generating circuit shown in FIG.", "17 is formed of first random signal generating unit 351, second random signal generating unit 352, selector 353 and distribution coefficient selection unit 115.Distribution coefficient unit 115 can be materialized in the same configuration as block 115 surrounded with dotted line in the first distribution coefficient generating circuit, as shown in FIG.", "6.First random signal generating unit 351 and second random signal generating unit 352 are different in ratio of generating one bit random signals “0” and “1.” For example, when two distribution coefficients in FIG.", "54B and FIG.", "54C are switched, first random signal generating unit 351 generates signals selecting the respective distribution coefficients statistically roughly half and half.", "Second random signal generating unit 352 is made to generate random signals in which either of the distribution coefficients is selected more.", "Selector 353 switches first random signal 354 and second random signal 355 on the basis of second processing condition signal 346.From distribution coefficient selection unit 115, selected distribution coefficient 347 (347A to 347D) is outputted.", "In the present embodiment, there are provided two random signal generating unit, that is, first random signal generating unit 351 and second random signal generating unit 352.The number of random signal generating unit is not limited to two.", "More than two may be provided to control minutely.", "Without a plurality of random signal generating units, one random signal might be controlled by the second processing condition signal outputted from processing condition determining means, in order to change a ratio of each distribution coefficients to be selected.", "The ratio of the value “1” to “0” could be changed by the logical synthesis with the OR or AND element of random signals, which are generated by delaying a random signal.", "In addition to controlling random signal, the selection of distribution coefficients may be controlled (selector 112 is controlled) (the same is applicable to the embodiments that will be described later).", "If distribution coefficients are switched in a random way, the occurrence of texture can be controlled, but the granularity will be poor.", "If distribution coefficients and random ratio are selected properly in view of the relation between granularity and texture, therefore, the picture quality will improve.", "In the case of highlight or shadow area, if dots are dispersed, the dot delay observed in the error diffusion method will be reduced and the picture quality will improve.", "Therefore, it is better for distribution coefficients to be switched at random.", "In the case of the minimum density level or maximum density level, occurrence of unnecessary dots can be prevented if all coefficients are turned to “0” so that no errors may propagate.", "For an area like character/line-drawing area where texture does not stand out, the picture quality will improve without rather than with changing distribution coefficients in a random manner.", "Furthermore, for an area with density level such as a near multi-valued level, it is preferable to make the quality of the granularity poor intentionally because the granularity extremely decreases there.", "Such an area could be observed according to the granularity after multi-valuation.", "If the distribution coefficients are switched so as to make the quality of the granularity poor, the impression of grain for the area will be balanced with the impression for areas with other density levels.", "Sixth Embodiment FIG.", "18 is a block diagram of an image processing apparatus in the sixth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "18 is formed of input correction unit 361, multi-valuation unit 362, difference operation unit 363, processing condition determining unit 364, error re-distribution determining unit 365, error distribution update unit 366, distribution coefficient generating unit 367 and error storing unit 368.Processing condition determining unit 364 detects a specific area from density level at or around the target pixel out of the tone density levels 371 which are sampled from the original image by pixels and outputs first processing condition signal 375 and second processing condition signal 374.Error re-distribution determining unit 365 separates accumulation error 382 for the target pixel position into first correction accumulation error 373 and second correction accumulation error 381 on the basis of error distribution control signal 383 and first processing condition signal 375.Input correction unit 361 adds first correction accumulation error 373 to tone density level 371 and generates correction level 372.Multi-valuation unit 362 generates multi-valued data 377 from correction level 372 and a plurality of specific threshold value 376.Difference operation unit 363 works out multi-valued data 378 from correction level 372 and multi-valued data 377.Distribution coefficient generating unit 367 generates distribution coefficient 379 at specific intervals and outputs the same to error distribution update unit 366.Then, the distribution coefficient of distribution coefficient generating unit 367 is controlled by second processing condition signal 374 outputted from processing condition determining unit 364.Error distribution update unit 366 distributes multi-valuation error 378 according to distribution coefficients 379, and adds each distributed error to the respective accumulation error 380 at the target pixel position for each unprocessed pixel adjacent to a target pixel stored in error storing unit 368 (or stored in error distribution update unit 366) and updates the accumulation error.", "In FIG.", "18, input correction unit 361, multi-valuation unit 362, difference operation unit 363, processing condition determining unit 364, error re-distribution determining unit 365, error distribution update unit 366, distribution coefficient generating unit 367 and error storing unit 368 can be materialized in the same configuration as described before.", "Processing condition determining unit 364 outputs first processing condition signal 375 and second processing condition signal 374.As an alternative to that, those signals may be outputted from a lookup table shown in FIG.", "14 as different processing condition signals or the processing condition signal.", "The present embodiment is so configured that error re-distribution determining unit 365 and distribution coefficient generating unit 367 are both controlled by processing condition determining unit 364.As an alternative to that, only either of them may be controlled.", "According to the present embodiment, dots are overstruck in character, line drawing area even if other color dots are present, and therefore the edge sharpness of characters, line drawing increases, and the picture quality in the character, line drawing area will improve.", "When the maximum density level or minimum density level is detected, the propagation of accumulation error can be prevented and occurrence of unnecessary noise can be kept down.", "Seventh Embodiment FIG.", "19 is a block diagram of an image processing apparatus in the seventh embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "19 is formed of input correction unit 391, multi-valuation unit 392, difference operation unit 393, processing condition determining unit 394, data addition unit 395, error distribution update unit 396 and error storing unit 397.Processing condition determining unit 394 outputs third processing condition signal 409 using the density level at or around the target pixel out of tone density levels 401 which are sampled from the original image by pixels.", "Data addition unit 395 adds the density level changing at specific intervals to density level 401 on the basis of third processing condition signal 409 and generates input level 402.Input correction unit 391 adds accumulation error 408 for the target pixel position to input level 402 and generates correction level 403.Multi-valuation unit 392 generates multi-valued data 405 from correction level 403 and a plurality of specific threshold values 404.Difference operation unit 393 works out multi-valuation error 406 from correction level 403 and multi-valued data 405.Error distribution update unit 396 distributes multi-valuation error 406 according to the distribution coefficients and adds each distributed error to the respective accumulation error 407 at the pixel position for each unprocessed pixel adjacent to a target pixel stored in error storing unit 397 (or stored in error distribution update unit 396), and updates accumulation error.", "In FIG.", "19, input correction unit 391, multi-valuation unit 392, difference operation unit 393, processing condition determining unit 394, error distribution update unit 396, and error storing unit 397 can be materialized in the same configuration as mentioned before.", "Therefore, there will be explained data addition unit 395.In this embodiment, third processing condition signal 409 is outputted from processing condition determining unit 394, but any of the aforesaid processing condition signals or the processing condition signal outputted from the lookup table shown in FIG.", "14 may be used.", "FIG.", "20 is a second data addition circuit, an example of data addition unit 395.The second data addition circuit shown in FIG.", "20 is formed of data generating unit 151, multiplier 411, adder 412, and selector 413.Data generating unit 151 can be formed of the same circuits as block 151 in the first data addition circuit shown in FIG.", "8.Third processing condition signal 409 outputted from processing condition determining unit 394 is inputted in selector 413 and becomes selective signals of multiplicators 417 to 419.A selected multiplicator is multiplied by addition data level 416 in multiplier 411, and the multiplying result or addition level 421 is outputted to adder 412.At adder 412, density level 401 and addition level 421 are added and input level 402 is generated.", "If one of multiplicators 417 to 419 is made “0”, the data can be added to specific density level alone.", "The present embodiment is so configured that density level 401 is corrected on the basis of third processing condition signal 409 by data addition unit 395.Therefore, data level to be added to density level 401 of the original image can be changed for every area of image and the granularity can be controlled minutely.", "Eighth Embodiment FIG.", "21 is a block diagram of an image processing apparatus in the eighth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "21 is formed of input correction unit 431, multi-valuation unit 432, difference operation unit 433, error distribution update unit 434, error re-distribution determining unit 435, processing condition determining unit 436, data addition unit 437, and error storing unit 438.Processing condition determining unit 436 outputs first processing condition signal 451 and third processing condition signal 452, using density level at or around the target pixel position out of tone density levels 441 which are sampled from the original image by pixels.", "Error re-distribution determining unit 435 separates accumulation error 448 for the target pixel position into first correction accumulation error 453 and second correction accumulation error 449 on the basis of error distribution control signal 450 and first processing condition signal 451.Data addition unit 437 adds a density level fluctuating at specific intervals to density level 441 on the basis of third processing condition signal 452.Input correction unit 431 adds first correction accumulation error 453 to input level 442 and generates correction level 443.Multi-valuation unit 432 generates multi-valued data 445 from correction level 443 and a plurality of specific threshold values 444.Difference operation unit 433 works out multi-valuation error 446 from correction level 443 and multi-valued data 445.Error distribution update unit 434 distributes multi-valuation error 446 according to the distribution coefficients, and adds each distributed error to the respective accumulation error 447 at the pixel position for each unprocessed pixel adjacent to a target pixel stored in error storing unit 438 (or stored in error distribution update unit 434), and updates the accumulation error.", "All units in FIG.", "21 can be materialized in the same configuration as described before.", "The present embodiment is so configured that both error re-distribution determining unit 435 and data addition unit 437 are controlled by processing condition determining unit 436.As an alternative to that, it may be so configured that either of them is controlled.", "Ninth Embodiment FIG.", "22 is a block diagram of an image processing apparatus in the ninth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "22 is formed of data addition unit 461, input correction unit 462, multi-valuation unit 463, difference operation unit 464, error distribution update unit 466, processing condition determining unit 465, distribution coefficient generating unit 467, and error storing unit 468.Processing condition determining unit 465 outputs second processing condition signal 478 and third processing condition signal 472, using the density level at or around the target pixel position out of tone density levels 471 which are sampled from the original image by pixels.", "Data addition unit 461 adds data level fluctuating at specific intervals on the basis of third processing condition signal 472 and generates input level.", "Input correction unit 462 adds accumulation error 480 for the target pixel position to input level 473 and generates correction level 474.Multi-valuation unit 463 generates multi-valued data 476 from correction level 474 and a plurality of specific threshold values 475.Difference operation unit 464 works out multi-valuation error 477 from correction level 474 and multi-valued data 476.Distribution coefficient generating unit 467 generates distribution coefficient 479 at specific intervals and outputs the results to error distribution update unit 466.Then, the distribution coefficient of distribution coefficient generating unit 467 is controlled by second processing condition signal 478 outputted from processing condition determining unit 465.Error distribution update unit 466 distributes multi-valuation error 477 according the distribution coefficients 479, and adds each distributed error to the respective accumulation error 481 at the pixel position for each unprocessed pixel adjacent to a target pixel stored in error storing unit 468 (or stored in error distribution update unit 466) and updates the accumulation error.", "All units in FIG.", "22 can be materialized in the same configuration as described earlier.", "The present embodiment is so configured that data addition unit 461 and distribution coefficient generating unit 467 are both controlled by processing condition determining unit 465.Instead, either of them may be controlled.", "Tenth Embodiment FIG.", "23 is a block diagram of an image processing apparatus in the tenth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "23 is formed of data addition unit 491, input correction unit 492, multi-valuation unit 493, difference operation unit 494, processing condition determining unit 495, error re-distribution determining unit 496, error distribution update unit 497, distribution coefficient generating unit 498, and error storing unit 499.Processing condition determining unit 495 outputs first processing condition signal 514, second processing condition signal 508 and third processing condition signal 503, using the density level at or around the target pixel out of tone density levels 501 which are sampled from the original image by pixels.", "Error re-distribution determining unit 496 separates accumulation error 511 for the target pixel position 514 into first correction accumulation error 509 and second correction accumulation error 510 on the basis of error distribution control signal 515 and first processing condition signal 514.Data addition unit 491 adds a density level fluctuating at specific intervals to density level 501 on the basis of third processing condition signal 503 and generates input level 502.Input correction unit 492 adds first correction accumulation error 509 to input level 502, and generates correction level 504.Multi-valuation unit 493 generates multi-valued data 506 from correction level 504 and a plurality of specific threshold values 505.Difference operation unit 494 works out multi-valuation error 507 from correction level 504 and multi-valued data 506.Distribution coefficient generating unit 498 generates distribution coefficient 512 at specific intervals and outputs the same to error distribution update unit 497.Then, distribution coefficient of distribution coefficient generating unit 498 is controlled by second correction accumulation error 508 outputted from processing condition determining unit 495.Error distribution update unit 497 distributes multi-valuation error 507 according to distribution coefficient 512, and adds each distributed error to the respective pixel position for each unprocessed pixel adjacent to a target pixel stored in error storing unit 499 (or stored in error re-distribution determining unit 497), and updates the accumulation error.", "In FIG.", "23, all units can be materialized in the same configuration as described earlier.", "It is noted that processing condition determining unit 495 outputs first processing condition signal 514, second processing condition signal 508 and third processing condition signal 503.These signals may be outputted as different processing condition signals or may be all as the same processing condition signals form the lockup table shown in FIG.", "14.The present embodiment is so configured that processing condition determining unit 495 controls error re-distribution determining unit 496, distribution coefficient generating unit 498, data addition unit 491.Instead, at least one of them only may be controlled.", "There will be described control of error re-distribution determining unit, distribution coefficient generating unit, data addition unit by 1 to 3 processing condition signals outputted from processing condition determining unit.", "In a preferred embodiment, when highlight area and shadow area are detected in processing condition determining circuit A shown in FIG.", "10, data addition unit 121 may increase addition density level.", "If the data level is a highlight or shadow level which indicates highlights or shadows not with three color components but with only one component.", "Error re-distribution determining unit may increase the ratio of the second correction accumulation error and the fluctuation in distribution parameter by distribution coefficient generating unit may be increased.", "Furthermore, when it is detected in processing condition determining circuit B that the target pixel is the maximum level or minimum level, data addition unit may make addition density level “0”, error re-distribution determining unit may make first correction accumulation error and second correction accumulation error “0” and distribution coefficient generating unit may make all distribution coefficients “0”.", "Furthermore, in case processing condition determining circuit C detects character, line drawing area, data addition unit may make addition density level “0”, error re-distribution determining unit may increase the ratio of first correction accumulation error, and distribution coefficient generating unit may make distribution coefficients not fluctuate.", "In addition, when an area is detected in processing condition determining circuit D where the granularity fluctuates violently as compared with the other area, data addition unit may increase additional density level, error re-distribution determining unit may increase the ratio of first correction accumulation error, distribution coefficient generating unit may fluctuate distribution coefficients violently.", "As described, a high quality picture can be obtained without texture, with high sharpness in character, drawing line and good continuity of the impression of grain.", "Eleventh Embodiment FIG.", "24 is a block diagram of an image processing apparatus in the eleventh embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "24 is formed of threshold value generating unit 521, input correction unit 522, multi-valuation unit 523, difference operation unit 524, error distribution update unit 525, processing condition determining unit 526, and error storing unit 527.Processing condition determining unit 526 outputs fourth processing condition signal 532 using the density level of the target pixel out of tone density levels 531 which are sampled from the original image by pixels.", "Threshold value generating unit 521 generates a plurality of threshold values 533 for multi-valuation using fourth processing condition signal 532 outputted from processing condition determining unit 526.Input correction unit 522 adds accumulation error 538 to input level 531, that is, density level of the target pixel and generates correction level 535.Multi-valuation unit 523 generates multi-valued data 534 from correction level 535 and a plurality of threshold values 533.Multi-valuation unit 523 generates multi-valued data 534 from correction level 535 and a plurality of threshold values 533.Difference operation unit 524 works out multi-valuation error 536 from correction level 535 and multi-valued data 534.Error distribution update unit 525 distributes multi-valuation error 536 according to the distribution coefficients, and adds each distributed error to the respective accumulation error 537 at the target pixel position for each unprocessed pixel adjacent to a target pixel stored in error storing unit 527 (or stored in error distribution update unit 525), and updates the accumulation error.", "In FIG.", "24, all units except for threshold value 521 can be materialized in the same configuration as described earlier.", "Fourth processing condition signal 532 outputted from processing condition determining unit 526 is a density level only at the target pixel in the present embodiment.", "FIG.", "25 is a block diagram of a threshold value generating circuit, an example of threshold value generating unit 521.Threshold value generating circuit shown in FIG.", "25 is formed of lookup tables 541 to 545, selector 547, adder 548, and random signal generator 546.Fourth processing condition signal 532 outputted from processing condition determining unit 526 is inputted into lookup tables 541 to 445.Lookup table 541 is a threshold value generating table for C (cyan) data, lookup table 542 is for M (magenta) and lookup table 543 is Y (yellow), and lookup table 544 is for K (black).", "Selector 547 selects any one of threshold values 551 to 554 outputted from lookup tables according to color information 555, and outputs the selected threshold.", "The signal line is shown in one line, but plural threshold values are outputted from the lookup tables.", "Lookup table 545 outputs noise data 558, which is random in case of a specific density level, for example, from fourth processing condition signal 532 and random signal 557 outputted from random signal generator 546.Except for the specific density level, value “0” (noiseless) is outputted from lookup table 545.FIG.", "26 is an explanatory diagram of threshold value in the present embodiment, in which data stored in the lookup table is shown in graph.", "The abscissa represents values of fourth processing condition ( be obtained.", "In the present embodiment, the density level at the target pixel alone as fourth processing condition signal is used.", "In other embodiments, the target pixel alone does not necessarily have to be used.", "The present embodiment is so configured that lookup tables 541 to 544 for four colors are prepared, and a threshold value for each color may be generated.", "It may so configured that a lookup table for one color alone is used.", "Also, the present embodiment is so configured that a plurality of lookup tables 541 to 544 and selector 547 and adder 548 are used.", "The present embodiment is not limited to that.", "For example, instead of these, one lookup table may be used.", "Furthermore, it may be so configured that one threshold value for one color alone is different, and the threshold values for other colors are identical.", "Twelfth Embodiment FIG.", "27 is a block diagram of an image processing apparatus in the twelfth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "27 is formed of threshold value generating unit 571, input correction unit 572, multi-valuation unit 573, difference operation unit 574, processing condition determining unit 575, error re-distribution determining unit 576, error distribution update unit 577 and error storing unit 578.Processing condition determining unit 575 outputs first processing condition signal 587 and fourth processing condition signal 582, using the density level at or around the target pixel pieces of out of tone density level 581 which are sampled from the original image by pixels.", "Threshold value generating unit 571 generates a plurality of threshold values 583 for multi-valuation using fourth processing condition signal 582 outputted from processing condition determining unit 575.Error re-distribution determining unit 576 separates accumulation error 590 for the target pixel position into first correction accumulation error 591 and second correction accumulation error 588 on the basis of error distribution control signal 589 and first processing condition signal 587.Input correction unit 572 adds first correction accumulation error 591 to the density level of the target pixel or input level 581 and generates correction level 585.Multi-valuation unit 573 generates multi-valued data 584 from correction level 585 and a plurality of threshold values 583.Difference operation unit 574 works out multi-valuation error 586 from correction level 585 and multi-valued data 584.Error distribution update unit 577 distributes multi-valuation error 586 according to the distribution coefficients, and adds each distributed error to the respective accumulation error 592 at the pixel position for each unprocessed pixel adjacent to a target pixel stored in error storing unit 578 (or stored in error distribution update unit 577), and updates the accumulation error.", "As described above the image processing apparatus, in the twelfth embodiment, consists of the threshold value generating unit 571, processing condition determining unit 575 and error re-distribution determining unit 576.In FIG.", "27, all units can be materialized in the same configuration as described earlier.", "Thirteenth Embodiment FIG.", "28 is a block diagram of an image processing apparatus in the thirteenth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "28 is formed of threshold value generating unit 601, input correction unit 602, multi-valuation unit 603, difference operation unit 604, processing condition determining unit 605, error distribution update unit 606, distribution coefficient generating unit 607, and error storing unit 608.Processing condition determining unit 605 outputs second processing condition signal 618 and fourth processing condition signal 612, using the density level at or around the target pixel position out of tone density level 611 which are sampled from the original image by pixels.", "Threshold value generating unit 601 generates a plurality of threshold values 613 for multi-valuation using fourth processing condition signal 612 outputted from processing condition determining unit 605.Input correction unit 602 generates correction level 616 by adding accumulation error 615 to input level 611, that is, the density level at the target pixel.", "Multi-valuation unit 603 generates multi-valued data 614 from correction level 616 and a plurality of threshold values 613.Difference operation unit 604 works out multi-valuation error 617 from correction level 616 and multi-valued data 614.Distribution coefficient generating unit 607 generates distribution coefficients 619 at specific intervals and outputs the distribution coefficients 619 to error distribution update unit 606.In this, the distribution coefficients of distribution coefficient generating unit 606 is controlled by second processing condition signal 618 outputted from processing condition determining unit 605.Error distribution update unit 606 distributes multi-valuation error 617 according to the distribution coefficients 619 and adds each distributed error to the respective accumulation error 620 at the pixel position corresponding to each unprocessed pixel adjacent to a target pixel stored in error storing unit (or stored in error distribution update unit 606), and updates the accumulation error.", "As described above the image processing apparatus, in the thirteenth embodiment, consists of the threshold value generating unit 601, processing condition determining unit 602 and error re-distribution determining unit 607.In FIG.", "28, all units can be materialized in the same configuration as described earlier.", "Fourteenth Embodiment FIG.", "29 is a block diagram of an image processing apparatus in the fourteenth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "29 is formed of threshold value generating unit 631, input correction unit 632, multi-valuation unit 633, difference operation unit 634, processing condition determining unit 635, error re-distribution determining unit 636, error distribution update unit 637, distribution coefficient generating unit 638 and error distribution update unit 639.Processing condition determining unit 635 outputs first processing condition signal 649, second processing condition signal 648, and fourth processing condition signal 642, using the density level at the target pixel or around the pixel position out of the tone density levels 641 which are sampled from the original image by pixels.", "Threshold value generating unit 631 generates a plurality of threshold values 643 for multi-valuation using fourth processing condition signal 642 outputted from processing condition determining unit 635.Error re-distribution determining unit 636 separates accumulation error 652 for the target pixel position into first correction accumulation error 645 and second correction accumulation error 651 on the basis of error distribution control signal 650 and first processing condition signal 649.Input correction unit 632 adds first correction accumulation error 645 to input level 641, that is, the density level of the target pixel and generates correction level 646.Difference operation unit 633 generates multi-valued data 644 from correction level 646 and a plurality of threshold values 643.Difference operation unit 634 works out multi-valuation error 647 from correction level 646 and multi-valued data 644.Distribution coefficient generating unit 638 generates distribution coefficient 653 at specific intervals and outputs the same to error distribution update unit 637.Then, distribution coefficient 653 of distribution coefficient generating unit 638 is controlled by second processing condition signal 648 outputted from processing condition determining unit 635.Error distribution update unit 637 distributes multi-valuation error 647 according to the distribution coefficients 653 and adds each distributed error to the respective accumulation error 654 at the pixel position corresponding to each unprocessed pixel adjacent to a target pixel stored in error storing unit 639 (or stored in error distribution update unit 637), and updates the accumulation error.", "As described above the image processing apparatus, in the fourteenth embodiment, consists of the threshold value generating unit 631, processing condition determining unit 635, error re-distribution determining unit 636 and distribution coefficient generating unit 638.In FIG.", "29, all units can be materialized in the same configurations as described earlier.", "The present embodiment is so configured that threshold value generating unit 631, error re-distribution determining unit 636, and distribution coefficient generating unit 638 are all controlled by processing condition determining unit 635.Instead, it may be so configured that threshold value generating unit 631 and at least one of other unit only are controlled.", "Fifteenth Embodiment FIG.", "30 is a block diagram of an image processing apparatus in the fifteenth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "30 is formed of threshold value generating unit 661, data addition unit 662, input correction unit 663, multi-valuation unit 664, difference operation unit 665, error distribution update unit 666, error re-distribution determining unit 667, and error storing unit 668.Processing condition determining unit 667 outputs third processing condition signal 677 and fourth processing condition signal 672, using the density level at or around the target pixel position out of the tone density levels 671 which are sampled from the original image by pixels.", "Threshold value generating unit 661 generates a plurality of threshold values 673 for multi-valuation using fourth processing condition signal 672 outputted from processing condition determining unit 667.Data addition unit 662 adds a density level fluctuating at specific intervals to density level 671 on the basis of third processing condition signal 677.Input correction unit 663 adds accumulation error 678 to input level 674 and generates correction level 675.Multi-valuation unit 664 generates multi-valued data 676 from correction level 675 and a plurality of threshold values 673.Difference operation unit 665 works out multi-valuation error 679 from correction level 675 and multi-valued data 676.Error distribution update unit 666 distributes multi-valuation error 679 according to the distribution coefficients, adds each distributed error to the respective accumulation error 680 at pixel position corresponding to unprocessed pixel adjacent to the target pixel stored in error storing unit 668 (or stored in error distribution update unit 666), and updates the accumulation error.", "As described above the image processing apparatus, in the fifteenth embodiment, consists of the threshold value generating unit 661, data addition unit 662 and processing condition determining unit 667.In FIG.", "30, all units can be materialized in the same configuration as described earlier.", "Sixteenth Embodiment FIG.", "31 is a block diagram of an image processing apparatus in the sixteenth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "31 is formed of threshold value generating unit 691, data addition unit 692, input correction unit 693, multi-valuation unit 694, difference operation unit 695, processing condition determining unit 696, error re-distribution determining unit 697, error distribution update unit 698, and error storing unit 699.Processing condition determining unit 696 outputs first processing condition signal 710, third processing condition signal 707 and fourth processing condition signal 702, using the density level at or around target pixel position out of the tone density levels 701 which are sampled from the original image by pixels.", "Threshold value generating unit 691 generates a plurality of threshold values 730 for multi-valuation using fourth processing condition signal 702 outputted from error re-distribution determining unit 696.Error re-distribution determining unit 697 separates accumulation error 713 corresponding to the target pixel position into error distribution control signal 711 and first processing condition signal 710.Data addition unit 692 adds a density level fluctuating at specific intervals to density level 701 on the basis of third processing condition signal 707, and generates input level 704.Input correction unit 693 adds first correction accumulation error 708 to input level 704 and generates correction level 705.Multi-valuation unit 694 generates multi-valued data 706 from correction level 705 and a plurality of threshold values 703.Difference operation unit 695 works out multi-valuation error 709 from correction level 705 and multi-valued data 706.Error distribution update unit 698 distributes multi-valuation error 709 according to the distribution coefficients, and add each distributed error to the respective accumulation error 714 at the pixel position for each unprocessed pixel adjacent to a target pixel stored in error storing unit (or stored in error distribution update unit 698), and updates the accumulation error.", "As described above the image processing apparatus, in the sixteenth embodiment, consists of the threshold value generating unit 691, data addition unit 692, processing condition determining unit 696, and error re-distribution determining unit 697.In FIG.", "31, all units can be materialized in the same configuration as described earlier.", "The present embodiment is so configured that threshold value generating unit 691, error re-distribution determining unit 697 and data addition unit 692 are all controlled by processing condition determining unit 696.Instead, it may be so configured that threshold value generating unit 691 and at least one unit alone are controlled.", "Seventeenth Embodiment FIG.", "32 is a block diagram of an image processing apparatus in the seventeenth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "32 is formed of threshold value generating unit 721, data addition unit 722, input correction unit 723, multi-valuation unit 724, difference operation unit 725, processing condition determining unit 726, error distribution update unit 727, distribution coefficient generating unit 728 and error storing unit 729.Processing condition determining unit 726 outputs second processing condition signal 740, third processing condition signal 737, and fourth processing condition signal 732, using the density level at or around the target pixel position out of the tone density levels 731 which are sampled from the original image by pixels.", "Threshold value generating unit 721 generates a plurality of threshold values 733 for multi-valuation using fourth processing condition signal 732 outputted from processing condition determining unit 726.Data addition unit 722 adds a density level fluctuating at specific intervals to density level 731 on the basis of third processing condition signal 737, and generates inputs level 734.Input correction unit adds accumulation error 738 to input level 734 and generates correction level 735.Multi-valuation unit 724 generates multi-valued data 736 from correction level 735 and a plurality of threshold values 733.Difference operation unit 739 works out multi-valuation error 739 from correction level 735 and multi-valued data 736.Distribution coefficient generating unit 728 generates distribution coefficient 741 at specific intervals, and outputs the same to error distribution update unit 727.Then, distribution coefficient 741 of distribution coefficient generating unit 728 is controlled by second processing condition signal 740 outputted from processing condition determining unit 726.Error distribution update unit 727 distributes multi-valuation error 739 according to the distribution coefficients 741, and adds each distributed error to the respective accumulation error 742 at the pixel position for each unprocessed pixel adjacent to a target pixel stored in error distribution update unit 729 (or stored in error distribution update unit 727), and updates the accumulation error.", "As described above the image processing apparatus, in the seventeenth embodiment, consists of the threshold value generating unit 721, data addition unit 722, processing condition determining unit 726, and distribution coefficient generating unit 728.In FIG.", "32, all units can be materialized in the same configurations as described earlier.", "The present embodiment is so configured that threshold value generating unit 721, distribution coefficient generating unit 728, and data addition unit 722 are all controlled by processing condition determining unit 726.Instead, it may so configured that threshold value generating unit 721 and at least one unit alone are controlled.", "Eighteenth Embodiment FIG.", "33 is a block diagram of an image processing apparatus in the eighteenth embodiment according to the present invention.", "The image processing apparatus shown in FIG.", "33 is formed of threshold value generating unit 751, data addition unit 752, input correction unit 753, multi-valuation unit 754, difference operation unit 755, processing condition determining unit 756, error re-distribution determining unit 757, error distribution update unit 758, distribution coefficient generating unit 759, and error storing unit 760.Processing condition determining unit 756 outputs first processing condition signal 771, second processing condition signal 770, third processing condition signal 767 and fourth processing condition signal 762, using density level at or around the target pixel out of the tone density levels 761 which are sampled from the original image by pixels.", "Threshold value generating unit 751 generates a plurality of threshold values 763 for multi-valuation using fourth processing condition signal 762 outputted from processing condition determining unit 756.Error re-distribution determining unit 772 separates accumulation error 774 for the target pixel position into first correction accumulation error 768 and second correction accumulation error 773 on the basis of error distribution control signal 772 and first processing condition signal 771.Data addition unit 752 adds a density level fluctuating at specific intervals to density level 761 on the basis of third processing condition signal 767, and generates input level 765.Input correction unit 753 adds first correction accumulation error 768 to input level 765 and generates correction level 766.Multi-valuation unit 754 generates multi-valued data 764 from correction level 766 and a plurality of threshold values 763.Difference operation unit 755 works out multi-valuation error 769 from correction level 766 and multi-valued data 764.Distribution coefficient generating unit 759 generates distribution coefficient 775 at specific intervals and outputs the same to error distribution update unit 758.Then, the distribution coefficient of distribution coefficient generating unit 759 is controlled by second processing condition signal 770 outputted from processing condition determining unit 756.Error distribution update unit 758 distributes multi-valuation error 769 according to distribution coefficients 775, and adds each distributed error to the respective accumulation error 776 at the pixel position corresponding to each unprocessed pixel adjacent to a target pixel stored in error storing unit 760 (or stored in error distribution update unit 758), and updates the accumulation error.", "As described above the image processing apparatus, in the eighteenth embodiment consists of the threshold value generating unit 751, data addition unit 752, processing condition determining unit 756, error re-distribution determining unit 757 and distribution coefficient generating unit 759.In FIG.", "33, all units can be materialized in the same configurations as described earlier.", "The present embodiment is so configured that threshold value generating unit 751, error re-distribution determining unit 757, distribution coefficient generating unit 759 and data addition unit 752 are all controlled by processing condition determining unit 756.Instead, it may be so configured that threshold value generating unit 751 and at least one alone of the other unit are controlled.", "Nineteenth Embodiment The nineteenth embodiment to thirty-sixth embodiment are materialized with the first embodiment to the eighteenth embodiment as software (program for image processing).", "FIG.", "34 is a block diagram of an MPU system to materialize the image processing method of the present invention by software.", "The MPU system shown in FIG.", "34 is formed of MPU (micro processing unit) 782, ROM (read only memory) 781, RAM (random access memory) 783, input/output port 784.This MPU system is well known, and will be described briefly.", "MPU 782 executes a program for image processing stored in ROM 781 using RAM 783, as a working memory.", "Input/output port 784 does input 796 and output 797 of an image.", "Image data is transferred from input/output port 784 to RAM 783.According to a program for image processing of ROM, image processing is executed.", "As an alternative to that, the program for image processing may be transferred from input/output port to RAM 783 and executed on RAM.", "When processing is over, image data is outputted through input/output port 784.Image processing may be performed on a personal computer.", "FIG.", "35 is a flow chart of an image processing method in the nineteenth embodiment according to the present invention.", "The image processing method in the nineteenth embodiment is a method that is implemented as image processing program by software to carry out the processing corresponding to the function of image processing apparatus.", "When the image processing method according to the present invention starts (Step 1), the density level of the target pixel is read in Step 2.In Step 3, the accumulation error for the target pixel position is separated into the first and second correction accumulation errors.", "As to the distribution ratio at which accumulation error is separated, information as to whether other color dots are stricken or not may be used.", "Also, when the first and second correction accumulation errors are worked out, second correction accumulation error may be generated only when the accumulation error is a positive number and smaller than a specific value.", "In Step 4, first correction accumulation error is added to the density level of the target pixel.", "The correction level obtained is multi-valuated in Step 5.In Step 6, multi-valuation error or difference between correction level and multi-valued level is worked out.", "In Step 7, second correction accumulation error is added to multi-valuation error obtained to generate a correction multi-valuation error.", "In step 8 the correction multi-valuation error is distributed according to distribution coefficients.", "And each accumulation error corresponding to each unprocessed pixel is updated by adding each distributed error to each accumulation error.", "When it is judged that the above Step 2 to 8 are repeated for each pixel (Step 9), the processing of this image ends (Step 10).", "As set forth above, since the accumulation error for the target pixel position is separated into first and second correction accumulation errors, and added the first correction accumulation error to the density level of the original image, the density level of the target pixel can be made not larger than the original image when another color dot is presented as long as the error is not accumulated more than a specific value.", "Therefore, the overlapping of color dots can be kept down, and dots disperse, whereby the granularity will improve.", "Twentieth Embodiment FIG.", "36 is a flow chart of the image processing method in the twentieth embodiment according to the present invention.", "The image processing method in the twentieth embodiment is a method which is turned into software with the processing contents of the image processing apparatus in the second embodiment.", "The program according to this method will be executed by MPU system.", "The present embodiment, as shown in FIG.", "36 can be materialized by adding a new step 20 to the flow chart of the image processing method of the nineteenth embodiment shown in FIG.", "35.Step 20 may be added before Step 8.In FIG.", "36, Step 20 is added after Step 7.That is, in Step 7, second correction accumulation error is added to multi-valuation error, and after a correction multi-valuation error is generated, the correction multi-valuation error is distributed according to the distribution coefficients which are determined using the random function in Step 20.Instead of using the random function, a random value may be taken out of a table in processing with the table prepared in advance.", "As set forth above, fluctuating the distribution coefficients can keep down occurrence of texture in addition to the effects of the nineteenth embodiment.", "Twenty-First Embodiment FIG.", "37 is a flow chart of the image processing method in the twenty-first embodiment according to the present invention.", "The image processing method in the twenty-first embodiment is a method which is turned into software with the processing contents of the image processing apparatus in the third embodiment.", "The program according to this method will be executed by MPU system.", "The present embodiment, as shown in FIG.", "36 can be materialized by adding Step 30 between Step 2 (in the twentieth embodiment) and Step 3.That is, a density fluctuating at specific intervals is added to the density level of the target pixel read in Step 2.It is desirable that if all density levels of the respective factors in an area of a specific size are added together, the total value will be “0.” As set forth above, if the density level at the target pixel and a different density level are added, it is possible to substantially keep down the texture for an image with small change in density and an image with a uniform density generated by computer in addition to effects of the twentieth embodiment.", "Twenty-Second Embodiment FIG.", "38 is a flow chart of the image processing method in the twenty-second embodiment according to the present invention.", "The image processing method in the twenty-second embodiment is a method which is turned into software with the processing contents of the image processing apparatus in the fourth embodiment.", "The program according to this method will be executed by MPU system.", "The present embodiment as shown in FIG.", "38 can be materialized by adding Step 40 between Step 2 (in the nineteenth embodiment) and Step 3.That is, processing conditions are determined using the density levels at and around the target pixel read in Step 2.To be specific, a specific area in an image is detected using the density level at or around the target pixel.", "Among the specific areas in the image are high light area, shadow area, maximum density level (area), minimum density level (area), character, line drawing area, granularity decreasing area (described in the fourth embodiment.", "According to the results, the separation of first and second correction accumulation errors are controlled.", "That permits minute control of overlapping of color dots and keeps down occurrence of unnecessary dots.", "Twenty-Third Embodiment FIG.", "39 is a flow chart of an image processing method in the twenty-third embodiment according to the present invention.", "The image processing method in the twenty-third embodiment is a method which is turned into software with the processing contents of the image processing apparatus in the fifth embodiment.", "The program according to this method will be executed by MPU system.", "When the image processing method of the present invention, as shown in FIG.", "39 starts (Step 1), the density level at the target pixel is read in Step 2.Then, processing conditions are determined in Step 40 using the target pixel.", "In Step 50, the accumulation error is added to the density level of the target pixel.", "The correction level obtained is multi-valuated in Step 5.In Step 6, the multi-valuation error, that is, difference between correction level and multi-valued level is worked out.", "In Step 20, the distribution coefficients according to which the multi-valuation error is distributed are determined using the random function.", "Then, as processing contents in the fifth embodiment, the method of generating distribution coefficients are changed depending on the processing conditions worked out in Step 40.In Step 51, multi-valuation error is distributed according to each distribution coefficient.", "And each distributed error is added to accumulation error for each position adjacent to the target pixel to update the accumulation error value.", "When processing of all pixels is over (Step 9), the present image processing ends (Step 10).", "Processing conditions are determined using the target pixel, and the distribution coefficients are changed, and therefore occurrence of texture can be further kept down.", "Twenty-Fourth Embodiment FIG.", "40 is a flow chart of an image processing method in the twenty-fourth embodiment according to the present invention.", "The image processing method in the twenty-fourth embodiment is a method which is turned into software with the processing conditions of the image processing apparatus in the sixth embodiment.", "The program according to this method will be executed by MPU system.", "The image processing method in the twenty-fourth embodiment is a method of the twentieth embodiment to which Step 40 is added as shown in FIG.", "40.The Step 40 is the same as above explained.", "Since the processing condition is determined at Step 40 after Step 2, that permits to control the separation of first or second multi-valuation error and the fluctuation of distribution coefficients.", "Therefore, that keeps down occurrence of unnecessary dots and occurrence of texture.", "Twenty-Fifth Embodiment FIG.", "41 is a flow chart of an image processing method in the twenty-fifth embodiment according to the present invention.", "The image processing method in the twenty-fifth embodiment is a method which is turned into software with the processing contents of the image processing apparatus in the seventh embodiment.", "The program according to this method will be executed by MPU system.", "In the twenty-fifth embodiment, control of distribution coefficients depending on processing conditions in the twenty-third embodiment, shown in FIG.", "41, is not performed but addition data to target pixel is controlled instead.", "This is materialized in Step 40 and Step 30.The data added in Step 30 is controlled by the processing condition determined in Step 40.Since addition data to the target pixel is changed depending on the processing conditions, addition data according to the density level of input can be generated, and diffusion of dots can be improved on low density level and high density level.", "Twenty-Sixth Embodiment FIG.", "42 is a flow chart of an image processing method in the twenty-sixth embodiment according to the present invention.", "The image processing method in the twenty-sixth embodiment is a method that is implemented as image processing program by software to carry out the processing corresponding to the function of image processing apparatus in the eighth embodiment.", "The program according to this method will be executed by MPU system.", "In the twenty-sixth embodiment, Step 30 is added between Step 40 and Step 3 of the twenty-second embodiment, thereby the addition data to target pixel is controlled by the processing conditions as shown in FIG.", "42.Therefore, addition data depending on the density level of input can be generated and diffusion of dots on low density level and high density level can be improved.", "Twenty-Seventh Embodiment FIG.", "43 is a flow chart of an image processing method in the twenty-seventh embodiment.", "The image processing method in the twenty-seventh embodiment is a method that is implemented as image processing program by software to carry out the processing corresponding to the function of image processing apparatus in the ninth embodiment.", "The program according to this method will be executed by MPU system.", "In the twenty-seventh embodiment, Step 30 is added between Step 40 and Step 50 of the twenty-third embodiment, whereby the addition data to target pixel is controlled as shown in FIG.", "43.Since addition data to the target pixel is changed depending on processing conditions, addition data according to the density level of input can be generated and diffusion of dots can be improved on low density level and high density level.", "Twenty-Eighth Embodiment FIG.", "44 is a flow chart of the image processing method in the twenty-eighth embodiment according to the present invention.", "The image processing method in the twenty-eighth embodiment is a method that is implemented as image processing program by software to carry out the processing corresponding to the function of image processing apparatus in the tenth embodiment.", "The program according to this method will be executed by MPU system.", "Step 20 is added between Step 7 and Step 8 of the twenty-sixth embodiment, thereby controlling the generation of distribution coefficients.", "Since the distribution coefficients are changed depending on processing conditions, the occurrence of texture can be further kept down.", "Twenty-Ninth Embodiment FIG.", "45 is a flow chart of an image processing method in the twenty-ninth embodiment.", "The image processing method in the twenty-ninth embodiment is a method that is implemented as image processing program by software to carry out the processing corresponding to the function of image processing apparatus in the twenty-ninth embodiment.", "The program according to this method will be executed by MPU system.", "The image processing method of the present invention, as shown in FIG.", "45, starts (Step 1), and then the density level of the target pixel is read in Step 2.In the next step or Step 40, processing conditions are determined using only the density level of the target pixel.", "In Step 60, a plurality of threshold values for multi-valuation in Step 5 are generated.", "In Step 50, accumulation error is added to the density level of the target pixel to generate correction level.", "In Step 5, the correction level obtained is multi-valuated using a plurality of threshold values generated in Step 60.In Step 6, multi-valuation error or difference between correction level and multi-valued level is worked out.", "In Step 51, the multi-valuation error is distributed according to the distribution coefficients.", "And a distributed error out of the distributed errors is added to a accumulation error for the position adjacent to the target pixel to update the accumulation error.", "When it is judged that the above Step 2 to 8 are repeated for each pixel (Step 9), the processing of this image ends (Step 10).", "Since the generation of the threshold value is controlled by processing conditions as described, the delay of dot can be kept down.", "Thirtieth Embodiment FIG.", "46 is a flow chart of an image processing method in the thirtieth embodiment according to the present invention.", "The method in the thirtieth embodiment is a method that is implemented as image processing program by software to carry out the processing corresponding to the function of image processing apparatus in the twelfth embodiment.", "The program according to this method will be executed by MPU system.", "The image processing method of the present invention as shown in FIG.", "46 starts (Step 1), then the density level of the target pixel is read in Step 2.Then in Step 40, processing conditions are determined using the target pixel.", "In Step 60, threshold value for multi-valuation in Step 5 is generated according to the processing conditions.", "In Step 3, the accumulation error for the target pixel position is separated into first and second correction accumulation errors.", "When the accumulation error is separated, information as to whether another color dot is stricken or not is used as distribution ratio.", "Also in calculating first and second correction accumulation errors, only when accumulation error is a positive number not larger than a specific value, second correction accumulation error only may be generated.", "Then, depending on the processing conditions obtained in Step 40, separation of first and second correction accumulation errors is controlled.", "In Step 4, the first correction accumulation error is added to the density level of the target pixel to generate a correction level.", "In Step 5, the correction level obtained is multi-valuated using a plurality of threshold values generated in Step 60.In Step 6, the multi-valuation error, that is, the difference between the correction level and multi-valued level is calculated.", "In Step 7, the second correction accumulation error is added to the multi-valuation error obtained to generate a correction multi-valuation error.", "In Step 8, the correction multi-valuation error is distributed according to the distribution coefficients of the target pixel and added each distributed error to each accumulation error corresponding to the position adjacent to the target pixel.", "Thus the accumulation error is updated.", "When all pixels are processed (Step 9), the processing of this image ends (Step 10).", "Since the generation of the threshold value is controlled by processing conditions as described, the delay of dot can be kept down.", "Furthermore, separation of first and second correction accumulation errors are controlled, and occurrence of unnecessary dots can be reduced.", "Thirty-First Embodiment FIG.", "47 is a flow chart of an image processing method in the thirty-first embodiment according to the present invention, a method that is implemented as image processing program by software to carry out the processing corresponding to the function of image processing apparatus in the thirty-first embodiment.", "The program according to this method will be executed by MPU system.", "In the image processing method of the thirty-first embodiment, Step 20 is added between Step 6 and Step 51 of the twenty-ninth embodiment as shown in FIG.", "47.In Step 20 the generation of the coefficients is controlled by the processing condition determined in Step 40.Since occurrence of distribution coefficients are controlled through processing conditions, the occurrence of texture can be further curbed.", "Thirty-Second Embodiment FIG.", "48 is a flow chart of an image processing method in the thirty-second embodiment.", "The method in the thirty-second embodiment is a method that is implemented as image processing program by software to carry out the processing corresponding to the function of image processing apparatus in the fourteenth embodiment.", "The program according to this method will be executed by MPU system.", "In the image processing method of the thirty-second embodiment, Step 20 is added between Step 7 and Step 8 of the thirtieth embodiment as shown in FIG.", "48.In Step 20 the generation of the coefficients are controlled by the processing condition determined in Step 40.Since occurrence of distribution coefficients are controlled through processing conditions, the occurrence of texture can be further kept down.", "The program according to this method will be executed by MPU system.", "Thirty-Third Embodiment FIG.", "49 is a flow chart of an image processing method in the thirty-third embodiment according to the present invention.", "The method in the thirty-third embodiment is a method that is implemented as image processing program by software to carry out the processing corresponding to the function of image processing apparatus in the fifteenth embodiment.", "In the image processing method of the thirty-third embodiment, Step 30 is added between Step 60 and Step 50 of the twenty-ninth embodiment as shown in FIG.", "49.The adding data obtained in Step 30 is controlled by the processing condition determined in Step 40.Since addition data to the target pixel is controlled through processing conditions, the diffusion of dot can be controlled minutely on the density level of target pixel.", "Thirty-Fourth Embodiment FIG.", "50 is a flow chart of an image processing method in the thirty-fourth embodiment.", "The method in the thirty-fourth embodiment is a method that is implemented as image processing program by software to carry out the processing corresponding to the function of image processing apparatus in the sixteenth embodiment.", "The program according to this method will be executed by MPU system.", "In the image processing method of the thirty-fourth embodiment, Step 30 is added between Step 60 and Step 3 of the thirteenth embodiment as shown in FIG.", "50.The adding data obtained in Step 30 is controlled by the processing condition determined in Step 40.Since addition data to the target pixel is controlled depending on processing conditions, the diffusion of dots can be controlled minutely on the density level of target pixel.", "Thirty-Fifth Embodiment FIG.", "51 is a flow chart of an image processing method in the thirty-fifth embodiment.", "The method in the thirty-fifth embodiment is a method that is implemented as image processing program by software to carry out the processing corresponding to the function of image processing apparatus in the seventeenth embodiment.", "The program according to this method will be executed by MPU system.", "In the image processing method of the thirty-fifth embodiment, Step 20 is added between Step 6 and Step 51 of the thirty-third embodiment as shown in FIG.", "51.In Step 20 the generation of the distribution coefficients is controlled by the processing condition determined in Step 40.Since the occurrence of distribution coefficients are controlled depending on processing conditions, occurrence of texture can be further curbed.", "Thirty-Sixth Embodiment FIG.", "52 is a flow chart of an image processing method in the thirty-sixth embodiment.", "The method in the thirty-sixth embodiment is a method that is implemented as image processing program by software to carry out the processing corresponding to the function of image processing apparatus in the eighteenth embodiment.", "The program according to this method will be executed by MPU system.", "In the image processing method of the thirty-sixth embodiment, Step 20 is added between Step 7 and Step 8 of the thirty-fourth embodiment as shown in FIG.", "52.In Step 20 the generation of the distribution coefficients is controlled by the processing condition determined in Step 40.Since the occurrence of distribution coefficients are controlled according to processing conditions, occurrence of texture can be further curbed.", "In all multi-valuations of the present invention, a plurality of threshold values and correction level are compared.", "The present invention is not limited to this method.", "As an alternative to that, multi-valued data may be worked out using a lookup table.", "The table is referred to on the basis of correction level.", "In the image processing apparatus in Embodiments according to the present invention, no synchronizing signal is shown.", "But the circuits may be synchronized as necessary to execute processing through pipeline.", "It is also noted that as example of distribution coefficients, distribution coefficients shown in FIGS.", "54C and 54B are used in embodiments according to the present invention.", "The present invention is not limited to that.", "As an alternative to that, distribution coefficients in FIG.", "54D and (e) with different filter sizes may be used.", "Also, the present invention may be so configured that more than two distribution coefficients are switched.", "In the processing condition determining circuit, that is, an embodiment of processing condition determining unit, processing conditions are determined using the target pixel and its adjacent pixels.", "Instead, using the target pixel alone, the processing conditions may be determined.", "In this case, the density level of the target pixel or the density level scope can be detected by combining a plurality of processing condition determining circuits B, and by information obtained, unit in the subsequent steps are controlled.", "Also, the present invention is described using density level etc.", "taking recording systems as an example.", "As an alternative to that, a display system may be used.", "In that case, not density level but display systems such as RGB level or luminance level may be used.", "In the Embodiments described, the present invention is applied to single image processing apparatus as central processing system.", "The present invention is not limited to that.", "For example, the present invention is applicable to an image processing system as diffusion processing system with an image output printer connected to a computer system such as image output printer.", "According to the present invention, as set forth above, accumulation error for the target pixel is separated into first correction accumulation error and second correction accumulation error to control occurrence of dots.", "When another color dot is present, therefore, it is possible to see that the density level of the target pixel will not be larger than the original image.", "Through that, the overlapping of color dots can be curbed and dots disperse with good granularity.", "The present invention is also effective in keeping down diffusion of accumulation error and keep down occurrence of unnecessary dots.", "Also, it is possible to keep down the occurrence of texture and improve the diffusion of dots by changing distribution coefficients of error at specific intervals and adding another data to data level on the target pixel.", "Also, since it is possible to minutely control separation of accumulation error, interval of multi-valuation error distribution coefficients, distribution coefficients value of multi-valuation error, interval of addition density level at which is added the density level of the target pixel, quantity of addition density level, threshold value etc.", "by detecting a specific image area, image processing can be performed which is suitable for an area where it is desired that the granularity of image be enlarged or reduced.", "Through that, the picture quality will improve in character, line drawing area, highlight area, shadow area, and the continuity impression of grain in half tone area will improve and high picture quality can be obtained." ] ]	Patent_10466603
[ [ "Promoting cell regeneration and/or cell differentiation with non-metabolizable sugar and a polymeric absorbent", "A composition comprising at least one absorbent and at least one non-metabolizable sugar, and a method for using the composition, for promoting cell regeneration and/or cell differentiation." ], [ "1-19.", "(canceled) 20.A composition for promoting cellular reconstruction and/or cellular differentiation, comprising at least one absorbent and at least one nonmetabolizable sugar.", "21.The composition as claimed in claim 20, wherein the composition is in an injectable form.", "22.The composition as claimed in claim 20, wherein the nonmetabolizable sugar is xylose, arabinose, rhamnose or fucose.", "23.The composition as claimed in claim 20, wherein the nonmetabolizable sugar is xylose or arabinose.", "24.The composition as claimed in claim 20, wherein the nonmetabolizable sugar is xylose.", "25.The composition as claimed in claim 20, wherein the absorbent is a polymer.", "26.The composition as claimed in claim 20, wherein the absorbent is a polyacrylate, a polymethacrylate, a dextran, a carboxyvinyl polymer or an alginate.", "27.The composition as claimed in claim 20, wherein the absorbent is sodium alginate, guar gum, polyacrylic acid, a carboxyvinyl polymer or carboxymethyl cellulose.", "28.The composition as claimed in claim 20, wherein the absorbent is a carboxyvinyl polymer.", "29.The composition as claimed in claim 20, wherein the composition is in the form of a powder.", "30.The composition as claimed in claim 20, wherein the composition further comprises at least one antibiotic, antiseptic or corticosteroid.", "31.The composition as claimed in claim 20, wherein the composition comprises between 50 and 95% by weight of the at least one nonmetabolizable sugar and between 5 and 50% by weight of the at least one absorbent.", "32.The composition as claimed in claim 20, wherein the composition comprises between 80 and 95% by weight of the at least one nonmetabolizable sugar and between 5 and 20% by weight of the at least one absorbent.", "33.A method for promoting cellular reconstruction and/or cellular differentiation, comprising administering the composition as claimed in claim 20 to an affected area in need of cellular reconstruction and/or cellular differentiation.", "34.The method as claimed in claim 33, wherein the affected area is a wrinkle, deficit of dermal tissue or tissue underlying skin.", "35.The method as claimed in claim 33, wherein the affected area is nerve tissue, bone, or cartilage in need of reconstruction.", "36.The method as claimed in claim 33, wherein the affected area is a burn in need of healing.", "37.The method as claimed in claim 33, wherein the affected area is a bedsore in need of alleviating or area susceptible to a bedsore.", "38.The method as claimed in claim 33, wherein the affected area is a solid tumor.", "39.The method as claimed in claim 33, wherein the affected area is a bottom of a cell culture dish to promote the attachment, differentiation, migration or growth of a cell.", "40.The method as claimed in claim 33, wherein the absorbent and nonmetabolizable sugar are administered simultaneously, separately or staggered over time." ], [ "The present invention relates to the use of a combination, comprising at least one absorbent and at least one compound selected from the group formed by nonmetabolizable sugars and polyols, for preparing a composition, in particular a medicinal composition, for promoting cellular reconstruction and/or cellular differentiation, in particular for promoting healing, advantageously the healing of bedsores.", "The prior art discloses that it is possible to use sugars in the treatment of bedsores, in particular for fighting bacterial infections.", "The prior art is also found to point out that hydrophilic polymers can be used for treating bedsores with the aim of absorbing exudates.", "The prior art which is presented below is extracted from the book “L'escarre-évaluation et prise en charge [The bedsore-assessment and care]”, which is coordinated by B. Barrois, D. Colin and S. Desjobert and published by Frison-Roche.", "Bedsores are localized areas of tissue necrosis which are due to ischemia of the subcutaneous tissues.", "The main reason for the development of bedsores is a compression of the soft tissues between a bony protuberance and an external surface over an extended period of time.", "In a general manner, the development of bedsores is explained by the prolonged pressure of said external surface on the skin and the underlying tissues and also by the phenomena of tissue hypoxia or anoxia which result from this.", "Nevertheless, it has been observed that wide clinical heterogeneity exists and that, in certain patients, such as old people, persons with medullary injuries or comatose patients, it is not possible to regard the appearance of bedsores as being simply the result of the conjunction of a defect in care and an increase in tissue pressure.", "The appearance of localized bedsores in an area which is not a point of compression is frequently observed in certain patients, in particular patients in intensive care units.", "Besides compression, other intrinsic factors are also known to play a role.", "These factors are, classically, shearing, maceration, infections and nutritional deficiency.", "Shearing is mainly observed in patients who remain in a semi-sitting position for an extended period; it is engendered by the conjunction of hypertension and a tangential force which is linked to sliding.", "Maceration is frequently the consequence of hyperthermia, which gives rise to increased transpiration and perspiration and which can be accompanied by sphincteral incontinence.", "Infections also play a role, which can be all the more a determining factor since the patient may be subject to an immune deficiency.", "Nutritional deficiency is manifested in tissue protein renewal, replenishment of the tissues with energy substrates and deficiency in vitamins or trace elements.", "The appearance of endogenous cachexia and, in particular, of diabetes can only aggravate this pre-existing situation.", "Various products and techniques have been proposed for treating bedsores; the main ones are presented below.", "Film dressings, which are thin, transparent, pliable and extendable sheets which are reserved for nonexudative bedsores.", "Greasy dressings, which are made of tulle or gauze impregnated with vaseline, and which can additionally include an antibiotic, an antiseptic or a corticosteroid.", "The major drawback of these greasy dressings is that they only control the exudate poorly.", "Hydrocolloids, on the contrary, absorb the exudate while gelling and maintain a humid environment.", "The problem posed by these products is that they disintegrate on contact with the exudates, producing a foul-smelling odor.", "Alginates have great absorption power; they are useful when the wound is very exudative.", "While hydrogels are less absorbent than alginates, their power of humidifying is very useful in the phase of cleaning the areas of necrosis, which phase precedes the healing phase.", "They suffer the disadvantage of being expensive.", "Hydrocell dressings are very absorbent and control the exudate without abolishing it; their only indication is granulation.", "The application of sugar to bedsores for the purpose of absorbing exudates has also been used as a treatment.", "However, this practice can suffer from drawbacks, in particular when the patient is diabetic, since, apart from the fact of increasing the blood sugar concentration in the patient, it has been found that, in these patients, healing is adversely affected.", "The consequence of supplying sugar to the wounds of these patients would be to aggravate this adverse effect on the healing process.", "Surprisingly, it was possible to demonstrate, in in-vitro tests carried out on cell cultures, that the combination comprising at least one absorbent and at least one compound selected from the group formed by nonmetabolizable sugars and polyols very strongly promoted cellular differentiation and tissue reconstruction without exhibiting the abovementioned drawbacks.", "The present invention therefore relates to the use of a combination, comprising at least one absorbent and at least one compound selected from the group formed by nonmetabolizable sugars and polyols, for preparing a composition, in particular a medicinal composition, for promoting cellular reconstruction and/or cellular differentiation.", "This composition is intended, first and foremost, to be a medical tool, more especially a dressing item.", "Within the meaning of the present application, “nonmetabolizable sugar” is understood as being a sugar which cannot be used in its entirety as an energy source by the human body.", "In particular, the present invention relates to the use of a combination, comprising at least one absorbent and at least one compound selected from the group formed by nonmetabolizable sugars and polyols, for preparing a composition, in particular a medicinal composition, for tissue reconstruction in human or veterinary medicine, examples of the application of which are as follows: Injection, for making good wrinkles or deficits of dermal tissue or tissue underlying the skin, for the purpose of esthetic correction, Reconstruction of nervous tissue: spinal cord trauma, Bone reconstruction: deficit of bone tissue following interventions or traumas, Reconstruction of cartilage: deficit associated with degenerative diseases or surgical interventions, Treatment of burns, Healing in general, advantageously prevention and treatment of bedsores.", "The present invention also relates to the use of a combination, comprising at least one absorbent and at least one compound selected from the group formed by nonmetabolizable sugars and polyols, for preparing a composition, in particular a medicinal composition, for promoting cellular differentiation in medicine or in experimental biology: Treatment of solid tumors, Treatment of the bottom of cell culture dishes with the aim of promoting the attachment, differentiation, migration and growth of cells by replacing the matrices which are generally used for the same purpose but which are much more expensive in use and less versatile, such as collagen, fibronectin, polylysine or laminin or else various commercially available combinations of these substances.", "Healing, including the treatment or prevention of bedsores.", "The compound selected from the group formed by nonmetabolizable sugars and polyols is preferably selected from the group formed by xylose, arabinose, rhamnose, fucose, mannitol and sorbitol.", "Even more preferably, the compound will be xylose or arabinose, advantageously it will be xylose, which is commonly used in human diets as a substitute for sugar and which has demonstrated its innocuousness.", "The absorbent is preferably a polymer; it can be selected, in particular, from polyacrylates, polymethacrylates, dextrans, alginates and carboxyvinyl polymer.", "Advantageously, the polymer is an acrylic polymer.", "In particular, it is selected from carboxyvinyl polymer (carbomer), sodium alginate, guar gum, polyacrylic acid, carboxymethyl cellulose, agar, agarose, xanthan gum, polyvinylpyrrolindone, methyl cellulose, poly(methyl methacrylate) and poly(acrylamide-co-acrylic acid).", "Advantageously, it is selected from carboxyvinyl polymer (carbomer), sodium alginate, guar gum, polyacrylic acid and carboxymethyl cellulose.", "Even more advantageously, the polymer is carboxyvinyl polymer (carbomer).", "Said composition is preferably present in the form of a powder.", "This formulation in the form of a powder thus exhibits the advantage of permitting an application which is easy and less painful than the applications of the prior art.", "Said composition in the form of a powder also exhibits the advantage of being strongly antiseptic by virtue of the combined effects of removal of the water which is required for bacterial survival, regular absorption of the exudates and the intense osmotic pressure which is achieved.", "The compositions will preferably comprise between 50 and 95%, and even more preferably between 80 and 95%, by weight, of at least one compound selected from the group formed by nonmetabolizable sugars and polyols and between 5 and 50%, and even more preferably between 5 and 20%, by weight, of an absorbent.", "This composition can also include an agent which is selected from antibiotics, antiseptics, corticosteroids, etc.", "In addition, it has been established that the efficiency of the composition remains constant irrespective of whether the absorbent and the compound selected from the group formed by nonmetabolizable sugars and polyols are administered simultaneously, separately or such that the administrations are staggered over time.", "The following examples are given by way of indication and are not limiting.", "EXAMPLE 1 Demonstration of the Effect of the Combination of Carboxyvinyl Polymer (carbomer 914) and Xylose on the Migration, Attachment and Cellular Differentiation of Melanocytes Derived from a Continuous Cell Line A 6-well culture dish is coated with an aqueous gel based on a mixture of carbomer 914 and xylose in the proportions 1-9 (w/w) to which distilled water is added so as to obtain a concentration of 1% (w/w).", "The gel which has been formed is neutralized to pH 7.00 with a N solution of sodium hydroxide and sterilized by autoclaving it at 120° C. for 20 minutes.", "The resulting sterile gel is diluted 1/3 in a sterile manner with PBS buffer to which 34 μg of gentamycin have been added/ml.", "1 ml of this diluted gel is placed in each well and the gel is allowed to dry overnight under a sterile laminar air flow.", "Seeding is then carried out using a suspension containing 50 000 melanocyte cells from the M4Beu cell line (Tumourigenic phenotypes of human melanoma cell lines in nude mice determine by an active antitumor mechanism; R. Jacubovich, H. Cabrillat, D. Gerlier, M. Bailly & J. F. Doré; Br.", "J.", "Cancer (1985), 51, 335-345) in 3 ml of MEM culture medium containing 10% calf serum, 1% vitamin solution (ref.", "Sigma M 6895), 1% sodium pyruvate solution (ref.", "Sigma S 8636), 1% amino acid solution (ref.", "M 7145) and 50 μg of gentamycin/ml.", "The cultures are placed in an incubator at 37° C. and 5% CO2.Their growth is then examined.", "Controls are carried out without coating the bottom of the wells.", "Pronounced cellular differentiation is observed in the case of the carboxyvinyl polymer and xylose mixture, with this differentiation being associated with the formation of cell masses reminiscent of tissue formations.", "Fairly good differentiation is also observed with sodium alginate and carboxymethyl cellulose on their own and in combination with xylose.", "Good results are also achieved when arabinose is used in place of the xylose.", "These results, which are visible on the basis of cell morphology, are also evidenced by the synthesis of melanin by the melanocytes, with this synthesis representing a good marker of differentiation.", "EXAMPLE 2 Demonstration of the Effect of the Combination of Polyacrylate and Xylose on the Treatment of Bedsores Tests were carried out on 20 patients, 4 of whom presented with bedsores on the heel while the other 16 presented with sacral bedsores.", "The bedsores on the heel measured from 1 to 3 cm in diameter at the beginning of the treatment.", "The sacral bedsores measured from 4 to 12 cm.", "In two of them, the sacrum was visible at the bottom of the lesion.", "All these bedsores were strongly exudative.", "The treatment consisted in applying one composition in a powder form, comprising, by weight, 90% xylose and 10% polyacrylate, both morning and evening in such a way as to cover the whole of the interior surface of the lesion with an approximately 2 mm layer of powder.", "A 75 to 100% reduction in the diameter of the bedsores, and complete disappearance of the pain, were observed in all cases without exception after one week of treatment.", "The bedsores on the heel disappeared completely within ten days, merely leaving a discreet inflammation without any pain or itching.", "The 11 least deep sacral bedsores all had a diameter of between 4 and 6 cm.", "These bedsores were almost entirely filled in within two weeks, leaving a simple depression which was discreetly inflammatory in the case of 3 of the bedsores or even without any inflammation in the case of the other 8 bedsores.", "While the 5 most serious bedsores did not disappear entirely, epithelialization within the interior of the lesion was complete within two weeks.", "The final result presented as a loss of manner, which was very much less than the loss of material observed at the beginning of treatment, in the form of a depression which was more or less regular and more or less accentuated.", "The diameter of the lesion was observed to be reduced by from 75 to 85%." ] ]	Patent_10466619
[ [ "Implant", "An artefact which is suitable for use as an implant is provided.", "The artefact includes a body having at least an outer surface layer of a calcium phosphate-based material.", "The outer surface layer has a surface area of at least 1,5m2/g.", "A plurality of micropores are provided in at least the outer surface layer of the body.", "The micropores have a maximum dimension of up to about 150 μm." ], [ "1.An artefact which is suitable for use as an implant, the artefact including a body having at least an outer surface layer of a calcium phosphate-based material, with the outer surface layer having a surface area of at least 1,5 m2/g; and a plurality of micropores in at least the outer surface layer of the body, with the micropores having a maximum dimension of up to about 150 μm.", "2.An artefact according to claim 1, wherein the calcium phosphate-based material is hydroxyapatite.", "3.An artefact according to claim 1, wherein the entire body is of the calcium phosphate-based ceramic material having the plurality of micropores 4.An artefact according to claim 1, wherein the body comprises a core of dense material, and the outer surface layer which covers the core.", "5.An artefact according to claim 4, wherein the core is substantially devoid of any micropores.", "6.An artefact according to claim 4, wherein the core is of a different material to that of the outer surface layer material.", "7.An artefact according to claim 4, wherein the core is of the same material as the outer surface layer save that it has a lower concentration of the micropores 8.An artefact according to claim 1, wherein the surface area of the outer surface layer of the body is at least 2,0 m2/g.", "9.An artefact according to claim 1, wherein macropores are provided in the body.", "10.An artefact according to claim 9, wherein the macropores are substantially spherical, and at least some of them are interconnected, with the macropores that are interconnected being of spherical, intercoalesced form so that adjacent macropores are coalesced together.", "11.An artefact according to claim 10, wherein the macropores are from 100 to 2000 microns in size.", "12.An artefact according to claim 9, wherein the majority of the macropores are of substantially the same size, and/or wherein the macropores occupy from 20% to 80% of the total volume of that portion of the body in which they occur, and/or wherein the macropores are randomly interspersed throughout that portion of the body in which they occur.", "13.An artefact according to claim 9, wherein substantially all of the macropores are in communication with the outer surface of the artefact by means of capillary passages.", "14.An artefact according to claim 9, wherein the maximum dimension of the micropores is from sub-micron to 150 μm.", "15.An artefact according to claim 14, wherein the majority of the micropores are substantially spherical.", "16.An artefact according to claim 14, wherein the majority of the micropores are of irregular shape.", "17.An artefact according to claim 9, wherein the micropores are randomly interspersed throughout the body; and/or wherein the micropores are separate from one another; and/or wherein the majority of the micropores are of substantially the same size; and/or wherein the micropores occupy 60% or less of the total volume of that portion of the body in which they occur, excluding the volume occupied by any macropores.", "18.An artefact according to claim 1, wherein substantially all of the micropores are in communication with the outer surface of the artefact by means of capillary passages so that the calcium phosphate-based ceramic material contains substantially no sealed or isolated micropores.", "19.An artefact according to claim 1, wherein hemispherical surface concavities are provided in the outer surface layer of the body, with the surface concavities having diameters of from 100 to 2000 microns and depths of 50 to 1000 microns.", "20.A method of making an artefact which is suitable for use as an implant, the method including mixing, at elevated temperature, calcium phosphate-based material in powder form with a thermoplastic binder, to produce a powder/binder mixture; granulating the powder/binder mixture; forming a green compact from the mixture; and sintering the green compact, with the maximum temperature during the sintering being ≦1050° C., thereby to obtain an artefact comprising a sintered body having a surface area of at least 1,5 m2/g and a plurality of micropores interspersed throughout the body, with the micropores having a maximum dimension of up to about 150 μm.", "21.A method of making an artefact which is suitable for use as an implant, the method including mixing, at elevated temperature, a mixture of a calcium phosphate-based material in powder form and a powdered solid substance which is oxidizable into gaseous form, with a thermoplastic binder, to produce a powder/binder mixture; granulating the powder/binder mixture; forming a green compact from the mixture; sintering the green compact at a temperature, T1, and in a wet reducing or inert atmosphere, to obtain an artefact precursor; cooling the precursor to a temperature, T2, at which no further sintering takes place, while maintaining the wet reducing or inert atmosphere; while maintaining the precursor at about T2, exposing it to an oxidizing environment, so as to oxidize at least some of the solid substance and render it into gaseous form, so that it is thereby substantially removed from the body, thereby to obtain an artefact comprising a sintered body having a surface area of at least 1,5 m2/g, with the spaces which were occupied by the solid substance thus being micropores interspersed throughout thc body and having a maximum dimension of up to about 150 μm.", "22.A method according to claim 21, wherein the powdered calcium phosphate-based material is hydroxyapatite, with the hydroxyapatite particles having a narrow size distribution and a mean particle size of about 1 μm.", "23.A method according to claim 22, wherein the powdered solid substance is carbon, with the carbon particles having a narrow size distribution and a mean particle size of about 5 μm.", "24.A method according to claim 23, wherein the formation of the green compact is effected by pressing, moulding or extruding the mixture; and/or wherein the temperature, T1, is above 1100° C.; and/or wherein the atmosphere in which the sintering is effected is a combination of a 5% hydrogen in nitrogen mixture, and steam; and/or wherein the temperature, T2, is about 900° C.; and/or wherein the oxidizing environment is air; and/or wherein the mass proportion of carbon to hydroxyapatite in the powder/binder mixture is about 1:3.25.A method according to claim 23, wherein the carbon particles are smaller than the hydroxyapatite particles so that the carbon particles in the artefact precursor occupy interstitial sites between hydroxyapatite particles.", "26.A method according to claim 23, wherein the carbon particles are of substantially the same size as the hydroxyapatite particles so that the resultant micropores are of similar shape and size to the starting carbon particles.", "27.A method of making an artefact which is suitable for use as an implant, the method including mixing, at elevated temperature, a mixture of a calcium phosphate-based material in powder form and a powdered solid substance which is oxidizable into gaseous form, with a thermoplastic binder, to produce a first powder/binder mixture; granulating the first powder/binder mixture; mixing, at elevated temperature, calcium phosphate-based material in powder form with a thermoplastic binder, to produce a second powder/binder mixture containing no oxidizable powdered solid substance; granulating the second powder/binder mixture; forming the second powder/binder mixture into a core; covering the core with an outer surface layer of the first powder/binder mixture, to obtain a green compact; sintering the green compact at a temperature, T1, and in a wet reducing or inert atmosphere, to obtain an artefact precursor; cooling the precursor to a temperature, T2, at which no further sintering takes place, while maintaining the wet reducing or inert atmosphere; while maintaining the precursor at about T2, exposing it to an oxidizing environment, so as to oxidize at least some of the solid substance and render it into gaseous form, so that it is thereby substantially removed from the body, thereby to obtain an artefact comprising a sintered body having an outer surface layer, with the outer surface layer having a surface area of at least 1,5 m2/g, with the spaces which were occupied by the solid substance thus being micropores interspersed throughout the surface layer and having a maximum dimension of up to about 150 μnm.", "28.A method according to claim 27, wherein the powdered calcium phosphate-based material is hydroxyapatite, with the hydroxyapatite particles having a narrow size distribution and a mean particle size of about 1 μm.", "29.A method according to claim 28, wherein the powdered solid substance is carbon, with the carbon particles having a narrow size distribution and a mean particle size of about 5 μm.", "30.A method according to claim 29, wherein the formation of the core and the covering thereof with the outer surface layer is effected by pressing, moulding or extruding the powder/binder mixture; and/or wherein the temperature, T1, is above 1100° C.; and/or wherein the atmosphere in which the sintering is effected is a combination of a 5% hydrogen in nitrogen mixture, and steam; and/or wherein the temperature, T2, is about 900° C.; and/or wherein the oxidizing environment is air; and/or wherein the mass proportion of carbon to hydroxyapatite in the powder/binder mixture is about 1:3.31.A method according to claim 27, wherein the granulation of the powder/binder mixtures is effected by crushing or milling the mixtures, and sieving them to the required granule or particle size.", "32.A method according to claim 27, wherein the mixing of the powder components is effected by homogenizing the components in a ball mill for an extended period of time.", "33.A method according to claim 27, wherein fugitive phase particles which have sizes of 100 to 2000 microns and which are heat decomposable, are mixed with the first powder/binder mixture and/or with the second powder/binder mixture, with the green compact, prior to sintering, being heated to above the decomposition temperature of the fugitive phase particles, thereby to form macropores.", "34.A method according to claim 33, wherein the fugitive phase particles are stearic acid particles which are substantially spherical, with the stearic acid particles having a size range of 500 to 1000 microns.", "35-36.", "(canceled)" ], [ "This invention relates to an implant.", "It relates in particular to an artefact which is suitable for use as an implant, and to a method of manufacture thereof.", "According to a first aspect of the invention, there is provided an artefact which is suitable for use as an implant, the artefact including a body having at least an outer surface layer of a calcium phosphate-based material, with the outer surface layer having a surface area of at least 1,5 m2/g; and a plurality of micropores in at least the outer surface layer of the body, with the micropores having a maximum dimension of up to about 150 μm.", "The Applicant believes that the artefact according to the invention will have.", "a sufficiently high degree of bioactivity so that it can be used as an implant, typically a bone implant.", "In particular, it is believed that the artefact will have enhanced bioactivity as compared to bone implants of calcium phosphate-based material but which do not have the high surface area and microporosity of the artefact according to the invention.", "Thus, the artefact will be osteoconductive, ie permitting bone growth onto its surface and into surface pores when it is in close proximity to viable bone.", "However, the artefact should preferably have sufficient bioactivity so that it is also osteoinductive, ie inducing bone growth onto its surface and into surface pores independently of the presence of viable bone near the implant, thereby rendering it particularly suitable for use as a bone implant.", "Furthermore, it is suitable for use as a soft tissue implant in a site where only soft tissue is in direct contact with the implant.", "The Applicant has determined that an osteoinductive bone implant must have the combination of a high surface area, ie a surface area of at least 1,5 m3/g, and strong capillary action when immersed in liquid, such as water.", "The presence of micropores having a maximum dimension of 150 μm as hereinbefore discussed, promotes a strong capillary action.", "The necessary microporosity can be achieved by, for example, employing low temperature sintering of the calcium phosphate-based material during manufacture of the artefact, ie while sintering the artefact, limiting the maximum sintering temperature, Tmax to ≦1050° C., with the micropores or interparticle pores thereby being formed.", "The calcium phosphate-based material may, in particular, be a ceramic material.", "Thus, it may be hydroxyapatite.", "Hydroxyapatite is a sintered bioactive ceramic biomaterial.", "In one embodiment of the invention, the entire body, ie not only the surface layer, may be of the calcium phosphate-based ceramic material having the microporosity, ie the plurality of micropores, as hereinbefore described.", "In other words, the entire body will then have a calcium phosphate-based ceramic structure.", "However, in another embodiment of the invention, the body may comprise a core of dense material, and the outer surface layer, as hereinbefore described, covering the core.", "By ‘dense material’ is meant a material which has fewer of the micropores than the outer surface layer, ie has a lower concentration of the micropores than the outer surface layer, so that it has greater mechanical strength than the outer surface layer.", "The core may even be substantially devoid of any micropores.", "In one version of the invention, the core may be of a different material to that of the outer surface material.", "Thus, the core may then be a material which is chemically different to that of the surface layer.", "In another embodiment of the invention, the core may be of the same material as the outer surface layer save that it will, as hereinbefore set out, have a lower concentration of the micropores.", "In other words, the artefact will then have a mixed or graded structure comprising the relatively dense core and the outer surface layer as hereinbefore described, with both the core and the outer surface layer having the same chemical composition.", "The surface area of the outer surface layer of the body may be from 2,0 m2/g to 2,5 m2/g, or even greater.", "Macropores or macroporous spaces may be provided in the body.", "The macropores may be substantially spherical, and at least some may be interconnected.", "In particular, the macropores that are interconnected may be of spherical, intercoalesced form, ie adjacent macropores are coalesced together and thus not interconnected by elongate passageways.", "The macropores may be from 100 to 2000 microns in size, ie may have diameters of 100 to 2000 microns, preferably 400 to 800 microns.", "All, or the majority of, the macropores may be of substantially the same size.", "The macropores may occupy from 20% to 80% of the total volume of that portion of the body in which they occur.", "For example, the macropores may occupy about 60% of the total volume of such portion of the body.", "The macropores may be randomly interspersed throughout said portion of the body.", "Thus, said portion of the body may have a network of interconnected coalesced rounded inner macroporous spaces.", "However, it is also to be appreciated that most, and preferably all, of the macropores will be in communication with the outer surface of the artefact, eg by means of capillary passages.", "Thus, there will be few, if any, sealed or isolated macropores.", "The micropores may have a maximum dimension of from sub-micron, eg about 50 nm, to 150 μm, typically from 1-10 μm.", "In one embodiment of the invention, the micropores, or some of the micropores, may be substantially spherical.", "However, in another embodiment of the invention, the majority, eg substantially all, of the micropores may be of irregular shape.", "The micropores may be randomly interspersed throughout the body.", "The micropores may be separate from one another, ie not connected together.", "The majority of the micropores may be of substantially the same size.", "The micropores may occupy 60% or less of the total volume of that portion of the body in which they occur, excluding the volume occupied by any macropores, ie the residual volume of that portion of the body after, the volume of any macropores has been excluded.", "Typically, the micropores may occupy about 40% of the residual body portion volume.", "It will be appreciated that most, and preferably substantially all, of the micropores will be in communication with the outer surface of the artefact, eg by means of capillary passages.", "In other words, the calcium phosphate-based ceramic material will contain few, if any, sealed or isolated micropores.", "The body may also, if desired, be provided with surface concavities, ie surface concavities in the outer surface layer.", "The surface concavities may have diameters of from 100 to 2000 microns, preferably 400 to 800 microns, and depths of 50 to 1000 microns, preferably 200 to 400 microns.", "The surface concavities may be hemispherical.", "The surface concavities may be interconnected with the macropores by being coalesced therewith.", "According to a second aspect of the invention, there is provided a method of making an artefact which is suitable for use as an implant, the method including mixing, at elevated temperature, calcium phosphate-based material in powder form with a thermoplastic binder, to produce a powder/binder mixture; granulating the powder/binder mixture; forming a green compact from the mixture; and sintering the green compact, with the maximum temperature during the sintering being ≦1050° C., thereby to obtain an artefact comprising a sintered body having a surface area of at least 1,5 m2/g and a plurality of micropores interspersed throughout the body, with the micropores having a maximum dimension of up to about 150 μm.", "The formation of the green compact may be effected by pressing, moulding or extruding the powder/binder mixture.", "When the formation of the green compact is by pressing, then the size of the granules of the powder/binder mixture is typically less than 500 μm.", "When the formation of the green compact is by moulding or extruding, then the size of the granules can either be less than 500 μm, or greater than 500 μm, eg up to several millimeters.", "According to a third aspect of the invention, there is provided a method of making an artefact which is suitable for use as an implant, the method including mixing, at elevated temperature, a mixture of a calcium, phosphate-based material in powder form and a powdered solid substance which is oxidizable into gaseous form, with a thermoplastic binder, to produce a powder/binder mixture; granulating the powder/binder mixture; forming a green compact from the mixture; sintering the green compact at a temperature, T1, and in a wet reducing or inert atmosphere, to obtain an artefact precursor; cooling the precursor to a temperature, T2, at which no further sintering takes place, while maintaining the wet reducing or inert atmosphere; while maintaining the precursor at about T2, exposing .", "it to an oxidizing environment, so as to oxidize at least some of the solid substance and render it into gaseous form, so that it is thereby substantially removed from the body, thereby to obtain an artefact comprising a sintered body having a surface area of at least 1,5 m2/g, with the spaces which were occupied by the solid.", "substance thus being micropores interspersed throughout the body and having a maximum dimension of up to about 150 μm.", "The formation of the green compact may be effected by pressing, moulding or extruding the powder/binder mixture.", "When the formation of the green compact is by pressing, then the size of the granules of the powder/binder mixture is typically less than 500 μm.", "When the formation of the green compact is by moulding or extruding, then the size of the granules can either be less than 500 μm, or greater than 500 μm, eg up to several millimeters.", "The powdered solid substance or oxidizable powder constituent is thus not oxidized during the sintering however, during the subsequent exposure-of the precursor to the oxidizing environment, at least some of this constituent is oxidized into gaseous form.", "Sufficient of the powdered solid substance may be used so that the mass proportion of powdered solid substance to calcium phosphate based material in the powder/binder mixture is up to 3:2, and preferably about 1:3.The calcium phosphate-based material or powder incorporated in the powder/binder mixture may have particle sizes from submicron, eg about 50 nm, to 100 μm.", "Preferably, the powdered calcium phosphate-based material has a narrow size distribution with a mean particle size of about 5 μm or less, eg about 1 μm.", "It is believed that this particle size distribution represents a balance between the powder being sufficiently fine to allow sintering yet being sufficiently coarse to permit achievement of high solids loading when mixed with the thermoplastic binder.", "The powdered solid substance may be carbon.", "The carbon.", "particle size may be from submicron, eg about 50 nm, to 150 μm.", "Preferably, the carbon has a narrow size distribution, ie the particles are of substantially the same size, with a mean particle size of about 5 μm.", "As set out hereinbefore, the calcium phosphate-based material may, in particular, be hydroxyapatite.", "The temperature at which hydroxyapatite powder sinters is dependent on its particle size.", "Typically, however, initial sintering occurs at about 950° C.-1000° C. Thus, T1 is typically above 1100° C., eg is about 1200° C. The atmosphere in which the sintering is effected may be a combination of a 5% hydrogen in nitrogen mixture, and steam.", "The temperature, T2, may be about 900° C. The oxidizing atmosphere may be air.", "It is believed that, in the method of the third aspect of the invention, high temperature sintering can be carried out without micropore closure or collapse.", "This is due the temporary presence of the carbon powder particles which inhibit or prevent micropore closure during the high temperature sintering, which allows sintering of adjacent calcium phosphate-based material to progress further.", "This in turn results in a stronger artefact.", "The method of the third aspect of the invention has the further advantage that the shape and size of the micropores can be tailored, as desired.", "Thus, in one embodiment of the invention, the carbon particles may be smaller than the calcium phosphate-based material particles.", "The carbon particles will then, in the artefact precursor, occupy interstitial sites between hydroxyapatite particles.", "However, in another embodiment of the invention, the carbon particles may be of substantially the same size as the calcium phosphate-based material particles.", "The resultant micropores will then be of similar shape and size to the starting carbon particles, and have fundamentally different characteristics when compared to the case where the micropores are substantially smaller.", "According to a fourth aspect of the invention, there is provided a method of making an artefact which is suitable for use as an implant, the method including mixing, at elevated temperature, a mixture of a calcium phosphate-based material in powder form and a powdered solid substance which is oxidizable into gaseous form, with a thermoplastic binder, to produce a first powder/binder mixture; granulating the first powder/binder mixture; mixing, at elevated temperature, calcium phosphate-based material in-powder form with a thermoplastic binder, to produce a second powder/binder mixture containing no oxidizable powdered solid substance; granulating the second powder/binder mixture; forming the second powder/binder mixture into a core; covering the core with an outer surface layer of the first powder/binder mixture, to obtain a green compact; sintering the green compact at a temperature, T1, and in a wet reducing or inert atmosphere, to obtain an artefact precursor; cooling the precursor to a temperature, T21, at which no further sintering takes place, while maintaining the wet reducing or inert atmosphere; while maintaining the precursor at about T2, exposing it to an oxidizing environment, so as to oxidize at least some of the solid substance and render it into gaseous form, so that it is thereby substantially removed from the body, thereby to obtain an artefact comprising a sintered body having an outer surface layer, with the outer surface layer having a surface area of at least 1,5 m2/g, with the spaces which were occupied by the solid substance thus being micropores interspersed throughout the surface layer and having a maximum dimension of up to about 150 μm.", "As before, the formation of the green compact, ie the forming of the core and the covering thereof wit the outer surface layer, may be effected by pressing, moulding or extruding the powder/binder mixtures.", "When the formation of the green compact is by pressing, then the size of the granules of the first and second powder/binder mixtures is typically less than 500 μm.", "When the formation of the green compact is by moulding or extruding, then the size of the granules in the powder/binder mixtures can be less than 500 μm, or greater than 500 μm, eg up to several millimeters.", "The powdered solid substance, T1, T3, the wet reducing or inert atmosphere, and the oxidizing atmosphere may thus be as hereinbefore described.", "The core thus has few, if any, of the micropores.", "Any suitable thermoplastic binder, such as a commercial polymeric binder used for extrusion or injection moulding of ceramic materials, may be used, provided it allows ambient temperature compaction of the granules to a strength adequate for further processing.", "The temperature at which the mixing of the powders with the thermoplastic binder to produce the powder/binder mixtures takes place depends on the thermoplastic binder used, but is typically around 120° C. The granulation of the powder/binder mixtures may be effected by crushing or milling the mixtures, and sieving them to the required granule or particle size.", "The mixing of powder components may be effected by homogenizing the components in a ball mill for an extended period of time, eg for a period of several hours.", "When it is desired to have macropores in the core and/or the outer surface layer of the artefact, fugitive phase particles which have sizes of 100 to 2000 microns and which are heat decomposable may be mixed with the relevant powder/binder mixture prior to the compaction of the green compact.", "Prior to sintering, the green compacts or bodies will then be heated to above the decomposition temperature of the fugitive phase particles, to form the macropores.", "The fugitive phase particles may be stearic acid particles, which may be substantially spherical.", "The stearic acid particles will be selected such that they provide macropores or macroporous spaces of a desired size in the artefact.", "Thus, typically, stearic acid particles having a size range of 500 to 1000 microns may be used.", "The relevant mixture is admixed with the fugitive phase particles in a desired mass ratio in order to provide a resultant artefact having a desired macropore volume.", "Thus, if a desired macropore volume of approximately 60% of the total artefact volume if desired, then the mass proportion of combined mixture to fugitive phase particles will be about 1:1,27 by mass.", "To form the green compact or body, the mixture may be compacted at a pressure of about 20 MPa, moulded or extruded and machined, if necessary.", "The temperature to which the green compacts or bodies are heated is dependent on the fugitive phase used.", "However, when stearic acid particles are used as the fugitive phase, the green compacts are typically heated to about 500° C., to allow melting and decomposition of the stearic acid, thereby forming in the green compacts or bodies, interconnected macropores produced by the decomposition of the stearic acid particles.", "When sintering in air without an oxidizable component, the sintering temperature and time is set or limited by the level of micropores required in the resultant implant.", "For example, to obtain a total microporosity level of 40% by volume in the resultant implant, sintering may be effected at about 1020° C. for one hour.", "The invention will now be described by way of non-limiting example, with reference to the accompanying drawings which show simplified views of artefacts according to the invention.", "IN THE DRAWINGS FIG.", "1 shows a cross-sectional view of an artefact according to a first embodiment of the invention, and FIG.", "2 Shows a sectional view of an artefact according to a second embodiment of the invention.", "Referring to FIG.", "1, reference numeral 10 generally indicates an artefact according to a first embodiment of the invention.", "The artefact 10 includes a body 12 of hydroxyapatite.", "The body 12 comprises a plurality of particles 14 of hydroxyapatite, which are sintered, ie fused, together in zones 16 where the particles touch each other, so that irregular shaped micropores 18 are formed between the particles 14.The micropores 18 have a maximum dimension of 10 μm at most, and typically have a maximum dimension in the range of 1-10 μm.", "The micropores 18 are interspersed throughout the body 12.The body 12 has an outer surface 20 in which are provided a plurality of surface concavities 22.The surface concavities 22 are hemispherical in cross-section, and may have dimensions of 400-800 μm, and depths of 200-400 μm.", "The concavities 22 are irregularly or randomly spaced in the outer surface 20.The outer surface 20, which thus includes the surfaces of the concavities 22, has a surface area of at least 1,5 m2/g, and preferably 2,0-2,5 m2/g.", "It is to be appreciated that a simplified view of the artefact is shown in FIG.", "1.In practice, the zones 16 will not be as clearly demarcated as shown in FIG.", "1.Instead, adjacent hydroxyapatite particles 14 will flow or merge into each other to greater or lesser extent, depending on degree of sinter of the adjacent particles.", "As a result, the micropores 18 will in practice not have the same shapes and sizes as indicated in the drawing; instead, their shapes and sizes will be dictated by the degree of sinter of adjacent particles.", "In other words, most, if not all, of the micropores 18 will be of different shape and/or size.", "Still further, the micropores 18 is will not normally, in practice, be arranged in a regular pattern as indicated in the drawing; instead, they will be randomly arranged depending on the degree of sinter of the particles.", "Thus, for example, a number of the particles 14 may be fully sintered and thus wholly integral with one another, with no micropores being defined between such particles.", "Additionally, substantially none of the micropores 18 will be sealed or isolated, ie substantially all of the micropores 18 will be in communication with the outer surface 20 by means of capillary passages (not shown).", "Referring now to FIG.", "2, reference numeral 50 generally indicates an artefact according to another embodiment of the invention.", "Parts of the artefact 50 which are the same or similar to those of the artefact 10 hereinbefore described with reference to FIG.", "1, are indicated with the same reference numerals.", "The body 12 of the artefact 50 comprises a core 52 of dense hydroxyapatite material, ie hydroxyapatite material that is devoid of any pores, particularly micropores 18.The core 52 is covered by an outer surface layer 54 of hydroxyapatite material having the particles 14 and the micropores 18.The surface layer 54 thus also has the outer surface 20 with the concavities 22.The artefacts 10, 50 are suitable for use as bone implants having both osteoconductivity and osteoinductivity.", "The implants 10, 50 thus exhibit high surface area and strong capillary action when immersed in body fluid such as blood, by virtue of the high level of microporosity of the implant surface.", "The artefacts 10, 50 can be manufactured in accordance with Examples 1 to 4 hereunder, with the artefact 10 being produced by the method of Examples 1 and 2, and the artefact 50 by the method of Examples 3 and 4.EXAMPLE 1 A green artefact is formed by compounding hydroxyapatite powder, having a narrow size distribution and a mean particle size of about 5 μm, with a commercial thermoplastic polymeric binder at a temperature of about 120° C., to produce a powder/polymer mixture.", "This mixture in crushed and sieved to a particle size smaller than 300 μm.", "In this fashion, a granular mixture is obtained.", "Any commercial thermoplastic polymeric binder suitable for extrusion or injection moulding of ceramic materials, may be used, provided it allows ambient temperature compaction of the granules of the mixture to a strength adequate for further processing.", "The mixture is pressed or compacted, in a suitable die or mould, at a pressure of 20 MPa, and machined if necessary.", "In this fashion green compacts are obtained.", "The die or mould will typically be provided with protrusions for forming the cavities 22 in the outer surfaces of the green compacts.", "The green compacts are heated, in a furnace, to a temperature of 1050° C. for sintering of the hydroxyapatite powder.", "Due to the low sintering temperature or undersintering, adjacent particles merely sinter together where they abut, ie there is an absence of total fusion or merging of particles into one another.", "Irregular shaped micropores having a maximum size or dimension of 1-10 μm, are thus formed between particles.", "The green artefact is sintered at a relatively low temperature of 1050° C. to obtain the artefact 10 of FIG.", "1.EXAMPLE 2 A green artefact is formed by following the same general procedure as described in Example 1 except that 25% by mass of the hydroxyapatite powder is replaced by carbon powder, which is thus intimately admixed with the hydroxyapatite powder.", "The green compact that is obtained is sintered at a relatively high temperature of 1200° C., under a slightly reducing atmosphere of the combination of 5% (by volume) hydrogen in nitrogen mixture, and steam.", "The body material is then allowed to cool to 900° C. in the furnace, with this temperature being too low for further sintering to take place.", "Air is admitted to the furnace at this temperature over an extended period of several hours.", "The air results in the carbon being oxidized and removed as a gas, thereby yielding the artefact 10 having the fine microporous structure and the high surface area as hereinbefore described.", "Due to the higher sintering temperature in this Example as compared to the sintering temperature used in Example 1, sintering between adjacent particles progresses to a greater degree, resulting in a stronger artefact, as hereinbefore described.", "Using the method of this Example, an artefact as illustrated in FIG.", "1 is obtained when the carbon powder particles also have a narrow size distribution, and with their mean particle size an order of magnitude smaller than the hydroxyapatite particles.", "When the mean particle size of the carbon powder particles is similar to that of the hydroxyapatite particles, the micropores 18 will be of similar size and shape to the carbon powder particles.", "EXAMPLE 3 In order to produce the artefact 50, a hydroxyapatite/carbon granular mixture as described in Example 2, is made up (‘Component 1’).", "A standard hydroxyapatite granular mixture as described in Example 1 is also prepared (‘Component 2’).", "An amount of Component 1 is introduced into a pressing die and slightly compacted into disc form.", "An amount of Component 2 is then deposited on top of the slightly compacted disc of Component 1, while still in the die, levelled and thereafter slightly compacted.", "A third layer of Component 1 is then added to the stack in the die.", "The stack is then compacted under hydraulic pressure to yield a green artefact comprising standard hydroxyapatite powder (Component 2) sandwiched between two outer layers of carbon containing hydroxyapatite powder (Component 1).", "This green artefact is then sintered at a high temperature of 1200° C. under a slightly reducing atmosphere of the combination of 5% (by volume) hydrogen in nitrogen mixture, and steam.", "The material is thereafter allowed to cool to 900° C. in the furnace, with this temperature being too low for further sintering to take place.", "Air is admitted into the furnace at this temperature over an extended period of several hours.", "The carbon in the outer layer of the artefact precursor in oxidized and removed as a gas.", "The resultant artefact comprises a relatively dense hydroxyapatite inner core 52 of high strength, with an outer layer 54 of microporous hydroxyapatite of high surface area and enhanced bioactivity as hereinbefore described.", "EXAMPLE 4 This example describes how an elongated artefact with cross-section similar to that of the compacted artefact 50 can be produced by means of extrusion.", "A hydroxyapatite/carbon powder mixture as described in Example 2 is compounded with the polymeric binder to produce an extrudable component (‘Feedstock 1’).", "A hydroxyapatite powder as described in Example 1, is a compounded with the binder to produce a second extrudable component (‘Feedstock 2’).", "Feedstocks 1 and 2 are co-extruded at an elevated temperature appropriate for the particular binder used, to yield an inner rod-like core of Feedstock 2; covered by an outer sleeve-like layer of Feedstock 1.This green artefact is then sintered, cooled and subjected to air treatment as described in Example 3, to yield an artefact having a relatively dense inner core 52 of high strength and a microporous outer surface layer 54 having enhanced bioactivity as hereinbefore described.", "Concavities 22 may also be introduced on the outer surface of the extruded body by repeatedly indenting the surface of the extrudate as it emerges from the extrusion nozzle.", "EXAMPLES 5 to 8 Examples 1 to 4 were repeated, in Examples 5 to 8 respectively, using identical constituents, parameters, etc as in Examples 1 to 4, except for the following: In Example 5 (which corresponds to Example 1), the hydroxyapatite powder had a mean particle size of about 1 μm In Example 5, the irregular shaped micropores that were formed between the particles had a maximum size or dimension of less then 10 μm; In Example 7 (which corresponds to Example 3), the amount of Component 1 that as introduced into the pressing die was slightly compacted into a cylindrical form.", "An amount of Component 2 was placed in a different die and slightly compacted to a disc form.", "The disc form manufactured of Component 2 was then placed in the cylinder form manufactured of Component 1, and the entire structure consolidated by compaction to a higher pressure of 20 MPa.", "The resulting green artefact then comprised a disc of standard hydroxyapatite powder (Component 2) enclosed by a ring of carbon containing hydroxyapatite powder (Component 1); In Example 8 (which corresponds to Example 4), the hydroxyapatite/carbon powder mixture of Example 6 (which corresponds to Example 2) and which thus included the hydroxyapatite powder of Example 5 rather than that of Example 1, was used." ] ]	Patent_10466659
[ [ "Myoelectrically activated respiratory leak sealing", "The method and system are for sealing/unsealing (regulating) airway leaks occuring between the ventilator circuit and respiratory airways during lung ventilatory support in response to myoelectrical activity of diaphgram.", "Myolectrical activity of a patient's respiratory-related muscle is sensed to detect respiratory effort, and to produce a myoelectrical signal representative of the sensed muscle myoelectrical activity.", "Respiratory flow and pressure can also be measured to produce respective respiratory pressure and respiratory flow signals.", "A logic trigger sealing/unsealing of airway leaks in relation to the myoelectrical signal, respiratory flow signal and/or respiratory pressure signal to assist respiration of the patient.", "The amplitude of the myoelectrical signal Is compare to a given threshold, and airway leaks are sealed when the amplitude of the myoelectrical signal is higher than this threshold.", "Increment of myoelectrical signal amplitude can be also detected to trigger the airway leak regulating device to seal the airway leaks, while decrement of the myoelectrical signal amplitude can be detected to unseal the airway leaks and thus permit air evacuation from the patient's lungs." ], [ "1.A method for controlling an air seal between a ventilator air circuit and a patient's respiratory airways, comprising: sensing myoelectrical activity of a respiratory-related muscle of the patient so as to yield at least one myoelectrical signal representative of respiratory effort of the patient; comparing said at least one myoelectrical signal to a predetermined value, so as to determine the highest value therebetween; and modifying the seal according to the highest value so as to control said leak.", "2.A method for controlling an air seal is recited in claim 1, wherein said respiratory-related muscle is taken from the group consisting of diaphragm, parasternal intercostal muscles, sternocleidomatoids, scalenes, and alae nasi.", "3.A method for controlling an air seal as recited in claim 2, wherein said respiratory-related muscle is the diaphragm.", "4.A method for controlling an air seal as recited in claim 3, wherein said myoelectrical activity is sensed in the electrically active region of said diaphragm (DDR).", "5.A method for controlling an air seal as recited in claim 4, wherein said myoelectrical activity is sensed near the centre of the DDR.", "6.A method for controlling an air seal as recited in claim 5, further comprising: sensing at least one signal above said DDR and at least one signal below said DDR, and subtracting these two signals to yield at least one myoelectrical signal representative of the respiratory effort of the patient.", "7.A method for controlling an air seal as recited in claim 1, further comprising filtering from said at least one myoelectrical signal at least one of the following disturbances: motion artefacts, electrocardiogram (ECG), electrical interference, and high frequency noise.", "8.A method for controlling an air seal as recited in claim 1, wherein said predetermined value is a predetermined threshold; said air seal being modified so as to: (a) seal the patient's respiratory airways when said at least one myoelectrical signal is said highest value, thereby avoiding gas leaks during respiratory effort of the patient, and (b) unseal the patient's airways when said threshold is the highest value, thereby allowing gas leaks during relaxation of the respiratory effort of the patient.", "9.A method for controlling an air seal as recited in claim 8, wherein two different thresholds are used in (a) and in (b).", "10.A method for controlling an air seal as recited in claim 8, wherein said threshold is predetermined by manual adjustment using visual feedback.", "11.A method for controlling an air seal as recited in claim 8, wherein said threshold is predetermined automatically by letting the level be relative to a predetermined noise level.", "12.A method for controlling an air seal as recited in claim 1, further comprising multiplying a current sample of said at least one myoelectrical signal by a predetermined constant to produce a multiplied sample; wherein said predetermined value corresponds to a prior sample of said at least one myoelectrical signal; said air seal being modified so as to: (a) seal the patient's respiratory airways when said current sample is said highest value, thereby avoiding gas leaks during respiratory effort of the patient, and (b) unseal the patient's airways when said prior sample of said at least one myoelectrical signal is the highest value, thereby allowing gas leaks during relaxation of the respiratory effort of the patient.", "13.A method for controlling an air seal as recited in claim 1, further comprising detecting the level of noise in said at least one myoelectrical signal; and determining whether the respiratory-related muscle of the patient is active in relation to the detected level of noise.", "14.A system for controlling an air seal between a ventilator air circuit and a patient's respiratory airways, comprising: a controller; a myoelectrical sensor connected to said controller, said sensor being configured to sense at least one myoelectrical signal representative of the respiratory effort of the patient; and a respiratory sealing device connected to said controller and configured to modify the air seal according to the at least one sensed myoelectrical signal.", "15.A system for controlling an air seal as recited in claim 14, wherein said respiratory sealing device is in the form of a sealing balloon.", "16.A system for controlling an air seal as recited in claim 15, wherein said sealing balloon is mounted on a ventilatory assist tube of the ventilator air circuit; said ventilatory assist tube including a first lumen in the form of an air passage from the ventilatory air circuit, and a second lumen in the form of a fluid passage for fluid communication between said sealing balloon and a balloon inflation device and pressure control of said balloon.", "17.A method for controlling an air seal as recited in claim 16, wherein two different thresholds are used in (a) and in (b).", "18.A system for controlling an air seal as recited in claim 14, wherein said respiratory sealing device is in the form of a face mask including a seal pressure lumen.", "19.A system for controlling an air seal as recited in claim 14, wherein said myoelectrical sensor is in the form of an array of electrodes.", "20.A system for controlling an air seal as recited in claim 19, wherein said array of electrodes is provided with a constant inter-electrode distance.", "21.A system for controlling an air seal as recited in claim 19, wherein said array of electrodes includes nine electrodes.", "22.A system for controlling an air seal as recited in claim 14, wherein said myoelectrical sensor is mounted on the free end of a catheter.", "23.A system for controlling an air seal as recited in claim 22, wherein said myoelectrical sensor includes a steel wire wound around said catheter.", "24.A system for controlling an air seal as recited in claim 23, wherein said wound steel wire is smoothed out by solder.", "25.A system for controlling an air seal as recited in claim 22, wherein said catheter is an oesophageal catheter.", "26.A system for controlling an air seal as recited in claim 14, wherein said myoelectrical sensor is mounted on the free end of a nasogastric tube.", "27.A system for controlling an air seal as recited in claim 19, further comprising at least one differential amplifier connected to both said electrodes and said controller.", "28.A system for controlling an air seal as recited in claim 27, wherein said at least one amplifier includes single-ended amplifiers, allowing monopolar readings.", "29.A system for controlling an air seal as recited in claim 27, wherein said at least one amplifier is connected to said electrodes via electric wires.", "30.A system for controlling an air seal as recited in claim 27, comprising a differential amplifier for each pair of electrodes.", "31.A system for controlling an air seal as recited in claim 27, wherein said at least one isolation amplifier is configured for sampling said at least one myoelectrical signal to form signal segments.", "32.A system for controlling an air seal as recited in claim 14, wherein said controller is in the form of a personal computer.", "33.A system for controlling an air seal between a ventilator air circuit and a patient's respiratory airways, comprising: means for sensing myoelectrical activity of a respiratory-related muscle of the patient; means for modifying the air seal; and means for controlling the air seal modifying means depending on the sensed myoelectrical activity.", "34.A system as recited in claim 29, further comprising means for filtering from said at least one myoelectrical signal at least one of the following disturbances: motion artefacts, ECG, electrical interference, and high frequency noise." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Inherent to methods of administrating ventilatory support via delivering inspiratory flow, volume, and/or pressure to the airways is the influence of airway leaks occurring between the ventilator circuit and respiratory airways.", "A poor seal between the device used for administration of ventilatory support (e.g., endotracheal tube, face/nasal mask) and the patient (e.g., airway, airway opening) introduces difficulties to deliver appropriate gas flow, volume, or pressure into the airway system in order to inflate the lungs." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>A present invention relates to a method and system for sealing/unsealing airway leaks between the patient's airways and a ventilatory support apparatus in response to a respiratory effort via the use of myoelectrical activity of the diaphragm (or other muscles associated with respiratory effort).", "Methods and systems according to the present invention allow synchronizing the activation of the seal between the respiratory airways and ventilator circuit with the neural activation of inspiratory muscles.", "Methods and systems according to the present invention further allow to reduce the problems related to the interface and the leaks occurring between the respiratory airways and ventilator circuit during the entire (or parts of) the period of neural inspiratory activation, which help to ensure adequate delivery of gas flow, volume and/or pressure into the lungs.", "Methods and systems according to the present invention also allow synchronizing the deactivation of the, seal between the respiratory airways and ventilator circuit with the neural deactivation of inspiratory muscles.", "More specifically, according to the present invention, there is provided a method for controlling an air seal between a ventilator air circuit and a patient's respiratory airways, comprising: sensing myoelectrical activity of a respiratory-related muscle of the patient so as to yield at least one myoelectrical signal representative of respiratory effort of the patient; comparing the at least one myoelectrical signal to a predetermined value, so as to determine the highest value therebetween; and modifying the seal according to the highest value so as to control the leak.", "According to another aspect of the present invention, there is provided a system for controlling an air seal between a ventilator air circuit and a patient's respiratory airways, comprising: a controller; a myoelectrical sensor connected to the controller, the sensor being configured to sense at least one myoelectrical signal representative of the respiratory effort of the patient; and a respiratory sealing device connected to the controller and configured to modify the air seal according to the at least one sensed myoelectrical signal.", "According to still another aspect of the present invention, there is provided a system for controlling an air seal between a ventilator air circuit and a patient's respiratory airways, comprising: means for sensing myoelectrical activity of a respiratory-related muscle of the patient; means for modifying the air seal; and means for controlling the air seal modifying means depending on the sensed myoelectrical activity.", "Other objects, advantages and features of the present invention will become more apparent upon reading the following non-restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings." ], [ "FIELD OF THE INVENTION The present invention relates to ventilatory support systems.", "More particularly, the present invention is concerned with a myoelectrically activated respiratory leak sealing method and system.", "BACKGROUND OF THE INVENTION Inherent to methods of administrating ventilatory support via delivering inspiratory flow, volume, and/or pressure to the airways is the influence of airway leaks occurring between the ventilator circuit and respiratory airways.", "A poor seal between the device used for administration of ventilatory support (e.g., endotracheal tube, face/nasal mask) and the patient (e.g., airway, airway opening) introduces difficulties to deliver appropriate gas flow, volume, or pressure into the airway system in order to inflate the lungs.", "OBJECTS OF THE INVENTION An object of the present invention is to use myoelectrical activity of the diaphragm or other respiratory-related muscles to activate and/or to deactivate a seal in order to regulate leaks between the ventilator circuit and respiratory airways.", "SUMMARY OF THE INVENTION A present invention relates to a method and system for sealing/unsealing airway leaks between the patient's airways and a ventilatory support apparatus in response to a respiratory effort via the use of myoelectrical activity of the diaphragm (or other muscles associated with respiratory effort).", "Methods and systems according to the present invention allow synchronizing the activation of the seal between the respiratory airways and ventilator circuit with the neural activation of inspiratory muscles.", "Methods and systems according to the present invention further allow to reduce the problems related to the interface and the leaks occurring between the respiratory airways and ventilator circuit during the entire (or parts of) the period of neural inspiratory activation, which help to ensure adequate delivery of gas flow, volume and/or pressure into the lungs.", "Methods and systems according to the present invention also allow synchronizing the deactivation of the, seal between the respiratory airways and ventilator circuit with the neural deactivation of inspiratory muscles.", "More specifically, according to the present invention, there is provided a method for controlling an air seal between a ventilator air circuit and a patient's respiratory airways, comprising: sensing myoelectrical activity of a respiratory-related muscle of the patient so as to yield at least one myoelectrical signal representative of respiratory effort of the patient; comparing the at least one myoelectrical signal to a predetermined value, so as to determine the highest value therebetween; and modifying the seal according to the highest value so as to control the leak.", "According to another aspect of the present invention, there is provided a system for controlling an air seal between a ventilator air circuit and a patient's respiratory airways, comprising: a controller; a myoelectrical sensor connected to the controller, the sensor being configured to sense at least one myoelectrical signal representative of the respiratory effort of the patient; and a respiratory sealing device connected to the controller and configured to modify the air seal according to the at least one sensed myoelectrical signal.", "According to still another aspect of the present invention, there is provided a system for controlling an air seal between a ventilator air circuit and a patient's respiratory airways, comprising: means for sensing myoelectrical activity of a respiratory-related muscle of the patient; means for modifying the air seal; and means for controlling the air seal modifying means depending on the sensed myoelectrical activity.", "Other objects, advantages and features of the present invention will become more apparent upon reading the following non-restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings.", "BRIEF DESCRIPTION OF THE DRAWINGS In the appended drawings: FIG.", "1 is a schematic view of a myoelectrically activated respiratory leak-sealing system, according to a first embodiment of the present invention, illustrated on a human patient; FIG.", "2 is a front elevational view of the myoelectrical sensor of FIG.", "1, according to a first embodiment of the present invention; FIG.", "3 is a front elevational view of the myoelectrical sensor of FIG.", "1, according to a second embodiment of the present invention; FIG.", "4 is a graph showing a set of EMG signals of the diaphragm (EMGdi signals) detected by pairs of successive electrodes of the sensor of FIG.", "2; FIG.", "5 is a cross-sectional view taken along line 5-5 of FIG.", "1; FIG.", "6 is a schematic view of a respiratory sealing device, according to a second embodiment of the present invention, illustrated inserted in a nasal air passage of the patient of FIG.", "1; FIG.", "7 is a schematic view of respiratory sealing device according to a third embodiment of the present invention, illustrated mounted on the face of the patient of FIG.", "1; FIG.", "8 is a flow chart of a myoelectrically activated respiratory leaksealing method according to an embodiment of, the present invention; FIGS.", "9a and 9b illustrate a flow chart of step 102 from FIG.", "8; FIG.", "10a is a graph showing the power density spectrum of electrode motion artefacts, the power density spectrum of electrocardiogram (ECG), and the power density spectrum of EMGdi signals; FIG.", "10b is a graph showing an example of transfer function for a filter to be used for filtering out the electrode motion artefacts, electrocardiogram (ECG), the 50 or 60 Hz disturbances from electrical mains and high frequency noise; FIG.", "11a is a graph of inspiratory and expiratory flow versus time for quiet breathing of a chronic obstructive pulmonary disease (COPD) patient; FIG.", "11b is a graph of the RMS value of EMG versus time for quiet breathing of a COPD patient, the graphs of FIGS.", "10a and 10b showing the time delay from EMG to airway inspiratory flow; FIG.", "12 is a graph showing the distribution of correlation coefficients calculated for determining the position of the centre of the depolarizing region of the diaphragm along the array of electrodes of FIG.", "2; FIG.", "13 is a schematic view with graphs illustrating, in the time domain, a double subtraction technique for improving the signal-to-noise ratio and to reduce an electrode-position-induced filter effect; FIG.", "14 is a schematic diagram, illustrating in the frequency domain, stabilization by the double subtraction technique of the centre frequency upon displacement of the centre of the depolarizing region of the diaphragm of FIG.", "1 along the array of electrodes of FIG.", "2; and FIG.", "15a is a graph of oesophageal and gastric pressure versus time for quiet breathing of a chronic obstructive pulmonary disease (COPD) patient; and FIG.", "15b is a graph of the RMS value of EMG versus time for quiet breathing of a COPD patient; the graphs of FIGS.", "15a and 15b show the relation between EMG and the oesophageal and gastric pressure.", "DESCRIPTION OF THE PREFERRED EMBODIMENT Turning to FIG.", "1 of the appended drawings, a myoelectrically activated respiratory leak sealing system 10 according to a first embodiment of the present invention is illustrated.", "The system 10 comprises a myoelectrical sensor 12 mounted on the free end section 14 of an oesophageal catheter 16, a respiratory sealing device in the form of a sealing balloon 18, and a controller 20.As illustrated in FIG.", "2, the myoelectrical sensor 12 is in the form of an array of electrodes 22 provided with a constant inter-electrode distance d, and allows measuring the electromyographic (EMG) activity of the diaphragm 24 (EMGdi) of a patient 26.The electrodes 22 are mounted on the free end section 14 of the catheter 16 by winding stainless steel wire (not shown) around the catheter 16.The wound stainless steel wire presents a rough surface smoothed out by solder, which in turn is electroplated with nickel, copper and then gold or silver.", "Of course, it is within the scope of the present invention to use other electrode structures.", "In the embodiment illustrated in FIGS.", "1 and 2, the free end section 14 of the catheter 16 is provided with an array of eight electrodes 22 defining seven pairs 1, 2, 3, 4, 5, 6 and 7 of successive electrodes 22 respectively collecting seven different EMGdi signals.", "Although it has been found that EMG activity of the diaphragm (EMGdi) can be measured accurately with an oesophageal catheter 16 provided on the free end section 14 thereof with an array of eight electrodes 22, a different number and/or configuration of pairs of electrodes 22 can be contemplated depending on the patient's anatomy and movement of the diaphragm 24.Also, the pairs 1-7 do not need to be pairs of successive electrodes; FIG.", "3 illustrates an array of nine electrodes to form seven overlapping pairs of electrodes 1′-7′.", "Alternatively, the electrodes 22 can possibly be applied to a nasogastric tube (not shown), which is routinely introduced in intensive-care unit (ICU) patients.", "Electric wires (not shown) interconnect each pair of successive electrodes such as 1-7 (FIG.", "2) with a respective one of a group of differential amplifiers 30 (FIG.", "1).", "Obviously, these electric wires follow the catheter 16 from the respective electrodes 22 to the corresponding amplifiers 30, and are preferably integrated to the catheter 16.The electric wires transmitting the EMGdi signals collected by the various pairs 1-7 of electrodes 22 are shielded to reduce the influence of external noise, in particular disturbance from the 50 or 60 Hz current and voltage of the electrical mains.", "The group of differential amplifiers 30 amplifies (first subtraction step of the double subtraction technique that will be described hereinbelow) and band-pass filters each EMGdi signal.", "This first subtraction step may also be carried out in the controller, which is in the form of a personal computer 20, when the amplifiers 16 are single-ended or equivalently designed amplifiers (monopolar readings).", "A common problem in recording EMGdi signals is to maintain the noise level as low and as constant as possible.", "Since the electric wires transmitting the EMGdi signals from the electrodes 22 to the differential amplifiers 30 act as an antenna, these electric wires are shielded to thereby protect the EMGdi signals from additional artefactual noise.", "Also, the package enclosing the differential amplifiers 30 is preferably made as small as possible (miniaturized) and is positioned in close proximity to the patient's nose to decrease as much as possible the distance between the electrodes 22 and the amplifiers 30.The personal computer 20 allows sampling the amplified EMGdi signals through respective isolation amplifiers of a unit 32, to form signal segments of fixed duration.", "Unit 32 supplies electric power to the various electronic components of the differential and isolation amplifiers while ensuring adequate isolation of the patient's body from such power supply.", "The unit 32 also incorporates bandpass filters included in the respective EMGdi signal channels to reduce the effects of aliasing.", "The successive EMGdi signal segments are then digitally processed into the personal computer 20 after analog-to-digital conversion thereof.", "An analog-to-digital converter implemented in the personal computer 20 conveniently carries out this analog-to-digital conversion.", "It is believed to be within the capacity of those of ordinary skill in the art to construct suitable differential amplifiers 30 and adequate isolation amplifiers and power supply unit 32.Accordingly, the amplifiers 30 and the unit 32 will not be further described in the present specification.", "As shown in FIG.", "1, the catheter 16 is introduced into the patient's oesophagus through one nostril or the mouth until the array of electrodes 22 is situated at the level of the gastroesophageal junction.", "Since the diaphragm 24 and/or the oesophagus slightly moves during breathing of the patient 26, the array of electrodes 22 also slightly moves about the diaphragm 24.As will be explained in the following description, automatic compensation for this displacement is advantageously provided for.", "An example of the seven EMGdi signal components (hereinafter EMGdi signals) collected by the pairs 1-7 of successive electrodes 22 (FIGS.", "1 and 2) and supplied to the computer 20 is illustrated in FIG.", "4.The sealing balloon 18 (FIG.", "1) is mounted on a ventilatory assist tube 34 thereabout.", "The tube 34 is an endotracheal tube that is to be inserted in the trachea 36 of the patient 26 via the mouth or the nose or tracheotomy.", "The tube 34 is part of the ventilator air circuit of a conventional ventilatory assistance system and is therefore connected to ventilator assist and sealing balloon controllers (both not shown).", "As shown in FIG.", "5, the ventilator assist tube 34 comprises two lumens: a ventilator assist lumen 38 and a seal pressure control lumen 40.The ventilatory assist lumen 48 is an air passage from the ventilator assist device (not shown) and the patient's lungs.", "The seal pressure control lumen 40 is an air or fluid passage from a balloon inflation device (not shown) to the sealing balloon 18 or mask 46 (see FIG.", "7).", "The balloon inflation device can be any device providing a known volume or a known pressure.", "The sealing balloon controller is connected to the computer 20 and its operation is controlled thereby.", "In operation, the ventilatory endotracheal ventilatory assist tube 34, with the sealing balloon 18 integrally mounted thereto, are inserted in the trachea 36 of the patient 26 via the mouth or the nose or tracheotomy.", "The oesophageal catheter 16 with the myoelectrical sensor 12 are introduced into the patient's oesophagus through one nostril or mouth until the array of electrodes 22 is located at the level of the gastroesophageal junction.", "As will be explained in further detail hereinbelow, upon inspiration of the patient 26, the change in EMG activity of the diaphragm 24 is detected by the sensor 12 and the detected signal is analysed by the computer 20 that commands the balloon controller to inflate the sealing balloon 18, thereby providing an air seal between the ventilatory assist tube 34 and the patient's respiratory airways (the trachea 36 in this exemplary embodiment).", "Upon expiration of the patient 26, the sensor 12 detects the change in EMG activity of the diaphragm 24 and the computer 20 commands the balloon controller to deflate the balloon 18, thereby allowing gas leaks around the ventilatory assist tube 34.FIGS.", "6 and 7 show two alternative embodiments of respiratory sealing devices.", "In the embodiment of FIG.", "6, the sealing balloon 18′ is so mounted to the ventilatory assist tube 34 as to be located, in operation, in the nasal passage 42 of the patient 26.In operation, upon inspiration of the patient 26, the sealing balloon 18′ inflates, thereby providing an air seal between the ventilatory assist tube 34 and the patient's respiratory airways (the nasal passage 42 in this exemplary embodiment).", "Upon expiration of the patient 26, the sealing balloon 18′ will deflate, thereby allowing gas leaks around the ventilatory assist tube 34, and also giving the patient 26 the ability to speak.", "FIG.", "7 shows the human patient 26 with a ventilatory assist facemask 44 over its mouth and nose.", "The facemask 44 is connected to the ventilatory assist tube 34.The ventilator assist tube 34 is in turn connected to ventilator assist and sealing balloon controllers (both not shown).", "In this particular embodiment, a seal 46 is provided at the edge portion of facemask 44.The seal 46 is fluidly connected to a ventilator assist tube seal pressure control lumen 40 (FIG.", "5) through the facemask seal pressure control lumen 48.Upon inspiration of the patient 26, the seal 46 inflates, thereby providing an air seal between the facemask 44 and the patient's respiratory airways (the patient's mouth and nose in this exemplary embodiment).", "Upon expiration of the patient 26, the seal 46 deflates, thereby allowing gas leaks around the facemask 62.Other features of the system 10 will become more apparent upon reading the following description of a myoelectrically activated respiratory leak sealing method 100, according to an embodiment of the present invention.", "As will now be described in more detail, the method 100 allows controlling the air seal 18.Generally stated, the method 100 comprises the following steps: 102—sensing the myoelectrical activity of the diaphragm 24; 104—comparing the myoelectrical signal to a predetermined value; and 106—modifying the state of the sealing device 18 according to the comparison result in step 104.Each of these steps will now be described in further detail.", "In step 102, the myoelectrical activity of the diaphragm is measured using sensor 12.The objective is to provide a myoelectrical signal representative of the respiratory effort of the patient 26.More specifically, a crural diaphragm EMG is recorded from a sheet of muscle whose fibre direction is generally perpendicular to an oesophageal bipolar electrode.", "The region from which the action potentials are elicited, the electrically active region of the diaphragm (DDR), and the centre of this region, the DDR centre, may vary during voluntary contractions, in terms of their position with respect to an oesophageal electrode.", "Depending on the position of the bipolar electrode with respect to the DDR centre, the EMGdi signal is filtered to different degrees.", "Based on experimental results and anatomical descriptions of the crural diaphragm, a transfer function for diaphragm EMG measured Perpendicular filtering ≈ ( K 0 ⁡ ( ω ⁡ ( h - d ) / v ) - K 0 ⁡ ( ω ⁡ ( h + d ) / v ) ) 2 K 0 2 ⁡ ( ω ⁢ ⁢ a / v ) with bipolar electrodes, such as electrodes 22, has been developed where, K( )=modified Bessel function, ω=angular frequency (i.e.", "2πf (f being the frequency), h=distance between the signal source and observation point, d=½ inter-electrode distance, v=conduction velocity, a=muscle fiber diameter.", "Based on this transfer function, a signal analysis procedure has been developed which involves: (a) locating the electrode pair at the centre of the diaphragm depolarizing region (DDR) (this region will be defined hereinbelow); (b) selecting the signals above and below the centre of the DDR (reversed in polarity) yielding the highest signal-to-noise ratio; and (c) subtracting these two signals (double subtraction technique).", "The double subtraction technique allows to reduce the influence of movement of the DDR centre relative to the electrode array 12 on the EMG power spectrum centre frequency and root mean square values, to increase the signal to noise ratio by 2 dB, and to increase the number of EMG samples that are accepted by the signal quality indices by 50%.", "A more detailed description of the above mentioned double subtraction technique is given hereinbelow.", "Step 102 will now be described in further detail with reference to FIG.", "9.The first operation (substep 202) performed by the computer 20 is a filtering operation to remove from all the EMGdi signals of FIG.", "4 electrode motion artefacts, ECG, 50 and 60 Hz interference from the electrical network, and high frequency noise.", "The graph of FIG.", "10a shows the power density spectrum of the above defined electrode motion artefacts, the power density spectrum of ECG, and the power density spectrum of EMGdi signals.", "It is to be noted that motion artefacts are induced by motion of the electrodes 22.More generally, motion artefacts are defined as a low frequency fluctuation of the EMGdi signals' DC level induced by mechanical alterations of the electrode metal to electrolyte interface i.e.", "changes in electrode contact area and/or changes in pressure that the tissue exerts on the electrode.", "The influence of ECG on the EMGdi signals can be suppressed or eliminated in different ways.", "Depending on the working mode, i.e.", "on-line or off-line analysis, time domain or frequency domain processing, different optimal signal conditioning methods can be chosen.", "In time-critical applications, an optimized filtering has been found advantageous.", "FIG.", "10b presents an optimal filter transfer function to isolate the EMGdi from a compound signal including ECG and also disturbed by background noise and electrode motion artefacts.", "In FIG.", "10b, the dashed line shows the optimal transfer function, while the solid line shows the transfer function implemented by the inventors.", "FIG.", "10b is therefore an example of filter transfer function that can be used in substep 202 for filtering out the electrode motion artefacts, ECG, the 50 or 60 Hz disturbance from the electrical mains, and the high frequency noise.", "Processing of the EMGdi signals by the computer 20 to follow, as closely as possible, the optimal transfer function of FIG.", "10b will provide adequate filtering in substep 202.An example of integrated EMGdi signal from a chronic obstructive pulmonary diseased (COPD) patient in relation to oesophageal and gastric pressure is depicted in FIGS.", "10a and 10b.", "Substep 204 involves the determination of the position of the centre of the DDR.", "As the diaphragm is generally perpendicular to the longitudinal axis of the oesophageal catheter 16 equipped with an array of electrodes 22, only a portion of the electrodes 22 are situated in the vicinity of the diaphragm 24.Determining the position of the diaphragm 24 with respect to the oesophageal electrode array 12 therefore provides for better results.", "The portion of the crural diaphragm 24, which forms the muscular tunnel through which the oesophageal catheter 16 is passed, is referred to the “diaphragm-depolarizing region” (DDR).", "The thickness of the DDR is about 20-30 mm.", "It is assumed that, within the DDR, the distribution of active muscle fibres has a centre from which the majority of the EMGdi signals originate, i.e.", "the “diaphragm-depolarizing region centre” (DDR centre).", "Therefore, EMGdi signals detected on opposite sides of the DDR centre will be reversed in polarity with no phase shift; i.e.", "EMGdi signals obtained along the electrode array 12 are reversing in polarity at the DDR centre.", "Moving centrally from the boundaries of the DDR, EMGdi power spectrums progressively attenuate and enhance in frequency.", "Reversal of signal polarity on either side of the electrode pair 4 with the most attenuated power spectrum confirms the position from which the EMGdi signals originate, the DDR centre.", "In step 204 of FIG.", "9a, the position of the centre of the DDR along the array of electrodes 22 is determined.", "The centre of the DDR is repeatedly updated, that is re-determined at predetermined time intervals.", "For that purpose, the EMGdi signals are cross-correlated in pairs in substep 204a to calculate cross-correlation coefficients r. As well known to those skilled in the art, cross-correlation is a statistical determination of the phase relationship between two signals and essentially calculates the similarity between two signals in terms of a correlation coefficient r. A negative correlation coefficient r indicates that the cross-correlated signals are of opposite polarities.", "FIG.", "12 shows curves of the value of the correlation coefficient r versus the midpoint between the pairs of electrodes 22 from which the correlated EMGdi signals originate.", "In this example, the inter-electrode distance d is 10 mm.", "Curves are drawn for distances between the correlated pairs of electrodes 22 of 5 mm (curve 52), 10 mm (curve 54), 15 mm (curve 56) and 20 mm (curve 58).", "One can appreciate from FIG.", "12, that negative correlation coefficients r are obtained when EMGdi signals from respective electrode pairs situated on opposite sides of the electrode pair 4 are cross-correlated.", "It therefore appears that the change in polarity occurs in the region of electrode pair 4, which is confirmed by the curves of FIG.", "4.Accordingly, it can be assumed that the centre of the DDR is situated substantially midway between the electrodes 22 forming pair 4.In substep 204b, the correlation coefficients are systematically compared to determine the centre of the DDR.", "For example, the centre of the DDR can be precisely determined by interpolation using a square law based fit of the three most negative correlation coefficients of curve 54 from FIG.", "12 obtained by successive cross-correlation of the EMGdi signal segments from each electrode pair to the EMGdi signal segments from the second next electrode pair.", "Association of the centre of the DDR to a pair of electrodes 22 provides a “reference position” from which to obtain EMGdi signal segments within the DDR.", "As mentioned in the foregoing description, the position of the DDR centre along the array of electrodes 22 is continuously updated, i.e.", "re-calculated at predetermined time intervals overlapping or not.", "In substep 204c, update of the position of the DDR centre is controlled by comparing the most negative correlation coefficient rNEG to a constant K3 (substep 204d).", "If rNEG<K3, it is considered that the EMGdi signal represents the diaphragm 24 and the position of the centre of the DDR is updated (substep 204e); if rNEG>K3, it is considered that the EMGdi signal does not represent the diaphragm 21 and the position of the centre of the DDR is not updated (substep 204f).", "The control carried out in substep 204c allows overcoming the artefactual influence on the EMGdi power spectrum or signal strength measurement.", "It has been experimentally demonstrated that EMGdi signals recorded in the oesophagus of adults are satisfactory as long as they are obtained from electrode pairs (with an inter-electrode distance situated between 5 and 20 mm) positioned at a distance situated between 5 and 30 mm on the opposite sides of the DDR centre (the inter-pair distance being therefore situated between 5 and 30 mm).", "With infants, this may change.", "Although EMGdi signals obtained from these positions offer a clear improvement in acceptance rates, the signal-to-noise ratio during quiet breathing still tends to remain unsatisfactorily low.", "For example, in FIG.", "4, the EMGdi signals originating from the electrode pairs 3 and 5, situated respectively 10 mm below and 10 mm above the DDR, are strongly inversely correlated at zero time delay.", "In contrast to the inversely correlated EMGdi signals, the noise components for electrode pairs 3 and 5 are likely to be positively correlated.", "Hence, as illustrated in FIG.", "13, subtraction of the EMGdi signals 60 and 62 from electrode pairs 3 and 5 will result in an addition of the corresponding EMGdi signals (see signal 64) and in a subtraction, that is, an elimination of the common noise components.", "This technique is referred to as “the double subtraction technique”.", "This second subtraction step of the double subtraction technique can be carried out either in the time domain, or after conversion of signals 60 and 62 into the frequency domain.", "A double subtraction technique can be performed by subtracting other combinations of signals, or by altering the polarities of electrode pairs.", "Two signals of opposite polarities obtained in the vicinity of the muscle on opposite sides of the DDR are subtracted, or if polarity is altered, on opposite sides of the DDR, to add signals from opposite sides of the DDR.", "Therefore, double-subtracted signal segments 206 are obtained at the output of step 206a by subtracting the EMGdi signal segments from the pair of electrodes 22 in optimal location above the diaphragm 24 from the EMGdi signal segments from the pair of electrodes 22 in optimal location below the diaphragm 24.The double subtraction technique compensates for the changes in signal strength and frequency caused by movement of the diaphragm 24 (FIG.", "1) and/or the oesophagus during breathing of the patient 26 causing movement of the array of electrodes 22 with respect to the diaphragm 24.Referring to FIG.", "14, off centre of the array of electrodes 22 (electrode-position-induced filter effect) causes a variation of centre frequency values (see curves 66 and 68) for the EMGdi signals from the electrode pairs 3 and 5.The double subtraction technique eliminates such variation of centre frequency values as indicated by curve 70 as well as variation of signal strength.", "Therefore, the reciprocal influence of the position of the DDR centre on the EMGdi signal frequency content is eliminated by the double subtraction technique.", "It has been found that the double subtraction technique may improve the signal-to-noise ratio by more than 2 dB and reduce an electrode-position-induced filter effect.", "Double subtraction technique also allows for a relative increase in acceptance rates by more than 50%.", "Cross-talk signals from adjacent muscles are strongly correlated at zero time delay and equal in polarity between all pairs of electrodes 22.Hence, these cross-talk signals appear as a common mode signal for all electrode pairs and therefore, are eliminated by the double subtraction technique.", "In substep 206, the strength of the EMGdi signal is calculated.", "In substep 206a, a pair of EMGdi signals (signals 1-7 of FIG.", "4) obtained from electrode pairs above and below the DDR centre are subtracted from each other and the RMS (Root-Mean-Square) value of the resulting signal is calculated and referred to as RMSsub (substep 206c).", "Measures of signal intensity, other than the RMS value, can also alternatively be used.", "In a substep 206b, the above mentioned pair of EMGdi signals (see signals 1-7 of FIG.", "4), obtained from electrode pairs above and below the DDR centre, are added to each other and the RMS (Root-Mean-Square) value of the resulting addition signal is calculated and referred to as RMSadd (substep 206d).", "Measures of signal intensity other than the RMS value can also potentially be used.", "In substep 208, a sufficient increment of the RMS signal amplitude RMSsub is detected.", "More specifically, in substep 208a, the RMS amplitude RMSsubn of the last EMGdi subtraction signal segment, as calculated by substep 206c, is compared with the RMSsubn−1 of EMGdi subtraction signal segment last accepted in substep 210c.", "If (RMSsubn×K1)<RMSsubn−1, no increment is detected and the system will wait until analysis of the next EMGdi subtraction signal segment is performed.", "On the contrary, if (RMSsubn×K1)>RMSsubn−1, an increment of the RMS intensity of the EMGdi signal is detected and detection of the common mode influence (substep 210) is activated.", "Of course, the multiplication operation (×K1) can be replaced by other suitable mathematical operations conducted on either the term RMSsubn or RMSsubn−1.Substep 210 enables detection of signal artefacts of non-diaphragmatic origin.", "As indicated in the foregoing description, EMGdi signals generated by the diaphragm and recorded on either side of the diaphragm 24 will have reversed polarity and no time delay.", "Accordingly, a subtraction signal, representative of the difference between these two EMGdi signals, will have a larger amplitude than an addition signal representing the sum of such EMGdi signals.", "In contrast, signals generated away from, and on the same side of the diaphragm 24, will have the same polarity on all electrode pairs and no time delay.", "As well, signals from the heart that are not obtained with electrode pairs located too far apart will have a similar shape but will have a time delay.", "Differing from signals with reversed polarity, subtracted signals with the same polarity will have smaller amplitudes than added signals.", "Hence the ratio or difference between the sum and difference between signals obtained from the same electrode pairs on either side of the diaphragm can indicate if a signal is of a diaphragm or an artefactual origin.", "For that purpose, in substep 210b, the amplitude RMSsubn is compared with the amplitude RMSaddn multiplied by a constant K2.It is to be noted that the indicia “n” is representative of the last EMGdi subtraction or addition signal segment.", "If RMSsubn<(RMSaddn×K2), the RMS signal amplitude is rejected (substep 210a ) and the two EMGdi signals are considered to have an artefactual origin.", "If RMSsubn>(RMSaddn×K2), the RMS signal amplitude is accepted (substep 210c) and the two EMGdi signals are considered to have a diaphragm origin.", "Of course, the multiplication operation (×K2) can be replaced by other suitable mathematical operations conducted on either the term RMSsubn or RMSaddn.", "In EMGdi signal replacement substep 216, a substep 216a determines whether the last RMS signal amplitude is accepted.", "If the last RMS signal amplitude is accepted, RMSsubn is kept (substep 216a).", "If the last RMS signal amplitude is not accepted, RMSsubn is replaced by RMSsubn−1 or with another prediction (substep 216c).", "An increase in amplitude of RMSsubn does not necessarily mean that the diaphragm 24 is the signal source.", "It is therefore advantageous to discriminate signals originating from the diaphragm 24 from signals of other origins.", "In the foregoing description, it has been described that a technique of sequential cross-correlation of the EMGdi signals from pairs of electrodes 22 can be used to determine the location of the diaphragm by the most negative correlation coefficient rNEG.", "Other simplified calculations of correlation can be used.", "The magnitude of the correlation coefficient rNEG is characteristic of each subject but is typically negative when the diaphragm is active.", "If the diaphragm is not active, the negative correlation coefficient rNEG is very low or the correlation coefficient is positive.", "The onset of diaphragm activation can therefore be detected through the amplitude of the correlation coefficient rNEG.", "To determine the mean level of noise RMSsubNOISE (step 218), a mean amplitude of RMSsubn is calculated.", "For that purpose, when rNEG>K4, K4 being a constant, this indicates that the diaphragm is not active (substep 218a) and the mean level of RMSsubn, i.e.", "RMSsubNOISE is calculated (substep 218b) and outputted.", "If rNEG<K4, the system 10 remains in an idle state (step 218c).", "An alternative to substep 218 is to detect the onset of inspiration through detection of airway inspiratory flow.", "Even though step 102 has been described by referring to the measurement of the myoelectrical activity of the diaphragm 24 using the system 10, the measurement of other respiratory-related EMG can be obtained with a suitable device placed in the vicinity of the respiratory-related muscle, inserted or implanted on the surface of or into the muscle of interest.", "Furthermore, other increases in EMGdi signal amplitude, its integrals or derivatives or combinations thereof, detected via an EMG recording of the diaphragm or other muscles associated with inspiration above a desired threshold level, and exceeding a desired duration, can be used to indicate the onset of an inspiratory effort.", "The magnitude of the signal itself may also be used.", "The signal can be applied, for example, in proportion to the signal times a constant and its maximum value up to a certain pressure or volume level.", "After a myoelectrical signal representative of the inspiratory effort of the patient 26 has been obtained, this signal is compared, in step 104, to a predetermined threshold so as to determine the highest value therebetween, and to send a control command to the respiratory sealing device 18 so as to modify the state of the sealing device according to a comparison result (step 106).", "Determination of the level to be exceeded (threshold) in terms of amplitude and duration can either be performed by manual adjustment supervised via visual feedback, or by automatically letting the level be relative to the above described mean noise level.", "An algorithm can further be used to trigger the respiratory sealing device 18 when the amplitude of an EMG signal segment of defined duration exceeds the threshold.", "The duration of time that the EMG amplitude remains above the threshold level can be used to decide the duration of the breath e.g.", "the ventilatory support system can start and deliver a full breath independent of the presence of EMG activity that exceeds the threshold level.", "The algorithm can also be adjusted to discontinue the ventilatory support if the EMG amplitude drops below the threshold level, or in response to a decrease in amplitude that exceeds a given magnitude (decrement).", "In step 104, the RMS amplitude RMSsubn may be compared to a predetermined parameter P5.If RMSsubn>P5, the RMS amplitude is higher than the threshold P5 and the sealing device 18 is activated so as to seal the air leak to avoid gas leaks during the respiratory effort of the patient 26.If, on the other hand, RMSsubn<P5 the RMS amplitude is below the threshold P5, and the sealing device 18 is activated so as to unseal the air leak to allow gas leaks during the relaxation of the patient's respiratory effort.", "P5 is a parameter equal to RMSsubNOISE×K7, K7 being a predetermined constant.", "It is to be noted that the parameter P5 would normally be different for triggering on and triggering off the sealing device 18 since the noise level is different in both cases.", "Again, the multiplication operation (×K7) can be replaced by other suitable mathematical operations conducted on term RMSsubNOISE.", "Alternatively or additionally to the comparison between the myoelectrical signal corrected amplitude RMSsub to a predetermined threshold, a RMSsub amplitude increment and decrement detection can be performed.", "The predetermined value to which the amplitude is compared is, in this particular case, a prior measured and corrected signal amplitude.", "The prior value RMSsubn−1 is compared to (RMSsubn×K6).", "If (RMSsubn×K6)<RMSsubn−1, the sealing device 18 remains in an idle state.", "If (RMSsubn×K6)>RMSsubn−1, this indicates an increment of the RMS amplitude, and sealing of the air leak by the sealing device 18 is requested through an increment counting/integrating to support the patient 26.The multiplication operation (×K6) can be replaced by other suitable mathematical operations conducted on either the term RMSsubn or RMSsubn−1.The function of the increment counting/integrating substep is to determine the time/magnitude response.", "The increment signal is averaged to adjust to sensitivity.", "The prior value RMSsubn−1 is also compared to (RMSsubn×(1/K6)).", "If (RMSsubn×(1/K6))>RMSsubn−1, the sealing device 18 remains in an idle state.", "If (RMSsubn×(1/K6))<RMSsubn−1, this indicates a decrement of the RMS amplitude and unsealing of the air leak is performed via the sealing device 18 through a decrement counting/integrating step.", "Of course, the multiplication operation (×(1/K6)) can be replaced by other suitable mathematical operations conducted on either the term RMSsubn or RMSsubn−1.The function of the decrement counting/integrating step is to determine the time/magnitude response.", "The decrement signal is averaged to adjust to sensitivity.", "In response to EMG signals, airway inspiratory flow and/or pressure control commands are sent by the computer 20 for triggering a ventilatory support system (ventilator) through an interface (not shown).", "Indeed, the system 10 advantageously comprises a digital-to-analog converter and/or other means for analog and digital interface.", "The decision for triggering will be made by a logic circuit on a “first come, first serve” basis.", "For example, if the diaphragm EMG (or EMG of other inspiratory related muscle) indicates an inspiratory effort before airway inspiratory flow and/or pressure indicates the onset of inspiration, the ventilatory support will be engaged.", "In the same fashion, the ventilatory support will be initiated if the inspiratory effort is detected by a threshold for airway inspiratory flow and/or pressure being exceeded before the EMG threshold is exceeded.", "Other changes in airway inspiratory flow and/or pressure, its integrals or derivatives or combinations thereof, in the inspiratory direction beyond a desired threshold level and detected via the inspiratory and/or expiratory lines can be used to indicate the onset of an inspiration.", "The graphs in FIGS.", "10a and 10b show, in the case of the quiet breathing of a COPD patient, that an EMG RMS signal will be detected approximately 200 ms prior to the onset of airway inspiratory flow.", "The graphs in FIGS.", "11a and 11b show, still in the case of the quiet breathing of a COPD patient, a similar relation between EMG RMS signal and the gastric and oesophageal pressure.", "In this particular example, sealing/unsealing in response to an EMG will enable the airleak regulating device to assist the patient directly at the onset of inspiration occurring 200 ms after detection of an EMG RMS amplitude signal.", "The method and device according to the invention is applicable to all patients (adults and infants) on ventilatory support and can enhance the possibilities of obtaining spontaneous breathing and optimizing patient ventilator interaction.", "The method and device applies to many kinds of ventilatory support systems used in intensive care unit settings and other wards where assisted ventilation is applied, and to other respiratory sealing devices (also referred to as an air leak regulating device).", "It is to be noted that substeps 204 and 210 of FIG.", "9 are part of the double subtraction technique and are therefore not necessarily executed with other signal analysis techniques.", "Moreover, substep 210 is optional even when using the double subtraction technique.", "Alternatively, the operation of a system according to the present invention can be based on the amplitude of the signals or the area under the curve (integration) of these signals, or other measures of signal strength.", "Although the preferred embodiment of the present invention will be described in relation to the use of an EMGdi signal obtained by means of a double subtracted signal, and representative of the myoelectrical activity of the diaphragm, it should be kept in mind that it is within the scope of the present invention to use another type of EMGdi signal, or to use a signal representative of the myoelectrical activity of muscles other than the diaphragm, yet associated with inspiratory effort to trigger the ventilatory support apparatus.", "Examples of other muscles are parasternal intercostal muscles, sternocleidomatoids, scalenes, alae nasi, etc.", "The myoelectrical activity of these muscles can eventually be detected by means of electrodes directly implanted in the muscle.", "Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified without departing from the spirit and nature of the subject invention, as defined in the appended claims." ] ]	Patent_10466699
[ [ "Photodynamic stimulation device and methods", "A treatment device which uses a light radiation of multiple wavelengths and pulse-shaped electromagnetic fields for the photodynamic stimulation of cells, especially cells of human tissue, and also for the activation and stimulation of light sensitive substances (PTD).", "The device produces energy radiation by the use of semiconductor and/or laser diodes, which emit light in several separate wavelengths due to a special operation mode and the use of tuneable diodes.", "The equipment consists of a stand, with which machine applicators are connected via a jointed arm.", "The stand is freely moveable on wheels and includes a control mechanism whereby the various parameters for therapy can be adjusted and switched on and off.", "The stand is also connected to a hand applicator for treatment of small tissue-areas, e.g., acupuncture points.", "Photodynamic substances are introduced into the tissue with a special hand applicator." ], [ "1.A device for the (PDT) photodynamic therapy and electromagnetic field stimulation of light sensitive substances and human cells, comprising: light sources with suitable strength, suitable wavelength, a light conductor, an applicator, an optic lens, a polarization filter, an electromagnetic field transmitter coil, and a control and power unit, characterized by the following: the light source consists of at least one semiconductor diode and/or at least one laser diode, the wavelength of the light source is adjustable to correspond with the light sensitive substance and the indication type, the supply of the light sources are adjustable and selectable in modes like a continuous and a pulsed mode, wherein the frequency, pulse-length and amplitude of the current pulses are selected to correspond with the type of indication and/or light sensitive substance, the frequency, pulse-shape and amplitude of the electromagnetic field pulses are adjustable to correspond with the type of indication and the light sensitive substance, wherein the light sensitive substances is are introduced to the tissue to be treated by help of a hand applicator of the inventive device using a method of air-pressure and/or iontophoresis and/or photophoresis.", "2.A device according to claim 1, wherein the light source contains at least one adjustable laser diode.", "3.A device according to claim 1, wherein the light source contains at least one semiconductor diode, which produces light of different wavelengths.", "4.A device according to claim 1, wherein the light-source contains several semiconductor and/or laser diodes, which emit light of different wavelengths.", "5.A device according to claim 1, wherein the light sources are individually selected—switched ON or OFF.", "6.A device according to claim 1, wherein the applicator contains a feedback photosensor for the light reflected from the skin surface.", "7.A device according to claim 1, wherein the applicator contains a feedback sensor measuring the temperature changes of the treated tissue.", "8.A device according to claims 4, 5 or 6, wherein the light sources and sensors are located on a printed circuit board.", "9.A device according to claim 8, wherein the electromagnetic field transmitter coil is placed in the same printed circuit board on which the light sources are placed.", "10.A device according to claim 1, wherein the at least one applicator is mounted to the control unit by means of a movable jointed arm.", "11.A device according to claim 10, wherein the at least one applicator comprises several single applicators hinged together so as to be adjustable at angles with respect to one another.", "12.A device according to claim 11, wherein the radiation outlet is covered by a polarization filter.", "13.A device according to claim 11, wherein the at least one applicator contains sensors connected to the control mechanism for measurement of reflected light for feedback control and automatic adjustment.", "14.A device according to claim 11, wherein the printed circuit board, on which the light sources is located, is moved linearly and/or rotated by help of a scan mechanism.", "15.A device according to claim 11, wherein at least one of the applicators is mounted with a light source emitting a fluorescent light for photo diagnosis (PD).", "16.A device according to claim 15, wherein the applicator contains an optic system with magnifier for photo diagnostic (PD).", "17.A device according to claim 1, further comprising a hand-held applicator containing at least one second light source connected to said pulse generator and at least one light outlet.", "18.A device according to claim 17, wherein the hand-held applicator is equipped with a shaft and a head and a printed circuit board equipped with semiconductor diodes.", "19.A device according to claim 17, wherein the at least one light outlet is equipped with a mounted lens and a polarization filter.", "20.A device according to claim 1, further comprising a hand-held applicator containing four selective light sources and a conductor for a light fibre cable.", "21.A device according to claim 20 wherein: on the circular printed circuit board four different light sources are placed at 90° intervals; there is at least one light source emitting a fluorescent light for the photodiagnosis (PD); the head comprises a light conductor rotatable in four steps to selectively conduct light for photo diagnosis (PD) or one of the three selectable and adjustable light sources of suitable wavelengths for therapy to said at least one light outlet.", "22.A device according to claim 21, wherein the conductor mounted with an expander includes a fibre optic cable suitable for dental use.", "23.A device according to claim 21, wherein the conductor is mounted with an expander with a flexible fibre cable for internal medical treatment.", "24.A device according claim 1, wherein the hand-held applicator is formed as a rectangle with a handle at the upper part equipped with a start/stop switch.", "25.A device according claim 1, wherein the applicator has a circular housing containing at least one light source equipped with a lens.", "26.A device according claim 25, wherein the applicator housing is equipped with a self-adhesive pad for placing the applicator on the patient's skin when radiating acupuncture points.", "27.A device according claim 1, wherein the applicator is formed as a rectangular tube containing the printed circuit boards with light sources placed at all four inner walls for intensive radiation of the blood in the inner rectangular tube.", "28.A device according to claim 1, wherein the applicator for whole body treatment is made of a lower part like a bed with a hinged upper part.", "29.A device according to claim 28, wherein the printed circuit boards mounted with multiple light sources are placed in a housing formed like a normal round light tube in the length of 2.15 m. 30.A device according claim 28, wherein the light tubes containing the multiple light sources are favourably produced in the form of a flat oval tube in the length of 2.15 m. 31.A device according to claim 28, wherein the light tubes containing the multiple light sources, including the electromagnetic transmitter coils, can also be built as a separate unit also containing the control unit.", "32.A device according to claim 28, wherein both under and upper parts are equipped with a multiple number of light tubes of 2.15 m length containing a multiple number of said light sources of which every second could be mounted with normal UV light tubes.", "33.A device according to claim 1 for introducing light sensitive substances into the tissues, comprising: a pressurized air-supply system connected by an air-supply tube to a hand applicator a chamber containing the light-sensitive substances, which is integrated in the hand applicator.", "34.A device according to claim 33, wherein the air pressure can be regulated and displayed on an instrument.", "35.A device according to claim 33, wherein the length of the air impulses can be regulated by means of an electronic or manual valve-system.", "36.A device according to claim 33, wherein the hand applicator contains a mechanical or electrical switch system to activate the treatment.", "37.A device according to claim 33, wherein the hand applicator contains a valve by the air inlet.", "38.A device according to claim 33, where the treatment head is exchangeable to suit the treatment area.", "39.A device according to claim 33, where the treatment head is equipped with a skin contact sensor system to protect from excessive treatment.", "40.A device according to claim 33, wherein the treatment head contains a valve-system which opens up automatically upon skin contact.", "41.A device according to claim 33, wherein a chamber containing the light sensitive substance is integrated in the side of the treatment head.", "42.A device according to claim 33, wherein the hand applicator contains a dosage pump for the light-sensitive substance.", "43.A device according to claim 33, where the housing of the hand applicator is made of insulating material and the treatment head is made of a conducting material.", "44.A device according to claim 33, where the treatment head is connected to the iontophoresis generator in the control mechanism and used as an iontophoresis electrode.", "45.A device according to claim 33, where the patient, during the iontophoresis treatment, holds an electric conductor handle in his hand.", "46.A device according to claim 33, where the iontophoresis amplitude and frequency can be regulated on the control mechanism.", "47.A device according to claim 33, wherein the hand applicator contains at least one second light source connected to said pulse generator and at least one light outlet 48.A device according claim 33, wherein the hand applicator contains a printed circuit board equipped with semiconductor diodes and a feedback sensor.", "49.A device according to claim 33, wherein the at least one light outlet is equipped with a mounted lens and/or polarization filter.", "50.A method of treating tissue, comprising the steps of: introducing a photosensitive substance to the tissue; determining when the tissue has absorbed a predetermined level of the photosensitive substance; and irradiating the tissue with a device according to claim 1.51.A method according to claim 50, wherein the photosensitive substance is one of photofrin, 5-aminolevulan acid, hematoporphyrin, verteporfin, chlorins, phthaldodyanines, phenothiazine, benzoporphyrin-derivative mono acid-A (A TMPn), L-Phenylalanin.", "52.A method according to claim 50, wherein the step of determining when the tissue has absorbed a predetermined level of the photosensitive substance consists in observing that the tissue undergoes a predetermined colour change when viewed under a predetermined illumination.", "53.A method according to claim 52, wherein the predetermined illumination consists of an optic system and a fluorescent light source.", "54.A device according to claim 28, wherein the printed circuit boards mounted with multiple light sources are placed in a housing formed like a normal round or oval light bulb." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>" ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a device with changeable applicators using a light and/or laser radiation of several wavelength ranges suited for the photodynamic stimulation of the cell energy in living cells, in particular human cells of both surface and underlying tissue.", "The light and/or laser radiation especially enhances vesicular respiration, most particularly stimulation of the ATP production in cells, thus increasing the therapeutic capabilities of the device.", "Furthermore, it is also possible to stimulate the activity of the cytochromes and the enzyme activity of the cells.", "The device consists of a stand, to which machine applicators are connected by means of a jointed arm.", "The stand, freely moveable on wheels, consists of a control mechanism, on which the desired therapy data can be adjusted and the device can be switched ON and OFF.", "The plain surface applicators can consist of several applicators placed side by side and flexibly connected with each other through hinges, whereby the applicators are suitable for the treatment of large-area tissues such as the human back.", "The applicators contain printed circuit boards mounted with semiconductor diodes and/or laser diodes (in large numbers), and the diodes are mounted with reflectors, which collect the radiation and bundle them in front of the applicator.", "The applicators also contain one or more transmitter coils for the emission of pulse-shaped electromagnetic radiation.", "The applicators are also equipped with an adjustable scan system, which permits an even and gap-free radiation of the surface with the multiple wavelengths of light.", "A diagnostic system (PD) containing a fluorescent light source and optics for photodiagnosis during the treatment is also included in the applicator.", "At least one of the applicator elements is equipped with feedback sensors for controlling the patient's response to the therapy, and via an automatic regulation system in the control mechanism it is possible to optimise the therapy results.", "The applicator contains a polarization filter, which is placed directly in front of the diodes.", "The control mechanism is also connected with a hand applicator, which is constructed for treatment of small tissue areas, e.g.", "acupuncture points and trigger points (pain points).", "The hand applicator includes a cylindrical shaft to which a headpiece is connected.", "A printed circuit board is fastened to the headpiece, mounted with semiconductor diodes or laser diodes.", "The light radiation is emitted from an axial opening in the front, equipped with a polarization filter and a lens for the focusing of the light rays.", "A second version hand applicator, which is especially invented for dental and/or invasive treatment, including (PD) diagnosis, contains at the front end of its shaft a printed circuit board, where 4 light and/or laser diodes of different wavelengths are placed at 90° intervals.", "One of these radiation sources can be selected as a fluorescent light for diagnostic purposes (PD) related to PDT therapy using light reactive biopharmaceuticals.", "The headpiece in front of the printed circuit board can be rotated in steps of 90° so that the expander, which is connectable with various types of optical fibres, can be positioned in front ofeither radiation source.", "The applicator may selectively emit blue light for the bonding and hardening of composite plastic fillings or infrared light for the treatment of dental pain, gingivitis, and wounds.", "In order to optimise bonding with the blue light, the output of the hand applicator is supplied at 25% of full power for the first ten seconds of the radiation time, and then is switched to full power.", "Acupuncture applicators made as small heads mounted on self-adhesive pads connected to the control mechanism, allow a certain number of applicators to be connected corresponding with the usual number of points utilised in classical acupuncture.", "The control mechanism can be programmed for a randomised acupuncture programme with changing frequency, modulation and amplitude instead of a programme with classical needling and Moxa treatment.", "Two applicator types are made for the stimulation of blood, either of venous blood or integrated in a heart/lung-machine.", "The first applicator allows radiation of blood passing the applicators' radiation sources in a 5 mm infusion lead, and the second version provides an intensive radiation of a quadrant tube, where the blood passes and receives radiation from 4 sides from light and/or laser diodes mounted on print-boards also containing transmitter coils radiating pulse-shaped electromagnetic emission.", "The applicators can also be designed as standard 2 meter and 15 cm long light tubes of the type commonly used in sun-beds for whole body therapy.", "Here it is advantageous to make the applicator in the form of a flat oval tube in order to achieve a better radiation surface.", "The tube applicators contain print-boards mounted with a suitable number of semiconductor light and/or laser-diodes as well as transmitter coils for the emission of pulse-shaped electromagnetic fields.", "The applicators are then mounted in a large body treatment arrangement like a sun-bed, where the patient lies on the lower part beneath a top part covering the whole body.", "Applicators of this type could be useful for treating office workers suffering from SAD disorders caused by too little exposure to natural light.", "The invention provides multiple wavelength stimulation that is also effective in conjunction with photodynamic therapy (PDT) chemicals.", "Such chemicals are applied or injected into or onto tissue to be treated, and subsequent photo-stimulation of them causes reactions in them that result in treatment of the tissue.", "Irradiation at multiple wavelengths enhances the effects of PDT chemicals while reducing discomfort to the patient.", "The present invention provides an apparatus including a semiconductor light source including a hand applicator.", "The hand applicator can selectively emit light of various wavelengths and introduce the light-sensitive substances into the tissue by means of air-pressure and electrical impulses (iontophoresis).", "The absorption time, depending on the type of light-reactive substances, may vary from 1 to 24 hours without this technique.", "Other advantages with the described technique are that the light-sensitive substances can be applied very precisely and the absorption dose can be improved and more accurately regulated.", "Other advantages of the invention will become evident from the following description of the invention and from the appended drawings, wherein:" ], [ "BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates in general to electrotherapy devices and more particularly to devices and methods for photodynamic and electromagnetic stimulation of living tissue, directly and also indirectly, by stimulation of photosensitive substances introduced into or onto living tissue.", "DESCRIPTION OF RELATED SCIENCE The mitochondria within the cells of protozoa and metazoa are sources of energy produced by cell respiration.", "They are moreover capable of synthesizing proteins, because they have a genetic system of DNA and RNA independent of the cell nucleus.", "The mitochondrias' main function, however, is vesicular respiration.", "This is the transformation within the cells of nutrients and oxygen (supplied, amongst other ways, via the bloodstream) into energy and endogenous substances, whereby through this transformation, waste products like water, carbon dioxide, alcohol and lactic acid are produced.", "Of great importance is adenosine-triphosphoric acid (ATP), which is synthesized by the mitochondria into adenosinediphosphoric acid (ADP) and orthophosphate.", "Complicated chemical compounds are of great importance as reaction catalysts.", "Stimulation of the vesicular respiration, especially a stimulation of the ATP production by cells, is used therapeutically to meet strong demands on cell energy during healing processes, and for weight-reduction, wound-healing and reduction of pain sensitivity due to illness or weakness caused by hypo- or depolarization of the cell membrane.", "In general, weakening of cells caused by an increase of vesicular respiration due to stress, illness or by old age can be counteracted.", "In order to achieve stimulation of the mitochondria through optical radiation, two conditions must be fulfilled.", "The radiation must be of appropriate wavelengths in order to be effective, and a pulse frequency must be chosen to penetrate to an appropriate tissue depth without causing tissue damage or pain.", "Moreover, pulsating electromagnetic fields have been shown to exert a positive influence on the bodies of both animals and humans.", "With the help of pulsating electromagnetic fields it is possible to send protons from electrolytic internal body fluids such as blood or lymph directly and in controlled measures into the surrounding vessel walls and membranes.", "This is normally not possible, since the lipids in the membranes of the blood vessel walls, which are in contact with the blood, carry a negative charge creating a surface potential which hinders the protons and ions from entering the vessel walls.", "The pulsating electromagnetic field enables the protons to enter the cell and vessel walls in spite of the barrier.", "When this occurs, the increased concentration of protons within the cell and vessel walls reverses the polarity of the barrier, thereby hindering the protons and ions from exiting through the cell and vessel walls again.", "In turn, this phenomenon causes a beneficial change in the local pH value, especially within the vessel walls.", "Additionally, prolonged exposure to pulsating electromagnetic fields has other effects, such as the electrical constriction of the membranes and vessel walls, the adjustment of polyvalent ion chains, the tangential displacement of absorbed counter ions, the force effect on dielectric bodies in homogeneous and non-homogenous fields, and electro-osmosis.", "A device is known (Patent DE-U-8-13852/Normed, E. Larsen).", "which uses infrared radiation for the photodynamic stimulation of energy in living cells, cells at the surface of the skin and especially cells lying deeper down.", "The device consists of a supply and control mechanism and an applicator on which infrared radiating (from 900 nm [1 nm=1 nanometer]) semiconductor diodes are mounted with reflectors for the bundling of the IR radiation from the applicator (IR=infrared).", "In this known device, a generator containing a control-mechanism supplies the semiconductor diodes with current pulses of a frequency within the range of 500-5000 Hz.", "A disadvantage of the known device is that the semiconductor diodes tend to overheat during use, which causes a decrease in the effectiveness of the device.", "The known device therefore does not deliver a constant effect during use.", "Another disadvantage is that only inared radiation within a range of 900 nm is available, while other wavelengths may be called for to achieve cell stimulation.", "Another device (Patent EPA 0568 666) is used for the photodynamic stimulation of cells.", "The semiconductor and/or laser diodes radiate light of different wavelengths.", "With the aid of light sensors the advanced control-system is able to test the patients for the required radiation dose in order to avoid over-stimulation.", "Furthermore, the radiation outlets in the applicators are covered with a polarization filter, which enhances absorption in the irradiated tissue.", "The basic equipment consists of a mobile stand, to which machine applicators are connected with a jointed arm.", "The machine applicators are adapted for the treatment of large tissue-areas, for example the back of humans.", "The device also includes a control-mechanism, whereby the various parameters for therapy can be adjusted and switched ON and OFF.", "The device is also connected to a hand applicator designed for the treatment of small tissue-areas, e.g.", "acupuncture points or dental treatment with the aid of a connectable fibre.", "Another device is (EPA Patent 0570 544), which uses electromagnetic fields for therapy on humans and animals.", "The pulse-shaped electromagnetic fields cause protons to migrate out of the electrolytic internal body fluids into the surrounding vessel walls and membranes.", "The device produces the electromagnetic pulse-bundles in a certain pulse-rhythm, in which each pulse-bundle is followed by a pause.", "The basic device consists of a generator for producing the electro-magnetic pulses, connected with a transmitter coil, whose windings are placed on the surface of the base plate.", "The base plates are manufactured from light, flexible insulating material and mounted in a flat applicator housing placed on a jointed arm connected to the basic device.", "In the fields of dermatology and rehabilitation, light is used as a stand-alone therapy for wounds, leg ulcers, eczema, burns, pain, rheumatic disorders etc., and as such is used to stimulate tissue directly.", "Techniques are known for introducing agents for altering the light absorbing qualities of tissue to enhance the effect of light (for example, U.S. Pat.", "No.", "5,226,907 to Tankovich teaches contamination ofhair follicles with a dark particulate material to enhance light-induced heating in the follicles for hair removal).", "Treatments have included the application of substances such as photoflim, 5-aminolevulan acid, hematoporphyrin, verteporfin, chlorins, phthalodyanines, phenothiazine, and benzoporphyrin-derivative monoacid-A (ATMPn) onto or into tissue for healing solar keratoses, basal cell carcinoma, melanomas, etc.", "Such substances are known as “biopharmaceuticals” and treatment with these substances has been called biopharmaceutical therapy.", "Therapy involving the application of biopharmaceuticals and their subsequent activation by light after they have been absorbed into tissue has been called photodynamic therapy (PDT).", "PDT has been used successfully in the treatment of internal inoperable cancers.", "A biopharmaceutical (specifically, hematoporphyrin) is injected into the tumor tissue, and an optical method known as photodynamic diagnosis (PD) is used to determine when the biopharmaceutical has been absorbed by the entire tumor.", "Then the tumor tissue is irradiated with light typical for a dye laser, which activates the photosensitive reactors in the hematoporphyrin, whereby singlet oxygen is liberated.", "Singlet oxygen is toxic to protein and phosphorlipids in the tumor tissue, whereby the tumor is destroyed without destroying the surrounding tissue.", "For treatment of skin keratosis (pre-cancerous tissue), trials with, for example, 5-aminolevulinic acid have shown that it can be used effectively in PDT if introduced into oil in a water suspension which is then applied to skin keratosis and then irradiated with a light source.", "A fast and cosmetically perfect healing has been attained with a very low rate of recurrence compared to conventional treatments, such as cryo-therapy.", "In view of these favourable test results, it is anticipated that pharmaceutical companies will be marketing the next generations of PDT chemicals in convenient fiorms, such as creams, suspensions, sprays, etc.", "The light source typically used to irradiate PDT chemicals is commonly known as the surgical laser, a solid-state laser which is bulky, and which is expensive both to purchase and to operate.", "Surgical lasers are designed primarily for cutting, i.e., they output very high energy in a very small spot, and are thus difficult to adapt to the requirements of irradiating a more generalized area for PDT.", "Further, they generally radiate at a single wavelength Radiation at several wavelengths is desirable in PDT, for several reasons: a single wavelength may cause the patient to experience burning pain in adjacent tissue during treatment; some photosensitive chemicals respond to two different wavelengths; and, some pigmented melanomas do not respond to visible radiation due to absorption in the pigment (typically melanin), and must be irradiated with near-infrared light.", "Common dermatological diseases like acne, warts, and onychomycosis (nail fungus) can successfully be treated with light as a stand-alone treatment, but recent work indicates that treatments using PDT (with ALA/5-aminolevulanic acid) give excellent results with only two or three treatments.", "In a recent pilot study using PDT to treat acne, the cosmetic results were excellent, and oil gland activity which causes acne, and the resultant inflammation, were reduced for as much as twenty weeks after a series of PDT treatments.", "(The PDT treatments precipitated immediate but short-term inflammatory reactions.)", "In general the photodynamic stimulation used in physiotherapy is producing very good results, but in the area of long-term chronic diseases such as gout, arthritis, etc.", "there is often a need for many treatments, as many as 12-20 treatments spaced over a period of time.", "Also, initial phases of such treatment often cause reactions, which in turn cause pain and discomfort.", "A recent trial study showed that using a light and/or laser radiation combined with an electromagnetic field emission resulted in better results, without reactions to the intensive therapy.", "It seems that the combined radiation has a better penetration due to the electromagnetic fields removing the blocking potential and the vasodilatation of the capillaries, whereby the increased ATP energy is better utilized.", "A recent trial in post-surgery light and/or laser therapy after coronary angioplasty and stenting, where the restenosis rate is normally quite high, showed promising results, and here again it is expected that the results can be improved using a light sensitive biopharmaceutical for regeneration and stabilisation of the vessel walls.", "Studies also support the theory that a light and/or laser radiation of blood can provide an effective therapy for chronic diseases such as leukemia and cancer, our tests on athletes also support the theory that this therapy improves the immune system and the vitality.", "A number of erothrocytes are often damaged in artificial heart-lung machines, but blood irradiated with light and/or laser showed less deformability and the ATP levels were significantly higher.", "Here too we expect an increased activity of the leukocytes and and lymfocytes by using light sensitive biopharmaceuticals.", "For many years large-surface therapy systems for dermatological diseases like psoriasis have been equipped with UV radiation sources, for example UV tubes.", "Prior to the treatment the patients have received various types of photo-chemical substances like 8-MOP (Oxsoralen), 5-MOP or Meladinine (bathing therapy).", "Due to the risk of skin cancer and other side effects the use of PUVA therapy has declined during recent years.", "When more studies have been completed it is expected that PDT will in future be the procedure of choice for treating most chronic dermatological diseases, due to its effectiveness and lack of side effects.", "Also due to the risk of skin cancer, tanning on sun-beds has declined much during recent years.", "Among other side effects is the erythema that follows the first treatments, and most patients, especially those with fair skin, find that their skin becomes very dry and irritated.", "Our tests have showed that by using a combination of UV light and photodynamic light produced by semiconductor diodes, we can avoid all the side effects of using sun-beds.", "It is also expected that the increased vitality (high ATP level) of the skin can counteract the risk of skin cancer.", "In classical acupuncture a technique called moxibustion is commonly used for the treatment of deep-lying acupuncture points, especially in chronic diseases.", "Needles with a special metal handle are used and, after the needles are inserted in the patient, a herbal substance is placed on the handle and combusted, whereby the needle is heated and leads the heat deep into the tissues.", "The effect is excellent, but western doctors do not like this praxis because of the strong smell, which may linger for several days.", "This method can now be replaced by the application of topical light-sensitive lotions over the acupuncture points, which are subsequently radiated with a suitable light and/or laser radiation.", "Looking at the current state of technology, devices are available for the photodynamic stimulation of human cell energy in the form of red and infrared radiation emitted by laser diodes and semiconductor diodes.", "These devices are not suitable for intensive, invasive and whole body treatments mainly due to the lack of applicators with suitable adjustable radiation sources for fill surface treatment with combined diagnostic abilities during treatment.", "The same can be said for existing devices for treatment with pulsating electromagnetic fields.", "Moreover, a combined treatment with both red/infrared and blue light together with electromagnetic fields is not possible with these devices for the stimulation of light sensitive substances.", "A device able to deliver an intensive light radiation with selective multiple wavelengths within the wavelength area of 300-2000 nm and electromagnetic fields is not at present available.", "Thus the invention is aimed at creating a device for intensive photodynamic therapy, which is capable of stimulating photodynamic energy of selective multiple light and/or laser radiation within a wavelength range of 300-2000 nanometers, capable of treatment with pulsating electromagnetic fields, and can also be used for stimulating light sensitive biopharmaceuticals.", "SUMMARY OF THE INVENTION The present invention provides a device with changeable applicators using a light and/or laser radiation of several wavelength ranges suited for the photodynamic stimulation of the cell energy in living cells, in particular human cells of both surface and underlying tissue.", "The light and/or laser radiation especially enhances vesicular respiration, most particularly stimulation of the ATP production in cells, thus increasing the therapeutic capabilities of the device.", "Furthermore, it is also possible to stimulate the activity of the cytochromes and the enzyme activity of the cells.", "The device consists of a stand, to which machine applicators are connected by means of a jointed arm.", "The stand, freely moveable on wheels, consists of a control mechanism, on which the desired therapy data can be adjusted and the device can be switched ON and OFF.", "The plain surface applicators can consist of several applicators placed side by side and flexibly connected with each other through hinges, whereby the applicators are suitable for the treatment of large-area tissues such as the human back.", "The applicators contain printed circuit boards mounted with semiconductor diodes and/or laser diodes (in large numbers), and the diodes are mounted with reflectors, which collect the radiation and bundle them in front of the applicator.", "The applicators also contain one or more transmitter coils for the emission of pulse-shaped electromagnetic radiation.", "The applicators are also equipped with an adjustable scan system, which permits an even and gap-free radiation of the surface with the multiple wavelengths of light.", "A diagnostic system (PD) containing a fluorescent light source and optics for photodiagnosis during the treatment is also included in the applicator.", "At least one of the applicator elements is equipped with feedback sensors for controlling the patient's response to the therapy, and via an automatic regulation system in the control mechanism it is possible to optimise the therapy results.", "The applicator contains a polarization filter, which is placed directly in front of the diodes.", "The control mechanism is also connected with a hand applicator, which is constructed for treatment of small tissue areas, e.g.", "acupuncture points and trigger points (pain points).", "The hand applicator includes a cylindrical shaft to which a headpiece is connected.", "A printed circuit board is fastened to the headpiece, mounted with semiconductor diodes or laser diodes.", "The light radiation is emitted from an axial opening in the front, equipped with a polarization filter and a lens for the focusing of the light rays.", "A second version hand applicator, which is especially invented for dental and/or invasive treatment, including (PD) diagnosis, contains at the front end of its shaft a printed circuit board, where 4 light and/or laser diodes of different wavelengths are placed at 90° intervals.", "One of these radiation sources can be selected as a fluorescent light for diagnostic purposes (PD) related to PDT therapy using light reactive biopharmaceuticals.", "The headpiece in front of the printed circuit board can be rotated in steps of 90° so that the expander, which is connectable with various types of optical fibres, can be positioned in front ofeither radiation source.", "The applicator may selectively emit blue light for the bonding and hardening of composite plastic fillings or infrared light for the treatment of dental pain, gingivitis, and wounds.", "In order to optimise bonding with the blue light, the output of the hand applicator is supplied at 25% of full power for the first ten seconds of the radiation time, and then is switched to full power.", "Acupuncture applicators made as small heads mounted on self-adhesive pads connected to the control mechanism, allow a certain number of applicators to be connected corresponding with the usual number of points utilised in classical acupuncture.", "The control mechanism can be programmed for a randomised acupuncture programme with changing frequency, modulation and amplitude instead of a programme with classical needling and Moxa treatment.", "Two applicator types are made for the stimulation of blood, either of venous blood or integrated in a heart/lung-machine.", "The first applicator allows radiation of blood passing the applicators' radiation sources in a 5 mm infusion lead, and the second version provides an intensive radiation of a quadrant tube, where the blood passes and receives radiation from 4 sides from light and/or laser diodes mounted on print-boards also containing transmitter coils radiating pulse-shaped electromagnetic emission.", "The applicators can also be designed as standard 2 meter and 15 cm long light tubes of the type commonly used in sun-beds for whole body therapy.", "Here it is advantageous to make the applicator in the form of a flat oval tube in order to achieve a better radiation surface.", "The tube applicators contain print-boards mounted with a suitable number of semiconductor light and/or laser-diodes as well as transmitter coils for the emission of pulse-shaped electromagnetic fields.", "The applicators are then mounted in a large body treatment arrangement like a sun-bed, where the patient lies on the lower part beneath a top part covering the whole body.", "Applicators of this type could be useful for treating office workers suffering from SAD disorders caused by too little exposure to natural light.", "The invention provides multiple wavelength stimulation that is also effective in conjunction with photodynamic therapy (PDT) chemicals.", "Such chemicals are applied or injected into or onto tissue to be treated, and subsequent photo-stimulation of them causes reactions in them that result in treatment of the tissue.", "Irradiation at multiple wavelengths enhances the effects of PDT chemicals while reducing discomfort to the patient.", "The present invention provides an apparatus including a semiconductor light source including a hand applicator.", "The hand applicator can selectively emit light of various wavelengths and introduce the light-sensitive substances into the tissue by means of air-pressure and electrical impulses (iontophoresis).", "The absorption time, depending on the type of light-reactive substances, may vary from 1 to 24 hours without this technique.", "Other advantages with the described technique are that the light-sensitive substances can be applied very precisely and the absorption dose can be improved and more accurately regulated.", "Other advantages of the invention will become evident from the following description of the invention and from the appended drawings, wherein: BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a perspective drawing of the invented device, FIGS.", "2a, 2b, 2c, 2d illustrate details of the machine applicator of the invented device; FIG.", "3 illustrates a jointed arm used for the movable connection of the machine applicators; FIG.", "4 is a circuit block diagram of a control unit, which supplies the applicators.", "FIG.", "5 depicts a hand applicator according to the present invention; FIG.", "6 depicts an applicator conforming to FIG.", "5 with axial light emission; FIG.", "7 depicts the light sources with a lens of FIG.", "6; FIG.", "8 an applicator with a rotary headpiece; FIG.", "9.shows details of a printed circuit board for the applicator of FIG.", "8; and FIG.", "10 depicts the flexible light fibre cable with adaptor, FIG.", "11 shows the air unit with the hand applicator belonging to this part of the invention.", "FIG.", "12 illustrates an exchangeable round head for the hand applicator.", "FIG.", "13a shows the hand applicator for light-sensitive substances viewed from below.", "FIG.", "13b illustrates the hand applicator in side view.", "FIG.", "14a shows the acupuncture applicator in top view, FIG.", "15b depicts the acupuncture applicator from the underside, showing the light sources.", "FIG.", "15a illustrates a rectangular hand-applicator in a top view; FIG.", "15b shows the hand applicator in side view with skin contact used for hair removal; FIG.", "15c illustrates the hand applicator viewed from below, showing the light sources; FIG.", "16a illustrates a tube applicator for the radiation of blood; FIG.", "16b shows the applicator-like quadratic tube viewed from the end; FIG.", "16c shows the quadratic tube used for blood radiation; FIG.", "17a shows the body applicator viewed from the end, in closed mode; FIG.", "17b shows the body applicator open, with the radiation surface of the lower and upper part; FIG.", "17c illustrates the closed body applicator in a side view.", "FIG.", "18a illustrates a round light tube as seen from above; FIG.", "18b illustrates the round light tube viewed from the end; FIG.", "18c shows the light tube formed as a flat oval and viewed from above; FIG.", "18d shows the flat oval light tube viewed from the end; DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT As shown in FIG.", "1, the invented device 10 for the stimulation of cells with the aid of (PDT) photodynamic light and electromagnetic fields combined with light sensitive substances consists of a stand 11, with which machine applicators 13 (in the following just called applicators 13) are connected through a jointed arm 12.The stand 11 is also connected by an electric circuit 14 with a hand applicator 15.The stand 11, freely movable on wheels, includes control mechanism 16 (described in FIG.", "4), whereby the function of the control mechanism 16 can be adjusted and switched ON/OFF at a control panel 30 (also called description equipment 30).", "The FIGS.", "2a, 2b and 2c show plain surfaced printed circuit boards with light sources mounted in the applicators 13.These can be used in the working model according to FIG.", "2a to 2c individually, side by side (in large numbers) or in combination with an applicator.", "Furthermore the printed circuit boards on which the light sources are mounted can favourably be produced as multilayer, also containing the electromagnetic field transmitter coils 65 shown in FIG.", "2d.", "According to FIG.", "2a, the applicators 13 in the working model are mounted in a shifting order with semiconductor diodes (LED) and/or laser diodes 17 and 17a Cm the following called light sources), whereby shifting the order of the light sources 17 means, that the respective light source 17a of one row is placed at the point of intersection of the two diagonals through the two respective light sources 17, which are placed adjoining on both sides.", "The light sources 17 and 17a are mounted with reflectors 18, which collect the radiation and bundle it in front of the applicator 13.The applicator contains a polarization filter, which is placed directly in front of the light sources 17 and 17a, whereby the radiation can be better absorbed by the irradiated tissue.", "According to FIGS.", "2b and 2c the light sources 17 are placed in regular row arrangements, i.e., 20 equidistant from each other, whereby, according to FIG.", "2c, one applicator 13, in addition to the diodes 17, has a light source 19.The light sources 17 are adjustable and can radiate light in at least three wavelengths within the wavelength area 300-2000 nm.", "The light source 19 formed as a tube can be selected for radiating blue light or fluorescent light within the wavelength area of 300-450 nm for photodiagnostics (PD).", "A diagnostic system (PD) containing optics 67 with a magnifier for photodiagnosis during the the treatment is also included in the applicator.", "The light sources 17a (FIG.", "2a) radiate light with a wavelength of 350-500 nm, i.e., blue light.", "For the treatment of large-area tissues according to FIG.", "1, several applicators 13 are flexibly connected to each other through hinges 10, respectively connecting one edge with the other, whereby the applicators are suitable for the treatment of for example, the backs of humans and so become adjustable for an equidistant positioning of the applicators 13 above the skin.", "In each applicator the printed circuit board carrying the light sources is connected to a small electrical scan engine 66, which can move the light sources with linear movements, whereby a correctly adjusted scan length and frequency gives the radiated surface a total radiation at the necessary wavelengths without leaving any unradiated gaps.", "The joint arm 12, shown in FIG.", "3, connects one or more applicator(s) 13 with the stand 11.The jointed arm 12 has three joint carriers 21, 22, 23, where the joint carrier 21, together with the stand 11 and the joint carrier 23 are moveable at a free end through a fixing joint 24 connected with one or more applicators 13.Another fixing joint 25 connects the joint carrier 23 with 21, while the joint carrier 22 is connected with the joint carrier 21 with a hinge 26.The joint carrier 21 is connected with the stand 11 through a joint 27 and one or more joints can also be produced as friction-adjustable ball-joints, which gives almost unlimited adjustment possibilities and user comfort.", "The jointed arm 12 thereby allows the positioning of the applicators 13 in front of, or above, a tissue area while maintaining a correct positioning distance.", "The jointed arm 12 also carries the electrical circuits 14 (not further described) from the control mechanism 16, which is integrated in stand 11, to the applicator(s) 13.According to FIG.", "4 the controller mechanism 16 consists of a generator 28, a timer 29, and a display 30.With help of the generator 28 the current impulses necessary to the production of light and the current pulses for the supply to the transmitter coils emitting the electromagnetic fields are contributed, while with the aid of timer 29, all time functions are adjustable, e.g.", "the duration of treatment.", "Display 30 shows pertinent treatment data, such as current pulse frequency, pulse length, pulse amplitude and pulse modulation.", "With the help of the control mechanism 16, the invented device is adjustable within a relatively large range with reference to duration, amplitude and frequency of surge of current, so that the semiconductor diodes 17, 17a and 18, as well as laser 17 diodes with the same realisation as semiconductor diode 17, can be used as light sources.", "For that purpose the control mechanism is equipped with a switch select system for operating different types of semiconductor diodes 17 and/or with laser diodes.", "Both semiconductor diodes and/or laser diodes with tuneable wavelengths can be operated, which is a great advantage, and therefore the printed circuit boards can be equipped with a more intensive radiation effect.", "The semiconductor diodes and laser diodes are tuneable in wavelength by means of various methods such as resonators, piezo elements or by the help of special current modes.", "The semiconductor diodes and/or laser diodes useable in this invention emit a light radiation with either SPE (single photon emission), TPE (Two photon emission) and/or MTE (multiple photon emission) within the wavelength area of 300-2000 nanometers in order to correspond with available light sensitive substances (PDT).", "The useable light sources in the form of semiconductor diodes and/or laser diodes are supplied with current pulse lengths of ms, ns and/or fs (femto-seconds) within a frequency range of 1 KHz-100 MHz.", "The transmitter coil (transducer) for transmitting the pulse-shaped electromagnetic fields is supplied with basic pulses having a frequency between 2 and 500 Hz; ON times of about four-tenths of a period; OFF times of about six-tenths of a period and non-instantaneous rise and fill times.", "Furthermore, the basic pulses can be superimposed with pulse-bundles at a frequency off about 10 KHz and, optionally, also with pulse-bundles of a frequency between 20-30 MHz.", "The applicators 13, according to the FIGS.", "2a, 2b and 2c are equipped with sensors 32 arranged between the semiconductor and/or laser diodes 17.For therapeutic uses it is typically intended to apply a given amount of energy (Joule/cm2) per irradiated surface of tissue, which can be adjusted at the control mechanism 16.Sensors 32 measure the amount of energy radiated away from the skin surface, which is indicative of the total energy penetrating into the tissue.", "Taking into account individual variations from patient to patient, the exposure can be determined according to the measurements taken by the sensors 32 so that the correct amount of therapeutic energy (Joule/cm2) reaches the tissue.", "An increase of the registered amount of energy can be achieved by the invented device by increasing the operating potential (and thereby the pulse amplitude) or the pulse frequency and/or prolonging the duration of the treatment time through an adjustment of the control mechanism 16.A sensor 32a is also contained in at least one of the machine applicators, measuring the temperature change of the radiated tissue, whereby the control unit can react with a feedback regulation of the radiation parameters depending on the therapy indication, location and (PDT) light sensitive substance used.", "While the applicators 13 according to FIGS.", "2a, 2b and 2c are constructed for the treatment of larger tissue areas, the hand applicators 15a, 15b according to FIGS.", "5 and 8 are constructed for the treatment of small tissue areas.", "The hand applicator 15a includes a cylindrical shaft 34, with a handle to which a headpiece 35 is connected.", "At the headpiece 35 a printed circuit board 36 is fastened with light sources 17 of tuneable wavelengths (not described).", "In headpiece 35, in front of the opening 39, are placed a lens 40 for the focusing of the light rays and a polarization filter 41.The device with this kind of light 38 emission is especially designed for the treatment of small tissue areas, e.g.", "acupuncture points and triggerpoints.", "FIG.", "8, in connection with FIG.", "9, describes a hand applicator 15b, which is especially intended for dental treatment and internal medical treatment.", "The applicator 15b shows at the front end of the shaft 42 a printed circuit board 43, where three different light sources 44 with tuneable wavelengths within the wavelength area of 300-2000 nm and a light source 45 for photodiagnostics (PD) are placed.", "In front of the printed circuit board 43 a headpiece 46 is placed, connected with an interchangeable fastened hollow expander 47, in which an optical fibre is sealed (not shown).", "The head piece 46 is in front of the printed circuit board 43 so it can be rotated 360° in steps of 90°, so that the expander 47 can be positioned in front of either one of the three light sources 44a, 44b, 44c, depending on the required wavelength for therapy, or in front of the light source 45 if fluorescent light for photodiagnostics (PD) is needed.", "If the expander 47 is positioned, for example, in front of the diode 44b, light within the infrared wavelength area is transmitted through the optical fibre in expander 47 and ultimately strikes the tissue, e.g.", "gum tissue, through which painful gingival diseases can be treated.", "Through a positioning of the expander 47 in front of the 44a, blue light with a wavelength of 470 nm is conducted through the expander 47, with which plastic fillings in teeth can be hardened.", "It is obvious that the light rays with this form of execution can also be conducted through polarization fibres.", "Furthermore, the two hand applicators are equipped with sensors 32 for the same purpose as described for the applicators 13.The hand applicator 15b can also be very useful in the case of internal medical diseases, where the flexible fibre cable can be used together with a video-cable, which is produced with an internal opening for instrumentation, laser fibre etc.", "In this situation the flexible light fibre cable is connected, whereby first the light source 45 is used for photodiagnosis and hereafter one of the light sources 44a, 44b, 44c is selected for the treatment.", "FIG.", "11 Shows a diagram of the air-pressure unit 48, which can either be built into the control mechanism of the device or produced as a separate device connectable with the invented photodynamic stimulation device.", "The air unit can either be produced as a rechargeable air-tank or as a small air-compressor with container.", "The container outlet is equipped with a reduction valve 51, combined with a pressure meter 52, of familiar sort.", "The electronic valve 51a in the air tube 49 leading to the hand applicator 50 can be switched on from the hand applicator and the ON impulses can be regulated at the control mechanism 53.The current impulses 63 and amplitude 64 for the iontophoresis treatment are also adjustable at the control mechanism.", "FIG.", "12 illustrates an interchangeable round head 56 for the hand applicator, which can be changed via a click in bracket (not illustrated).", "The treatment head 56 is interchangeable according to the purpose of radiation, so that a round head could be used for treating round spots, while a rectangular head 56a would be preferable for treating wrinkles.", "In FIG.", "13a the hand applicator is illustrated seen from below, mounted with a rectangular treatment head 56a Next to treatment head the semiconductor and or laser diodes 17, 17a, 18 are placed in rows covered by a polarization filter 37 and/or lens system.", "A sensor 32 for feedback measurement is also integrated.", "FIG.", "13b shows a drawing of the hand applicator 50, which can be used for the following purposes: Introducing light-reactive substances to the tissue with the aid of air-pressure pulses Introducing light-reactive substances to the tissue with the aid of iontophoresis Radiating the tissue with a mixture of light radiation, which can be selected on the control mechanism.", "The hand applicator contains a valve 55 placed just after the air inlet, prohibiting the substances from running back into the air tube 49.The treatment head is mounted with a sensor 57 permitting exposure only on skin contact and furthermore the head also contains a valve-system 58, which opens only on skin contact, thus preventing the substances from running out of the head before it touches the skin.", "The chamber 59 containing the substances is placed next to the air duct in the hand applicator 50 and the chamber is connected with a dosage pump 60, so that the amount of substance per air-shot can be dosed very accurately.", "The hand applicator 50 housing is made of an insulating material, and the treatment head 56 is made of an electrically conductive material so that it can also be used for iontophoresis treatment combined with air-pressure treatment in order to attain maximum absorption.", "Around the treatment head the semiconductor and/or laser diodes 17, 17a, 18 are placed for the selective light radiation.", "The ON/OFF switches 54, 61, 62 for operating the hand applicator are placed on the top of the applicator.", "During the iontophoresis treatment the patient must hold an electric conductor 65 handle in his hand.", "FIG.", "14a illustrates an acupuncture applicator made with a small head containing the light sources mounted on a self-adhesive pad connected to the control mechanism, which allows a certain number of applicators to be connected corresponding with the usual number of needles used in a classical acupuncture treatment.", "FIG.", "14b shows the acupuncture applicator from the radiation side and the lens placed in this version over the three light sources 17, 17a, 18.The control mechanism can be programmed for a randomised acupuncture programme with changing frequency, modulation and amplitude.", "This method can easily replace the well-known Moxa method; Western doctors do not like Moxa treatment because of the smell it produces, although it is very effective for treating chronic diseases.", "This form of light acupuncture is without any risk of infection as no needles are used.", "It is completely pain free and the benefit can be greatly augmented by applying a topical light sensitive lotion before radiation (PDT).", "The radiation oftrigger points and/or acupuncture points with strong light sources can cause pain, but by choosing low frequencies and intensities in the start phase and successively increasing the frequency and intensity, the treatment is pain free and more efficient.", "FIG.", "15a shows a rectangular hand applicator with start-stop switch connected to the control mechanism with a cable.", "FIG.", "15b shows an illustration of the hand applicator in side view, where the upper part is formed as a handle and the lower part is rounded for application directly onto the tissue to be treated, for example hair removal after application oflight sensitive substances (PDT-hair reduction).", "FIG.", "15c depicts the hand applicator from the application side, where the rectangular optic covers the printed circuit board mounted with multiple various selectable light sources 17, 17a, 18.The size and form make it very suitable for hair reduction treatment where the radiation outlet can cover the whole area above the upper lip.", "FIG.", "16a illustrates an applicator, connected with a cable to the control mechanism, produced in the form of a quadrant tube, through which the blood to be treated passes and receives radiation from all four sides of the quadrant.", "FIG.", "16b shows how the inner sides are equipped with printed circuit boards with light sources 17, 17a, 18 also containing transmitter coils, radiating pulse-shaped electromagnetic emission.", "FIG.", "16c illustrates the blood tube designed for inner radiation room in the applicator, through which the blood is passed during the treatment.", "The illustrated version is designed for the radiation of venous blood, for example in combination with infusions of light sensitive biopharmaceuticals (PDT therapy), but other applicator types are also available for use in artificial heart/lung machines (not illustrated).", "FIG.", "17a shows a large body applicator connected with a cable to the control mechanism.", "The applicator is made of a lower and upper part hinged together and, in this illustration, shown closed, in an end view, ready for treatment.", "FIG.", "17b is an illustration where the applicator is opened and the radiation surface shows the light sources available for therapy.", "This model shows in the upper part every second light source as a standard UV light tube, and in between, the flattened oval tubes containing the light sources for the photodynamic therapy (PDT).", "The lower part is equipped only with the light tubes containing the light sources for the photodynamic therapy (PDT).", "FIG.", "17c illustrates the body applicator in a side view, where the applicator is closed and placed on the ground, with a solid socket system.", "FIG.", "18a illustrates one version of an emitter for the body applicator, shaped like an ordinary round light tube of standard length, 2.15 meters, with a connector in each end for the supply.", "FIG.", "18b shows how the printed circuit board equipped with the light sources 17, 17a, 18 is placed in the round tube.", "FIG.", "18c illustrates another version of an emitter for the body applicator, preferably formed as a flattened oval tube in a standard length of 2.15 meters and equipped with a standard connector in each end for the supply.", "FIG.", "18d shows an end view of the tube with the printed circuit board equipped with light sources 17, 17a, 18.The same printed circuit board can also contain a transmitter coil for the emission of pulse-shaped electromagnetic fields.", "The light tube is preferably mounted with a polarization 41 filter on the emitting side.", "In the field of dermatology, light is used as a stand-alone therapy for wounds, leg ulcers, eczema, burns, etc., and as such is used to stimulate tissue directly.", "Light and the emission of pulse-shaped electromagnetic fields may also be used to treat tissue using photodynamic therapy (PDT) by activating chemical reactions in photosensitive chemicals introduced into or onto the tissue, such as photofrin, 5-aminolevulan acid, hematoporphyrin, verteporfin, chlorins, phthalodyanines, phenothiazine, and benzoporphyrin-derivative monoacid-A (A TMPn) etc.", "for healing solar keratoses, basal cell carcinoma, melanomas, etc.", "PDT substances may be administered in various forms: lotion or cream for topical application, tablets or capsules for oral injection, and local injection of solutions or infusion.", "Dimethylsulfoxide (DMSO) is a solution, which has the property of breaking down the barrier of the skin and is often used before administering PDT substances in order to increase the absorption thereof.", "Alternatively, PDT substances may be mixed with DMSO for application to the skin.", "An instrument consisting of a handle with a head, wherein a number of needles are connected to a spring arrangement, can be used to pierce small, closely distanced holes in the upper layer of the skin before the PDT substances are applied, in order to increase and accelerate the absorption.", "Treatment by light irradiation with the invented device should not commence until sufficient absorption by the target tissue is obtained.", "Simply waiting for empirically determined times to elapse can suffice, or photodynamic diagnostics (PD) may be employed to determine absorption.", "PD comprises viewing the target area under illumination of a particular spectral content (such as from a fluorescent light) and observing apparent colour change of the target tissue.", "High-intensity treatments (higher doses of PDT substances and strong irradiation) are used where it is desired to destroy tissue, as in destroying tumor tissue to cure cancer, or in hair removal where it is desired to destroy the hair follicle.", "Low-intensity treatments are used where it is desired to energize affected cells and to stimulate the local immune system, as in the rehabilitation of epicondylitis, tendinitis, arthritis, arthroses, gout, and pulmonary diseases; or in the treatment of acne, actinic keratoses, warts, onychomycosis, psoriasis, dennatitis, and basal carcinoma; and in improving the appearance ofwrinkles, cellulite, and fat deposits.", "Low-intensity treatments have been observed to activate aspects of the local immune system such as the macrophages, which produce prostaglandin E2 (PGE2) and TNF (pro-inflammatory cytokines).", "There have also been observed an accumulation of leucocytes in the venules, and higher activity of the lymphocytes and plasma cells in the skin.", "The residual 5 content TNF-α of pro-inflammatory cytokines has been detected in the urine of patients after having PDT treatment.", "Treatment with the invented device further enhances the efficacy of medicinal substances by photophoresis, a process of propelling fluids into the skin or tissue and propelling molecules through cell walls.", "The absorption process is accelerated, and the amount of PDT substance absorbed is increased.", "Other methods of phoresis are in use, such as galvanic iontophoresis, exchange phoresis, and phono-phoresis.", "These methods create a concentration gradient across the skin, and a resultant Brownian molecular motion creates a thermal influence which enhances transfer of medicaments.", "Photofrin is a PDT substance which is administered by injection, at a dosage of 1-2 mg. per kg.", "of the patient's weight.", "48 hours is allowed for absorption of the photofrin by the tissue to be treated, during which time the patient is kept in dim light The treatment consists of irradiation by the invented device.", "The patient remains photosensitive for 6 to 8 weeks, and should avoid strong light and direct sunlight during that time.", "ALA (5-Aminolavulinacid) is externally applied as a 10 to 20 percent mixture in an oil in water emulsion or in a cream.", "4 to 6 hours is allowed for absorption, during which time the patient should remain in dim light.", "After treatment by irradiation from the invented device, the patient remains photosensitive for 24 to 48 hours, during which time he should avoid strong light and direct sunlight.", "L-Phenylalanin is applied in liquid form as a lotion or a spray or in a cream form, in a 5 to 30 percent mixture according to the severity of the condition to be treated.", "Optical irradiation with the invented device may begin almost immediately.", "Alternatively, doses of 50 to 100 mg may be taken orally 30 to 60 minutes before irradiation.", "The patient is photosensitive for 24 hours after application.", "PDT has been used successfully in the treatment of internal inoperable cancers.", "A biopharmaceutical (specifically, hematoporphyrin) is injected into the tumor tissue, and an optical method known as photodynamic diagnostics (PD) is used to determine when the biopharmaceutical has been absorbed by the entire tumor.", "Then the tumor tissue is irradiated with light typical for a dye laser, which activates the photosensitive reactors in the hematoporphyrin, whereby singlet oxygen is liberated.", "Singlet oxygen is toxic to protein and phosphor lipids in the tumor tissue, whereby the tumor is destroyed without destroying the surrounding tissue.", "For treatment of skin keratosis (precancerous tissue), trials with, for example, 5-aminolevulinic acid have shown that it can be used effectively in PDT if introduced into oil in a water suspension which is then applied to skin keratosis and then irradiated with a light source.", "A fast and cosmetically perfect healing has been attained with a very low rate of recurrence compared to conventional treatments, such as cryotherapy.", "Common dermatological conditions, such as acne, warts, onychomycosis (nail fungus) and wrinkles, can be successfully and effectively treated using PDT (with ALA/5-aminolevulanic acid) at a lower concentration thm has conventionally been used.", "The treatment works not by causing cell death as light treatment has historically done, but instead works by stimulating the immune system so as to enable it to better control the inflammatory reaction to oil gland activity.", "The irradiation at multiple wavelengths as provided by the present invention enhances the efficacy of treatment in this manner.", "Stimulating the immune system so as to reduce inflammatory reactions has also been found effective in the therapy of many other conditions, for example, epicondylitis (tennis elbow), tendinitis, gout, arthritis, arthroses, pulmonary diseases, and numerous other muscular and joint symptoms.", "Good results have been obtained with PDT in conjunction with the present invention's multiple wavelength output.", "Studies indicate that the patient often is pain-free after only one treatment, and the number of treatments can be reduced to 3-4, instead of 12-20 as required without the invented therapy.", "The PDT substance is applied topically as cream or oil in water suspension, typically a 10-20 percent solution.", "Augmented action may be obtained by use of injection instead of or in addition to topical application.", "A large joint such as the knee requires 10-12 subcutaneous or intra-muscular injections, preferably at the trigger points, while for a smallerjoint such as the elbow 5-6 injections is sufficient.", "First the trigger points are found and irradiated for 30 seconds with the hand applicator of the present invention.", "This gives an anaesthetic effect, which is useful for lessening discomfort from the injections.", "(Injection of the trigger points is a known method for pain reduction).", "Then, after determination thatthe PDT substance has been absorbed by the target tissue, the surface applicator of the present invention is folded around the target joint and irradiation takes place for 30 minutes.", "Good results have also been obtained in physiotherapy and physical rehabilitation with the present invention's ability to radiate visible light together with several wavelengths of infrared light and pulse-shaped electromagnetic radiation which, in combination, give a much better effect in deep tissue affected by chronic disorders.", "Thus, while the fundamental novel features of the invention have been shown and described in this prototypic application, it should be understood that various omissions and substitutions and changes in the form and details of the devices illustrated, and in their operation, may be made by those skilled in the art, without departing from the spirit of the invention.", "For example, it is expressly intended that all combinations of those elements and/or method-steps that perform substantially the same function in substantially the same way to achieve the same results are within the scope of the invention.", "Moreover, it should be recognized that structures and/or elements and/or procedures shown and/or described in connection with any disclosed form or embodiment of the invention may be incorporated in any other disclosed or described or suggested form or embodiment as a general matter of design choice.", "It is the intention, therefore, to be limited only as indicated by the scope of the claims appended hereto.", "The invention is intended for medical/dental invasive treatment, physiotherapy/rehabilitation therapy, dermatological and cosmetic skin treatment." ] ]	Patent_10466734
[ [ "Miniature precision bearings for minisystems or microsystems and method for assembling such systems", "The aim of the invention is to obtain a cost-effective solution for providing a microsystem with bearings, which have sufficiently high precision and long-term stress resistance.", "The invention proposes a method for manufacturing, adapting or adjusting a bearing portion in a fluidal microcomponent (M) comprising a stator (30) and a rotor (40,2).", "Said rotor is rotatably supported on the at least one bearing portion (L10,L11) relative to said stator.", "Said rotor (40,2) is rotatably supported by a sleeve (10, 11) inserted into said stator (30), for forming said bearing portion, the at least one sleeve being inserted in said stator as a bearing sleeve and comprising an inner surface and an outer surface (10i, 10a; 11i, 11a).", "Before being inserted in said stator, said bearing sleeve (10, 11) is a separate bearing component comprising an inner surface (10i, 11i) as an inner bearing surface, which is mechanically micro-finished before being inserted into said stator (30).", "The outer surface (10a, 11a) of said bearing component (10, 11) is mechanically permanently connected with said stator (30)." ], [ "1.Method for at least one of manufacturing, adapting and adjusting at least one bearing portion in a fluidal mini to micro component (M), said component comprising a stator (30) and at least one rotor (40,2), said rotor being supported at said at least one bearing portion (L10,L11) relative to said stator to be rotatable and rotatably supported; characterized in that (a) said rotor (40,2) is rotatably supported by a sleeve (10,11) inserted in said stator (30), for forming said bearing portion, said at least one sleeve is inserted in said stator as a bearing sleeve and comprises an inner surface and an outer surface (10i,10a; 11i,11a); (b) said bearing sleeve (10,11), prior to inserting in said stator, is a separate bearing component, comprising an inner bearing surface as said inner surface (10i,11i), and said bearing sleeve is mechanically micro-finished on at least said surface prior to inserting in said stator (30); (c) said outer surface (10a,11a) of said bearing component (10,11) is brought into a mechanically permanent connection with said stator (30).", "2.The method of claim 1, wherein said outer surface (10a,11a) of said bearing component (10,11) is inserted by friction setting into a receiving portion (30i,30i′) of said stator, said receiving portion having a smaller inner dimension, whereby a particularly radial displacement of a surface portion of a softer material of said stator is effected by said bearing sleeve (10,11), for providing a mechanically permanent connection with said stator (30).", "3.The method of claim 1, wherein said outer surface (10a,11a) of said bearing component (11,10) is inserted into a receiving portion of said stator (30), said receiving portion having a larger inner dimension, and a gap (13) or an irregular interspace between said bearing component and said receiving portion is provided with a hardenable filling material (12), said filling material hardening after being filled in, for a mechanically permanent connection of said bearing component (10,11) with said stator (30).", "4.The method of claim 3, wherein said hardening is obtained by one of cooling down, particularly when a solder is used as filling material, and a chemical reaction, particularly when an adhesive substance is used as filling material (12).", "5.The method of claim 1, wherein at least two axially spaced bearing portions (L10,L11; 10,11) are provided in said stator (30), one of said bearing portions being formed by a first sleeve (10) and the other of said bearing portions being formed by a further sleeve (11), said first sleeve being fixed and said second sleeve being fixed to said stator (30) and relative to each other.", "6.The method of claim 1, wherein said mechanical micro-finishing of said inner surface (10i,11i) of said bearing component is one of grinding, honing, and lapping.", "7.The method of claim 2, wherein during the entire inserting operation, said bearing component is guided and supported (50) by a mechanical contact, to result in an accurately positioned bearing component in said stator (30) during said friction setting.", "8.The method of claim 3, wherein said bearing component (10,11) is supported in an accurate position during hardening, for obtaining an accurately positioned alignment of said supported bearing component in said stator (30) after said hardening.", "9.The method of claim 1, wherein said bearing portion is a unilateral bearing, particularly being positioned in said stator (30) as housing, such that said bearing portion is closer to a drive than said microsystem (M).", "10.The method of claim 1, wherein said at least one bearing component (10,11) has a cylindrical shape, particularly also an outer cylindrical shape.", "11.The method of claim 1, wherein a first and a second bearing body (10,11) each being shaped as a sleeve, and each defining an axis (100,101), said two sleeves being mounted in a housing (30,31) as stator, to have eccentric or radially offset (dr) axes relative to each other, and to be axially offset, for obtaining a bearing support at an axial distance (dL) or non-identical axial positions (L11,L10) for a shaft (40) and a bearing support for an outer rotor (2) as two rotors.", "12.The method of claim 1, wherein two bearing components are inserted into said stator, into two axially spaced portions (30i,30i′) of an inner opening (31) of said stator (30), and wherein said two portions of said opening (31) are designed eccentrically with respect to each other, for attributing each of two rotors to one of said two bearing components for being rotatably supported.", "13.The method of claim 1, wherein at least two axially spaced bearing portions (L10,L11; 10,11) are provided in said stator (30), one of said bearing portions being formed by a first sleeve (10) and the other of said bearing portions being formed by a further sleeve (11), said first sleeve being fixed and said second sleeve being fixed relative to said stator (30) and relative to each other; and wherein two bearing components are inserted into said stator, into two axially spaced portions (30i,30i′) of an inner opening (31) of said stator (30), and wherein said two portions of said opening (31) are designed eccentrically with respect to each other, for attributing each of two rotors to one of said two bearing components for being rotatably supported; and wherein said first bearing component (10) is provided for supporting said shaft (40), and said second bearing component (11) is provided for supporting said rotor (2).", "14.The method of claim 12, wherein said first bearing component (10) has an outer diameter of an outer surface (10a) with a first diameter (d10a), and said second bearing component has an inner diameter of an inner bearing surface (11i) with an inner diameter (d11i), said inner diameter being smaller than said outer diameter, for an axial supporting surface (10c) between said bearing bodies (10,11) in a difference portion; for an axial bearing surface (b,10b) within said inner diameter.", "15.The method of claim 1, wherein said at least one bearing component is a freely shaped bearing body, having an inner surface (11i,10i) suitable for a bearing support.", "16.The method of claim 11, wherein said radial offset (dr) and said inner diameter and said outer diameter (d10a,d11i) are coordinated such that said two bearing bodies contact each other circumferentially continuously at a face end, at a support surface ring (10c).", "17.The method of claim 1, wherein a first and a second bearing body (10,11) each being shaped as a sleeve, and each defining an axis (100,101), said two sleeves being mounted in a housing (30,31) as stator, to have eccentric or radially offset (dr) axes relative to each other, and to be axially offset, for obtaining a bearing support at an axial distance (dL) or non-identical axial positions (L11,L10) for a shaft (40) and a bearing support for an outer rotor (2) as two rotors; and wherein two bearing components are inserted into said stator, into two axially spaced portions (30i,30i′) of an inner opening (31) of said stator (30), and wherein said two portions of said opening (31) are designed eccentrically with respect to each other, for attributing each of two rotors to one of said two bearing components for being rotatable supported; and wherein said first bearing component (10) has an outer diameter of an outer surface (10a) with a first diameter (d10a), and said second bearing component has an inner diameter of an inner bearing surface (11i) with an inner diameter (d11i), said inner diameter being smaller than said outer diameter.", "for an axial supporting surface (10c) between said bearing bodies (10,11) in a difference portion; for an axial bearing surface (b,10b) within said inner diameter; and wherein said radial offset (dr), said inner diameter, and said outer diameter of a respective sleeve are coordinated such that a circumferentially extending face end or strip portion (10c,10b,b) is provided, as one of an axial support when permanently fixing said second bearing body and an operational bearing (b) of at least one rotatable part of said microsystem (M), particularly of one of said outer rotor and said inner rotor.", "18.The method of claim 17, wherein said strip portion as a face end surface (10b) does not have a constant width (b) along its circumferential extension.", "19.The method of claim 1, wherein the bearing portion is constructed of one of hardened steel, ceramic, and hard metal.", "20.Microsystem with a fluidal flow, said microsystem comprising a first portion for one of an inlet and an outlet of a fluid (F) and a second portion having at least one bearing portion (10,11), wherein (a) a rotor (40,2) is rotatably supported relative to a stator (30) by at least one bearing body (10,11), said bearing body being prefabricated of a hard material; (b) said stator (30) comprises an inner surface portion (30i,30i′) receiving said bearing body (10,11), said inner surface portion being made of a softer material than said bearing body.", "21.The microsystem of claim 20, wherein two bearing bodies (10,11) are made of said hard material and positioned in said stator.", "22.The microsystem of claim 21, wherein two bearing bodies are radially offset relative to each other and are arranged in said stator such that a respective center axis (100,101) of a respective bearing body (10,11) has a radial distance from the other (dr) axis.", "23.The microsystem of claim 20, comprising two axially offset (dL), but closely neighboring bearing positions as separate bearing bodies (10,11) in said stator (30).", "24.The microsystem of claim 20, wherein said stator (30) comprises as an inner portion an initially not fitting receiving portion (30i′, 30i) for said bearing body (10,11).", "25.The microsystem of claim 24, wherein, when inserting said bearing body into said not fitting portion, said initially not fitting portion and said at least one bearing body (10,11) form a gap, said gap having a width of larger than zero, and a hardening filling material is introduced into said gap (13), for fixing said bearing body relative to said stator after a hardening of said filling material (12).", "26.The microsystem of claim 20, wherein said initially not fitting portion is an undersize of said receiving portion (30i′,30i) of said stator, into which a bearing body (10,11) is mechanically pressed by friction setting, said bearing body being radially larger than said receiving portion and made of a harder material, thus displacing part of said receiving portion of said stator (30), but at least modifying said receiving portion with respect to its surface structure.", "27.Method for at least one of manufacturing, adapting and adjusting at least one bearing portion in a fluidal mini to micro system (M), said system comprising a stator (30) and at least one rotor (40,2), said rotor being rotatably supported at said at least one bearing portion (L10,L11) to be rotatable relative to said stator, characterized in that (a) said stator, prior to inserting at least one separate bearing body, comprises a portion (30i,30i′) not suitable for a bearing support, said portion being made of a softer material than said bearing body (10,11) (as a non-fitting portion or misfit); (b) said non-fitting portion, by inserting, particularly by pressing in or gluing in, said bearing body made of a harder material with respect to the material of said stator, is provided as a mechanical assembly and adjusting portion, for spatially-geometrically, high-precisely determining an inner surface (11i,10i) defined by said bearing body as a bearing surface for rotatably supporting said rotor (40,2).", "28.The method of claim 27, wherein said pressing in is effected by displacing, but at least by modifying an inner surface (30i) of said bearing body.", "29.The method of claim 28, wherein a hardening material (12) is introduced into one of a gap and, subsequently to said mechanical displacement, the remaining interspaces, for obtaining a mechanical fixing and a spatial/geometrical positioning of said bearing body as a bearing portion after hardening of said filling material (12).", "30.The method of claim 1, wherein said bearing component (10,11) has at least one of an outer diameter of less than 15 mm, particularly less than 10 mm, and an inner diameter of less than 5 mm, particularly less than 2 mm, for supporting an outer rotor (2), particularly a shaft (40).", "31.The method of claim 27, wherein two bearing portions (L10,L11) are determined successively, one by friction setting (10a,10i) and a further one by soldering, gluing in (11a,11i) or friction setting.", "32.The method of claim 31, wherein initially friction setting and thereafter gluing in takes place.", "33.The method of claim 27; wherein two bearing portions (L10,L11) are determined successively, one by friction setting (10a,10i) and a further one by soldering, gluing in (11a,11i) or friction setting; and wherein initially friction setting and thereafter gluing in takes place; and wherein said first bearing portion inserted by friction setting is used as an auxiliary bearing portion determined relatively to said stator (30), for spatially/geometrically positioning said second bearing portion (L11) prior to fixing/locating it by said hardening material (12).", "34.The method of claim 27, wherein two bearing portions (L10,L11) are determined successively, one by friction setting (10a,10i) and a further one by soldering, gluing in (11a,11i) or friction setting; and wherein said first bearing portion inserted by friction setting is used as an auxiliary bearing portion determined relatively to said stator (30), for spatially/geometrically positioning said second bearing portion (L11) prior to fixing/locating it by said hardening material (12); and wherein said second bearing portion (11;11a,11i) is positioned in at least one of an axial (10b) and a radial (10i,11i) direction, supported by said first bearing portion (10).", "35.Method for manufacturing a first and a second bearing portion for two rotating bodies (2,40) and forming a joint system of a stator and rotors, rotatable relative to said stator, wherein said mechanically precise joint system is obtained from two bearing portions (L10,L11) and corresponding two rotors (2,40) by simple, but mechanically/geometrically precise bodies (10,11) and a stator (30) not precise enough for a bearing support, and by a permanent connecting technology, permanently fixing said precise bodies with respect to each other and with respect to said stator; by subsequently inserting and bearingly supporting said two rotors (2,40) in said stator (30;10,11) adapted for a bearing support by said connecting technology and said precise bodies (10,11)." ], [ "The invention relates to a method for at least one of manufacturing, adapting and adjusting a bearing portion in a mini to microsystem, such a microsystem being disclosed in WO 97/12147 (Fraunhofer-Gesellschaft) for example as micropump or micromotor for conveying a fluid or for being driven by a fluid.", "Due to the small nominal sizes for microsystems, in respect to which reference is made to the scale drawing of FIG.", "1, bearings of outer wheels, inner wheels or shafts, which all together are designated as “rotors”, have to meet exacting requirements.", "Particularly for transporting (conveying or driving) non-lubricating media, it is necessary to use very hard and simultaneously corrosion-resistant materials, such as ceramic or hard metal.", "The application of said materials is useful for all tribologically stressed functional components of a microsystem, to avoid the use of soft or corrosive materials with a continuous or stronger abrasion.", "Abrasion in bearing portions, particularly having small and miniature dimensions in a millimeter range (mini to micro system), quickly results in a breakdown of the whole system.", "Further, in view of such small nominal sizes, production engineering faces difficulties to constantly keep to the required high-precision dimensions.", "These dimensional accuracies are in a micrometer range, required accuracy being in the range of 1 to 2 μm.", "Particularly the use of eccentrically operating microsystems, comprising two rotors meshing with each other, so-called micropumps having internal teeth according to the gerotor principle, require a high-precise observation of the eccentricity, said eccentricity being obtained by two eccentrically positioned bearing portions.", "In addition to their radial offset, said bearings have an axial offset, but are located axially closely to each other.", "Thus, for principle reasons, the axes are eccentrically offset relative to each other.", "Said eccentricity requires a precision in micrometer range, said precision being expensive and complex, if not impossible from the production-engineering point of view, when using metal cutting manufacturing methods with a usual housing structure.", "It is therefore an object of the invention to propose a cost-effective solution for providing a microsystem of the kind illustrated for example in FIG.", "1 with bearings having the required maximum precision and a long-term stress resistance, particularly when operated with non-lubricating fluids.", "Said almost impossible object is achieved by a microsystem according to claim 20 or a manufacturing method, an adapting method, or an adjusting method according to one of claims 1, 35 and 27, for manufacturing at least one bearing portion of said microsystem.", "According to the invention, a mechanically precise joint system comprising simple, precise bodies (sleeves) and a “not precisely” manufactured housing (stator) is cost-effectively assembled by a connecting technique (soldering, gluing, friction-setting), particularly in connection with two axially spaced apart bearings or bearing portions and in a dimension of the “rotors” to be bearingly supported in a diameter range of below 15 mm, whereby larger embodiments shall not be excluded, but smaller diameters meet an increased attention.", "Within the scope of the specification of the invention, reference is made to both, a microsystem and a method for manufacturing a bearing portion of said microsystem, said method describing the microsystem negatively with respect to spaces and bearing portions being provided, into which said microsystem can then be positively inserted.", "As far as the microsystem itself is concerned, the finished state is described, in which the manufacturing method is only indirectly perceivable, as can be seen from the subsequent specification in support of the claims.", "With regard to claims 1, 35 and 27, it is to be first mentioned that the term of a hard bearing material is compared to that of a “soft” stator material.", "Said terms are to be understood such that said hard bearing material is for example ceramic or hard metal, for ensuring a long-term stress resistance and a long-term precision of said at least one bearing portion.", "Said softer stator materials, which can be processed more easily by cutting and which can be obtained at a lower cost and processed more easily from the production-engineering point of view, are understood in contrast to said hard materials.", "The softer stator materials receive the substantially small bearing components that provide the precision and abrasion resistance required for achieving the inventive object (claim 20).", "The stator comprises at least one portion made of a material that can be processed more easily by metal cutting, said stator receiving at least one bearing body made of a hard material.", "In said bearing body, preferably a sleeve, a rotor is bearingly supported either as shaft, or as outer rotor, or as inner rotor.", "Between said hard material and said stator material, there is a portion providing the mechanically permanent connection, which portion can be obtained in three ways.", "When a portion of the housing material in the bearing portion is displaced by a mechanical friction-setting operation, a direct, mechanically permanent connection is obtained for bearingly supporting the bearing component such that after insertion, a mechanically permanent connection between said bearing component and said stator is obtained and that said bearing component is precisely aligned (claim 2).", "An alternative variant for obtaining said mechanically permanent connection is a hardening of a filling material during a period of time, said filling material being inserted into a gap, which is present between said bearing component and a slightly larger inner size of the receiving portion of said stator (claim 3).", "Said gap can be in a range of between 20 μm and 70 μm, particularly below 100 μm.", "After said hardening, a connection of materials is obtained, which is manufactured mechanically permanent, with a long-term stress resistance, and precisely with respect to its position.", "Further, the realization of said at least one bearing portion thus formed is cost-effective.", "A third variant is a combination of the two above-described methods, when two axially spaced bearings are provided.", "Then, said friction setting with a mechanical pressing operation with a mechanically direct permanent connection for a bearing support of said first bearing component can be combined with a hardening of a filling material between said second bearing component and said stator.", "Initially, the first bearing is inserted by friction setting, mechanically displacing said soft material.", "Subsequently, the second bearing is put initially loosely into the stator, supported by said mechanically already permanent bearing, the center of which is axially spaced apart.", "Subsequently, a positioning of said second bearing relative to said first bearing, and thus an absolute positioning of said second bearing relative to said stator follows, and a hardening filling material is inserted between said second bearing and said stator, with which filling material the hardening and permanently fixing during a period of time is realized.", "An adhesive effect is obtained in a gap, which is left between said second bearing and said stator, as described above.", "Preferably, the first bearing portion, which is positioned mechanically by displacing a surface portion of the stator material, is that of a shaft, the outer diameter of the sleeve, which forms said bearing portion, being smaller than the sleeve, which forms the subsequently determined second bearing portion, which is accurately positioned by the hardening of a filling material (one of claims 5, 31 and 32).", "The displacement or the portion filled up with a hardening material is the portion, which is to be described as “non-fitting portion”, “not properly dimensioned fit” or “misfit” (compare claim 27).", "During the manufacturing process, said misfit or not properly dimensioned fit becomes a fit.", "The non-fitting portion is obtained either by a mechanical displacement of a portion of the stator material (claim 24, 26), or said misfit becomes a mechanically permanent connection by providing a hardening intermediate material, which, as filling material realizes said mechanically permanent connection.", "During friction setting, said bearing body is guided high-precisely during the entire friction setting operation, to obtain an accurate position in said stator (claim 7).", "During said mechanical guiding and displacement process, at least the surface of the stator portion, which receives said bearing component, is modified, particularly more than the surface or a radial portion is displaced (claim 7, claim 26).", "The hardening of a filling material (claim 25, claim 3 and 4) operates without displacement, the bearing component being supported accurately positioned during hardening, to make said mechanically permanent connection an accurately positioned precise connection.", "Said at least one bearing body, which, before finishing the fabrication, was a bearing body separate from said stator and made from a different material, is processed by mechanical micro-finishing of the inner surface, for example by grinding, honing or lapping (claim 6), to obtain a suitable bearing support surface for the shaft or the outer rotor.", "Particularly rotationally symmetrical bearing bodies are suited for grinding operations, such as centerless grinding, and can be manufactured comparably inexpensively in the required precision.", "Additionally, grinding allows processing of hard materials without restriction, the material selection thus not being limited.", "After high-precisely manufacturing the bearing support surfaces, the mechanical connection with the stator is carried out, the insertion of the bearing sleeves and their relative alignment, particularly by gluing or friction setting, being effected with a separate arrangement, said arrangement defining the position and orientation of said one bearing portion, particularly two eccentric bearing portions (claim 21, 22 and 23), and realizing the required tolerances with a comparably low effort or cost.", "Prior to a hardening of the solder or the adhesive substance, the sleeves can be adjusted relative to each other, so that said sleeves initially float and are aligned in said gap filled with adhesive.", "A supporting arrangement serves for stabilizing the position and for securing it during the proceeding hardening of said solder or adhesive.", "The manufacturing method advantageously limits the variety of parts of a modular system of parts comprising different rotor sizes of the tooth ring pump, since identical bearing bodies can be used for different tooth systems—defined by the eccentricity and the tooth parameters.", "Friction setting is done with a slight press fit, the manufacturing tolerance of a “not sufficiently precise” stator, e.g.", "a stator manufactured by cutting (by a metal cutting method) defining the oversize of the fit.", "As the tolerance of the position of the negative mold in the housing does usually not correspond to the position of the corresponding bearing bodies, the material is displaced during the friction setting operation.", "In most cases, said operation occurs asymmetrically and is enabled by the roughness and by a defined small supporting portion on the surface of the negative mold.", "The roughness of the surface to be produced is adjusted such that tips of the surface, which carry the bearing body to be pressed in, can be displaced relatively easily.", "Alternatively, the surface results from a defined axial or radial structure (comparable to a peg).", "The radial offset to be compensated between the bearing body and the portion of the stator receiving said bearing body can be around 10 μm to 20 μm.", "The principle of said bearing support can also be transferred to other mechanical systems with defined bearings, such as pumps having external teeth, etc., so that the invention does not imperatively relate to axes with two bearing portions only.", "A rough determination of the position of the bearing bodies is inexpensively predetermined by metal cutting methods (lathing, milling or the like) or basic (original) shaping (e.g.", "by injection molding), reshaping or other manufacturing methods.", "The recesses (negative molds) have dimensions of a limited precision, thus possibly having larger tolerances than directly provided bearing portions.", "This already reduces the manufacturing cost, to subsequently obtain the precise and accurate position of the bearing bodies relative to each other by using the assembly arrangement, which high-precisely positions the hard bearing bodies in the comparably soft stator and determines their relative position and alignment with a micrometer accuracy.", "A separate substantial assembly arrangement, which is described hereinafter, is of decisive influence for all assembly operations.", "Due to its geometry, which is of micrometer accuracy, said assembly arrangement defines the eccentric position of the sleeve axes relative to each other and stabilizes said position during the assembly operation, either during friction setting or during the supporting time, during which the adhesive material hardens.", "The bearing support is designed to correspond to a so-called flying (unilateral) bearing support (claim 9).", "Said unilateral bearing portion is closer to the drive means than the backside of the bearing support, which is occupied by the microsystem.", "Said unilateral bearing support allows reducing the number of bearing portions requiring precision.", "Therefore, by using a bearing sleeve receiving a rotor (outer rotor, inner rotor or shaft), a radial bearing support of the rotating functional element can be ensured.", "When two bearing supports that are arranged eccentrically relative to each other are provided, the bearing body serves as an axial support for the outer rotor of the microsystem for forming the shaft bearing (claim 15 to 18).", "With respect thereto, the inner diameter of the bearing body for the shaft is smaller than the inner diameter of the bearing body for the outer rotor of the microsystem.", "As also the outer diameter of the bearing body for the shaft is larger than the inner diameter of the bearing body for the outer rotor, an axial bearing surface is obtained.", "Thus, the outer rotor (and the inner rotor) is in surface contact With the axial face end surface of the bearing component having the smallest inner diameter.", "A strip is formed between said two bearing components (claim 17), said strip not having a constant width in a circumferential direction due to the eccentricity (claim 18).", "The eccentric sleeves are in surface contact with each other along their complete circumference (on at least one inner surface) (claim 16) and are particularly mounted on an axial end portion, i.e.", "on a face end surface of the stator.", "On the other end of the stator, a coupling arrangement is provided, said coupling arrangement establishing a connection with a motor arrangement in the sense of a drive means.", "When speaking of a radially offset bearing support and of an axially offset bearing support, reference can be made to the respective centers.", "As far as said radial offset is concerned, the axes are offset relative to each other, said offset being represented by the parameter dr. An axial offset corresponds to a distance of the centers of the bearing portions, said distance being designated dL.", "Said two bearing portions themselves, however, have a final axial length and are closely neighboring each other, particularly are directly adjacent to each other (claim 15).", "The only spatially limited dimension of the bearing portions also allows the use of highly special and expensive materials for said bearing portions, without insubordinately increasing the cost of the entire system.", "In a mechanical micro-finishing operation relating to the separate bearing component, which prior to inserting comprises a surface suitable as an inner bearing surface, a rectangularity of said inner bearing surface relative to a face end of said bearing component can be observed.", "Rectangularity is advantageous for an additional auxiliary support in the sense of a mechanical support portion during the assembly of the bearing portions (claim 14, 17 and 34).", "Another adjusting possibility is given in an axial direction, when a first supporting portion as a first bearing portion is already finished (claim 31 to 33, claim 5).", "The height (measured in an axial direction) of the bearing portion for receiving said rotor, i.e.", "the second bearing portion, can be adjusted by manufacturing engineering relative to the stator to obtain a defined face end clearance.", "Said face end clearance refers to the rotor inserted later, which is rotatably supported in said second bearing portion.", "With said face end clearance, the friction and the fluidal bearing can be predetermined.", "The inner opening of the stator, into which said at least one bearing portion, preferably two axially spaced apart bearing portions are inserted, comprises two portions (claim 12), each forming an inward directing surface.", "Said surfaces are the surface portions not yet suited for a bearing support, onto which the bearing portions are mounted by said bearing sleeves, which from a production-engineering point of view are more precise, namely by a gluing or pressing method or by a combination of said connecting techniques.", "Said two surface portions of the raw bearing are already eccentrically aligned relative to each other to form a respective axis each, said axes having an axial distance “dr” in a radial direction.", "The inner receiving portion therefore has two functional portions for receiving two functionally different bearing portions, each comprising a respective bearing body.", "A compensating function by friction setting or gluing has an effect only in a very small dimensional range, an eccentricity being dependent on the toothing, for example 180 μm, in which example a gluing gap is in a range of 70 μm at maximum and a pressing oversize is around 10 μm.", "The claimed invention is described in detail and supplemented by embodiments.", "FIG.", "1 is a full scale illustration (1:1) of the complete microsystem 1, said micro system comprising a fluid connection F, the proper fluid transporting micro component M, e.g.", "as pump having a motor drive A, or as fluidal motor M having a drive means A.", "FIG.", "1a is an exploded and considerably enlarged view of FIG.", "1, illustrating all components, which are to be described in more detail in the following, said micro component M comprising an inner rotor 3 and an outer rotor 2, said inner rotor being mounted on a shaft 40.Said micro component is described in more detail in the above-mentioned WO-document and is therefore designated as gerotor system or as tooth ring system having internal teeth, said teeth being in a meshing engagement during rotation.", "FIG.", "2 is a sectional view along a main axis of FIG.", "1a and illustrates an assembly of a tooth ring system with all components.", "FIG.", "3 illustrates a section along a center axis of the system according to the above figures, only a stator 30 being schematically displayed as a housing, for illustrating sleeves 10,11 mounted therein as bearing portions.", "FIG.", "3a illustrates surfaces 30i and 10a of a bearing sleeve 10 and a stator 30 before and after inserting said sleeve.", "FIG.", "4 shows a supporting and positioning system 50 for inserting said sleeves 10,11 according to FIG.", "3.FIG.", "5 is a perspective view of FIG.", "3, showing said stator and, still spaced apart, i.e.", "before being mounted, a first sleeve part 10 and a second sleeve part 11 for receiving said shaft 40 in a shaft space W and an outer rotor of said micro system in a rotor space R. Said two parts are inserted into a provided inner space 31 of said stator in a direction s. FIG.", "6 illustrates an alternative adjustment and permanent fixing of said sleeves 10,11 of FIG.", "5, compared to FIG.", "3a.", "FIG.", "7 is a top plan view of FIG.", "3 in an axial direction, still without an inserted rotor and without an inserted shaft 40, for illustrating an axial bearing and supporting surface 10b.", "The full-scale illustration of the microsystem according to FIG.", "1 shows the requirements with regard to a miniaturization and the necessity of manufacturing bearings provided in said system with an extremely high precision and of ensuring their stress resistance and abrasion resistance.", "FIGS.", "1a and 2 shall be described together for providing an insight into the microsystem illustrated in FIG.", "1.The largest portion is occupied by a drive system A, which is connected with a micro component over a flange portion.", "A shaft of a motor is connected with a shaft 40 of said micro component over a coupling means 23, said connection being non-rotating.", "An inner space 32, provided for this purpose, is limited by a sleeve 21, said sleeve having a longer axial extension than a length of said coupling means 23.At said shaft 40, a first hat-shaped gasket 24 is provided, said gasket having a collar-shaped protruding thin flange portion and an opening for a passage of said shaft 40.Said gasket is positioned in an axial inner space 31, in which also a first bearing sleeve 10 is located, said bearing sleeve also having an inner opening, in which said shaft 40 is suitably supported for rotation.", "Above said first sleeve 10, a second sleeve 11 is provided, said second sleeve having a larger outer diameter and a lager inner opening, for receiving a rotor or rotors 2,3 of said microsystem M, one of which rotors being non-rotatably positioned over a pin 40a on said shaft 40.When rotating said shaft, both internally toothed rotors rotate with said shaft, for which rotation an outer bearing support of the outer toothed ring at said second sleeve 11 is provided.", "Said second sleeve 11 has a considerably shorter axial extension, but a larger radial inner opening, whereas said first sleeve 10 comprises a small opening suitable for said shaft, but extending over a larger axial length.", "The described micro component is generally marked with M, but it comprises said two internally toothed rotors 2 and 3, as illustrated in FIG.", "1a.", "Said sleeves 10,11, said gasket 24, and said shaft 40 are received in a stator 30, which can be regarded as a portion of the housing.", "Said stator comprises a longitudinally extending flange portion 30b, extending outside over a distance sleeve 21 and engaging at an edge of said drive A for fixation, and a further above located portion 30a in which said micro system M and said shaft 40 are supported.", "The stator 30 is directly screwed up with the motor.", "For this purpose, small size electric motors comprise standard threads or connection holes, over which motor drives are usually fastened.", "The inner opening of said second sleeve 11 for receiving said micro system M is disposed in said stator at a face end thereof.", "Said sleeve can be mounted flush with respect to the face end of said stator 30.Preferably, however, a slight projection can be provided to obtain a better sealing effect at the rotors, when a portion 29,29′, located above said rotors and comprising a fluid guiding means towards the connections F, is pressed with a higher pressure over a screwed flange 28 against said stator 30 with an intermediate sealing ring 25 and a kidney plate 25a.", "Between said screwed flange 28 and said stator 30, preferably a left-hand thread is provided, which is disposed outside.", "A special claw tool is used for screwing, said claw tool engaging in a lateral bore.", "Thus, an unauthorized opening is avoided.", "Said portion 29,29′ comprises fluidal control contours (inlet opening and outlet opening) and is aligned exactly (radially and circumferentially) with its lower portion 29′ over a cylindrical pin 22 for engaging in a fit opening 22a in said stator 30, and, if required, in a collar at said stator 30.The described flush contact of the lower portion 29′ of the fluid transporting portion 29,29′ with its surface extending towards said drive with said rotors of the fluidal system M is improved by providing a compensating ring 27 between the clamping arrangement 28 and said fluid transporting portion 29.Said compensating ring 27 is made of a soft material, for example aluminum, copper or plastic, and provides a plane-parallel and flush contact of said portion 29′ with said stator, which is also provided with an O-shaped gasket 25 or an additional disk or plate 25a with fluid transporting kidneys, particularly also contacting the outward-directing face end surfaces of said rotors, for obtaining a better sealing effect.", "Said better sealing effect is achieved by a higher surface pressure (a more solid seat/contact) of said fluid transporting portion 29′ against said second sleeve 11, said better sealing effect being favored by said soft compensating ring 27.From the above description, three constructive portions can be taken.", "A fluid transporting portion F comprising the components 28,29,29′, which can also be regarded as stator.", "The proper stator 30 at a portion 30a, for receiving a microsystem, said stator comprising an adjacent coupling portion 23 of a shaft 40 at a portion 30b.", "Said portion is attached to a drive portion A.", "It is emphasized that in said stator, a separation of the fluid transporting portion 28,29,29′, F from said microsystem takes place, said separation being provided by the assembly and positioned at a face end of the rotors of the microsystem, said face end directing away from the bearing side.", "In other words, the stator 30 is structured such that the bearing support is positioned flush at a face end directing away from said drive A, so that a mounting of said fluid transporting portion 29,29′ is directly adjacent to the fluidal micro component and ensures a fluid transport and functional operation of said micro component M by a provided fluid conveying structure comprising kidneys and bores.", "The above general view is intended to increase the understanding of the constructive design and structure of a microsystem according to FIG.", "1.In the following, details are explained, which particularly describe the mounting and assembly of the first and second sleeve 10,11 according to FIG.", "2, reference insofar being made to FIG.", "3.FIG.", "3 is a section through an axis of the system according to FIG.", "2, two axes 100 and 101 being shown, said axes being offset relative to each other.", "Said offset of axes is marked with dr. Said axis 100 is the axis of the first sleeve 10, said sleeve having a length L10.Said sleeve is made of a hard material, e.g.", "hard metal or ceramic.", "Initially, it is not inserted in the stator 30, said stator having an elongated opening 31 for receiving said sleeve, a lower portion of said opening having an inner surface 30i.", "Said inner surface is schematically illustrated in FIG.", "3a (in the lower part of the illustration).", "Said surface is of considerable roughness, which can be obtained by a metal cutting method.", "Said surface does not have to be particularly precise and can be embodied even larger than illustrated in FIG.", "6.Likewise, a further receiving portion is provided, said receiving portion being disposed axially above in said stator 30 as part of said opening 31, for receiving said second sleeve 11, which can also be made of a hard material, such as ceramic or hard metal.", "Said sleeve, too, is initially not inserted.", "The use of hard materials in contrast to “soft” materials of said stator 30 protects the bearing sleeves against abrasion.", "Said bearing sleeves are of small spatial extension, so that also expensive materials can be used.", "Said bearing sleeves are preferably designed as hollow cylinders and comprise an inner space each, for receiving the respective “rotor”.", "Said first sleeve 10 has an inner space with an inner surface 10i for receiving a shaft 40.Said inner space is marked with W and has a longitudinal extension corresponding to said sleeve length L10.The axially adjacent second sleeve 11 is provided for receiving and supporting the outer rotor 2.In respect thereto, said sleeve has a rotor-receiving portion R, a diameter d11i of said rotor-receiving portion being larger than a diameter d10i of said shaft space W. An inner surface 11i is designed to allow a bearing support of said rotor.", "The inner surface 10i of the first sleeve 10 is also designed to allow a bearing support of said shaft 40.Both surfaces have a high precision and are designed for their respective bearing support function by grinding, eroding, honing, or lapping.", "An inserting arrangement according to FIG.", "4 is provided for inserting said two bearing sleeves into the respective axial portion of the opening 31 of said stator 30 with said inner surface 30i and said inner radially larger surface 30i′.", "The two sleeves 10,11 are spatially geometrically aligned relative to each other by place holders 53,52, thus ensuring a high precision.", "Said two place holders 52,53 are spatially fixed relative to a support plate 51.The placeholder 52 for the outer rotor receives said second sleeve 11, said placeholder filling up the rotor geometry of the rotor space R. The second placeholder 53 for the shaft 40 is axially longer.", "Said second placeholder fills up the shaft space W and locates the first sleeve 10 spatially geometrically, to obtain the two spaced apart axes 100,101 for an eccentric bearing support of said microsystem M comprising two rotors.", "A not illustrated pin at said support plate 51 provides an absolute determination of the position of said support plate in relation to said stator 30, for engaging in an opening 22a.", "After mounting said sleeves 10,11 on said inserting arrangement 50 and said two placeholders 52,53 which are radially offset by “dr”, a mechanical arrangement (not illustrated) is used for axially moving said inserting arrangement into said opening 31 of said stator 30, said movement being geometrical and precise, even high precise with regard to the masses.", "The movement path s or the movement direction s, is shown in FIGS.", "5 and 3a.", "Due to the dimensioning and the surface structure of the two sleeves 10,11 and of the inner surfaces 30i′ and 30i of the stator, a modification of at least the inner surfaces of the stator 30 occurs, said modification being visible in FIG.", "3a prior to and after inserting said sleeve part 10.The rough surface of the not high-precisely manufactured inner surfaces is leveled or even removed or displaced, the soft material being modified on the surface, but simultaneously applying mechanical forces for spatially geometrically fixing said pressed-in sleeves 10,11, which serve as bearing support pieces.", "The inner surfaces 11i,10i of said two sleeves are high precise, and after inserting, geometrically precisely fixed to achieve their bearing function.", "The outer surfaces 10a and 11a of the two sleeves enter into a mechanical connection with the inner surfaces 30i′ and 30i of the stator, when the inserting arrangement 50 is axially introduced under pressure.", "An alternative fixation can be provided by a hardening substance 12, when the inner surfaces 30i′ and/or 30i are designed to have a slightly larger spatial geometry than the outer surfaces 11a and/or 10a of said sleeves 10 and/or 11, as illustrated in FIG.", "6.In this case, said inserting arrangement cares for an attribution of the eccentrically offset axes 100,101 of said two sleeves, and positions them in the inner space 31 with the two eccentric portions 30i,30i′ of the stator 30 until an introduced hardening substance 12 fills up a gap 13 for fixing it, and mechanically fixes said sleeves.", "A solder or a bonding agent can be used as hardening substance; said first material hardens by a decreasing temperature, said second material by a chemical reaction.", "One function of said inserting arrangement is to take over the mechanical attribution during the axial friction setting.", "Regarding the variant of fixation with a hardening substance in a gap 13 (also as an irregular interspace), said gap having a size of between 20 μm and 70 μm, said inserting arrangement takes over the geometrical fixation of the sleeves during hardening, therefore, during insertion, said inserting arrangement does not have to apply an additional mechanical force in a direction s. The second sleeve 11 is axially shorter and has an axial length L11.The total stator length is L. Said stator 30 having an axial length L, the total of said two sleeve lengths L11 and L10 is still shorter than said stator length.", "The distance of the centers of said two sleeves is dL, which represents an axial offset, the face end surfaces of said two sleeves 10,11, however, contacting each other.", "Said contact of the two face end surfaces is described with reference to FIG.", "7.FIG.", "7 illustrates a top plan view in an axial direction 100,101 from above (regarded from FIG.", "3 or FIG.", "6), the inner spaces R and W for the outer rotor and the shaft still being open, thus no shaft 40 and no rotor 2 or 3 of a microsystem M being inserted yet.", "A face-end bearing surface 10b is visible, which is also marked in FIG.", "3 and in FIG.", "6.Said bearing surface has a width b, said width not being constant in a circumferential direction, which results from the offset dr or Δr of said two axes 100,101, and from the two selected diameters of the sleeves, here the outer diameter d10a of the longer sleeve 10 and the inner diameter d11i of the shorter sleeve 11.Said diameters and the corresponding radii as respective half diameters, as well as the radial offset (eccentricity) are selected such that one of the hard bearing components 10,11 forms an annular axial support surface 10c, which is outside of a surface 10b and completely continuous also in a circumferential direction, and on which the other hard bearing component 11 is supported to have surface contact.", "When observing said radial offset dr, the outer diameter d10a of said sleeve 10 is as much larger as the inner diameter d11i of said sleeve 11 that at no circumferential position, the soft material of said stator 30 as a portion of said support surface 10b for said rotor 2 according to FIG.", "1a and possibly also for said inner rotor 3 according to FIG.", "1a—regarded in an axial direction—appears or is of importance.", "The rotor or the rotors are—inserted in said rotor space R—then axially safely supported, geometrically precisely fixed, and a good sealing is obtained at the surface 10b, whereas the annular portion 10c, which supports said sleeves 10 and 11 relative to each other and aligns them orthogonally, is no longer visible from outside.", "Inner surfaces 11i and 10i form bearing surfaces for the shaft 40 and the outer rotor of the fluidal microcomponent M, for serving as a slide bearing.", "Said annular surfaces 10c and 10b together form the axially directing face end surface of the complete bearing component 10 provided for said shaft.", "Said inner portion 10b serves for supporting and aligning the microsystem, and the surrounding outer portion 10c, which is located on the same level, serves for aligning and supporting said second bearing component 11.The top plan view according to FIG.", "7 also illustrates the gap 13 according to FIG.", "6, said gap already being filled up with an adhesive or a solder 12, for fixing the inserted sleeve 11 relative to the softer material of said stator 30.Before said solder or adhesive hardens, said sleeve 11 was aligned by contacting at said outer annular surface 10c of said lower sleeve 10, so that the axis 101 of said sleeve is also aligned precisely in parallel to the axis 100.Said precise alignment results from a high-precise manufacturing of the face end surfaces, which extend exactly perpendicularly to said axes and are thus adapted to have a direct effect on the positioning and exact position.", "In an embodiment of specific dimensions, which, however, are not to be understood as restricting, a sleeve 10 was manufactured having an outer diameter of 5 mm and an inner diameter of 1.2 mm.", "An outer rotor 2 has an outer dimension of 3.8 mm, and is therefore—also when the selected eccentricity of the two axes 100,101 is considered—within the outer dimension of 5.0 mm of said sleeve 10, axially supporting said rotor for providing a rotatable bearing support.", "From said dimension, also the inner size d11i of said second sleeve 11 is visible, corresponding to the outer size of said rotor, for radially supporting said rotor with an annular bearing.", "Both bearing supports, which are perpendicular relative to each other, the inner wall surface 11i and the axially directing support surface of the sleeve 10 provide a precise alignment and precise bearing support of the rotor component 2.A gap 13, which for explanatory purposes is illustrated in an oversize in FIG.", "7, results from the difference between the radius of the inner surface 30i′ of the stator 30, compare FIG.", "3, and the outer dimension of the outer surface 11a of the hard bearing sleeve 11.For a bonding, the size of said gap is preferably between 50 μm and 70 μm, which, when illustrated to scale, would not be visible in the illustration according to FIG.", "7, if it had not been represented at a substantially enlarged scale.", "FIG.", "5 is a perspective view showing the insertion of the two bearing sleeves 10,11, used for an assembly and adjustment of the sleeves with an adhesive substance.", "An adhesive substance 12 is introduced into a gap 13 having a size of between 20 μm and 70 μm with reference to a respective inner diameter of said stator 30 at the surfaces 30i and 30i′.", "The inner space 31 for receiving said first sleeve 10 is longer than said bearing sleeve 10.The corresponding difference—as illustrated in FIG.", "2—is occupied by a radial shaft sealing 24, which is fixed against said motor A by a distance sleeve 21.An inserting path s of the two bearing sleeves 10,11, supported by an inserting arrangement 50 according to FIG.", "4, provides a precise positioning.", "After filling in an adhesive substance 12, which can also be present at said inner surfaces according to FIG.", "5 already prior to said insertion, a spatial geometrical attribution and an absolute positioning of said sleeves 10,11 is maintained for at least the hardening time of said adhesive substance or solder, until a mechanical hardening occurs.", "Also visible from FIG.", "5 is a receiving portion 22a, in which a positioning pin 22 according to FIG.", "2 engages, when mounting a fluid transporting portion 28,29,29′.", "A radially offset stepped bore 22 at the inner side of the surfaces 30i′ and 30i, respectively, of said stator 30 is provided.", "Circumferentially spaced apart from said receiving portion 22a, said bore offers a possibility of using a fluid in a small quantity as slide bearing lubrication or in an annular flow after inserting and mounting said bearing sleeves 10,11, when operating said system M. Said bore 22b has a minimum depth of L10+L11.Said stepped bore 22b, which is also illustrated in FIG.", "1a, is located with a portion of its bore depth in the surface portion 30i′ (compare FIG.", "3) and with a further portion in a surface portion 30i.", "By said bore, the annular flow of the fluid, which flows through the shaft bearing, is obtained.", "By said stepped bore, a discharge of the fluid present between the sealing and the shaft bearing in a direction towards the suction side of the microsystem is obtained, said microsystem in the present embodiment being provided as a pump.", "The fluid from said stepped bore 22b, insofar operating as a channel for said fluid, is again taken up in the fluid conducting portion 28,29,29′, here in a portion 29′ directing towards said microsystem by covering said channel, and is returned into said microsystem 2,3.It is to be mentioned, that the spatial-geometrical high-precise bearing support is only provided unilaterally with respect to the shaft W, but that also a second bearing support can be provided in said fluid conveying portion 29, said second bearing support, however, not having to be as precise as said first bearing support in said sleeve 10, which is additionally effective on an axially larger length L10.The bearing portions can be manufactured as sleeves in a rotationally symmetrical simple manner.", "They can also have a different geometry with regard to their outer diameter, only their inner diameter and their inner surface have to be aligned such that the rotors 40,2 (shafts and outer rotor of the microsystem) can be bearing-supported geometrically precisely and resistant to abrasion.", "The described methods of inserting and positioning can also be combined.", "A less solid mechanical connection can be provided for an insertion by pressing in or friction setting the sleeves 10,11, determined by a corresponding adaptation of the diameter geometries of inner space and outer surface of the sleeves.", "After said friction setting operation, an alignment and subsequently a bonding can be effected by an additional arrangement, so that the two methods can also be used in combination.", "The combined inserting method can also be effected temporally successively.", "The first receiving portion with the inner surface 30i in the first portion of the opening 31 of the stator can be connected by a mechanical friction setting operation, in which the sleeve is precisely positioned, as shown in FIG.", "3a.", "Relative to said first sleeve fixed by said method, which sleeve can then serve as an auxiliary bearing or an auxiliary arrangement, the second bearing portion (here with the sleeve 11) can be positioned in the portion L11 with the arrangement according to FIG.", "4, a gap 13, illustrated in FIG.", "6 being filled with an adhesive substance 12 at a circumference between the outer surface 11a and the inner surface 30i′.", "When said first sleeve 10 fits solidly, said second sleeve can be positioned and bonded relative to said first sleeve and consequently relative to said stator.", "As an alternative to said bonding operation, a pressing operation can also be used for said second operation, which corresponds to the variant described before, only temporally successively.", "The arrangement according is to FIG.", "4 can be used for all these variants.", "A combination of pressing (friction setting) and gluing (bonding) turned out to be particularly precise.", "Initially, the first sleeve 10 is pressed into the stator 30, the two opening portions 30i,30i′ being provided as two portions of the complete opening 31, said portions being positioned eccentrically with respect to each other.", "After pressing in, the second support portion 11 is formed by inserting a high-precisely manufactured bearing sleeve into the housing, said bearing sleeve being in a flush surface contact with said first sleeve, at a face end surface portion 10c thereof.", "Subsequently, the position of the second sleeve relative to said first sleeve is defined by using the arrangement according to FIG.", "4.Subsequently, an adhesive substance 12 is introduced into the gap 13 at the outer surface 11a of said second sleeve and hardened, to fix said bearing portion, i.e.", "to permanently connect it with said stator 30.The rectangularity of the preceding mechanical micro-finishing of said sleeve 11 and of said sleeve 10 can provide two auxiliary bearing portions for positioning and fixing.", "An axial support surface 10c and a circumferential inner surface 10i which, over the arrangement according to FIG.", "4, can directly influence the precise positioning of said second inserted sleeve.", "The mounting order of the two sleeves 10 and 11 can also be exchanged.", "Firstly, said sleeve 11, which is larger in diameter, subsequently—axially supported over the support surface portion 10c—the longer sleeve 10 for the shaft 40.In this case, said second sleeve 10 is inserted into the lower receiving portion of the opening 31 from a coupling space 32.It is mentioned that said mechanically precise positioning in the sense of a spatial-geometrical fixing concerns two substantial dimensions.", "On the one hand, the amount of the eccentricity vector “dr” as radial offset.", "On the other hand, the correct absolute positioning of the two bearing sleeves 10,11 in the stator 30, thus their position/alignment relative to the housing.", "Said position is obtained over a pin, which is mounted in the plate 51 of the arrangement 50 according to FIG.", "4 and engages in said housing instead of a pin 22a, when mounting said bearing sleeves 10,11.Said pin is not illustrated in FIG.", "4, but it is evident from the context and from the spatial/geometrical positioning of the receiving means 22a of FIG.", "2, in which the pin 22, providing the finished assembly is marked.", "Said pin takes over the circumferential fixing of the fluid conveying portion 28,29,29′ relative to the housing 30, which is designated as stator." ] ]	Patent_10466792
[ [ "Vectors capable of imparting herbicide resistance and viral cross protection and methods", "A nucleic acid vector for concurrently imparting herbicide resistance to a plant and cross protecting the plant.", "The vector includes sufficient potyvirus nucleic acid sequence to permit viral replication and spread.", "The vector further includes mutations which attenuate symptoms of viral infection in the plant and which abolish transmission of the virus by an insect vector.", "The vector further includes an additional nucleic acid sequence encoding a protein which imparts resistance to an herbicide when expressed in the infected plant.", "Further disclosed is a method of concurrently imparting herbicide resistance to a plant and cross protecting the plant against at least one potyvirus comprising inoculating at least a portion of the plant with the vector.", "Further disclosed are plant cells treated according to the method and virions derived from the vector." ], [ "1.A nucleic acid vector for concurrently imparting herbicide resistance to a plant and cross protecting the plant, the vector comprising; (a) sufficient potyvirus nucleic acid sequence to permit a potyvirus to replicate and spread within the plant infected by the vector; (b) a first mutation in said potyvirus nucleic acid sequence which attenuates symptoms of said potyvirus in the plant infected by the vector; (c) a second mutation in said potyvirus nucleic acid sequence which abolishes transmission of said potyvirus by an insect vector; and (d) an additional nucleic acid sequence encoding a protein which imparts resistance to an herbicide when expressed in the plant infected by the vector.", "2.The nucleic acid vector of claim 1, wherein said first mutation includes an amino acid substitution of the conserved FRNK box in the HC-Pro gene (SEQ ID NO.", ": 4) of said potyvirus nucleic acid sequence.", "3.The nucleic acid vector of claim 2, wherein said amino acid substitution in the conserved FRNK box of the potyvirus nucleic acid sequence includes a substitution of a Arg to Ile at position 180 within the HC-pro gene product (SEQ ID NO.", ": 5) of said potyvirus.", "4.The nucleic acid vector of claim 1, wherein said second mutation includes an alteration of a conserved DAG triplet at position 8 in an N terminal region of the coat protein (SEQ ID NO.", ": 3) of said potyvirus.", "5.The nucleic acid vector of claim 4, wherein said alteration of said DAG triplet includes a substitution for an alanine residue in said DAG triplet.", "6.The nucleic acid vector of claim 1, wherein said insect vector is an aphid.", "7.The nucleic acid vector of claim 1, wherein said sufficient potyvirus nucleic acid sequence is derived from zucchini yellow mosaic virus (ZYMV).", "8.The nucleic acid vector of claim 1, wherein said additional nucleic acid sequence is at least a portion of a phosphinothricin acetyltransferase coding sequence.", "9.The nucleic acid vector of claim 1, wherein said protein is at least a functional portion of a phosphinothricin acetyltransferase.", "10.A method of concurrently imparting herbicide resistance to a plant and cross protecting the plant against at least one potyvirus, the method comprising inoculating at least a portion of the plant with a vector comprising; (a) sufficient potyvirus nucleic acid sequence to permit a potyvirus to replicate and spread within the plant infected by the vector; (b) a first mutation in said potyvirus nucleic acid sequence which attenuates symptoms of said potyvirus in the plant infected by the vector; (c) a second mutation in said potyvirus nucleic acid sequence which eliminates transmission of said potyvirus by an insect vector; and (d) an additional nucleic acid sequence encoding a protein which imparts resistance to an herbicide when expressed in the plant infected by the vector.", "11.The method of claim 10, wherein said first mutation includes an amino acid substitution of the conserved FRNK box in the HC-Pro gene (SEQ ID NO.", ": 4) of the potyvirus nucleic acid sequence.", "12.The method of claim 10, wherein said amino acid substitution of the conserved FRNK box in the HC-Pro gene of the potyvirus nucleic acid sequence includes a substitution of a Arg to Ile at position 180 within the HC-pro gene product (SEQ ID NO.", ": 5) of said potyvirus.", "13.The method of claim 10, wherein said second mutation includes an alteration of a conserved DAG triplet at position 8 in an N terminal region of the coat protein (SEQ ID NO.", ": 3) of said potyvirus.", "14.The method of claim 13, wherein said alteration of said DAG triplet includes a substitution for an alanine residue in said DAG triplet.", "15.The method of claim 10, wherein said insect vector is an aphid.", "16.The method of claim 10, wherein said sufficient potyvirus nucleic acid sequence is derived from zucchini yellow mosaic virus (zymv).", "17.The method of claim 10, wherein said additional nucleic acid sequence is at least a portion of a phosphinothricin acetyltransferase coding sequence.", "18.The method of claim 10, wherein said protein is at least a functional portion of a phosphinothricin acetyltransferase.", "19.The method of claim 10, wherein said at least a portion of a plant includes an item selected from the group consisting of at least one cell in the plant and at least one plant cell in tissue culture.", "20.At least a portion of a plant treated according to the method of claim 10.21.An infectious virion harvested from a plant treated according to the method of claim 10." ], [ "<SOH> FIELD AND BACKGROUND OF THE INVENTION <EOH>The present invention relates to nucleic acid vectors capable of imparting herbicide resistance and viral cross protection, methods of use thereof and plants expressing same and, more particularly, to vectors based on sequences derived from attenuated potyvirus sequences and further including sequences which impart resistance to a chosen herbicide.", "Zucchini yellow mosaic virus is a member of the potyviridae family (Shukla et al.", "(1989) Adv.", "Virus Res.", "36:273-314).", "Potyviridae is the largest group of plant viruses and its members infect most commercial or cultivated crops.", "Worldwide, ZYMV is one of the most devastating diseases of cucurbit species (e.g., squash, melon, watermelon, cucumber etc.", "; Desbiez and Lecoq, (1997) Plant Pathol.", "46:809-829).", "As in all potyviruses, the ZYMV genome consists of a single messenger-polarity RNA molecule of about 9.6 kb, encapsidated by ˜2000 units of coat protein (CP), forming a helical, flexuous, filamentous particle of about 750 nm long and 11 nm wide (Desbiez, and Lecoq, (1997) Plant Pathol.", "46:809-829 and Lisa et al.", "(1981) Phytopathology 71:667-672).", "Means for attenuating potyviruses in general, and ZYMV in particular, have been described in WO 99/51749 and in Gal-On (2000, Phytopathology 90:467-473).", "However, these earlier teachings contain neither a hint nor a suggestion that a single vector might be employed to concurrently cross protect a plant and render the plant resistant to an herbicide.", "U.S. Pat.", "No.", "5,958,422 as well as WO9602649 and WO9218618 teach modified plant viruses as vectors for heterologous peptides, including peptides useful for vaccination.", "However, these patents relate to use of plants as bioreactors for vaccine production and do not teach cross protection of the plants themselves.", "Further, these patents do not teach introduction of herbicide resistance genes into the plants.", "While use of Glufosinate resistance genes in commercial agriculture is well known (e.g.", "AgrEvo's Liberty-Link-Oilseed Rape, -Canola, -Maize etc.", "), this resistance has typically been accomplished by germ line transformation of plants.", "Such germ line transformation raises concerns about unwanted spread of herbicide resistant plants and or transfer of the herbicide resistance gene to wild plant relatives via pollination (Quist D. and Chapela I. H. (2001) Nature 414(6863): 541-3.)", "Whitham et al.", "(Proc.", "Natl.", "Acad.", "Sci.", "USA (1999) 96: 772-777) teaches use of a potyvirus expressing an herbicide resistance gene in plants in order to identify plant mutants that affect viral replication, cell to cell and long distance movement within a plant.", "The teachings of Whitham preclude use of attenuated strains of virus to impart cross protection against subsequent wild type viral infection.", "In addition, use of the teachings of Whitham in commercial agriculture is infeasible because of the pathogenic outcome of viral infection on agriculturally important plants and the high probability of transmission by insect vectors in the field.", "U.S. Pat.", "No.", "6,303,848 to Kumagai et al.", "teaches use of nucleic acid vectors to impart herbicide resistance to crops using a tobamovirus vector.", "The teachings of Kumagai require use of a subgenomic plant viral promoter which precludes application of his teachings to potyvirus.", "Further, the teachings of Kumagai do not include expression of a phosphinothricin acetyltransferase gene which confers resistance to glufosinate ammonium based herbicides.", "Further, Kumagai teaches use of a tobamavirus which is devastating to plants so that its use in commercial agriculture is infeasible.", "Further, Kumagai does not teach mutants which are impaired in their ability to be transmitted from plant to plant by their normal mode of transmission i.e.", "mechanically via infected tissue and contaminated soil.", "Thus spread of viral vectors according to the teachings of Kumagai cannot be controlled increasing the likelihood of uncontrolled infection in untreated plants.", "U.S. Pat.", "No.", "5,766,885 to Carrington et al.", "teaches expression of foreign genes in a potyvirus vector.", "However, Carrington fails to teach use of attenuated strains of potyvirus in order to minimize viral symptoms.", "Further, Carrington fails to teach use of potyvirus vectors to impart herbicide resistance.", "The scope of the teachings of Carrington is limited to use of plants as bioreactors and added value agricultural traits are not found in his teachings.", "There is thus a widely recognized need for, and it would be highly advantageous to have, non-insect transmissible nucleic acid vectors capable of concurrently imparting herbicide resistance and cross protection and, methods of use thereof and plants expressing same devoid of the above limitations." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>According to one aspect of the present invention there is provided a nucleic acid vector for concurrently imparting herbicide resistance to a plant and cross protecting the plant.", "The vector includes: (a) sufficient potyvirus nucleic acid sequence to permit a potyvirus to replicate and spread within the plant infected by the vector; (b) a first mutation in the potyvirus nucleic acid sequence which attenuates symptoms of the potyvirus in the plant infected by the vector; (c) a second mutation in the potyvirus nucleic acid sequence which abolishes transmission of the potyvirus by an insect vector; and (d) an additional nucleic acid sequence encoding a protein which imparts resistance to an herbicide when expressed in the plant infected by the vector.", "According to another aspect of the present invention there is provided a method of concurrently imparting herbicide resistance to a plant and cross protecting the plant against at least one potyvirus and.", "The method includes inoculating at least a portion of the plant with a vector including; (a) sufficient potyvirus nucleic acid sequence to permit a potyvirus to replicate and spread within the plant infected by the vector; (b) a first mutation in the potyvirus nucleic acid sequence which attenuates symptoms of the potyvirus in the plant infected by the vector; (c) a second mutation in the potyvirus nucleic acid sequence which eliminates transmission of the potyvirus by an insect vector; and (d) an additional nucleic acid sequence encoding a protein which imparts resistance to an herbicide when expressed in the plant infected by the vector.", "According to further features in preferred embodiments of the invention described below, the first mutation includes an amino acid substitution in the HC-Pro gene (SEQ ID NO.", ": 4) of the conserved FRNK box of the potyvirus nucleic acid sequence.", "According to still further features in the described preferred embodiments the amino acid substitution in the HC-Pro gene of the conserved FRNK box of the potyvirus nucleic acid sequence includes a substitution of an Arg to Ile at position 180 within the HC-pro gene product (SEQ ID NO.", ": 5) of the potyvirus.", "According to still further features in the described preferred embodiments the insect vector is an aphid.", "According to still further features in the described preferred embodiments the sufficient potyvirus nucleic acid sequence is derived from zucchini yellow mosaic virus (ZYMV).", "According to still further features in the described preferred embodiments the additional nucleic acid sequence is at least a portion of a phosphinothricin acetyltransferase coding sequence.", "According to still further features in the described preferred embodiments the protein is at least a functional portion of a phosphinothricin acetyltransferase.", "According to still further features in the described preferred embodiments at least a portion of a plant treated according to the claimed method is an integral part of the invention, as are progeny of at least a portion of a plant treated according to the method.", "According to still further features in the described preferred embodiments an infectious virion harvested from a plant treated according to the claimed method is an integral part of the invention.", "The present invention successfully addresses the shortcomings of the presently known configurations by providing a vector for concurrently imparting herbicide resistance and cross protection against wild type virus.", "Concurrent receipt of these two effects from a single treatment contributes to increased crop yield and significantly reduces production costs.", "Reduction in production costs stein from both elimination of crop damage and from ease of introducing these traits into a wide variety of commercial plant strains." ], [ "FIELD AND BACKGROUND OF THE INVENTION The present invention relates to nucleic acid vectors capable of imparting herbicide resistance and viral cross protection, methods of use thereof and plants expressing same and, more particularly, to vectors based on sequences derived from attenuated potyvirus sequences and further including sequences which impart resistance to a chosen herbicide.", "Zucchini yellow mosaic virus is a member of the potyviridae family (Shukla et al.", "(1989) Adv.", "Virus Res.", "36:273-314).", "Potyviridae is the largest group of plant viruses and its members infect most commercial or cultivated crops.", "Worldwide, ZYMV is one of the most devastating diseases of cucurbit species (e.g., squash, melon, watermelon, cucumber etc.", "; Desbiez and Lecoq, (1997) Plant Pathol.", "46:809-829).", "As in all potyviruses, the ZYMV genome consists of a single messenger-polarity RNA molecule of about 9.6 kb, encapsidated by ˜2000 units of coat protein (CP), forming a helical, flexuous, filamentous particle of about 750 nm long and 11 nm wide (Desbiez, and Lecoq, (1997) Plant Pathol.", "46:809-829 and Lisa et al.", "(1981) Phytopathology 71:667-672).", "Means for attenuating potyviruses in general, and ZYMV in particular, have been described in WO 99/51749 and in Gal-On (2000, Phytopathology 90:467-473).", "However, these earlier teachings contain neither a hint nor a suggestion that a single vector might be employed to concurrently cross protect a plant and render the plant resistant to an herbicide.", "U.S. Pat.", "No.", "5,958,422 as well as WO9602649 and WO9218618 teach modified plant viruses as vectors for heterologous peptides, including peptides useful for vaccination.", "However, these patents relate to use of plants as bioreactors for vaccine production and do not teach cross protection of the plants themselves.", "Further, these patents do not teach introduction of herbicide resistance genes into the plants.", "While use of Glufosinate resistance genes in commercial agriculture is well known (e.g.", "AgrEvo's Liberty-Link-Oilseed Rape, -Canola, -Maize etc.", "), this resistance has typically been accomplished by germ line transformation of plants.", "Such germ line transformation raises concerns about unwanted spread of herbicide resistant plants and or transfer of the herbicide resistance gene to wild plant relatives via pollination (Quist D. and Chapela I. H. (2001) Nature 414(6863): 541-3.)", "Whitham et al.", "(Proc.", "Natl.", "Acad.", "Sci.", "USA (1999) 96: 772-777) teaches use of a potyvirus expressing an herbicide resistance gene in plants in order to identify plant mutants that affect viral replication, cell to cell and long distance movement within a plant.", "The teachings of Whitham preclude use of attenuated strains of virus to impart cross protection against subsequent wild type viral infection.", "In addition, use of the teachings of Whitham in commercial agriculture is infeasible because of the pathogenic outcome of viral infection on agriculturally important plants and the high probability of transmission by insect vectors in the field.", "U.S. Pat.", "No.", "6,303,848 to Kumagai et al.", "teaches use of nucleic acid vectors to impart herbicide resistance to crops using a tobamovirus vector.", "The teachings of Kumagai require use of a subgenomic plant viral promoter which precludes application of his teachings to potyvirus.", "Further, the teachings of Kumagai do not include expression of a phosphinothricin acetyltransferase gene which confers resistance to glufosinate ammonium based herbicides.", "Further, Kumagai teaches use of a tobamavirus which is devastating to plants so that its use in commercial agriculture is infeasible.", "Further, Kumagai does not teach mutants which are impaired in their ability to be transmitted from plant to plant by their normal mode of transmission i.e.", "mechanically via infected tissue and contaminated soil.", "Thus spread of viral vectors according to the teachings of Kumagai cannot be controlled increasing the likelihood of uncontrolled infection in untreated plants.", "U.S. Pat.", "No.", "5,766,885 to Carrington et al.", "teaches expression of foreign genes in a potyvirus vector.", "However, Carrington fails to teach use of attenuated strains of potyvirus in order to minimize viral symptoms.", "Further, Carrington fails to teach use of potyvirus vectors to impart herbicide resistance.", "The scope of the teachings of Carrington is limited to use of plants as bioreactors and added value agricultural traits are not found in his teachings.", "There is thus a widely recognized need for, and it would be highly advantageous to have, non-insect transmissible nucleic acid vectors capable of concurrently imparting herbicide resistance and cross protection and, methods of use thereof and plants expressing same devoid of the above limitations.", "SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided a nucleic acid vector for concurrently imparting herbicide resistance to a plant and cross protecting the plant.", "The vector includes: (a) sufficient potyvirus nucleic acid sequence to permit a potyvirus to replicate and spread within the plant infected by the vector; (b) a first mutation in the potyvirus nucleic acid sequence which attenuates symptoms of the potyvirus in the plant infected by the vector; (c) a second mutation in the potyvirus nucleic acid sequence which abolishes transmission of the potyvirus by an insect vector; and (d) an additional nucleic acid sequence encoding a protein which imparts resistance to an herbicide when expressed in the plant infected by the vector.", "According to another aspect of the present invention there is provided a method of concurrently imparting herbicide resistance to a plant and cross protecting the plant against at least one potyvirus and.", "The method includes inoculating at least a portion of the plant with a vector including; (a) sufficient potyvirus nucleic acid sequence to permit a potyvirus to replicate and spread within the plant infected by the vector; (b) a first mutation in the potyvirus nucleic acid sequence which attenuates symptoms of the potyvirus in the plant infected by the vector; (c) a second mutation in the potyvirus nucleic acid sequence which eliminates transmission of the potyvirus by an insect vector; and (d) an additional nucleic acid sequence encoding a protein which imparts resistance to an herbicide when expressed in the plant infected by the vector.", "According to further features in preferred embodiments of the invention described below, the first mutation includes an amino acid substitution in the HC-Pro gene (SEQ ID NO.", ": 4) of the conserved FRNK box of the potyvirus nucleic acid sequence.", "According to still further features in the described preferred embodiments the amino acid substitution in the HC-Pro gene of the conserved FRNK box of the potyvirus nucleic acid sequence includes a substitution of an Arg to Ile at position 180 within the HC-pro gene product (SEQ ID NO.", ": 5) of the potyvirus.", "According to still further features in the described preferred embodiments the insect vector is an aphid.", "According to still further features in the described preferred embodiments the sufficient potyvirus nucleic acid sequence is derived from zucchini yellow mosaic virus (ZYMV).", "According to still further features in the described preferred embodiments the additional nucleic acid sequence is at least a portion of a phosphinothricin acetyltransferase coding sequence.", "According to still further features in the described preferred embodiments the protein is at least a functional portion of a phosphinothricin acetyltransferase.", "According to still further features in the described preferred embodiments at least a portion of a plant treated according to the claimed method is an integral part of the invention, as are progeny of at least a portion of a plant treated according to the method.", "According to still further features in the described preferred embodiments an infectious virion harvested from a plant treated according to the claimed method is an integral part of the invention.", "The present invention successfully addresses the shortcomings of the presently known configurations by providing a vector for concurrently imparting herbicide resistance and cross protection against wild type virus.", "Concurrent receipt of these two effects from a single treatment contributes to increased crop yield and significantly reduces production costs.", "Reduction in production costs stein from both elimination of crop damage and from ease of introducing these traits into a wide variety of commercial plant strains.", "BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings.", "With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention.", "In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.", "In the drawings: FIGS.", "1a-d depict construction and use of a vector according to the present invention.", "FIG.", "1a is a schematic representation of the AGII genome.", "AGII non-coding (shaded), coding (open boxes) regions, and the bar gene (bar) are shown.", "Arrows indicate NIa protease involved in proteolysis of the bar gene product.", "NIa cleavage sites are indicated by /.", "Amino acid sequences corresponding to the NIa protease recognition motif are indicated in bold.", "The termini of the bar protein sequence are indicated by italics.", "FIG.", "1b depicts an RT-PCR analysis of AGII-Bar viral progeny RNAs.", "Total RNA was extracted from leaves of AGII-Bar and AGII systemically infected plants or from virus-free plants, and subjected to RT-PCR with primers flanking the bar gene insertion point.", "Positions of RT-PCR primers relative to AGII-Bar genome are shown schematically below.", "The expected size (bp) of the fragment with (1025) or without (476) the bar gene is marked by an arrow.", "HindIII-EcoRI-digested Lambda DNA was used as a molecular weight marker (M).", "FIG.", "1c is a histogram illustrating accumulation of AGII-Bar and AGII viruses in systemically infected squash 14 and 26 dpi.", "The level of each virus was determined by quantitative DAS-ELISA, and is the average of three independent samples taken from three different plants.", "FIG.", "1d shows representative leaves of squash, systemically infected with AGII-Bar or AGII four days after spraying with 0.25% Basta herbicide.", "FIGS.", "2a-c demonstrate that functional expression of bar via a vector according to the present invention confers resistance to glufosinate ammonium in-planta.", "FIGS.", "2a and 2b illustrate Squash (2a) and melon (2b) plants inoculated with either AGII-Bar (left pots) or AGII (right pots) and sprayed to runoff with 0.5% Basta at 24 or 10 dpi respectively.", "FIG.", "2c shows greenhouse grown cucumbers inoculated with AGII or AGII-Bar (as indicated) sprayed to runoff with 1% Basta at about 45 dpi.", "Days 0 and 10 indicate time after spraying.", "FIG.", "2d shows hydroponically grown squash inoculated with either AGII-Bar (left pot) or AGII (right pot).", "Basta (1%) was added in the water, 1 week after inoculation.", "Photographs were taken 7 days after Basta treatment.", "FIGS.", "3a-d illustrate Basta resistance of a variety of cucurbit species inoculated with a vector according to the present invention in the field.", "Watermelon Seedless (FIG.", "3a), Melon Ananas-type (FIG.", "3b), squash (FIG.", "3c), and cucumber (FIG.", "3d) were infected with AGII (right panel) or with AGII-Bar (left panel).", "Plants were sprayed 14 days after planting with 0.5% Basta.", "Pictures of representative plants were taken 5 days after spraying.", "FIGS.", "4a-c depict resistance of melons affected with a vector according to the present invention to herbicide in the field.", "FIG.", "4a shows melons (Ananas-type) infected with AGII (right panel) or with AGII-Bar (left panel).", "Plants were sprayed with 0.5% Basta 14 days after planting.", "Pictures of representative plants were taken 5 days after spraying.", "FIGS.", "4b and 4c depict weed eradication in a field of Galia-type melons inoculated with a vector according to the present invention.", "Photographs were taken 5 days (FIG.", "4b) and 18 days (FIG.", "4c) after spraying, with the indicated concentration of Basta or with water.", "FIGS.", "5a-c are histograms illustrating the effect of practice of a method according to the present invention on crop yield and fruit number.", "Data are the averages of 13 plants per treatment.", "FIG.", "5a shows numbers of squash fruit per plant collected during an 18-day period as a function of applied Basta concentration.", "FIGS.", "5b and 5c show the effect of increasing Basta concentration on Ananas and Galia type Melons inoculated with a vector according to the present invention.", "After 18 days of Basta treatment, potentially marketable sized fruit was weighed (5b) and all fruit was counted (5c).", "The percentage of Basta sprayed is indicated under each column.", "FIGS.", "6a-d illustrate viral cross protection in squash with a vector according to the present invention.", "6a shows leaves infected with a virulent strain of ZYMV (ZYMV-JV); 6b shows leaves not infected with virus (H); 6c shows leaves inoculated first with the attenuated ZYMV-AGII and then with the virulent ZYMV-JV.", "6d shows leaves inoculated first with the attenuated ZYMV-AGII carrying the heterologous human interferon alpha 2a (IFN) gene and then with the virulent ZYMV-JV.", "FIG.", "7 illustrates, by RT-PCR analysis, that ZYMV-AG carrying a foreign gene (IFN), prevents virulent ZYMV-JV RNA accumulation in Squash.", "Total RNA was extracted from leaves of squash plants inoculated with indicated viruses 28 days post ZYMV-JV challenge, and subjected to RT-PCR with primers flanking the IFN insertion point.", "pAGII and pIFN plasmids containing AGII and IFN cDNAs respectively.", "Mix-reaction mixture devoid of RNA.", "The expected size (bp) of the fragment with (955) or without (476) the IFN gene is marked by an arrow.", "HindIII-EcoRI-digested Lambda DNA was used as a molecular weight marker (M).", "DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of nucleic acid vectors capable of concurrently imparting herbicide resistance and viral cross protection, methods of use thereof and plants inoculated with same which can be used in commercial agriculture.", "Specifically, the present invention can be used to impart herbicide resistance to a crop while concurrently affording viral cross protection to the crop.", "The principles and operation of nucleic acid vectors capable of imparting herbicide resistance and viral cross protection, methods of use thereof and plants inoculated with same according to the present invention may be better understood with reference to the drawings and accompanying descriptions.", "Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings.", "The invention is capable of other embodiments or of being practiced or carried out in various ways.", "Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.", "For purposes of this specification and the accompanying claims, the terms “cross protection” and “cross protecting” refer to inoculation of a plant with an attenuated strain of a virus in order to impart protection against subsequent infection with a wild type strain of the same virus.", "Typically, cross protection protects more than 50% of the plants inoculated with the attenuated virus.", "This definition is essentially as described by H. Lecoq in “Control of plant virus diseases by cross protection” (Plant Virus Disease Control (1998) Hadidi A., Khetarpal R. K. and Koganezawa H.", "Eds., APS Press) For purposes of this specification and the accompanying claims, the term “inoculate” refers to any administration of a virally derived material which results in infection of a plant.", "Thus, inoculation may refer equally to administration of vector nucleic acid and to administration of virions.", "FIG.", "1a illustrates an example of a nucleic acid vector according to the present invention.", "These nucleic acid vectors are designed and constructed to concurrently impart herbicide resistance to a plant and cross protect the plant and.", "For purposes of this specification and the accompanying claims, the terms “concurrent” and “concurrently” refer to results which share a common causative event.", "Thus, herbicide resistance may occur before or after appearance of viral cross protection in a plant and the two traits may still be deemed to have been “concurrently imparted” if they are the result of inoculation with the same vector or virus.", "The vector includes sufficient potyvirus nucleic acid sequence to permit a potyvirus to replicate and spread within the plant infected by the vector.", "Preferably, the sufficient potyvirus nucleic acid sequence is derived from zucchini yellow mosaic virus (ZYMV).", "Vectors according to the present invention are infectious and accumulate titers similar to AGII (FIG.", "1c), although they are attenuated almost to the point of being asymptomatic (FIG.", "6).", "The vector further includes a first mutation in the potyvirus nucleic acid sequence which attenuates symptoms of the potyvirus in the plant infected by the vector.", "The first mutation may include, for example, an amino acid substitution in the HC-Pro gene (SEQ ID NO.", ": 4) of the conserved FRNK box of the potyvirus nucleic acid sequence.", "The amino acid substitution in the HC-Pro gene of the conserved FRNK box of the potyvirus nucleic acid sequence may be, for example, a substitution of a Arg to Ile at position 180 within the HC-pro gene product (SEQ ID NO.", ": 5) of the potyvirus.", "The vector further includes a second mutation in the potyvirus nucleic acid sequence which abolishes transmission of the potyvirus by an insect vector (Gal-On et al.", "(1992) J Gen Virol.", "73: 2183-2187).", "According to preferred embodiments of the invention, the second mutation includes an alteration of a conserved DAG triplet at position 8 in the coat protein (SEQ ID NOs.", ": 2 and 3) of the potyvirus.", "This alteration of the DAG preferably includes a substitution for an alanine residue in the DAG triplet.", "Alternately, the second mutation may include deletion of the DAG triplet altogether.", "This may be accomplished, for example, by removal or replacement of the Coat Protein N-terminal domain or a portion thereof (Arazi, T., et al.", "(2001) J Virol.", "75(14): 6329-36).", "Preferably, the insect vector is an aphid.", "The vector further includes additional nucleic acid sequence encoding a protein which imparts resistance to an herbicide when expressed in the plant infected by the vector.", "Preferably the additional nucleic acid sequence is at least a portion of a phosphinothricin acetyltransferase coding sequence and the protein is at least a functional portion of a phosphinothricin acetyltransferase.", "According to another aspect of the present invention there is provided a method of concurrently imparting herbicide resistance to a plant (FIGS.", "2, 3 and 4) and cross protecting the plant (FIG.", "6) against at least one potyvirus.", "The method includes inoculating at least a portion of the plant with a vector as described hereinabove As used in this specification and the accompanying claims, the phrase “at least a portion of the plant” may include, but is not limited to, at least one cell in the plant, at least one plant cell in tissue culture.", "The claimed invention is further embodied by at least a portion of a plant treated according to the claimed method.", "Progeny of at least a portion of a plant treated according to the method are also within the scope of the claimed invention, as are infectious virions harvested from a plant treated according to the claimed method.", "It will be appreciated that, prior to the advent of the claimed invention, cross protection was not generally practiced in commercial agriculture.", "However, because the claimed invention offers prevention of yield loss by both herbicide resistance for weed eradication in the field (FIG.", "5) and prevention of yield loss from viral infection in a single application, it is likely to increase the prevalence of viral cross protection in commercial settings.", "Together, these features offer unprecedented increases in crop yield.", "This is because both viruses and weeds are major sources of economic loss in commercial production of cucurbit crops.", "Further, the herbicide resistance conferring and cross protecting vectors of the present invention may readily be used to infect many different species and cultivars without the costly and time consuming process of generating transgenic or transformed plants.", "Therefore, any new cultivar on the market can be protected without additional breeding, seed collection and screening.", "Protection may be induced, for example, before planting or in the field.", "Further, vectors of the present invention are not germ line incorporated.", "Therefore, maximum selection pressure may be applied to desired traits without including selection for vector-born traits in the breeding plan.", "Further, vectors according to the present invention are not seed or pollen transmitted.", "This insures that the recombinant nucleic acid of the vector is not accidentally transmitted to progeny or to other plant species.", "This feature addresses issues of containment, which have slowed development of many plant biotechnology products.", "Further, vectors according to the present invention overcome the major problems previously associated with potyvirus vectors, i.e.", "loss due to disease and uncontrolled spread by insect vectors.", "Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting.", "Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.", "EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion.", "The following materials and methods were employed in executing the experiments described in the examples presented hereinbelow.", "Materials and Methods AGII-Bar Construction To construct AGII-bar, the bar gene (GenBank acc.", "nr.", "X17220) was amplified from pME509, by using a Taq polymerase and the oligonucleotide primers: sense 5′-ATGCTGCAGATGAGCCCAGAACGACGC-3′ (SEQ ID NO.", ": 7) and antisense 5′-AGTCTCGAGGATCTCGGTGACGGGCAG-3′ (SEQ ID NO.", ": 8) which added PstI and XhoI sites (underlined) to the 5′ and 3′ ends of the bar sequence respectively.", "The amplified fragment was double digested by PstI and XhoI and cloned into the PstI and SalI sites of the partial clone pKSΔSacI-PstI-poly (Arazi et al.", "(2001) Journal of Biotechnology 92:37-46).", "pKSΔSacI-PstI-poly-Bar clone was double-digested by SacI and MluI, and the resulting fragment containing the bar gene were cloned into the AGII genome (Arazi et al.", "(2001) Journal of Biotechnology 92:37-46) between the coat protein (CP) and the NIb coding regions, to create AGII-Bar (FIG.", "1).", "Plant Growth, Inoculation, and Evaluation of Virus Infection Potted squash and melon plants were grown in a growth chamber under continuous light at 23 degrees C. Cucumbers (cv.", "Muhassan) were grown in 20 Liter pails with automatic irrigation and fertilization, in an insect-proof net-house.", "Hydroponically grown squash were seeded in vermiculite that was placed on nylon nets floated in a water-filled container.", "Particle bombardment with a hand held device, the HandGun (Gal-On et al.", "(1997) Journal of Virology Methods 64:103-110), was used to propel micro-projectiles containing a plasmid with AGII-Bar cDNA into the fully expanded cotyledons of different cucurbits.", "Mechanical inoculation of seedlings and enzyme-linked immunosorbent assay (ELISA) with anti-ZYMV CP polyclonal antibody, performed on infected plant material, were performed as described previously by Antignus et al.", "(1989; Phytoparasitica 17: 289-298).", "ELISA Assays for Evaluation of Infectivity and Viral Titer Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with anti-ZYMV CP polyclonal antibody (1:2000), was performed on infected plant material, as described previously by Antignus et al.", "(1989; Phytoparasitica 17, 289-298).", "For quantitative analysis, three squash plants from each treatment, AGII and AGII-Bar were chosen.", "Eight leaf discs per plant were taken from two different leaves 14 and 26 days post inoculation (dpi), combined, and the homogenized tissue samples were subjected to DAS-ELISA.", "All samples were collected from developmentally equivalent leaves at the indicated dpi.", "The significance of the differences in the accumulation of the AGII and AGII-Bar was determined by ANOVA statistical analysis with Statview statistical software package.", "RT-PCR Analysis of Recombinant Virus Progeny RT-PCR of viral progeny was conducted in a one-tube single-step method modified from Seliner et al.", "(1992; Nucleic Acids Research 20:1487-1490).", "Briefly, a 50-μl volume was used containing the polylinker flanking primers 5′-AGCTCCATACATAGCTGAGACA-3′ (SEQ ID NO.", ": 9) and 5′-TGGTTGAACCAAGAGGCGAA-3′ (SEQ ID NO.", ": 10) in the following mixture: 1.5 mM MgCl2; 125 μM dNTPs; 1× Sellner buffer: [10× Sellner buffer contains: 670 mM Tris-HCl; 170 mM (NH4)2SO4; 10 mM beta-mercapto-ethanol; 2 mg/ml gelatin (Aldrich, calf skin 225 bloom); 60 μM EDTA pH 8.0 (Sellner et al., 1992)]; 100 ng of each specific primer; 2 units of Taq polymerase; 5 units of AMV-RT (Chimerex USA); 2-5 μg total RNA.", "RT-PCR cycles were as follows: 46 degrees C. 30 min; 94 degrees C. 2 min, followed by 33 cycles at 94 degrees C., 60 degrees C. and 72 degrees C., each of 30 s., and one final cycle of 5 min at 72 degrees C. Field Experiment Two varieties of melon (Galia-type cv.", "5080 and Ananas-type cv.", "Ofir), two varieties of watermelon (cvs Crimson and Seedless 313), squash (cv.", "Maayan) and cucumber (cv.", "Shimshon) were seeded in Speedling type trays.", "Seedlings were inoculated by bombardment of cotyledons with the AGII-Bar or AGII construct.", "Plants were planted in a 500 m2 field, in a sandy loam soil, at 0.5 m intervals in six 2 m wide rows and irrigated by a single line of 0.5 m interval dripline.", "Each row included 15 plants of each of the five-cucurbit varieties, 13 plants inoculated with AGII-Bar and two inoculated with AGII as a control.", "Irrigation was initiated a week previously to boost weeds and facilitate planting.", "Rows were sprayed 13 days after planting with a motorized back-mounted sprayer (Solo, Germany), installed with a boom with 4 overlapping (distance 0.5 m, total width 2 m) T-jet 11002 nozzles (Spraying Systems, USA).", "Spray height was 0.5 m; pressure 40 PSI, with an actual average of 300 L/hectare.", "Plants were sprayed with different dosages of the glufosinate-ammonium herbicide Basta 20 (AgrEvo, Germany) containing 200 g/L active ingredient.", "The active ingredient was calculated per hectare (g a.i./ha) on basis of ground speed measured separately for each treatment.", "Treatments were given per row: “1.5% Basta”—930 g a.i./ha, “1% Basta”—600 g a.i./ha, “0.5% Basta+AMS”—300 g a.i./ha augmented with ammonium sulphate (AMS) 1.5 g/L., “0.5% Basta”—270 g a.i./ha (without AMS), “0.25% Basta”—165 g a.i./ha and finally “Water” as untreated control.", "Squash fruit were picked and counted daily.", "Melon fruit were picked once 66 days after planting and were manually sorted into potentially marketable and unmarketable sizes.", "All AGII-Bar inoculated plants were picked from each of the six treatment rows.", "Each group was counted and then weighed collectively.", "Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques.", "Such techniques are thoroughly explained in the literature.", "See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.", "(1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md.", "(1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al.", "(eds) “Genome Analysis: A Laboratory Manual Series”, Vols.", "1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat.", "Nos.", "4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed.", "(1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y.", "(1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed.", "(1994); Stites et al.", "(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat.", "Nos.", "3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.", "(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds.", "(1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds.", "(1994); “Animal Cell Culture” Freshney, R. I., ed.", "(1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.", "1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); and “Using Antibodies: A Laboratory Manual” (Ed Harlow, David Lane eds., Cold Spring Harbor Laboratory Press (1999)) all of which are incorporated by reference as if fully set forth herein.", "Other general references are provided throughout this document.", "The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.", "All the information contained therein is incorporated herein by reference.", "EXAMPLE 1 Construction of the AGII-Bar Vector In order to express the phosphinothricin acetyltransferase gene (hereinafter bar gene; SEQ ID NO.", ": 1) in the AGII virus-vector, the gene was inserted between the NIb (Genbank accession number L29569) and CP (SEQ ID NO.", ": 2) genes using a polylinker-cloning site next to the NIa proteinase cleavage site in the NIb 3′ end of AGII (FIG.", "1a).", "The inserted gene was designed to create an in-frame translational fusion with the flanking NIa processing sites.", "Proteolysis of the nascent AGII-Bar polyprotein by NIa protease in trans was predicted to yield the bar gene product (SEQ ID NO.", ": 6) with seven additional amino acid residues (VDTVMLQ) at its C′-terminus (FIG.", "1a; SEQ ID NO.", ": 6).", "EXAMPLE 2 Assay of Infectivity of the AGII-Bar Vector AGII-Bar was 100% infectious on susceptible squash.", "Symptoms appeared 7-8 days post-inoculation (dpi) with similar characteristics to those of the parental AGII virus.", "Squash was employed as the test plant because it is the only cucurbit in which the attenuated AGII symptoms are visible.", "No symptoms at all are seen on cucumber, melon, pumpkin and watermelon.", "AGII symptoms in squash include slight vein clearing in young leaves and light patches on older leaves.", "Slight dark patches appear on fruit of light colored varieties of squash.", "No deformation or filiform leaf appearance characteristic of the wild type ZYMV are visible.", "Wild type ZYMV infected plants are highly deformed in leaf and fruit, foliar symptoms consisting of a prominent yellow mosaic, necrosis, distortion, and stunting.", "Fruits remain small, greatly malformed, and green mottled causing total loss of yield.", "In sharp contrast, AGII infected plants, whether carrying a foreign gene or not, yield fruit in number, weight and quality equivalent to virus free plants in the field or in the greenhouse.", "100% infectivity was also observed in cucumber, melon and watermelon, though no symptoms were visible in these plants.", "The presence of the intact bar coding sequence was verified by RT-PCR of the viral progeny (FIG.", "1b) and direct sequencing of the amplified product.", "AGII-Bar, accumulated to levels similar to the parental AGII (no significant difference) 14 and 26 dpi in squash, as determined by quantitative DAS-ELISA (FIG.", "1c).", "The resistance of AGII-Bar infected plants to 0.25% Basta treatment was easily discernible (FIG.", "1d).", "EXAMPLE 3 Induction of Gluofosinate Ammonium (Basta) Resistance in Curcurbit Species Using the Vector The biological activity of the bar gene product translated from the inoculated vector was tested in the greenhouse.", "Various concentrations of the glufosinate ammonium based herbicide Basta (Hoechst-Schering AgrEvo, Berlin, Germany) were applied to foliage of squash plants mechanically inoculated with AGII or AGII-Bar from second-generation infected plants.", "26 dpi, plants were sprayed till runoff with Basta.", "All sprayed AGII inoculated plants developed widespread necrosis and died in less than 48 Hrs including plants sprayed with the lowest dose used, 0.125% (Table 1).", "In contrast, all AGII-Bar plants survived and no necrosis was observed on young and newly emerged leaves even at the highest concentration, 1.0% (Table 1).", "A representative squash plant is shown in FIG.", "2a.", "Some herbicide mediated necrosis was observed, mainly on cotyledons and on the first 2-3 leaves, and was positively correlated to the Basta concentration (Table 1).", "TABLE 1 Responses of potted squash plants inoculated with AGII-Bar or with AGII to foliar application of glufosinate ammonium (Basta) in the greenhouse.", "AGII AGII-Bar % Basta Survival a Survival Necrosis index b Unsprayed 3/3 5/5 − 0.125 0/3 10/10 + 0.25 0/3 10/10 ++ 0.5 0/3 11/11 +++ 1.0 0/3 8/8 ++++ a Survival from total tested plants b Necrosis on first 2-3 leaves and cotyledons: − no necrosis or lesions; + few and mild lesions; ++ few patchy lesions; +++ large necrotic areas; ++++ two thirds or more of the leaf area are necrotic.", "Similar results were obtained with melon seedlings (FIG.", "2b).", "As squash and melon were found to be resistant to 1.0%, this concentration was tested on a commercial variety of parthenocarpic cucumber.", "Cucumber seedlings were inoculated with AGII-Bar or AGII and planted in a nethouse.", "One and a half months post inoculation, when these plants were fully developed and fruiting, they were sprayed with 1.0% Basta.", "Forty-eight hours after spraying, leaves of AGII inoculated plants were completely shriveled and dry (FIG.", "2c, at 10 days after spray).", "AGII-Bar plants did not sustain observable damage and continued to develop normally (FIG.", "2c).", "In an effort to discern the extent of protection afforded by the bar gene, squash plants were grown in a hydroponic system and inoculated with AGII-Bar or control AGII construct.", "Seven days post inoculation 0.2% ammonium glufosinate (1% Basta) was added to the water in which the roots were immersed.", "Two days later AGII inoculated plants had died whereas AGII-Bar plants continued to develop though some necrosis was observed, especially to older leaves (FIG.", "2d).", "EXAMPLE 4 Assay of Glufosinate Ammonium (Basta) Resistance in the Field In order to verify the applicability of AGII-Bar to commercial cucurbit cultivation in a field prone to weed infestation an experiment including more than 450 plants was conducted.", "Plants were inoculated prior to planting by bombardment with AGII-Bar (FIGS.", "3a, 3b, 3c and 3d; left hand photograph and FIGS.", "4b and 4c), or with AGII (FIGS.", "3a, 3b, 3c and 3d; right hand photograph) as a control.", "Two varieties of melon, two varieties of watermelon, squash and cucumber were planted in the field.", "The field was pre-irrigated for one week by drippers to increase weed proliferation and facilitate planting.", "Ten day post inoculation, the infection rate was determined by ELISA of 50 random plant samples and found to be 100%.", "Two weeks after planting the field was sprayed by rows with different doses of Basta, with or without AMS (FIGS.", "4a, 4b and 4c), used as an adjuvant (Maschhoff, J. R., (2000).", "Weed Sci.", "48, 2-6).", "The control row was sprayed with water.", "After just 24 hours the damage to AGII inoculated plants and weeds became evident, and in 3-5 days all AGII inoculated plants and most weeds had browned, shriveled and died (FIGS.", "4a and 3 a-d; right hand photograph).", "This response was dose dependent as expected (FIG.", "4c).", "In sharp contrast, all AGII-Bar inoculated plants survived, although some minor necrotic lesions occurred on the first leaves, at high dosages of Basta.", "This damage did not adversely affect the vigorous new growth (FIGS.", "4a-c and 3a-d).", "The fact that the weed density in the field would have been high in the absence of herbicide application is demonstrated by the water sprayed rows in Galia type melon (FIGS.", "4b and 4c) All weeds including nutgrass (Cyperus rotundus L.), little hogweed (Portulaca oleracea L.), pigweed (Amaranthus sp.)", "and several annual graminaceous weeds were eliminated in 1-1.5% Basta (FIG.", "4c), and most weeds were eliminated or suppressed at 0.25-0.5% as well (FIG.", "4c).", "AMS did not exert a detectable difference on herbicidal activity (FIGS.", "4b and c).", "Herbicide dose-related weed suppression was sustained for the duration of the experiment, 53 days from spray to harvest.", "The dose effect of applied glufosinate ammonium on yields of weed-infested fields of squash and of Galia-type and Ananas-type melons was also examined (FIGS.", "5a-c).", "Squash cv.", "Maayan fruit were picked regularly, collectively by treatment, during an 18-day period.", "An average count of all thirteen plants picked per row was calculated (FIG.", "5a).", "In the control water-sprayed row, the number of fruit per plant, 3.8, was 1.5-2.5 times less than in 0.25%-1.0% Basta sprayed rows respectively (FIG.", "5a).", "The highest average number of fruit per plant was counted at 1% and 1.5%, 9.8 and 9 fruit respectively.", "Melons were picked several days prematurely, 66 days from planting.", "Potentially marketable yields of 0.25-1.5% Basta sprayed rows, of both melon types, were about 2.5 to more than 4-fold higher than water sprayed control, and showed a positive dose dependant response till a maximum achieved at 1% (FIG.", "5b).", "As all the melons were picked at once, unselectively, a second parameter for potential yield, fruit number, which included undersized fruits, was assessed.", "The number of fruit, which had set in the Ananas-type cultivar at the 0.25% Basta treatment (FIG.", "5c, white bars), was about the same as the water sprayed control.", "The highest number of fruit set at the 1.5% Basta treatment, twice as high as the control.", "However, the number of fruit which had set in the Galia-type cultivar (FIG.", "5c, gray bars), which was distinctly positively dose-related, was markedly higher than the water sprayed control, even at the lower concentrations of Basta.", "In this cultivar, in the 0.25% Basta treatment, twice as many fruit set than in the control, and the number of fruit which set in the 1.5% treatment, which had the highest fruit count of all treatments, was about fourfold higher than the control.", "These results confirm the ability of the AGII-Bar vector to impart herbicide resistance determined in Example 2 in the field and further indicate that the imparted herbicide resistance allows application of an herbicide during the production cycle so that crop yield may be significantly increased.", "EXAMPLE 5 Assay of Recombinant AGII Vector Cross Protection In order to establish that an attenuated ZYMV vector with impaired aphid transmissibility could impart viral cross protection, and that this ability was not diminished by introduction of a heterologous gene into the vector, 20 squash plants (cv Maayan) were seeded in pots and grown in a green house.", "Nine days after mechanical pre-inoculation with AGII or AGII-IFN (an AGII construct carrying a foreign gene (human Interferon alpha 2a; Arazi T., et al.", "(2001) J Biotechnol.", "87(1): 67-82.", "), plants were challenged by mechanical inoculation with wild type virulent ZYMV-JV.", "Development of symptoms was monitored and recorded (see FIGS.", "6 a-d and table 2).", "Only ZYMV-JV infected leaves are deformed (FIG.", "6a).", "Leaves from plants pre inoculated with AGII (FIG.", "6c) or AGII-IFN (FIG.", "6d) challenged with ZYMV-JV resembled leaves from virus free plants (FIG.", "6b).", "RT-PCR analysis of extracts prepared 28 days post ZYMV-JV challenge from all plants was performed.", "Results are presented in FIG.", "7.AGII or ZYMV-JV infection of plants is expected to yield a 436 bp PCR fragment.", "The AGII-IFN construct is expected to yield a 955 bp fragment.", "FIG.", "7 clearly demonstrates that all plants pre-inoculated with AGII-IFN were protected from the virulent wild type ZYMV-JV virus (lanes AGII-IFN+ZYMV-JV).", "These plants showed no disease symptoms (FIG.", "6a-d) and AGII-IFN plants had only one PCR band at 955 bp as expected (FIG.", "7 and table 2).", "TABLE 2 Viral cross protection experiment treatment protocol and results DNA fragment Visible size (amplified results 26 by RT-PCR, 28 Primary Secondary days post- days post- Treatment inoculation inoculation challenge challenge ) AGII AGII No 2/2 436 bp Attenuated AGII-IFN AGII-IFN No 2/2 955 bp Attenuated AGII + AGII ZYMV-JV 6/6 436 bp ZYMV-JV Attenuated AGII-IFN + AGII-IFN ZYMV-JV 6/6 955 bp ZYMV-JV Attenuated ZYMV-JV No ZYMV-JV 2/2 436 bp Blisters, mosaic, filiform leaves Healthy No No 2/2 Healthy None Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art.", "Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.", "All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.", "In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention." ] ]	Patent_10466794
[ [ "Virus causing respiratory tract illness in susceptible mammals", "The invention relates to the field of virology.", "The invention provides an isolated essentially mammalian negative-sense single stranded RNA virus (MPV) within the sub-family Pneumovirinae of the family Paramyxoviridae and identifiable as phylogenetically corresponding to the genus Metapneumovirus and components thereof." ], [ "1.An isolated essentially mammalian negative-sense single stranded RNA virus (MPV) belonging to the sub-family Pneumovirinae of the family Paramyxouiridae and identifiable as phylogenetically corresponding to the genus Metapneumovirus 2.An isolated negative-sense single stranded RNA virus (MPV) belonging to the sub-family Pneumovirinae of the family Paramyxovirdae and identifiable as phylogenetically corresponding to the genus Metapneumovirus by determining a nucleic acid sequence of said virus and testing it in phylogenetic tree analyses wherein maximum likelihood trees are generated using 100 bootstraps and 3 jumbles and finding it to be more closely phylogenetically corresponding to a virus isolate deposited as I-2614 with CNCM, Paris than it is corresponding to a virus isolate of avian pneumovirus (APV) also known as turkey rhinotracheitis virus (TRTV), the aetiological agent of avian rhinotracheitis.", "3.A virus according to claim 2 wherein said avian pneumovirus comprises APV type C (APV-C).", "4.A virus according to claim 1 to 3 wherein said nucleic acid sequence comprises an open reading frame (ORF) encoding a viral protein of said virus.", "5.A virus according to claim 4 wherein said open reading frame is selected from the group of ORFs encoding the N, P, M, and F proteins.", "6.A virus according to claim 5 wherein said open reading frame is selected from the group of ORFs encoding the SH or G proteins.", "7.A virus according to anyone of claims 1 to 6 comprising a nucleic acid or functional fragment phylogenetically corresponding to a sequence shown in FIG.", "6.8.A virus according to anyone of claims 1 to 7 comprising an MPV isolate deposited as I-2614 with CNCM, Institute Pasteur, Paris or a virus isolate phylogenetically corresponding therewith.", "9.A virus according to claim 8 isolatable from a human with respiratory tract illness.", "10.An isolated or recombinant nucleic acid or MPV-specific functional fragment thereof obtainable from a virus according to anyone of claims 1 to 9.11.A vector comprising a nucleic acid according to claim 10.12.A host cell comprising a nucleic acid according to claim 10 or a vector according to claim 11.13.An isolated or recombinant proteinaceous molecule or MPV-specific functional fragment thereof encoded by a nucleic acid according to claim 10.14.An antigen comprising a proteinaceous molecule or MPV-specific functional fragment thereof according to claim 13.15.An antibody specifically directed against an antigen according to claim 14.16.A method for identifying a viral isolate as an MPV comprising reacting said viral isolate or a component thereof with an antibody according to claim 15.17.A method for identifying a viral isolate as an MPV comprising reacting said viral isolate or a component thereof with a nucleic acid according to claim 10.18.A method according to claim 16 or 17 wherein said MPV comprises a human MPV.", "19.A viral isolate identifiable with a method according to anyone of claims 16 to 18 as a mammalian negative-sense single stranded RNA virus within the sub-family Pneumovirinae of the family Paramyxoviridae and identifiable as phylogenetically corresponding to the genus Metapneumovirus.", "20.A method for virologically diagnosing an MPV infection of a mammal comprising determining in a sample of said mammal the presence of a viral isolate or component thereof by reacting said sample with a nucleic acid according to claim 10 or an antibody according to claim 15.21.A method for serologically diagnosing an MPV infection of a mammal comprising determining in a sample of said mammal the presence of an antibody specifically directed against an MPV or component thereof by reacting said sample with a proteinaceous molecule or fragment thereof according to claim 13 or an antigen according to claim 14.22.A diagnostic kit for diagnosing an MPV infection comprising a virus according to anyone of claims 1 to 9, a nucleic acid according to claim 10, a proteinaceous molecule or fragment thereof according to claim 13, an antigen according to claim 14 and/or an antibody according to claim 15.23.Use of a virus according to any one claims 1 to 9, a nucleic acid according to claim 10, a vector according to claim 11, a host cell according to claim 12, a proteinaceous molecule or fragment thereof according to claim 13, an antigen according to claim 14, or an antibody according to claim 15 for the production of a pharmaceutical composition.", "24.Use according to claim 23 for the production of a pharmaceutical composition for the treatment or prevention of an MPV infection.", "25.Use according to claim 23 or 24 for the production of a pharmaceutical composition for the treatment or prevention of respiratory tract illnesses.", "26.A pharmaceutical composition comprising a virus according to any one claims 1 to 9, a nucleic acid according to claim 10, a vector according to claim 11, a host cell according to claim 12, a proteinaceous molecule or fragment thereof according to claim 13, an antigen according to claim 14, or an antibody according to claim 15.27.A method for the treatment or prevention of an MPV infection comprising providing an individual with a pharmaceutical composition according to claim 26.28.A method for the treatment or prevention of a respiratory illness comprising providing an individual with a pharmaceutical composition according to claim 26.29.A method according to claim 27 or 28 wherein said individual comprises a human.", "30.A method to obtain an antiviral agent useful in the treatment of respiratory tract illness comprising establishing a cell culture or experimental animal comprising a virus according to any one of claims 1 to 9, treating said culture or animal with an candidate antiviral agent, determining the effect of said agent on said virus or its infection of said culture or animal, and selecting an anitviral agent with the desired effect.", "31.An antiviral agent obtainable according to the method of claim 30.32.Use of an antiviral agent according to claim 31 for the preparation of a pharmaceutical composition.", "33.Use according to claim 33 for the preparation of a pharmaceutical composition for the treatment of respiratory tract illness.", "34.Use according to claim 32 or 33 for the preparation of a pharmaceutical composition for the treatment of an MPV infection.", "35.A pharmaceutical composition comprising an antiviral agent according to claim 31.36.A method for the treatment or prevention of an MPV infection comprising providing an individual with a pharmaceutical composition according to claim 35.37.A method for the treatment or prevention of a respiratory illness comprising providing an individual with a pharmaceutical composition according to claim 35.38.A method according to claim 36 or 37 wherein said individual comprises a human.", "39.A method for virologically diagnosing an MPV infection of an animal comprising determining in a sample of said animal the presence of a viral isolate or component thereof by reacting said sample with a nucleic acid or an antibody specifically reactive with a component of an avian pneumovirus (APV), said nucleic acid or antibody being cross-reactive with a component MPV.", "40.A method for serologically diagnosing an MPV infection of an animal comprising determining in a sample of said animal the presence of an antibody directed against an MPV or component thereof by reacting said sample with a proteinaceous molecule or fragment thereof or antigen derived from an APV isolate or component thereof, said molecule, fragment or antigen selected for being essentially homologous with a component of MPV.", "41.A method for virologically diagnosing an APV infection of a bird comprising determining in a sample of said bird the presence of a viral isolate or component thereof by reacting said sample with a nucleic acid according to claim 10 or an antibody according to claim 15 said nucleic acid or antibody being cross-reactive with a component of APV.", "42.A method for serologically diagnosing an APV infection of a bird comprising determining in a sample of said bird the presence of an antibody specifically directed against an APV or component thereof by reacting said sample with a proteinaceous molecule or fragment thereof according to claim 13 or an antigen according to claim 14, said molecule, fragment or antigen selected for being essentially homologous with a component of APV.", "43.A method according to anyone of claims 39 to 42 wherein said APV comprises APV-C. 44.Use of a diagnostic test designed to detect APV specific antibodies for the detection of an antibody directed against MPV.", "45 Use according to claim 44 wherein said test comprises an enzyme immune assay (EIA).", "46.A method for the detection of an antibody directed against MPV in a sample comprising testing said sample in a diagnostic test designed to detect APV specific antibodies.", "47.A method according to claim 46 wherein said test comprises an enzyme immune assay (EIA)." ], [ "The invention relates to the field of virology.", "In the past decades several etiological agents of mammalian disease, in particular of respiratory tract illnesses (RTI), in particular of humans, have been identified7.Classical etiological agents of RTI with mammals are respiratory syncytial viruses belonging to the genus Pneumovirus found with humans (hRSV) and ruminants such as cattle or sheep (bRSV and/or oRSV).", "In human RSV differences in reciprocal cross neutralization assays, reactivity of the G proteins in immunological assays and nucleotide sequences of the G gene are used to define 2 hRSV antigenic subgroups.", "Within the subgroups the aa sequences show 94% (subgroup A) or 98% (subgroup B) identity, while only 53% aa sequence identity is found between the subgroups.", "Additional variability is observed within subgroups based on monoclonal antibodies, RT-PCR assays and RNAse protection assays.", "Viruses from both subgroups have a worldwide distribution and may occur during a single season.", "Infection may occur in presence of pre-existing immunity and the antigenic variation is not strictly required to allow re-infection.", "See for example Sullender, W. M., Respiratory Syncytial Virus Genetic and Antigenic Diversity.", "Clinical Microbiology Reviews, 2000.13(1): p. 1-15; Collins, P. L., McIntosh, K. and Chanock, R. M., Respiratory syncytial virus.", "Fields virology, ed.", "B. N. Knipe, Howley, P. M. 1996, Philadelphia: lippencott-raven.", "1313-1351; Johnson, P. R., et al., The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: extensive sequence divergence between antigenically related proteins.", "Proc Natl Acad Sci USA, 1987.84(16): p. 5625-9; Collins, P. L., The molecular Biology of Human Respiratory Syncytial Virus (RSV) of the Genus Pneumovirus, in The Paramyxoviruses, D. W. Kingsbury, Editor.", "1991, Plenum Press: New York.", "p. 103-153.Another classical Pneumovirus is the pneumonia virus of mice (PVM), in general only found with laboratory mice.", "However, a proportion of the illnesses observed among mammals can still not be attributed to known pathogens.", "The invention provides an isolated essentially mammalian negative-sense single stranded RNA virus (MPV) belonging to the sub-family Pneumovirinae of the family Paramyxoviridae and identifiable as phylogenetically corresponding to the genus Metapneumovirus.", "Said virus is identifiable as phylogenetically corresponding to the genus Metapneumouvirus by determining a nucleic acid sequence of said virus and testing it in phylogenetic analyses, for example wherein maximum likelihood trees are generated using 100 bootstraps and 3 jumbles and finding it to be more closely phylogenetically corresponding to a virus isolate deposited as I-2614 with CNCM, Paris than it is corresponding to a essentially avian virus isolate of avian pneumovirus (APV) also known as turkey rhinotracheitis virus (TRTV), the aetiological agent of avian rhinotracheitis.", "For said phylogenetic analyses it is most useful to obtain the nucleic acid sequence of a non-MPV as outgroup to be compared with, a very useful outgroup isolate can be obtained from avian pneumovirus serotype C (APV-C), as is for example demonstrated in FIG.", "5 herein.", "Although phylogenetic analyses provides a convenient method of identifying a virus as an MPV several other possibly more straightforward albeit somewhat more course methods for identifying said virus or viral proteins or nucleic acids from said virus are herein also provided.", "As a rule of thumb an MPV can be identified by the percentages of a homology of the virus, proteins or nucleic acids to be identified in comparison with isolates, viral proteins, or nucleic acids identified herein by sequence or deposit.", "It is generally known that virus species, especially RNA virus species, often constitute a quasi species wherein a cluster of said viruses displays heterogeneity among its members.", "Thus it is expected that each isolate may have a somewhat different percentage relationship with one of the various isolates as provided herein.", "When one wishes to compare with the deposited virus I-2614, the invention provides an isolated essentially mammalian negative-sense single stranded RNA virus (MPV) belonging to the sub-family Pneumovirinae of the family Paramyxoviridae and identifiable as phylogenetically corresponding to the genus Metapneumovirus by determining an amino acid sequence of said virus and determining that said amino acid sequence has a percentage amino acid homology to a virus isolate deposited as I-2614 with CNCM, Paris which is essentially higher than the percentages provided herein for the L protein, the M protein, the N protein, the P protein, or the F protein, in comparison with APV-C or, likewise, an isolated essentially mammalian negative-sense single stranded RNA virus (MPV) belonging to the sub-family Pneumovirinae of the family Paramyxoviridae is provided as identifiable as phylogenetically corresponding to the genus Metapneumovirus by determining a nucleic acid sequence of said virus and determining that said nucleic acid sequence has a percentage nucleic acid identity to a virus isolate deposited as I-2614 with CNCM, Paris which is essentially higher than the percentages identified herein for the nucleic acids encoding the L protein, the M protein, the N protein, the P protein, or the F protein as identified herein below in comparison with APV-C. Again as a rule of thumb one may consider an MPV as belonging to one of the two serological groups of MPV as identified herein when the isolates or the viral proteins or nuclear acids of the isolates that need to be identified have percentages homology that fall within the bounds and metes of the percentages of homology identified herein for both separate groups, taking isolates 00-1 or 99-1 as the respective isolates of comparison.", "However, when the percentages of homology are smaller or there is more need to distinguish the viral isolates from for example APV-C it is better advised to resort to the phylogenetic analyses as identified herein.", "Again one should keep in mind that said percentages can vary somewhat when other isolates are selected in the determination of the percentage of homology.", "With the provision of this MPV, the invention provides diagnostic means and methods and therapeutic means and methods to be employed in the diagnosis and/or treatment of disease, in particular of respiratory disease, in particular of mammals, more in particular in humans.", "However, due to the, albeit distant, genetic relationship of the essentially mammalian MPV with the essentially avian APV, in particular with APV-C, the invention also provides means and methods to be employed in the diagnosis and treatment of avian disease.", "In virology, it is most advisory that diagnosis and/or treatment of a specific viral infection is performed with reagents that are most specific for said specific virus causing said infection.", "In this case this means that it is preferred that said diagnosis and/or treatment of an MPV infection is performed with reagents that are most specific for MPV.", "This by no means however excludes the possibility that less specific, but sufficiently cross-reactive reagents are used instead, for example because they are more easily available and sufficiently address the task at hand.", "Herein it is for example provided to perform virological and/or serological diagnosis of MPV infections in mammals with reagents derived from APV, in particular with reagents derived from APV-C, in the detailed description herein it is for example shown that sufficiently trustworthy serological diagnosis of MPV infections in mammals can be achieved by using an ELISA specifically designed to detect APV antibodies in birds.", "A particular useful test for this purpose is an ELISA test designed for the detection of APV antibodies (e.g in serum or egg yolk), one commercialy available version of which is known as APV-Ab SVANOVIR® which is manufactured by SVANOVA Biotech AB, Uppsal Science Park Glunten SE-751 83 Uppsala Sweden.", "The reverse situation is also the case, herein it is for example provided to perform virological and/or serological diagnosis of APV infections in mammals with reagents derived from MPV, in the detailed description herein it is for example shown that sufficiently trustworthy serological diagnosis of APV infections in birds can be achieved by using an ELISA designed to detect MPV antibodies.", "Considering that antigens and antibodies have a lock-and-key relationship, detection of the various antigens can be achieved by selecting the appropriate antibody having sufficient cross-reactivity.", "Of course, for relying on such cross-reactivity, it is best to select the reagents (such as antigens or antibodies) under guidance of the amino acid homologies that exist between the various (glyco)proteins of the various viruses, whereby reagents relating to the most homologous proteins will be most useful to be used in tests relying on said cross-reactivity.", "For nucleic aciddetection, it is even more straightforward, instead of designing primers or probes based on heterologous nucleic acid sequences of the various viruses and thus that detect differences between the essentially mammalian or avian Metapneumoviruses, it suffices to design or select primers or probes based on those stretches of virus-specific nucleic acid sequences that show high homology.", "In general, for nucleic acid sequences, homology percentages of 90% or higher guarantee sufficient cross-reactivity to be relied upon in diagnostic tests utilizing stingent conditions of hybridisation.", "The invention for example provides a method for virologically diagnosing a MPV infection of an animal, in particular of a mammal, more in particular of a human being, comprising determining in a sample of said animal the presence of a viral isolate or component thereof by reacting said sample with a MPV specific nucleic acid a or antibody according to the invention, and a method for serologically diagnosing an MPV infection of a mammal comprising determining in a sample of said mammal the presence of an antibody specifically directed against an MPV or component thereof by reacting said sample with a MPV-specific proteinaceous molecule or fragment thereof or an antigen according to the invention.", "The invention also provides a diagnostic kit for diagnosing an MPV infection comprising an MPV, an MPV-specific nucleic acid, proteinaceous molecule or fragment thereof, antigen and/or an antibody according to the invention, and preferably a means for detecting said MPV, MPV-specific nucleic acid, proteinaceous molecule or fragment thereof, antigen and/or an antibody, said means for example comprising an excitable group such as a fluorophore or enzymatic detection system used in the art (examples of suitable diagnostic kit format comprise IF, ELISA, neutralization assay, RT-PCR assay).", "To determine whether an as yet unidentified virus component or synthetic analogue thereof such as nucleic acid, proteinaceous molecule or fragment thereof can be identified as MPV-specific, it suffices to analyse the nucleic acid or amino acid sequence of said component, for example for a stretch of said nucleic acid or amino acid, preferably of at least 10, more preferably at least 25, more preferably at least 40 nucleotides or amino acids (respectively), by sequence homology comparison with known MPV sequences and with known non-MPV sequences APV-C is preferably used) using for example phylogenetic analyses as provided herein.", "Depending on the degree of relationship with said MPV or non-MPV sequences, the component or synthetic analogue can be identified.", "The invention also provides method for virologically diagnosing an MPV infection of a mammal comprising determining in a sample of said mammal the presence of a viral isolate or component thereof by reacting said sample with a cross-reactive nucleic acid derived from APV (preferably serotype C) or a cross-reactive antibody reactive with said APV, and a method for serologically diagnosing an MPV infection of a mammal comprising determining in a sample of said mammal the presence of a cross-reactive antibody that is also directed against an APV or component thereof by reacting said sample with a proteinaceous molecule or fragment thereof or an antigen derived from APV.", "Furthermore, the invention provides the use of a diagnostic kit initially designed for AVP or AVP-antibody detection for diagnosing an MPV infection, in particular for detecting said MPV infection in humans.", "The invention also provides method for virologically diagnosing an APV infection in a bird comprising determining in a sample of said bird the presence of a viral isolate or component thereof by reacting said sample with a cross-reactive nucleic acid derived from MPV or a cross-reactive antibody reactive with said MPV, and a method for serologically diagnosing an APV infection of a bird comprising determining in a sample of said bird the presence of a cross-reactive antibody that is also directed against an MPV or component thereof by reacting said sample with a proteinaceous molecule or fragment thereof or an antigen derived from MPV.", "Furthermore, the invention provides the use of a diagnostic kit initially designed for MPV or MPV-antibody detection for diagnosing an APV infection, in particular for detecting said APV infection in poultry such as a chicken, duck or turkey.", "As said, with treatment, similar use can be made of the cross-reactivity found, in particular when circumstances at hand make the use of the more homologous approach less straightforward.", "Vaccinations that can not wait, such as emergency vaccinations against MPV infections can for example be performed with vaccine-preparations derived from APV(preferably type C) isolates when a more homologous MPV vaccine is not available, and, vice versa, vaccinations against APV infections can be contemplated with vaccine preparations derived from MPV.", "Also, reverse genetic techniques make it possible to generate chimeric APV-MPV virus constructs that are useful as a vaccine, being sufficiently dissimilar to field isolates of each of the respective strains to be attenuated to a desirable level.", "Similar reverse genetic techniques will make it also possible to generate chimeric paramyxovirus-metapneumovirus constructs, such as RSV-MPV or PI3-MPV constructs for us in a vaccine preparation.", "Such constructs are particularly useful as a combination vaccine to combat respiratory tract illnesses.", "The invention thus provides a novel etiological agent, an isolated essentially mammalian negative-sense single stranded RNA virus (herein also called MTV) belonging to the subfamily Pneumovirinae of the family Paramyxoviridae but not identifiable as a classical pneumovirus, and belonging to the genus Metapneumovirus, and MPV-specific components or synthetic analogues thereof.", "Mammalian viruses resembling metapneumoviruses, i.e.", "metapneumoviruses isolatable from mammals that essentially function as natural host for said virus or cause disease in said mammals, have until now not been found.", "Metapneumoviruses, in general thought to be essentially restricted to poultry as natural host or aetiological agent of disease, are also known as avian pneumoviruses.", "Recently, an APV isolate of duck was described (FR 2 801 607), further demonstrating that APV infections are essentially restricted to birds as natural hosts.", "The invention provides an isolated mammalian pneumovirus (herein also called MPV) comprising a gene order and amino acid sequence distinct from that of the genus Pneumovirus and which is closely related and considering its phylogenetic relatedness likely belonging to the genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae.", "Although until now, metapneumoviruses have only been isolated from birds, it is now shown that related, albeit materially distinct, viruses can be identified in other animal species such as mammals.", "Herein we show repeated isolation of MPV from humans, whereas no such reports exists for APV.", "Furthermore, unlike APV, MPV essentially does not or only little replicates in chickens and turkeys where it easily does in cynomolgous macaques.", "No reports have been found on replication of APV in mammals.", "In addition, whereas specific anti-sera raised against MPV neutralize MPV, anti-sera raised against APV A, B or C do not neutralize MPV to the same extent, and this lack of full cross reactivity provides another proof for MPV being a different metapneumovirus.", "Furthermore, where APV and MPV share a similar gene order, the G and SH proteins of MPV are largely different from the ones known of APV in that they show no significant sequence homologies on both the amino acid or nucleic acid level.", "Diagnostic assays to discriminate between APV and MPV isolates or antibodies directed against these different viruses can advantageously be developed based on one or both of these proteins (examples are IF, ELISA, neutralization assay, RT-PCR assay).", "However, also sequence and/or antigenic information obtained from the more related N, P, M, F and L proteins of MPV and analyses of sequence homologies with the respective proteins of APV, can also be used to discriminate between APV and MPV.", "For example, phylogenetic analyses of sequence information obtained from MPV revealed that MPV and APV are two different viruses.", "In particular, the phylogenetic trees show that APV and MPV are two different lineages of virus.", "We have also shown that MPV is circulating in the human population for at least 50 years, therefore interspecies transmission has probably taken place at least 50 years ago and is not an everyday event.", "Since MPV CPE was virtually indistinguishable from that caused by hRSV or hPIV-1 in tMK or other cell cultures, the MPV may have well gone unnoticed until now.", "tMK (tertiary money kidney cells, i.e.", "MK cells in a third passage in cell culture) are preferably used due to their lower costs in comparison to primary or secondary culltures.", "The CPE is, as well as with some of the classical Paramyxoviridae, characterized by syncytium formation after which the cells showed rapid internal disruption, followed by detachment of the cells from the monolayer.", "The cells usually (but not always) displayed CPE after three passages of virus from original material, at day 10 to 14 post inoculation, somewhat later than CPE caused by other viruses such as hRSV or hPIV-1.Classically, as devastating agents of disease, paramyxoviruses account for many animal and human deaths worldwide each year.", "The Paramyxouiridae form a family within the order of Mononegavirales (negative-sense single stranded RNA viruses), consisting of the sub-familys Paramyxovirinae and Pneumovirinae.", "The latter sub-family is at present taxonomically divided in the genera Pneumovirus and Metapneumovirus1 .", "Human respiratory syncytial virus (hRSV), the type species of the Pneumovirus genus, is the single most important cause of lower respiratory tract infections during infancy and early childhood worldwide2.Other members of the Pneumovirus genus include the bovine and ovine respiratory syncytial viruses and pneumonia virus of mice (PVM).", "Avian pneumovirus (APV) also known as turkey rhinotracheitis virus (TRTV), the aetiological agent of avian rhinotracheitis, an upper respiratory tract infection of turkeys3, is the sole member of the recently assigned Metapneumovirus genus, which, as said was until now not associated with infections, or what is more, with disease of mammals.", "Serological subgroups of APV can be differentiated on the basis of nucleotide or amino acid sequences of the G glycoprotein and neutralization tests using monoclonal antibodies that also recognize the G glycoprotein, Within subgroups A, B and D the G protein shows 98.6 to 99.7% aa sequence identity within subgroups while between the subgroups only 31.2-38% aa identity is observed.", "See for example Collins, M. S., Gough, R. E. and Alexander, D. J., Antigenic differentiation of avian pneumouirus isolates using polyclonal antisera and mouse monoclonal antibodies.", "Avian Pathology, 1993.22: p. 469-479.; Cook, J. K. A., Jones, B. V., Ellis, M. M., Antigenic differentiation of strains of turkey rhinotracheitis virus using monoclonal antibodies.", "Avian Pathology, 1993.22: p. 257-273; Bayon-Auboyer, M. H., et al., Nucleotide sequences of the F, L and G protein genes of two non-A/non-B avian pneumouiruses (APV) reveal a novel APV subgroup.", "J Gen Virol, 2000.81(Pt 11): p. 2723-33; Seal, B. S., Matrix protein gene nucleotide and predicted amino acid sequence demonstrate that the first US avian pneumovirus isolate is distinct from European strains.", "Virus Res, 1998.58(1-2): p. 45-52; Bayon-Auboyer, M. H., et al., Comparison of F-, G- and N-based RT-PCR protocols with conventional virological procedures for the detection and typing of turkey rhinotracheitis virus.", "Arch Virol, 1999.144(6): p. 1091-109; Juhasz, K and A. J. Easton, Extensive sequence variation in the attachment (G) protein gene of avian pneumovirus: evidence for two distinct subgroups.", "J Gen Virol, 1994.75(Pt 11): p. 2873-80.A further serotype of APV is provided in WO00/20600, which describes the Colorado isolate of APV and compared it to known APV or TRT strains with in vitro serum neutralization tests.", "First, the Colorado isolate was tested against monospecific polyclonal antisera to recognized TRT isolates.", "The Colorado isolate was not neutralized by monospecific antisera to any of the TRT strains.", "It was, however, neutralized by a hyperimmune antiserum raised against a subgroup A strain.", "This antiserum neutralized the homologous virus to a titre of 1:400 and the Colorado isolate to a titer of 1: 80.Using the above method, the Colorado isolate was then tested against TRT monoclonal antibodies.", "In each case, the reciprocal neutralization titer was<10.Monospecific antiserum raised to the Colorado isolate was also tested against TRT strains of both subgroups.", "None of the TRT strains tested were neutralized by the antiserum to the Colorado isolate.", "The Colorado strain of APV does not protect SPF chicks against challenge with either a subgroup A or a subgroup B strain of TRT virus.", "These results suggest that the Colorado isolate may be the first example of a further serotype of avian pneumovirus, as also suggested by Bayon-Auboyer et al (J. Gen. Vir.", "81:2723-2733 (2000).", "In a preferred embodiment, the invention provides an isolated MPV taxonomically corresponding to a (hereto unknown mammalian) metapneumovirus comprising a gene order distinct from that of the pneumoviruses within the sub-family Pneumovirinae of the family Paramyxoviridae.", "The classification of the two genera is based primarily on their gene constellation; metapneumoviruses generally lack non-structural proteins such NS1 or NS2 (see also Randhawa et al., J. Vir.", "71:9849-9854 (1997) and the gene order is different from that of pneumoviruses (RSV: '3-NS1-NS2-N-P-M-SH-G-F-M2-L-5′, APV: '3-N-P-M-F-M2-SH-G-L-5′)4,5,6.MPV as provided by the invention or a virus isolate taxonomically corresponding therewith is upon EM analysis revealed by paramyxovirus-like particles.", "Consistent with the classification, MPV or virus isolates phylogenetically corresponding or taxonomically corresponding therewith are sensitive to treatment with chloroform; are cultured optimally on tMK cells or cells functionally equivalent thereto and are essentially trypsine dependent in most cell cultures.", "Furthermore, the typical CPE and lack of haemagglutinating activity with most classically used red blood cells suggested that a virus as provided herein is, albeit only distantly, related to classical pneumoviruses such as RSV.", "Although most paramyxoviruses have haemagglutinating acitivity, most of the pneumoviruses do not13.An MPV according to the invention also contains a second overlapping ORF (M2-2) in the nucleic acid fragment encoding the M2 protein, as in general most other pneumoviruses such as for example also demonstrated in Ahmadian et al., J. Gen. Vir.", "80:2011-2016 (1999).", "To find further viral isolates as provided by the invention it suffices to test a sample, optionally obtained from a diseased animal or human, for the presence of a virus of the sub-family Pneumovirinae, and test a thus obtained virus for the presence of genes encoding (functional) NS1 or NS2 or essentially demonstrate a gene order that is different from that of pneumoviruses such as RSV as already discussed above.", "Furthermore, a virus isolate phylogenetically corresponding and thus taxonomically corresponding with MPV may be found by cross-hybridisation experiments using nucleic acid from a here provided MPV isolate, or in classical cross-serology experiments using monoclonal antibodies specifically directed against and/or antigens and/or immunogens specifically derived from an MPV isolate.", "Newly isolated viruses are phylogenetically corresponding to and thus taxonomically corresponding to MPV when comprising a gene order and/or amino acid sequence sufficiently similar to our prototypic MPV isolate(s), or are structurally corresponding therewith, and show close relatedness to the genus Metapneumovirus within the subfamily Pneumovirinae.", "The highest amino sequence homology, and defining the structural correspondence on the individual protein level, between MPV and any of the known other viruses of the same family to date (APV subtype C) is for matrix 87%, for nucleoprotein 88%, for phosphoprotein 68%, for fusionprotein 81% and for parts of the polymerase protein 56-64%, as can be deduced when comparing the sequences given in FIG.", "6 with sequences of other viruses, in particular of AVP-C.", "Individual proteins or whole virus isolates with, respectively, higher homology to these mentioned maximum values are considered phylogenetically corresponding and thus taxonomically corresponding to MPV, and comprise a nucleic acid sequence structurally corresponding with a sequence as shown in FIG.", "6.Herewith the invention provides a virus phylogenetically corresponding to the deposited virus.", "It should be noted that, similar to other viruses, a certain degree of variation is found between different isolated essentially mammalian negative-sense single stranded RNA virus isolates as provided herein.", "In phylogenetic trees, we have identified at least 2 genetic clusters of virus isolates based on comparitive sequence analyses of parts of the L, M, N and F genes.", "Based on nucleotide and amino-acid differences in the viral nucleic acid or amino acid sequences (the viral sequences), and in analogy to other pneumoviruses such as RSV, these MPV genotypes represent subtypes of MPV.", "Within each of the genetic clusters of MPV isolates, the percentage identity at the nucleotide level was found to be 94-100 for L, 91-100 for M, 90-100 for N and 93-100 for F and at the amino acid level the percentage identity was found to be 91-100 for L, 98-100 for M, 96-100 for N and 98-100 for F. A further comparison can be found in FIGS.", "18 to 28.The minimum percentage identity at the nucleotide level for the entire group of isolated essentially mammalian negative-sense single stranded RNA virus as provided herein (MPV isolates) identified so far was 81 for L and M, 83 for N and 82 for F. At the amino acid level, this percentage was 91 for L and N, 94 for M, and 95 for F. The viral sequence of a MPV isolate or an isolated MPV F gene as provided herein for example shows less than 81% nucleotide sequence identity or less than 82%(amino acid sequence identity with the respective nucleotide or amino acid sequence of an APV-C fusion (F) gene as for example provided by Seal et al., Vir.", "Res.", "66:139147 (2000).", "Also, the viral sequence of a MPV isolate or an an isolated MPV L gene as provided herein for example shows less than 61% nucleotide sequence identity or less than 63% amino acid sequence identity with the respective nucleotide or amino acid sequence of an APV-A polymerase gene as for example provided by Randhawa et al., J. Gen. Vir.", "77:3047-3051 (1996).", "Sequence divergence of MPV strains around the world may be somewhat higher, in analogy with other viruses.", "Consequently, two potential genetic clusters are identified by analyses of partial nucleotide sequences in the N, M, F and L ORFs of 9 virus isolates.", "90-100% nucleotide identity was observed within a cluster, and 81-88% identity was observed between the clusters.", "Sequence information obtained on more virus isolates confirmed the existence of two genotypes.", "Virus isolate ned/00/01 as prototype of cluster A, and virus isolate ned/99/01 as prototype of cluster B have been used in cross neutralization assays to test whether the genotypes are related to different serotypes or subgroups.", "From these data we conclude that essentially mammalian virus isolates displaying percentage amino acid homology higher than 64 for L, 87 for M, 88 for N, 68 for P, 81 for F 84 for M2-1 or 58 for M2-2 to isolate I-2614 may be classified as an isolated essentially mammalian negative-sense single stranded RNA virus as provided herein.", "In particular those virus isolates in general that have a minimum percentage identity at the nucleotide sequence level with a prototype MPV isolate as provided herein of 81 for L and M, 83 for N and/or 82 for F are members of the group of MPV isolates as provided herein.", "At the amino acid level, these percentage are 91 for L and N, 94 for M, and/or 95 for F. When the percentage amino acid sequence homology for a given virus isolate is higher than 90 for L and N, 93 for M, or 94 for F, the virus isolate is similar to the group of MPV isolates displayed in FIG.", "5.When the percentage amino acid sequence homology for a given virus isolate is higher than 94 for L, 95 for N or 97 for M and F the virus isolate can be identified to belong to one of the genotype clusters represented in FIG.", "5.It should be noted that these percentages of homology, by which genetic clusters are defined, are similar to the degree of homology found among genetic clusters in the corresponding genes of RSV.", "In short, the invention provides an isolated essentially mammalian negative-sense single stranded RNA virus (MPV) belonging to the sub-family Pneumovirinae of the family Paramyxoviridae and identifiable as phylogenetically corresponding to the genus Metapneumovirus by determining a nucleic acid sequence of a suitable fragment of the genome of said virus and testing it in phylogenetic tree analyses wherein maximum likelihood trees are generated using 100 bootstraps and 3 jumbles and finding it to be more closely phylogenetically corresponding to a virus isolate deposited as I-2614 with CNCM, Paris than it is corresponding to a virus isolate of avian pneumovirus (APV) also known as turkey rhinotracheitis virus (TRTV), the aetiological agent of avian rhinotracheitis.", "Suitable nucleic acid genome fragments each useful for such phylogenetic tree analyses are for example any of the RAP-PCR fragments 1 to 10 as disclosed herein in the detailed description, leading to the various phylogenetic tree analyses as disclosed herein in FIG.", "4 or 5.Phylogenetic tree analyses of the nucleoprotein (N), phosphoprotein (P), matrixprotein (M) and fusion protein (1) genes of MPV revealed the highest degree of sequence homology with APV serotype C, the avian pneumovirus found primarily in birds in the United States.", "In a preferred embodiment, the invention provides an isolated essentially mammalian negative-sense single stranded RNA virus (MPV) belonging to the sub-family Pneumovirinae of the family Paramyxoviridae and identifiable as phylogenetically corresponding to the genus Metapneumovirus by determining a nucleic acid sequence of a suitable fragment of the genome of said virus and testing it in phylogenetic tree analyses wherein maximum likelihood trees are generated using 100 bootstraps and 3 jumbles and finding it to be more closely phylogenetically corresponding to a virus isolate deposited as I-2614 with CNCM, Paris than it is corresponding to a virus isolate of avian pneumovirus (APV) also known as turkey rhinotracheitis virus (TRTV), the aetiological agent of avian rhinotracheitis, wherein said suitable fragment comprises an open reading frame encoding a viral protein of said virus.", "A suitable open reading frame (ORF) comprises the ORF encoding the N protein.", "When an overall amino acid identity of at least 91%, preferably of at least 95% of the analysed N-protein with the N-protein of isolate I-2614 is found, the analysed virus isolate comprises a preferred MPV isolate according to the invention.", "As shown, the first gene in the genomic map of MPV codes for a 394 amino acid (aa) protein and shows extensive homology with the N protein of other pneumoviruses.", "The length of the N ORF is identical to the length of the N ORF of APV-C (table 5) and is smaller than those of other paramyxoviruses (Barr et al., 1991).", "Analysis of the amino acid sequence revealed the highest homology with APV-C (88%), and only 7-11% with other paramyxoviruses (Table 6).", "Barr et al (1991) identified 3 regions of similarity between viruses belonging to the order Mononegauirales: A, B and C (FIG.", "8).", "Although similarities are highest within a virus family, these regions are highly conserved between virus familys.", "In all three regions MPV revealed 97% aa sequence identity with APV-C, 89% with APV-B, 92 with APV-A, and 66-73% with RSV and PVM.", "The region between aa residues 160 and 340 appears to be highly conserved among metapneumoviruses and to a somewhat lesser extent the Pneumovirinae (Miyahara et al., 1992; Li et al., 1996; Barr et al., 1991).", "This is in agreement with MPV being a metapneumovirus, this particular region showing 99% similarity with APV C. Another suitable open reading frame (ORF) useful in phylogenetic analyses comprises the ORF encoding the P protein.", "When an overall amino acid identity of at least 70%, preferably of at least 85% of the analysed P-protein with the P-protein of isolate 1-2614 is found, the analysed virus isolate comprises a preferred MPV isolate according to the invention.", "The second ORF in the genome map codes for a 294 aa protein which shares 68% aa sequence homology with the P protein of APV-C, and only 22-26% with the P protein of RSV (Table 6).", "The P gene of MPV contains one substantial ORF and in that respect is similar to P from many other paramyxoviruses (Reviewed in Lamb and Kolakofsky, 1996; Sedlmeier et al., 1998).", "In contrast to APV A and B and PVM and similar to RSV and APV-C the MPV P ORF lacks cysteine residues.", "Ling (1995) suggested that a region of high similarity between all pneumoviruses (aa 185-241) plays a role in either the RNA synthesis process or in maintaining the structural integrity of the nucleocapsid complex.", "This region of high similarity is also found in MPV (FIG.", "9) especifically when conservative substitutions are taken in account, showing 100% similarity with APV-C, 93% with APV-A and B, and approximately 81% with RSV.", "The C-terminus of the MPV P protein is rich in glutamate residues as has been described for APVs (Ling et al., 1995).", "Another suitable open reading frame (ORF) useful in phylogenetic analyses comprises the ORF encoding the M protein.", "When an overall amino acid identity of at least 94%, preferably of at least 97% of the analysed M-protein with the M-protein of isolate I-2614 is found, the analysed virus isolate comprises a preferred MPV isolate according to the invention.", "The third ORF of the MPV genome encodes a 254 aa protein, which resembles the M ORFs of other pneumoviruses.", "The M ORF of MPV has exactly the same size as the M ORFs of other metapneumoviruses (Table 5) and shows high aa sequence homology with the matrix proteins of APV (76-87%) lower homology with those of RSV and PVM (37-38%) and 10% or less homology with those of other paramyxoviruses (Table 6).", "Easton (1997) compared the sequences of matrix proteins of all pneumoviruses and found a conserved hexapeptide at residue 14 to 19 that is also conserved in MPV (FIG.", "10).", "For RSV, PVM and APV small secondary ORFs within or overlapping with the major ORF of M have been identified (52 aa and 51 aa in bRSV, 75 aa in RSV, 46 aa in PVM and 51 aa in APV) (Yu et al., 1992; Easton et al., 1997; Samal et al., 1991; Satake et al., 1984).", "We noticed two small ORFs in the M ORF of MPV.", "One small ORF of 54 aa residues was found within the major M ORF, starting at nucleotide 2281 and one small ORF of 33 aa residues was found overlapping with the major ORF of M starting at nucleotide 2893 (data not shown).", "Similar to the secondary ORFs of RSV and APV there is no significant homology between these secondary ORFs and secondary ORFs of the other pneumoviruses, and apparent start or stop signals are lacking.", "In addition, evidence for the synthesis of proteins corresponding to these secondary ORFs of APV and RSV has not been reported.", "Another suitable open reading frame (ORF) useful in phylogenetic analyses comprises the ORF encoding the F protein.", "When an overall amino acid identity of at least 95%, preferably of at least 97% of the analysed F-protein with the F-protein of isolate I-2614 is found, the analysed virus isolate comprises a preferred MPV isolate according to the invention.", "The F ORF of MPV is located adjacent to the M ORF, which is characteristic for members of the Metapneumovirus genus.", "The F gene of MPV encodes a 539 aa protein, which is two aa residues longer than F of APV-C (Table 5).", "Analysis of the aa sequence revealed 81% homology with APV-C, 67% with APV-A and B, 33-39% with pneumovirus F proteins and only 10-18% with other paramyxoviruses (Table 6).", "One of the conserved features among F proteins of paramyxoviruses, and also seen in MPV is the distribution of cysteine residues (Morrison, 1988; Yu et al., 1991).", "The metapneumoviruses share 12 cysteine residues in F1 (7 are conserved among all paramyxoviruses), and two in F2 (1 is conserved among all paramyxoviruses).", "Of the 3 potential N-linked glycosylation sites present in the F ORF of MPV, none are shared with RSV and two (position 66 and 389) are shared with APV.", "The third, unique, potential N-linked glycosylation site for MPV is located at position 206 FIG.", "11).", "Despite the low sequence homology with other paramyxoviruses, the F protein of MPV revealed typical fusion protein characteristics consistent with those described for the F proteins of other Paramyxoviridae family members (Morrison, 1988).", "F proteins of Paramyxoviridae members are synthesized as inactive precursors (F0) that are cleaved by host cell proteases which generate amino terminal F2 subunits and large carboxy terminal F1 subunits.", "The proposed cleavage site (Collins et al., 1996) is conserved among all members of the Paramyxoviridae family.", "The cleavage site of MPV contains the residues RQSR.", "Both arginine (R) residues are shared with APV and RSV, but the glutamine (Q) and serine (S) residues are shared with other paramyxoviruses such as human parainfluenza virus type 1, Sendai virus and morbilliviruses (data not shown).", "The hydrophobic region at the amino terminus of F1 is thought to function as the membrane fusion domain and shows high sequence similarity among paramyxoviruses and morbilliviruses and to a lesser extent the pneumoviruses (Morrison, 1988).", "These 26 residues (position 137-163, FIG.", "11) are conserved between MPV and APV C, which is in agreement with this region being highly conserved among the metapneumoviruses (Naylor et al., 1998; Seal et al., 2000).", "As is seen for the F2 subunits of APV and other paramyxoviruses, MPV revealed a deletion of 22 aa residues compared with RSV (position 107-128, FIG.", "11).", "Furthermore, for RSV and APV, the signal peptide and anchor domain were found to be conserved within subtypes and displayed high variability between subtypes (Plows et al., 1995; Naylor et al., 1998).", "The signal peptide of MPV (aa 10-35, FIG.", "11) at the amino terminus of F2 exhibits some sequence similarity with APV-C (18 out of 26 aa residues are similar) and less conservation with other APVs or RSV.", "Much more variability is seen in the membrane anchor domain at the carboxy terminus of F1, although some homology is still seen with APV-C. Another suitable open reading frame (ORF) useful in phylogenetic analyses comprises the ORF encoding the M2 protein.", "When an overall amino acid identity of at least 85%, preferably of at least 90% of the analysed M2-protein with the M2-protein of isolate I-2614 is found, the analysed virus isolate comprises a preferred MPV isolate according to the invention.", "M2 gene is unique to the Pneumovirinae and two overlapping ORFs have been observed in all pneumoviruses.", "The first major ORF represents the M2-1 protein which enhances the processivity of the viral polymerase (Collins et al., 1995; Collins, 1996) and its readthrough of intergenic regions (Hardy et al., 1998; Fearns et al., 1999).", "The M2-1 gene for MPV, located adjacent to the F gene, encodes a 187 aa protein (Table 5), and reveals the highest (84%) homology with M2-1 of APV-C (Table 6).", "Comparison of all pneumovirus M2-1 proteins revealed the highest conservation in the amino-terminal half of the protein (Collins et al., 1990; Zamora et al., 1992; Ahmadian et al., 1999), which is in agreement with the observation that MPV displays 100% similarity with APV-C in the first 80 aa residues of the protein (FIG.", "12A).", "The MPV M2-1 protein contains 3 cysteine residues located within the first 30 aa residues that are conserved among all pneumoviruses.", "Such a concentration of cysteines is frequently found in zinc-binding proteins (Ahmadian et al., 1991; Cuesta et al., 2000).", "The secondary ORFs M2-2) that overlap with the M2-1 ORFs of pneumoviruses are conserved in location but not in sequence and are thought to be involved in the control of the switch between virus RNA replication and transcription (Collins et al., 1985; Elango et al., 1985; Baybutt et al., 1987; Collins et al., 1990; Ling et al., 1992; Zamora et al., 1992; Alansari et al., 1994; Ahmadian et al., 1999; Bermingham et al., 1999).", "For MPV, the M2-2 ORF starts at nucleotide 512 in the M2-1 ORF (FIG.", "7), which is exactly the same start position as for APV-C.", "The length of the M2-2 ORFs are the same for APV-C and MPV, 71 aa residues (Table 5).", "Sequence comparison of the M2-2 ORF (FIG.", "12B) revealed 56% aa sequence homology between MPV and APV-C and only 26-27% aa sequence homology between MPV and APV-A and B (Table 6).", "Another suitable open reading frame (ORE) useful in phylogenetic analyses comprises the ORF encoding the L protein.", "When an overall amino acid identity of at least 91%, preferably of at least 95% of the analysed L-protein with the L-protein of isolate I-2614 is found, the analysed virus isolate comprises a preferred MPV isolate according to the invention.", "In analogy to other negative strand viruses, the last ORF of the MPV genome is the RNA-dependent RNA polymerase component of the replication and transcription complexes.", "The L gene of MPV encodes a 2005 aa protein, which is 1 residue longer than the APV-A protein (Table 5).", "The L protein of MPV shares 64% homology with APV-A, 42-44% with RSV, and approximately 13% with other paramyxoviruses (Table 6).", "Poch et al.", "(1989; 1990) identified six conserved domains within the L proteins of non-segmented negative strand RNA viruses, from which domain III contained the four core polymerase motifs that are thought to be essential for polymerase function.", "These motifs (A, B, C and D) are well conserved in the MPV L protein: in motifs A, B and C: MPV shares 100% similarity with all pneumoviruses and in motif D MPV shares 100% similarity with APV and 92% with RSV's.", "For the entire domain III (aa 625-847 in the L ORF), MPV shares 83% identity with APV, 67-68% with RSV and 26-30% with other paramyxoviruses (FIG.", "15).", "In addition to the polymerase motifs the pneumovirus L proteins contain a sequence which conforms to a consensus ATP binding motif K(X)21GEGAGN(X)20K (Stec, 1991).", "The MPV L ORF contains a similar motif as APV, in which the spacing of the intermediate residues is off by one: K(x)22GEGAGN(X)19K.", "A much preferred suitable open reading frame (ORE) useful in phylogenetic analyses comprises the ORF encoding the SH protein.", "When an overall amino acid identity of at least 30%, preferably of at least 50%, more preferably of at least 75% of the analysed SH-protein with the SH-protein of isolate I-2614 is found, the analysed virus isolate comprises a preferred MPV isolate according to the invention.", "The gene located adjacent to M2 of MPV encodes a 183 aa protein (FIG.", "7).", "Analysis of the nucleotide sequence and its deduced amino acid sequence revealed no discernible homology with other RNA virus genes or gene products.", "The SH ORF of MPV is the longest SH ORF known to date (Table 5).", "The composition of the aa residues of the SH ORF is relatively similar to that of APV, RSV and PVM, with a high percentage of threonine and serine (22%, 18%, 19%, 20.0%, 21% and 28% serine/threonine content for MPV, APV, RSV A, RSV B, bRSV and PVM respectively).", "The SH ORF of MPV contains 10 cysteine residues, whereas APV SH contains 16 cysteine residues.", "All pneumoviruses have similar numbers of potential N-glycosylation sites (MPV 2, APV 1, RSV 2, bRSV 3, PVM 4).", "The hydrophobicity profiles for the MPV SH protein and SH of APV and RSV revealed similar structural characteristics (FIG.", "13B).", "The SH ORFs of APV and MPV have a hydrophylic N-terminus (aa 1-30), a central hydrophobic domain (aa 30-53) which can serve as a potential membrane spanning domain, a second hydrophobic domain around residue 160 and a hydrophilic C-terminus.", "In contrast, RSV SH appears to lack the C-terminal half of the APV and MPV ORFs.", "In all pneumovirus SH proteins the hydrophobic domain is flanked by basic amino acids, which are also found in the SH ORF for MPV (aa 29 and 54).", "Another much preferred suitable open reading frame (ORF) useful in phylogenetic analyses comprises the ORF encoding the G protein.", "When an overall amino acid identity of at least 30%, preferably of at least 50%, more preferably of at least 75% of the analysed G-protein with the G-protein of isolate I-2614 is found, the analysed virus isolate comprises a preferred MPV isolate according to the invention.", "The G ORF of MPV is located adjacent to the SH gene and encodes a 236 amino acid protein.", "A secondary small ORF is found immediately following this ORF, potentially coding for 68 aa residues (pos.", "6973-7179,), but lacking a start codon.", "A third major ORF, in a different reading frame, of 194 aa residues (fragment 4, FIG.", "7) is overlapping with both of these ORFs, but also lacks a startcodon (nucleotide 6416-7000).", "This major ORF is followed by a fourth ORF in the same reading frame (nt 7001-7198), possibly coding for 65 aa residues but again lacking a start codon.", "Finally, a potential ORF of 97 aa residues (but lacking a startcodon) is found in the third reading frame (nt 6444-6737, FIG.", "1).", "Unlike the first ORF, the other ORFs do not have apparent gene start or gene end sequences (see below).", "Although the 236 aa residue G ORF probably represents at least a part of the MPV attachment protein it can not be excluded that the additional coding sequences are expressed as separate proteins or as part of the attachment protein through some RNA editing event.", "It should be noted that for APV and RSV no secondary ORFs after the primary G ORF have been identified but that both APV and RSV have secondary ORFs within the major ORF of G. However, evidence for expression of these ORFs is lacking and there is no homology between the predicted aa sequences for different viruses (Ling et al., 1992).", "The secondary ORFs in MPV G do not reveal characteristics of other G proteins and whether the additional ORFs are expressed requires further investigation.", "BLAST analyses with all four ORFs revealed no discernible homology at the nucleotide or aa sequence level with other known virus genes or gene products.", "This is in agreement with the low sequence homologies found for other G proteins such as hRSV A and B (53%) (Johnson et al., 1987) and APV A and B (38%) (Juhasz et al., 1994).", "Whereas most of the MPV ORFs resemble those of APV both in length and sequence, the G ORF of MPV is considerably smaller than the G ORF of APV (Table 5).", "The aa sequence revealed a serine and threonine content of 34%, which is even higher than the 32% for RSV and 24% for APV.", "The G ORF also contains 8.5% proline residues, which is higher than the 8% for RSV and 7% for APV.", "The unusual abundance of proline residues in the G proteins of APV, RSV and MPV has also been observed in glycoproteins of mucinous origin where it is a major determinant of the proteins three dimensional structure (Collins et al., 1983; Wertz et al., 1985; Jentoft, 1990).", "The number of potential N-linked glycosylation sites in G of MPV is similar to other pneumoviruses: MPV has 5, whereas hRSV has 7, bRSV has 5, and APV has 3 to 5.The predicted hydrophobicity profile of MPV G revealed characteristics similar to the other pneumoviruses.", "The amino-terminus contains a hydrophylic region followed by a short hydrophobic area (aa 33-53) and a mainly hydrophilic carboxy terminus (FIG.", "14B).", "This overall organisation is consistent with that of an anchored type II transmembrane protein and corresponds well with these regions in the G protein of APV and RSV.", "The G ORF of MPV contains only 1 cysteine residue in contrast to RSV and APV (5 and 20 respectively).", "According to classical serological analyses as for example known from Francki, R. I.", "B., Fauquet, C. M., Knudson, D. L., and Brown, F., Classification and nomenclature of viruses.", "Fifth report of the international Committee on Taxonomy of Viruses.", "Arch Virol, 1991.Supplement 2: p. 140-144.an MPV isolate is also identifiable as belonging to a serotype as provided herein, being defined on the basis of its immunological distinctiveness, as determined by quantitative neutralization with animal antisera (obtained from for example ferrets or guinnea pigs as provided in the detailed description).", "Such a serotype has either no cross-reaction with others or shows a homologous-to heterologous titer ratio>16 in both directions.", "If neutralization shows a certain degree of cross-reaction between two viruses in either or both directions (homologous-to-heterologous tier ration of eight or 16), distinctiveness of serotype is assumed if substantial biophysical/biochemical differences of DNA's exist.", "If neutralization shows a distinct degree of cross-reaction between two viruses in either or both directions (homologous-to-heterologous tier ration of smaller than eight), identity of serotype of the isolates under study is assumed.", "As said, useful prototype isolates, such as isolate 1-2614, herein also known as MPV isolate 00-1, are provided herein.", "A further classification of a virus as an isolated essentially mammalian negative-sense single stranded RNA virus as provided herein can be made on the basis of homology to the G and/or SH proteins.", "Where in general the overall amino acid sequence identity between APV (isolated from birds) and MPV (isolated from humans) N, P, M, F, M2 and L ORFs was 64 to 88 percent, and nucleotide sequence homology was also found between the non-coding regions of the APV and MPV genomes, essentially no discernable amino acid sequence homology was found between two of the ORFs of the human isolate (MPV) and any of the ORFs of other paramyxoviruses.", "The amino acid content, hydrophobicity profiles and location of these ORFs in the viral genome show that they represent G and SH protein analogues.", "The sequence homology between APV and MPV, their similar genomic organization (3′-N-P-M-F-M2-SH-G-L-5′) as well as phylogenetic analyses provide further evidence for the proposed classification of MPV as the first mammalian metapneumovirus.", "New MPV isolates are for thus example identified as such by virus isolation and characterisation on tMK or other cells, by RT-PCR and/or sequence analysis followed by phylogenetic tree analyses, and by serologic techniques such as virus neutralisation assays, indirect immunofluorescence assays, direct immunofluorescence assays, FACs analyses or other immunological techniques.", "Preferably these techniques are directed at the SH and/or G protein analogues.", "For example the invention provides herein a method to identify further isolates of MPV as provided herein, the method comprising inoculating a essentially MPV-uninfected or specific-pathogen-free guinea pig or ferret (in the detailed description the animal is inoculated intranasally but other ways of inoculation such as intramuscular or intradermal inoculation, and using an other experimental animal, is also feasible) with the prototype isolate I-2614 or related isolates.", "Sera are collected from the animal at day zero, two weeks and three weeks post inoculation.", "The animal specifically seroconverted as measured in virus neutralisation (VN) assay and indirect.", "IFA against the respective isolate I-2614 and the sera from the seroconverted animal are used in the immunological detection of said further isolates.", "As an example, the invention provides the characterisation of a new member in the family of Paramyxoviridae, a human metapneumovirus or metapneumovirus-like virus (since its final taxonomy awaits discussion by a viral taxonomy committee the MPV is herein for example described as taxonomically corresponding to APV) (MPV) which may cause severe RTI in humans.", "The clinical signs of the disease caused by MPV are essentially similar to those caused by hRSV, such as cough, myalgia, vomiting, fever, broncheolitis or pneumonia, possible conjunctivitis, or combinations thereof.", "As is seen with hRSV infected children, especifically very young children may require hospitalisation.", "As an example an MPV which was deposited Jan. 19, 2001 as I-2614 with CNCM, Institute Pasteur, Paris or a virus isolate phylogenetically corresponding therewith is herewith provided.", "Therewith, the invention provides a virus comprising a nucleic acid or functional fragment phylogenetically corresponding to a nucleic acid sequence shown in FIG.", "6a, 6b, 6c, or structurally corresponding therewith.", "In particular the invention provides a virus characterised in that after testing it in phylogenetic tree analyses wherein maximum likelihood trees are generated using 100 bootstraps and 3 jumbles it is found to be more closely phylogenetically corresponding to a virus isolate deposited as I-2614 with CNCM, Paris than it is related to a virus isolate of avian pneumovirus (APV) also known as turkey rhinotracheitis virus (TRTV), the aetiological agent of avian rhinotracheitis.", "It is particularly useful to use an AVP-C virus isolate as outgroup in said phylogenetic tree analyses, it being the closest relative, albeit being an essentially non-mammalian virus.", "We propose the new human virus to be named human metapneumovirus or metapneumovirus-like virus (MPV) based on several observations.", "EM analysis revealed paramyxovirus-like particles.", "Consistent with the classification, MPV appeared to be sensitive to treatment with chloroform.", "MPV is cultured optimal on tMK cells and is trypsine dependent.", "The clinical symptoms caused by MPV as well as the typical CPE and lack of haemagglutinating activity suggested that this virus is closely related to hRSV.", "Although most paramyxoviruses have haemaglutinating acitivity, most of the pneumoviruses do not13.As an example, the invention provides a not previously identified paramyxovirus from nasopharyngeal aspirate samples taken from 28 children suffering from severe RTI.", "The clinical symptoms of these children were largely similar to those caused by hRSV.", "Twenty-seven of the patients were children below the age of five years and half of these were between 1 and 12 months old.", "The other patient was 18 years old.", "All individuals suffered from upper RTI, with symptoms ranging from cough, myalgia, vomiting and fever to broncheolitis and severe pneumonia.", "The majority of these patients were hospitalised for one to two weeks.", "The virus isolates from these patients had the paramyxovirus morphology in negative contrast electron microscopy but did not react with specific antisera against known human and animal paramyxoviruses.", "They were all closely related to one another as determined by indirect immunofluorescence assays (IFA) with sera raised against two of the isolates.", "Sequence analyses of nine of these isolates revealed that the virus is somewhat related to APV.", "Based on virological data, sequence homology as well as the genomic organisation we propose that the virus is a member of Metapneumovirus genus.", "Serological surveys showed that this virus is a relatively common pathogen since the seroprevalence in the Netherlands approaches 100% of humans by the age of five years.", "Moreover, the seroprevelance was found to be equally high in sera collected from humans in 1958, indicating this virus has been circulating in the human population for more than 40 years.", "The identification of this proposed new member of the Metapneumovirus genus now also provides for the development of means and methods for diagnostic assays or test kits and vaccines or serum or antibody compositions for viral respiratory tract infections, and for methods to test or screen for antiviral agents useful in the treatment of MPV infections.", "To this extent, the invention provides among others an isolated or recombinant nucleic acid or virus-specific functional fragment thereof obtainable from a virus according to the invention.", "In particular, the invention provides primers and/or probes suitable for identifying an MPV nucleic acid.", "Furthermore, the invention provides a vector comprising a nucleic acid according to the invention.", "To begin with, vectors such as plasmid vectors containing (parts of) the genome of MPV, virus vectors containing (parts of) the genome of MPV.", "(For example, but not limited to other paramyxoviruses, vaccinia virus, retroviruses, baculovirus), or MPV containing (parts of) the genome of other viruse or other pathogens are provided.", "Furthermore, a number of reverse genetics techniques have been described for the generation of recombinant negative strand viruses, based on two critical parameters.", "First, the production of such virus relies on the replication of a partial or full-length copy of the negative sense viral RNA (vRNA) genome or a complementary copy thereof (cRNA).", "This vRNA or cRNA can be isolated from infectious virus, produced upon in-vitro transcription, or produced in cells upon transfection of nucleic acids.", "Second, the production of recombinant negative strand virus relies on a functional polymerase complex.", "Typically, the polymerase complex of pneumoviruses consists of N, P, L and possibly M2 proteins, but is not necessarily limited thereto.", "Polymerase complexes or components thereof can be isolated from virus particles, isolated from cells expressing one or more of the components, or produced upon transfection of specific expression vectors.", "Infectious copies of MPV can be obtained when the above mentioned vRNA, cRNA, or vectors expressing these RNAs are replicated by the above mentioned polymerase complex16,17.18,19,20,21,22.For the generation of minireplicons or, a reverse genetics system for generating a full-length copy comprising most or all of the genome of MPV it suffices to use 3′end and/or 5′end nucleic acid sequences obtainable from for example APV (Randhawa et al., 1997) or MPV itself.", "Also, the invention provides a host cell comprising a nucleic acid or a vector according to the invention.", "Plasmid or viral vectors containing the polymerase components of MPV (presumably N, P, L and M2, but not necessarily limited thereto) are generated in prokaryotic cells for the expression of the components in relevant cell types (bacteria, insect cells, eukaryotic cells).", "Plasmid or viral vectors containing full-length or partial copies of the MPV genome will be generated in prokarotic cells for the expression of viral nucleic acids in-vitro or in-vivo.", "The latter vectors may contain other viral sequences for the generation of chimeric viruses or chimeric virus proteins, may lack parts of the viral genome for the generation of replication defective virus, and may contain mutations, deletions or insertions for the generation of attenuated viruses.", "Infectious copies of MPV (being wild type, attenuated, replication-defective or chimeric) can be produced upon co-expression of the polymerase components according to the state-of-the-art technologies described above.", "In addition, eukaryotic cells, transiently or stably expressing one or more full-length or partial MPV proteins can be used.", "Such cells can be made by transfection (proteins or nucleic acid vectors), infection (viral vectors) or transduction (viral vectors) and may be useful for complementation of mentioned wild type, attenuated, replication-defective or chimeric viruses.", "A chimeric virus may be of particular use for the generation of recombinant vaccines protecting against two or more viruses23,24,26.For example, it can be envisaged that a MPV virus vector expressing one or more proteins of RSV or a RSV vector expressing one or more proteins of MPV will protect individuals vaccinated with such vector against both virus infections.", "A similar approach can be envisaged for PI3 or other paramyxoviruses.", "Attenuated and replication-defective viruses may be of use for vaccination purposes with live vaccines as has been suggested for other viruses25.26.In a preferred embodiment, the invention provides a proteinaceous molecule or metapneumovirus-specific viral protein or functional fragment thereof encoded by a nucleic acid according to the invention.", "Useful proteinaceous molecules are for example derived from any of the genes or genomic fragments derivable from a virus according to the invention.", "Such molecules, or antigenic fragments thereof, as provided herein, are for example useful in diagnostic methods or kits and in pharmaceutical compositions such as sub-unit vaccines.", "Particularly useful are the F, SH and/or G protein or antigenic fragments thereof for inclusion as antigen or subunit immunogen, but inactivated whole virus can also be used.", "Particulary useful are also those proteinaceous substances that are encoded by recombinant nucleic acid fragments that are identified for phylogenetic analyses, of course preferred are those that are within the preferred bounds and metes of ORFs useful in phylogenetic analyses, in particular for eliciting MPV specific antibodies, whether in vivo (e.g.", "for protective puposes or for providing diagnostic antibodies) or in vitro (e.g.", "by phage display technology or another technique useful for generating synthetic antibodies).", "Also provided herein are antibodies, be it natural polyclonal or monoclonal, or synthetic (e.g.", "(phage) library-derived binding molecules) antibodies that specifically react with an antigen comprising a proteinaceous molecule or MPV-specific functional fragment thereof according to the invention.", "Such antibodies are useful in a method for identifying a viral isolate as an MPV comprising reacting said viral isolate or a component thereof with an antibody as provided herein.", "This can for example be achieved by using purified or non-purified MPV or parts thereof (proteins, peptides) using ELISA, RIA, FACS or similar formats of antigen detection assays (Current Protocols in Immunology).", "Alternatively, infected cells or cell cultures may be used to identify viral antigens using classical immunofluorescence or immunohistochemical techniques.", "Other methods for identifying a viral isolate as a MPV comprise reacting said viral isolate or a component thereof with a virus specific nucleic acid according to the invention, in particular where said mammalian virus comprises a human virus.", "In this way the invention provides a viral isolate identifiable with a method according to the invention as a mammalian virus taxonomically corresponding to a negative-sense single stranded RNA virus identifiable as likely belonging to the genus Metapneumovirus within the sub-family Paeumovirinae of the family Paramyxoviridae.", "The method is useful in a method for virologically diagnosing an MPV infection of a mammal, said method for example comprising determining in a sample of said mammal the presence of a viral isolate or component thereof by reacting said sample with a nucleic acid or an antibody according to the invention.", "Examples are further given in the detailed description, such as the use of PCR (or other amplification or hybridization techniques well known in the art) or the use of immunofluorescence detection (or other immunological techniques known in the art).", "The invention also provides a method for serologically diagnosing a MPV infection of a mammal comprising determining in a sample of said mammal the presence of an antibody specifically directed against a MPV or component thereof by reacting said sample with a proteinaceous molecule or fragment thereof or an antigen according to the invention.", "Methods and means provided herein are particularly useful in a diagnostic kit for diagnosing a MPV infection, be it by virological or serological diagnosis.", "Such kits or assays may for example comprise a virus, a nucleic acid, a proteinaceous molecule or fragment thereof, an antigen and/or an antibody according to the invention.", "Use of a virus, a nucleic acid, a proteinaceous molecule or fragment thereof, an antigen and/or an antibody according to the invention is also provided for the production of a pharmaceutical composition, for example for the treatment or prevention of MPV infections and/or for the treatment or prevention of respiratory tract illnesses, in particular in humans.", "Attenuation of the virus can be achieved by established methods developed for this purpose, including but not limited to the use of related viruses of other species, serial passages through laboratory animals or/and tissue/cell cultures, site directed mutagenesis of molecular clones and exchange of genes or gene fragments between related viruses.", "A pharmaceutical composition comprising a virus, a nucleic acid, a proteinaceous molecule or fragment thereof, an antigen and/or an antibody according to the invention can for example be used in a method for the treatment or prevention of a MPV infection and/or a respiratory illness comprising providing an individual with a pharmaceutical composition according to the invention.", "This is most useful when said individual comprises a human, especifically when said human is below 5 years of age, since such infants and young children are most likely to be infected by a human MPV as provided herein.", "Generally, in the acute phase patients will suffer from upper respiratory symptoms predisposing for other respiratory and other diseases.", "Also lower respiratory illnesses may occur, predisposing for more and other serious conditions.", "The invention also provides method to obtain an antiviral agent useful in the treatment of respiratory tract illness comprising establishing a cell culture or experimental animal comprising a virus according to the invention, treating said culture or animal with an candidate antiviral agent, and determining the effect of said agent on said virus or its infection of said culture or animal.", "An example of such an antiviral agent comprises a MPV-neutralising antibody, or functional component thereof, as provided herein, but antiviral agents of other nature are obtained as well.", "The invention also provides use of an antiviral agent according to the invention for the preparation of a pharmaceutical composition, in particular for the preparation of a pharmaceutical composition for the treatment of respiratory tract illness, especifically when caused by an MPV infection, and provides a pharmaceutical composition comprising an antiviral agent according to the invention, useful in a method for the treatment or prevention of an MPV infection or respiratory illness, said method comprising providing an individual with such a pharmaceutical composition.", "The invention is further explained in the detailed description without limiting it thereto.", "FIGURE LEGENDS FIG.", "1A comprises table 1: Percentage homology found between the amino acid sequence of isolate 00-1 and other members of the Pneumovirinae.", "Percentages (×100) are given for the amino acid sequences of N, P, M, F and two RAP-PCR fragments in L (8 and 9/10).", "Accession numbers used for the analyses are described in the materials and methods section.", "FIG.", "1B comprises table 2: Seroprevalence of MPV in humans categorised by age group using immunofluorescence and virus neutralisation assays.", "FIG.", "2: Schematic representation of the genome of APV with the location and size of the fragments obtained with RAP-PCR and RT-PCR on virus isolate 00-1.Fragments 1 to 10 were obtained using RAP-PCR.", "Fragment A was obtained with a primer in RAP-PCR fragment 1 and 2 and a primer designed based on alignment of leader and trailer sequences of APV and RSV6.Fragment B was obtained using primers designed in RAP-PCR fragment 1 and 2 and RAP-PCR fragment 3.Fragment C was obtained with primers designed in RAP-PCR fragment 3 and RAP-PCR fragment 4,5,6 and 7.For all phylogenetic trees, (FIGS.", "3-5) DNA sequences were aligned using the ClustalW software package and maximum likelihood trees were generated using the DNA-ML software package of the Phylip 3.5 program using 100 bootstraps and 3 jumbles15.Previously published sequences that were used for the generation of phylogenetic trees are available from Genbank under accessions numbers: For all ORFs: hRSV: NC001781; bRSV: NC001989; For the F ORF: PVM, D11128; APV-A, D00850; APV-B, Y14292; APV-C, AF187152; For the N ORF: PVM, D10331; APV-A, U39295; APV-B, U39296; APV-C, AF176590; For the M ORF: PMV, U66893; APV-A, X58639; APV-B, U37586; APV-C, AF262571; For the P ORF: PVM, 09649; APV-A, U22110, APV-C, AF176591.Phylogenetic analyses for the nine different virus isolates of MPV were performed with APV strain C as outgroup.", "Abbreviations used in figures: hRSV: human RSV; bRSV: bovine RSV; PVM: pneumonia virus of mice; APV-A,B, and C: avian pneumovirus typa A, B and C. FIG.", "3 Comparison of the N, P, M and F ORF's of members of the subfamily Pneumovirinae and virus isolate 00-1.The alignment shows the amino acid sequence of the complete N, P, M and F proteins and partial L proteins of virus isolate 00-1.Amino acids that differ between isolate 00-1 and the other viruses are shown, identical amino acids are represented by periods, gaps are represented as dashes.", "Numbers correspond to amino acid positions in the proteins.", "Accession numbers used for the analyses are described in the materials and methods section.", "APV-A, B or C: Avian Pneumovirus type A, B or C, b-or hRSV: bovine or human respiratory syncytial virus, PVM: pneumonia virus of mice.", "L8: fragment 8 obtained with RAP-PCR located in L, L9/10: consensus of fragment 9 and 10 obtained with RAP-PCR, located in L. For the P allignment, no APV-B sequence was available from the Genebank, For the L alligment only bRSV, hRSV and APV-A sequences were available.", "FIG.", "4: Phylogenetic analyses of the N, P, M, and F ORF's of members of the genus Pneumovirinae and virus isolate 00-1.Phylogenetic analysis was performed on viral sequences from the following genes: F (panel A), N (panel B), M (panel C), and P (panel D).", "The phylogenetic trees are based on maximum likelyhood analyses using 100 bootstraps and 3 jumbles.", "The scale representing the number of nucleotide changes is shown for each tree.", "FIG.", "5: Phylogenetic relationship for parts of the F (panel A), N (panel B), M (panel C) and L panel D) ORFs of nine of the primary MPV isolates with APV-C, it's closest relative genetically.", "The phylogenetic trees are based on maximum likelyhood analyses.", "The scale representing the number of nucleotide changes is shown for each tree.", "Accesion numbers for APV-C: panel A: D00850; panel B: U39295; panel C: X58639; and panel D: U65312.FIG.", "6A: Nucleotide and amino acid sequence information from the 3′end of the genome of MPV isolate 00-ORF's are given.", "N: ORF for nucleoprotein; P: ORF for phosphoprotein; M: ORF for matrix protein; F: ORF for fusion protein; GE: gene end; GS: gene start.", "FIGS.", "6B and C: Nucleotide and amino acid sequence information from obtained fragments in the polymerase gene (L) of MPV isolates 00-1.Positioning of the fragments in L is based on protein homologies with APV-C (accession number U65312).", "The translated fragment 8 (FIG.", "6B.)", "is located at amino acid number 8 to 243, and the consensus of fragments 9 and 10 (FIG.", "6C) is located at amino acid number 1358 to 1464 of the APV-C L ORF.", "FIG.", "7 Genomic map of MPV isolate 00-1.The nucleotide positions of the start and stop codons are indicated under each ORF.", "The double lines which cross the L ORF indicate the shortened representation of the L gene.", "The three reading frames below the map indicate the primary G ORF (nt 6262-6972) and overlapping potential secondary ORFs.", "FIG.", "8: Alignment of the predicted amino acid sequence of the nucleoprotein of MPV with those of other pneumoviruses.", "The conserved regions identified by Barr (1991) are represented by boxes and labelled A, B, and C. The conserved region among pneumoviruses (Li, 1996) is shown gray shaded.", "Gaps are represented by dashes, periods indicate the positions of identical amino acid residues compared to MPV.", "FIG.", "9: Amino acid sequence comparison of the phosphoprotein of MPV with those of other pneumoviruses.", "The region of high similarity (Ling, 1995) is boxed, and the glutamate rich region is grey shaded.", "Gaps are represented by dashes and periods indicate the position of identical amino acid residues compared to MPV.", "FIG.", "10: Comparison of the deduced amino acid sequence of the matrix protein of MPV with those of other pneumoviruses.", "The conserved hexapeptidesequence (Easton, 1997) is grey shaded.", "Gaps are represented by dashes and periods indicate the position of identical amino acid residues relative to MPV.", "FIG.", "11: Allignment of the predicted amino acid sequence of the fusion protein of MPV with those of other pneumoviruses.", "The conserved cysteine residues are boxed, N-linked glycosylation sites are underlined, the cleavage site of F0 is double underlined, the fusion peptide, signal peptide and membrane anchor domain are shown grey shaded.", "Gaps are represented by dashes and periods indicate the position of identical amino acids relative to MPV.", "FIG.", "12 Comparison of amino acid sequence of the M2 ORFs of MPV with those of other pneumoviruses.", "The alignment of M2-1 ORFs is shown in panel A, with the conserved amino terminus (Collins, 1990; Zamora, 1999) shown grey shaded.", "The three conserved cysteine residues are printed bold face and indicated by #.", "The alignment of M2-2 ORFs is shown in panel B.", "Gaps are represented by dashes and periods indicate the position of identical amino acids relative to MPV.", "FIG.", "13 Amino acid sequence analyses of the SH ORF of MPV.", "(A) Amino acid sequence of the SH ORF of MPV, with the serine and threonine residues grey shaded, cysteine residues in bold face and the hydrophobic region double underlined.", "Potential N-linked glycosylation sites are single underlined.", "Numbers indicate the positions of the basic amino acids flanking the hydrophobic domain.", "(B) Alignment of the hydrophobicity plots of the SH proteins of MPV, APV-A and hRSV-B.", "The procedure of Kyte and Doolittle (1982) was used with a window of 17 amino acids.", "Arrows indicate a strong hydrophobic domain.", "Positions within the ORF are given on the X-axis.", "FIG.", "14 Amino acid sequence analyses of the G ORF of MPV.", "(A) Amino acid sequence of the G ORF of MPV, with serine, threonine and proline residues grey shaded, the cysteine residue is in bold face and the hydrophobic region double underlined.", "The potential N-linked glycosylation sites are single underlined.", "(B) Alingment of the hydrophobicity plots of the G proteins of MPV, APV-A and hRSV-B.", "The procedure of Kyte and Doolittle (1982) was used with a window of 17 amino acids.", "Arrows indicate the hydrophobic region, and positions within the ORF are given at the X-axis.", "FIG.", "15 Comparison of the amino acid sequences of a conserved domain of the polymerase gene of MPV and other paramyxoviruses.", "Domain III is shown with the four conserved polymerase motifs (A, B, C, D) in domain III (Poch 1998, 1999) boxed.", "Gaps are represented by dashes and periods indicate the position of identical amino acid residues relative to MPV.", "hPIV3: human parainfluenza virus type 3; SV: sendai virus; hPIV-2: human parainfluenza virus type 2; NDV: New castle disease virus; MV: measles virus; nipah: Nipah virus.", "FIG.", "16: Phylogenetic analyses of the M2-1 and L ORFs of MPV and selected paramyxoviruses.", "The M2-1 ORF was aligned with the M2-1 ORFs of other members of the genus Pneumovirinae (A) and the L ORF was aligned with L ORFs members of the genus pneumovirinae and selected other paramyxoviruses as described in the legends of FIG.", "15(B).", "Phylogenetic trees were generated by maximum likelihood analyses using 100 bootstraps and 3 jumbles.", "The scale representing the number of nucleotide changes is shown for each tree.", "Numbers in the trees represent bootstrap values based on the consensus trees.", "FIG.", "17: Noncoding sequences of hMPV isolate 00-1.", "(A) The noncoding sequences between the ORFs and at the genomic termini are shown in the positive sense.", "From left to right, stop codons of indicated ORFs are shown, followed by the noncoding sequences, the gene start signals and start codons of the indicated subsequent ORFs.", "Numbers indicate the first position of start and stop codons in the hMPV map.", "Sequences that display similarity to published gene end signals are underlined and sequences that display similarity to UAAAAAU/A/C are represented with a line above the sequence.", "(B) Nucleotide sequences of the genomic termini of hMPV.", "The genomic termini of hMPV are aligned with each other and with those of APV.", "Underlined regions represent the primer sequences used in RT-PCR assays which are based on the 3′ and 5′ end sequences of APV and RSV (Randhawa et al., 1997; Mink et al., 1991).", "Bold italicalized nucleotides are part of the gene start signal of the N gene.", "Le: leader, Tr: trailer.", "FIG.", "18: Comparison of two prototypic hMPV isolates with APV-A and APV-C; DNA similarity matrices for nucleic acids encoding the various viral proteins.", "FIG.", "19: Comparison of two prototypic hMPV isolates with APV-A and APV-C; protein similarity matrices for the various viral proteins.", "FIG.", "20: Amino acid alignment of the nucleoprotein of two prototype hMPV isolates FIG.", "21: Amino acid alignment of the phosphoprotein of two prototype hMPV isolates FIG.", "22: Amino acid alignment of the matrix protein of two prototype hMPV isolates FIG.", "23: Amino acid alignment of the fusion protein of two prototype hMPV isolates FIG.", "24: Amino acid alignment of the M2-1 protein of two prototype hMPV isolates FIG.", "25: Amino acid alignment of the M2-2 protein of two prototype hMPV isolates FIG.", "26: Amino acid alignment of the short hydrophobic protein of two prototype hMPV isolates FIG.", "27: Amino acid alignment of the attachement glycoprotein of two prototype hMPV isolates FIG.", "28: Amino acid alignment of the N-terminus of the polymerase protein of two prototype hMPV isolates FIG.", "29: Results of RT-PCR assays on throat and nose swabs of 12 guinea pigs inoculated with ned/00/01 and/or ned/99/01.FIG.", "30A: IgG response against ned/00/01 and ned/99/01 for guinea pigs infected with ned/00/01 and re-infected with ned/00/01 (GP 4, 5 and 6) or ned/99/01 (GP 1 and 3).", "FIG.", "30B: IgG response against ned/00/01 and ned/99/01 for guinea pigs infected with ned/99/01 and re-infected with either ned/00/01 (GP's 8 and 9) or with ned/99/01 (GP's 10, 11, 12).", "FIG.", "31: Specificity of the ned/00/01 and ned/99/01 ELISA on sera taken from guinea pigs infected with either ned/00/01 or ned/99/01.FIG.", "32: Mean IgG response against ned/00/01 and ned/99/01 ELISA of 3 homologous (00-1/00-1), 2 homologous (99-1/99-1), 2 heterologous (99-1/00-1) and 2 heterologous (00-1/99-1) infected guinea pigs.", "FIG.", "33: Mean percentage of APV inhibition of hMPV infected guinea pigs.", "FIG.", "34: Virus neutralisation titers of ned/O/1 and ned/99101 infected guinea pigs against ned/00/01, ned/99/01 and APV-C.", "FIG.", "35: Results of RT-PCR assays on throat swabs of cynomolgous macaques inoculated (twice) with ned/00/01.FIG.", "36A (top two panels): IgA, IgM and IgG response against ned/10/01 of 2 cynomologous macaques (re)infected with ned/00/01.FIG.", "36B (bottom panels) IgG response against APV of 2 cynbomologous macaques infected with ned/00/01.FIG.", "37: Comparison of the use of the hMPV ELISA and the APV inhibition ELISA for the detection of IgG antibodies in human sera.", "DETAILED DESCRIPTION Virus Isolation and Characterisation From 1980 till 2000 we found 28 unidentified virus isolates from patients with severe Respiratory disease.", "These 28 unidentified virus isolates grew slowly in tMK cells, poorly in VERO cells and A549 cells and could not or only little be propagated in MDCK or chicken embryonated fibroblast cells.", "Most of these virus isolates induced CPE after three passages on tMK cells, between day ten and fourteen.", "The CPE was virtually indistinguishable from that caused by hRSV or hPIV in tMK or other cell cultures, characterised by syncytium formation after which the cells showed rapid internal disruption, followed by detachment of the cells from the monolayer.", "The cells usually (sometimes later) displayed CPE after three passages of virus from original material, at day 10 to 14 post inoculation, somewhat later than CPE caused by other viruses such as hRSV or hPIV.", "We used the supernatants of infected tMK cells for EM analysis which revealed the presence of paramyxovirus-like virus particles ranging from 150 to 600 nanometer, with short envelope projections ranging from 13 to 17 nanaometer.", "Consistent with the biochemical properties of enveloped viruses such as the Paramyxoviridae, standard chloroform or ether treatment8 resulted in>104 TCID50 reduction of infectivity for tMK cells.", "Virus-infected tMK cell culture supernatants did not display heamagglutinating activity with turkey, chicken and guinea pig erythrocytes.", "During culture, the virus replication appeared to be trypsine dependent on the cells tested.", "These combined virological data allowed that the newly identified virus was taxonomically classified as a member of the Paramyxoviridae family.", "We isolated RNA from tMK cells infected with 15 of the unidentified virus isolates for reverse transcription and polymerase chain reaction (RT-PCR) analyses using primer-sets specific for Paramyxovirinae9, hPIV 1-4, sendai virus, simian virus type 5, New-Castle disease virus, hRSV, morbilli, mumps, Nipah, Hendra, Tupaia and Mapuera viruses.", "RT-PCR assays were carried out at low stringency in order to detect potentially related viruses and RNA isolated from homologous virus stocks were used as controls.", "Whereas the available controls reacted positive with the respective virus-specific primers, the newly identified virus isolates did not react with any primer set, indicating the virus was not closely related to the viruses tested.", "We used two of the virus-infected tMK cell culture supernatants to inoculate guinea pigs and ferrets intranasaly.", "Sera were collected from these animals at day zero, two weeks and three weeks post inoculation.", "The animals displayed no clinical symptoms but all seroconverted as measured in virus neutralization (VN) assays and indirect IFA against the homologous viruses.", "The sera did not react in indirect IFA with any of the known paramyxoviruses described above and with PVM.", "Next, we screened the so far unidentified virus isolates using the guinea pig and ferret pre- and post-infection sera, of which 28 were clearly positive by indirect IFA with the post-infection sera suggesting they were serological closely related or identical.", "RAP PCR To obtain sequence information on the unknown virus isolates, we used a random PCR amplification strategy known as RAP-PCR10.To this end, tMK cells were infected with one of the virus isolates (isolate 00-1) as well as with hPIV-1 which served as a control.", "After both cultures displayed similar levels of CPE, virus in the culture supernatants was purified on continuous 20-60% sucrose gradients.", "The gradient fractions were inspected for virus-like particles by EM, and RNA was isolated from the fraction containing approximately 50% sucrose, in which nucleocapsids were observed.", "Equivalent amounts of RNA isolated from both virus fractions were used for RAP-PCR, after which samples were run side by side on a 3% NuSieve agarose gel.", "Twenty differentially displayed bands specific for the unidentified virus were subsequently purified from the gel, cloned in plasmid pCR2.1 (Invitrogen) and sequenced with vector-specific primers.", "When we used these sequences to search for homologies against sequences in the Genbank database using the BLAST software (www.ncbi.nlm.nih.gov/BLAST/) 10 out of 20 fragments displayed resemblance to APV/TRTV sequences.", "These 10 fragments were located in the genes coding for the nucleoprotein (N; fragment 1 and 2), the matrix protein (M; fragment 3), the fusion protein (F; fragment 4, 5, 6, 7,) and the polymerase protein (A; fragment 8,9,10) (FIG.", "2).", "We next designed PCR primers to complete the sequence information for the 3′ end of the viral genome based on our RAP PCR fragments as well as published leader and trailer sequences for the Pneumovirinae6.Three fragments were amplified, of which fragment A spanned the extreme 3′ end of the N open reading frame (ORF), fragment B spanned the phosphoprotein (P) ORF and fragment C closed the gap between the M and F ORFs (FIG.", "2).", "Sequence analyses of these three fragments revealed the absence of NS1 and NS2 ORFs at the extreme 3′ end of the viral genome and positioning of the F ORF immediately adjacent to the M ORF.", "This genomic organisation resembles that of the metapneumovirus APV, which is also consistent with the sequence homology.", "Overall the translated sequences for the N, P, M and F ORFs showed an average of 30-33% homology with members of the genus Pneumovirus and 66-68% with members of the genus Metapneumovirus.", "For the SH and G ORF's no discernable homology was found with members of either of the genera.", "The amino acid homologies found for N showed about 40% homology with hRSV and 88% with APV-C, its closest relative genetically, as for example can be deduced by comparing the amino acid sequence of FIG.", "3 with the amino acid sequence of the respective N proteins of other viruses.", "The amino acid sequence for P showed about 25% homology with hRSV and about 66-68% with APV-C, M showed about 36-39% with hRSV and about 87-89% with APV-C, F showed about 40% homology with hRSV and about 81% with APV-C, M2-1 showed about 34-36% homology with pneumoviruses and 84-86% with APV-C, M2-2 showed 15-17% homology with pneumoviruses and 56% with APV-C and the fragments obtained in L showed an average of 44% with pneumoviruses and 64% with APV-C. Phylogeny Although BLAST searches using nucleotide sequences obtained from the unidentified virus isolate revealed homologies primarily with members of the Pneumovirinae, homologies based on protein sequences revealed some resemblance with other paramyxoviruses as well (data not shown).", "As an indication for the relation between the newly identified virus isolate and members of the Pneumovirinae, phylogenetic trees were constructed based on the N, P, M and F ORFs of these viruses.", "In all four phylogenetic trees, the newly identified virus isolate was most closely related to APV (FIG.", "4).", "From the four serotypes of APV that have been described11, APV serotype C, the metapneumovirus found primarily in birds in the USA, showed the closest resemblance to the newly identified virus.", "It should be noted however, that only partial sequence information for APV serotype D is available.", "To determine the relationship of our various newly identified virus isolates, we constructed phylogenetic trees based on sequence information obtained from eight to nine isolates (8 for F, 9 for N, M and L).", "To this end, we used RT-PCR with primers designed to amplify short fragments in the N, M, F and L ORFs, that were subsequently sequenced directly.", "The nine virus isolates that were previously found to be related in serological terms (see above) were also found to be closely related genetically.", "In fact, all nine isolates were more closely related to one another than to APV.", "Although the sequence information used for these phylogenetic trees was limited, it appears that the nine isolates can be divided in two groups, with isolate 94-1, 99-1 and 99-2 clustering in one group and the other six isolates (94-2; 93-1; 93-2; 93-3; 93-4; 00-1) in the other (FIG.", "5).", "Seroprevalence To study the seroprevalence of this virus in the human population, we tested sera from humans in different age categories by indirect IFA using tMK cells infected with one of the unidentified virus isolates.", "This analysis revealed that 25% of the children between six and twelve months had antibodies to the virus, and by the age of five nearly 100% of the children were seropositive.", "In total 56 serum samples tested by indirect IFA were tested by VN assay.", "For 51 (91%) of the samples the results of the VN assay (titre>8) coincided with the results obtained with indirect IFA (titre>32).", "Four samples that were found positive in IFA, were negative by VN test (titre<8) whereas one serum reacted negative in IFA (titre<32) and positive in the VN test (titre 16) (table 2).", "IFA conducted with 72 sera taken from humans in 1958 (ages ranging from 8-99 years)12,27 revealed a 100% seroprevalence, indicating the virus has been circulating in the human population for more than 40 years.", "In addition a number of these sera were used in VN assays to confirm the IFA data (table 2).", "Genetic analyses of the N, M, P and F genes revealed that MPV has higher sequence homology to the recently proposed genus Metapneumovirinae (average of 63%) as compared to the genus Pneumovirinae (average of 30%) and thus demonstrates a genomic organization similar to and resembling that of APV/TRTV.", "In contrast to the genomic organisation of the RSVs (‘3-NS1-NS2-N-P-M-SH-G-F-M2-L-5’), metapneumoviruses lack NS1 and NS2 genes and have a different positioning of the genes between M and L ('3-N-P-M-F-M2-SH-G-15′).", "The lack of ORFs between the M and F genes in our virus isolates and the lack of NS1 and NS2 adjacent to to N, and the high amino acid sequence homology found with APV are reasons to propose the classification of MPV isolated from humans as a first member of the Metapneumovirus genus of mammalian, in particular of human origin.", "Phylogenetic analyses revealed that the nine MPV isolates from which sequence information was obtained are closely related.", "Although sequence information was limited, they were in fact more closely related to one another than to any of the avian metapneumoviruses.", "Of the four serotypes of APV that have been described, serotype C was most closely related to MPV based on the N, P, M and F genes.", "It should be noted however that for serotype D only partial sequences for the F gene were available from Genbank and for serotype B only M, N and F sequences were available.", "Our MPV isolates formed two clusters in phylogenetic trees.", "For both hRSV and APV different genetic and serological subtypes have been described.", "Whether the two genetic clusters of MPV isolates represent serogical subgroups that are also functionally different remains unknown at presentOur serological surveys showed that MPV is a common human pathogen.", "The repeated isolation of this virus from clinical samples from children with severe RTI indicates that the clinical and economical impact of MPV may be high.", "New diagnostic assays based on virus detection and serology will allow a more detailed analysis of the incidence and clinical and economical impact of this viral pathogen.", "The slight differences between the IFA and VN results (5 samples) maybe due to the fact that in the IFA only IgG serum antibodies were detected whereas the VN assay detects both classes and sub-classes of antibodies or differences may be due to the differences in sensitivity between both assays.", "For IFA a cut off value of 16 is used, whereas for VN a cut off value of 8 is used.", "On the other hand, differences between IFA versus VN assay may also indicate possible differences between different serotypes of this newly identified virus.", "Since MPV seems most closely related to APV, we speculate that the human virus may have originated from birds.", "Analysis of serum samples taken from humans in 1958 revealed that MPV has been widespread in the human population for more then 40 years indicating that a tentative zoonosis event must have taken place long before 1958.Materials and Methods Specimen Collection Over the past decades our laboratory has collected nasopharyngeal aspirates from children suffering from RTI, which are routinely tested for the presence of viruses.", "All nasopharyngeal aspirates were tested by direct immunofluorescence assays (DIF) using fluorescence labelled antibodies against influenza virus types A, and B, hRSV and human parainfluenza virus (hPIV) types 1 to 3.The nasopharyngeal aspirates were also processed for virus isolation using rapid shell vial techniques14 on various cellines including VERO cells, tertiary cynomolgous monkey kidney (tMK) cells, human endothelial lung (HEL) cells and marbin dock kidney (MDCK) cells.", "Samples showing cytophatic effects (CPE) after two to three passages, and which were negative in DIF, were tested by indirect immunofluorescence assays (IFA) using virus specific antibodies against influenza virus types A, B and C, hRSV types A and B, measles virus, mumps virus, human parainfluenza virus (hPIV) types 1 to 4, sendai virus, simian virus type 5, and New-Castle disease virus.", "Although for many cases the aetiological agent could be identified, some specimens were negative for all these viruses tested.", "Direct Immunofluorescence Assay (DIF) Nasopharyngeal aspirate samples from patients suffering from RTI were used for DIF and virus isolation as described14,15.Samples were stored at −70° C. In brief, nasopharyngeal aspirates were diluted with 5 ml Dulbecco MEM (BioWhittaker, Walkersville, Md.)", "and thoroughly mixed on a vortex mixer for one minute.", "The suspension was thus centrifuged for ten minutes at 840×g.", "The sediment was spread on a multispot slide (Nutacon, Leimuiden, The Netherlands), the supernatant was used for virus isolation.", "After drying, the cells were fixed in aceton for 1 minute at room temperature.", "After washing the slides were incubated for 15 minutes at 37° C. with commercial available FITC-labelled virus specific anti-sera such as influenza A and B, hRSV and hPIV 1 to 3 (Dako, Glostrup, Denmark).", "After three washings in PBS and one in tap water, the slides were included in a glycerol/PBS solution (Citifluor, UKC, Canterbury, UK) and covered.", "The slides were analysed using a Axioscop fluorescence microscope (Carl Zeiss B. V, Weesp, the Netherlands.", "Virus isolation For virus isolation tMK cells (RIVM, Bilthoven, The Netherlands) were cultured in 24 well plates containing glass slides (Costar, Cambridge, UK, with the medium described below supplemented with 10% fetal bovine serum (BioWhittaker, Vervier, Belgium).", "Before inoculation the plates were washed with PBS and supplied with Eagle's MEM with Hanks' salt (ICN, Costa mesa, CA) of which half a litre was supplemented with 0.26 gram HaHCO3, 0.025 M Hepes (Biowhittaker), 2 mM L-glutamine (Biowhittaker), 100 units penicilline, 100 μg streptomycine (Biowhittaker), 0.5 gram lactalbumine (Sigma-Aldrich, Zwijndrecht, The Netherlands), 1.0 gram D-glucose (Merck, Amsterdam, The Netherlands), 5.0 gram peptone (Oxoid, Haarlem, The Netherlands) and 0.02% trypsine (Mfe Technologies, Bethesda, Md.).", "The plates were inoculated with supernatant of the nasopharyngeal aspirate samples, 0,2 ml per well in triplicate, followed by centrifuging at 840×g for one hour.", "After inoculation the plates were incubated at 37° C. for a maximum of 14 days changing the medium once a week and cultures were checked daily for CPE.", "After 14 days cells were scraped from the second passage and incubated 14 days.", "This step was repeated for the third passage.", "The glass slides were used to demonstrate the presence of the virus by indirect IFA as described below.", "Animal Immunisation Ferret and guinea pig specific antisera for the newly discovered virus were generated by experimental intranasal infection of two specific pathogen free ferrets and two guinea pigs, housed in separate pressurised glove boxes.", "Two to three weeks later all the animals were bled by cardiac puncture, and their sera were used as reference sera.", "The sera were tested for all previous described viruses with indirect IFA as described below.", "Antigen Detection by Indirect IFA We performed indirect IFA on slides containing infected tMK cells.", "After washing with PBS the slides were incubated for 30 minutes at 37° C. with virus specific anti-sera.", "We used monoclonal antibodies in DIF against influenza A, B and C, hPIV type 1 to 3 and hRSV as described above.", "For hPIV type 4, mumps virus, measles virus, sendai virus, simian virus type 5, New-Castle Disease virus polyclonal antibodies (RIVM) and ferret and guinea pig reference sera were used.", "After three washings with PBS and one wash with tap water, the slides were stained with a secondary antibodies directed against the sera used in the first incubation.", "Secondary antibodies for the polyclonal anti sera were goat-anti-ferret (KPL, Guilford, UK, 40 fold diluted), mouse-anti-rabbit (Dako, Glostrup, Denmark, 20 fold diluted), rabbit-anti-chicken (KPL, 20 fold dilution) and mouse-anti-guinea pig (Dako, 20 fold diluted).", "Slides were processed as described for DIF.", "Detection of Antibodies in Humans by Indirect IFA For the detection of virus specific antibodies, infected tMK cells were fixed with cold acetone on coverslips, washed with PBS and stained with serum samples at a 1 to 16 dilution.", "Subsequently, samples were stained with FITC-labelled rabbit anti human antibodies 80 times diluted in PBS (Dako).", "Slides were processed as described above.", "Virus Culture of MPV Sub-confluent mono-layers of tMK cells in media as described above were inoculated with supernatants of samples that displayed CPE after two or three passages in the 24 well plates.", "Cultures were checked for CPE daily and the media was changed once a week.", "Since CPE differed for each isolate, all cultures were tested at day 12 to 14 with indirect IFA using ferret antibodies against the new virus isolate.", "Positive cultures were freeze-thawed three times, after which the supernatants were clarified by low-speed centrifugation, aliquoted and stored frozen at −70° C. The 50% tissue culture infectious doses (TCID50) of virus in the culture supernatants were determined as described16.Virus Neutralisation Assay VN assays were performed with serial two-fold dilutions of human and animal sera starting at an eight-fold dilution.", "Diluted sera were incubated for one hour with 100 TCID50 of virus before inoculation of tMK cells grown in 96 well plates, after which the plates were centrifuged at 840×g.", "The media was changed after three and six days and IFA was conducted with ferret antibodies against MPV 8 days after inoculation.", "The VN titre was defined as the lowest dilution of the serum sample resulting in negative IFA and inhibition of CPE in cell cultures.", "Virus Characterisation Haemagglutination assays and chloroform sensitivity tests were performed as described8,14.For EM analyses, virus was concentrated from infected cell culture supernatants in a micro-centrifuge at 4° C. at 17000×g, after which the pellet was resuspended in PBS and inspected by negative contrast EM.", "For RAP-PCR, virus was concentrated from infected tMK cell supernatants by ultra-centrifugation on a 60% sucrose cussion (2 hours at 150000×g, 4° C.).", "The 60% sucrose interphase was subsequently diluted with PBS and layered on top of a 20-60% continuous sucrose gradient which was centrifuged for 16 hours at 275000×g at 4° C. Sucrose gradient fractions were inspected for the presence of virus-like particles by EM and poly-acrylamide gel electrophoresis followed by silver staining.", "The approximately 50% sucrose fractions that appeared to contain nucleocapsids were used for RNA isolation and RAP-PCR.", "RNA Isolation RNA was isolated from the supernatant of infected cell cultures or sucrose gradient fractions using a High Pure RNA Isolation kit according to instructions from the manufacturer (Roche Diagnostics, Almere, The Netherlands).", "RT-PCR Virus-specific oligonucleotide sequences for RT-PCR assays on known paramyxoviruses are described in addenda 1.A one-step RT-PCR was performed in 50 μl reactions containing 50 mM Tris.HCl pH 8.5, 50 mM NaCl, 4 mM MgCl2, 2 mM dithiotreitol, 200 μM each dNTP, 10 units recombinant RNAsin (Promega, Leiden, the Netherlands), 10 units AMV RT (Promega, Leiden, The Netherlands), 5 units Amplitaq Gold DNA polymerase (PE Biosystems, Nieuwerkerk aan de IjsseL The Netherlands) and 5 μl RNA.", "Cycling conditions were 45 min.", "at 42° C. and 7 min.", "at 95° C. once, 1 min at 95° C., 2 min.", "at 42° C. and 3 min.", "at 72° C. repeated 40 times and 10 min.", "at 72° C. once.", "RAP-PCR RAP-PCR was performed essentially as described10.The oligonucleotide sequences are described in addenda 2.For the RT reaction, 2 μl RNA was used in a 10 μl reaction containing 10 ng/μl oligonucleotide, 10 mM dithiotreitol, 500 μm each dNTP, 25 mM Tris-HCl pH 8.3, 75 mM KCl and 3 mM MgCl2.The reaction mixture was incubated for 5 min.", "at 70° C. and 5 min.", "at 37° C., after which 200 units Superscript RT enzyme (LifeTechnologies) were added.", "The incubation at 37° C. was continued for 55 min.", "and the reaction terminated by a 5 min.", "incubation at 72° C. The RT mixture was diluted to give a 50 μl PCR reaction containing 8 ng/μl oligonucleotide, 300 μm each dNTP, 15 mM Tris-HCL pH 8.3, 65 mM KCl, 3.0 mM MgCl2 and 5 units Taq DNA polymerase (PE Biosystems).", "Cycling conditions were 5 min.", "at 94° C., 5 min.", "at 40° C. and 1 min.", "at 72° C. once, followed by 1 min.", "at 94° C., 2 min.", "at 56° C. and 1 min.", "at 72° C. repeated 40 times and 5 min.", "at 72° C. once.", "After RAP-PCR, 15 μl the RT-PCR products were run side by side on a 3% NuSieve agarose gel (FMC BioProducts, Heerhugowaard, The Netherlands).", "Differentially displayed fragments specific for MPV were purified from the gel with Qiaquick Gel Extraction kit (Qiagen, Leusden, The Netherlands) and cloned in pCR2.1 vector (Invitrogen, Groningen, The Netherlands) according to instructions from the manufacterer.", "Sequence Analysis RAP-PCR products cloned in vector pCR2.1 (Invitrogen) were sequenced with M13-specific oligonucleotides.", "DNA fragments obtained by RT-PCR were purified from agarose gels using Qiaquick Gel Extraction kit (Qiagen, Leusden, The Netherlands), and sequenced directly with the same oligonucleotides used for PCR.", "Sequence analyses were performed using a Dyenamic ET terminator sequencing kit (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) and an ABI 373 automatic DNA sequencer (PE Biosystem).", "All techniques were performed according to the instructions of the manufacturer.", "Generating Genomic Fragments of MPV by RT-PCR To generate PCR fragments spanning gaps A, B and C between the RAP-PCR fragments (FIG.", "2) we used RT-PCR assays as described before on RNA isolated from virus isolate 00-1.The following primers were used: For fragment A: TR1 designed in the leader: (5′-AAAGAATTCACGAGAAAAAAACGC-3′) and N1 designed at the 3′end of the RAP-PCR fragments obtained in N (5′-CTGTGGTCTCTAGTCCCACTTC-3′) For fragment B: N2 designed at the 5′end of the RAP-PCR fragments obtained in N: (5′-CATGCAAGCTTATGGGGC-3′) and M1 designed at the 3′end of the RAP-PCR fragments obtained in M: (5′-CAGAGTGGTTATTGTCAGGGT-3).", "For fragment C: M2 designed at the 5′end of the RAP-PCR fragment obtained in M: (5′-GTAGAACTAGGAGCATATG-3′) and F1 designed at the 3′end of the RAP-PCR fragments obtained in F: (5′-TCCCCAATGTAGATACTGCTTC-3′).", "Fragments were purified from the gel, cloned and sequenced as described before.", "RT-PCR for Diagnosing MPV.", "For the amplification and sequencing of parts of the N, M, F and L ORFs of nine of the MPV isolates, we used primers N3 (5′-GCACTCAAGAGATACCCTAG-3′) and N4 (5′-AGACTTTCTGCTTTGCTGCCTG-3′), amplifying a 151 nucleotide fragments, M3 (5′-CCCTGACAATAACCACTCTG-3′) and M4 (5′-GCCAACTGATTTGGCTGAGCTC-3′) amplifying a 252 nucleotide fragment, F7 (5′-TGCACTATCTCCTCTTGGGGCTTTG-3) and F8 (5′-TCAAAGCTGCTTGACACTGGCC-3′) amplifying a 221 nucleotide fragment and L6 (5′-CATGCCCACTATAAAAGGTCAG-3′) and L7 (5′-CACCCCAGTCTTTCTTGAAA-3) amplifying a 173 nucleotide fragment respectively.", "RT-PCR, gel purification and direct sequencing were performed as described above.", "Furthermore, probes used were: Probe used in M: 5′-TGC TTG TAC TTC CCA AAG-3′ Probe used in N: 5′-TAT TTG AAC AAA AAG TGT-3′ Probe used in L: 5′-TGGTGTGGGATATTAACAG-3′ Phylogenetic Analyses For all phylogenetic trees, DNA sequences were alligned using the ClustalW software package and maximum likelihood trees were generated using the DNA-ML software package of the Phylip 3.5 program using 100 bootstraps and 3 jumblesl15.Previously published sequences that were used for the generation of phylogenetic trees are available from Genbank under accessions numbers: For all ORFs: hRSV: NC001781; bRSV: NC001989; For the F ORF: PVM, D11128; APV-A, D00850; APV-B, Y14292; APV-C, AF187152; For the N ORF: PVM, D10331; APV-A, U39295; APV-B, U39296; APV-C, AF176590; For the M ORF: PMV, U66893; APV-A, X58639; APV-B, U37586; APV-C, AF262571; For the P ORF: PVM, 09649; APV-A, U22110, APV-C, AF176591.Phylogenetic analyses for the nine different virus isolates of MPV were performed with APV strain C as outgroup.", "Abbreviations used in figures: hRSV: human RSV; bRSV: bovine RSV; PVM: pneumonia virus of mice; APV-A, B, and C: avian pneumovirus typ A, B and C. Examples of Methods to Identify MPV Specimen Collection In order to find virus isolates nasopharyngeal aspirates, throat and nasal swabs, broncheo alveolar lavages preferably from mammals such as humans, carnivores (dogs, cats, mustellits, seals etc.", "), horses, ruminants (cattle, sheep, goats etc.", "), pigs, rabbits, birds (poultry, ostriches, etc) should be examined.", "From birds cloaca swabs and droppings can be examined as well Sera should be collected for immunological assays, such as ELISA and virus neutralisation assays.", "Collected virus specimens were diluted with 5 ml Dulbecco MEM medium (BioWhittaker, Walkersville, Md.)", "and thoroughly mixed on a vortex mixer for one minute.", "The suspension was thus centrifuged for ten minutes at 840×g.", "The sediment was spread on a multispot slide (Nutacon, Leimuiden, The Netherlands) for immunofluorescence techniques, and the supernatant was used for virus isolation.", "Virus Isolation For virus isolation tMK cells (RIVM, Bilthoven, The Netherlands) were cultured in 24 well plates containing glass slides (Costar, Cambridge, UK), with the medium described below supplemented with 10% fetal bovine serum BioWhittaker, Vervier, Belgium).", "Before inoculation the plates were washed with PBS and supplied with Eagle's MEM with Hanks' salt (ICN, Costa mesa, CA) supplemented with 0.52/liter gram NaHCO3, 0.025 M Hepes (Biowhittaker), 2 mM L-glutamine (Biowhittaker), 200 units/liter penicilline, 200 μg/liter streptomycine (Biowhittaker), 1 gram/liter lactalbumine (Sigma-Aldrich, Zwijndrecht, The Netherlands), 2.0 gram/liter D-glucose (Merck, Amsterdam, The Netherlands), 10 gram/liter peptone (Oxoid, Haarlem, The Netherlands) and 0.02% trypsine (Life Technologies, Bethesda, M).", "The plates were inoculated with supernatant of the nasopharyngeal aspirate samples, 0,2 ml per well in triplicate, followed by centrifuging at 840×g for one hour.", "After inoculation the plates were incubated at 37° C. for a maximum of 14 days changing the medium once a week and cultures were checked daily for CPE.", "After 14 days, cells were scraped from the second passage and incubated for another 14 days.", "This step was repeated for the third passage.", "The glass slides were used to demonstrate the presence of the virus by indirect IFA as described below.", "CPE was generally observed after the third passage, at day 8 to 14 depending on the isolate.", "The CPE was virtually indistinghuisable from that caused by hRSV or hPIV in tMK or other cell cultures.", "However, hRSV induces CPE starting around day 4.CPE was characterised by syncytia formation, after which the cells showed rapid internal disruption, followed by detachment of cells from the monolayer.", "For some isolates CPE was difficult to observe, and IFA was used to confirm the presence of the virus in these cultures.", "Virus culture of MPV Sub-confluent monolayers of tMK cells in media as described above were inoculated with supernatants of samples that displayed CPE after two or three passages in the 24 well plates.", "Cultures were checked for CPE daily and the media was changed once a week.", "Since CPE differed for each isolate, all cultures were tested at day 12 to 14with indirect IFA using ferret antibodies against the new virus isolate.", "Positive cultures were freeze-thawed three times, after which the supernatants were clarified by low-speed centrifugation, aliquoted and stored frozen at −70° C. The 50% tissue culture infectious doses (TCID50) of virus in the culture supernatants were determined following established techniques used in the field16.Virus Characterisation Haemagglutination assays and chloroform sensitivity tests were performed following well established and described techniques used in the field14.For EM analyses, virus was concentrated from infected cell culture supernatants in a micro-centrifuge at 4° C at 17000×g, after which the pellet was resuspended in PBS and inspected by negative contrast EM.", "Antigen Detection by Indirect IRA Collected specimens were processed as described and sediment of the samples was spread on a multispot slide.", "After drying, the cells were fixed in aceton for 1 minute at room temperature.", "Alternatively, virus was cultured on tMK cells in 24 well slides containing glass slides.", "These glass slides were washed with PBS and fixed in aceton for 1 minute at room temperature.", "After washing with PBS the slides were incubated for 30 minutes at 37° C. with polyclonal antibodies at a dilution of 1:50 to 1:100 in PBS.", "We used immunised ferrets and guinea pigs to obtain polyclonal antibodies, but these antibodies can be raised in various animals, and the working dilution of the polyclonal antibody can vary for each immunisation.", "After three washes with PBS and one wash with tap water, the slides were incubated at 37° C. for 30 minutes with FITC labeled goat-anti-ferret antibodies (KPL, Guilford, UK, 40 fold diluted).", "After three washes in PBS and one in tap water, the slides were included in a glycerol/PBS solution (Citifluor, UKC, Canterbury, UK) and covered.", "The slides were analysed using an Axioscop fluorescence microscope (Carl Zeiss B. V., Weesp, the Netherlands).", "Detection of Antibodies in Humans, Mammals, Ruminants or Other Animals by Indirect IFA For the detection of virus specific antibodies, infected tMK cells with MPV were fixed with acetone on coverslips (as described above), washed with PBS and incubated 30 minutes at 37° C. with serum samples at a 1 to 16 dilution.", "After two washes with PBS and one with tap water, the slides were incubated 30 minutes at 37° C. with FITC-labelled secondary antibodies to the species used (Dako).", "Slides were processed as described above.", "Antibodies can be labelled directly with a fluorescent dye, which will result in a direct immuno fluorescence assay.", "FITC can be replaced with any fluorescent dye.", "Animal Immunisation Ferret and guinea pig specific antisera for the newly discovered virus were generated by experimental intranasal infection of two specific pathogen free ferrets and two guinea pigs, housed in separate pressurised glove boxes.", "Two to three weeks later the animals were bled by cardiac puncture, and their sera were used as reference sera.", "The sera were tested for all previous described viruses with indirect IFA as described below.", "Other animal species are also suitable for the generation of specific antibody preparations and other antigen preparations may be used.", "Virus Neutralisation Assay (VN Assay) VN assays were performed with serial two-fold dilutions of human and animal sera starting at an eight-fold dilution.", "Diluted sera were incubated for one hour with 100 TCID50 of virus before inoculation of tMK cells grown in 96 well plates, after which the plates were centrifuged at 840×g.", "The same culture media as described above was used.", "The media was changed after three and six days, and after 8 days IFA was performed (see above).", "The VN titre was defined as the lowest dilution of the serum sample resulting in negative IFA and inhibition of CPE in cell cultures.", "RNA Isolation RNA was isolated from the supernatant of infected cell cultures or sucrose gradient fractions using a High Pure RNA Isolation kit according to instructions from the manufacturer (Roche Diagnostics, Almere, The Netherlands).", "RNA can also be isolated following other procedures known in the field (Current Protocols in Molecular Biology).", "RT-PCR A one-step RT-PCR was performed in 50 μl reactions containing 50 mM Tris.HCl pH 8.5, 50 mM NaCl, 4 mM MgCl2, 2 mM dithiotreitol, 200 μM each dNTP, 10 units recombinant RNAsin (Promega, Leiden, the Netherlands), 10 units AMV RT (Promega, Leiden, The Netherlands), 5 units Amplitaq Gold DNA polymerase (PE Biosystems, Nieuwerkerk aan de Ijssel, The Netherlands) and 5 μl RNA.", "Cycling conditions were 45 min.", "at 42 CC and 7 min.", "at 95° C. once, 1 min at 95° C., 2 min.", "at 42° C. and 3 min.", "at 72° C. repeated 40 times and 10 min.", "at 72° C. once.", "Primers used for diagnostic PCR: In the nucleoprotein: N3 (5′-GCACTCAAGAGATACCCTAG 3′) and N4 (5′-AGACTTTCTGCTTTGCTGCCTG-3′), amplifying a 151 nucleotide fragment.", "In the matrixprotein: M3 (5′-CCCTGACAATAACCACTCTG-3′) and M4 (5′-GCCAACTGATTTGGCTGAGCTC-3′) amplifying a 252 nucleotide fragment In the polymerase protein: L6 (5′-CATGCCCACTATAAAAGGTCAG-3′) and L7 (5′-CACCCCAGTCTTTCTTGAAA-3′) amplifying a 173 nucleotide fragment.", "Other primers can be designed based on MPV sequences, and different buffers and assay conditions may be used for specific purposes.", "Sequence Analysis Sequence analyses were performed using a Dyenamic ET terminator sequencing kit (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) and an ABI 373 automatic DNA sequencer (PE Biosystem).", "All techniques were performed according to the instructions of the manufacturer.", "PCR fragments were sequenced directly with the same oligonucleotides used for PCR, or the fragments were purified from the gel with Qiaquick Gel Extraction kit (Qiagen, Leusden, The Netherlands) and cloned in pCR2.1 vector (Invitrogen, Groningen, The Netherlands) according to instructions from the manufacturer and subsequently sequenced with M13-specific oligonucleotides.", "Oligonucleotides Used for Analysing the 3′end of the Genome (Absence of NS1I/NS2).", "Primer TR1 (5′-AAAGAATTCACGAGAAAAAAACGC-3) was designed based on published sequences of the trailer and leader for hRSV and APV, published by Randhawa (1997) and primer N1 ('5‘-CTGTGGTCTCTAGTCCCACTTC-3’) was designed based on obtained sequences in the N protein.", "The RT-PCR assay and sequencing was performed as described above.", "The RT-PCR gave a product of approximately 500 base pairs which is to small to contain information for two ORFS, and translation of these sequences did not reveal an ORF.", "Detection of Antibodies in Humans, Mammals, Ruminants or Other Animals by ELISA In Paramyxoviridae, the N protein is the most abundant protein, and the immune response to this protein occurs early in infection.", "For these reasons, a recombinant source of the N proteins is preferably used for developing an ELISA assay for detection of antibodies to MPV.", "Antigens suitable for antibody detection include any MPV protein that combines with any MPV-specific antibody of a patient exposed to or infected with MPV virus.", "Preferred antigens of the invention include those that predominantly engender the immune response in patients exposed to MPV, which therefore, typically are recognised most readily by antibodies of a patient.", "Particularly preferred antigens include the N, F and G proteins of MPV.", "Antigens used for immunological techniques can be native antigens or can be modified versions thereof.", "Well known techniques of molecular biology can be used to alter the amino acid sequence of a MPV antigen to produce modified versions of the antigen that may be used in immunologic techniques.", "Methods for cloning genes, for manipulating the genes to and from expression vectors, and for expressing the protein encoded by the gene in a heterologous host are well-known, and these techniques can be used to provide the expression vectors, host cells, and the for expressing cloned genes encoding antigens in a host to produce recombinant antigens for use in diagnostic assays.", "See for instance: Molecular cloning, A laboratory manual and Current Protocols in Molecular Biology.", "A variety of expression systems may be used to produce MPV antigens.", "For instance, a variety of expression vectors suitable to produce proteins in E. Coli, B. subtilis, yeast, insect cells and mammalian cells have been described, any of which might be used to produce a MPV antigen suitable to detect anti-MPV antibodies in exposed patients.", "The baculovirus expression system has the advantage of providing necessary processing of proteins, and is therefor preferred.", "The system utilizes the polyhedrin promoter to direct expression of MPV antigens.", "(Matsuura et al.", "1987, J. Gen. Virol.", "68: 1233-1250).", "Antigens produced by recombinant baculo-viruses can be used in a variety of immunological assays to detect anti-MPV antibodies in a patient.", "It is well established, that recombinant antigens can be used in place of natural virus in practically any immunological assay for detection of virus specific antibodies.", "The assays include direct and indirect assays, sandwich assays, solid phase assays such as those using plates or beads among others, and liquid phase assays.", "Assays suitable include those that use primary and secondary antibodies, and those that use antibody binding reagents such as protein A.", "Moreover, a variety of detection methods can be used in the invention, including calorimetric, fluorescent, phosphorescent, chemiluminescent, luminescent and radioactive methods.", "EXAMPLE 1 Of Indirect Anti-MPV IgG EIA Using Recombinant N Protein An indirect IgG EIA using a recombinant N protein (produced with recombinant baculo-virus in insect (Sf9) cells) as antigen can be performed.", "For antigen preparation, Sf9 cells are infected with the recombinant baculovirus and harvested 3-7 days post infection.", "The cell suspension is washed twice in PBS, pH 7.2, adjusted to a cell density of 5.0×106 cells/ml, and freeze-thawed three times.", "Large cellular debris is pelleted by low speed centrifugation (500×g for 15 min.)", "and the supernatant is collected and stored at −70° C. until use.", "Uninfected cells are processed similarly for negative control antigen.", "100 μl of a freeze-thaw lysate is used to coat microtiter plates, at dilutions ranging from 1:50 to 1:1000.An uninfected cell lysate is run in duplicate wells and serves as a negative control.", "After incubation overnight, plates are washed twice with PBS/0.05%Tween.", "Test sera are diluted 1:50 to 1:200 in ELISA buffer PBS, supplemented to 2% with normal goat sera, and with 0.5% bovine serum albumine and 0.1% milk), followed by incubation wells for 1 hour at 37° C. Plates are washed two times with PBS/0.05% Tween.", "Horseradish peroxidase labelled goat anti-human (or against other species) IgG, diluted 1:3000 to 1:5000 in ELISA buffer, added to wells, and incubated for 1 hour at 37°.", "The plates are then washed two times with PBS/0.05% Tween and once with tap water, incubated for 15 minutes at room temperature with the enzyme substrate TMB, 3,3′,5,5′ tetramethylbenzidine, such as that obtained from Sigma, and the reaction is stopped with 100 μl of 2 M phosphoric acid.", "Colorimetric readings are measured at 450 nm using an automated microtiter plate reader.", "EXAMPLE 2 Capture anti-MPV IgM EIA Using a Recombinant Nucleoprotein A capture IgM EIA using the recombinant nucleoprotein or any other recombinant protein as antigen can be performed by modification of assays as previously described by Erdman et al (1990) J. Clin.", "Microb.", "29: 1466-1471.Affinity purified anti-human IgM capture antibody (or against other species), such as that obtained from Dako, is added to wells of a microtiter plate in a concentration of 250 ng per well in 0.1 M carbonate buffer pH 9.6.After overnight incubation at room temperature, the plates are washed two times with PBS/0.05% Tween.", "100 μl of test serum diluted 1:200 to 1:1000 in ELISA buffer is added to triplicate wells and incubated for 1 hour at 37° C. The plates are then washed two times with in PBS/0.05% Tween.", "The freeze-thawed (infected with recombinant virus) Sf21 cell lysate is diluted 1:100 to 1: 500 in ELISA buffer is added to the wells and incubated for 2 hours at 37° C. Uninfected cell lysate serves as a negative control and is run in duplicate wells.", "The plates are then washed three times in PBS/0.06% Tween and incubated for 1 hour at 37° C. with 100 μl of a polyclonal antibody against MPV in a optimal dilution in ELISA buffer.", "After 2 washes with PBS/0.05% Tween, the plates are incubated with horseradish peroxide labeled secondary antibody (such as rabbit anti ferret), and the plates are incubated 20 minutes at 37° C. The plates are then washed five times in PBS/0105% Tween, incubated for 15 minutes at room temperature with the enzyme substrate TMB, 3,3′,5,6′ tetramethylbenzidine, as, for instance obtained from “Sigma”, and the reaction is stopped with 100 μl of 2M phosphoric acid.", "Colormetric readings are measured at 450 nm using automated microtiter plate reader.", "The sensitivities of the capture IgM EIAs using the recombinant nucleoprotein (or other recombinant protein) and whole MPV virus are compared using acute-and convalescent-phase serum pairs form persons with clinical MPV virus infection.", "The specificity of the recombinant nucleoprotein capture EIA is determined by testing serum specimens from healthy persons and persons with other paramyxovirus infections.", "Potential for EIAs for using recombinant MPV fusion and glycoprotein proteins produced by the baculovirus expression.", "The glycoproteins G and F are the two transmembraneous envelope glycoproteins of the MPV virion and represent the major neutralisation and protective antigens.", "The expression of these glycoproteins in a vector virus system sych as a baculovirus system provides a source of recombinant antigens for use in assays for detection of MPV specific antibodies.", "Moreover, their use in combination with the nucleoprotein, for instance, further enhances the sensitivity of enzyme immunoassays in the detection of antibodies against MPV.", "A variety of other immunological assays (Current Protocols in Immunology) may be used as alternative methods to those described here.", "In order to find virus isolates nasopharyngeal aspirates, throat and nasal swabs, broncheo alveolar lavages and throat swabs preferable from but not limited to humans, carnivores (dogs, cats, seals etc.", "), horses, ruminants (cattle, sheep, goats etc.", "), pigs, rabbits, birds (poultry, ostridges, etc) can be examined.", "From birds, cloaca and intestinal swabs and droppings can be examined as well.", "For all samples, serology (antibody and antigen detection etc.", "), virus isolation and nucleic acid detection techniques can be performed for the detection of virus.", "Monoclonal antibodies can be generated by immunising mice (or other animals) with purified MPV or parts thereof (proteins, peptides) and subsequently using established hybridoma technology (Current protocols in Immunology).", "Alternatively, phage display technology can be used for this purpose (Current protocols in Immunology).", "Similarly, polyclonal antibodies can be obtained from infected humans or animals, or from immunised humans or animals (Current protocols in Immunology).", "The detection of the presence or absence of NS1 and NS2 proteins can be performed using western-blotting, IFA, immuno precipitation techniques using a variety of antibody preparations.", "The detection of the presence or absence of NS1 and NS2 genes or homologues thereof in virus isolates can be performed using PCR with primer sets designed on the basis of known NS1 and/or NS2 genes as well as with a variety of nucleic acid hybridisation techniques.", "To determine whether NS1 and NS2 genes are present at the 3′ end of the viral genome, a PCR can be performed with primers specific for this 3′ end of the genome.", "In our case, we used a primer specific for the 3′ untranslated region of the viral genome and a primer in the N ORF.", "Other primers may be designed for the same purpose.", "The absence of the NS1/NS2 genes is revealed by the length and/or nucleotide sequence of the PCR product.", "Primers specific for NS1 and/or NS2 genes may be used in combination with primers specific for other parts of the 3′ end of the viral genome (such as the untranslated region or N, M or F ORFs) to allow a positive identification of the presence of NS1 or NS2 genes.", "In addition to PCR, a variety of techniques such as molecular cloning, nucleic acid hybridisation may be used for the same purpose.", "EXAMPLE 3 Different Serotypes/Subgroups of MPV Two potential genetic clusters are identified by analyses of partial nucleotide sequences in the N, M, F and L ORFs of 9 virus isolates.", "90-100% nucleotide identity was observed within a cluster, and 81-88% identity was observed between the clusters.", "Sequence information obtained on more virus isolates confirmed the existence of two genotypes.", "Virus isolate ned/00/01 as prototype of cluster A, and virus isolate ned/99/01 as prototype of cluster B have been used in cross neutralization assays to test whether the genotypes are related to different serotypes or subgroups.", "Results Using RT-PCR assays with primers located in the polymerase gene, we identified 30 additional virus isolates from nasopharyngeal aspirate samples.", "Sequence information of parts of the matrix and polymerase genes of these new isolates together with those of the previous 9 isolates were used to construct phylogenetic trees (FIG.", "16).", "Analyses of these trees confirmed the presence of two genetic clusters, with virus isolate ned/00/00-1 as the prototype virus in group A and virus isolate ned/99/01 as the prototype virus in group B.", "The nucleotide sequence identity within a group was more than 92%, while between the clusters the identity was 81-85%.", "Virus isolates ned/00/01 and ned/99/01 have been used to inoculate ferrets to raise virus-specific antisera.", "These antisera were used in virus neutralization assays with both viruses.", "TABLE 3 Virus neutralization titers isolate 00-1 isolate 99-1 preserum □2 □2 ferret A (00-1) ferret A 64 □2 22 dpi (00-1) preserum □2 □2 ferret B (99-1) ferret B 4 64 22 dpi (99-1) For isolate 00-1 the titer differs 32 (64/2) fold For isolate 99-1 the titer differs 16 (64/4) fold In addition, 6 guinea pigs have been inoculated with either one of the viruses (ned/00/01 and ned/99/01).", "RT-PCR assays on nasopharyngeal aspirate samples showed virus replication from day 2 till day 10 post infection.", "At day 70 post infection the guinea pigs have been challenged with either the homologous or the heterologous virus, and for in all four cases virus replication has been noticed.", "TABLE 4 primary virus secondary virus infection replication infection replication guinea pig 1-3 00-1 2 out of 3 99-1 1 out of 2 guinea pig 4-6 00-1 3 out of 3 00-1 1 out of 3 guinea pig 7-9 99-1 3 out of 3 00-1 2 out of 2 guinea pig 10-12 99-1 3 out of 3 99-1 1 out of 3 note: for the secondary infection guinea pig 2 and 9 were not there any more.", "Virus neutralization assays with anti sera after the first challenge showed essentially the same results as in the VN assays performed with the ferrets (>16-fold difference in VN titer).", "The results presented in this example confirm the existence of two genotypes, which correspond to two serotypes of MPV, and show the possibility of repeated infection with heterologous and homologous virus.", "EXAMPLE 4 Further Sequence Determination This example describes the further analysis of the sequences of MPV open reading frames (ORFs) and intergenic sequences as well as partial sequences of the genomic termini.", "Sequence analyses of the nucleoprotein (N), phosphoprotein (P), matrixprotein (M) and fusion protein (F) genes of MPV revealed the highest degree of sequence homology with APV serotype C, the avian pneumovirus found primarily in birds in the United States.", "These analyses also revealed the absence of non-structural proteins NS1 and NS2 at the 3′ end of the viral genome and positioning of the fusion protein immediately adjacent to the matrix protein.", "Here we present the sequences of the 22K (M2) protein, the small hydrophobic (SH) protein, the attachment (G) protein and the polymerase (L) protein genes, the intergenic regions and the trailer sequence.", "In combination with the sequences described previously the sequences presented here complete the genomic sequence of MPV with the exception of the extreme 12-15 nucleotides of the genomic termini and establish the genomic organisation of MPV.", "Side by side comparisons of the sequences of the MPV genome with those of APV subtype A, B and C, RSV subtype A and B, PVM and other paramyxoviruses provides strong evidence for the classification of MPV in the Metapneumovirus genus.", "Results Sequence Strategy MPV isolate 00-1 (van den Hoogen et al., 2001) was propagated in tertiary monkey kidney (tMK cells and RNA isolated from the supernatant 3 weeks after inoculation was used as template for RT-PCR analyses.", "Primers were designed on the basis of the partial sequence information available for MTV 00-1 (van den Hoogen et al., 2001) as well as the leader and trailer sequences of APV and RSV (Randhawa et al., 1997; Mink et al., 1991).", "Initially, fragments between the previously obtained products, ranging in size from 500 bp to 4 Kb in length, were generated by RT-PCR amplification and sequenced directly.", "The genomic sequence was subsequently confirmed by generating a series of overlapping RT-PCR fragments ranging in size from 500 to 800 bp that represented the entire MPV genome.", "For all PCR fragments, both strands were sequenced directly to minimize amplification and sequencing errors.", "The nucleotide and amino acid sequences were used to search for homologies with sequences in the Genbank database using the BLAST software (www.ncbi.nlm.nih.gov/BLAST).", "protein names were assigned to open reading frames (ORFs) based on homology with known viral genes as well as their location in the genome.", "Based on this information, a genomic map for MPV was constructed FIG.", "7).", "The MPV genome is 13378 nucleotides in length and its organization is similar to the genomic organization of APV.", "Below, we present a comparison between the ORFs and non-coding sequences of MPV and those of other paramyxoviruses and discuss the important similarities and differences.", "The Nucleoprotein (N) Gene As shown, the first gene in the genomic map of MPV codes for a 394 amino acid (aa) protein and shows extensive homology with the N protein of other pneumoviruses.", "The length of the N ORF is identical to the length of the N ORF of APV-C (Table 5) and is smaller than those of other paramyxoviruses (Barr et al., 1991).", "Analysis of the amino acid sequence revealed the highest homology with APV-C (88%), and only 7-11% with other paramyxoviruses (Table 6).", "Barr et al (1991) identified 3 regions of similarity between viruses belonging to the order Mononegavirales: A, B and C (FIG.", "8).", "Although similarities are highest within a virus family, these regions are highly conserved between virus familys.", "In all three regions MPV revealed 97% aa sequence identity with APV-C, 89% with APV-B, 92% with APV-A, and 66-73% with RSV and PVM.", "The region between aa residues 160 and 340 appears to be highly conserved among metapneumoviruses and to a somewhat lesser extent the Pneumovirinae (Miyahara et al., 1992; Li et al., 1996; Barr et al., 1991).", "This is in agreement with MPV being a metapneumovirus, showing 100% similarity with APV C. The Phosphoprotein (P) Gene The second ORF in the genome map codes for a 294 aa protein which shares 68% aa sequence homology with the P protein of APV-C, and only 22-26% with the P protein of RSV (Table 6).", "The P gene of MPV contains one substantial ORF and in that respect is similar to P from many other paramyxoviruses (Reviewed in Lamb and Kolakofsky, 1996; Sedlmeier et al., 1998).", "In contrast to APV A and B and PVM and similar to RSV and APV-C the MPV P ORF lacks cysteine residues.", "Ling (1995) suggested that a region of high similarity between all pneumoviruses (aa 185-241) plays a role in either the RNA synthesis process or in maintaining the structural integrity of the nucleocapsid complex.", "This region of high similarity is also found in MPV (FIG.", "9) especifically when conservative substitutions are taken in account, showing 100% similarity with APV-C, 93% with APV-A and B, and approximately 81% with RSV.", "The C-terminus of the MPV P protein is rich in glutamate residues as has been described for APVs (Ling et al., 1995).", "The Matrix (M) Protein Gene The third ORF of the MPV genome encodes a 254 aa protein, which resembles the M ORFs of other pneumoviruses.", "The M ORF of MPV has exactly the same size as the M ORFs of other metapneumoviruses (Table 5) and shows high aa sequence homology with the matrix proteins of APV (78-87%), lower homology with those of RSV and PVM (37-38%) and 10% or less homology with those of other paramyxoviruses (Table 6).", "Easton (1997) compared the sequences of matrix proteins of all pneumoviruses and found a conserved heptadpeptide at residue 14 to 19 that is also conserved in MPV (FIG.", "10).", "For RSV, PVM and APV small secondary ORFs within or overlapping with the major ORF of M have been identified (52 aa and 51 aa in bRSV, 75 aa in RSV, 46 aa in PVM and 51 aa in APV) (Yu et al., 1992; Easton et al., 1997; Samal et al., 1991; Satake et al., 1984).", "We noticed two small ORFs in the M ORF of MPV.", "One small ORF of 54 aa residues was found within the major M ORF (fragment 1, FIG.", "7), starting at nucleotide 2281 and one small ORF of 33 aa residues was found overlapping with the major ORF of M starting at nucleotide 2893 (fragment 2, FIG.", "7).", "Similar to the secondary ORFs of RSV and APV there is no significant homology between these secondary ORFs and secondary ORFs of the other pneumoviruses, and apparent start or stop signals are lacking.", "In addition, evidence for the synthesis of proteins corresponding to these secondary ORFs of APV and RSV has not been reported.", "The Fusion Protein (F) Gene The F ORF of MPV is located adjacent to the M ORF, which is characteristic for members of the Metapneumovirus genus.", "The F gene of MPV encodes a 539 aa protein, which is two aa residues longer than F of APV-C (Table 5).", "Analysis of the aa sequence revealed 81% homology with APV-C, 67% with APV-A and B, 33-39% with pneumovirus F proteins and only 10-18% with other paramyxoviruses (Table 6).", "One of the conserved features among F proteins of paramyxoviruses, and also seen in MPV is the distribution of cysteine residues (Morrison, 1988; Yu et al., 1991).", "The metapneumoviruses share 12 cysteine residues in F1 (7 are conserved among all paramyxoviruses), and two in F2 (1 is conserved among all paramyxoviruses).", "Of the 3 potential N-linked glycosylation sites present in the F ORF of MPV, none are shared with RSV and two (position 74 and 389) are shared with APV.", "The third, unique, potential N-linked glycosylation site for MPV is located at position 206 (FIG.", "11).", "Despite the low sequence homology with other paramyxoviruses, the F protein of MPV revealed typical fusion protein characteristics consistent with those described for the F proteins of other Paramyxoviridae family members (Morrison, 1988).", "F proteins of Paramyxoviridae members are synthesized as inactive precursors (F0) that are cleaved by host cell proteases which generate amino terminal F2 subunits and large carboxy terminal F1 subunits.", "The proposed cleavage site (Collins et al., 1996) is conserved among all members of the Paramyxoviridae family.", "The cleavage site of MPV contains the residues RQSR.", "Both arginine (R) residues are shared with APV and RSV, but the glutamine (Q) and serine (S) residues are shared with other paramyxoviruses such as human parainfluenza virus type 1, Sendai virus and morbilliviruses (data not shown).", "The hydrophobic region at the amino terminus of F1 is thought to function as the membrane fusion domain and shows high sequence similarity among paramyxoviruses and morbilliviruses and to a lesser extent the pneumoviruses (Morrison, 1988).", "These 26 residues (position 137-163, FIG.", "11) are conserved between MPV and APV-C, which is in agreement with this region being highly conserved among the metapneumoviruses (Naylor et al., 1998; Seal et al., 2000).", "As is seen for the F2 subunits of APV and other paramyxoviruses, MPV revealed a deletion of 22 aa residues compared with RSV (position 107-128, FIG.", "11).", "Furthermore, for RSV and APV, the signal peptide and anchor domain were found to be conserved within subtypes and displayed high variability between subtypes Plows et al., 1995; Naylor et al., 1998).", "The signal peptide of MPV (aa 10-35, FIG.", "11) at the amino terminus of F2 exhibits some sequence similarity with APV-C (18 out of 26 aa residues are similar), and less conservation with other APVs or RSV.", "Much more variability is seen in the membrane anchor domain at the carboxy terminus of F1, although some homology is still seen with APV-C.", "The 22K (M2) Protein The M2 gene is unique to the Pneumovirinae and two overlapping ORFs have been observed in all pneumoviruses.", "The first major ORF represents the M2-1 protein which enhances the processivity of the viral polymerase (Collins et al., 1995; Collins, 1996) and its readthrough of intergenic regions (Hardy et al., 1998; Fearns et al., 1999).", "The M2-1 gene for MPV, located adjacent to the F gene, encodes a 187 aa protein (Table 5), and reveals the highest (84%) homology with M2-1 of APV-C (Table 6).", "Comparison of all pneumovirus M2-1 proteins revealed the highest conservation in the amino-terminal half of the protein (Collins et al., 1990; Zamora et al., 1992; Ahmadian et al., 1999), which is in agreement with the observation that MPV displays 100% similarity with APV-C in the first 80 aa residues of the protein (FIG.", "12A).", "The MPV M2-1 protein contains 3 cysteine residues located within the first 30 aa residues that are conserved among all pneumoviruses.", "Such a concentration of cysteines is frequently found in zinc-binding proteins (Ahmadian et al., 1991; Cuesta et al., 2000).", "The secondary ORFs (M2-2) that overlap with the M2-1 ORFs of pneumoviruses are conserved in location but not in sequence and are thought to be involved in the control of the switch between virus RNA replication and transcription (Collins et al., 1985; Elango et al., 1985; Baybutt et al., 1987; Collins et al., 1990; Ling et al., 1992; Zamora et al., 1992; Alansari et al., 1994; Ahmadian et al., 1999; Bermingham et al., 1999).", "For MPV, the M2-2 ORF starts at nucleotide 512 in the M2-1 ORF (FIG.", "7), which is exactly the same start position as for APV-C.", "The length of the M2-2 ORFs are the same for APV-C and MPV, 71 aa residues (Table 5).", "Sequence comparison of the M2-2 ORF (FIG.", "12B) revealed 64% aa sequence homology between MPV and APV-C and only 44-48% aa sequence homology between MPV and APV-A and B (Table 6).", "The Small Hydrophobic Protein (SH)ORF The gene located adjacent to M2 of hMPV probably encodes a 183 aa SH protein (FIGS.", "1 and 7).", "There is no discernible sequence identity between this ORF and other RNA virus genes or gene products.", "This is not surprising since sequence similarity between pneumovirus SH proteins is generally low.", "The putative SH ORF of hMPV is the longest SR ORF known to date (Table 1).", "The aa composition of the SR ORF is relatively similar to that of APV, RSV and PVM, with a high percentage of threonine and serine residues (22%, 18%, 19%, 20.0%, 21% and 28% for hMPV, APV, RSV A, RSV B, bRSV and PVM respectively).", "The SR ORF of hMPV contains 10 cysteine residues, whereas APV SR contains 16 cysteine residues.", "The SH ORF of hMPV contains two potential N-linked glycosylation sites (aa 76 and 121), whereas APV has one, RSV has two or three and PVM has four.", "The hydrophilicity profiles for the putative hMPV SE protein and SH of APV and RSV revealed similar characteristics (FIG.", "7B).", "The SH ORFs of APV and hMPV have a hydrophilic N-terminus, a central hydrophobic domain which can serve as a potential membrane spanning domain (aa 30-53 for hMPV), a second hydrophobic domain (aa 155-170) and a hydrophilic C-terminus.", "In contrast, RSV SH appears to lack the C-terminal part of the APV and hMPV ORFs.", "In all pneumovirus SH proteins the hydrophobic domain is flanked by basic aa residues, which are also found in the SR ORF for hMPV (aa 29 and 54).", "The Attachment Glycoprotein (G) ORF The putative G ORF of hMPV is located adjacent to the putative SR gene and encodes a 236 aa protein (nt 6262-6972, FIG.", "1).", "A secondary small ORF is found immediately following this ORF, potentially coding for 68 aa residues (nt 6973-7179) but lacking a start codon.", "A third potential ORF in the second reading frame of 194 aa residues is overlapping with both of these ORFs but also lacks a start codon (nt 6416-7000).", "This ORF is followed by a potential fourth ORF of 65 aa residues in the same reading frame (nt 7001-7198), again lacking a start codon.", "Finally, a potential ORF of 97 aa residues (but lacking a start codon) is found in the third reading frame (nt 6444-6737, FIG.", "1).", "Unlike the first ORF, the other ORFs do not have apparent gene start or gene end sequences (see below).", "Although the 236 aa G ORF probably represents at least a part of the hMPV attachment protein it can not be excluded that the additional coding sequences ae expressed as separate proteins or as part of the attachment protein through some RNA editing event.", "It should be noted that for APV and RSV no secondary ORFs after the primary G ORF have been identified but that both APV and RSV have secondary ORFs within the major ORF of G. However, evidence for expression of these ORFs is lacking and there is no sequence identity between the predicted aa sequences for different viruses (Ling et al., 1992).", "The secondary ORFs in hMPV G do not reveal characteristics of other G proteins and whether the additional ORFs are expressed requires further investigation.", "BLAST analyses with all ORFs revealed no discernible sequence identity at the nucleotide or aa sequence level with other known virus genes or gene products.", "This is in agreement with the low percentage sequence identity found for other G proteins such as those of hRSV A and B (53%) (Johnson et al., 1987) and APV A and B (38%) (Juhasz and Easton, 1994).", "Whereas most of the hMPV ORFs resemble those of APV both in length and sequence, the putative G ORF of 236 aa residues of hMPV is considerably smaller than the G ORF of APV (Table 1).", "The as sequence revealed a serine and threonine content of 34%, which is even higher than the 32% for RSV and 24% for APV.", "The putative G ORF also contains 8.5% proline residues, which is higher than the 8% for RSV and 7% for APV.", "The unusual abundance of proline residues in the G proteins of APV, RSV and hMPV has also been observed in glycoproteins of mucinous origin where it is a major determinant of the proteins three dimensional structure (Collins and Wertz, 1983; Wertz et al., 1985; Jentoft, 1990).", "The G ORF of hMPV contains five potential N-linked glycosylation sites, whereas hRSV has seven, bRSV has five and APV has three to five.", "The predicted hydrophilicity profile of hMPV G revealed characteristics similar to the other pneumoviruses.", "The N-terminus contains a hydrophilic region followed by a short hydrophobic area (aa 33-53 for hMPV) and a mainly hydrophilic C-terminus (FIG.", "8B).", "This overall organization is consistent with that of an anchored type II transmembrane protein and corresponds well with these regions in the G protein of APV and RSV.", "The putative G ORF of hMPV contains only 1 cysteine residue in contrast to RSV and APV (5 and 20 respectively).", "Of note, only two of the four secondary ORFs in the G gene contained one additional cysteine residue and these four potential ORFs revealed 12-20% serine and threonine residues and 6-11% proline residues.", "The Polymerase Gene (L) In analogy to other negative strand viruses, the last ORF of the MPV genome is the RNA-dependent RNA polymerase component of the replication and transcription complexes.", "The L gene of MPV encodes a 2005 aa protein, which is 1 residue longer than the APV-A protein (Table 5).", "The L protein of MPV shares 64% homology with APV-A, 42-44% with RSV, and approximately 13% with other paramyxoviruses (Table 6).", "Poch et al.", "(1989; 1990) identified six conserved domains within the L proteins of non-segmented negative strand RNA viruses, from which domain III contained the four core polymerase motifs that are thought to be essential for polymerase function.", "These motifs (A, B, C and D) are well conserved in the MPV L protein: in motifs A, B and C: MPV shares 100% similarity with all pneumoviruses and in motif D MPV shares 100% similarity with APV and 92% with RSVs.", "For the entire domain III (aa 627-903 in the L ORF), MPV shares 77% identity with APV, 61-62% with RSV and 23-27% with other paramyxoviruses (FIG.", "15).", "In addition to the polymerase motifs the pneumovirus L proteins contain a sequence which conforms to a consensus ATP binding motif K(X)21GEGAGN(X)20K (Stec, 1991).", "The MPV L ORF contains a similar motif as APV, in which the spacing of the intermediate residues is off by one: K(X)22GEGAGN(X)19K.", "Phylogenetic Analyses As an indicator for the relationship between MPV and members of the Pneumovirinae, phylogenetic trees based on the N, P, M, and F ORFs have been constructed previously (van den Hoogen et al., 2001) and revealed a close relationship between MPV and APV-C. Because of the low homology of the MPV SH and G genes with those of other paramyxoviruses, reliable phylogenetic trees for these genes can not be constructed.", "In addition, the distinct genomic organization between members of the Pneumovirus and Metapneumovirus genera make it impossible to generate phylogenetic trees based on the entire genomic sequence.", "We therefore only constructed phylogenetic trees for the M2 and L genes in addition to those previously published.", "Both these trees confirmed the close relation between APV and MPV within the Pneumovirinae subfamily (FIG.", "16).", "MPV Non-Coding Sequences The gene junctions of the genomes of paramyxoviruses contain short and highly conserved nucleotide sequences at the beginning and end of each gene (gene start and gene end signals), possibly playing a role in initiation and termination of transcription (Curran et al., 1999).", "Comparing the intergenic sequences between all genes of MPV revealed a consensus sequence for the gene start signal of the N, P, M, F, M2 and G: GGGACAAGU (FIG.", "17A), which is identical to the consensus gene start signal of the metapneumoviruses (ling et al., 1992; Yu et al., 1992; Li et al., 1996; Bäyon-Auboyer et al., 2000).", "The gene start signals for the SH and L genes of MPV were found to be slightly different from this consensus (SH: GGGAUAAAU, L: GAGACAAAU).", "For APV the gene start signal of L was also found to be different from the consensus: AGGACCAAT (APV-A) (Randhawa et al., 1996) and GGGACCAGT (APV-D) (Bäyon-Auboyer et al., 2000).", "In contrast to the similar gene start sequences of MPV and APV, the consensus gene end sequence of APV, UAGUUAAU (Randhawa et al., 1996), could not be found in the MPV intergenic sequences.", "The repeated sequence found in most genes, except the G-L intergenic region, was U AAAAA U/A/C, which could possibly act as gene end signal.", "However, since we sequenced viral RNA rather than mRNA, definitive gene end signals could not be assigned and thus requires further investigation.", "The intergenic regions of pneumoviruses vary in size and sequence (Curran et al., 1999; Blumberg et al., 1991; Collins et al., 1983;).", "The intergenic regions of MPV did not reveal homology with those of APV and RSV and range in size from 10 to 228 nucleotides (FIG.", "17B).", "The intergenic region between the M and F ORFs of MPV contains part of a secondary ORF, which starts in the primary M ORF (see above).", "The intergenic region between SH and G contains 192 nucleotides, and does not appear to have coding potential based on the presence of numerous stop-codons in all three reading frames.", "The intergenic region between G and L contains 241 nucleotides, which may include additional ORFs (see above).", "Interestingly, the start of the L ORF is located in these secondary ORFs.", "Whereas the L gene of APV does not start in the preceding G ORF, the L ORF of RSV also starts in the preceding M2 gene.", "At the 3′ and 5′extremities of the genome of paramyxoviruses short extragenic region are referred to as the leader and trailer sequences, and approximately the first 12 nucleotides of the leader and last 12 nucleotides of the trailer are complementary, probably because they each contain basic elements of the viral promoter (Curran et al., 1999; Blumberg et al., 1991; Mink et al., 1986).", "The 3′leader of MPV and APV are both 41 nucleotides in length, and some homology is seen in the region between nucleotide 16 and 41 of both viruses (18 out of 26 nucleotides) (FIG.", "17B).", "As mentioned before the first 15 nucleotides of the MPV genomic map are based on a primer sequence based on the APV genome.", "The length of the 5′trailer of MPV (188 nucleotides) resembles the size of the RSV 5′trailer (155 nucleotides), which is considerably longer than that of APV (40 nucleotides).", "Alignments of the extreme 40 nucleotides of the trailer of MPV and the trailer of APV revealed 21 out of 32 nucleotides homology, apart from the extreme 12 nucleotides which represent primer sequences based on the genomic sequence of APV.", "Our sequence analyses revealed the absence of NS1 and NS2 genes at the 3′end of the genome and a genomic organisation resembling the organisation of metapneumoviruses (3′-N-P-M-F-M2-SH-G-L5′).", "The high sequence homology found between MPV and APV genes further emphasises the close relationship between these two viruses.", "For the N, P, M, F, M2-1 and M2-2 genes of MPV an overall amino acid homology of 79% is found with APV-C.", "In fact, for these genes APV-C and MPV revealed sequence homologies which are in the same range as sequence homologies found between subgroups of other genera, such as RSV-A and B or APV-A and B.", "This close relationship between APV-C and MPV is also seen in the phylogenetic analyses which revealed MPV and APV-C always in the same branch, separate from the branch containing APV-A and B.", "The identical genomic organisation, the sequence homologies and phylogentic analyses are all in favour of the classification of MPV as the first member in the Metapneumovirus genus that is isolatable from mammals.", "It should be noted that the found sequence variation between different virus isolates of MPV in the N, M, F and L genes revealed the possible existence of different genotypes (van den Hoogen et al., 2001).", "The close relationship between MPV and APV-C is not reflected in the host range, since APV infects birds in contrast to MPV (van den Hoogen et al., 2001).", "This difference in host range may be determined by the differences between the SH and G proteins of both viruses that are highly divergent.", "The SH and G proteins of MPV did not reveal significant aa sequence homology with SH and G proteins of any other virus.", "Although the amino acid content and hydrophobicity plots are in favour of defining these ORFs as SH and G, experimental data are required to assess their function.", "Such analyses will also shed light on the role of the additional overlapping ORFs in these SH and G genes.", "In addition, sequence analyses on the SH and G genes of APV-C might provide more insight in the function of the SH and G proteins of MPV and their relationship with those of APV-C.", "The noncoding regions of MPV were found to be fairly similar to those of APV.", "The 3′leader and 5′ trailer sequences of APV and MPV displayed a high degree of homology.", "Although the lengths of the intergenic regions were not always the same for APV and MPV, the consensus gene start signals of most of the ORFs were found to be identical.", "In contrast, the gene end signals of APV were not found in the MPV genome.", "Although we did find a repetitive sequence (U AAAAA U/A/C) in most intergenic regions, sequence analysis of viral mRNAs is required to formally delineate those gene end sequences.", "It should be noted that sequence information for 15 nucleotides at the extreme 3′end and 12 nucleotides at the extreme 5′end is obtained by using modified rapid amplification of cDNA ends (RACE) procedures.", "This technique has been proven to be successful by others for related viruses (Randhawa, J. S. et al., Rescue of synthetic minireplicons establishes the absence of the NS1 and NS2 genes from avian pneumovirus.", "J. Virol, 71, 9849-9854 (1997); Mink, M. A., et al.", "Nucleotide sequences of the 3′ leader and 5′ trailer regions of human respiratory syncytial virus genomic RNA.", "Virology 185, 615-24 (1991).)", "To determine the sequence of the 3′ vRNA leader sequence, a homopolymer A tail is added to purified vRNA using poly-A-polymerase and the leader sequence subsequently amplified by PCR using a poly-T primer and a primer in the N gene.", "To determine the sequence of the 5′ vRNA trailer sequence, a cDNA copy of the trailer sequence is made using reverse transcriptase and a primer in the L gene, followed by homopolymer dG tailing of the cDNA with terminal transferase.", "Subsequently, the trailer region is amplified using a poly-C primer and a primer in the L gene.", "As an alternative strategy, vRNA is ligated to itself or synthetic linkers, after which the leader and trailer regions are amplified using primers in the L and N genes and linker-specific primers.", "For the 5′ trailer sequence direct dideoxynucleotide sequencing of purified vRNA is also feasible (Randhawa, 1997).", "Using these approaches, we can analyse the exact sequence of the ends of the hMPV genome.", "The sequence information provided here is of importance for the generation of diagnostic tests, vaccines and antivirals for MPV and MPV infections.", "Materials and Methods Sequence analysis Virus isolate 00-1 was propagated to high titers (approximately 10,000 TCID50/ml) on tertiary monkey kidney cells as described previously (van den Hoogen et al., 2001).", "Viral RNA was isolated from supernatants from infected cells using a High Pure RNA Isolating Kit according to instructions from the manufacturer (Roch Diagnostics, Almere, The Netherlands).", "Primers were designed based on sequences published previously (van den Hoogen et al., 2001) in addition to sequences published for the leader and trailer of APV/RSV (Randhawa et al., 1997; Mink et al., 1991) and are available upon request.", "RT-PCR assays were conducted with viral RNA, using a one-tube assay in a total volume of 50 μl with 50 mM Tris pH 8.5, 50 mM NaCl, 4.5 mM MgCl2, 2 mM DTT, 1 μM forward primer, 1M reverse primer, 0.6 mM dNTPs, 20 units RNAsin (Promega, Leiden, The Netherlands), 10 U AMV reverse transcriptase (Promega, Leiden, The Netherlands), and 5 units Taq Polymerase (PE Applied Biosystems, Nieuwerkerk aan de IJssel, The Netherlands).", "Reverse transcription was conducted at 42° C. for 30 minutes, followed by 8 minutes inactivation at 95° C. The cDNA was amplified during 40 cycles of 95° C., 1 min.", "; 42° C., 2 min.", "72° C., 3 min.", "with a final extension at 72° C. for 10 minutes.", "After examination on a 1% agarose gel, the RT-PCR products were purified from the gel using a Qiaquick Gel Extraction kit (Qiagen, Leusden, The Netherlands) and sequenced directly using a Dyenamic ET terminator sequencing kit (Amersham Pharmacia Biotech, Roosendaal, the Netherlands) and an ABI 373 automatic DNA sequencer (PE Applied Biosystem, Nieuwerkerk aan den IJssel, the Netherlands), according to the instructions of the manufacturer.", "Sequence alignments were made using the clustal software package available in the software package of BioEdit version5.0.6.", "(http://jwbrown.mbio.ncsu.edu/ Bioedit//bioedit.html; Hall, 1999).", "Phylogenetic Analysis To construct phylogenetic trees, DNA sequences were aligned using the ClustalW software package and maximum likelihood trees were generated using the DNA-ML software package of the Phylip 3.5 program using 100 bootstraps and 3 jumbles.", "Bootstrap values were computed for consensus trees created with the consense package (Felsenstein, 1989).", "The MPV genomic sequence is available from Genbank under accession number AF371337.All other sequences used here are available from Genbank under accession numbers AB046218 (measles virus, all ORFs), NC-001796 human parainfluenza virus type 3, all ORFs), NC-001552 (Sendai virus, all ORFs), X57559 (human parainfluenza virus type 2, all ORFs), NC-002617 (New Castle Disease virus, all ORFs), NC-002728 (Nipah virus, all ORFs), NC-001989 (bRSV, all ORFs), M11486 RSV A, all ORFs except L), NC-001803 (hRSV, L ORF), NC-001781 (hRSV B, all ORFs), D10331 (PVM, N ORF), U09649 (PVM, P ORF), U66893 (PVM, M ORF), U66893 (PVM, SH ORF), D11130 (PVM, G ORF), D11128 (F ORF).", "The PVM M2 ORF was taken from Ahmadian (1999), AF176590 (APV-C, N ORF), U39295 (APV-A, N ORF), U39296 (APV-B, N ORF), AF262571 (APV-C, M ORF), U37586 (APV-B, M ORF), X58639 (APV-A, M ORF), AF176591 (APV-C, P ORF), AF325443 (APV-B, P ORF), U22110 (APV-A, P ORF), AF187152 (APV-C, F ORF), Y14292 (APV-B, F ORF), D00850 (APV-A, F ORF), AF176592 (APV-C, M2 ORF), AF35650 (APV-B, M2 ORF), X63408 (APV-A, M2 ORF), U65312 (APV-A, L ORF), S40185 (APV-A, SH ORF).", "TABLE 5 Lengths of the ORFs of MPV and other paramyxoviruses.", "N1 P M F M2-1 M2-2 SH G L MPV 394 294 254 539 187 71 183 236 2005 APV A 391 278 254 538 186 73 174 391 2004 APV B 391 279 254 538 186 73 —2 414 —2 APV C 394 294 254 537 184 71 —2 —2 —2 APV D —2 —2 —2 —2 —2 —2 —2 389 —2 hRSV A 391 241 256 574 194 90 64 298 2165 hRSV B 391 241 249 574 195 93 65 299 2166 bRSV 391 241 256 569 186 93 81 257 2162 PVM 393 295 257 537 176 77 92 396 —2 others3 418-542 225-709 335-393 539-565 —4 —4 —4 —4 2183-2262 Footnotes: 1length in amino acid residues.", "2sequences not available 3others: human parainfluenza virus type 2 and 3, Sendai virus, measles virus, nipah virus, phocine distemper virus, and New Castle Disease virus.", "4ORF not present in viral genome TABLE 6 Amino acid sequence identity between the ORFs of MPV and those of other paramyxoviruses1.N P M F M2-1 M2-2 L APV A 69 55 78 67 72 26 64 APV B 69 51 76 67 71 27 —2 APV C 88 68 87 81 84 56 —2 hRSV A 42 24 38 34 36 18 42 hRSV B 41 23 37 33 35 19 44 bRSV 42 22 38 34 35 13 44 PVM 45 26 37 39 33 12 —2 others3 7-11 4-9 7-10 10-18 —4 —4 13-14 Footnotes: 1No sequence homologies were found with known G and SH proteins and were thus excluded 2Sequences not available.", "3See list in table 5, footnote 3.4ORF absent in viral genome.", "REFERENCES Current Protocols in Molecular Biology, volume 1-3 (1994-1998).", "Ed.", "by Ausubel, F. 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(1991).", "Deduced amino acid sequence of the fusion glycoprotein of turkey rhinotracheitis virus has greater identity with that of human respiratory syncytial virus, a pneumovirus, than that of paramyxoviruses and morbilliviruses.", "J Gen Virol 72, 75-81.YU, Q., DAVIS, P. J., LI, J., and CAVANAGH, D. (1992).", "Cloning and sequencing of the matrix protein (M) gene of turkey rhinotracheitis virus reveal a gene order different from that of respiratory syncytial virus.", "Virology 186, 426-34.ZAMORA, M., and SAMAL, S. K. (1992).", "Sequence analysis of M2 mRNA of bovine respiratory syncytial virus obtained from an F-M2 dicistronic mRNA suggests structural homology with that of human respiratory syncytial virus.", "J Gen Virol 73, 737-41.Primers used for RT-PCR detection of known paramyxo-viruses.", "Primers for hPIV-1 to 4, mumps, measles, Tupaia, Mapuera and Hendra are developed in house and based on allignments of available sequences.", "Primers for New Castle Disease Virus are taken from Seal, J., J. et al, Clin.", "Microb., 2624-2630, 1995.Primers for Nipah and general paramyxovirus-PCR are taken from: Chua, K. B., et al; Science, 288 26 may 2000 Virus primers located in protein HPIV-1 fwd 5′-TGTTGTCGAGACTATTCCAA-3′ HN Rev 5′-TGTTG(T/A)ACCAGTTGCAGTCT-3′ HIPV-2 Fwd 5′-TGCTGCTTCTATTGAGAAACGCC-3′ N Rev 5′-GGTGAC/T TC(T/C)AATAGGGCCA-3′ HPIV-3 Fwd 5′-CTCGAGGTTGTCAGGATATAG-3′ HN Rev 5′-CTTTGGGAGTTGAACACAGTT-3′ HPIV-4 Fwd 5′-TTC(A/G)GTTTTAGCTGCTTACG-3′ N Rev 5′-AGGCAAATCTCTGGATAATGC-3′ Mumps Fwd 5′-TCGTAACGTCTCGTGACC-3′ SH Rev 5′-GGAGATCTTTCTAGAGTGAG-3′ NDV Fwd 5′-CCTTGGTGAiTCTATCCIAG-3′ F Rev 5′-CTGCCACTGCTAGTTGiGATAATCC-3′ Tupaia Fwd 5′-GGGCTTCTAAGCGACCCAGATCTTG-3′ N Rev 5′-GAATTTCCTTATGGACAAGCTCTGTGC-3′ Mapuera Fwd 5′-GGAGCAGGAACTCCAAGACCTGGAG-3′ N Rev: 5′-GCTCAACCTCATCACATACTAACCC-3′ Hendra Fwd 5′-GAGATGGGCGGGCAAGTGCGGCAACAG-3′ N Rev 5′-GCCTTTGCAATCAGGATCCAAATTTGGG-3′ Nipah Fwd 5′-CTGCTGCAGTTCAGGAAACATCAG-3′ N Rev 5′-ACCGGATGTGCTCACAGAACTG-3′ HRSV Fwd 5′-TTTGTTATAGGCATATCATTG-3′ F Rev 5′-TTAACCAGCAAAGTGTTA-3′ Measles Fwd 5′-TTAGGGCAAGAGATGGTAAGG-3′ N Rev 5′-TTATAACAATGATGGAGGG-3′ General Paramyxoviridae: Fwd 5′-CATTAAAAAGGGCACAGACGC-3′ P Rev 5′-TGGACATTCTCCGCAGT-3′ Primers for RAP-PCR: ZF1: 5′-CCCACCACCAGAGAGAAA-3′ ZF4: 5′-ACCACCAGAGAGAAACCC-3′ ZF7: 5′-ACCAGAGAGAAACCCACC-3′ ZF10: 5′-AGAGAGAAACCCACCACC-3′ ZF13: 5′-GAGAAACCCACCACCAGA-3′ ZF16: 5′-AAACCCACCACCAGAGAG-3′ CS1: 5′-GGAGGCAAGCGAACGCAA-3′ CS4: 5′-GGCAAGCGAACGCAAGGA-3′ CS7: 5′-AAGCGAACGCAAGGAGGC-3′ CS10: 5′-CGAACGCAAGGAGGCAAG-3′ CS13: 5′-ACGCAAGGAGGCAAGCGA-3′ CS16: 5′-GAAGGAGGCAAGCGAACG-3′ 20 fragments successfully purified and sequenced: 10 fragments found with sequence homology in APV Fragment 1 ZF 7, 335 bp N gene Fragment 2 ZF 10, 235 bp N gene Fragment 3 ZF 10, 800 bp M gene Fragment 4 CS 1, 1250 bp F gene Fragment 5 CS 10, 400 bp F gene Fragment 6 CS 13, 1450 bp F gene Fragment 7 CS 13, 750 bp F gene Fragment 8 ZF 4, 780 bp L gene (protein level) Fragment 9 ZF 10, 330 bp L gene (protein level) Fragment 10 ZE 10, 250 bp L gene (protein level) Primers used for RAP-PCR amplification of nucleic acids from the prototype isolate.", "EXAMPLE 5 Further Exploration of the Two Subtypes of hMPV Based on phylogenetic analysis of the different isolates of hMPV obtained so far, two genotypes have been identified with virus isolate 00-1 being the prototype of genotype A and isolate 99-1 the prototype of genotype B.", "We hypothesise that the genotypes are related to subtypes and that re-infection with viruses from both subgroups occur in the presence of pre-existing immunity and the antigenic variation may not be strictly required to allow re-infection.", "Furthermore, hMPV appears to be closely related to avian pneumovirus, a virus primarily found in poultry.", "The nucleotide sequences of both viruses show high percentages of homology, with the exception of the SH and G proteins.", "Here we show that the viruses are cross-reacting in tests, which are based primarily on the nucleoprotein and matrixprotein, but they respond differently in tests, which are based on the attachment proteins.", "The differences in virus neutralisation titers provide further proof that the two genotypes of hMPV are two different serotypes of one virus, where APV is a different virus.", "The Cross Reaction Between the Two Serotypes and the Cross Reaction Between APV an hMPV Methods Protocol for IgG, IgA and IgM antibody detection for hMPV: The indirect IgG EIA for hMPV was performed in microtitre plates essentially as described previously (Rothbarth, P. H. et al., 1999; Influenza virus serology-a comparative study.", "J. of Vir.", "Methods 78 (1999) 163-169.Briefly, concentrated hMPV was solubilized by treatment with 1% Triton X-100 an coated for 16 hr at room temperature into ,microtitre plates in PBS after determination of the optimal working dilution by checkerboard titration.", "Subsequently, 100 ul volumes of 1:100 diluted human serum samples in EIA buffer were added to the wells and incubated for 1 h at 37C.", "Binding of human IgG was detected by adding a goat anti-human IgG peroxidase conjugate (Biosource, USA).", "Adding TMB as substrate developed plates and OD was measured at 450 nm.", "the results were expressed as the S(ignal)/N(egative) ratio of the OD.", "A serum was considered positive for IgG, if the SIN ratio was beyond the negative control plus three times the standard.", "hMPV antibodies of the IgM and IgA classes were detected in sera by capture EIA essentially as described previously (Rothbarth, P. H et al.", "1999; Influenza virus serolgy-a comparative study.", "J. Vir.", "methods 78 (1999) 163-169.For the detection of IgA and IgM commercially available microtiter plates coated with anti human IgM or IgA specific monoclonal antibodies were used.", "Sera were diluted 1:100 and after incubation of 1 hr at 37C., an optimal working dilution of hMPV is added at each well (100 ul).", "Incubated 1 hr 37C.. After washing polyclonal anti hMPV labeled with peroxidase was added, the plate was incubated 1 hr 37C.", "Adding TMB as substrate developed plates and OD was measured at 450 nm.", "the results were expressed as the S(ignal)/N(egative) ratio of the OD.", "A serum was considered positive for IgG, if the S/N ratio was beyond the negative control plus three times the standard.", "AVP antibodies were detected in an AVP inhibition assay.", "Protocol for APV inhibition test is included the APV-Ab SVANOVIR® enzyme immunoassay which is manufactured by SVANOVA Biotech AB, Uppsal Science Park Glunten SE-751 83 Uppsala Sweden.", "The results were expressed as the S(ignal)/N(egative) ratio of the OD.", "A serum was considered positive for IgG, if the S/N ratio was beyond the negative control plus three times the standard.", "1.Guinea Pigs A.", "(re) Infection of Guinea Digs with Both Subtypes of hMPV Virus isolates ned/00/01 (subtype A) and ned/99/01 (subtype B) have been used to inoculate 6 guinea pigs per subtype (intratracheal, nose and eyes).", "6 GP's infected with hMPV 00-1 (10e6,5 TCID50) 6 GPs infected with hMPV 99-1 (10e4,1 TCID50) 54 Days after the primary infection, the guinea pigs have been inoculated with the homologous and heterologous subtypes (10e4 TCID50/ml): 2 guinea pigs: 1st infection 00-1; 2nd 99-1 (heterologous) 3 guinea pigs: 1st infection 00-1; 2nd 00-1 (homologous) 2 guinea pigs: 1st infection 99-1; 2nd 00-1 (heterologous) 3 guinea pigs: 1st infection 99-1; 2nd 99-1 homologous) Throat and nose swabs have been collected for 12 days (1st infection) or 8 days (2nd infection) post infection, and have been tested for presence of the virus by RT-PCR assays.", "Results of RT-PCR Assay: FIG.", "29 Summary of results: guinea pigs inoculated with virus isolate ned/00/01 show infection of the upper respiratory tract day 1 to 10 post infection.", "Guinea pigs inoculated with ned/99/01 show infection of the upper respiratory tract day 1 to 5 post infection.", "Infection with ned/99/01 appears to be less severe than infection with ned/00/01.A second inoculation of the guinea pigs with the heterologous virus results in re-infection in 3 out of 4 guinea pigs and with the homologous virus in 2 out of 6 guinea pigs.", "No or only little clinical symptoms were noted in those animals that became re-infected, and no clinical symptoms were seen in those animals that were protected against the re-infections, demonstrating that even with wild-type virus, a protective effect of the first infection is evident, showing the possible use of heterologous (and of course homologues) isolates as a vaccine, even in an unattenuated form.", "Both subtypes of hMPV are able to infect guinea pigs, although infection with subtype B (ned/99/01) seems less severe (shorter period of presence of the virus in nose and throat) than infection with subtype A (ned/00/01).", "This may be due to the higher dose given for subtype A, or to the lower virulence of subtype B.", "Although the presence of pre-existing immunity does not completely protect against re-infection with both the homologous and heterologous virus, the infection appears to be less prominent in that a shorter period of presence of virus was noted and not all animals became virus positive.", "B. Serology of Guinea Pigs Infected with Both Subtypes of hMPV At day 0, 52, 70, 80, 90, 110, 126 and 160 sera were collected from the guinea pigs and tested at a 1:100 dilution in a whole virus ELISA against ned/00/01 and ned/99/01 antigen.", "FIGS.", "30A and B: IgG response against ned/00/01 and ned/99/01 for each individual guinea pig FIG.", "31: Specificity of the ned/00/01 and ned/99/01 ELISA Only data from homologous reinfected guinea pigs have been used.", "FIG.", "32: Mean IgG response against ned/00/01 and ned/99/01 ELISA of 3 homologous (00-1100-1), 2 homologous (99-1/99-1), 2 heterologous (99-1100-1) and 2 heterologous (00-1/99-1) infected guinea pigs.", "Summary of Results: Only a minor difference in response to the two different ELISA's is observed.", "Whole virus ELISA against 00-1 or 99-1 cannot be used to discriminate between the two subtypes.", "C. Reactivity of Sera Raised Against hMPV in Guinea Pigs with APV Antigen Sera collected from the infected guinea pigs have been tested with an APV inhibition ELISA FIG.", "33: Mean percentage of APV inhibition of hMPV infected guinea pigs.", "Summary of Results: Sera raised against hMPV in guinea pigs, react in the APV inhibition test in a same manner as they react in the hMPV IgG ELISA's.", "Sera raised against ned/99/01 reveal a lower percentage of inhibition in the APV inhibition ELISA than sera raised against ned/00/01.Guinea pigs infected with ned/99/01 might have a lower titer (as is seen in the hMPV ELISA's) or the cross-reaction of ned/99/01 with APV is less than that of ned/00/01.Nevertheless, the APV-Ab inhibition ELISA can be used to detect hMPV antibodies in guinea pigs.", "{D. Virus Neutralisation Assays with Sera Raised Against hMPV in Guinea Pigs.", "Sera collected at day 0, day 52, 70 and 80 post infection were used in a virus (cross) neutralisation assay with ned/00/01, ned/99/01 and APV-C.", "Starting dilution was 1 to 10 and 100 TCID50 virus per well was used.", "After neutralisation, the virus was brought on tMK cells, 15 min.", "centrifuged at 3500 RPM, after which the media was refreshed.", "The APV tests were grown for 4 days and the hMPV tests were grown for 7 days.", "Cells were fixed with 80% aceton, and IFA's were conducted with monkey-anti hMPV fitc labeled.", "Wells that were negative in the staining were considered as the neutralising titer.", "For each virus a 10-log titration of the virus stock and 2 fold titration of the working solution was included.", "FIG.", "34: Virus neutralisation titers of ned/00/01 and ned/99/01 infected guinea pigs against ned/00/01, ned/99/01 and APV-C. 2.Cynomologous Macaques A.", "(Re) Infection of Cynomologous Macagues with Both Subtypes of hMPV Virus isolates ned/00/01 (subtype A) and ned/99/01 (subtype B) (1e5 TCID50) have been used to inoculate 2 cynomologous macaques per subtype (intratracheal, nose and eyes).", "Six months after the primary infection, the macaque have been inoculated for the second time with ned/00/01.Throat swabs have been collected for 14 days (1st infection) or 8 days (2nd infection) post infection, and have been tested for presence of the virus by RT-PCR assays.", "FIG.", "36: Results of RT-PCR assays on throat swabs of cynomolgous macaques inoculated (twice) with ned/00/01.Summary of Results: Summary of results: cynomologous macaques inoculated with virus isolate ned/00/01 show infection of the upper respiratory tract day 1 to 10 post infection.", "Clinical symptoms included a suppurative rhinitis.", "A second inoculation of the macaques with the homologous virus results in re-infection, as demonstrated by PCR, however, no clinical symptoms were seen.", "B. Serology on Sera Collected of hMPV Infected Cynomologous Macaques.", "From the macaques which received ned/00/01 sera were collected during 6 months after the primary infection (re-infcetion occurred at day 240 for monkey 3 and day 239 for monkey 6).", "Sera were used to test for the presence of IgG antibodies against either ned/00/01 or APV, and for the presence against IgA and IgM antibodies against ned/00/01.Results: FIG.", "36A IgA, IgM and IgG response against ned/00/01 of 2 cynomologous macaques (re)infected with ned/00/01.FIG.", "36B IgG response against APV of 2 cynbomologous macaques infected with ned/00101.Summary of Results: Two macaques have been succesfully infected with ned/00101 and in the presence of antibodies against ned/00/01 been reinfected with the homologous virus.", "The response to IgA and IgM antibodies shows the raise in IgM antibodies after the first infection, and the absence of it after the reinfection.", "IgA antibodies are only detected after the re-infection, showing the immediacy of the immune response after a first infection.", "Sera raised against hMPV in macaques which were tested in an APV inhibition ELISA show a similar response as to the hMPV IgG ELISA.", "Discussion/Conclusion hMPV antibodies in cynomologous macaques are detected with the APV inhibition ELISA with a similar sensitivity as with an hMPV ELISA, and therefore the APV inhibition EIA is suitable for testing human samples for the presence of hMPV antibodies.", "C. Virus (Cross) Neutralisation Assays with Sera Collected from hMPV Infected Cynomologous Macaques Summary of results: The sera taken from day 0 to day 229 post primary infection show only low virus neutralisation titers against ned/00/01 (0-80), the sera taken after the secondary infection show high neutralisation titers against ned/00/01:>1280.Only sera taken after the secondary infection show neutralisation titers against ned/99/01 (80-640), and none of the sera neutralise the APV C virus.", "There is no cross reaction between APV-C and hMPV in virus (cross)neutralisation assays, where there is a cross reaction between ned/00/01 and ned/99/01 after a boost of the antibody response.", "3.Humans Sera of patients ranging in age of<6 months to>20 years of age have previously been tested in IFA and virus neutralisation assays against ned/00/01.", "(See tabel 1 of patent).", "Here we have tested a number of these sera for the presence of IgG, IgM and IgA antibodies in an ELISA against ned/00/01, and we tested the samples in the APV inhibition ELISA.", "Results: FIG.", "37 Comparison of the use of the hMPV ELISA and the APV inhibition ELISA for the detection of IgG antibodies in human sera, there is a strong correlation between the IgG hMPV test and the APV-Ab test, therefore the APV-Ab test is essentially able to detect IgG antibodies to hmPV in humans.", "4.Poultry 96 chickens have been tested in both the APV inhibition ELISA and the ned/00/01 ELISA for the presence of IgG antibodies against APV.", "Summary of results: Both the hMPV ELISA and the APV inhibition ELISA detect antibodies against APV (data not shown).", "Summary of Results.", "We found two genotypes of hMPV with ned/00/01 being the prototype of subgroup A and ned/99/01 the prototype of subgroup B.", "“According to classical serogical analyses (as for example known Francki, R. I.", "B., Fauquet, C. M., Knudson, D. L., and Brown, F., Classification and nomenclature of viruses.", "Fifth report of the international Committee on Taxonomy of Viruses.", "Arch Virol, 1991.Supplement 2: p. 140-144), two subtypes can be defined on the basis of its immunological distinctiveness, as determined by quantitative neutralization assays with animal antisera.", "Two distinct serotypes have either no cross-reaction with each other or show a homologous-to heterologous titer ratio>16 in both directions.", "If neutralization shows a certain degree of cross-reaction between two viruses in either or both directions (homologous-to-heterologous titer ration of eight or 16), distinctiveness of serotype is assumed if substantial biophysical/biochemical differences of DNA's exist.", "If neutralization shows a distinct degree of cross-reaction between two viruses in either or both directions (homologous-to-heterologous titer ration of smaller than eight), identity of serotype of the isolates under study is assumed.” For RSV it is known that re-infection occurs in the presence of pre-existing immunity (both homologous and heterologous).", "Infection of guinea pigs and cynomologous macaques with both the homologous and heterologous serotypes of hMPV revealed that this is also true for hMPV.", "In addition, IgA and IgM ELISA's against hMPV revealed the reaction of IgA antibodies only occurs after re-infection.", "Sera raised against hMPV or APV respond in an equal way in APV and hMPV ELISAs.", "From the nucleotide sequence comparisons, it is known that the viruses show about 80% amino acid homology for the N, P, M, and F genes.", "In ELISA's the N and M proteins are the main antigens to react.", "Virus neutralisation assays (known to react against the surface glycoproteins G, SH and F) show a difference between the two different sera.", "Although APV en hMPV cross react in ELISAs, phylogenetic analyses of the nucleotide sequences of hMPV and APV, the differences in virus neutralisation titers of sera raised against the two different viruses, and the differences in host usage again reveal that APV-C and hMPV are two different viruses.", "Based on the results we speculate that hMPV infection in mammals is possible a result of a zoonotic event from birds to mammals.", "But the virus has adapted in such a way (i.e.", "the G and SH proteins) that a return (from mammals to birds) zoonotic event seems unlikely, considering the presence of AVP in birds.", "Addendum Background Information on Pneumovirinae The family of Paramyxoviridae contains two subfamilys: the Paramyxovirinae and the Pneumovirinae.", "The subfamily Pneumovirinae consists of two genera: Pneumovirus and Metapneumovirus.", "The genus Pneumovirus contains the human, bovine, ovine and caprine respiratory syncytial viruses and the pneumonia virus of mice (PVM).", "The genus Metapneumovirus contains the avian pneumoviruses (APV, also referred to as TRTV).", "The classification of the genera in the subfamily Pneumovirinae is based on classical virus characteristics, gene order and gene constellation.", "Viruses of the genus Pneumovirus are unique in the family of Paramyxoviridae in having two nonstructural proteins at the 3′end of the genome (3′-NS1-NS2-N-P-M-SH-G-F-M2-L-5′).", "In contrast, viruses in the genus Metapneumovirus lack the NS1 and NS2 genes and the organisation of genes between the M and L coding regions is different: 3′-N-P-M-F-M2-SH-G-L-5′.", "All members of the subfamily Paramyxovirinae have haemagluttinating activity, but this function is not a defining feature for the subfamily Pneumovirinae, being absent in RSV and APV but present in PMV.", "Neuraminidase activity is present in members of the genera Paramyxovirus and Rubulavirus (subfamily Paramyxovirinae) but is absent in the genus Morbillivirus (subfamily Paramyxovirinae) and the genera Pneumovirus and Metapneumovirus (subfamily Pneumovirinae).", "A second distinguishing feature of the subfamily Pneumovirinae is the apparent limited utilization of alternative ORFs within mRNA by RSV.", "In contrast, several members of the subfamily Paramyxovirinae, such as Sendai and Measles viruses, access alternative ORFs within the mRNA encoding the phosphoprotein (P) to direct the synthesis of a novel protein.", "The G protein of the Pneumovirinae does not have sequence relatedness or structural similarity to the HN or H proteins of Paramyxovirinae and is only approximately half the size of their chain length.", "In addition, the N and P proteins are smaller than their counterparts in the Paramyxovirinae and lack unambigous sequence homology.", "Most nonsegmented negative stranded RNA viruses have a single matrix (M) protein.", "Members of the subfamily Pneumovirinae are an exception in having two such proteins, M and M2.The M protein is smaller than its Paramyxovirinae counterparts and lacks sequence relatedness with Paramyxovirinae.", "When grown in cell cultures, members of the subfamily Pneumovirinae show typical cytopathic effects; they induce characteristic syncytia formation of cells.", "(Collins, 1996).", "The subfamily Pneumovirinae, genus Pneumovirus hRSV is the type-species of the genus Pneumovirus and is a major and widespread cause of lower respiratory tract illness during infancy and early childhood (Selwyn, 1990).", "In addition, hRSV is increasingly recognised as an important pathogen in other patient groups, including immune compromised individuals and the elderly.", "RSV is also an important cause of community-acquired pneumonia among hospitalised adults of all ages (Englund, 1991; Falsey, 2000; Dowell, 1996).", "Two major antigenic types for RSV (A and B) have been identified based on differences in their reactivity with monoclonal and polyclonal antibodies and by nucleic acid sequence analyses (Anderson, 1985; Johnson, 1987; Sullender, 2000).", "In particular the G protein is used in distinguishing the two subtypes.", "RSV-A and B share only 53% amino acid sequence homology in G, whereas the other proteins show higher homologies between the subtypes (table 1) (Collins, 1996).", "Detection of RSV infections has been described using monoclonal and polyclonal antibodies in immunofluorescence techniques (DIF, IFA), virus neutralisation assays and ELISA or RT-PCR assays (Rothbarth, 1988; Van Milaan, 1994; Coggins, 1998).", "Closely related to hRSV are the bovine (bRSV), ovine (oRSV) and caprine RSV (oRSV), from which bRSV has been studied most extensively.", "Based on sequence homology with hRSV, the ruminant RSVs are classified within the Pneumovirus genus, subfamily Pneumovirinae (Collins, 1996).", "Diagnosis of ruminant RSV infection and subtyping is based on the combined use of serology, antigen detection, virus isolation and RT-PCR assays (Uttenthal, 1996; Valarcher, 1999; Oberst, 1993; Vilcek, 1994).", "Several analyses on the molecular organisation of bRSV have been performed using human and bovine antisera, monoclonal antibodies and cDNA probes.", "These analyses revealed that the protein composition of hRSV and bRSV are very similar and the genomic organisation of bRSV resembles that of hRSV.", "For both bRSV and hRSV, the G and F proteins represent the major neutralisation and protective antigens.", "The G protein is highly variable between the hRSV subtypes and between hRSV and bRSV (53 and 28% respectively) (Prozzi, 1997; Lerch, 1990).", "The F proteins of hRSV and bRSV strains present comparable structural characteristics and antigenic relatedness.", "The F protein of bRSV shows 80-81% homology with hRSV, while the two hRSV subtypes share 90% homology in F (Walravens, K. 1990).", "Studies based on the use of hRSV and bRSV specific monoclonal antibodies have suggested the existence of different antigenic subtypes of bRSV.", "Subtypes A, B, and AB are distinguished based on reaction patterns of monoclonal antibodies specific for the G protein (Furze, 1994; Prozzi, 1997; Elvander, 1998).", "The epidemiology of bRSV is very similar to that of hRSV.", "Spontaneous infection in young cattle is frequently associated with severe respiratory signs, whereas experimental infection generally results in milder disease with slight pathologic changes Elvander, 1996).", "RSV has also been isolated from naturally infected sheep (oRSV) (LeaMaster, 1983) and goats (cRSV) (Lehmkuhl, 1980).", "Both strains share 96% nucleotide sequence with the bovine RSV and are antigenically crossreacting.", "Therefore, these viruses are also classified within the Pneumovirus genus.", "A distinct member of the subfamily Pneumovirinae, genus Pneumovirus is the Pneumonia virus of mice (PVM).", "PVM is a common pathogen in laboratory animal colonies, particularly those containing atymic mice.", "The naturally acquired infection is thought to be asymptomatic, though passage of virus in mouse lungs resulted in overt signs of disease ranging from an upper respiratory tract infection to a fatal pneumonia (Richter, 1988; Weir, 1988).", "Restricted serological crossreactivity between the nucleocapsid protein (N) and the phosphoprotein (P) of PVM and hRSV has been described but none of the external proteins show cross-reactivity, and the viruses can be distinguished from each other in virus neutralisation assays (Chambers, 1990a; Gimenez, 1984; Ling, 1989a).", "The glycoproteins of PVM appear to differ from those of other paramyxoviruses and resemble those of RSV in terms of their pattern of glycosylation.", "They differ, however, in terms of processing.", "Unlike RSV, but similar to the other paramyxoviruses, PVM has haemagglutinating activity with murine erythrocytes, for which the G protein appears to be responsible since a monoclonal antibody to this protein inhibits haemagglutination (Ling, 1989b).", "The genome of PVM resembles that of hRSV, including two nonstructural proteins at its 3′end and a similar genomic organisation (Chambers, 1990a; Chambers, 1990b).", "The nucleotide sequences of the PVM NS1/NS2 genes are not detectably homologous with those of hRSV (Chambers, 1991).", "Some proteins of PVM show strong homology with hRSV (N: 60%, and F: 38 to 40%) while G is distinctly different (the amino acid sequence is 31% longer) (Barr, 1991; Barr, 1994; Chambers, 1992).", "The PVM P gene, but not that of RSV or APV, has been reported to encode a second ORF, representing a unique PVM protein (Collins, 1996).", "New PVM isolates are identified by virus isolation, heamagglutination assays, virus neutralisation assay and various immuno-fluorescence techniques.", "Table with addedum: Amino acid homology between the different viruses within the within the genus Pneumovirus of the subfamilyPneumovirinae.", "oRSV v. bRSV v. bRSV v. PVM vs. Gene hRSV's bRSV's hRSV hRSV oRSV hRSV NS1 87 68-69 89 * NS2 92 83-84 87 * N 96 93 60 P — 81 M — 89 F 89 80-81 38-40 G 53 88-100 21-29 38-41 60-62 * M2 92 94 41 SH 76 45-50 56 L — * No detectable sequence homology The Genus Metapneumovirus Avian pneumoviruses (APV) has been identified as the aetiological agent of turkey rhinotracheitis (McDougall, 1986; Collins, 1988) and is therefore often referred to as turkey rhinotracheitis virus (TRTV).", "The disease is an upper respiratory tract infection of turkeys, resulting in high morbidity and variable, but often high, mortality.", "In turkey hens, the virus can also induce substantial reductions in egg production.", "The same virus can also infect chickens, but in this species, the role of the virus as a primary pathogen is less clearly defined, although it is commonly associated with swollen head syndrome (SHS) in breeder chicken (Cook, 2000).", "The virions are pleiomorphic, though mainly spherical, with sizes ranging from 70 to 600 nm and the nucleocapsid, containing the linear, non-segmented, negative-sense RNA genome, shows helical symmetry (Collins, 1986; Giraud, 1986).", "This morphology resembles that of members of the family Paramyxoviridae.", "Analyses of the APV-encoded proteins and RNAs suggested that of the two subfamilys of this family (Paramyxovirinae and Pneumovirinae), APV most closely resembled the Pneumovirinae (Collins, 1988; Ling, 1988; Cavanagh, 1988).", "APV has no non-structural proteins (NS1 and NS2) and the gene order (3′-N—P-M-F-M2-SH-G-L-5′) is different from that of mammalian pneumoviruses such as RSV.", "APV has therefore recently been classified as the type species for the new genus Metapneumovirus (Pringle, 1999).", "Differences in neutralisation patterns, ELISA and reactivity with monoclonal antibodies have revealed the existence of different antigenic types of APV.", "Nucleotide sequencing of the G gene led to the definition of two virus subtypes (A and B), which share only 38% amino acid homology (Collins, 1993; Juhasz, 1994).", "An APV isolated from Colorado, USA (Cook, 1999), was shown to cross-neutralize poorly with subtype A and B viruses and based on sequence information was designated to a novel subtype, C (Seal, 1998; Seal 2000).", "Two non-A/non-B APVs were isolated in France, and were shown to be antigenically distinct from subtypes A, B and C. Based on amino acid sequences of the F, L and G genes, these viruses were classified again as a novel subtype, D (Bayon-Auboyer, 2000).", "Diagnosis of APV infection can be achieved by virus isolation in chicken or turkey tracheal organ cultures (TOCs) or in Vero cell cultures.", "A cytopathic effect (CPE) is generally observed after one or two additional passages.", "This CPE is characterised by scattered focal areas of cell rounding leading to synctyial formation (Buys, 1989).", "A number of serology assays, including IF and virus neutralisation assays have been developed.", "Detection of antibodies to APV by ELISA is the most commonly used method (O'Loan, 1989; Gulati, 2000).", "Recently, the polymerase chain reaction (PCR) has been used to diagnose APV infections.", "Swabs taken from the oesophagus can be used as the starting material (Bayon-Auboyer, 1999; Shin, 2000) Alansari, H. and Potgieter, L. N. D. 1994.Nucleotide and predicted amino acid sequence analysis of the ovine respiratory syncytial virus non-structural 1C and 1B genes and the small hydrophobic protein gene.", "J. Gen. Virol.", "75: 401-404.Alansari, H., Duncan R. B., Baker, J. C. and Potgieter, L. N. 1999.Analysis of ruminant respiratory syncytial virus isolates by RNAse protection of the G glycoprotein transcripts.", "J. Vet.", "Diagn.", "Invest.", "11: 215-20 Anderson, L. J, Hierholzer, J. C., Tsou, C., Hendry, R. M., Fernic, B. F., Stone, Y. and McIntosh, K 1985.Antigenic characterisation of respiratory syncytial virus strains with monoclonal antibodies.", "J. Inf.", "Dis.", "151: 626-633.Barr, J., Chambers, Pringle, C. R., Easton, A. J.", "1991.Sequence of the major nucleocapsid protein gene of pneumonia virus of mice: sequence comparisons suggest structural homology between nucleocapsid proteins of pneumoviruses, paramyxoviruses, rhabdoviruses and filoviruses.", "J. Gen. 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Pringle CR, Easton A J.", "1992.Sequence analysis of the gene encding the fusion glycoprotein of pneumonia virus of mice suggests possible conserved secondary structure elements in pramyxovirus fusion glycoproteins.", "J. Gen. Virol.", "73: 1717-1724.Coggins, W. B., Lefkowitz, E. J. and Sullender, W. M. 1998.Genetic variability among group A and group B respiratory syncytial viruses in a children's hospital.", "J. Clin.", "Microbiol.", "36: 3552-3557.Collins, M. S. and Gough, R. E., Lister, SA., Chettle, N. and Eddy, R. 1986.Further characterisation of a virus associated with turkey rhiotracheitis.", "Vet.", "Rec.", "119: 606.Collins, M. S. and Gough, R. E. 1988.Characterisation of a virus associated with turkey rhinotracheitis.", "J. Gen. Virol.", "69: 909-916.Collins, M. S., Gough, R. E., and Alexander, D. J.", "1993.Antigenic differentiation of avian pneumovirus isolates using polyclonal antisera and mouse monoclonal antibodies.", "Avian Pathology 22: 469-479.Collins, P. L., McIntosh, K., Chanock, R. M. 1996.Respiratory syncytial virus.", "P. 1313-1351.In: B. N. Fields, D. M. Knipe, and P. M. Howley (ed.).", "Fields virology, 3rd ed., vol.", "1 Lippincott-Raven, Philadelphia, Pa., USA Cook, J. K. A., Huggins, M. B., Orbell, S. J. and Senne, D. A.", "1999.Preliminary antigenic characterization of an avian pneumovirus isolated from commercial turkeys in Colorado, USA.", "Avian pathol.", "28: 607-617.Cook, J. K. A.", "2000.Avian rhinotracheitis.", "Rev.", "Sci.", "tech.", "off int.", "Epiz.", "19: 602-613.Dowell, S. F., Anderson, L. J., Gary, H. E., Erdman, D. D., Plouffe, J. F., File, T. M., Marston, B. J. and Breiman, R. F. 1996.Respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults.", "J. 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T. 1984.Monoclonal antibodies to human respiratory syncytial virus and their use in comparison of different virus isolates.", "J. Gen. Virol.", "65: 963-971.Gulati, B. R., Cameron, K. T., Seal, B.", "S, Goyal, S. M., Halvorson, D. A. and Njenga, M. K 2000.Development of a highly sensitive and specific enzyme-linked immunosorbent assay based on recombinant matrix protein for detection of avian pneumovirus antibodies.", "J. Clin.", "Microbiol.", "38: 4010-4.Johnson, P. R., Spriggs M. K, Olmsted, R. A. and Collins, P. L. 1987.The G glycoprotein of human respiratory syncytial virus subgroups A and B: extensive sequence divergence between antigenically related proteins.", "Proc.", "Natl.", "Acad.", "Sd.", "USA 84: 5625-5629.Juhasz, K. and Easton, A. J.", "1994.Extensive sequence variation in the attachment (G) protein gene of avian pneumovirus: evidence for two distinct subgroups.", "J. Gen. Virol.", "75: 2873-2880.LeaMaster, B. R., Evermann, J. F., Mueller, M. K., Prieur, M. 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[ [ "Use of the slug gene, or of the transcription or expression products thereof in the detection and/or treatment of cancerous cells", "Method for the detection of cancerous cells, in particular, mesenchymal tumoral cells, in a biological sample suspected of containing said malignant cells.", "The methods are based on the aberrant expression of the Slug gene or transcription or expression products thereof.", "Also disclosed are methods of treatment that based on the alteration of the transcription or expression of the Slug gene and methods for screening the compounds that have cancer treating activities.", "Pharmaceutical compositions comprising the anti-cancer compounds are also disclosed." ], [ "1-21.", "(canceled) 22.A method for detecting the presence of cancerous cells in a test sample, based on the aberrant expression of a Slug gene, which comprises: 1) obtaining a test sample for a vertebrate suspected of having cancerous cells; 2) evaluating the expression of the Slug gene in cells contained in said test sample; and 3) comparing the expression of the Slug gene in the cells of the sample with the expression of the Slug gene in the cells of a control sample; in which the presence of an aberrant expression of the Slug gene in the cells of the test sample, when compared with the expression of the Slug gene in cells of the control sample, is indicative of the presence of cancerous cells in said test sample.", "23.A method according to claim 22, in which said cancerous cells are cells of mesenchymal tumor.", "24.A method according to claim 23, in which said mesenchymal tumor is leukemia or sarcoma.", "25.A method according to claim 22, in which said vertebrate suspected of having cancerous cells is a human being.", "26.A method according to claim 22, in which said control sample is (i) a biological sample of a vertebrate that does not have cancerous cells, or (ii) a biological sample of a vertebrate that has cancerous cells.", "27.A method according to claim 22, in which the evaluation of the expression of the Slug gene is performed by determining the number of copies of the Slug gene.", "28.A method according to claim 27, in which determination of the number of copies of the Slug gene is performed by visualizing double extrachromosomal fragments, visualizing homogeneously integrated staining regions, or by hybridization techniques.", "29.A method according to claim 22, in which the evaluation of the expression of the Slug gene is performed by determining the level of mRNA corresponding to the Slug gene (SLUG mRNA).", "30.A method according to claim 29, in which the determination of the level of SLUG mRNA is performed by Northern blot analysis, mapping with S1 nuclease, polymerase chain reaction (PCR), retrotranscription in combination with polymerase chain reaction (RT-PCR), retro-transcription in combination with the ligase chain reaction (RT-LCR), hybridization or microarrays.", "31.A method for detecting the presence of cancerous cells in a test sample, based on the level of the expression product of the Slug gene (SLUG protein), which comprises: 1) obtaining a biological test of a vertebrate suspected of having cancerous cells; 2) evaluating the level of the SLUG protein in the cells contained in said test sample; and 3) comparing the level of the SLUG protein in the cells of the sample with the level of SLUG protein in cells of a control sample; in which the presence of an aberrant level of the SLUG protein in the cells of the test sample, when compared with the level of the SLUG protein in cells of the control sample, is indicative of the presence of cancerous cells in said test sample.", "32.A method according to claim 31, in which said cancerous cells are cells of mesenchymal tumor.", "33.A method according to claim 32, in which said mesenchymal tumor is leukemia or sarcoma.", "34.A method according to claim 31, in which said vertebrate suspected of having cancerous cells is a human being.", "35.A method according to claim 31, in which said control sample is (i) a biological sample of a vertebrate that does not have cancerous cells, or (ii) a biological sample of a vertebrates that has cancerous cells.", "36.A pharmaceutical composition that comprises a therapeutically effective quantity of an antibody that recognizes the SLUG protein, or a fragment thereof, or of a compound that interferes with the Slug function at the DNA level, or at the RNA level or at the protein level (SLUG), and a pharmaceutically acceptable excipient.", "37.A composition according to claim 36, in which said antibody recognizes the human SLUG protein, or a fragment thereof.", "38.A composition according to claim 36, in which said antibody that recognizes the SLUG protein, or a fragment thereof, is a monoclonal or polyclonal antibody.", "39.A composition according to claim 36, in which said compound that interferes with the Slug function at the DNA level, or at the RNA level or at the protein level (SLUG) is a compound, or a mixture of compounds, that impedes the expression of the Slug gene, or which deactivate the SLUG mRNA, or that deactivates the SLUG protein or that decreases the effect of said protein.", "40.A method for in vitro screening of an antitumoral agent based on the regulation of the expression of the Slug gene, wherein the method comprises (i) developing a cell system which, under suitable conditions, expresses the Slug gene, (ii) bringing said cell system into contact with a compound to be tested, and (iii) evaluating the expression of the Slug gene, such that if the Slug gene is not expressed, or its expression is inhibited, the compound tested is a potential antitumoral agent.", "41.A method for treating cancer, comprising administering an effective amount of a pharmaceutical composition of claim 36 to a patient in need thereof." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The recent advances in the treatment of cancer have shown that in order to plan a suitable cancer treatment and to determine a precise prognosis, it is necessary to have sensitive methods available for detecting the presence of cancer, the type of cancer and the stage thereof in order to determine its specific location and its possible spread to other tissues.", "A precise diagnosis of the cancer can contribute to a reduction in the number of deaths due to this disease and improve the quality of life of patients, given that it allows the most appropriate treatment to be chosen (chemotherapy, surgical removal, etc.)", "and helps reduce the inconveniences for the patient by defining the end point of the therapeutic treatment.", "The prognostic markers provide important information for the treatment and development of cancer in patients.", "In fact, for the application of systemic adjuvant therapy in the treatment of some types of primary cancers, identification of patients at high and low risk is one of the main goals.", "Several prognostic markers are known, both classical, such as tumor size, state of lymph nodule, histopathology, state of steroid receptor, and second generation markers, such as rate of proliferation, DNA ploidy, oncogenes, growth factor receptors and some glycoprotein receptors, which are useful for taking therapeutic decisions (McGuire, W. L., Prognostic Factors for Recurrence and Survival, in “Educational Booklet American Society of Clinical Oncology,” 25 th Annual Meeting, 89-92 (1989); Contesso et al., Eur.", "J. Clin.", "Oncol., 25: 403-4′9 (1989)).", "Although none of the known prognostic markers completely satisfy the objective of distinguishing between patients of high and low risk, the combination of different markers can improve the prediction of the prognosis of the patient, and so the search continues for new prognosis markers that can be added to the current ones to aid in the corroboration of the prognosis of the cancer, its progression and the residual disease after treatment.", "On the other hand, most of the methods used for the detection of cancerous cells have a limited sensitivity, although the molecular methods based on analysis of nucleic acids have improved said sensitivity (Burchill S. A.", "& Selby P. J., J. Pathol.", "190: 6-14 (2000)).", "Nevertheless, none of these strategies allows an invasive tumor cell to be distinguished from a non-invasive tumor cell.", "The Slug gene is a gene present in vertebrates.", "It codes for a transcription factor of the “zinc fingers” type (SLUG), implicated in epithelial-mesenchymal transitions (Nieto et al., Science 264: 835-849 (1994))." ], [ "<SOH> BRIEF DESCRIPTION OF THE INVENTION <EOH>The invention addresses, in general, the problem of finding a marker for the detection of cancerous cells, such as mesenchymal tumor cells.", "The solution provided by this invention is based on the fact that the inventors have surprisingly discovered that expression of the Slug gene is related to the presence of cancerous cells, such as mesenchymal tumor cells, as they have been able to observe that malignant cells express significantly high levels of the Slug gene and/or of its transcription and expression products when they are compared to normal cells.", "Different trials performed by the inventors have shown that the product of the SLUG gene is expressed in cells of tissue samples that present mesenchymal tumor cells but it is not expressed, or expressed at almost inappreciable levels, in samples of normal tissues.", "In a particular embodiment, the inventors have observed that the Slug gene regulates the disseminative capacity of the leukemia cells with BCR-ABL, which indicates that said Slug gene plays a role in tumor invasion.", "Among the possible applications derived from the aforementioned discovery is the possibility of using the Slug gene, or transcription or expression products thereof, for detecting tumor invasiveness and/or as a therapeutic target in the treatment of cancer.", "Therefore, an object of this invention constitutes a method for detecting the presence of cancerous cells in a test sample based on the evaluation of the expression of the Slug gene or transcription or expression products thereof (RNA or protein).", "An additional object of this invention constitutes the use of the Slug gene or transcription or expression products thereof, as a therapeutic target in the treatment of cancer." ], [ "FIELD OF THE INVENTION The invention relates to the use of the Slug gene for detecting the presence of cancerous cells in a biological sample, based on the aberrant expression of said Slug gene, or transcription or expression products thereof.", "The invention also relates to the use of the Slug gene, or transcription or expression products thereof (RNA or proteins) in the treatment of cancer.", "BACKGROUND OF THE INVENTION The recent advances in the treatment of cancer have shown that in order to plan a suitable cancer treatment and to determine a precise prognosis, it is necessary to have sensitive methods available for detecting the presence of cancer, the type of cancer and the stage thereof in order to determine its specific location and its possible spread to other tissues.", "A precise diagnosis of the cancer can contribute to a reduction in the number of deaths due to this disease and improve the quality of life of patients, given that it allows the most appropriate treatment to be chosen (chemotherapy, surgical removal, etc.)", "and helps reduce the inconveniences for the patient by defining the end point of the therapeutic treatment.", "The prognostic markers provide important information for the treatment and development of cancer in patients.", "In fact, for the application of systemic adjuvant therapy in the treatment of some types of primary cancers, identification of patients at high and low risk is one of the main goals.", "Several prognostic markers are known, both classical, such as tumor size, state of lymph nodule, histopathology, state of steroid receptor, and second generation markers, such as rate of proliferation, DNA ploidy, oncogenes, growth factor receptors and some glycoprotein receptors, which are useful for taking therapeutic decisions (McGuire, W. L., Prognostic Factors for Recurrence and Survival, in “Educational Booklet American Society of Clinical Oncology,” 25th Annual Meeting, 89-92 (1989); Contesso et al., Eur.", "J. Clin.", "Oncol., 25: 403-4′9 (1989)).", "Although none of the known prognostic markers completely satisfy the objective of distinguishing between patients of high and low risk, the combination of different markers can improve the prediction of the prognosis of the patient, and so the search continues for new prognosis markers that can be added to the current ones to aid in the corroboration of the prognosis of the cancer, its progression and the residual disease after treatment.", "On the other hand, most of the methods used for the detection of cancerous cells have a limited sensitivity, although the molecular methods based on analysis of nucleic acids have improved said sensitivity (Burchill S. A.", "& Selby P. J., J. Pathol.", "190: 6-14 (2000)).", "Nevertheless, none of these strategies allows an invasive tumor cell to be distinguished from a non-invasive tumor cell.", "The Slug gene is a gene present in vertebrates.", "It codes for a transcription factor of the “zinc fingers” type (SLUG), implicated in epithelial-mesenchymal transitions (Nieto et al., Science 264: 835-849 (1994)).", "BRIEF DESCRIPTION OF THE INVENTION The invention addresses, in general, the problem of finding a marker for the detection of cancerous cells, such as mesenchymal tumor cells.", "The solution provided by this invention is based on the fact that the inventors have surprisingly discovered that expression of the Slug gene is related to the presence of cancerous cells, such as mesenchymal tumor cells, as they have been able to observe that malignant cells express significantly high levels of the Slug gene and/or of its transcription and expression products when they are compared to normal cells.", "Different trials performed by the inventors have shown that the product of the SLUG gene is expressed in cells of tissue samples that present mesenchymal tumor cells but it is not expressed, or expressed at almost inappreciable levels, in samples of normal tissues.", "In a particular embodiment, the inventors have observed that the Slug gene regulates the disseminative capacity of the leukemia cells with BCR-ABL, which indicates that said Slug gene plays a role in tumor invasion.", "Among the possible applications derived from the aforementioned discovery is the possibility of using the Slug gene, or transcription or expression products thereof, for detecting tumor invasiveness and/or as a therapeutic target in the treatment of cancer.", "Therefore, an object of this invention constitutes a method for detecting the presence of cancerous cells in a test sample based on the evaluation of the expression of the Slug gene or transcription or expression products thereof (RNA or protein).", "An additional object of this invention constitutes the use of the Slug gene or transcription or expression products thereof, as a therapeutic target in the treatment of cancer.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1 illustrates the positive regulation of the Slug gene by BCR-ABL.", "FIG.", "1A shows the result of Northern blot analysis of total RNA isolated from Ba/F3 (band 1), Ba/F3+p190 (band 2) and Ba/F3+p210 (band 3) cells.", "Control experiments were performed on Ba/F3 cells transfected with an empty vector.", "The nylon membrane was hybridized with a fragment of SLUG cDNA and then purified and rehybridized with a β-actin probe.", "FIG.", "1B shows the result of Northern blot analysis of total RNA isolated from Ba/F3+Combi p190 cells developed in the absence (band 1) or presence of tetracycline (band 2), Ba/F3+p190 (band 2) and Ba/F3 control cells (band 3).", "The blot was hybridized with SLUG cDNA fragments, Bcl-2 and myc, and then purified and rehybridized with a β-actin probe.", "FIG.", "2 shows the expression of SLUG mRNA in tissues of normal mice.", "FIG.", "2A shows the result of Northern blot analysis of total RNA isolated from different mouse tissues [lung, (PB) peripheral blood, heart, testicle, brain, intestine, kidney, muscle, liver, spleen, thymus and BM (bone marrow)], hybridized with a Slug cDNA probe and then purified and rehybridized with a β-actin probe.", "The mobility of the 28S and 18S RNA are indicated (PB, peripheral blood; BM, bone marrow).", "FIG.", "2B illustrates the expression of SLUG mRNA in BCR-ABLP190 and BCR-ABLP210 transgenic mice and collects the results from Northern blot analysis of total RNA isolated from peripheral blood of control mice(band 1), and from BCR-ABLP190 transgenic mice (bands 2 to 4) and BCR-ABLP210 transgenic mice (bands 5 to 6), hybridized with a cDNA probe and then purified and rehybridized with a P-actin probe.", "The mobility of the 28S and 18S RNA are indicated.", "FIG.", "3 illustrates that the endogenous SLUG is present in human invasive cell lines.", "Each RNA molecule is transcribed by means of retrotranscription (RT) and the PCR products are transferred to a nylon membrane and analyzed by hybridization with internal oligonucleotide probes labeled specifically at the terminus for each gene.", "The RNA were evaluated for the non-leukemia haematopoietic cell line 707 (band 1), for the myeloid leukemia cells U937 (band 2), for ALL-SIL leukemia T cell lines (band 3) and KOPTI-KI (band 4), for the pre-B leukemia 697 cell lines (band 5), for samples of patients with t(9;22) (bands 6 to 8) and for human leukemia cell lines positive for t(9;22) K562 (band 9), TOM-1 (band 190) and Nalm-1 (band 11).", "FIG.", "4 illustrates the effect of the Slug gene on the survival of Ba/F3 cells in absence of growth factor.", "FIG.", "4A shows the result of Northern blot analysis of total RNA isolated from Ba/F3 cells transfected with SLUG cDNA from mouse (band 1) and Ba/F3 control cells (band 2).", "The nylon membrane is hybridized with a fragment of SLUG cDNA and then purified and rehybridized with a probe of β-actin.", "FIG.", "4B is a graph that illustrates the survival of Ba/F3 cells that express the SLUG protein in absence of IL-3.The cells that grew exponentially in supplemented medium with IL-3 were adjusted to 5×105 cells/ml on day 0 and were then cultured after elimination of IL-3.The number of viable cells is shown for the Ba/F3 cells transfected with BCR-ABL and with SLUG in the absence of IL-3.FIG.", "4C illustrates that cell death is accompanied by the appearance of the DNA ladder by internucleosomal cleavage after deprivation of IL-3.Low molecular weight DNA was isolated 24 hours after deprivation of IL-3 from Ba/F3 +Slug cells (band 1), Ba/F3+p210 cells (band 2), Ba/F3+p190 cells (band 3) and from control Ba/F3 cells (band 4).", "The DNA was labeled terminally, resolved by 2% agarose gel electrophoresis and developed by autoradiography.", "FIG.", "5 illustrates the repression of SLUG mRNA in Ba/F3 cells that express BCR-ABL with antisense cDNA versus SLUG FIG.", "5A shows the results of the isolation of total RNA from Ba/F3+p210 cells (band 1) and Ba/F3+p190 cells (band 2) transfected with a vector that expressed the Slugh gene (Slug gene of mouse) without sense.", "The cellular RNA was hybridized with a cDNA probe of mouse Slugh.", "The filter was denatured and rehybridized with ABL and β-actin.", "FIG.", "5B shows the result of the hybridization of Northern filter of Ba/F3 cells that expressed transfected BCR-ABL (band 1, Ba/F3+p210+ASlug; band 3, Ba/F3+p190+ASlug) and non-transfected (bands 2, Ba/F3+p210; band 4, Ba/F3+p190).", "As a probe, an antisense Slugh oligonucleotide that comprises the first 49 bases of the sequence coding for Slugh cDNA of mouse was used.", "FIG.", "5C illustrates that the lymphoid and myeloid differentiation specific to the B cells induced in Ba/F3 cells by BCR-ABLP190 and BCR-ABLP210 oncogenes, respectively, is not influenced by the suppression of Slug.", "In FIG.", "5C, the expression profiles are shown of Ba/F3+p190 cells (upper left panel) Ba/F3+p190+antisense Slug (upper right panel), Ba/F3+p210 (lower left panel) and Ba/F3+p210+antisense Slug (lower right panel).", "The cells were stained with B220 monoclonal antibodies (specific B cell marker) and Gr-1 (specific marker of myeloids) and they were analyzed by flow cytometry.", "FIG.", "6 shows the requirements of Slug in the tumorigenicity of Ba/F3 cells that express BCR-ABL.", "The initial Ba/F3 cells that expressed BCR-ABL oncogenes grew as tumors in naked mice (REF).", "By analogy, it was discovered that the formation of tumors was already observed at five days after injections of the cell lines that expressed BCR-ABL.", "In contrast to these controls, the Ba/F3 cells that expressed BCR-ABL and antisense Slug are much less tumorigenic.", "The tumors were sliced to perform a macroscopic analysis on day 20 after cell injection.", "On average, the weight of the tumors found in the mice that had been injected with Ba/F3 cells that expressed BCR-ABL were twice as high as that of the tumors induced by cells that expressed BCR-ABL and the antisense Slug construct.", "FIG.", "7 illustrates the effect of Slug on the development of tumors by BCR-ABL cells.", "FIG.", "7A shows the result of a macroscopic analysis of tumors induced by Ba/F3+p190 cells, while FIGS.", "7B and 7C show the histological aspect of the tumors induced in naked mice.", "The section stained with hematoxylin-eosin of the mouse tumor developed after injection of the Ba/F3+p190 cells in which the expression of Slug had be specifically suppressed (FIG.", "7C).", "The Ba/F3+p190 cells that express Slugh disobey the social order of organ boundaries and migrate as individual cells, producing metastasis in different regions.", "Similar results were observed in multiple sections of the two tumors.", "The images of FIGS.", "7B and 7C are amplified 40 times.", "FIG.", "8 is a representation that illustrates the model of the role of Slug in the development of cancer.", "FIG.", "8A shows that in the haematopoietic system, the normal, non-committed, progenitor cells with capacity for self-renewal are differentiated from the mature cells.", "During this transition, the expression of Slug is down regulated.", "These normal non-committed progenitor cells are responsible for signals of the medium that regulate the number of mature cells produced and limit the self-renewal of the primitive cells.", "When, in physiological situations, these normal, non-committed progenitor cells migrate, the Slug gene could promote their survival, allowing them to perform their function.", "If this is not achieved in a specific period of time, they will undergo apoptosis, as they have been denied the necessary external signals.", "In FIG.", "8B, the situation is illustrated in the case of leukemogenesis; in this case, the target cell in which there is a chromosomal abnormality is a non-committed progenitor (Cobaleda et al., Blood 95: 1007-1113 (2000); Sánchez-García et al., Current Genomics, 1: 71-80 (2000)).", "As a result, the differentiation of the target cell is blocked, but the inhibition of the differentiation is not sufficient for the transformation, because the survival and proliferation of the target cells would be restricted to a particular microenvironment.", "Thus, there must be other genetic changes that allow the growth of the cells outside their normal environment, in addition to mutations that block differentiation.", "The fusion oncogenes associated with mesenchymal cells (both leukemias and solid tumors) block differentiation and have the capacity to activate target genes such as Slug that promote survival (regardless of the external signals required) and the migration of the defective target cells to different environments.", "These data reinforce the idea that the transformation may occur as a result of the creation/activation of a single oncogene.", "DETAILED DESCRIPTION OF THE INVENTION The invention relates, in general, to the use of the Slug gene or transcription or expression products thereof (RNA or protein) in the detection and/or treatment of cancerous cells.", "The Slug gene codes for a transcription factor of the “zinc fingers” type (SLUG) implicated in epithelial-mesenchymal transitions.", "It has now been discovered that cancerous cells, in particular, mesenchymal tumor cells, express significantly elevated levels of the Slug gene and/or transcription or expression products thereof (RNA or protein), in comparison with normal cells, which allows a method to be established for the detection of said malignant cells based on the aberrant expression of the Slug gene or transcription or expression products thereof (RNA or protein).", "1.Detection of Cancerous Cells In a first aspect, the invention provides a method for the detection of cancerous cells in a test sample, based on the evaluation of the aberrant expression of the Slug gene or transcription or expression products thereof (RNA or proteins), in comparison with the expression of said gene or transcription or expression products thereof (RNA or protein) in a control sample.", "This method can be applied to any vertebrate suspected of having said cells, in particular, in mammals, for example, in human beings.", "The method provided by this invention is suitable for the detection of cancerous cells that express said Slug gene or transcription or expression products thereof (RNA or protein), such as mesenchymal tumor cells, for example, leukemia or sarcoma, and therefore, it is of use in the diagnosis of disease caused by said cells, as well as in the evaluation of its progression and in the determination of residual disease after treatment, essential aspects in the treatment of said disease.", "In the sense used in this description, the term “test sample” refers to a biological sample of a vertebrate suspected of having mesenchymal or cancerous tumor cells.", "The term “control sample,” as it is used here includes (i) biological samples of vertebrates that do not have mesenchymal or cancerous tumor cells, and (ii) biological samples of vertebrates that have mesenchymal or cancerous tumor cells in order to obtain information relating to the prognosis of the disease in vertebrates that contain said cells.", "By “aberrant expression,” as it is used in this specification, it should be understood, in general, the altered expression of a gene or transcription or expression products thereof (RNA or protein) in cells from a tumorigenic tissue with respect to the expression of said gene or transcription or expression products thereof (RNA or protein) in normal cells of the same non-tumorigenic tissue.", "The aberrant expression of a gene includes the amplification of the gene, the over-expression of the gene and the expression of the gene in cells that normally do not express it.", "1.A Detection Based on the Aberrant Expression of the Slug Gene The invention provides a method for detecting the presence of cancerous cells in a test sample, based on the aberrant expression of the Slug gene, which comprises: 1) obtaining a biological sample of a vertebrate suspected of having cancerous cells, by which a test sample is obtained; 2) evaluating the expression of the Slug gene in the cells contained in said test sample; and 3) comparing the expression of the Slug gene in the cells of the test sample with the expression of the Slug gene in the cells of a control sample; in which the presence of an aberrant expression, of the Slug gene in the cells of the test sample, when it is compared with the expression of the Slug gene in the cells of the control sample, is indicative of the presence of cancerous cells in said test sample.", "In a particular embodiment, the method for the detection of cancerous cells based on the aberrant expression of the Slug gene provided by this invention is applied to a human being suspected of having said cells.", "The cDNA sequence of the human Slug gene as well as the amino acid sequence derived from the cDNA have been described by Nieto and others (Nieto et al., 1994, cited above).", "In a particular embodiment, the cancerous cells to be detected are mesenchymal tumor cells, for example, leukemias or sarcomas.", "The test sample is obtained from a biological sample of the vertebrate to be tested.", "Said biological sample can be obtained by any conventional method, for example, by biopsy of the tissue or blood extraction.", "The aberrant expression of the Slug gene in mesenchymal or cancerous tumor cells includes amplification of the Slug gene, overexpression of said gene and expression thereof in cells that normally do not express it.", "Currently, it is accepted that the amplification of DNA plays a crucial role in the progression of tumors, allowing the cancerous cells to regulate numerous genes.", "On the other hand, the frequency of the amplification of the DNA as well as the increase in the number of copies during progression of different cancers, generally in patients who do not respond to treatment, suggests that the overexpression of the amplified target genes confers a selective advantage on the malignant cells.", "The expression of the Slug gene may be evaluated by any appropriate conventional method, for example, determining the level of mRNA corresponding to the Slug gene (SLUG mRNA), or determining the number of copies of Slug gene produced.", "In the sense used in this description “determining the level of SLUG mRNA” includes any method that allows qualitative or quantitative measurement or estimation of the level of mRNA that can be translated into the SLUG protein in cells of a test sample, either directly or relatively by comparing it with the level of SLUG mRNA in cells in a control sample.", "Similarly, “determine the number of copies of Slug gene produced” includes any method that allows qualitative or quantitative measurement of the number of copies produced of the Slug gene in cells of a test sample, either directly or relatively, by comparing it with the number of copies of the Slug gene produced in cells of a control sample.", "In a particular embodiment, the level of SLUG mRNA or the number of copies of the Slug gene produced in the cells of the test sample is measured or estimated and then compared with the level of SLUG mRNA or with the number of copies of the Slug gene produced in the cells of the control sample.", "In a particular embodiment, the control sample can be a biological sample of a vertebrate that does not have cancerous cells, for example, mesenchymal tumor cells.", "In this case, once the level of SLUG mRNA or the number of copies of the Slug gene is known in the control sample, the resulting information can be used repeatedly as a standard for purposes of comparison.", "Alternatively, the control sample can be a biological sample of a vertebrate that has cancerous cells, for example, mesenchymal tumor cells, whereby, in this case, the level of SLUG mRNA or the number of copies of the Slug gene will provide information relating to the prognosis of the disease among vertebrates that contain said cells.", "The determination of the number of copies of the Slug gene can be performed by any appropriate conventional method, for example, visualizing extrachromosomal double minutes (dmin) or integrated homogeneously staining regions (hsrs) (Gebhart et al., Breast Cancer Res.", "Treat.", "8: 125 (1986); Dutrillaux et al., Cancer Genet.", "Cytogenet.", "49: 203 (1990)), by means of hybridization techniques using appropriate probes obtained by conventional methods in view of the nucleotide sequence of the Slug gene, etc.", "Similarly, the level of SLUG mRNA can be determined by any appropriate conventional method, for example, by Northern blot analysis (Harada et al., Cell 63: 303-312 (1990)), mapping with S1 nuclease (Fujita et al., Cell 49: 357-367 (1987)), polymerase chain reaction (PCR) (U.S. Pat.", "Nos.", "4,683,195, U.S. Pat.", "No.", "4,683,202, and U.S. Pat.", "No.", "4,965,188), retrotranscription in combination with polymerase chain reaction (RT-PCR) (Makino et al., Technique 2: 295-301 (1990)), microarray hybridization techniques (Wooster R., Trends in Genetics 16: 327-329 (2000), etc.", "In a particular embodiment, the expression of Slug mRNA has been evaluated by means of Northern Blot analysis and RT-PCR (see Materials and Methods).", "In accordance with the invention, the presence of an aberrant expression of the Slug gene in the test sample, when compared with expression of the Slug gene in the control sample, is indicative of the presence of mesenchymal or cancerous tumor cells in said test sample.", "1.B Detection Based on the Expression Product of the Slug Gene The invention also provides a method for detecting the presence of cancerous cells in a test sample, based on the expression of the expression product of the Slug gene (SLUG protein), which comprises: a) obtaining a biological sample of a vertebrate suspected of having cancerous cells, with which a test sample is obtained; b) evaluating the expression of the SLUG protein in cells of said test sample; and c) comparing the expression of the SLUG protein in the cells of the test sample with the expression of the SLUG protein in cells of a control sample; in which the presence of an aberrant expression of the SLUG protein in the cells of the test sample, when compared with the expression of the SLUG protein in the cells of the control sample, is indicative of the presence of cancerous cells in same test sample.", "In a particular embodiment, the cancerous cells to be detected are mesenchymal tumor cells, for example, leukemias or sarcomas.", "Just as in the preceding alternative [1.A], the test sample is obtained from a biological sample of the vertebrate to test, for example, a human being, by any conventional method, for example, by biopsy of the tissue or extraction of blood.", "The expression of the SLUG protein can be evaluated by any appropriate conventional method, for example, by means of techniques based on the use of antibodies, techniques based on in vivo image diagnosis, flow cytometry, proteomics, etc.", "By way of example, the expression of SLUG protein in tissues can be studied by using classical histological immunological methods, in which the specific recognition is provided by an anti-SLUG antibody while the detection system can use secondary antibodies labeled with appropriate markers.", "The expression of the SLUG protein in a tissue can also be studied by Western Blot or dot/slot analysis (Jalkanen et al., J.", "Cell Biol.", "101:976-985 (1985); Jalkanen et al., J.", "Cell Biol.", "105: 3087-3096 (1987)), by immunoassays, such as ELISA (enzyme linked immunabsorbent assay) or RIA (radioimmunoassay).", "In addition, the expression of the SLUG protein can be detected in vivo by means of image diagnostic techniques employing anti-SLUG antibodies bound to appropriate markers, for example, markers detectable by X-rays, nuclear magnetic resonance (NMR), etc.", "A review of the imaging diagnosis of tumors can be found in Tumor Imaging: The Radiochemical Detection of Cancer (S. W. Burchiel & B.", "A. Rhodes, eds., Masson Publishing Inc. (1982)).", "Anti-SLUG antibodies that can be used in the method provided by this invention include antibodies against the intact SLUG protein or against an antigenic fragment thereof, optionally conjugated to a carrier.", "The antibodies can be polyclonal or, preferably, monoclonal, which can be obtained by hybridoma technology (Kohler et al., Nature 256: 495 (1975)).", "Alternatively, antibody fragments can be used, for example, Fab, F(ab′)2, etc.", "Other methods for determining the expression of the SLUG protein in tissues include flow cytometry (Ward M. S., Pathology 31: 382-392 (1999), proteomics (Pandey M. & Mann M., Nature 405: 837-846 (2000); Chambers et al., J. Pathol.", "192: 280-288 (2000), etc.", "In accordance with the invention, the aberrant expression of the SLUG protein in cells of the test sample, when compared with the expression of the SLUG protein in cells of the control sample, is indicative of the presence of cancerous cells in said test samples.", "2.Treatment of Cancer In a second aspect, the invention relates to the use of the Slug gene or transcription or expression products thereof, in the treatment of cancer, in particular, in the treatment of pathologies related to the presence of cancerous cells, such as mesenchymal tumor cells, for example leukemia or sarcoma.", "In the sense used in this description, “the use of the Slug gene or transcription or expression products thereof in the treatment of cancer” includes any method that allows qualitative or quantitative interference with the Slug gene, the level of mRNA that can be translated into the SLUG protein, or with the SLUG protein itself.", "In this sense, the invention provides a pharmaceutical composition that comprises a therapeutically effective quantity of an antibody that recognizes the SLUG protein, or a fragment thereof, or a compound that interferes in the function of Slug at the DNA level, or at the RNA level, or at the protein level (SLUG), optionally along with the pharmaceutically acceptable excipients.", "The anti-SLUG antibodies that can be used in the elaboration of the pharmaceutical composition provided by this invention can be antibodies against the intact SLUG protein or against an antigene fragment thereof, optionally conjugated to a carrier.", "The antibodies can be obtained using the hybridoma technology (Kohler et al., Nature 256: 495 (1975)).", "Alternatively antibody fragments can be used, for example, Fab, F(ab′)2, etc., which can be obtained by conventional methods.", "The compound that interferes with the Slug function, both at the DNA level and at the RNA or protein level (SLUG) can be any compound, or mixture of compounds, which impede expression of the Slug gene, or which deactivate the SLUG mRNA that is generated, for example, by anti-sense oligonucleotides or ribosomes, or which deactivate the SLUG protein, for example, by use of antibodies or which compete for the effect of said protein with negative dominants (Choo, Y, et al.", "J. Mol.", "Biol.", "273: 525-532, (1997); Cobaleda, C., & Sánchez-García, I., Blood 95: 731-737 (2000)).", "The excipients that may be present in the pharmaceutical composition of the invention will depend, among other things, on the administration route of said pharmaceutical composition.", "A review of the different administration routes of active substances, the excipients to use and their manufacturing processes can be found in the Treatise on Formulation Pharmacy, C. Fauli i Trillo, Luzan 5, S. A. of Editors, 1993.The invention also relates to a method for in vitro screening of antitumoral agents based on the regulation of the expression of the Slug gene that comprises (i) developing a cellular system that, in certain conditions, expresses the Slug gene, (ii) bringing said cell system into contact with the compound to be tested, and (iii) evaluating the expression of the Slug gene, such that if the Slug gene is not expressed, the compound tested is a potential antitumoral agent.", "Interference with the function of the Slug gene can be performed at the DNA level by impeding its expression, deactivating the mRNA that is generated by antisense oligonucleotides or ribosomes, deactivating the protein by using antibodies or competing with the effect of said protein with negative dominants (Choo, Y, et al.", "J. Mol.", "Biol.", "273: 525-532, (1997); Cobaleda, C., & Sánchez-García, I., Blood 95: 731-737 (2000)).", "The invention will now be illustrated by means of an assay performed by the inventors, which shows that the Slug gene regulates the disseminative capacity of the leukemia cells with BCR-ABL.", "I.", "Materials and Methods Cell Cultures The cell lines used include BalF3 cells (Palacios and Steinmetz, Cell 41: 727 (1985)) and Ba/F3 cells that express the human proteins BCR-ABLP190 (Ba/F3+p190) and BCR-ABLP210 (Ba/F3+p210) (Sánchez-García and Grütz, Proc.", "Natl.", "Acad.", "Sci.", "USA, 92: 5287-5291 (1995)).", "The cells were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS).", "When it was necessary, 10% of medium was added conditioned with WEHI-3B as source of IL-3 (interleukin-3).", "Representational Difference Analysis The RDA was performed as described by Hubank and Schatz (Hubank and Schatz, Nucleic Acid Res.", "22: 5640-5648 (1994)).", "In summary, mRNA was isolated from Ba/F3, Ba/F3+p190 and Ba/F3+p210 cells.", "A sample of 10 mg of mRNA was extracted from each cell population for the RDA.", "cDNA was synthesized from the mRNA and digested with Dpn II.", "Adaptors were added to the cDNA digested with Dpn II composed of two oligonucleotides: (R-24) [SEQ ID NO: 1] and (R-12) [SEQ ID NO: 2].", "The resulting mixture was amplified by PCR with R-24 oligonucleotides [SEQ ID NO: 1] and the adaptors were cleaved with Dpn II.", "Then, a second pair of adaptors were bound: (J-24) [SEQ ID NO: 3] and (J-12) [SEQ ID NO: 4] to the amplified fragments from the Ba/F3+p190 and Ba/F3+p210 (tester) cells.", "These were then hybridized with the amplified cDNA fragments with R-24 [SEQ ID NO: 1] from Ba/F3 cells (inductor) with a ratio of 1:100 for 20 hours.", "The hybridization mixture was used as a template for the PCR based amplification.", "A second round of removal was performed by withdrawing the J adaptors [SEQ ID NO: 3 and SEQ ID NO: 4] from an aliquot of the product of the first round of PCR, binding a third pair of oligonucleotide adaptors: (N-24) [SEQ ID NO: 5] and (N-12) [SEQ ID NO: 6] and hybridising with induction amplicons at a ratio of 1:1000.The PCR products were separated on 2.0% agarose gel and the individual bands subcloned and examined by Northern Blot Analysis with respect to the differential expression.", "Retrotranscription Polymerase Chain Reaction (RT-PCR) In order to analyse the expression of SLUG in human cell lines and in peripheral blood samples in patients positive for Ph-1, an RT was performed in accordance with the manufacturer's protocol in a reaction of 20 μl that contained 50 ng of random hexamers, 3 μg of total RNA and 200 units of Superscript II RNAse H-reverse transcriptase (GIBCO/BRL).", "The parameters of the thermal cycles for the PCR and the sequences of the specific primers were as follows: SLUG, 30 cycles at 94° C. for 1 minute, 56° C. for 1 minute and 72° C. for 2 minutes, primer in correct sense [SEQ ID NO: 7] and antisense primer [SEQ ID NO: 8]; c-ABL, 30 cycles at 94° C. for 1 minute, 56° C. for 1 minute and 72° C. for 2 minutes with the correct sense [SEQ ID NO: 9] and antisense primer [SEQ ID NO: 10].", "The amplification of the mRNA of c-ABL served as a control to evaluate the quality of each sample of RNA.", "The sequences of the internal probes were the following: SLUG: SEQ ID NO: 11, and c-ABL: SEC.", "ID.", "NO.", "12.Cloning of cDNA of Slug of Complete Length by RT-PCR The Slug cDNA of mouse was cloned by RT-PCR using the advance primer (SEQ ID NO: 13) and the reverse primer (SEQ ID NO: 14) (GenBank accession number U70550).", "Analysis of the RNA The total cytoplasmic RNA (10 μg) was glyoxylated and fractioned on gels with 1.4% agarose in 10 mM Na2HPO4 buffer (pH 7.0).", "After electrophoresis, the gel was transferred to Hybond-N nylon membranes (Amersham), which were treated with UV light and hybridized with probes labeled with 32P.", "The charge was controlled, re-treating the filters with a cDNA probe of β-actin.", "The antisense Slugh oligonucleotide used as a probe comprises the first 34 bases of the sequence coding for the Slugh cDNA of mouse.", "Plasmid Construct for In Vivo Studies The Slug cDNA of mouse was amplified by PCR to facilitate cloning by adding restriction enzymes, using primers that hybridize with the 5′ and 3′ terminals of cDNA and amplifying a region that included the entire coding region of the gene.", "The Slugh cDNA of mouse was cloned both in the sense and antisense orientation in the pEF-BOS vector (denominated BOS-Slug and BOS-antiSlug, respectively), which contained sequences of the EF1-α promoter followed by a region of polyengarce bound to the signal of poly(A)adenylation of the cDNA of human G-CSF, as has been described (Mizushima and Nagata, Nucleic Acid Res.", "18: 4322 (1990)).", "The Combi-p190 vector was obtained by replacing the cDNA of the luciferase of the Combi-tTA plasmid (Schultze et al., Nature Biotechnology 14: 499-503 (1996)) with the cDNA of BCR-ABLP190.The authenticity of the constructs was confirmed by DNA sequencing.", "Cellular Transfection Ba/F3 cells were transfected by electroporation (960 μF, 220 V) with 20 μg of Combi-p190, respectively, along with 1 μg of MC1-neo expression vector.", "In the cells (Ba/F3+combi p190), the expression of BCR-ABL was analyzed by Northern Blot in the presence and in absence of tetracycline (20 ng/ml).", "These cells were resistant to the absence of IL-3 when they were developed in the presence of tetracycline.", "Expression of the Murine Slug Gene in Cells that Express Ba/F3 and BCR-ABL and Cell Survival Assay Ba/F3, Ba/F3+p190 and Ba/F3+p210 cells were transfected by electroporation (960 μF, 220 V) with 20 μg of BOS-Slug and BOS-antiSlug, respectively, along with 1 μg of pure MCl expression vector.", "In the cell lines, Slug expression was analyzed by Northern blot.", "The cells were selected with respect to the resistance to the absence of IL-3 and cell viability was determined by exclusion with trypan blue.", "DNA Analysis The low-molecular weight DNA was isolated as indicated below.", "The cells were collected in 1.5 ml of culture medium, micro-centrifuged for 1 minute at 1500 rpm (400×g) and the sediments suspended in 300 μl of proteinase K buffer.", "After an incubation period of one night at 55° C., the DNA was precipitated with ethanol, suspended in 200 μl of TE buffer that contained 50 μl/ml of RNAse A and incubated at 37° C. for 2 h. The DNA was extracted with phenol and chloroform and precipitated with ethanol.", "DNA aliquots (2 μg) were labeled at the termini with α32-dCTP and submitted to electrophoresis on 2% agarose gel.", "After electrophoresis, the gel was transferred to a Hybond N (Amersham) and autoradiographed for 2hat-70° C. Phenotype Analysis For the cytometry staining, the following monoclonal anti-mouse antibodies from Pharmingen were used: the lymphoid marker CD45R/B220 and the myeloid marker Gr-1.Suspension of a single cell of the different cell lines obtained by routine techniques with anti-mouse CD32/CD16 (Pharmingen), purified to block binding by Fc receptors, were incubated with an appropriate dilution of different antibodies at room temperature or at 4° C., respectively.", "The samples were washed twice with PBS and re-suspended in PBS.", "Dead cells were excluded by staining with propidium iodide.", "The samples and the data were analyzed by FACScan using the CellQuest software (Becton Dickinson).", "Tumorigenicity Assay In order to assay the tumorigenicity of the different cell lines, 106 cells re-suspended in 200 μl of saline solution buffered with phosphate were injected subcutaneously into both sides of male athymic (naked) mice aged 2 to 4 weeks old.", "The formation of tumors in the animals was examined during the course of two months.", "Histological Analysis The tumor samples were fixed in 10% formaline overnight.", "They were then processed and set in paraffin blocks.", "Sections of 6 μm were stained with haematoxylin and eosin, examined histologically and photographed.", "All the sections were taken from homogeneous and viable portions of the tumors submitted for dissection.", "II.", "Results BCR-ABL Positively Regulates the Expression of Slug in Haematopoietic Cells Molecular characterization of certain chromosomal abnormalities associated with cancer in humans has shown that the main consequences are tumor-specific fusion proteins.", "A common idea put forward is that the creation of these proteins alters the normal development of the tumor-specific target cells blocking apoptosis (Cobaleda et al., BioAssays 20; 922 (1998)), but the most threatening aspects for life due to the oncogenic process are invasion and metastasis.", "Although the clinical significance of such expression of the malignant phenotype has been correctly appreciated, the lack of understanding of the molecular mechanisms implicated in the invasion have delayed other development work in the field of cancer research.", "Thus, an initial critical stage for understanding the malignant transformation mediated by the products of the chromosomal abnormalities associated with leukemia is the identification of reporter genes downstream from those that direct these proteins.", "The BCR-ABL oncogenes are being used as an experimental model and benefit has been obtained from a cell system that uses a murine haematopoietic precursor Ba/F3 cell line transduced with vectors that code for the BCR-ABL fusion protein (Sánchez-García and Grütz, 1995, cited earlier).", "The expression of BCR-ABL in Ba/F3 induces cell transformation, confers an independent growth of cytokines and blocks apoptosis (Daley and Baltimore, Proc.", "Natl.", "Acad.", "Sci.", "USA 85: 9312 (1988); Sánchez-García and Grütz, 1995, cited earlier).", "In order to identify potential targets for the action of BCR-ABL, a substrate process has been chosen for the representational difference analysis (RDA).", "Using mRNA molecules from Ba/F3 cells, and Ba/F3 cells that expressed BCR-ABLp190 (Ba/F3+p190) and Ba/F3 cells that expressed BCR-ABLp210 (Ba/F3+p210), cDNA was prepared and submitted to three cycles of sequential hybridization and PCR amplification in accordance with the RDA protocol of Hubank and Schatz (Hubank and Schatz, 1994, cited earlier).", "Several fragments of differentially amplified cDNA fragments were individually subcloned and assayed with respect to differential gene expression by Northern Blot analysis of Ba/F3 RNA.", "A fragment of cDNA was identified that represented a gene regulated differentially and denoted I-1.Comparison of sequences showed that the 527 nucleotide I-1 fragment corresponded to a part of the coding region and the untranslated 3′ region of murine SLUG cDNA (Nieto et al, 1994, cited earlier), identified with the BLAST search utility (National Center for Biotechnology Information).", "The capacity of the BCR-ABL oncogenes to stimulate production of SLUG mRNA was assayed by hybridization with Northern mRNA Ba/F3, Ba/F3+p190 and Ba/F3+p210 cell filters (FIG.", "1A).", "Northern blot analysis showed the expression of SLUG in Ba/F3+p190 and Ba/F3+p210 cells.", "This assay shows that the cells positive for BCR-ABL express SLUG mRNA, but that the control cells do not.", "Therefore, these data indicate a clear relationship between activation of the Slug gene and the expression of BCR-ABL in this system.", "The Presence of BR-ABL is Required for Aberrant Expression of SLUG The data described above show the aberrant expression of SLUG I cells that contain the fusion gene BCR-ABL.", "In order to determine whether BCR-ABL is required for transcription of the Slug gene, Ba/F3 cells were created in which expression of the BCR-ABL gene could be exogenously regulated.", "The Combi-tTa system was used, which has the transactivator and minimum promoter of the tet operator that directs the gene expression unit in a single plasmid (Schultze et al., 1996, cited above).", "The repressor tet protein fused to the transactivator domain of viral VP 16 was bound, in the absence of tetracycline, to a minimum promoter of the tet operator obtained by genetic engineering and activated the transcription of BCR-ABL (Combi-p190).", "In the presence of the effecter molecule, the binding was cancelled and the promoter silenced (FIG.", "1B).", "In this sense, although BCR-ABLP190 was detected in Ba/F3+Combip190 without tetracycline, mRNA was not found in the presence of tetracycline (20 ng/ml).", "These data indicate that the induction of BCR-ABL can be completely suppressed by tetracycline.", "The specificity of the activation of the Slug gene was analyzed by Norther hybridization of Northern filters in Ba/F3+Combip190 cells.", "After two days of culturing, in the presence or absence of tetracycline, SLUG expression was determined.", "As shown in FIG.", "1B, SLUG expression was rapidly reduced, coinciding with the reduction of the expression of mRNA of BCR-ABLPp190.In addition, in these cells, it was analyzed whether the expression of two well-known downstream effectors of the BCR-ABL oncogenes, Myc (Sawyers et al., Cell 70: 901-910 (1992)) and Bcl-2 (Sánchez-García and Grütz, 1995 cited above; Sánchez-García and Martin-Zanca, J. Mol.", "Biol.", "267: 225 (1997)) were dependent on the presence of BCR-ABL.", "As shown in FIG.", "1B, although expression of both Bcl2 and Myc was detected in Ba/F3+Combip190 without tetracycline, no mRNA could be found in presence of tetracycline (FIG.", "1B).", "This observation confirms that the changes observed in the gene expression were stimulated by NCR-ABL.", "Slug Expression is Present in Cell Lines and Peripheral Blood Cells of Patients with Leukemia Positive for the Philadelphia Chromosome The discovery that BCR-ABL positively regulates expression of Slug in Ba/F3 cells led the inventors to test whether the levels of expression of Slug are also positively regulated as a consequence of expression of BCR-ABL in primary parent cells, the natural target of the oncogenic BCR-ABL fusion protein (Cobaleda et al., 2000; Sánchez-García, 2000, cited above).", "Northern blot analysis of a spectrum of mouse tissue indicated that, with rare exceptions, SLUG is widely expressed in mouse tissue (FIG.", "2A).", "However, SLUG is not expressed by the peripheral blood leukocytes (FIG.", "2A).", "Knowing that SLUG is not expressed in peripheral blood of normal mice, the possible effects of expression of BCR-ABL on the levels of SLUG mRNA in peripheral blood of transgenic BCR-ABL mice with leukemia were tested (Castellanos et al., Blood 90: 2168 (1997)).", "The parent cells of mouse that expressed BCR-ABL by hybridization with Northern filters were examined (FIG.", "2B).", "Each band of RNA was evaluated with respect to integrity and charge by hybridization with a probe of β-actin (FIG.", "2B, lower section).", "In accordance with the results observed in Ba/F3 cells, the primitive cells with BCR-ABL produced an increase in the expression of SLUG Afterwards, it was tested whether the expression of SLUG was also present in primitive cells of patients with human leukemias positive for Ph1.It was shown that the expression product of the Slug gene was absent in cells from normal human samples (FIG.", "3, band 1).", "On the contrary, the expression of SLUG was present in cells of patients with the Philadelphia chromosome (FIG.", "3, bands 6-8).", "The cell lines K562, Nalm-1 and TOM-1, positive for t(9; 22), come from patients with LMC and from LLA patients positive for Ph1.Therefore, these cell lines were compared with a human non-leukaemic cell line with respect to expression of SLUG Examination of the mRNA for expression of SLUG by reverse transcriptase PCR showed that Slug is present in all these cell lines positive for Ph1 (FIG.", "3, bands 9-11).", "The discovery that expression of SLUG is present in cells after expression of transformation BCR-ABL, along with the discovery that the Slug gene is present in cell lines and in bone marrow cells isolated from patients with leukemia positive for BCR-ABL, suggests that the presence of the Slug gene could be a component in the invasion of leukemias positive for BCR-ABL.", "Slug is Present in Cell Lines of Leukemia Patients with Other Chromosomal Abnormalities As SLUG is a member of the Snail family of proteins implicated in the formation of the mesoderm (Nieto et al., 1994, cited above) and its expression is somewhat uncontrolled (FIG.", "2), the expression of the Slug gene was then analyzed in other mesenchymal tumors.", "FIG.", "3 shows examples of RNA from a further four leukemia cell lines that lack t(9; 22), including the lineage B primitive 697 (FIG.", "3, band 5), the myeloid U937 (FIG.", "3, band 3) and KOPTI-KI (FIG.", "2, band 4).", "As shown in FIG.", "3, in all leukemia cell lines, expression of the Slug gene was shown.", "In this sense, recent discoveries show that SLUG is also expressed in t(17; 19) leukemia cells (Inukai et al., Molecular Cell 4: 343-352 (1999)) and in radbomyosarcoma cells that express the PAX3-FKHR translocation (Khan et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 96: 13264-13269 (1999)).", "Thus, these results combined indicate that expression of Slug is not rare in mesenchymal tumors (both leukemia or sarcoma) transformed under the auspices of other genetic alterations and suggest that the Slug gene could be a component of the biology of cancer, not only in leukemias positive for BCR-ABL, but possibly also in other mesenchymal cancers.", "Antiapoptotic Activity of SLUG In order to understand better the function of SLUG in the biology of cancer, the Ba/F3 cell lines transduced with vectors that code for the BCR-ABL fusion protein were also used (Sánchez-García and Grütz, 1995, cited above).", "The expression of BCR-ABL in Ba/F3 confers a growth independent of cytokines and blocks apoptosis (Daley and Baltimore, 1988; Sánchez-García and Grütz, 1995, cited above).", "Thus, the inventors were asked whether SLUG could perform these functions in the absence of the oncoprotein.", "Therefore, Ba/F3 cells were transfected with a vector that expressed Slug (FIG.", "4A) and the cell viability was analyzed in absence of IL-3.As shown in FIG.", "4, the Ba/F3 cells that expressed the BCR-ABL oncogenes survived with little loss of viability and those that expressed Slug did not undergo apoptosis after elimination of IL-3 from the growth medium (FIGS.", "4B, 4C).", "This observation confirms the previous results (Inukai et al., 1999, cited above) and indicates that Slug blocks apoptosis in the test cell system.", "Thus, its deregulation by BCR-ABL in Ph+ cells probably contributes to the properties of prolonged survival of the t(9; 22) cells.", "The Slug gene could replace BCR-ABL in the promotion of the survival of murine Ba/F3 cells deprived of growth factor, which suggests that it is more probable that it is implicated in the resistance to cell death characteristic of Ph+ leukemias and a key stage towards the development of leukemias positive for t(9; 22).", "Suppression of the Slug Gene Compromises the Tumorigenicity of BCR-ABL Oncogenes Affecting the Capacity for Dissemination of BCR-ABL Leukemia Cells The previous results show that expression of BCR-ABL protects the cells from apoptosis, inducing the Slug gene.", "In this case, it would be expected that some modification would be produced in the growth and tumorigenicity of the Ba/F3 cells that express BCR-ABL when the level of expression of Slug is altered.", "Ba/F3 cells that expressed BCR-ABL were transfected with a vector that expressed cDNA of antisense mouse Slugh (BOS-antiSlug) and clones were established (FIG.", "5A).", "The RNA was extracted and the levels of mRNA of Slugh were compared with those present in the untransfected control cells (FIG.", "5B).", "In cells transfected with BOS-antiSlug, antisense Slugh was detected and in the Ba/F3 cells that expressed BCR-ABL transfected with BOS-antiSlug the level of BCR-ABL was not affected (FIG.", "5A).", "On the other hand, SLUG mRNA was not detected in cells transfected with antisense Slugh, although it was present in Ba/F3-p190 and Ba/F3-p210 cells (FIG.", "5B).", "The consequences of the suppression of Slugh expression were evaluated by examining the differentiation program imposed by BCR-ABL oncogenes in Ba/F3 cells and the in vivo tumorigenicity of the different cell lines by injection into naked mice.", "In order to determine whether Slug expression is a critical component per se in the differentiation program induced by the BCR-ABL oncogenes, the inventors then measured the capacity to differentiate haematopoietic precursors that expressed BCR-ABL transfected in those in which expression of Slug had been specifically suppressed.", "The effects of the suppression of Slugh expression on cell differentiation in Ba/F3 cells that expressed BCR-ABL were evaluated by analysing the expression of specific haematopoietic markers in the different cell lines.", "As shown in FIG.", "5C, the initial Ba/F3 cells that express BCR-ABL-p190 or BCR-ABL-p210 oncogenes are specifically differentiated in myeloid and lymphoid cells, as defined by the presence of the myeloid marker Gr-1 or the lymphoid marker B220, respectively.", "By analogy, differentiation of Ba/F3 cells that expressed BCR-ABL in those that expression of Slug had been specifically suppressed was not affected (FIG.", "5C).", "These results show that the effect of the BCR-ABL oncogenes on the differentiation does not depend on the expression of Slug.", "Later, the present investigators examined the in vivo tumorigenic capacity of the different cell lines.", "The initial Ba/F3 cells that expressed the BCR-ABL oncogenes grew as tumors in naked mice (Sánchez-García and Grütz, 1995, cited earlier).", "By analogy, it was described that the formation of tumors was observed just five days after injection of the cell lines that expressed BCR-ABL.", "In contrast to these controls, the Ba/F3 cells that expressed BCR-ABL and antisense Slugh were much less tumorigenic (FIG.", "6).", "The tumors were cleaved to perform the macroscopic analysis on day 20 after injection of the cells.", "On average, the weight of the tumors found in mice that had been injected with Ba/F3 cells that expressed BCR-ABL was twice as large as the weight of tumors of mice that had been injected with cells that expressed BCR-ABL and the antisense Slugh construct (FIG.", "6).", "During the development of the in vivo invasive tumors, the tumor cells disobeyed the social order of the boundaries of the organs and penetrated foreign tissues.", "Thus, later, a histological examination was performed of the BCR-ABL tumors generated in presence or absence of Slug.", "In the tumors originating as a result of Ba/F3 cells that expressed BCR-ABL in which the expression of Slugh had been suppressed, the cells accumulated, but the proliferation was systematically limited to the permissive medium (FIG.", "7).", "In contrast, in tumors generated as a result of Ba/F3 cells that expressed BCR-ABL in which the expression of Slug had been modified, the previously established limits of the permissive medium extended and, thus, the tumor progressed.", "As shown in FIG.", "7, Slug clearly confers a metastatic behavior on the BCR-ABL cells, allowing the migration of tumor cells as individual cells.", "Similar results were found in multiple sections of tumors and for the two forms (p190 and p210) of the BCR-ABL oncogene.", "Thus, these results show that the Slug gene plays a fundamental role in the transition of a tumor from in situ to invasive, allowing the migration of tumor cells as individual cells.", "III.", "Discussion SLUG Regulates the Invasive Capacity of BCR-ABL Cells: Consequences for the Pathogenesis of Mesenchymal Tumors In leukeamogenesis, the future tumor cells are primitive parent cells in which differentiation is blocked (Cobaleda et al., 2000; Sánchez-García, 2000, cited earlier), but the inhibition of the differentiation is not sufficient for transformation because the survival and proliferation of the target cells is restricted to a particular micro-environment.", "Thus, the transformation must depend on genetic changes that allow the cells to develop outside their normal environment in addition to mutations that block differentiation.", "In order to validate this hypothesis, it was attempted to identify the target downstream of the genes BCR-ABL fused by t(9; 22) (q34; q11) in the haematopoietic cells.", "Identification of the gene products downstream that confer the neoplasic nature is prerequisite for full understanding of the mechanism of leukemogenesis.", "In the present study, the Slug gene has been identified as a downstream target gene implicated in the leukemogenic process induced by the chimeric BCR-ABL fusion gene.", "The studies of the present investigators do not establish whether the Slug promoter is a direct target of BCR-ABL or whether it is more likely that this gene is implicated as an element in certain biochemical cascades that respond to modulation by the chimeric protein.", "The discovery that expression of Slug is present in cells after expression of transforming BCR-ABL, along with the discovery that Slug is present in cell lines and in bone marrow cells isolated from patients with leukemias positive for BCR-ABL, indicate that the presence of Slug has to be an important component of the malignant nature of the leukemias positive with respect to BCR-ABL.", "This suggests the possibility the Slug can contribute to blocking differentiation or can be responsible for the disseminative capacity of this disease.", "Other discoveries of the inventors define the biological function of Slug in the context of cell transformation dependent on BCR-ABL oncogenes.", "The data presented here show that Slug, which is activated by expression of BCR-ABL oncogenes, mediates the prevention of apoptosis in the Ba/F3 system.", "In addition, a modification of the growth and tumorigenicity of the cells that express BCR-ABL is produced, altering the level of expression of Slug.", "In in vivo tumors originating as a result of BCR-ABL cells in which expression of Slug had been eliminated previously, the cells accumulated, but the proliferation was limited systematically to the permissive environment.", "In contrast, in tumors generated as a result of BCR-ABL cells in which expression of Slug had not been previously modified, the previous boundaries of the permissive environment spread and, as a result, the tumor progresses, disobeying the order of boundaries and infiltrating foreign tissue.", "Thus, Slug clearly confers metastatic behavior on the BCR-ABL cells in transition from in situ to invasive, allowing the migration of tumor cells as individual cells.", "The discoveries of the inventors are consistent with a model in which the parent cells that carry the fusion protein BCR-ABL would constitutively express Slug, promoting both the aberrant survival of the tumoral target cell, regardless of the external signals required, which allows the cell to grow outside its normal environment, and migration of defective target cells into different environments (FIG.", "8).", "However, an increase in expression of Slug cannot be the only event implicated in the transformation by BCR-ABL oncogenes.", "The reason is as follows: although the expression of constitutive Slug replaces the signals of survival induced through growth receptors and allows the cells to grow outside their normal micro-environment, the results of the present investigators show that the effect of the BCR-ABL oncogenes on differentiation does not depend on the expression of Slug.", "Thus, other components are required in addition to Slug in order to reconstitute the BCR-ABL transformation signal.", "The inventors have shown that the BCR-ABL oncogenes are sufficient in their own right to induce the expression of Slug, and that Slug is required for the biological activity of these oncogenes in cell invasion.", "As Slug is a protein implicated in the formation of the mesoderm (Nieto et al., 1994, cited above; Savagner et al., J.", "Cell Biology 137: 1403-1491 (1997)) and its expression is fairly uncontrolled (FIG.", "2), it may have a more general role in the biology of the cancer than that associated specifically with transformation of BCR-ABL.", "Thus, expression of Slug could serve as a mechanism of cell invasion for fusion proteins found in other leukemias and proteins associated with sarcomas.", "In fact, expression of Slug is not rare in mesenchymal tumors (both leukemia or sarcoma) transformed under the auspices of other genetic alterations (Inukai et al., 1999; Khan et al., 1999, cited earlier), which suggests that Slug can be a component of the tumoral invasion, not only of leukemias positive for BCR-ABL, but also possibly of other mesenchymal cancers.", "Therefore, Slug appears to be a mechanism of tumoral invasion, both for leukemias and for sarcomas.", "In conclusion, with the present work, a mechanism has been established responsible for the invasive capacity of BCR-ABL and indications have been presented that suggest that Slug could represent a potentially broad tumoral invasion mechanism for mesenchymal tumors.", "Therefore, Slug itself could constitute an attractive target for treatment (it could be considered a marker of malignity) and therapeutic modulation of the invasive capacity in the treatment of human cancer.", "Metastasis and Tumorigenicity may have the same Genetic Control The leukaemic conversion of the tumoral target cells (Cobaleda et al., 2000; Sánchez-García, 2000, cited earlier) to cells with an autonomous growth state implies that certain specific genes have to be activated in order to decouple control of proliferation/differentiation and generate intracellular signals that can replace the requirements of the growth factors during invasion and cell dissemination.", "During the development of invasive tumors, the tumor cells disobey the social order of boundaries of organs and infiltrate foreign tissues.", "Only during transition of a tumor in situ to an invasive tumor, the tumor cells penetrate in the basal epithelial membrane and enter the underlying interstitial stroma, interacting with the stromatic cells.", "Thus, a definition of behavior of the metastatic tumor cells is the tendency to cross tissue compartment boundaries and intermix with different types of cell.", "The inventors have shown that BCR-ABL cells behave like metastatic tumor cells.", "It is assumed that blocking the differentiation is a consequence of the alteration of the properties of the chimeric fusion proteins (Sánchez-García, Annu.", "Rev.", "Genetics 31: 429-453 (1997)), BCR-ABL in the present case.", "It has been suspected that genes other than the oncogenes can produce metastasis that have been shown to be relevant for tumorigenesis.", "However, the results show clearly that transfection of BCR-ABL oncogenes into an appropriate receptor cell can induce the complete phenotype of tumorigenicity and invasion.", "These findings show that a fusion protein imposes an altered differentiation program on the target cell and a specific inductor agent of invasion that is required for the tumor cell to be transformed into an invasive cell.", "The model of the inventors also shows that the effecter genes of the invasion can be regulated independently of those that confer tumorigenicity by the same fusion gene, which represents a new oncogenic mechanism of action.", "Taking these data together, the idea is reinforced that transformation can be produced as a result of the creation/activation of a single oncogene (Sánchez-García, 1997, cited earlier).", "An interesting topic for future investigation will be identification of the factors that modulate expression of Slug and the possible implication of other oncoproteins that induce its expression.", "Slug Associates Invasion and Tumoral Development SLUG is a member of the Snail family of transcription factors with “zinc fingers” that play a conserved role from the evolutionary point of view in the formation of the mesoderm in invertebrates and vertebrates (Nieto et al., 1994, cited earlier).", "In chicken, Slug is expressed by ectodermal epithelial cells during their transition to mesenchymal cells.", "The chicken embryos treated with anti-sense oligonucleotide directed against Slug show inappropriate formation of the mesoderm related to defects in cellular migration in compartments of transition from epithelial cells to mesenchymal cells (Nieto et al., 1994, cited earlier).", "Thus, Slug induces cell migration in the epithelial-mesenchymal transition in the formation of the mesoderm and in the migration of cells from the neural crest (Fuse et al., Genes & Development 8, 2270-2281 (1994)).", "However, in mice, activation of the epithelial-mesenchymal transitions is under the control of Snail (Cano et al., Nature Cell Biology 2;: 76-83 (2000)), which allows migration from the primitive mesoderm in the form of epithelial cells.", "As a result, in mice without Slug, they develop normally (Jiang et al., Development Biology 18: 277-285 (1998)).", "In addition, it has been shown that the gene Snail activates the EMT associated with the acquisition of the invasive phenotype in solid epithelial tumors (Batlle et al., Nature Cell Biology 2: 84-89 (2000); Cano et al., 2000, cited earlier), where it contributes to the first event of the metastatic process.", "Thus, induction of EMT appears to be a function associated specifically with the Snail gene in mouse (Cano et al., 2000, cited earlier).", "The data presented here show that Slug is an important regulator of the capacity of invasion during the progression of mesenchymal tumors in which the EMT are not required.", "This idea could be extended to the role of Slug in the acquisition on the part of parent mesenchymal cells of the capacity to migrate.", "In support of this theory, the experiments of the investigators indicate that, in vivo, Slug is not expressed in the peripheral blood of BCR-ABL transgenic mice with leukemia, defining an undifferentiated, pluripotent and migratory phenotype in the mesenchymal cells.", "Thus, it is conceivable that the presence of Slug is required in certain phases of normal development for an appropriate expansion and for the survival of the primitive haematopoietic cells (FIG.", "8).", "At the same time, the results provide a molecular association between a regulator of the initial stages of development and the leukaemiogenesis, and offer clues for understanding the molecular alterations that lead to the invasive behavior of leukemias, in particular, and mesenchymal tumors, in general." ] ]	Patent_10466817
[ [ "Audit system and method", "The invention provides a system and method for reducing and potentially eliminating the review of source documents by auditors to determine whether there is compliance of an audited subject area with a predetermined set of rules.", "The inventive system and method provides one or more questions directed to personnel that are familiar with the subject area being audited.", "It may be accompanied by cross-checking question and/or verification of the responses.", "The responses and cross-checks or verifications, if any, are compared for consistency and rule compliance.", "Where non-compliance is determined to exist, or where there is a question whether non-compliance exists, an audit alert is generated.", "This may be followed by evaluating the audit alert through a review of source documents, thereby eliminating the need for a review of source documents unless an audit alert is determined to exist, and limiting the review of source documents to documents relevant to the audit alert." ], [ "1) A method for auditing a subject area, the audit being adapted to ascertain whether the audited subject area is being administered in compliance with a set of rules without the need to review source documents, the methods comprising the steps of: a) identifying an element of the set of rules that is relevant to the subject area to be audited; b) creating a first question designed to elicit an answer relevant to the subject area to be audited; c) obtaining a response to the first question from a first person; d) obtaining a verification of the response to the first question from a second person; e) flagging an audit alert if the response to the first question reveals that the audited subject area is not being administered in compliance with the set of rules; f) flagging the audit alert if the verification of the response to the first question is negative; and g) if the audit alert has been flagged, providing notice of the flagged audit alert.", "2) The method claimed in claim 1, wherein the step of obtaining a response to the first question comprises: a) electronically delivering the first question to the first person; and b) electronically receiving the response to the first question from the first person.", "3) A method for auditing a subject area, the audit being adapted to provide notification of an audit alert if audited subject area is not being administered in compliance with a set of rules, the audit being conducted without review source documents, the methods comprising the steps of: a) identifying an element of the set of rules that is relevant to the subject area to be audited; b) creating a first multiple-choice question having a first response; c) creating a second multiple-choice question having a second response, the second response being adapted such that the selection of the second response to the second multiple-choice question and the selection of the first response to the first multiple-choice question would reveal that the audited subject area is not being administered in compliance with the set of rules; d) obtaining a response to the first question; e) obtaining a response to the second question; f) providing notification of an audit alert if the response obtained to the first question is the first response and the response obtained to the second question is the second response.", "4) The method claimed in claim 3, wherein the step of obtaining a response to the first question comprises: a) electronically delivering the first question to a first person; and b) electronically receiving the response to a first question from the first person.", "5) The method claimed in claim 4, wherein the step of obtaining a response to the second question comprises: a) electronically delivering the second question to the first person; and b) electronically receiving the response to the second question from the first person.", "6) The method claimed in claim 4, wherein the step of obtaining a response to the second question comprises: a) electronically delivering the second question to a second person; and b) electronically receiving the response to the second question from a second person.", "7) A method of performing an audit to determine non-compliance with a set of rules without the need for review of source documents, the method comprising the steps of: a) creating a first and a second audit question; b) determining a logical relationship between at least one response to the first audit question and at least one response to the second audit question, the logical relationship being adapted to reflect the existence of non-compliance with the set of rules; c) obtaining responses to each of the first and second audit questions; d) determining whether an audit alert exists due to the satisfaction of the logical relationship between the at least one response to the first audit question and the at least one response to the second audit question; and e) providing a report of the audit alert determined to exist.", "8) The method claimed in claim 7, wherein the step of obtaining a response to each of the first and second audit questions comprises the steps of: a) electronically delivering the first question to a first person; b) obtaining a response to the first question from the first person; c) electronically delivering the second question to a second person; and d) obtaining a response from the second person to the second question.", "9) The method claimed in claim 7, wherein the step of obtaining a response to each of the first and second audit questions comprises the steps of: a) electronically delivering the first question to a first person; b) obtaining a response to the first question from the first person; c) electronically delivering the second question to the first person; and d) obtaining a response to the second question from the first person.", "10) A method of performing an audit to determine non-compliance with a set of rules without the need for review of source documents, the method comprising the steps of: a) creating a first and a second audit question; b) determining a logical relationship between at least one response to the first audit question and at least one response to the second audit question, the logical relationship being adapted to reflect the existence of an internal inconsistency between one of the at least one response to the first audit question and one of the at least one response to the second audit question; c) obtaining responses to each of the first and second audit questions; d) generating an audit alert in the event that the responses to the first and second audit questions reflect the existence of an internal inconsistency; and e) providing a report of the generated audit alert.", "11) The method claimed in claim 10, wherein the step of obtaining a response to each of the first and second audit questions comprises the steps of: a) electronically delivering the first question to a first person; b) obtaining a response to the first question from the first person; c) electronically delivering the second question to a second person; and d) obtaining a response to the second question from the second person.", "12) The method claimed in claim 10, wherein the step of obtaining a response to each of the first and second audit questions comprises the steps of: a) electronically delivering the first question to a first person; b) obtaining a response to the first question from the first person; c) electronically delivering the second question to the first person; and d) obtaining a response to the second question from the first person.", "13) A method of performing an audit to determine non-compliance with a set of rules without the need for review of source documents, the method comprising the steps of: a) creating an audit question; b) determining at least one response to the audit question that reflects the existence of an audit alert; c) obtaining a response to the audit question; and d) comparing the at least one response determined in step b to the response to the audit question obtained in step c, and providing a notification of an audit alert if one of the at least one response determined in step b is the same as the response to the audit question obtained in step c. 14) The method claimed in claim 13, wherein the step of obtaining an answer to the audit question comprises: a) electronically delivering the audit question to a first person; b) obtaining a response to the audit question from the first person.", "15) A method of performing an audit to determine non-compliance with a set of rules the method comprising the steps of: a) creating a first and a second audit question; b) determining a logical relationship between a first audit question and a second audit question, the logical relationship being adapted to reflect the existence of non-compliance with the set of rules; c) obtaining a response to each of the first and second audit questions; d) determining whether an audit alert exists due to the satisfaction of the logical relationship between at least the first answer and the second answer; e) providing a report of the audit alert determined to exist; and f) evaluating the audit alert by a review of source documents, thereby eliminating the need for a review of source documents unless an audit alert is determined to exist, and limiting the review of source documents to documents relevant to the audit alert." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>In 1974, Congress enacted a law, the Employee Retirement Income Security Act (ERISA) which contains fiduciary rules, employee protection provisions and amendments to the Internal Revenue Code (IRC) that require employee benefit plan sponsors (employers or trustees in the case of multi-employer plans) to operate employee benefit plans in accordance with their terms and in accordance with the law.", "Failure to do so can result in personal liability through litigation or governmental action or the imposition of monetary sanctions on plan sponsors in the case of operational or plan document violations-relating to retirement plans.", "In order to avoid liability, the IRS and Department of Labor (“DOL”) have established programs that require plan sponsors to establish a self-audit compliance process that identifies and corrects operational and plan document violations prior to an audit by IRS or DOL.", "The evaluation of compliance with ERISA and the IRC is accomplished through an investigation of documents and personnel records that normally involve a review of: (i) employee benefit plans, trusts, summary plan description brochures, administrative manuals, employee communications and other related documents; (ii) annual financial returns filed on behalf of employee benefit plans; (iii) personnel records which reflect the extent of compliance with procedures relating to employee enrollment, participation, vesting, change in employment status, contributions and benefit accrual, joint and survivor payment and notice requirements for married employees, proper calculation and payment of benefits and a myriad of other legal and regulatory requirements; and (iv) compliance with IRS requirements that prohibit discriminations in favor of highly compensated employees with respect to contributions and/or benefits provided by the employee benefit plan.", "This review is primarily conducted on-site at the location of the documents and personnel records across the country.", "The accomplishment of such a review of operational and plan document compliance is a Herculean task that involves a myriad of professional disciplines including legal, accounting/auditing, actuarial/consulting, plan administration, investment management, communications, and other services.", "It is also very costly and time-consuming.", "This is further exacerbated by the expense and administrative burden of an on-site review.", "As a result, plan sponsor employers and trustees of multi-employer plans are reluctant to perform such a compliance review even though it is required by ERISA and IRS policy and procedures.", "There is a need for a system and method that identifies areas of noncompliance on a cost-efficient basis." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Accordingly, it is an object of the present invention to provide an audit system and method that mitigates the need for review of source documents and personnel records.", "It is another object of the present invention to provide a system and method for the analysis of responses to questions in order to ascertain whether a plan sponsor has adequate procedures in place to comply with the requirements of the law, regulations or other requirements.", "It is yet another object of the invention to permit the identification of operational and plan document noncompliance without the need for an on-site document review and audit.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "CROSS REFERENCE TO RELATED APPLICATION This application claims priority from U.S.", "Provisional Application No.", "60/240,215, which is herein incorporated by reference in its entirety.", "FIELD OF THE INVENTION The present invention relates to an audit system and method, and more particularly to a system and method for performing an audit without the need for review or spot-check of the source documents.", "BACKGROUND OF THE INVENTION In 1974, Congress enacted a law, the Employee Retirement Income Security Act (ERISA) which contains fiduciary rules, employee protection provisions and amendments to the Internal Revenue Code (IRC) that require employee benefit plan sponsors (employers or trustees in the case of multi-employer plans) to operate employee benefit plans in accordance with their terms and in accordance with the law.", "Failure to do so can result in personal liability through litigation or governmental action or the imposition of monetary sanctions on plan sponsors in the case of operational or plan document violations-relating to retirement plans.", "In order to avoid liability, the IRS and Department of Labor (“DOL”) have established programs that require plan sponsors to establish a self-audit compliance process that identifies and corrects operational and plan document violations prior to an audit by IRS or DOL.", "The evaluation of compliance with ERISA and the IRC is accomplished through an investigation of documents and personnel records that normally involve a review of: (i) employee benefit plans, trusts, summary plan description brochures, administrative manuals, employee communications and other related documents; (ii) annual financial returns filed on behalf of employee benefit plans; (iii) personnel records which reflect the extent of compliance with procedures relating to employee enrollment, participation, vesting, change in employment status, contributions and benefit accrual, joint and survivor payment and notice requirements for married employees, proper calculation and payment of benefits and a myriad of other legal and regulatory requirements; and (iv) compliance with IRS requirements that prohibit discriminations in favor of highly compensated employees with respect to contributions and/or benefits provided by the employee benefit plan.", "This review is primarily conducted on-site at the location of the documents and personnel records across the country.", "The accomplishment of such a review of operational and plan document compliance is a Herculean task that involves a myriad of professional disciplines including legal, accounting/auditing, actuarial/consulting, plan administration, investment management, communications, and other services.", "It is also very costly and time-consuming.", "This is further exacerbated by the expense and administrative burden of an on-site review.", "As a result, plan sponsor employers and trustees of multi-employer plans are reluctant to perform such a compliance review even though it is required by ERISA and IRS policy and procedures.", "There is a need for a system and method that identifies areas of noncompliance on a cost-efficient basis.", "SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide an audit system and method that mitigates the need for review of source documents and personnel records.", "It is another object of the present invention to provide a system and method for the analysis of responses to questions in order to ascertain whether a plan sponsor has adequate procedures in place to comply with the requirements of the law, regulations or other requirements.", "It is yet another object of the invention to permit the identification of operational and plan document noncompliance without the need for an on-site document review and audit.", "DESCRIPTION OF PREFERRED EMBODIMENTS The present invention is a system and method for providing audit capability without the need to review source documents.", "In one embodiment, the system and method may be applied to ERISA compliance, and used to: (i) assist plan sponsors (employers and, in the case of multi-employer plans, trustees) and prototype plan sponsors (a business entity that provides employee benefit plan documents to employers and trustees who use their plan administration and/or investment management services) in monitoring the operation of their employee benefit plans (employer-provided benefits including retirement, health, disability, dependent care and cafeteria plans) in a manner that is consistent with the requirements of the Employee Retirement Income Security Act of 1974 (ERISA) and the Internal Revenue Code (IRC), (ii) identify areas of noncompliance with ERISA and the IRC and (iii) be cost-efficient by eliminating the need for review of most source documents in an on-site investigation.", "In one embodiment, the inventive system and method consists of three main parts: (i) an investigative audit questionnaire which is designed to ascertain whether the audited subject area is administered in compliance with a set of rules; (ii) a comparison of the responses to the questionnaire with the plan documents to ascertain whether there are any apparent areas of noncompliance; and (iii) the preparation of an audit report which describes any areas of noncompliance that are identified by the audit.", "In a preferred embodiment, the inventive system and method may consist of: (i) an investigative questionnaire designed to ascertain how an ERISA plan is administered through a series of easy to respond to questions on each aspect of plan administration and legal compliance; (ii) an analysis of the completed questionnaire and comparison to the plan documents to ascertain whether the operation and administration of the plan is being carried out in conformity with the plan documents; and (iii) the preparation of an annual fiduciary audit report which describes any areas of noncompliance that are identified by the analysis.", "The questionnaire may be delivered in a traditional paper format, or through a computer based system technology.", "In a preferred embodiment, the entire audit, including the presentation of a questionnaire, the gathering of responses to the questionnaire that reflect the administration of the plan in comparison with the plan documents, the analysis of the responses, and the preparation of a non-compliance report, may be conducted without on-site review of most source documents.", "Exemplary ERISA Audit In an exemplary embodiment of the present invention, a questionnaire is provided that contains a series of questions, the questions being based on each aspect of an ERISA plan document and its administration and legal compliance.", "The questionnaire is completed by the target of the audit and its agents.", "Many of the questions may be responded to by the plan administrator or other professional advisor, although some questions should be completed only by the employer or trustee plan sponsor.", "Any particular question from the questionnaire should be responded to by personnel having first hand knowledge of the correct response to the question, and preferably, by the person with the most-knowledge with respect to the subject matter of the question.", "In a preferred embodiment, the employer/trustee will be required to verify the accuracy of at least some of the responses of others, such as, for example, the plan administrator.", "In one embodiment, verification may be obtained by placing a checkmark in a box that is placed next to each question.", "For example, the plan administrator is asked to complete the question: “How many hours of service are required to receive a year of vesting credit?” The employer/trustee is then required to review the plan administrator's response to that question and verify that the response is correct in light of how the employer/trustee is administering the plan by placing a checkmark in the box located directly below the question.", "Alternatively, in another embodiment, two different people—such as, for example, the plan administrator and the employer/trustee—may be asked the same question, and the verification results from consistency between the two responses.", "It is also contemplated that, within a single audit, for the questions that are verified, some questions will be verified by a review and check-mark procedure, while other questions may be verified by asking the same question to two different people and comparing the responses.", "Although any question may be asked, in a preferred questionnaire, most questions require only that the respondent check a “yes” or “no” box or elect one or more of the available responses.", "Thus, a response to a given question may preferably be provided by checking one or more boxes provided for the response to that question.", "Some questions may require a short written response, e.g., 1,000 hours or age 21.Some questions may require a descriptive response, particularly if a response to a question is in the nature of a selection of “Other” for a multiple choice type question.", "In the latter case, a longer descriptive answer is usually required to be provided by the respondent.", "The responses to the questionnaire are used to identify areas of noncompliance, if any, using a variety of methods to verify the accuracy of the response (as described above and further described below) and to verify compliance or noncompliance with a plan, as further described below.", "As will be apparent to a person of ordinary skill in the art, the questionnaire is preferably designed in such a way as to permit ready identification of the areas of noncompliance.", "One method of determining areas of noncompliance comprises identifying responses that are not verified.", "This could result where the employer/trustee is unable to verify that the plan administrator's response to a question is correct.", "Similarly, it could result from the employer/trustee and the plan administrator providing different responses to the same question.", "See item 3A below which illustrates an exemplary employer/trustee verification process.", "A second method of determining areas of noncompliance comprises the comparison of the response to one question with the response to another question.", "In this situation, various questions are tied (i.e., related) to one or more other questions so that a response to one question can be verified by the response to one or more other questions.", "If responses to such related questions are inconsistent, the inconsistencies are identified as possible areas of noncompliance.", "As an example, the question may be provided: “Does the plan require a specific number of hours an employee must complete before becoming a participant?” If the plan administrator responds “yes”, the number of hours must also be provided.", "Another question may ask for the same information in a different manner.", "That question provides “How many hours of service are required to become a participant?” The plan administrator must provide a numerical response.", "If, for example, the plan administrator indicates that the plan does not have control procedures in place to determine the number of hours of service earned by a participant, or the plan administrator describes a procedure that is inadequate to determine a participant's hours of service, an area of noncompliance may be identified.", "In other words, if the response to the first question is not consistent with the response to the second question, an area of noncompliance may be identified.", "A third method of determining areas of noncompliance comprises flagging of responses such as “other”, “none” or a noncompliant response.", "In other words, many of the questions may provide a response which allows the respondent to choose a response that would be, at least initially, identified as an area of noncompliance.", "For example, where the respondent selects “Other” because none of the available options contained in the questionnaire adequately describe plan administration, the “other” response may be treated initially as an area of noncompliance until it is analyzed and evaluated by a reviewer.", "An evaluation may be necessary because the “other” selection is preferably accompanied by a written response that may show compliance.", "Similarly, where a “none” selection is made that evidences that there are no plan provisions or procedures with respect to the compliance area illustrated by the question, this would preferably be treated as an area of noncompliance, at least until it is further evaluated.", "Moreover, some questions may provide a response which is known as a noncompliant response.", "In such a case, the selection of that response may be treated as an area of noncompliance.", "To illustrate, a question may provide: “Has the plan established procedures to determine when employees have met the Plan's participation and vesting requirements?", "[ ] Yes, payroll records are reviewed periodically in accordance with the plan's entry dates to verify a participant's hours of service.", "[ ] Other (Description of the plan's procedures) ______.", "[ ] No, there are no procedures for determining when an employee has met the participation requirements.", "If the “No” response is selected, an area of noncompliance will be identified.", "If the “Other” response is selected, an area of noncompliance will be identified and the descriptive response will be evaluated to determine if compliance is adequate.", "Reference to item 3C below may be had, which illustrates some circumstances under which a response will be evaluated.", "In addition to the above-described methodology contained in the questionnaire, the inventive method and apparatus will enable the performance of other tasks that may make it easier to identify areas of noncompliance.", "For example: 1.It will be possible to identify questions that frequently generate areas of noncompliance.", "2.It will also be possible to identify questions that frequently generate an area of noncompliance in the audit report but upon further evaluation are determined not to be an area of noncompliance.", "3.It will also enable the comparison of responses contained in the questionnaire with the basic terms of the plan document, other pertinent information contained in employee communications and annual returns filed on behalf of the plan by creating questions that will elicit such information for each plan, or making an entry into an automated system for implementing the inventive method that reflects such information.", "In a preferred, computerized implementation of the inventive method, the automated system would have the capacity to perform items 1 and 2 above on a system-wide basis or on behalf of a subset of questionnaires, such as for each prototype plan sponsor.", "Illustrations of the Process A. Employer/Trustee Verification of Plan Administrator's Response If the employer/trustee indicates that the plan's administrative procedure is different from the plan administrator's response by checking the “Not Correct” box, an area of noncompliance will be identified.", "For example: Question: How many hours of service are required to receive a year of vesting credit?", "______ Employer/Trustee verification: □ Correct □ Not Correct B.", "Consistent Response Required Between Related Questions The following two questions are related since both questions relate to procedures for identifying rehired employees who are eligible to participate in the plan.", "The responses to both questions must be consistent.", "For example: Question: Has the plan established procedures to ascertain whether new or rehired employees who have met the plan's eligibility requirements are included in the plan on a timely basis (e.g., comparison of payroll data to list of new participants; verification of hours through payroll records)?", "Yes □ No □ is preferably related to: Question: Has the plan established procedures to determine whether the employee has had prior employment with an affiliated employer (“affiliated employment”) or previously terminated under the plan (e.g., review by the Plan Administrator of personnel records which are transferred when an employee is transferred to another affiliate)?", "Yes □ No □ The question below is related to the next eight questions since all of the questions relate to the plan's age and service requirements for either becoming a participant or receiving vesting credit.", "Responses to all of these questions must be consistent.", "Any inconsistency will result in an area of noncompliance being identified.", "For example: Question: Has the plan established procedures to determine when employees have met the plan's participation and vesting requirements (e.g., verification of hours of service and age requirement through review of payroll records)?", "Yes □ No □ is related to all of the following questions: Question: Does the plan require a specific number of hours an employee must complete before becoming a participant?", "Yes □ No □ If so, how many?", "□□□□□ Question: Is a record of hours worked by employees maintained to establish membership?", "Yes □ No □ Question: Are participants credited for periods of time during which no duties are performed and for which the employee is paid (e.g., vacation, holiday, illness, disability, jury duty, military duty or leave of absence) in order to satisfy the hours of service requirements?", "Yes □ No □ Question: From what age is an employee's service counted to: a. become a participant □□.□□ b. receive a year of vesting credit □□.□□ Question: How many months of service are required to: a. become a participant □□.□□ b. receive a year of vesting credit □□.□□ Question: How many hours of service are required to: a. become a participant □□.□□ b. receive a year of vesting credit □□.□□ Question: For participation and vesting purposes, is service counted on: □ Plan year basis or □ The anniversary of an employee's initial employment date Question: If eligibility service is counted on a plan year basis rather than from the anniversary date of an employee's initial employment date, is an employee who meets the plan's eligibility requirements on both his/her anniversary date and the end of the first plan year credited with 2 years of service for purposes of eligibility to participate?", "Yes □ No □ C. Evaluation of Written Response or “No” Response Question: Has the plan established procedures to determine when employees have met the plan's participation and vesting requirements?", "□ Yes, payroll records are reviewed periodically in accordance with the plan's entry dates to verify a participant's hours of service.", "□ Other (Description of plan's procedures) □ No, there are no procedures for determining when an employee has met the participation requirements.", "Employee/Trustee verification: □ Correct □ Incorrect Where an “Other” response is selected, the description provided by the respondent in connection with the “Other” response will be evaluated to determine if compliance is adequate.", "Where “No” is selected, an area of noncompliance will be identified.", "While the foregoing describes and illustrates the preferred embodiment of the present invention and suggests certain modifications thereto, those of ordinary skill in the art will recognize that still further changes and modifications may be made therein without departing from the spirit and scope of the invention.", "Accordingly, the above description should be construed as illustrative and not in a limiting sense, the scope of the invention being defined by the following claims." ] ]	Patent_10466831
[ [ "Method and apparatus for solid or solution phase reaction under ambient or inert conditions", "The present invention generally provides a novel, automation-compatible solid or solution phase reaction vessel, as well as methods for using such a vessel.", "Generally, the reaction vessel comprises a microplate assembly with a modular solid phase included within the individual reaction wells.", "The reaction vessel of the invention allows for the integration of solid phase chemistry with the processing abilities of solution phase chemistry.", "According to the invention, the microplate assembly and the solid phases are configured so as to integrate together into a single reaction vessel.", "The combination enables solid phase reactions in a single vessel with full compatibility to liquid handling automation.", "Further, the combination enables novel methods for performing combination solution phase/solid phase reactions under inert conditions." ], [ "1.A reaction vessel assembly comprising: a microplate having a rigid body with a plurality of open reaction wells disposed therein, each of said open reaction wells comprising a fluid vessel with an opening and an interior volume with a sample-holding space located therein; a funnel cap inserted into each of the open reaction wells for at least partially sealing the open well while allowing for venting of the well through a vent passage; a modular solid phase disposed within the interior volume of each of the open wells such that the modular solid phase does not block the passage of the funnel cap; whereby the reaction vessel is accessible through the funnel cap at all times.", "2.The reaction vessel of claim 1, wherein the solid phase is an exposed polymer surface object comprising a rigid or polymer-containing, unreactive base, wherein the polymer is attached or contained by the base.", "3.The reaction vessel of claim 2, wherein the active polymer attached to the unreactive base is selected from the group consisting of polyethylene, polypropylene, and polytetrafluoroethylene.", "4.The reaction vessel of claim 1, wherein each funnel cap comprises a sealing plug and a vent tube; wherein the sealing plug forms a seal at the mouth of the open wells; and the vent tube forms the vent passage, attaches to the sealing plug and terminates in a vent opening.", "5.The reaction vessel of claim 4, wherein the solid phase is disposed in the lower portion of the reaction well below the vent tube such that the solid phase does not block-the vent opening.", "6.The reaction vessel of claim 4, wherein the solid phase is immobilized in the upper portion of the reaction well above the vent opening such that the solid phase does not block the vent opening.", "7.The reaction vessel of claim 1, wherein the funnel cap is configured so as to substantially prevent the escape of a liquid sample contained within the interior volume of the reaction well.", "8.The reaction vessel of claim 7, wherein the solid phase is immobilized in the lower portion of the reaction well such that the solid phase does not block the vent passage.", "9.The reaction vessel of claim 7, wherein the solid phase is disposed in the upper portion of the reaction well such that the solid phase does not block the vent passage.", "10.The reaction vessel of claim 1, wherein the solid phase is directly disposed on at least a portion of the interior walls of the reaction wells, whereby the solid phase adheres to the at least portion of the interior walls of the reaction wells.", "11.The reaction vessel of claim 10, wherein the solid phase is disposed on a lower portion of the interior walls of the reaction wells relative to the vent passage.", "12.The reaction vessel of claim 10, wherein the solid phase is disposed on an upper portion of the interior walls of the reaction wells relative to the vent passage.", "13.The reaction vessel of claim 10, wherein the solid phase covers the entire surface of the interior walls of the reaction wells below the vent passage.", "14.The reaction vessel of claim 10, wherein the solid phase covers the entire surface of the interior walls of the reaction wells above the vent passage.", "15.The reaction vessel of claim 1, further comprising an upper inert atmosphere cap configured so as to provide a constant positive pressure of inert gas while allowing for access to the reaction wells.", "16.The reaction vessel of claim 15, wherein the solid phase is shaped to press fit into the funnel caps inserted into the open reaction wells.", "17.The reaction vessel of claim 15, wherein the solid phase is directly disposed on at least a portion of the interior walls of the reaction wells, whereby the solid phase adheres to the at least portion of the interior walls of the reaction wells.", "18.The reaction vessel of claim 17, wherein the solid phase is disposed on a lower portion of the interior walls of the reaction wells relative to the vent passage.", "19.The reaction vessel of claim 17, wherein the solid phase is disposed on an upper portion of the interior walls of the reaction wells relative to the vent passage.", "20.The reaction vessel of claim 17, wherein the solid phase covers the entire surface of the interior walls of the reaction wells below the vent passage.", "21.The reaction vessel of claim 17, wherein the solid phase covers the entire surface of the interior walls of the reaction wells above the vent passage.", "22.A reaction vessel assembly comprising: a lower microplate assembly having a rigid body comprising a plurality of open reaction wells disposed therein and a funnel vent associated with each open reaction well for at least partially sealing the open well while allowing for venting of the well; and an upper inert atmosphere cap configured so as to provide a constant positive pressure of inert gas while allowing for access to the open reaction wells.", "23.The reaction vessel of claim 22, wherein the funnel vent is configured so as to substantially prevent the escape of a liquid sample contained within an interior volume of the reaction well.", "24.A reaction vessel assembly comprising: a microplate having a rigid body with a plurality of open reaction wells disposed therein, each of said open reaction wells comprising a fluid vessel with an opening and an interior volume with a sample-holding space located therein and a solid phase disposed within the interior volume of each of the reaction wells, wherein the solid phase is directly disposed on at least a portion of the interior walls of the reaction wells, and whereby the solid phase adheres to the at least portion of the interior walls of the reaction wells.", "25.The reaction vessel of claim 24, wherein the solid phase is disposed on a lower portion of the interior walls of the reaction wells.", "26.The reaction vessel of claim 24, wherein the solid phase is disposed on an upper portion of the interior walls of the reaction wells.", "27.The reaction vessel of claim 24, wherein the solid phase substantially covers the entire surface of the interior walls of the reaction wells.", "28.The reaction vessel of claim 24, further comprising an upper inert atmosphere cap configured so as to provide a constant positive pressure of inert gas while allowing for access to the reaction wells.", "29.A method for performing a combination solution phase/solid phase reaction using the reaction vessel of claim 8 comprising the steps of: (a) inserting solution phase reagents into the interior volume of the reaction well such that the solution phase reagent mixture is in contact with the solid phase; and (b) allowing a solid phase reaction to proceed to form a product attached to the solid phase.", "30.The method of claim 29, further comprising the steps of (c) removing the solution phase mixture from the wells; (d) introducing into the wells a solution capable of cleaving the product of the solid phase reaction from the solid phase and allowing for the cleavage of the product from the solid phase to proceed; and (e) recovering the product cleaved from the solid phase.", "31.A method for performing a catalyzed solution reaction using the reaction vessel of claim 8 comprising the steps of: (a) inserting solution phase reagents into the interior volume of the reaction well such that the solution phase reagent mixture is in contact with the solid phase; and (b) allowing a solution phase reaction catalyzed by the solid phase to proceed to form a product in the solution phase.", "32.A method for performing a solution phase reaction using the reaction vessel of claim 8 comprising the steps of: (a) inserting solution phase reagents into the interior volume of the reaction well such that the solution phase reagent mixture is in contact with the solid phase; and (b) allowing a solution phase reaction to proceed to form a primary product and one or more secondary products; wherein at least one of the secondary products attaches to the solid phase; and (c) separating the solution phase from the solid phase with one or more secondary products attached to the solid phase.", "33.A method for performing a combination solution phase/solid phase reaction using the reaction vessel of claim 11 comprising the steps of: (a) inserting solution phase reagents into the interior volume of the reaction well such that the solution phase reagent mixture is in contact with the solid phase; and (b) allowing a solid phase reaction to proceed to form a product attached to the solid phase.", "34.The method of claim 33, further comprising the steps of (c) removing the solution phase mixture from the wells; (d) introducing into the wells a solution capable of cleaving the product of the solid phase reaction from the solid phase and allowing for the cleavage of the product from the solid phase to proceed; and (e) recovering the product cleaved from the solid phase.", "35.A method for performing a catalyzed solution reaction using the reaction vessel of claim 11 comprising the steps of: (a) inserting solution phase reagents into the interior volume of the reaction well such that the solution phase reagent mixture is in contact with the solid phase; and (b) allowing a solution phase reaction catalyzed by the solid phase to proceed to form a product in the solution phase.", "36.A method for performing a solution phase reaction using the reaction vessel of claim 11 comprising the steps of: (a) inserting solution phase reagents into the interior volume of the reaction well such that the solution phase reagent mixture is in contact with the solid phase; and (b) allowing a solution phase reaction to proceed to form a primary product and one or more secondary products; wherein at least one of the secondary products attaches to the solid phase; and (c) separating the solution phase from the solid phase with one or more secondary products attached to the solid phase.", "37.A method for performing a combination solution phase/solid phase reaction using the reaction vessel of claim 18 comprising the steps of: (a) inserting solution phase reagents into the interior volume of the reaction well such that the solution phase reagent mixture is in contact with the solid phase adhered to the interior walls of the reaction wells; and (b) allowing a solid phase reaction to proceed to form a product attached to the solid phase.", "38.The method of claim 37, further comprising the steps of (c) removing the solution phase mixture from the wells; (d) introducing into the wells a solution capable of cleaving the product of the solid phase reaction from the solid phase and allowing for the cleavage of the product from the solid phase to proceed; and (e) recovering the product cleaved from the solid phase.", "39.A method for performing a catalyzed solution reaction using the reaction vessel of claim 18 comprising the steps of: (a) inserting solution phase reagents into the interior volume of the reaction well such that the solution phase reagent mixture is in contact with the solid phase; and (b) allowing a solution phase reaction catalyzed by the solid phase to proceed to form a product in the solution phase.", "40.A method for performing a solution phase reaction using the reaction vessel of claim 18 comprising the steps of: (a) inserting solution phase reagents into the interior volume of the reaction well such that the solution phase reagent mixture is in contact with the solid phase; and (b) allowing a solution phase reaction to proceed to form a primary product and one or more secondary products; wherein at least one of the secondary products attaches to the solid phase; and (c) separating the solution phase from the solid phase with one or more secondary products attached to the solid phase.", "41.A method for performing a combination solution phase/solid phase reaction using the reaction vessel of claim 9 comprising the steps of: (a) inserting starting material reagents into the interior volume of the reaction well and inverting the reaction vessel such that the reagents contact the solid phase; (b) allowing a solid phase reaction to proceed to thereby form a solid phase product; (c) turning the reaction vessel to an upright position such that the solution phase is no longer in contact with the solid phase; (d) removing the solution phase from the reaction wells and introducing into the reaction wells a cleaving solution capable of separating the solid phase product from the solid phase; (e) inverting the vessel such that the cleaving solution separates the product from the solid phase.", "42.The method of claim 41, further comprising inverting the vessel after the separation of the solid phase product from the solid phase, introducing into the vessel a second reagent solution; and allowing a solution phase reaction to proceed between the solid phase product and the second solution reagent to form a solution phase product.", "43.A method for performing a combination solution phase/solid phase reaction using the reaction vessel of claim 19 comprising the steps of: (a) inserting starting material reagents into the interior volume of the reaction well and inverting the reaction vessel such that the reagents contact the solid phase; (b) allowing a solid phase reaction to proceed to thereby form a solid phase product; (c) turning the reaction vessel to an upright position such that the solution phase is no longer in contact with the solid phase; (d) removing the solution phase from the reaction wells and introducing into the reaction wells a cleaving solution capable of separating the solid phase product from the solid phase; (e) inverting the vessel such that the cleaving solution separates the product from the solid phase.", "44.The method of claim 43, further comprising inverting the vessel after the separation of the solid phase product from the solid phase, introducing into the vessel a second reagent solution; and allowing a solution phase reaction to proceed between the solid phase product and the second solution reagent to form a solution phase product." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Combinatorial chemistry generally relates to a set of techniques for creating a multiplicity of compounds and then testing them for desired activity.", "More specifically, combinatorial chemistry involves the formation of large libraries of molecules en masse, instead of the synthesis of compounds one by one as had been done traditionally.", "Once the libraries are obtained, the most promising lead pharmaceutical compounds are identified by high-throughput screening for further evaluation.", "Generally, combinatorial compounds are created either by solution-phase synthesis or by producing compounds bound covalently to solid phase particles.", "Solid phase synthesis can make multi-step reactions easier to perform and more reliably allows one to drive reactions to completion because excess reagents can be added and then easily washed away after each reaction step.", "Further, solid phase synthesis allows for the use of split synthesis, a technique that produces large support-bound libraries in which each solid phase particle holds a single compound.", "Soluble libraries can then be produced by cleavage of the compounds from the solid support.", "Nonetheless, a much wider range of organic reactions can be available for solution phase synthesis, and products in solution can often be more easily identified and characterized.", "As such, solution phase synthesis can still be preferable in some situations.", "Regardless of the method employed, combinatorial synthesis methods can either be manually performed, or can be automated.", "Manual synthesis requires repetitions of several relatively simple operations—addition of reagents, incubation and separation of solid and liquid phases, and removal of liquids.", "This character of the synthetic process renders it optimal for automation.", "Several designs of automated instruments for combinatorial synthesis have appeared in the patent and non-patent literature.", "The combinatorial approach to the synthesis of new drug entities has stimulated the development of a wide range of technologies for parallel processing.", "These have ranged from simple heated agitation systems to fully automated multi-probe synthesizers.", "Many have been developed to meet the demand for new drug candidates, but the drive towards parallel processing in all areas of laboratory development has also expanded significantly.", "Historically the discovery and optimization of candidate compounds for development as drugs has been extraordinarily expensive and time-consuming.", "Although the relatively new approach of “rational drug design” has promise for the future, the pharmaceutical industry has generally relied on mass screening of many-membered “libraries” of chemical compounds for the identification of “lead” compounds worthy of further study and structure-activity relationship (SAR) work.", "To meet this need high-throughput screening (HTS) technology has been developed that permits pharmaceutical companies to evaluate hundreds of thousands of individual chemical entities per year.", "Typically, these screens involve measuring some interaction (e.g., binding) between a biological target such as an enzyme or receptor and chemical compounds under test.", "The screens generally commence with the addition of individual compounds (or mixtures of compounds) to the individual wells in a 96 or higher-well “microtiter” plate that contains the biological target of interest (e.g., a receptor, enzyme or other protein).", "Ligand/receptor binding or other interaction events are then deduced by, for instance, various spectrophotometric techniques.", "Those chemical entities that exhibit promise in initial screens (e.g., that bind a biological target with some threshold affinity) are then subjected to chemical optimization, SAR work, other types of testing, and, if warranted, eventual development as drugs.", "Now that HTS has simplified and made more cost-effective the task of determining whether large chemical libraries contain promising lead compounds or “hits”, many pharmaceutical companies are limited not by their ability to screen candidate compounds but rather by their ability to synthesize them in the first place.", "At one point, most pharmaceutical companies relied on their historical collections of natural products and individually synthesized chemical entities as compound libraries to be subjected to mass screening.", "However, expanding these libraries—especially with a view toward increasing the “diversity” of the chemical space that they probe—has proven problematic.", "For instance the cost of having a synthetic organic or medicinal chemist synthesizes individual molecules in a serial fashion has been estimated to be several thousand dollars, and this is obviously a painstakingly slow process.", "Thus, the advent of high-throughput screening has created a need for correspondingly high-throughput chemical synthesis (HTCS) to feed this activity.", "“Combinatorial chemistry” and related techniques for high-throughput parallel syntheses of large chemical libraries were created in response to this need.", "To simplify the separation of intermediate compounds during multistep organic syntheses, much of this chemistry is generally performed while the compound being synthesized is covalently immobilized on a solid support such as a bead.", "Once the chemical building blocks have been properly assembled, the desired compounds are usually cleaved from their supports (often highly swellable polymeric resins) before being carried through to HTS.", "Various definitions of “combinatorial chemistry” and “combinatorial synthesis” have been proposed and are in current use.", "Some synthesis strategies (e.g., “split-and-mix”) are truly “combinatorial” in nature and have as their hallmark the ability to produce very large libraries; indeed, as many as a million library members can be synthesized in a modest number of reactions (and correspondingly small number of reaction vessels) by virtue of the exponential mathematics involved.", "One of the several limitations of such approaches, however, is the difficulty of identifying the particular individual chemical species responsible for any activity measured in an assay of what is generally a mixture of compounds.", "Other approaches such as high-throughput parallel synthesis are typically used to produce somewhat smaller chemical libraries containing, for example, from several hundred to several hundred thousand individual compounds.", "Here, discrete compounds (and occasionally mixtures) are spatially segregated during chemical synthesis so no ambiguity exists as to the identity of any compound producing a “hit.” However, parallel synthesis requires that chemical reactions be conducted in parallel in a relatively large number of reaction vessels, thus placing a premium on the ability to automate and improve the speed and efficiency of the synthetic process.", "Most high-throughput chemical syntheses (HTCS) performed in the context of combinatorial chemistry and parallel synthesis are presently conducted in multi-vessel reaction assemblies often referred to as “reaction blocks” by virtue of their monolithic construction.", "In most solid-phase syntheses, the compound being constructed is covalently attached to resin beads and so many of these multi-vessel reaction blocks include provision for a porous frit to retain the polymer resin beads (and compounds attached thereto) in the reaction vessel during the multiple resin washing steps that are used to remove excess reagents (e.g., building blocks, solvents, catalysts, etc.)", "after individual reaction steps.", "Constructions based on specialized reactors connected permanently (or semipermanently) to containers for the storage of reagents are strongly limited in their throughput.", "The productivity of automated instruments can be dramatically improved by use of disposable reaction vessels, (such as multititer plates or test tube arrays) into which reagents are added by pipetting, or by direct delivery from storage containers.", "The optimal storage vehicle is a syringe-like apparatus of a material inert to the chemical reactants, etc., e.g., a glass syringe, allowing the storage of the solution without any exposure to the atmosphere, and capable of serving as a delivery mechanism at the same time.", "See U.S. Pat.", "No.", "6,045,755 issued on Apr.", "4, 2000.Liquid removal from the reaction vessel (reactor) is usually accomplished by filtration through a filter-type material.", "The drawback of this method is the potential clogging of the filter by the solid phase support material, leading to extremely slow liquid removal, or to contamination of adjacent reactor compartments.", "An alternative technique based on the removal of liquid by suction from the surface above the sedimented solid phase is limited due to incomplete removal of the liquid from the reaction volume.", "See U.S. Pat.", "No.", "6,045,755 issued on Apr.", "4, 2000.U.S.", "Pat.", "Nos.", "5,202,418; 5,338,831; and 5,342,585 describe methods for liquid removal involving the placement of resin in polypropylene mesh packets, and removal of liquid through the openings of these packets (therefore this process is basically filtration), or removal of the liquid from the pieces of porous textile-like material by centrifugation.", "Liquid removal by centrifugation was also described in U.S. Pat.", "No.", "6,12,054 issued on Sep. 19, 2000.The method described therein generally involves the use of widely available solid phase organic synthetic protocols and disposable reaction vessel arrays such as microtiter style plates.", "The reaction vessel array is spun around its axis to create a “pocket” in which the solid material is retained.", "None of the prior art contemplates the removal of liquid by creation of “pockets” from which material cannot be removed by centrifugal force.", "Microtiter plates provide convenient handling systems for processing, shipping, and storing small liquid samples.", "Such devices are especially useful in high throughput screening and combinatorial chemistry applications and are well suited for use with robotic automation systems, which are adapted to selectively deliver various substances into different individual wells of the microtiter plate.", "As such, microtiter plates have proven especially useful in various biological, pharmacological, and related processes, which analyze and/or synthesize large numbers of small liquid samples.", "Standard multi-well microtiter plates come in a range of sizes, with shallow well plates having well volumes on the order of 200 to 300 microliters, with deep well plates typically having well volumes of 1.2 ml or 2.0 ml.", "A common example of a multi-well microtiter plate system is the standard 96-well microplate.", "Such microplates are typically fabricated from a variety of materials including polystyrene, polycarbonate, polypropylene, PTFE, glass, ceramics, and quartz.", "Unfortunately, standard microtiter plates suffer from a number of limitations, particularly with regard to chemical synthesis.", "For example, spillage, leakage, evaporation loss, airborne contamination of well contents, and inter-well cross-contamination of liquid samples are some of the common deficiencies that limit the application of standard microtiter plate assemblies in high throughput synthesis systems.", "Various techniques such as the inclusion of sealing layers or septums have been used in an attempt to overcome some of these shortcomings.", "For instance, WO 00/03805 discloses a microtiter reaction system comprising a support rack having an array of reaction wells disposed therein.", "The microtiter reaction system further includes a porous gas-permeable layer positioned over support rack, wherein the gas-permeable layer has an array of holes therein with each hole being positioned over each of the plurality of reaction wells.", "Finally, the assembly includes a gasket positioned over the porous gas-permeable layer and a top cover positioned over gasket.", "While effective to some degree, such microtiter assemblies are complicated, difficult to seal and reseal, and are generally expensive to manufacture.", "There still remains a need for a simple, efficient means of performing solid phase synthesis, particularly a method and apparatus amenable to use with automated methods for such synthesis.", "There also remains a need for a simple, efficient means for preventing spillage, leakage, evaporation loss, airborne contamination of well contents, and inter-well cross-contamination of liquid samples in microtiter reaction systems suitable for use in conjunction with automated solid phase/liquid synthesis." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention allows for the integration of solid phase chemistry with the process of solution phase chemistry.", "Generally, the present invention provides a novel method and apparatus for solid phase, combination solution/solid phase, and solution phase reactions.", "In one aspect of the invention, a reaction vessel assembly is provided which comprises a microplate having a rigid body with a plurality of open reaction wells mounted therein, a funnel cap inserted into each of the open reaction wells for at least partially sealing the open wells while allowing for venting of the well through a vent passage, and a modular solid phase immobilized within the interior volume of each of the open wells such that the modular solid phase does not block the passage of the funnel cap.", "In a preferred embodiment of the invention, the funnel cap is configured so as to substantially prevent the escape of a liquid sample contained within the interior volume of the reaction wells.", "Further, the discrete solid phase includes a support which is preferably a rigid or self-contained polymer object comprising a rigid or self-contained, unreactive base with an active polymer attached or within containment of the unreactive base, allowing for very high exposed polymer surface area.", "Examples of this polymer object can be in the form of tea-bags [1], crowns [2] , Irori Kans [3] , lanterns [4] , or sintered resins [5] .", "As long as the polymer object is rigid (not free-flowing), its support is unreactive, and it can be shaped to fit the funnel insert, it is a candidate for use within this reactor.", "In another aspect of the invention, a method for performing a combination solution phase/solid phase reaction using the reaction vessel of the invention where the solid support is immobilized in the lower portion of the reaction is provided comprising the steps of: a.", "Solid phase reaction wherein i. Reactants are added to the vessel and contacted with the solid support ii.", "A solid phase reaction occurs and the product is attached to the solid phase material iii.", "The reaction solution is removed from the vessel iv.", "A Cleaving solution is added v. The solid phase product is released in the solution phase b.", "Solution phase as a catalyst of a solution phase reaction wherein i. Reactants are added to the vessel and contacted with the solid support ii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iii.", "The solution phase product is then released in the solution phase c. Solution phase reaction wherein the solid phase is functionalized to serve as a scavenger wherein i. Reactants are added to the vessel and contacted with the solid support ii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iii.", "By products bond to the solid support iv.", "The solution phase product is recovered in the solution phase, while the by products are left behind on the solid support In another aspect of the invention, a method for performing a combination solution phase/solid phase reaction using the reaction vessel of the invention where the solid support is immobilized in the upper portion of the reaction is provided comprising the steps of: d. Solid Phase Reaction.", "i. Reactants are added to the vessel and initially do not contact the solid support ii.", "Reaction vessel is inverted to contact reactants with solid support iii.", "A solid phase reaction occurs with the solid support iv.", "Reaction vessel is turned upright and the reaction mixture is removed v. Cleavage solution is added to the vessel vi.", "Reaction vessel is inverted to contact the cleavage solution with the solid support vii.", "Solid phase product is cleaved into the solution viii.", "Reaction vessel is returned to upright position and product can be removed e. Solution phase reaction (catalyst) i. Reactants are added to vessel and do not contact the solid support ii.", "Reaction vessel is inverted to contact reactants with solid support iii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iv.", "Reaction vessel is returned to upright position.", "v. The solution phase product is then removed in the solution phase f. Solution phase reaction (solid phase functionalized to serve as a scavenger) i. Reactants are added to vessel and do not contact the solid support ii.", "Reaction vessel is inverted to contact reactants with solid support iii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iv.", "By products bond to the solid support v. Reaction vessel is returned to upright position vi.", "The solution phase product can now be removed in the solution phase and while the by products are left behind on the solid support In another aspect, the invention provides a novel microtiter chemical reaction system which allows for reactions to be carried out in an inert atmosphere while minimizing spillage, leakage, evaporation, and cross-contamination.", "In one embodiment of the invention, a reaction vessel assembly is provided which comprises a lower reaction well microplate assembly having a rigid body with a plurality of open reaction wells disposed therein, and a funnel cap inserted into each of the open reaction wells for at least partially sealing the open well while allowing for venting of the well through a vent passage.", "The reaction vessel further comprises an upper inert atmosphere cap, which is configured so as to provide a constant positive pressure of inert gas while allowing access to the lower reaction wells.", "In a preferred embodiment of the invention, the funnel cap is configured so as to substantially prevent the escape of a liquid sample contained within the interior volume of the reaction well.", "The reaction system can be used as an inert solution phase reactor where reactants are dispensed through the funnel of the upper inert atmosphere cap and through the funnel of the lower reaction vessel.", "The liquid reaction is contained in the lower reaction vessel while the atmosphere is controlled by the positive pressure of inert gas flowing in and out of the entire vessel through the upper cap.", "In another embodiment of the invention, the inert reaction system can be used for solid phase reactions using the solid support in the lower reaction vessel.", "Within that vessel the solid support can be immobilized at the bottom or top of the vessel.", "A method for performing a combination solution phase/solid phase reaction using the reaction vessel of the invention where the solid support is immobilized in the lower portion of the reaction is provided comprising the steps of: g. Solid phase reaction i. Reactants are added to vessel and contact the solid support ii.", "A solid phase reaction occurs and the product is attached to the solid phase material iii.", "The reaction solution is removed from the vessel iv.", "A Cleaving solution is added v. The solid phase product can now be removed in the solution phase h. Solution phase reaction (catalyst) i. Reactants are added to vessel and contact the solid support ii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iii.", "The solution phase product can now be removed in the solution phase i.", "Solution phase reaction (solid phase functionalized to serve as a scavenger) i. Reactants are added to vessel and contact the solid support ii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iii.", "By products bond to the solid support iv.", "The solution phase product can now be removed in the solution phase and while the by products are left behind on the solid support In another aspect of the invention, a method for performing a combination solution phase/solid phase reaction using the reaction vessel of the invention where the solid support is immobilized in the upper portion of the reaction is provided comprising the steps of: Inert reaction vessel, solid support is located at the upper end of the reaction vessel.", "j.", "Solid Phase Reaction.", "i. Reactants are added to vessel and do not contact the solid support ii.", "Reaction vessel is inverted to contact reactants with solid support iii.", "A solid phase reaction occurs with the solid support iv.", "Reaction vessel is turned upright and the reaction mixture is removed v. Cleavage solution is added to the vessel vi.", "Reaction vessel is inverted to expose cleavage solution with solid support vii.", "Solid phase product is cleaved into the solution viii.", "Reaction vessel is returned to upright position and product can be removed k. Solution phase reaction (catalyst) i. Reactants are added to vessel and do not contact the solid support ii.", "Reaction vessel is inverted to contact reactants with solid support iii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iv.", "Reaction vessel is returned to upright position.", "v. The solution phase product can now be removed in the solution phase l. Solution phase reaction (solid phase functionalized to serve as a scavenger) i. Reactants are added to vessel and do not contact the solid support ii.", "Reaction vessel is inverted to contact reactants with solid support iii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iv.", "By products bond to the solid support v. Reaction vessel is returned to upright position The solution phase product can now be removed in the solution phase while the by products are left behind on the solid support.", "In another embodiment of the invention, the polymer object can also be shaped to fit into the reaction vessel in such a way that it does not interfere with the action of aspirating or dispensing from this vessel.", "The polymer object would take the shape of a cylinder or donut and reside on the outer wall of the reaction vessel.", "The funnel cap can be used to ensure that the accessing device does not contact the polymer object.", "In another embodiment of the invention, the polymer can be directly attached to the walls of the reaction vessel, perhaps through sintering [5] , whereas the solid support would be the reaction vessel itself.", "The polymer object would reside on the perimeter of the reaction vessel to avoid interference with the accessing device.", "Again, the funnel cap could be used to ensure that the accessing device does not contact the polymer object.", "In yet another embodiment the inert reaction system can utilize the shaped polymer placed into the lower reaction vessels or the polymer can be attached to the walls of the lower reaction vessel." ], [ "CROSS REFERENCE TO PRIOR APPLICATIONS The present application claims priority to U.S.", "Provisional Application Ser.", "No.", "60/263,762 filed Jan. 25, 2001, and to U.S.", "Provisional Application Ser.", "No.", "60/300,103 filed Jun.", "25, 2001.The contents of both Provisional Applications are hereby incorporated by reference in their entirety.", "FIELD OF THE INVENTION The present invention relates generally to an apparatus and method for chemical synthesis.", "More particularly, the present invention relates to an apparatus and method for high-throughput, solid or solution phase organic synthesis.", "The invention also relates to a microtiter chemical system particularly suitable for the apparatus and method of the invention.", "BACKGROUND OF THE INVENTION Combinatorial chemistry generally relates to a set of techniques for creating a multiplicity of compounds and then testing them for desired activity.", "More specifically, combinatorial chemistry involves the formation of large libraries of molecules en masse, instead of the synthesis of compounds one by one as had been done traditionally.", "Once the libraries are obtained, the most promising lead pharmaceutical compounds are identified by high-throughput screening for further evaluation.", "Generally, combinatorial compounds are created either by solution-phase synthesis or by producing compounds bound covalently to solid phase particles.", "Solid phase synthesis can make multi-step reactions easier to perform and more reliably allows one to drive reactions to completion because excess reagents can be added and then easily washed away after each reaction step.", "Further, solid phase synthesis allows for the use of split synthesis, a technique that produces large support-bound libraries in which each solid phase particle holds a single compound.", "Soluble libraries can then be produced by cleavage of the compounds from the solid support.", "Nonetheless, a much wider range of organic reactions can be available for solution phase synthesis, and products in solution can often be more easily identified and characterized.", "As such, solution phase synthesis can still be preferable in some situations.", "Regardless of the method employed, combinatorial synthesis methods can either be manually performed, or can be automated.", "Manual synthesis requires repetitions of several relatively simple operations—addition of reagents, incubation and separation of solid and liquid phases, and removal of liquids.", "This character of the synthetic process renders it optimal for automation.", "Several designs of automated instruments for combinatorial synthesis have appeared in the patent and non-patent literature.", "The combinatorial approach to the synthesis of new drug entities has stimulated the development of a wide range of technologies for parallel processing.", "These have ranged from simple heated agitation systems to fully automated multi-probe synthesizers.", "Many have been developed to meet the demand for new drug candidates, but the drive towards parallel processing in all areas of laboratory development has also expanded significantly.", "Historically the discovery and optimization of candidate compounds for development as drugs has been extraordinarily expensive and time-consuming.", "Although the relatively new approach of “rational drug design” has promise for the future, the pharmaceutical industry has generally relied on mass screening of many-membered “libraries” of chemical compounds for the identification of “lead” compounds worthy of further study and structure-activity relationship (SAR) work.", "To meet this need high-throughput screening (HTS) technology has been developed that permits pharmaceutical companies to evaluate hundreds of thousands of individual chemical entities per year.", "Typically, these screens involve measuring some interaction (e.g., binding) between a biological target such as an enzyme or receptor and chemical compounds under test.", "The screens generally commence with the addition of individual compounds (or mixtures of compounds) to the individual wells in a 96 or higher-well “microtiter” plate that contains the biological target of interest (e.g., a receptor, enzyme or other protein).", "Ligand/receptor binding or other interaction events are then deduced by, for instance, various spectrophotometric techniques.", "Those chemical entities that exhibit promise in initial screens (e.g., that bind a biological target with some threshold affinity) are then subjected to chemical optimization, SAR work, other types of testing, and, if warranted, eventual development as drugs.", "Now that HTS has simplified and made more cost-effective the task of determining whether large chemical libraries contain promising lead compounds or “hits”, many pharmaceutical companies are limited not by their ability to screen candidate compounds but rather by their ability to synthesize them in the first place.", "At one point, most pharmaceutical companies relied on their historical collections of natural products and individually synthesized chemical entities as compound libraries to be subjected to mass screening.", "However, expanding these libraries—especially with a view toward increasing the “diversity” of the chemical space that they probe—has proven problematic.", "For instance the cost of having a synthetic organic or medicinal chemist synthesizes individual molecules in a serial fashion has been estimated to be several thousand dollars, and this is obviously a painstakingly slow process.", "Thus, the advent of high-throughput screening has created a need for correspondingly high-throughput chemical synthesis (HTCS) to feed this activity.", "“Combinatorial chemistry” and related techniques for high-throughput parallel syntheses of large chemical libraries were created in response to this need.", "To simplify the separation of intermediate compounds during multistep organic syntheses, much of this chemistry is generally performed while the compound being synthesized is covalently immobilized on a solid support such as a bead.", "Once the chemical building blocks have been properly assembled, the desired compounds are usually cleaved from their supports (often highly swellable polymeric resins) before being carried through to HTS.", "Various definitions of “combinatorial chemistry” and “combinatorial synthesis” have been proposed and are in current use.", "Some synthesis strategies (e.g., “split-and-mix”) are truly “combinatorial” in nature and have as their hallmark the ability to produce very large libraries; indeed, as many as a million library members can be synthesized in a modest number of reactions (and correspondingly small number of reaction vessels) by virtue of the exponential mathematics involved.", "One of the several limitations of such approaches, however, is the difficulty of identifying the particular individual chemical species responsible for any activity measured in an assay of what is generally a mixture of compounds.", "Other approaches such as high-throughput parallel synthesis are typically used to produce somewhat smaller chemical libraries containing, for example, from several hundred to several hundred thousand individual compounds.", "Here, discrete compounds (and occasionally mixtures) are spatially segregated during chemical synthesis so no ambiguity exists as to the identity of any compound producing a “hit.” However, parallel synthesis requires that chemical reactions be conducted in parallel in a relatively large number of reaction vessels, thus placing a premium on the ability to automate and improve the speed and efficiency of the synthetic process.", "Most high-throughput chemical syntheses (HTCS) performed in the context of combinatorial chemistry and parallel synthesis are presently conducted in multi-vessel reaction assemblies often referred to as “reaction blocks” by virtue of their monolithic construction.", "In most solid-phase syntheses, the compound being constructed is covalently attached to resin beads and so many of these multi-vessel reaction blocks include provision for a porous frit to retain the polymer resin beads (and compounds attached thereto) in the reaction vessel during the multiple resin washing steps that are used to remove excess reagents (e.g., building blocks, solvents, catalysts, etc.)", "after individual reaction steps.", "Constructions based on specialized reactors connected permanently (or semipermanently) to containers for the storage of reagents are strongly limited in their throughput.", "The productivity of automated instruments can be dramatically improved by use of disposable reaction vessels, (such as multititer plates or test tube arrays) into which reagents are added by pipetting, or by direct delivery from storage containers.", "The optimal storage vehicle is a syringe-like apparatus of a material inert to the chemical reactants, etc., e.g., a glass syringe, allowing the storage of the solution without any exposure to the atmosphere, and capable of serving as a delivery mechanism at the same time.", "See U.S. Pat.", "No.", "6,045,755 issued on Apr.", "4, 2000.Liquid removal from the reaction vessel (reactor) is usually accomplished by filtration through a filter-type material.", "The drawback of this method is the potential clogging of the filter by the solid phase support material, leading to extremely slow liquid removal, or to contamination of adjacent reactor compartments.", "An alternative technique based on the removal of liquid by suction from the surface above the sedimented solid phase is limited due to incomplete removal of the liquid from the reaction volume.", "See U.S. Pat.", "No.", "6,045,755 issued on Apr.", "4, 2000.U.S.", "Pat.", "Nos.", "5,202,418; 5,338,831; and 5,342,585 describe methods for liquid removal involving the placement of resin in polypropylene mesh packets, and removal of liquid through the openings of these packets (therefore this process is basically filtration), or removal of the liquid from the pieces of porous textile-like material by centrifugation.", "Liquid removal by centrifugation was also described in U.S. Pat.", "No.", "6,12,054 issued on Sep. 19, 2000.The method described therein generally involves the use of widely available solid phase organic synthetic protocols and disposable reaction vessel arrays such as microtiter style plates.", "The reaction vessel array is spun around its axis to create a “pocket” in which the solid material is retained.", "None of the prior art contemplates the removal of liquid by creation of “pockets” from which material cannot be removed by centrifugal force.", "Microtiter plates provide convenient handling systems for processing, shipping, and storing small liquid samples.", "Such devices are especially useful in high throughput screening and combinatorial chemistry applications and are well suited for use with robotic automation systems, which are adapted to selectively deliver various substances into different individual wells of the microtiter plate.", "As such, microtiter plates have proven especially useful in various biological, pharmacological, and related processes, which analyze and/or synthesize large numbers of small liquid samples.", "Standard multi-well microtiter plates come in a range of sizes, with shallow well plates having well volumes on the order of 200 to 300 microliters, with deep well plates typically having well volumes of 1.2 ml or 2.0 ml.", "A common example of a multi-well microtiter plate system is the standard 96-well microplate.", "Such microplates are typically fabricated from a variety of materials including polystyrene, polycarbonate, polypropylene, PTFE, glass, ceramics, and quartz.", "Unfortunately, standard microtiter plates suffer from a number of limitations, particularly with regard to chemical synthesis.", "For example, spillage, leakage, evaporation loss, airborne contamination of well contents, and inter-well cross-contamination of liquid samples are some of the common deficiencies that limit the application of standard microtiter plate assemblies in high throughput synthesis systems.", "Various techniques such as the inclusion of sealing layers or septums have been used in an attempt to overcome some of these shortcomings.", "For instance, WO 00/03805 discloses a microtiter reaction system comprising a support rack having an array of reaction wells disposed therein.", "The microtiter reaction system further includes a porous gas-permeable layer positioned over support rack, wherein the gas-permeable layer has an array of holes therein with each hole being positioned over each of the plurality of reaction wells.", "Finally, the assembly includes a gasket positioned over the porous gas-permeable layer and a top cover positioned over gasket.", "While effective to some degree, such microtiter assemblies are complicated, difficult to seal and reseal, and are generally expensive to manufacture.", "There still remains a need for a simple, efficient means of performing solid phase synthesis, particularly a method and apparatus amenable to use with automated methods for such synthesis.", "There also remains a need for a simple, efficient means for preventing spillage, leakage, evaporation loss, airborne contamination of well contents, and inter-well cross-contamination of liquid samples in microtiter reaction systems suitable for use in conjunction with automated solid phase/liquid synthesis.", "SUMMARY OF THE INVENTION The present invention allows for the integration of solid phase chemistry with the process of solution phase chemistry.", "Generally, the present invention provides a novel method and apparatus for solid phase, combination solution/solid phase, and solution phase reactions.", "In one aspect of the invention, a reaction vessel assembly is provided which comprises a microplate having a rigid body with a plurality of open reaction wells mounted therein, a funnel cap inserted into each of the open reaction wells for at least partially sealing the open wells while allowing for venting of the well through a vent passage, and a modular solid phase immobilized within the interior volume of each of the open wells such that the modular solid phase does not block the passage of the funnel cap.", "In a preferred embodiment of the invention, the funnel cap is configured so as to substantially prevent the escape of a liquid sample contained within the interior volume of the reaction wells.", "Further, the discrete solid phase includes a support which is preferably a rigid or self-contained polymer object comprising a rigid or self-contained, unreactive base with an active polymer attached or within containment of the unreactive base, allowing for very high exposed polymer surface area.", "Examples of this polymer object can be in the form of tea-bags[1], crowns[2], Irori Kans[3], lanterns[4], or sintered resins[5].", "As long as the polymer object is rigid (not free-flowing), its support is unreactive, and it can be shaped to fit the funnel insert, it is a candidate for use within this reactor.", "In another aspect of the invention, a method for performing a combination solution phase/solid phase reaction using the reaction vessel of the invention where the solid support is immobilized in the lower portion of the reaction is provided comprising the steps of: a.", "Solid phase reaction wherein i. Reactants are added to the vessel and contacted with the solid support ii.", "A solid phase reaction occurs and the product is attached to the solid phase material iii.", "The reaction solution is removed from the vessel iv.", "A Cleaving solution is added v. The solid phase product is released in the solution phase b.", "Solution phase as a catalyst of a solution phase reaction wherein i. Reactants are added to the vessel and contacted with the solid support ii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iii.", "The solution phase product is then released in the solution phase c. Solution phase reaction wherein the solid phase is functionalized to serve as a scavenger wherein i. Reactants are added to the vessel and contacted with the solid support ii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iii.", "By products bond to the solid support iv.", "The solution phase product is recovered in the solution phase, while the by products are left behind on the solid support In another aspect of the invention, a method for performing a combination solution phase/solid phase reaction using the reaction vessel of the invention where the solid support is immobilized in the upper portion of the reaction is provided comprising the steps of: d. Solid Phase Reaction.", "i. Reactants are added to the vessel and initially do not contact the solid support ii.", "Reaction vessel is inverted to contact reactants with solid support iii.", "A solid phase reaction occurs with the solid support iv.", "Reaction vessel is turned upright and the reaction mixture is removed v. Cleavage solution is added to the vessel vi.", "Reaction vessel is inverted to contact the cleavage solution with the solid support vii.", "Solid phase product is cleaved into the solution viii.", "Reaction vessel is returned to upright position and product can be removed e. Solution phase reaction (catalyst) i. Reactants are added to vessel and do not contact the solid support ii.", "Reaction vessel is inverted to contact reactants with solid support iii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iv.", "Reaction vessel is returned to upright position.", "v. The solution phase product is then removed in the solution phase f. Solution phase reaction (solid phase functionalized to serve as a scavenger) i. Reactants are added to vessel and do not contact the solid support ii.", "Reaction vessel is inverted to contact reactants with solid support iii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iv.", "By products bond to the solid support v. Reaction vessel is returned to upright position vi.", "The solution phase product can now be removed in the solution phase and while the by products are left behind on the solid support In another aspect, the invention provides a novel microtiter chemical reaction system which allows for reactions to be carried out in an inert atmosphere while minimizing spillage, leakage, evaporation, and cross-contamination.", "In one embodiment of the invention, a reaction vessel assembly is provided which comprises a lower reaction well microplate assembly having a rigid body with a plurality of open reaction wells disposed therein, and a funnel cap inserted into each of the open reaction wells for at least partially sealing the open well while allowing for venting of the well through a vent passage.", "The reaction vessel further comprises an upper inert atmosphere cap, which is configured so as to provide a constant positive pressure of inert gas while allowing access to the lower reaction wells.", "In a preferred embodiment of the invention, the funnel cap is configured so as to substantially prevent the escape of a liquid sample contained within the interior volume of the reaction well.", "The reaction system can be used as an inert solution phase reactor where reactants are dispensed through the funnel of the upper inert atmosphere cap and through the funnel of the lower reaction vessel.", "The liquid reaction is contained in the lower reaction vessel while the atmosphere is controlled by the positive pressure of inert gas flowing in and out of the entire vessel through the upper cap.", "In another embodiment of the invention, the inert reaction system can be used for solid phase reactions using the solid support in the lower reaction vessel.", "Within that vessel the solid support can be immobilized at the bottom or top of the vessel.", "A method for performing a combination solution phase/solid phase reaction using the reaction vessel of the invention where the solid support is immobilized in the lower portion of the reaction is provided comprising the steps of: g. Solid phase reaction i. Reactants are added to vessel and contact the solid support ii.", "A solid phase reaction occurs and the product is attached to the solid phase material iii.", "The reaction solution is removed from the vessel iv.", "A Cleaving solution is added v. The solid phase product can now be removed in the solution phase h. Solution phase reaction (catalyst) i. Reactants are added to vessel and contact the solid support ii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iii.", "The solution phase product can now be removed in the solution phase i.", "Solution phase reaction (solid phase functionalized to serve as a scavenger) i. Reactants are added to vessel and contact the solid support ii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iii.", "By products bond to the solid support iv.", "The solution phase product can now be removed in the solution phase and while the by products are left behind on the solid support In another aspect of the invention, a method for performing a combination solution phase/solid phase reaction using the reaction vessel of the invention where the solid support is immobilized in the upper portion of the reaction is provided comprising the steps of: Inert reaction vessel, solid support is located at the upper end of the reaction vessel.", "j.", "Solid Phase Reaction.", "i. Reactants are added to vessel and do not contact the solid support ii.", "Reaction vessel is inverted to contact reactants with solid support iii.", "A solid phase reaction occurs with the solid support iv.", "Reaction vessel is turned upright and the reaction mixture is removed v. Cleavage solution is added to the vessel vi.", "Reaction vessel is inverted to expose cleavage solution with solid support vii.", "Solid phase product is cleaved into the solution viii.", "Reaction vessel is returned to upright position and product can be removed k. Solution phase reaction (catalyst) i. Reactants are added to vessel and do not contact the solid support ii.", "Reaction vessel is inverted to contact reactants with solid support iii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iv.", "Reaction vessel is returned to upright position.", "v. The solution phase product can now be removed in the solution phase l. Solution phase reaction (solid phase functionalized to serve as a scavenger) i. Reactants are added to vessel and do not contact the solid support ii.", "Reaction vessel is inverted to contact reactants with solid support iii.", "A solution phase reaction occurs with the solid support and the product remains in the solution phase iv.", "By products bond to the solid support v. Reaction vessel is returned to upright position The solution phase product can now be removed in the solution phase while the by products are left behind on the solid support.", "In another embodiment of the invention, the polymer object can also be shaped to fit into the reaction vessel in such a way that it does not interfere with the action of aspirating or dispensing from this vessel.", "The polymer object would take the shape of a cylinder or donut and reside on the outer wall of the reaction vessel.", "The funnel cap can be used to ensure that the accessing device does not contact the polymer object.", "In another embodiment of the invention, the polymer can be directly attached to the walls of the reaction vessel, perhaps through sintering[5], whereas the solid support would be the reaction vessel itself.", "The polymer object would reside on the perimeter of the reaction vessel to avoid interference with the accessing device.", "Again, the funnel cap could be used to ensure that the accessing device does not contact the polymer object.", "In yet another embodiment the inert reaction system can utilize the shaped polymer placed into the lower reaction vessels or the polymer can be attached to the walls of the lower reaction vessel.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 illustrates details of a preferred spill proof microplate assembly for use with the reaction vessel of the present invention.", "FIG.", "2 shows an embodiment of an individual reaction vessel well of the present invention with the solid phase support immobilized near the bottom of the reaction well.", "FIG.", "3 shows an individual reaction vessel well of the present invention with an injection needle inserted through a vent passageway.", "FIG.", "4 shows the individual reaction vessel well of FIG.", "3 with the injection needle removed.", "FIG.", "5 shows another embodiment of an individual reaction vessel well of the present invention with the solid phase support immobilized near the top of the reaction well.", "FIG.", "6 shows the individual reaction vessel well of FIG.", "5 in an inverted position.", "FIG.", "7 illustrates details of one embodiment of the inert microtiter chemical reaction system of the present invention.", "FIG.", "8 illustrates the inert microtiter chemical reaction system of the present invention with the solid phase inserts at the bottom of the funnels in the lower vessel.", "FIG.", "9 illustrates the inert microtiter chemical reaction system of the present invention with the solid phase inserts at the top of the funnels in the lower vessel.", "FIG.", "10 illustrate the inert microtiter chemical reaction system of the present invention with the solid phase material sintered or press fit into the lower vessel.", "FIG.", "11 illustrate a reaction vessel with no insert and solid support fused or press fit into a reaction vessel.", "DETAILED DESCRIPTION OF THE INVENTION By way of introduction, in solid phase synthesis, final compounds are synthesized attached to solid-phase supports that permit the use of simple mechanical means to separate intermediate or partially synthesized intermediate compounds between synthetic steps.", "Conventional solid-phase supports generally include beads, including microbeads, of 30 microns to 300 microns in diameter, which are functionalized in order to covalently attach intermediate or final compounds, and are made of, e.g., various glasses, plastics, or resins.", "Solid-phase combinatorial synthesis typically proceeds according to the following steps.", "In a first step, reaction vessels are charged with a solid-phase support, typically a slurry of functionalized beads suspended in a solvent.", "These beads are then preconditioned by incubating them in an appropriate solvent, and the first of a plurality of building blocks, or a linker moiety, is covalently linked to the functionalized beads.", "Subsequently, a sequence of reaction steps is performed in a sequence chosen to synthesize the desired compound in a manner as follows.", "First, a sufficient quantity of a solution containing the building block moiety selected for addition is accurately added to the reaction vessels so that the building block moiety is present in a molar excess to the intermediate compound.", "The reaction is triggered and promoted by activating reagents and other reagents and solvents, which are also added to the reaction vessel.", "The reaction vessel is then incubated at a controlled temperature for a time, typically between 5 minutes and 24 hours, sufficient for the building block addition reaction or transformation to go to substantial completion.", "Optionally, during this incubation, the reaction vessel can be intermittently agitated or stirred.", "Finally, in a last substep of building block addition, the reaction vessel containing the solid phase support with attached intermediate compound is prepared for addition of the next building block by removing the reaction fluid and thorough washing and reconditioning the solid-phase support.", "Washing typically involves three to seven cycles of adding and removing a wash solvent.", "Optionally, during the addition steps, multiple building blocks can be added to one reaction vessel in order to synthesize a mixture of compound intermediates attached to one solid-phase support, or alternatively, the contents of separate reaction vessels can be combined and partitioned in order that multiple compounds can be synthesized in one reaction vessel with each microbead having only one attached final compound.", "After the desired number of building block addition steps, the final compound is present in the reaction vessel attached to the solid-phase support.", "The final compounds can be utilized either directly attached to the synthetic supports, or alternatively, can be cleaved from the supports and extracted into a liquid phase.", "With this process in mind, the present invention generally provides a novel, automation-compatible solid phase reaction vessel, as well as methods for using such a vessel.", "Generally, the reaction vessel comprises a microplate assembly with a modular solid phase support included within the individual reaction wells.", "The reaction vessel of the invention allows for the integration of solid phase chemistry with the processing abilities of solution phase chemistry.", "Thereby providing more flexibility in the synthesis steps that can be carried out and therefore significantly increasing the ability to probe complex synthesis mechanisms.", "According to the invention, the microplate assembly and the solid phase supports are configured so as to integrate together into a single reaction vessel.", "The combination enables solid phase reactions in a single vessel with full compatibility to liquid handling automation.", "Microplate Assembly The present invention can employ any microplate assembly known in the art.", "For instance, a standard microplate assembly can comprises a microplate having a plurality of open wells and a closure device for sealing the wells shut.", "Commonly available microplates generally embody a unitary molded structure comprising a rigid frame for housing a plurality of open wells arranged in a rectangular array.", "Standard well closures include resilient, press-fit stoppers, rigid screw caps, adhesive films, and the like.", "Microplates come in a range of sizes; a well may be sized to hold as high as five milliliters or as low as only a few microliters of liquid or even sub-micro quantities of liquid (e.g.", "in the range of few nanoliters).", "In addition, microplates come in a variety of materials, such as polystyrene, polycarbonate, polypropylene, Teflon, glass, ceramics, and quartz.", "Microplates found in many high-throughput systems comprise a 96-well geometry molded into an 8×12 rectangular array of open wells.", "Microplates with lower well densities (e.g., 24 and 48 wells) and higher well densities (e.g., 384 and 864 wells) are also available.", "More preferably however, the microplate assembly of the present invention is a spill proof microplate assembly having a plurality of open wells, such as those disclosed in U.S. Pat.", "No.", "6,027,694, which is incorporated herein by reference.", "Each of the wells comprises a vessel with an interior volume.", "A seal is coupled to the wells for sealing the wells so that liquid in the interior volume is prevented from exiting the wells.", "A vent equalizes the pressure of the wells with the ambient pressure.", "The structure and function of the preferred embodiments of the invention can best be understood by reference to the drawings.", "It will be noted that the same reference numerals appear in multiple figures.", "Where this is the case, the numerals refer to the same or corresponding structure in the figures.", "It should further be noted that many of the general functions and operations described below in connection with particular embodiments of the apparatus of the present invention may be realized equally well by a number of alternative mechanical designs that will suggest themselves to those of skill in the art.", "Such functionally equivalent alternatives, similar in concept but different in mechanical detail, are within the scope of the present invention.", "In one embodiment of the invention, the spill proof microplate assembly 10 comprises a multi-well microplate 11, a plurality of funnel caps 12 and an optional porous vent film 13 (see FIG.", "1).", "The microplate 11 houses a plurality of open wells 17 in a rectangular array.", "The funnel caps 12 seal and vent the wells 17.When the funnel caps 12 are coupled to the wells, an interior volume 30 is formed in each well 17.The wells are thus configured to accommodate liquid samples 19 within predetermined spaces of the interior volumes 30.The liquid samples 19 remain within the predetermined space for all orientations of the microplate assembly.", "In one embodiment, the funnel caps 12 comprise sealing plugs 28 and vent tubes 29, which can optionally be interconnected by a porous perforated web 13.The sealing plugs 28 form a seal at the mouth of the open wells 17.The vent tubes 29 attach to the sealing plugs 28 and terminate in vents 34.The vents 34 communicate with the interior volumes 30 outside the predetermined spaces, which can accommodate liquid samples 19.The vents 34 permit the pressure within the interior volume 30 to be equalized with the ambient pressure via a passage that runs through the vent tube 29 and the sealing plugs 28.Material may be added to or removed from the wells 17 via the passages 34.The optional perforated web 13 can have an adhesive coating which adheres the web to the funnel caps 12 while covering the passages 34, thereby inhibiting evaporation of tile liquid samples.", "Consequently, vent caps 12 function as multiple vented seals for interior volumes 30 of wells 17.Each well insert 20 couples with a different well 17 such that plug 28 forms a tight press-fit seal with the edge of the mouth of the well 17.With vent cap 12 properly coupled to wells 17, each plug 28 prevents liquid sample 19 from exiting the interior volume 30 via the seam at the interface between plug 28 and tile mouth of the well 17.In addition, each vent 34 will permit the pressure within interior volume 30 to be equalized with tile ambient pressure via passage 32, thereby avoiding forces that may dislodge plug 28.As mentioned above, manufactures typically fabricate microplates from polystyrene, polycarbonate, polypropylene, Teflon, glass, ceramics, or quartz.", "As such, vent caps 12 may be readily molded from a variety of compatible materials.", "In this regard, the materials of funnel cap 12 must be such that plugs 28 will have sufficient resiliency to form a good press-fit seal with the mouth of well 17.In addition, optional web 13 preferably can flex to allow for easy positioning and removal of vent cap 12.Further, the microplate assembly of the invention can be configured as a microfilter plate such that liquid reagent samples can be removed through the filter upon the application of suction, as is known in the art.", "Solid Phase Supports The solid phase support of the present invention may include any of the many different known types of solid phase supports and is not limited by the nature of any functional group(s) linked to the support.", "The only requirements are that the solid phase support should include a discrete, modular structure, and should be substantially insoluble in aqueous and organic solvents.", "Further, the solid phase support should be substantially inert to the reaction conditions needed to employ the solid support in chemical synthesis; any modular, immobilizable solid phase support known in the art can be used.", "For instance, organic polymer resins, silica based compounds, and composites are within the scope of the invention so long as they arc incorporated into a modular base which can be inserted and immobilized within the individual wells of the microplate assembly.", "By employing such solid phase supports with a modular base, solid phase synthesis techniques can be carried out without the filtration, weighing, handling, and cleavage problems generally associated with conventional resin techniques.", "Generally, solid phase supports include various linkers.", "Linkers are solid-phase protecting groups, which allow attachment of a scaffold or template molecule to a solid phase; support.", "Attachment of the scaffold or template undergoing chemical modifications to a solid phase support provides a practical method for removal of excess reagents and starting materials via extensive washing and filtration without loss of product.", "After suitable chemical modifications, the scaffold or template can be cleaved from the solid phase support under selective conditions that will not alter the modified scaffold or template.", "Linkers are molecules that are attached to a solid support and to which the desired members of a library of chemical compounds may in turn be attached.", "When the construction of the library is complete, the linker allows clean separation of the target compounds from the solid support without harm to the compounds and preferably without damage to the support.", "Several linkers have been described in the literature.", "Their value is constrained by the need to have sufficient stability, which allows the steps of combinatorial synthesis under conditions that will not cleave the linker.", "An additional constraint is the need to have a fairly high liability under at least one set of conditions that is not employed in the chemical synthesis.", "For example, if an acid labile linker is employed, then the combinatorial synthesis must be restricted to reactions that do not require the presence of an acid of sufficient strength to endanger the integrity of the linker.", "Likewise, when a photocleavable linker is employed, conditions that exclude light are necessary to avoid untimely cleavage of the compound from the resin.", "This sort of balancing act often imposes serious constraints on the reactions that are chosen for preparation of the library.", "For example, 4-[4-(hydroxymethyl)-3-methoxyphenoxy] butyryl residue is a known linker, which is attached to a solid support having amino groups by forming an amide with the carboxyl of the butyric acid chain.", "N-Protected amino acids are attached to the hydroxyl of the 4-hydroxymethyl group via their carboxyl to form 2,4-dialkoxybenzyl esters, which can be readily cleaved in acid media when the synthesis is complete.", "The drawback to such 2,4-dialkoxybenzyl esters is their instability with many of the reagents that are available for use in combinatorial synthesis resulting in cleavage of the ester.", "A somewhat more stable ester is formed from 4-[4-(hydroxymethyl) phenoxy] butyric acid, described in European published application EP 445915.In this case, the ester was cleaved with a 90:5:5 mixture of trifluoroacetic acid, dimethyl sulfide and thioanisole.", "When the desired product is a peptide amide, the 4-[4-(formyl)-3,5-dimethoxyphenoxy] butyryl residue has been employed as a linker.", "This particular linker is attached to a solid phase substrate via the carboxyl of the butyric acid chain, and the 4-formyl group is reductively aminated.", "N-Protected amino acids are then reacted with the alkylamine via their carboxyl to form 2,4,6-trialkoxybenzylamides.", "These may be cleaved by 1:1 trifluoroacetic acid in dichloromethane (PCT application WO97/23508).", "If a photocleavable linker is used to attach chemical compounds to the main support, milder photolytic conditions of cleavage can be used which complement traditional acidic or basic cleavage techniques.", "A wider range of combinatorial synthetic conditions will be tolerated by photocleavable linkers.", "Other examples of linkers include a phenacyl based linking group that is photocleavable.", "The 4-bromomethyl-3-nitrobenzoyl residue has been widely employed as a photocleavable linker for both peptide acids and amides.", "Photocleavable linkers such as the 3-bromomethyl-4-nitro-6-methoxyphenoxyacetyl residue are stable to acidic or basic conditions yet, are rapidly cleavable under mild conditions and do not generate highly reactive byproducts (U.S. Pat.", "No.", "5,739,386, issued Apr.", "14, 1998).", "More particularly, in a preferred embodiment, the solid phase support includes polyethylene, polypropylene, polytetrafluoroethylene supports.", "Generally these supports are comprised of a mobile polymer, such as polystyrene, polyacrylamide or polyacrylic acid, attached onto a rigid unreactive base polymer core.", "The active surface polymer can optionally have functional groups attached along the backbone, including amines, alcohols, and other linkers.", "The modular base polymer core can be shaped to maximize surface area while allowing efficient drainage of liquid when the reaction mixture is removed.", "In a particularly preferred embodiment, the solid phase support is cylindrical in appearance for compatibility with microplate assembly well dimensions.", "As mentioned above, the solid phase supports of the invention can optionally be functionalized with one or more functional groups.", "That is, the supports can have one or more functional groups usually covalently linked thereto.", "The functional groups may be incorporated into the active surface polymer, or may be covalently attached to the surface of the polymer.", "The functional groups can provide a reactive site for attachment of an optional spacer group or linker.", "Several solid phase particles having functional groups covalently linked thereto have been described in the chemical and biochemical literature.", "For example, see E. Atherton and R. C. Sheppard, “Solid Phase Synthesis: A Practical Approach” Oxford University Press, 1989, and E. C. Blossey, “Solid Phase Synthesis,” Dowden Hutchinson & Ross Publishers.", "The solid phase support of the invention can also include an optional spacer group.", "The spacer group can serve to provide the connection between the solid support and a linker.", "The spacer can function to tether the linker away from the solid phase support, thereby minimizing the effect of the neighboring solid phase support on the chemical reactivity of the linker.", "The spacer group may consist of a chain of atoms between (to 1,000 atoms in total.", "In some instances, it is desirable for no spacer group to be employed.", "When employed, the spacer group typically consists of an alkyl, cycloalkyl, or aryl grouping of atoms.", "Tills grouping may contain branching and or may contain heteroatoms.", "The spacer group may also consist of a combination of alkyl, cycloalkyl, and aryl.", "As mentioned above, the solid phase supports of the invention can be provided with a derivatized surface and/or linkers, as is known in the art.", "For instance, the surface can be aminomethylated, chloromethylated, or hydroxymethylated.", "Further, the surface can include a rink amide linker, a hydroxymethylphenoxy linker, a trityl alcohol linker, a hyperliabile linker, or a backbone amide linker.", "The solid phase supports useful in the present invention should be substantially insoluble in both organic and aqueous solvents.", "Selection of organic solvent is described below.", "Generally, less than 20% of 1 g of the support should solubilize in 1000 g of an aqueous or organic solvent at 40° C. and atmospheric pressure.", "More typically, less than 15% of 1 g of the support will solubilize in 1000 g of aqueous or organic solvent at 40° C. and atmospheric pressure.", "Preferably, less than 10% of 1 g of the support will solubilize in 1000 g of aqueous or organic solvent at 40° C. and atmospheric pressure.", "An important aspect of the present invention is that the solid support is substantially insoluble in the organic solvents with which it will be used.", "Organic solvents suitable for the present invention include, but are not limited to the ones listed in Table 1 below: TABLE 1 Examples Of Organic Solvents For Use With Solid Phase Supports Alcohols: Methanol, ethanol, isopropanol, n-propanol, n- butanol, iso-butanol, amyl alcohol, hexafluoroisopropyl alcohol, benzyl alcohol, phenol, diethylcne glycol, propylene glycol Ketones Acetone, methyl ethyl ketone, methyl isopropyl ketone, methyl isobutyl ketone, cyclohexanone Halocarbons Dichloromethane, chloroform, trichloroethylene, tetrachloroethylene, [1,1,1]-trichloroethane, trichlorotrifluorethane, carbon tetrachloride), hydrocarbons (pentane, hexane, heptane, octane) Aromatic Benzene, toluene, xylene, m-cresol, hydrocarbons chlorobenzene, trifluoromethyl benzene), amides (dimethyl formamide, dimethyl acetamide, N- methylpyrrolidinone), sulfoxides/sulfones (dimethyl sulfoxides, dimethyl sulfone, sullblanc) Nitriles Acetonitrile, ethyl nitrile ethers (tetrahydrofuran, diethyl ether, [1,4]-dioxane) Organic acids Acetic acid, formic acid Amines Pyridine, aniline, triethanolamine Esters Butyl acetate, ethyl acetate, trimethyl phosphate Nitro Nitromethane, nitrobenzene compounds Reaction Vessel Referring back to the drawings, in a particularly preferred embodiment of the invention, microplate assembly 10 comprises microplate 11 and funnel caps 12.Microplate 11 includes an array of wells 17.FIGS.", "2-5 depict an individual well 17 of the invention, which functions as a receptacle for the solid phase support 14 and any liquid reagent samples 19.The wells 17 of the invention can be shaped like a conventional test tube.", "However, as will become apparent from the following description, the present invention is applicable to a variety of conventional microplate configurations and well shapes.", "With reference to FIG.", "2, funnel caps 12 each comprise a well insert 20 optionally interconnected by perforated web (not shown).", "Each well insert 20 includes sealing plug 28 with attached vent tube 29.Passage 32 extends through vent tube 29 and sealing plug 28.Passage 32 terminates in vent 34 at its lower end.", "Vent tube 29, sealing plug 28 and the interior walls of well 17 form interior volume 30 in which solid phase support 14 is immobilized, and liquid reagent sample 19 can be deposited.", "The solid phase support 14 can be immobilized anywhere within interior volume 30.For instance, as shown in FIG.", "2, the solid phase support 14 can be shaped as a hollowed cylinder, and can be immobilized at the base of vent tube 29 such that the interior passage of the solid phase support 14 is coincident with passage 32 to thereby result in a continuous vent passage which extends the length of the vent tube 29 and the solid phase support 14.Liquid reagent sample 19 can then occupy and remain confined to a liquid-holding space within interior volume 30 for all orientations of well 17 until removed by aspiration, filtration or other removal method known in the art.", "Alternatively, as shown in FIG.", "5, the solid phase support 14 can be immobilized within the upper portion of the interior volume 30 of the well 17.Again, in one embodiment, the solid phase support can be configured as a hollow cylinder, which can be positioned about the exterior circumference of the funnel cap well insert 20.Such a reaction vessel configuration is particularly suited for combination solution phase/solid phase reaction methodologies as described below.", "Manufacturers may readily choose appropriate dimensions for vent caps 12 so that the solid phase support 14 can be in contact with liquid reagent sample 19.Further, sealing plug 28 and its associated vent tube 29 can be shaped so as to prevent loss of liquid reagent sample 19.For instance, the shape and size of vent 34 and passage 32 can be such that it is difficult for liquid to exit passage 32 due to fluid surface tension.", "Therefore, during all but the most violent movements of microplate assembly 10, liquid reagent sample 19 can remain in its liquid-holding space.", "Microplate assembly 10 includes features that make it suitable for use in a variety of processes.", "Passage 32 permit the addition and removal of material to and from the interior volume 30 without requiring that vent caps 12 be removed, altered or otherwise manipulated.", "As shown in FIGS.", "3 and 4, such materials may be added to or removed from wells 17 as a liquid, a gas, or a solid.", "In the later case, of course, the solid must be dimensioned to permit movement through passage 32.As illustrated in FIGS.", "34, liquids may be injected into or removed from wells 17 with the aid of injection probe 24.Likewise, solids, e.g., pellets or a powder, may also be deposited or removed via passages 32.Gases may also be directed into wells 17 via passages 32 using probes or other gas injection apparatus to provide, for example, a special environment in volume 30.Microplate assembly 10 can be used in either manual or automatic processes.", "For instance, passages 32 provide a convenient avenue through which material may be inserted manually into wells 17, with or without the use of a probe or other apparatus.", "In this regard, passages 32 may act as funnels to help lead the material into interior volume 30.On the other hand, most automation processes use one or more probes or needles 24 to add material or remove material via suction.", "In this instance, fluted apertures can aid the automation process by acting as self-centering guides that can easily direct probe 24 into passages 32.A splined probe or one that is narrower than vent 34 will allow venting to occur during liquid injection or aspiration.", "Alternatively, vents 34 may be fabricated with polygonal cross-sections to prevent round probes from inhibiting venting of interior volume 30.Novel Microtiter While the subject invention can be practiced with any suitable automation-compatible reaction vessel, one preferred embodiment employs a novel inert microtiter according to one aspect of the subject invention.", "As indicated above, one aspect of the invention provides a novel, automation-compatible inert chemical reaction vessel, as well as methods for using such a vessel.", "Generally, as shown in FIG.", "7, the reaction vessel 210 according to this aspect of the invention comprises a lower reaction well microplate assembly 211 and an upper inert atmosphere cap 212.The reaction vessel 210 is configured so as to achieve an inert atmosphere by constantly flushing the upper inert atmosphere cap 212 with an outward flow of inert gas while providing for access to the lower reaction well microplate assembly 211.More particularly, the lower reaction well microplate assembly 211 provides individual reaction well volumes 250 while the upper inert atmosphere cap 212 maintains a common gas volume 201.Lower Reaction Well Microplate Assembly The present invention can employ any microplate assembly known in the art as the lower reaction well microplate assembly 211.For instance, a standard microplate assembly can comprises a microplate having a rigid body with a plurality of open wells.", "Commonly available microplates generally embody a unitary molded structure comprising a rigid frame for housing a plurality of open wells arranged in a rectangular array.", "Microplates come in a range of sizes; a well may be sized to hold as high as five milliliters or as low as only a few microliters of liquid.", "In addition, microplates come in a variety of materials, such as polystyrene, polycarbonate, polypropylene, Teflon, glass, ceramics, and quartz.", "Microplates found in many high-throughput systems comprise a 96-well geometry molded into an 8×12 rectangular array of open wells.", "Microplates with lower well densities (e.g., 24 and 48 wells) and higher well densities (e.g., 384 and 864 wells) are also available.", "In one embodiment, the microplate assembly of the present invention is a spill proof microplate assembly having a plurality of open wells similar to that disclosed in U.S. Pat.", "No.", "6,027,694, which is herein, incorporated by reference.", "Each of the wells comprises a vessel with an interior volume.", "In a preferred embodiment, the lower reaction well microplate assembly 211 comprises a multi-well microplate with an array of individual reaction wells 250, and a plurality of registered funnel vents 260.When the funnel vents 260 are coupled to the individual reaction wells 250, an interior volume 270 is formed in each well 250.The wells are thus configured to accommodate liquid samples within predetermined spaces of the interior volumes 270.The funnel caps 260 are configured such that liquid samples remain within the predetermined space for all orientations of the microplate assembly 211.See U.S. Pat.", "No.", "6,027,694 for further detail on the spill-proof design of funnels vents 260.In one embodiment, the funnel vents 260 comprise vent tubes 280, which can optionally be interconnected by a porous perforated web (not shown).", "The vent tubes 280 terminate in vents 290, which communicate with the interior volumes 270 outside the predetermined spaces which, can accommodate liquid samples.", "The vents 290 permit the pressure within the interior volume 270 to be equalized with the pressure of the common gas volume of the upper inert atmosphere cap 212 via a passage that runs through the vent tube 280.Material may be added to or removed from the wells 250 via the passage through vent tube 280.The optional perforated web 213 can have an adhesive coating which adheres the web to the funnel vents 260 while covering the passages of vent tubes 280, thereby further inhibiting evaporation of the liquid samples.", "Further, the microplate assembly of the invention can be configured as a microfilter plate such that liquid reagent samples can be removed through the filter upon the application of suction, as is known in the art.", "Upper Inert Atmosphere Cap The upper inert atmosphere cap 212 of the invention generally is configured so as to provide a common gas volume 201 above lower reaction well microplate assembly 211 while allowing for access to individual reaction wells 250.In one embodiment, the upper inert atmosphere cap 212 comprises at least one inert gas inlet 203 in communication with common gas volume 201, and a plurality of inert gas outlets 202 in registration with the plurality of funnel caps 260.The inert gas outlets 202 allow for an outward flow of inert gas through outlet vent tube 280 to provide a positive pressure of inert gas in common gas volume 201.Further, when inert gas outlets 202 are in registration with funnel vents 260, an access passageway is provided through outlets 202 and vents 260 into interior volumes 270 of reaction wells 250.The upper inert atmosphere cap 212 is thus configured so as to provide a constant positive pressure of inert gas while allow for access to the lower reaction wells.", "For instance, in an automated setting, robot needle 213 can access interior volume 270 through inert gas outlets 202 and funnel vents 202.In another aspect of the invention, a method for achieving an inert atmosphere in reaction vessel of the invention is provided.", "Such a method generally comprises providing an inert gas flow through inert gas inlet 203 such that an outward gas glow of at least about 5 mm/sec through inert gas outlets 202 is achieved.", "By way of example, when the lower reaction well microplate assembly is configured as a 96-well plate with funnel vents and inert gas outlets having a 2 mm aperture, 6 liters of inert gas per hour is required to maintain a 5 mm/sec outward gas flow.", "Such a gas flow is able to maintain an inert atmosphere within reaction wells 250 for at least 24 hours.", "Solid Phase Adhered to the Interior Walls of the Reaction Wells One aspect of the subject invention provides a general reactor design, which affords many of the advantages inherent in solid phase synthesis, such as ease of purification, and simple work-up, without the difficulties associated with the transfer of solid support materials or devices.", "The reactor takes well-defined structural materials common in reactor design such as but not limited to polypropylene, glass, or Teflon, and incorporates a support material for chemical synthesis.", "These support materials can include but are not limited to many of the common supports used in solid phase synthesis, such as polystyrene based supports like Wang resin, Merrifield resin, Rink resin, REM resin etc, as well as other less commonly used supports such as PEGylated resins, CLEAR resin and other custom designed supports.", "As illustrated in FIG.", "11 the reactor and the resin are fused or bound into a single unit.", "The binding process occurs only at the interior surface of the reactor and provides an interior surface that has very high surface area and incorporates the desired functionalized support material.", "An added advantage to this type of binding process is much more stringent control over the physical characteristics of the synthesis resin.", "For example, swelling upon exposure to various solvents (as well as shrinkage) can be controlled due to the presence of the support material.", "This entire process leaves the exterior surface of the reactor intact, thus ensuring that structural integrity of the reactor remains uncompromised.", "The new reactor design has many additional advantages.", "The simplicity of the formation of the reactor allows for mass production in microtiter plate or similar format, thus providing lower cost.", "It allows for de novo synthesis of compounds in discrete reactors, utilizing the ‘Cherry Picking’ strategy without the need to manually transfer the solid support material or device.", "The binding methodology can be scaled according to reactor design and size.", "Therefore, the reactor or reactors can exist as individual reactors for larger scale work or as an array for discovery type work depending on the scale and the application.", "Thus, simply by using a larger reactor one can use the same chemistry methodology for initial discovery and scale-up.", "Examples and Reaction Methodologies The reaction vessel of the invention can be used to perform solid phase synthesis with solution phase liquid handling automation generally by guiding the tips of injection probes and needles through the funnel cap and into the interior volume of the individual wells of the reaction vessel.", "The solid phase support can be positioned on the funnel cap so as not to interfere with the automation.", "Reagents can be added to the wells of the reaction vessel as if performing a solution phase reaction, but reactions take place on the solid phase support.", "Reaction solutions can then be removed and wash solutions can be added using standard liquid handling automation without the drawbacks associated with traditional resin based solid phase supports.", "In another embodiment of the invention solid phase reaction steps can be combined with solution phase reaction steps.", "For example, the solid phase support can be used in only certain reaction steps, while allowing other reaction steps to proceed in the solution phase.", "More particularly, as shown in FIGS.", "5-6, the solid phase support can be placed near the top of the vent tube of the funnel cap.", "In such an embodiment, an intermediate (or final) reaction product can be formed by solution-phase synthesis with the reaction vessel in an upright position (FIG.", "5).", "The reaction vessel can then be inverted so that the solution phase reaction mixture is contacted with the solid phase support located in the upper portion of the interior volume of the reaction well (FIG.", "6).", "The intermediate (or final) reaction products contained in the solution phase can then be reacted on the solid phase support.", "In this manner, the solid phase support can serve, e.g., as a capture step, a catalyst, or can contain a scaffold template molecule with which the solution phase intermediate reaction product is reacted to forth a final reaction product bound to the solid phase support.", "Alternatively, a solid phase reaction can be performed in a first step, the solid phase reaction product can be cleaved from the solid phase support, the reaction vessel can be inverted, and a solution phase reaction can be performed with the cleaved solid phase reaction product as a starting material.", "By way of example, such a combination reaction methodology can be used with covalent scavenger technology, to immobilize solid phase catalyst for use in only certain reaction steps, or as a capture step to remove unreacted reagents or reaction products.", "The combined liquid/solid phase capability in a high through put synthesis context as provided by the present invention is greatly advantageous in that more complex synthesis mechanisms can now be explored thereby allowing more versatility in the synthesis of novel molecules.", "The present invention is also advantageous in that it provides a tool for synthesizing novel molecules with increased speed.", "The advantages of the present invention are particularly beneficial in the context of the rational design of new molecules de novo where feasible and economical synthesis pathways are not readily available.", "It is expected that the present invention will reduce the bottleneck impediments associated with present combinatorial chemistry synthesis methods.", "Of course, various other modifications and variations are contemplated and may obviously be resorted to in light of the present disclosure.", "It is to be understood, therefore, that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.", "References [1] R. A. Houghten, Proc.", "Natl.", "Acad.", "Sci 1985, 82, 5131 [2] a) R. M. Valerio, A. M. Bray, N. J. Maegi, Int.", "J. Pept.", "Protein Res.", "1994, 44, 158; b) A. M. Bray, D. S. Chiefari, R. M. Valerio, N. J. Maeji.", "Tetrahedron Lett.", "1995, 36, 5081; c) R. M. Valerio, A. M. Bray, H. Patsiouras, Tetrahendron Lett, 1996, 37, 3019; d) A.", "A. Virgilio, J.", "A. Ellman, J.", "Am Chem.", "Soc.", "1994, 116, 11580; e) B.", "A. Bunin, M. J. Plunkett, J.", "A. Ellman, New J. Chem.", "1997, 21, 125; f) A.", "A. Virgilio, A.", "A. Bray, W. Zhang, L. Trinh, M. Snyder, M. M. Morrissey, J.", "A. Ellman, Tretrahedron 1997, 53, 6635; g) B.", "A. Bunin, M. J. Blunkett Proc.", "Natl.", "Acad.", "Sci.", "USA 1994, 91, 4708; j) B.", "A. Bunin, J.", "A. Ellman, J.Am.", "Chem.", "Soc.", "1992, 114, 10997.", "[3] a) K. C. Nicolaou, N. Winssinger, D. Vourloumis, T. Ohshima, S. Kim, J. Pfefferkorn, J.-Y.", "Xu, T. Li, J.", "Am.", "Chem.", "Soc.", "1998, 120, 10814; b) S. Shi, X.-Y.", "Xiao, A. W. Czarnik, Biotechnol.", "Bioeng.", "1998, 61, 7; c) C. J. Anres, R. T. Swann, K. Grant-Young.", "S. V. D″Andrea, M. S. Desh-pande, Comob.", "Chem.", "High Throughput Screening 1999, 2, 29; d) K. C. Nicolaou, X.-Y.", "Xiao, Z. parandoosh, A. Senyei, M. P. Nova, Angew.", "Chem.", "1995, 107, 2476; Andgew.", "Chem.", "Int.", "Ed.", "Engl.", "1995, 34, 2289.", "[4] Rasoul F, Ercole F, Pham Y, Bui C T, Wu Z, James S N, Trainor R W, Wickham G, Maeji N J. Biopolymers 2000; 55(3): 207-16, Grafted supports in solid-phase synthesis.", "Mimotopes Pty Ltd., 11 Duerdin Street, Clayton, Victoria, 3168, Australia.", "[5] Patent application GB99/03406 Porvair Sciences Ltd." ] ]	Patent_10466867
[ [ "Setting real time clocks in communications netwroks", "A request requesting a real time clock (RTC) value in a communications network is sent from a network element to a management system via a data communications network.", "The time taken from the sending of the request to the receipt of the RTC value is compared with a predetermined maximum and, if less than or equal to the maximum, the network element RTC is updated.", "If above the maximum, the RTC value is discarded, and a fresh request is sent.", "The received acceptable RTC value may be corrected by subtracting either the minimum transmission time or half the actual transmission time." ], [ "1.A method of setting a real time clock in a network element of a data communications network having a management system communicating with the network element across the network, the method characterised by: sending a message from the network element to the management system requesting a real time clock (RTC) value; receiving the RTC value at the network element; measuring the time taken between the sending of the RTC request message and receipt of the RTC value; comparing the measured time with a predetermined acceptable time; and if the measured value is acceptable: setting the network element real time clock with the received value.", "2.A method according to claim 1, wherein if the comparison of the measured value and the acceptable value shows the measured value to be unacceptable, the received RTC value is rejected and a further RTC value request message is sent by the network to the management system.", "3.A method according to claim 2, wherein upon determination that a received RTC value is unacceptable, the network element compares the number of unacceptable values received with a maximum permissible number and, if the number of unacceptable values received equals the maximum permissible number, sends an alarm to the management system.", "4.A method according to any preceding claim, wherein the step of setting the network element real time clock with an acceptable received value comprises correcting the received value to compensate for transmission time.", "5.A method according to claim 4, wherein the step of correcting the received RTC value comprises subtracting a minimum transmission time from the received value.", "6.A method according to claim 4, wherein the step of correcting the received RTC value comprises subtracting half the measured time taken between sending of the RTC request message and receipt of the RTC value.", "7.A network element for a data communications network having a management system communicating with the network element across the network, the network element comprising: a real time clock; a message generator for generating and sending real time clock (RTC) value requests to the management system; a message receiver for receiving requested RTC values from the management system; a timer for measuring the time between sending of an RTC request message and receipt of the RTC value; a comparator for comparing the measured time with a predetermined acceptable time; and means for setting the network element real time clock with the received RTC value if that value is acceptable.", "8.A data communications system comprising at least one network element and a management system, the network element and the management system communicating across the communications network; the system comprising at the network element: a real time clock set by the management system; a message generator for generating and sending real time clock (RTC) value requests to the management system; a message receiver for receiving requested RTC values from the management system; a timer for measuring the time between sending of an RTC request message and receipt of the RTC value; a comparator for comparing the measured time with a predetermined acceptable time; and means for setting the network element real time clock with the received value if that value is acceptable; and at the management system: means for receiving RTC value request messages from the network element and for sending RTC values to the network element in response thereto.", "9.A network element or data communications system according to claim 7 or claim 8, and further comprising: means for rejecting the received RTC value if the measured time is unacceptable.", "10.A network element or data communications system according to claim 9, wherein the network element comprises a further comparator for comparing the number of unacceptable received RTC values with a predetermined maximum, and an alarm generator for generating and sending an alarm to the management system if the predetermined maximum number of unacceptable values has been reached.", "11.A network element or data communications system according to any of claims 7 to 10, wherein the network element comprises a real time clock value corrector for correcting received acceptable real time clock values to compensate for transmission time from the management system.", "12.A network element or data communications system according to claim 11, wherein the real time clock value corrector comprises a subtractor for subtracting from the acceptable RTC value the minimum time between sending the RTC value request message and receiving the RTC value.", "13.A network element or data communications system according to claim 11, wherein the real time clock value corrector comprises a subtractor for subtracting half the measured time taken between sending of the RTC request message and receipt of the RTC value.", "14.A method of setting a real time clock in a network element of a data communications network comprising: requesting a real time clock value RTC from a remote RTC source; measuring the period between the RTC request and receipt of an RTC value; and updating the real time clock of the network value if the measured period is within a predetermined acceptable range." ], [ "This invention relates to communications networks, and in particular to the setting of real time clocks in such networks.", "Many networks include a management layer that is used to control and configure elements within the network.", "An example of such a network is the Synchronous Digital Hierarchy (SDH) transmission network.", "FIG.", "1 shows a typical situation within such a network.", "A management system 10 and network elements 12, 14 communicate with each other over a data communications network (DCN) 16.The management system carries out a number of functions including configuration, collection of performance metrics and collection of alarm states.", "In order to enable robust communications, and allow for correct routing of data, standard protocols such as OSI (open systems interconnect) and TCP/IP (transmission control protocol/internet protocol) are used across the network.", "The network which carries the management layer may be completely independent of the network elements, referred to as out of band, or carried within the traffic that they process, referred to as in band.", "In the example of SDH, this is typically carried in the data communications channel (DCC) within the traffic section overhead (SOH) as defined in the SDH standards.", "Where the management system is collecting performance and alarm state data it is important to know either over what time period the data was collected and/or the precise time a given event occurred.", "This is particularly important when verifying the quality of service delivered to customers and when fault finding in the network.", "In the case of alarms, it is important to correlate accurately alarms to assist in fault finding to establish quickly any traffic loss impact to specific customers.", "Management systems typically have a Global Positioning System (GPS) link 18 (FIG.", "1) to provide a highly accurate time reference.", "This enables the time, as a real time clock, to be set within the network elements so that the time that performance data or alarms were collected over can be specified accurately.", "Once obtained from the GPS, the management system distributes the time over the DCN to all the network elements.", "Depending on the running accuracy of the real time clock (RTC) within the network element, the management system can update the network element RTC at regular intervals which, typically, vary from hours to days as appropriate.", "It is well understood in the art that there are problems associated with setting the real time clock accurately within network elements.", "These problems arise for three main reasons: transit delays over the data communications network; setting delay when the time is received at a given network element; and data communications network protocol re-transmissions.", "The first of these, transit delays, are due to the routing of the messages over the DCN.", "In a large network, messages may travel over a number of elements each of which stores, processes and forwards the message.", "The transit delay is typically small, being in the order of 5 ms. As the transit delay is fairly stable and predictable it can be compensated for by simple subtraction.", "The setting delay only applies at the receiving network element and is the time taken for the termination of the received message and the setting of the internal RTC.", "As with the transit delay this is controllable and can be reduced to a few hundred ms by giving the RTC setting a high software priority.", "The most problematic source of delay is in DCN protocol re-transmissions.", "The DCN network is robust and caters for message transmission failures.", "The protocols used, typically in the transport layer, allow for the detection of failure and the re-transmission of messages.", "Since failures are likely to be caused by a temporary overload in the DCN network, the retransmission protocols have a back-off mechanism in which there is a waiting time before re-transmission.", "This may be appreciated from a consideration of FIG.", "2 which is a schematic timing diagram showing the sequence of events from the management system, data communications network and network element.", "A message 20 is sent by the management system to the network element via the DCN.", "The first three attempts by the DCN to send the message received from the management system on to the network element fail and the message is only sent on the fourth attempt.", "The delay between successive attempts increases to increase the chances of success.", "Thus, the delay between the first and second attempts is four seconds; between the second and third attempts is eight seconds; and between the third and fourth attempts is 16 seconds giving rise to a total delay of 28 seconds.", "The most significant problem arising from this delay is that neither the management system nor the network element has knowledge of it as re-transmission is handled by protocols within the DCN.", "Moreover, the message re-transmitted by the DCN protocol is always the same message as supplied by the management system.", "Thus, in the example given, the time value received at the network element will be 28 seconds slow with respect to real time.", "As a result of this delay accumulation, network elements in a large network can vary in time value by a large range.", "This may be up to 64 seconds depending on the re-transmission times set for the DCN protocols.", "Many attempts have been made to solve the above discussed problem.", "However, none of the solutions proposed are satisfactory.", "The existing attempts at solving the problem rely on a constant requesting and comparing of times across the network by the management or main control system.", "Differences are calculated and adjustments made to times sent as required.", "Such an approach works well on a reasonably controlled network, but reliability is reduced over large networks, where re-transmission may occur often.", "They are also complex to run on large networks.", "Other solutions proposed rely on sending out some form of RTC indication with the traffic carried by the network elements.", "This propagates over the network quickly allowing for accurate RTC settings.", "However, such an approach uses up valuable traffic bandwidth and requires a complex interconnection of traffic to ensure that all network elements within a network receive it.", "The invention aims, therefore, to provide an improved approach to setting real time clocks which overcomes or ameliorates the disadvantages mentioned above.", "In its broadest form, the invention overcomes, at least in part, the above mentioned problems by sending RTC request messages from the network element to the RTC source.", "This enables the time for the round trip to be monitored.", "If it is not within a predefined range, the RTC value received is rejected and a fresh request made.", "More specifically, there is provided a method of setting a real time clock in a network element of a data communications network having a management system communicating with the network element across the network, the method characterised by: sending a message from the network element to the management system requesting a real time clock (RTC) value; receiving the RTC value at the network element; measuring the time taken between the sending of the RTC request message and receipt of the RTC value; comparing the measured time with a predetermined acceptable time; and if the measured value is acceptable: setting the network element real time clock with the received value.", "The invention also provides a network element for a data communications network having a management system communicating with the network element across the network, the network element comprising: a real time clock; a message generator for generating and sending real time clock (RTC) value requests to the management system; a message receiver for receiving requested RTC values from the management system; a timer for measuring the time between sending of an RTC request message and receipt of the RTC value; a comparator for comparing the measured time with a predetermined acceptable time; and means for setting the network element real time clock with the received RTC value if that value is acceptable.", "The invention further provides a data communications system comprising: at least one network element and a management system, the network element and the management system communicating across the communications network; characterised in that the system comprising at the network element: a real time clock set by the management system; a message generator for generating and sending real time clock (RTC) value requests to the management system; a message receiver for receiving requested RTC values from the management system; a timer for measuring the time between sending of an RTC request message and receipt of the RTC value; a comparator for comparing the measured time with a predetermined acceptable time; and means for setting the network element real time clock with the received value if that value is acceptable; and at the management system: means for receiving RTC value request messages from the network element and for sending RTC values to the network element in response thereto.", "The invention still further provides a method of setting a real time clock in a network element of a data communications network comprising: requesting a real time clock value RTC from a remote RTC source; measuring the period between the RTC request and receipt of an RTC value; and updating the real time clock of the network value if the measured period is within a predetermined acceptable range.", "Embodiments of the invention have the advantage that by departing from the prior art arrangement of the management system sending out requests without the knowledge of the network element, the time taken to receive the RTC value can be measured.", "If it is too high, the RTC value can be discarded.", "Preferably, if the number of discarded RTC values exceeds a predetermined number, an alarm is sent to the management system.", "This has the advantage of alerting the management system to a persistent fault or problem.", "Preferably, the acceptable RTC values are modified to take into account the transmission time from the management system.", "In one preferred embodiment this is achieved by subtracting the minimum transmission time and in another preferred embodiment by subtracting half the measured transmission time.", "This has the advantage that the real time clock set at the network element is more accurate.", "Embodiments of the invention will now be described, by way of example only, and with reference to the accompanying drawings, in which: FIG.", "1, discussed earlier, is a schematic view of a typical network configuration; FIG.", "2, discussed earlier, shows how a message sent from the management system to a network element can accumulate a substantial delay; FIG.", "3 illustrates the principle of the present invention in terms of message flow between management system and network element; FIG.", "4 shows how the FIG.", "3 example works with message re-transmission from network element to management system; FIG.", "5 is a similar view to FIG.", "4 but with message re-transmission from management system to network element; FIG.", "6 is a flow chart showing the steps occurring at the network element; FIG.", "7 is a flow chart showing a first method of correcting the received RTC; and FIG.", "8 is a flow chart showing a second method of correcting the received RTC.", "The inventors have appreciated that the problems in the prior art systems discussed above arise because the management system and network element have no knowledge of the re-transmission within the DCN network.", "This is because messages are sent to the network element over the DCN and a reply awaited.", "This ensures reliable transmission but delays occurring within the DCN are undetectable.", "In the embodiment to be described, the process is reversed.", "Rather than sending RTC settings from the management system at some predetermined time, the RTC is sent in response to a specific request from the network element.", "The network element can time the period between the issue of the response and the receipt of the RTC and determine the validity of the RTC value by comparing the measured period with the accuracy required within the network element.", "The control of the sequence is thus within the network element, which can control and monitor the entire process.", "As an additional benefit, processing issues related to the extra work involved are distributed across the network rather than handled by one process such as in the management system.", "The network element requests the RTC value from the management system and times the period until receipt.", "This is a relative time and is not related to any inherent accuracy of the current RTC setting, but only the timing of a given period of time.", "Thus, the validity of the RTC time value can be determined.", "If the time taken for the response exceeds a maximum acceptable, the network element sends another request after a back off period to allow the DCN to clear.", "FIG.", "3 shows how this works for a successful request with no re-transmission.", "It may be assumed, for the purposes of explanation, that the acceptable accuracy of the RTC is to within 5 seconds.", "That is, the time taken between the network element issuing an RTC request to the management system and the receipt of that RTC by the network element must not exceed 5 seconds.", "Each of the propagation legs will have a predictable delay.", "It may be assumed that the transit time between the network element and the management system is 300 ms in both directions and that the management system has a response time of 500 ms.", "Thus, in FIG.", "3 at 30 the network element starts a timer and sends an RTC request to the management system across the data communications network.", "The first attempt to send the message succeeds as does the response from the management system to the network element.", "The timer is stopped when the RTC is received at the network element at 32.In this case the measured time will be 300 ms+500 ms+300 ms=1.1 seconds.", "This is within the acceptable accuracy of 5 seconds and so the network element will accept it.", "In FIGS.", "4 and 5, the sequences are shown where there are re-transmissions.", "In FIG.", "4, the re-transmission is from the network element to the management system.", "In the example shown the first attempt fails and the second attempt, 4 seconds later, succeeds.", "The total time for the RTC to reach the management system is (300×2 ms)+400 ms=4600 ms (4.6 seconds).", "The return path from the management system is successful at the first attempt so that the total time between starting and stopping the timer is 4600 ms+500 ms management response time+300 ms management system to network element propagation time.", "This gives a total of 5.4 seconds.", "This time is unacceptable and the network element will therefore reject the RTC value and send a new request.", "It will be appreciated that in the FIG.", "4 example the RTC received is accurate as there is no delay between the management system and the network element beyond the normal 300 ms delay.", "However, the network element can only measure the total time for the round trip and cannot determine where the delay has occurred.", "The network element must therefore reject the received RTC value.", "FIG.", "5 differs from FIG.", "4 only in that the RTC request sent from the network element to the management system is successful at the first attempt but the RTC message sent back to the network element is successful only at the second attempt, inserting a 4 second additional delay.", "The total time recorded by the network element timer is again 5.4 seconds and the RTC value must be rejected.", "In this example it will be appreciated that the RTC value actually is inaccurate as the substantial delay has occurred after it was sent by the management system.", "Where a received RTC is rejected, the network element continues to use its existing RTC until an RTC request is answered within the acceptable time period.", "After the network element has rejected a predetermined number of consecutive RTC values, a time-out will occur and the network element will raise an alarm to the management system to allow intervention by the management system.", "FIG.", "6 is a flow chart showing the steps in the process at the network element.", "At step 100 the network element sends out the RTC value to the management system and starts the timer.", "At step 102 the network element records the RTC value and stops the timer.", "At step 104 the network element compares the elapsed time.", "If it is within or equal to the predetermined maximum, the network element resets its RTC at 106.If the elapsed time is greater than the predetermined maximum, the system rejects the RTC value at 108 and the process loops back to the beginning with a fresh RTC request being sent.", "Following rejection of the RTC, a rejection counter is incremented at 110 and at 112 the value of the counter is compared with a predetermined number.", "If the counter value is equal to that number an alarm is sent to the management system at 114.The network element knows the time taken to receive the RTC message from the management system.", "This knowledge can be used to correct the real time clock.", "A first method is to define the minimum response time achievable.", "In the example given above, this is likely to be about 1 second.", "The network element then adds 1 second to the received time to compensate for the time taken to receive the message.", "A second method bases the correction on the period of time taken for the message to be received.", "The network element adds half the total time taken to receive the response to reduce any error caused by transmission through the network.", "If the delay occurs on the return leg, the FIG.", "5 example, but the total time is within the acceptable maximum, the error could be increased slightly but still within the acceptable limit.", "These two alternatives are illustrated in the flow diagrams of FIGS.", "7 and 8 which expand the reset network element step 106 of FIG.", "6.In FIG.", "7, the acceptable RTC is received at step 200.At 210 the minimum response time is subtracted and at 212 the network element RTC is updated.", "In FIG.", "8 the acceptable RTC is received at 300.At 302 the time counter is halved and at 304 substracted from the received RTC.", "At 306 the new value is used to update the RTC.", "Many variations and modifications to the embodiments described are possible and will occur to those in the art without departing from the scope of the invention which is defined by the claims appended hereto." ] ]	Patent_10466880
[ [ "Sample carrier", "The invention relates to a sample carrier which is used to receive chemical and/or biological samples, comprising a receiving part (30) having recesses (32) for receiving the samples.", "The recesses (32) are sealed by means of a base part (38).", "The base part (38) is glued to the receiving part (30).", "The receiving part has a slit-shaped recess (44) for receiving excess glue in order to prevent glue from spreading to the outside of the base part (38)." ], [ "1.A sample carrier for receiving chemical and/or biological samples, comprising a receiving part (30) provided with deepened portions (32) for receiving the samples, and a base part (38) closing the deepened portions (32), the base part (38) being connected to the receiving part (30) at connection sites by means of a bonding agent, characterized in that the receiving part (30) and/or the base part (38) are provided, at the connection sites, with at least one recess (44,52,60) for receiving excess bonding agent.", "2.The sample carrier according to claim 1, characterized in that the recess (44) is provided in the edge region of the base part (38) and/or of the receiving part (30).", "3.The sample carrier according to claim 1 or 2, characterized in that the recess (44) is formed by a stepped portion (42) provided in the receiving part (30) outside the deepened portions (32).", "4.The sample carrier according to claim 3, characterized in that the base part (38) extends beyond the stepped portion (42) to form a gap-shaped recess (44).", "5.The sample carrier according to claim 4, characterized in that the gap-shaped recess (44) is formed substantially continuously along the edge of the base part (38).", "6.The sample carrier according to any one of claims 1 to 5, characterized in that the recess (44) is open in the direction of an outer edge of the receiving part (30).", "7.The sample carrier according to any one of claims 1 to 6, characterized in that the base part (38) is permeable to radiation to allow for the examination of the sample.", "8.The sample carrier according to any one of claims 1 to 7, characterized in that the base part (38) comprises a plastic film or glass.", "9.The sample carrier according to any one of claims 1 to 8, characterized in that the thickness of the base part (38) is smaller than 500 μm, particularly smaller than 300 μm.", "10.The sample carrier according to any one of claims 1 to 9, characterized in that the receiving part (30) is provided with centering elements (48) determining the position of the base part." ], [ "The invention relates to a sample carrier for receiving chemical and/or biological samples.", "Sample carriers of the above type comprise a receiving part with deepened portions formed therein.", "The samples provided for examination will be introduced into the deepened portions.", "Part of the deepened portions are formed to extend wholly through the receiving part; therefore, these deepened portions are closed by a base part.", "The base part is provided e.g.", "as a glass plate or a transparent film.", "Normally, the base part is permeable to light of specific wavelengths so that the samples accommodated in the continuous deepened portions can be examined using a microscope or the like.", "Especially for the application of screening methods, the base part is permeable to visible light.", "In examination methods such as the screening method, use is made also of sample carriers wherein the base parts are not transparent to visible light.", "Such sample carriers will then comprise base parts is which are permeable to specific wavelength ranges in which e.g.", "a fluorescence of sample components will occur.", "The sample carriers can be e.g.", "titration plates having their individual deepened portions arranged at uniform distances from each other.", "Particularly, the samples received in the deepened portions are minimum-sized samples of a volume smaller than 1 ml.", "Further, titration plates are already known which are suited for the examination of sample quantities in the microliter or even sub-microliter range.", "Further, the sample carriers can be provided as chips.", "In chip-like sample carriers, the deepened portions are normally formed as one or a plurality of channels, and the chips contain one or a plurality of liquid reservoirs.", "For instance, the chips can be microfluidic chips comprising preferably two reservoirs connected to each other via a channel.", "Between the two reservoirs, a fluid exchange will occur which can be controlled by suitable valves, membranes and/or ion barriers.", "Such chips are useful, for instance, for controlling the mixing behavior of liquids, e.g.", "under the influence of electromagnetic forces.", "To allow for the examination of the behavior of the liquids, the bottom of a liquid reservoir and/or of a chip channel is preferably transparent.", "For this purpose, in turn, a base part is provided in order to close the deepened portions such as e.g.", "the reservoirs or the channel.", "The base part preferably is a glass plate or a transparent film.", "Attachment of the base parts to the receiving part comprising the deepened portions is performed e.g.", "by bonding.", "For this purpose, the receiving part 10 (FIG.", "3) is provided with a rectangular deepened portion 12 into which a glass plate 14 is inserted.", "The glass plate 14 will thus be centered in the rectangular deepened portion 12.The bonding agent is applied e.g.", "by tampon pressure onto the inner side 16 of base part 14 and onto the bottom side 18 of receiving part 10 comprising the deepened portions 20.Since the base part 14 must close the deepened portions 20 tightly, the base part 14 is firmly pressed onto the bottom side 18 of receiving part 10.Further, it has to be safeguarded that bonding agent is applied between all of the webs of the deepened portions 20 serving as connection points.", "In such known sample carriers, bonding agent 22 will be squeezed out along the outer edge of base part 14.This excess bonding agent 22 reaches a lower edge 24 of base part 14.In the above process, it may happen that bonding agent 22 flows so far into the interior region that outer deepened portions 20 will be at least partially covered by the bonding agent 22.This has the consequence that an optical examination of the samples located in these deepened portions 20 is adulterated because the light signal is e.g.", "refracted by the bonding agent.", "Thus, after the base part 14 has been attached by bonding, it is required to clean the base part in the edge region.", "Cleaning is performed e.g.", "by a piece of cloth soaked with alcohol or the like.", "In this case, residues remain on the outer side 24 of base part 14.These will cause adulterations of the measurement results.", "As a result, the outer deepened portions are often unfit for use.", "It is an object of the invention to provide a sample carrier for receiving chemical and/or biological samples wherein a contamination of the base part which would impair the examination of the samples is avoided.", "According to the invention, the above object is achieved by the features of claim 1.The sample carrier comprises a receiving part provided with deepened portions for receiving the samples.", "Connected to the receiving part is a base part closing the deepened portions.", "The connection is performed by bonding attachment of the receiving part to the base part along connection sites.", "For the connection sites, use is made e.g.", "of the webs arranged between the deepened portions.", "According to the invention, the receiving part and/or the base part are provided with at least one recess for receiving excess bonding agent.", "The recess can be provided e.g.", "in the manner of bulged or deepened regions in the receiving part's bottom side facing towards the base part.", "Since, according to the invention, it is to be precluded that bonding agent is squeezed out laterally on the edge of the base part and reaches the outer side, the recesses are preferably arranged in the edge region of the base part and/or in the edge region of the receiving part.", "The size and/or the number of the recesses is preferably selected to the effect that the maximum amount of excess bonding agent can be accommodated.", "The provision of the recesses of the invention offers the advantage that no bonding agent will reach the outer side of the base part facing towards the outside.", "Thus, it is precluded that measurement results are adulterated or affected due to layers of bonding agent or residues of bonding agent.", "Further, there is obviated the need for the working step of wiping off the outer side for removing excess bonding agent.", "Thereby, the production costs are reduced and the quality of the sample carriers, particularly with respect to the their optical characteristics, is noticeably improved.", "Preferably, the sample carrier comprises a plurality of recesses arranged in such a manner that, in the critical edge region, a sufficient quantity of excess bonding agent can be received.", "Further, a recess can be provided in the form of a channel closed in itself and surrounding all of the deepened portions along the edge region of the receiving part.", "By way of alternative or additionally, recesses can be provided between the deepened portions.", "This offers the advantage that excess bonding agent will be received also in this region and no disturbing quantities of bonding agent will enter the deepened portions.", "Further, the inventive provision of such recesses has the advantage that the dosage of the bonding agent quantity is allowed to be less precise.", "Preferably, the recess is formed by a stepped portion provided in the receiving part outside of the deepened portions.", "Thus, when assembling the receiving part with the base part, the bonding agent can in the edge region escape away from the edge region.", "Preferably, the stepped portion extends in the manner of a frame around all of the deepened portion so that plastic which is pressed in all four direction towards the outside can escape through the recess formed in the stepped portion.", "If desired, the stepped portion can be interrupted by centering webs used for the centering of the base part during the bonding-attachment process.", "In a preferred embodiment, the base part is configured to project beyond the stepped portion, thus forming a gap-shaped recess.", "This offers the advantage that bonding agent which is pressed into the recess formed by the stepped portion, cannot be pressed into the direction of the outer face of the base part or flow in this direction.", "Instead, the bonding agent is retained in the gap-shaped recess by means of the projecting base part and will harden in the recess.", "This has the additional advantage that the excess bonding agent is used for effecting an additional fixation of the base part to the receiving part.", "Preferably, the gap-shaped recess is formed to extend substantially continuously along the edge of the base part so that, during the bonding attachment of the receiving part, all of the bonding agent which is pressed out into all four directions on the base part can be taken up.", "For safeguarding that also in case of large quantities of excess bonding agent, the latter will not advance to the outer side of the base part, the recess is preferably open in the direction of an outer edge.", "The base part is preferably provided as a base part which is permeable to radiation to allow for the examination of the sample.", "Particularly, the base part is transparent to wavelength ranges used in FCS-spectroscopy and/or to visible light.", "Particularly useful for such purposes is a base part made of glass or a suited plastic film.", "The thickness of the base part is preferably smaller than 500 μm, preferably smaller than 300 μm.", "The invention will be explained in greater detail hereunder with reference to the drawings.", "FIG.", "1 is a schematic bottom view of a sample carrier formed as a titration plate, FIG.", "2 is a schematic sectional view taken along the line II-II in FIG.", "1, FIG.", "3 is a sectional view of a titration plate according to the state of the art, FIG.", "4 is a schematic plan view of a further embodiment of a receiving portion configured according to the invention, FIG.", "5 is a sectional view taken along the line V-V in FIG.", "4, and FIG.", "6 is a schematic plan view of a further embodiment of a receiving part according to the invention.", "For reasons of clarification, the titration plates are illustrated with the base part facing upwards.", "Normally, the titration plates are produced in this orientation.", "A sample carrier comprises a receiving part 30.The receiving part 30 is provided with deepened portions 32 arranged in columns and rows, presenting a matrix-like configuration.", "The deepened portions 32 are substantially cylindrical and extend from an upper side 34 of the receiving part 30 to a bottom side 36 through the receiving part 30.The deepened portions 32 are closed by a base part 38.For this purpose, the base part 38 is attached by bonding onto the bottom side 36 of receiving part 30.The base part 38 is e.g.", "a glass plate having a thickness of less than 500 μm.", "The receiving part comprises a stepped portion 42 formed in the edge region 40 of base part 38.The stepped portion 42 extends in a frame-like manner along the whole outer edge of receiving part 30.The base part 38 is arranged to extend beyond the outer side on all four directions.", "Thus, 30 the edge region 40 of base part 38 substantially does not rest on the outer side 36 of receiving part 30.Because of the projecting portion of base part 38, a gap-shaped recess 44 is formed.", "Along all of its outer edge, the gap 44 extends in the manner of a frame around the recesses 32.Thus, during the bonding attachment of the receiving part 30 to the base part 38, the boding agent 46 applied onto the base part 38 or the outer side 36 of receiving part 30 cannot escape into the gap-shaped recess 44.Further, the illustrated sample carrier is provided with centering elements 48.The centering elements 48 serve for the centering of the base part 38 during the bonding attachment of the base part 38 and the receiving part 30.By the centering elements 48, it is avoided that the base part 38 slides out of position during the bonding process.", "The centering elements can be formed as webs which on the side facing towards the base part 38 are formed with a taper, thus providing substantially only a line-shaped contact between the centering elements 48 and the base part 38.Alternatively, the centering elements can be provided e.g.", "as cylindrical centering pins for placement of the base part 38 therebetween.", "Further, the centering elements 48 can be configured to allow them to be removed after the base part 38 has been bonded to the receiving part 30.For this purpose, the centering elements 48 can be provided with a predetermined weakening line so that they will break off when bent repeatedly back and forth.", "This offers the advantage that, once the centering elements 48 have been removed, the carrier plate will have a plane underside and that no centering elements or other parts of the carrier plate will project from the underside of the carrier plate.", "Further, it is possible to select the height of the centering elements 48 in such a manner that, with the base part 38 inserted, they will not project from the underside of the carrier plate.", "Preferably, the centering elements 48 end at about half the height of base part 38.In the embodiment shown in FIGS.", "4 and 5, only the receiving part 30 is illustrated.", "The receiving part 30 is closed by a base part such as a glass plate, with the base part in FIG.", "5 being arranged above the shown receiving part 30 by being bonded onto the latter.", "The receiving part 30 is provided with deepened portions 32 corresponding to the above described titration plates.", "In the illustrated embodiment, the titration plates are surrounded by annular protrusions 50.The individual annular protrusions 50 do not contact each other in the embodiment shown.", "In this manner, a recess 52 is formed between the annular protrusions 50.The recess 52 is provided to receive bonding agent, as has been the case for recess 44 (FIG.", "2).", "As illustrated in FIG.", "5, the annular projections 50 are formed to have such a cross-sectional configuration that the inner side facing towards the deepened portions 32 comprises a flank or rounded portion 54.Thus, due to the bonded base part 38 (FIG.", "2), the annular projections 50 and the base part 38 have a wedge-shaped slot formed between them into which the bonding agent is sucked by the occurring capillary forces.", "This further contributes to the prevention of a contamination by bonding agent in the base part in the region of the deepened portions 32.The embodiment shown in FIG.", "6 is an embodiment similar to the one shown in FIGS.", "4 and 5, wherein, instead of the annular projections 50, annular projections 58 with a larger width are provided.", "Thereby, adjacent annular projections 58 contact and partially overlap each other, respectively.", "In case of such annular projections 58, rhomboid recesses 60 are formed between the projections for receiving excess bonding agent.", "The edge of the annular projections 58 facing towards the deepened portions 32 can be formed corresponding to the embodiment shown in FIG.", "5.Further, the possibility exists to provide the annular projections 58 with recesses and the like, not illustrated, which also serve for taking up bonding agent." ] ]	Patent_10466883
[ [ "Medical image segmentation apparatus and method thereof", "An apparatus and method for segmenting a medical image are provided.", "The apparatus includes a user interface unit, which provides interface with a user, a volume data input unit, which inputs medical image information; a two-dimensional image display control unit, which receives the volume data, generates and displays a two-dimensional image corresponding to a predetermined position in the medical image, and performs segmentation of the two-dimensional image; and a three-dimensional image display control unit, which receives the volume data, generates and displays a three-dimensional image with respect to the medical image, performs segmentation of the three-dimensional image, transmits the result of the segmentation to the two-dimensional image display control unit, receives position information of the two-dimensional image and the result of the segmentation of the two-dimensional image form the two-dimensional image display control unit, and displaying them on the three-dimensional image." ], [ "1.An apparatus for segmenting a medical image, comprising: a user interface unit, which provides interface with a user; a volume data input unit, which inputs medical image information in the form of volume data expressed as functions defined in a three-dimensional space; a two-dimensional image display control unit, which receives the volume data of a medical image from the volume data input unit, generates and displays a two-dimensional image corresponding to a predetermined position in the medical image, and performs segmentation of the two-dimensional image; a three-dimensional image display control unit, which receives the volume data of the medical image from the volume data input unit, generates and displays a three-dimensional image with respect to the medical image, performs segmentation of the three-dimensional image, transmits the result of the segmentation to the two-dimensional image display control unit, receives position information of the two-dimensional image and the result of the segmentation of the two-dimensional image from the two-dimensional image display control unit, and displaying them on the three-dimensional image; and a controller, which controls the two-dimensional image display control unit and the three-dimensional image display control unit based on a user's operating signals input through the user interface unit.", "2.The apparatus of claim 1, wherein the two-dimensional image display control unit displays the result of segmentation of a three-dimensional image, which is received from the three-dimensional image display control unit, on a currently displayed two-dimensional image.", "3.The apparatus of claim 1, wherein the two-dimensional image display control unit comprises: a two-dimensional segmentation block, which performs segmentation of a two-dimensional image based on selection information selected through the user interface unit; and a common manager, which separately manages operating environment information commonly applied to all types of segmentation for the two-dimensional segmentation block and the results of segmenting two-dimensional images using different types of segmentation.", "4.The apparatus of claim 3, wherein the two-dimensional segmentations block segments an adjacent region to a target region, which is set to be segmented by the selection information, from the two-dimensional image, then sets the segmented adjacent region as a protective region, and then segments the target region.", "5.The apparatus of claim 3, wherein the two-dimensional image display control unit displays further comprises: a modification block, which when a predetermined region in the two-dimensional image is modified based on selection information selected through the user interface unit, performs segmentation only a part of the modified predetermined region that satisfies predetermined modification conditions; and a condition manager, which stores and manages the predetermined modification conditions and provides the predetermined modification conditions to the modification block at the request of the modification block.", "6.A method of segmenting a medical image, comprising: (a) receiving medical image information in the form of volume data expressed as functions defined in a three-dimensional space; (b) generating a two-dimensional image and a three-dimensional image with respect to a medical image from the volume data and displaying the two- and three-dimensional images on a screen; (c) when it is requested to segment a predetermine target region from one of the displayed two- and three-dimensional images per forming segmentation in response to the request and applying the result of the segmentation to both the tow- and three-dimensional images; and (d) when it is requested to modify a predetermined region in the segmented image, performing modification in, response to the request and performing segmentation only a part of the modified region that satisfies predetermined modification conditions.", "7.The method of claim 6, wherein step (c) comprises segmenting an adjacent region to the target region, setting the segmented adjacent region as a protective region, and segmenting the target region.", "8.The method of claim 6, wherein step (c) further comprises when the segmentation is performed on the two-dimensional image, separately storing and managing segmentation information selected for the segmentation of the two-dimensional image in order to refer to it during next two-dimensional segmentation.", "9.The method of claim 6, wherein step (c) further comprises separately storing and managing segmentation information, which is selected for the segmentation, and the result of the segmentation by types of segmentation in order to refer to them during next segmentation." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>In general image segmentation methods, for an image with a particular number of dimensions, segmentation and verification are performed using visualization information of only the particular number of dimensions.", "For example, when a two-dimensional image is segmented, visualization information of only the two-dimensional image is used.", "When a three-dimensional image is segmented, visualization information of only the third-dimensional image is used.", "That is, according to conventional image segmentation methods, although the geometrical characteristics of a segmented region is used for segmentation of a two dimensional image, visualization information of a three-dimensional image is not used for verification of the segmented two-dimensional image.", "Moreover, according to the conventional image segmentation methods, the result of segmentation using visualization information of a three-dimensional image is not applied to segmentation of a two-dimensional image.", "Accordingly, the conventional image segmentation methods are disadvantageous in that a user interface is restrictively provided.", "In addition, according to the conventional image segmentation methods, segmentation tools by segmentation types and a drawing tool for modification operate independently, so segmentation and modification are performed inefficiently.", "In other words, operating environments must be separately set for the individual segmentation tools and drawing tool.", "Since the segmentation and modification results exist independently, a segmentation method using each segmentation tool and the segmentation result are not organically coordinated with each other, so synergy effects cannot be created." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>To solve the above problems, it is an object of the present invention to provided an apparatus and method for segmenting a medical image, in which when an image with a particular number of dimensions is segmented, visualization information of the particular number of dimensions and visualization information of other number of dimensions can be used, and segmentation tools by segmentation types and a drawing tool for modification can operate organically.", "To achieve the above or other objects, there is provided an apparatus—for segmenting a medical image.", "The apparatus includes a user interface unit, which inputs operating signals generated by a user; a volume data input unit, which inputs medical image information in the form of volume data expressed as functions defined in a three-dimensional space; a two-dimensional image display control unit, which receives the volume data of a medical image from the volume data input unit, generates and displays a two-dimensional image corresponding to a predetermined position in the medical image, and performs segmentation of the two-dimensional image; a three-dimensional image display control unit, which receives the volume data of the medical image from the volume data input unit, generates and displays a three-dimensional image with respect to the medical image, performs segmentation of the three-dimensional image, transmits the result of the segmentation to the two-dimensional image display control unit, receives position information of the two-dimensional image and the result of the segmentation of the two-dimensional image from the two-dimensional image display control unit, and displaying them on the three-dimensional image; and a controller, which controls the two-dimensional image display control unit and the three-dimensional image display control unit based on a user's operating signals input through the user interface unit.", "There is also provided a method of segmenting a medical image.", "The method includes receiving medical image information in the form of volume data expressed as functions defined in a three-dimensional space; generating a two-dimensional image and a three-dimensional image with respect to a medical image from the volume data and displaying the two- and three-dimensional images on a screen; when it is requested to segment a predetermine target region from one of the displayed two- and three-dimensional images, performing segmentation in response to the request and applying the result of the segmentation to both the two- and three-dimensional images; and when it is requested to modify a predetermined region in the segmented image, performing modification in response to the request and applying only a part of the modified region that satisfies predetermined modification conditions to the segmented image." ], [ "TECHNICAL FIELD The present invention relates to an image processing technique, and more particularly, to a segmentation method for separating a region of interest from a medical image.", "An image processing technique is a method including a series of processes, such as receiving multi-dimensional image data and separating a region of interest from a background image, extracting feature information from the image data in order to obtain necessary information, or restoring or reprocessing an image in order to improve image quality.", "In this image processing, a technique of separating a region of interest from a background image is referred, to as a segmentation technique.", "This segmentation technique is used to separate a region of interest from a background image and display the region of interest in the form of a two- or three-dimensional image during medical diagnosis or study, thereby increasing the accuracy or efficiency of the result of diagnosis or study.", "Accordingly, the segmentation technique is an essential image processing technique in the field of medical image processing.", "BACKGROUND OF THE INVENTION In general image segmentation methods, for an image with a particular number of dimensions, segmentation and verification are performed using visualization information of only the particular number of dimensions.", "For example, when a two-dimensional image is segmented, visualization information of only the two-dimensional image is used.", "When a three-dimensional image is segmented, visualization information of only the third-dimensional image is used.", "That is, according to conventional image segmentation methods, although the geometrical characteristics of a segmented region is used for segmentation of a two dimensional image, visualization information of a three-dimensional image is not used for verification of the segmented two-dimensional image.", "Moreover, according to the conventional image segmentation methods, the result of segmentation using visualization information of a three-dimensional image is not applied to segmentation of a two-dimensional image.", "Accordingly, the conventional image segmentation methods are disadvantageous in that a user interface is restrictively provided.", "In addition, according to the conventional image segmentation methods, segmentation tools by segmentation types and a drawing tool for modification operate independently, so segmentation and modification are performed inefficiently.", "In other words, operating environments must be separately set for the individual segmentation tools and drawing tool.", "Since the segmentation and modification results exist independently, a segmentation method using each segmentation tool and the segmentation result are not organically coordinated with each other, so synergy effects cannot be created.", "SUMMARY OF THE INVENTION To solve the above problems, it is an object of the present invention to provided an apparatus and method for segmenting a medical image, in which when an image with a particular number of dimensions is segmented, visualization information of the particular number of dimensions and visualization information of other number of dimensions can be used, and segmentation tools by segmentation types and a drawing tool for modification can operate organically.", "To achieve the above or other objects, there is provided an apparatus—for segmenting a medical image.", "The apparatus includes a user interface unit, which inputs operating signals generated by a user; a volume data input unit, which inputs medical image information in the form of volume data expressed as functions defined in a three-dimensional space; a two-dimensional image display control unit, which receives the volume data of a medical image from the volume data input unit, generates and displays a two-dimensional image corresponding to a predetermined position in the medical image, and performs segmentation of the two-dimensional image; a three-dimensional image display control unit, which receives the volume data of the medical image from the volume data input unit, generates and displays a three-dimensional image with respect to the medical image, performs segmentation of the three-dimensional image, transmits the result of the segmentation to the two-dimensional image display control unit, receives position information of the two-dimensional image and the result of the segmentation of the two-dimensional image from the two-dimensional image display control unit, and displaying them on the three-dimensional image; and a controller, which controls the two-dimensional image display control unit and the three-dimensional image display control unit based on a user's operating signals input through the user interface unit.", "There is also provided a method of segmenting a medical image.", "The method includes receiving medical image information in the form of volume data expressed as functions defined in a three-dimensional space; generating a two-dimensional image and a three-dimensional image with respect to a medical image from the volume data and displaying the two- and three-dimensional images on a screen; when it is requested to segment a predetermine target region from one of the displayed two- and three-dimensional images, performing segmentation in response to the request and applying the result of the segmentation to both the two- and three-dimensional images; and when it is requested to modify a predetermined region in the segmented image, performing modification in response to the request and applying only a part of the modified region that satisfies predetermined modification conditions to the segmented image.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a schematic block diagram of an apparatus for segmenting a medical image according to an embodiment of the present invention.", "FIG.", "2 is a schematic block diagram of a two-dimensional image display control unit according to the embodiment of the present invention.", "FIG.", "3 is a schematic block diagram of a three-dimensional image display control unit according to the embodiment of the present invention.", "FIG.", "4 is a flowchart of a method of segmenting a medical image according to an embodiment of the present invention.", "FIG.", "5 is a flowchart of a procedure of performing two-dimensional segmentation according to the embodiment of the present invention.", "FIG.", "6 is a flowchart of a procedure of performing conditional modification according to the embodiment of the present invention.", "FIG.", "7 is a diagram showing an exemplary screen, on which both two- and three-dimensional images are displayed, in an apparatus for segmenting a medical image according to the embodiment of the present invention.", "FIGS.", "8A through 8C are diagrams showing exemplary screens, on which the result of segmenting a three-dimensional image is applied to a two-dimensional image, in an apparatus for segmenting a medical image according to the embodiment of the present invention.", "FIG.", "9 is a diagram showing an exemplary screen for explaining an example in which segmentation information is transferred to two consecutive slice images in an apparatus for segmenting a medical image according to the embodiment of the present invention.", "FIGS.", "10A and 10B are diagrams showing exemplary screens for explaining an example in which options are shared among different segmentation methods in an apparatus for segmenting a medical image according to the embodiment of the present invention.", "FIGS.", "11A and 11B are diagrams showing exemplary screens for explaining an example in which conditional modification is performed in an apparatus for segmenting a medical image according to the embodiment of the present invention.", "FIGS.", "12A through 12E are diagrams showing exemplary screens for explaining an example in which an image is segmented using a region protection function in an apparatus for segmenting a medical image according to the embodiment of the present invention.", "DETAILED DESCRIPTION Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings.", "FIG.", "1 is a schematic block diagram of an apparatus for segmenting a medical image according to an embodiment of the present invention.", "Referring to FIG.", "1, the apparatus for segmenting a medical image includes a user interface unit 100, a controller 200, a volume data input unit 300, a two-dimensional image display control unit 400, and a three-dimensional image display control unit 500.The volume data input unit 300 inputs information on a medical image in the form of volume data expressed as functions defined in a three-dimensional space.", "For example, in a case where volume data, which is generated as the result of computerized tomography (CT) scan or magnetic resonance imaging (MRI), is directly received from each device and stored, the volume data input unit 300 can be composed of connection hardware connected to devices to receive volume data and a hard disc or memory device storing the received volume data.", "Alternatively, in a case where data, which has already been measured and stored in other storage media (for example, a floppy disc and a CD-ROM), is read, the volume data input unit 300 can be composed of connection hardware connected to internal/external storage devices of systems for these storage media to receive the data and a hard disc or memory-device storing the received data.", "The two-dimensional image display control unit 400 receives volume data of a medical image from the volume data input unit 300, generates and displays a two-dimensional image with respect to a predetermined position of the medical image, and performs segmentation of the two-dimensional image.", "Here, the two-dimensional image display control unit 400 receives the result of segmenting a three-dimensional image from the three-dimensional image display control unit 500 and displays the received result on a currently displayed two-dimensional image.", "In other words, the two-dimensional image display control unit 400 receives three-dimensional original image information with respect to a three-dimensional image and the result of segmenting the three-dimensional image and reflects the result of the segmentation on an original image so as to generate a new three-dimensional image.", "Then, the two-dimensional image display control unit 400 generates a two-dimensional image based on information on the new three-dimensional image and displays the generated two-dimensional image.", "In addition, the two-dimensional image display control unit 400 transmits the result of segmenting a two-dimensional image to the three-dimensional image display control unit 500 so that the result of segmenting the two-dimensional image can be applied to a three-dimensional image.", "The three-dimensional image display control unit 500 receives volume data of a medical image from the volume data input unit 300, generates and displays a three-dimensional image with respect to the medical image, performs segmentation of the three-dimensional image, and transmits.", "the result of segmentation to the two-dimensional image display control unit 400.In the meantime, the three-dimensional image display control unit 500 receives position information of a currently displayed two-dimensional image and the result of segmenting the two-dimensional image from the two-dimensional image display control unit 400, deduces three-dimensional image information from the received position information and result of segmentation, and displays the deduced three-dimensional image information on the three-dimensional image.", "In other words, the result of segmenting a two-dimensional image is applied to a three-dimensional image.", "The user interface unit 100 provides interface with a user.", "The controller 200 controls the operations of the two-dimensional image display control unit 400 and the three-dimensional image display control unit 500 according to signals, which are generated by the user's operation and input through the user interface unit 100.FIG.", "2 is a schematic block diagram of the two-dimensional image display control unit 400 according to the embodiment of the present invention.", "Referring to FIG.", "2, the two-dimensional image display control unit 400 includes a two-dimensional image generator 405, a position selector 410, a two-dimensional segmentation block 415, a parameter manager 420, a local parameter buffer 425, a common parameter buffer 430, an object manager 435, an object buffer 440, a protection manager 445, a protective region storage block 450, a drawing tool 455, a conditional parameter buffer 460, a three-dimensional interface 465, and a display controller 470.The three-dimensional interface 465 provides interface for data exchange with the three-dimensional image display control unit 500 shown in FIG.", "1.The two-dimensional image generator 405 generates a two-dimensional image to be displayed on a screen using volume data received from the volume data input unit 300 shown in FIG.", "1, and displays segmentation information, which is obtained from the result of three-dimensional segmentation received through the three-dimensional interface 465, on the currently displayed two-dimensional image.", "In other words, the result of three-dimensional segmentation is applied to the two-dimensional image.", "Since a three-dimensional image has a data structure composed of consecutive two-dimensional images, a k-th two-dimensional image can be derived from the result of three-dimensional segmentation by bringing as much data as an image size from (k−1)*image size.", "In the meantime, when an anatomical region to be segmented is well visualized in a three-dimensional image and easily separated from a background, it is easier to perform three-dimensional segmentation than two-dimensional segmentation.", "For example, the spine and the ribs are well visualized in a three-dimensional image, and when a contrast medium is not injected into the blood vessels, they can be easily separated from a background using a drawing tool because they are positioned at the outer ring of the three-dimensional image.", "The position selector 410 receives slice position information, i.e., a slice number, displayed on a three-dimensional image from the three-dimensional image display control unit 500, converts image data corresponding to the slice number to a bitmap structure, and displays the result of conversion on a screen.", "Here, a slice is a cross section of a three-dimensional image at a predetermined position and is a display unit of a two-dimensional image.", "Accordingly, the position selector 410 selects two-dimensional image information corresponding to slice information received from the three-dimensional image display control unit 500 and displays the selected two-dimensional image information on the screen.", "The two-dimensional segmentation block 415 performs segmentation of a two-dimensional image based on selection information selected through the user interface unit 100 shown in FIG.", "1.For example, if a user selects a particular region on a two-dimensional image using, for example, a mouse, the two-dimensional segmentation block 415 performs segmentation of the selected region.", "Segmentation methods for medical images can be divided into three different types: a method using the features of a region, a method using the features of a boundary, and a method using the features of both a region and a boundary.", "In order to effectively perform an operation, the two-dimensional image display control unit 400 separately manages operating environment information, which is commonly applied to the above-described different segmentation methods, for the two-dimensional segmentation block 415 and the results of two-dimensional segmentation, which are obtained in the respective segmentation methods.", "For this management, the two-dimensional image display control unit 400 according to the embodiment of the present invention shown in FIG.", "2 includes the common parameter buffer 430, which stores and manages common parameters commonly applied to all types of segmentation, and the local parameter buffer 425, which stores and manages local parameters separately applied to the individual types of segmentation.", "The common parameter buffer 430 and the local parameter buffer 425 are controlled by the parameter manager 420.The parameter manager 420 provides the parameters managed by the common parameter buffer 430 and the local parameter buffer 425 to the two-dimensional segmentation block 415 or the drawing tool 455 at the request of the two-dimensional segmentation block 415 or the drawing tool 455.These parameters set for the segmentation may be parameters defining maximum and minimum brightness, maximum intensity, the numbers of morphological dilations and erosions, maximum and minimum thresholds, tolerance, fill holes/no fill holes, a region-of-interest (ROI) type, a paint type, a pint brush width, and a propagation direction.", "Among these parameters, the parameters defining maximum and minimum brightness, maximum intensity, the numbers of morphological dilations and erosions, and maximum and minimum thresholds are the common parameters.", "The parameter defining tolerance is a local parameter applied to only two-dimensional/three-dimensional growth.", "The parameter defining fill holes/no fill holes is a local parameter applied to only thresholding and two-dimensional/three-dimensional growth.", "The parameters defining paint type and paint brush width are local parameters applied to only painting.", "The parameter defining propagation direction is a local parameter applied to only ROI and two-dimensional growth.", "The drawing tool 455 is provided for a user intending to manually modify the result of segmentation performed in response to the user's selection, and operates according to selection information input by the user through the user interface unit 100.The object buffer 440 stores objects used for each type of segmentation.", "The object manager 435 manages the objects stored in the object buffer 440.The object buffer 440 stores segmentation information of the objects in units of slices.", "Segmentation information with respect to one slice is influenced by segmentation information with respect to adjacent slices.", "For example, segmentation information with respect to a previous slice can be appropriately applied to segmentation of a current slice.", "Segmentation information that is consecutively applied to segmentation of the next slice includes the position of a seed point and ROI information.", "In the meantime, when the two-dimensional segmentation block 415 segments a target region selected by a user, an adjacent region to the target region is segmented, then the segmented adjacent region is set as a protective region, and then the target region is segmented, in order to prevent information of the target region from leaking into other regions, that is, to protect the target region.", "For this operation, the two-dimensional image display control unit 400 according to the embodiment of the present invention shown in FIG.", "2 includes the protective region storage block 450 for separately managing regions set as a protective region and the protection manager 445 managing the protective region storage block 450.When the target region is selected through the two-dimensional segmentation block 415, the protection manager 445 detects the position of the boundary of the target region, detects adjacent regions to the position of the boundary, segments the adjacent regions, and sets the segmented adjacent regions as protective regions.", "In other words, the protection manager 445 labels the segmented adjacent regions and stores them as the protective regions.", "Thereafter, if segmentation is requested through the two-dimensional segmentation block 415, the protection manager 445 checks whether a target region of the segmentation is a region that is stored in the protective region storage block 450 and allows the segmentation of the target region only when the target region is not a region stored in the protective region storage block 450.When a two-dimensional image of a predetermined region is modified based on selection information selected through the user interface unit 100, the drawing tool 455 applies only a portion satisfying predetermined modification conditions in the modified region to segmentation.", "In other words, the drawing tool 455 performs conditional modification.", "The conditional parameter buffer 460 stores and manages modification conditions and provides corresponding modification conditions to the drawing tool 455 at the request of the drawing tool 455.For example, brightness is a modification condition.", "For the conditional modification, the drawing tool 455 inspects the conditions of a pixel during modification.", "The display controller 470 controls two-dimensional images resulting from the operations of the two-dimensional image generator 405, the position selector 410, the two-dimensional segmentation block 415, and the drawing tool 455 to be displayed on a screen.", "FIG.", "3 is a schematic block diagram of the three-dimensional image display control unit 500 according to the embodiment of the present invention.", "Referring to FIG.", "3, the three-dimensional image display control unit 500 according to the embodiment of the present invention includes a three-dimensional visualizer 510, a position display block 520, a three-dimensional segmentation block 530, a two-dimensional interface 540, and a display controller 550.The two-dimensional interface 540 provides interface for data exchange with the two-dimensional image display control unit 400.The three-dimensional visualizer 510 generates a three-dimensional image to be displayed on a screen using volume data received from the volume data input unit 300 shown in FIG.", "1, and displays segmentation information, which is obtained from the result of two-dimensional segmentation received through the two-dimensional interface 540, on the currently displayed three-dimensional image.", "In other words, the result of two-dimensional segmentation is applied to the three-dimensional image.", "The result of two-dimensional segmentation is an image having a predetermined image size and is stored in a form of an array which can be displayed.", "In applying the result of two-dimensional segmentation to a three dimensional image, arrays of two-dimensional images are sequentially connected to one another starting from an array of the first two-dimensional image to generate the result of three-dimensional segmentation, and a segmented three-dimensional image is visualized using the result of three-dimensional segmentation.", "Here, only a volume corresponding to the result of three-dimensional segmentation can be selected and visualized as a three-dimensional image, or the volume data except the volume corresponding to the result of three-dimensional segmentation can be selected and visualized as a three-dimensional image.", "As a visualization engine used to display a three-dimensional image on a screen, a volume rendering (VR) engine, a maximum intensity projection (MIP) engine, or a minimum intensity projection (MinIP) engine can be used.", "The VIR engine makes an image, which is formed by performing volume rendering, such as ray tracing, on three-dimensional data, into a bitmap and shows the bitmap on a screen.", "The MIP engine makes a bitmap on the basis of the brightness of a pixel on a screen, on which among all three-dimensional voxels meeting a ray during ray tracing, a voxel having a maximum brightness is projected, and displays the bitmap on the screen.", "The MinIP engine makes a bitmap on the basis of the brightness of a pixel on a screen, on which lo among all three-dimensional voxels meeting a ray during ray tracing, a voxel having a minimum brightness is projected, and displays the bitmap on the screen.", "The position display block 520 displays the position of a two-dimensional image of a slice, which is displayed on the two-dimensional image display control unit 400, as a straight line on the three-dimensional image.", "The position display block 520 exchanges slice position information (i.e., slice numbers) with the position selector 410 shown in FIG.", "2 of the two-dimensional image display control unit 400.Accordingly, when an image displayed in the two-dimensional image display control unit 400 is changed, the position display block 520 moves a position display straight line on the three-dimensional image based on slice position information of the changed two-dimensional image.", "When the position display block 520 moves a position display straight line, an image displayed in the two-dimensional image display control unit 400 is changed based on slice position information, which is generated upon moving the position display straight line.", "The three-dimensional segmentation block 530 performs segmentation of a three-dimensional image based on selection information selected through the user interface unit 100 shown in FIG.", "1.For example, if a user selects a particular region on a three-dimensional image using, for example, a mouse, the three-dimensional segmentation block 530 segments the selected region.", "Here, the three-dimensional segmentation block 530 performs segmentation using the properties of three-dimensional volume itself and stores the result of the segmentation in an array of segmented results.", "In other words, the three-dimensional segmentation block 530 segments a three-dimensional volume satisfying connectivity and homogeneity properties as a separated object and stores the ID of the segmented object at a position corresponding to the object in the array of the results of three-dimensional segmentation.", "In addition, the three-dimensional segmentation block 530 transmits the result of three-dimensional segmentation to the two-dimensional image display control unit 400 so that the result of three-dimensional segmentation can be applied to a two-dimensional image.", "Accordingly, the two-dimensional interface 540 transmits two-dimensional image information from the two-dimensional image display control unit 400 to the three-dimensional visualizer 510, transmits two-dimensional slice position information from the two-dimensional image display control unit 400 to the position display block 520, and transmits the result of three-dimensional segmentation from the three-dimensional segmentation block 530 to the two-dimensional image display control unit 400.The display controller 550 controls three-dimensional images, which result from the operations of the three-dimensional visualizer 510, the position display block 520, and the three-dimensional segmentation block 530, to be displayed on a screen.", "FIG.", "4 is a flowchart of a method of segmenting a medical image according to an embodiment of the present invention.", "Referring to FIG.", "1f medical image information to be segmented is input in the form of volume data expressed as a function of three-dimensional position in step S100, two- and three-dimensional images are detected from the medical image information and displayed through separate display blocks in step S110.Here, the volume data is generated as the result of CT scan or MRI.", "In the meantime, specific procedures for detecting the two- and three-dimensional images from the medical image information have been widely known, and thus detailed descriptions thereof will be omitted.", "If segmentation of a predetermined region from one of the two- and three-dimensional images is requested in step S120, segmentation is performed and the result of the segmentation is applied to both the two- and three-dimensional images in steps S130 through S190.More specifically, if a user selects the predetermined region as a target region to be segmented and requests segmentation of the target region in step S120, it is determined which image the target region is selected from in step S130.If the target region is determined as being selected from the two-dimensional image, two-dimensional segmentation is performed in step S140.If the target region is determined as being selected from the three-dimensional image, three-dimensional segmentation is performed in step S170.The result of the two-dimensional segmentation and the result of the three-dimensional segmentation are respectively applied to the images with different numbers of dimensions in steps S150 and S180.In other words, the result of the two-dimensional segmentation is applied to the three-dimensional image in step S150, and the result of the three-dimensional segmentation is applied to the two-dimensional image in step S180.When the two-dimensional segmentation is performed, segmentation information is separately stored and managed in units of slices in step S160.When a slice is selected in order to segment a two dimensional image, segmentation information of a previous slice to the selected slice can be referred into for segmentation of the selected slice.", "If the user requests manual modification after the segmentation of the target region is completed, modification is performed.", "Here, a region selected to be modified is not entirely modified, but only a part of the selected region, which satisfies predetermined modification conditions, is modified.", "In other words, conditional modification is performed in step S190.For example, when brightness is selected as a modification condition, only pixels having brightness in a range of the maximum brightness value through the minimum brightness value are modified.", "In other words, only pixels whose brightness satisfies the modification condition are modified.", "FIG.", "5 is a flowchart of step S140 of performing two-dimensional segmentation according to the embodiment of the present invention.", "Referring to FIG.", "5, if it is determined that the user selects a protection function for the target region to be segmented in step S141, an adjacent region to the target region is segmented in step S142.The segmented adjacent region is labeled and separately managed as a protective region in step S143.Next, segmentation is performed on the target region in step S144.Here, the adjacent region managed as a protective region in step S142 and S143 is excluded from segmentation.", "Therefore, when the target region is segmented, a region other than the target region is prevented from being included in a segment.", "In FIG.", "5, only step S140 of performing two-dimensional segmentation is described, but the procedure shown in FIG.", "5 can be applied to step S170 of performing three-dimensional segmentation.", "During two-dimensional segmentation and three-dimensional segmentation, if different types of segmentation are performed, segmentation information set for the different types of segmentation is separately managed.", "Although some of the segmentation information set for segmentations is applied to each particular type of segmentation, some of the segmentation information is commonly applied to all types of segmentation.", "Accordingly, by separately managing the segmentation information by the types of segmentation, segmentation can be efficiently performed.", "FIG.", "6 is a flowchart of step S190 of performing conditional modification according to the embodiment of the present invention.", "Referring to FIG.", "6, if it is determined that the user has performed modification in step S191, a part of the modified region satisfying the modification condition is selected in step S192.Only modified content of the selected part is applied to the result of segmentation in step S193.FIGS.", "7 through 12E show exemplary screens for explaining the operations of an apparatus for segmenting a medical image according to the embodiment of the present invention.", "Referring to FIG.", "7 a two-dimensional image is displayed in an image display area on the left of a screen, and a three-dimensional image is displayed in an image display area on the right of the screen.", "Since the two-dimensional image and the three-dimensional image are simultaneously displayed on one screen, a user can increasingly accurately diagnose diseases.", "Referring to FIG.", "8A, multi planar reconstructed (MPR) images obtained from CT scan or MRI are displayed in three divided areas on the right of a screen, and a three-dimensional image with respect to the MPR images on the right is displayed in an area on the left of the screen.", "FIG.", "8B shows an exemplary screen displaying the result of performing segmentation of the three-dimensional image shown in FIG.", "Referring to FIG.", "8B, when a predetermined region of interest is selected from the three-dimensional image shown in FIG.", "8A, only the selected region is segmented from the three-dimensional image and displayed in an areas on the left of the screen.", "FIG.", "8C shows an exemplary screen on which the results of three-dimensional segmentations shown in FIG.", "8B are displayed as an two-dimensional images.", "In FIG.", "8C, two-dimensional images showing cross-sections of the three-dimensional image shown in FIG.", "8B, taken along different horizontal lines, are sequentially displayed.", "FIG.", "9 shows an exemplary screen for explaining an example in which segmentation information is transferred to two consecutive slice images.", "In FIG.", "9, a current slice image is displayed on the left of the screen, and a next slice image is displayed on the right of the screen.", "Referring to FIG.", "9, when a seeded region growing (SRG) method, which is a segmentation method applied to the current slice on the left, is transferred to the next slice on the right, a seed point A set in the left of the screen for performing the SRG method is transferred to the right of the screen, and thus the image on the right is automatically segmented based on the seed point A.", "Referring to FIGS.", "10A and 10B, option parameters set for thresholding segmentation in FIG.", "10A are used for two-dimensional region growing segmentation in FIG.", "10B.", "In other words, the same option values are set in option setup regions B positioned below each medical image display area in FIGS.", "10A and 10B.", "FIG.", "11A shows an exemplary screen when a region of interest is selected, and FIG.", "11B shows an exemplary screen when only a part of 15 the region of interest is applied to segmentation.", "In FIG.", "11A, a white-colored portion is the region of interest, and vertebrae and muscle are included in the region of interest.", "In FIG.", "11B, only the vertebrae are excluded from the region of interest.", "FIGS.", "12A through 12E show exemplary screens for explaining an example in which an image is segmented using a region protection function.", "In other words, FIGS.", "12A through 12E illustrate a case where a region protection function is selected in order to segment only a vertebra area from a medical image.", "FIG.", "12A shows the result of segmenting a blood vessel area connected to the vertebrae.", "FIG.", "12B shows the result of setting the segmented blood vessel area as a protective region using the region protection function.", "FIG.", "12C shows the result of three-dimensionally segmenting the vertebra area using an SRG method.", "FIG.", "12D shows the result of releasing the blood vessel area from the protective region shown in FIG.", "12B.", "FIG.", "12E shows the result of segmenting and removing the vertebra area from the medical image.", "The above description just concerns embodiments of the present invention.", "The present invention is not restricted to the above embodiments, and various modifications can be made thereto within the scope defined by the attached claims.", "For example, the shape and structure of each member specified in the embodiments can be changed.", "INDUSTRIAL APPLICABILITY As described above, in an apparatus and method for segmenting a medical image according to the present invention, when an image with a particular number of dimensions is segmented, visualization information of the particular number of dimensions and visualization information of other number of dimensions can be used, thereby providing wide-ranging user interface.", "For example, when a two-dimensional image is segmented according to the present invention, both visualization information of a two-dimensional image and visualization information of a three-dimensional image can be used.", "In addition, the present invention allows segmentation tools by segmentation types and a drawing tool for modification to operate organically thereby efficiently performing image segmentation and modification.", "For example, operating environments for the segmentation tools by segmentation types and the drawing tool can be simultaneously set, and the processing results by the segmentation tools and the drawing tool are synthetically managed, so a synergy effect can be maximized." ] ]	Patent_10466884
[ [ "Method and circuit for forming an atm cell", "A pressure pulse generator for use in transmitting pressure signals to surface in a fluid-based drilling system having a housing having an inlet to admit drilling fluid to the interior of housing, and an outlet to discharge fluid from the interior of the housing; a control element slidably mounted in the housing between an open and a closed position, the control element generating a pressure pulse in the supply of pressure fluid when the control element takes-up the closed position; a control passage extending through the control elements and closable by a pilot valve element exposed to the pressure of the fluid in the passage; and an actuator coupled with the valve element to generate a pressure signal, to move the valve element to a position closing said passage and thereby to cause movement of the control element towards the closed position; the coupling between the actuator and the valve element includes a yieldable biassing element which provides control of the amplitude of the pressure signals produces by the generator." ], [ "1.A pressure pulse generator for use in transmitting pressure signals to surface in a fluid-based drilling system, said generator being arranged in use in the path of a pressurized fluid to operate a drilling assembly and being capable of being actuated to generate pressure signals in such fluid for transmission to surface pressure monitoring equipment, in which the pulse generator comprises: a housing positionable in the path of the supply of pressurized fluid, said housing having an inlet arrangement for admitting a portion of the fluid to the interior of the housing, and an outlet arrangement for discharging fluid from the interior of the housing for supply of the drilling assembly; a control element slidably mounted in the housing for movement between an open and a closed position with respect to said inlet arrangement, said control element being operative to generate a pressure pulse in the supply of pressure fluid when the control element takes-up the closed position; a control passage extending through the control elements and closable by a pilot valve element arranged to be exposed to the pressure of the fluid in the passage; and an actuator coupled with the valve element and operative, when the pressure generator is activated to generate a pressure signal, to move the valve element to a position closing said passage and thereby to cause movement of the control element towards the closed position; in which the coupling between the actuator and the valve element includes a yieldable biassing element which provides control of the amplitude of the pressure signals produces by the generator.", "2.A pressure pulse generator according to claim 1, in which the biasing element comprises a spring arrangement.", "3.A pressure pulse generator according to claim 1, in which the biassing element comprises a floating piston assembly incorporated within an actuator link between the actuator and the valve element.", "4.A pressure pulse generator according to claim 1 in which the inlet arrangement comprises a ring mounted internally of the housing at an upstream end thereof, and which defines a restricted inlet passage with the valve element.", "5.A pressure pulse generator according to claim 4, in which a set of rings is provided, having different internal bores, and selectable for use according to by-pass requirements.", "6.A pressure pulse generator according to claim 4, in which the ring includes at least one by-pass port.", "7.A pressure pulse generator according to claim 1 in which the actuator comprises an electromagnetic actuator, and the coupling comprises an actuator shaft connected to the actuator a second actuator shaft connected to the valve element, and with said yieldable biassing element (20) located between first actuator shaft and the second actuator shaft." ], [ "This invention relates to a system of communication employed during the drilling of boreholes in the earth for purposes such as oil or gas exploration and production, the preparation of subterranean services ducts, and in other civil engineering applications.", "Taking the drilling of oil and gas wells as an example, it is highly desirable both for economic and for engineering reasons, to obtain information about the progress of the borehole and the strata which the drilling bit is penetrating from instruments positioned near the drilling bit, and to transmit such information back to the surface of the earth without interruption to the drilling of the borehole.", "The generic name associated with such techniques is “Measurement-while-Drilling” (MWD).", "Substantial developments have taken place in MWD technology during the last twenty-five years.", "One of the principal problems in MWD technology is that of reliably telemetering data from the bottom of a borehole, which may lie several thousand metres below the earth's surface.", "There are several established methods for overcoming this problem, one of which is to transmit the data, suitably encoded, as a series of pressure pulses in the drilling fluid; this method is known as “mud pulse telemetry”.", "A typical arrangement of a mud pulse MWD system is shown schematically in FIG.", "1.A drilling rig (50) supports a drillstring (51) in the borehole (52).", "Drilling fluid, which has several important functions in the drilling operation, is drawn from a tank (53) and pumped, by pump (54) down the centre of the drillstring (55) returning by way of the annular space (56) between the drillstring and the borehole (52).", "The MWD equipment (58) that is installed near the drill bit (59) includes a means for generating pressure pulses in the drilling fluid.", "The pressure pulses travel up the centre of the drilistring and are received at the earth's surface by a pressure transducer (57).", "Processing equipment (60) decodes the pulses and recovers the data that was transmitted from downhole.", "In one means of generating pressure pulses at a downhole location, the fluid flowpath through the drillstring is transiently restricted by the operation of a valve.", "This creates a pulse, the leading edge of which is a rise in pressure; hence the method is colloquially, although rather loosely, known as “positive mud pulse telemetry”.", "In contradistinction the term “negative mud pulse telemetry” is used to describe those systems in which a valve transiently opens a passage to the lower pressure environment outside the drillstring, thus generating a pulse having a falling leading edge.", "Devices for the generation of pulses for positive mud pulse telemetry have been described in, for example, U.S. Pat.", "Nos.", "3,958,217, 4,905,778, 4,914,637 and 5,040,155.The above references represent only a few of the very many pulse generating devices that have been developed over a relatively long period of time.", "In U.S. Pat.", "No.", "5,040,155, there is described a type of fluid pulse generator in which the operating energy is derived by creating a pressure drop in the flowing drilling fluid: this differential pressure is used to actuate a main valve element under the control of a pilot valve.", "The present application describes an invention which advantageously controls the amplitude of the pressure pulse in a pulser of a generally similar type to that described in U.S. Pat.", "No.", "5,040,155.According to the invention there is provided a pressure pulse generator as defined in claim 1.The biasing element may comprise a compliant spring or other suitable biasing device, and enables greater control of the amplitude (height) of the pressure signals which are produced, despite the possible variations which occur in practice in the pressure of the fluid which is provided to operate a drilling system.", "In the accompanying drawings: FIG.", "1 is a schematic illustration of a typical drilling installation with which a pressure pulse generator according to the invention may be used; FIG.", "2 is a detailed illustration of a pressure pulse generator of known design, which will be described to provide background to the invention; FIG.", "3 is a view, similar to FIG.", "2, of a preferred embodiment of pressure pulse generator according to the invention; FIG.", "3a is a detail view of part of FIG.", "3 and showing a resilient biassing arrangement provided in a 2 part actuator link extending between an electromagnetic actuator and a pilot valve; and, FIG.", "4 is a detail view of a modified inlet arrangement to the pressure pulse generator of FIG.", "3.First, the basic construction and operation of a known pulse generator will be reviewed with reference to FIG.", "2 of the accompanying drawings.", "This will serve to make clearer the advantages of the invention which will be detailed in the second part of the description, with reference to a preferred embodiment shown in FIG.", "3 of the accompanying drawings.", "FIG.", "2 shows a cross-section of a generally cylindrical pressure pulse generating device.", "The pulse generator 1 is installed in a drill string 2 of which only a part is shown.", "The flow of drilling fluid within the drill string is downwards in relation to the drawing orientation.", "The pressure pulse generator is shown terminated by electrical and mechanical connectors 3 and 4 respectively, for the connection of other pressure housings which would contain, for example, power supplies, instrumentation for acquisition of the data to be transmitted and a means for controlling the operation of the pulse generator itself.", "Such sub-units form a normal part of an MWD system and will not be further described herein.", "The pulse generator has an outer housing designated generally by reference 100 which is mounted and supported in the drill string element 2 by upper and lower centraliser rings 5 and 6 respectively.", "The centralisers have a number, typically three, of radial ribs between an inner and outer ring.", "The spaces between the ribs allow the passage of drilling fluid.", "The ribs may be profiled in such a way as to minimise the effects of fluid erosion.", "The lower centraliser 6 rests on a shoulder 7 in the drill string element.", "A spacer sleeve 8 supports a ring 9 and protects the bore of the drill string element from fluid erosion.", "The ring 9 together with a main valve element 10 define an inlet arrangement to the housing 100 and which will be described in more detail later, and form a significant restriction to the passage of fluid.", "The pulse generator is locked into the drillstring element 2 by conventional means (not shown) to prevent it rotating or reciprocating under the influence of shock and vibration from the drilling operation.", "Considering for the moment only the main flow, drilling fluid, supplied from the previously described storage tanks and pumps at surface, passes the upper centraliser 5, the ring 9, a main valve assembly 11 and the lower centraliser 6 before proceeding downwardly via an outlet arrangement of the housing 100 and towards the drill bit.", "As is well known, the drilling fluid returns to surface by way of the annular space between the drilling assembly and the generally cylindrical wall of the hole being created in the earth by the drill bit.", "The flow of drilling fluid through the restriction formed by the ring 9 and the main valve element 10 creates a significant pressure drop across the restriction.", "The absolute pressure at a point P1 is principally composed of the hydrostatic pressure due to the vertical head of fluid above that point together with the sum of the dynamic pressure losses created by the flowing fluid as it traverses all the remaining parts of the system back to the surface storage tanks.", "There are other minor sources of pressure loss and gain which do not need to be described in detail here.", "It should be noted that the surface pumps are invariably of a positive displacement type and therefore the flow through the system is essentially constant for a given pump speed, provided that the total resistance to flow in the whole system also remains essentially constant.", "Even when the total resistance to flow does change, the consequent change in flow is relatively small, being determined only by the change in the pump efficiency as the discharge pressure is raised or lowered, provided of course that the design capability of the pumps is not exceeded.", "The pressure at a point such as P2 is lower than that at P1 only by the pressure loss in the restriction described above, the change in hydrostatic head being negligible in comparison with the vertical height of the wellbore.", "Although some pressure recovery occurs, as is well known, in the region where the flow area widens out, at 12 in FIG.", "1, the main restriction at the ring 9 and the main valve 10 nevertheless causes a clear pressure differential, proportional approximately to the square of the flow rate, to appear across the points indicated.", "The inner assembly contains an electromagnetic actuator with coil 13, yoke 14, armature 15, and return spring 16.A shaft 17 connects the actuator to a pilot valve element 21, and extends continuously as a solid link from the actuator to the valve element.", "As is customary in apparatus of this kind, there are parts of the assembly that are preferably to be protected from ingress of the drilling fluid, which usually contains a high proportion of particulate matter and is electrically conductive.", "In FIG.", "2 the volumes indicated by the letter F are filled with a suitable fluid such as a mineral oil, and there is communication between these volumes by passageways and clearances not shown in detail.", "It is important for the operation of the pulse generator that the pressure in the oil-filled spaces should be held always equal to that of the drilling fluid surrounding it.", "Were this not so, the differential pressure between the two regions would lead to an unwanted axial force in one or other direction on shaft 17.The compliant element 22 provides this pressure equalising function, as does the compliant bellows 23.Between them these two elements allow the internal volume of the oil-filled space to change, either by expansion of the oil with temperature, or by axial movement of the bellows, without significantly affecting the force acting on shaft 19.This volume-compensated oil fill technique is well known.", "At the top of the pulse generator there is a probe 24 that carries a cylindrical filter element 25.", "(The profile of the top of the probe is designed to allow a retrieval tool to be latched to it, and is not otherwise significant to the subject of this application.)", "There is fluid communication from the inside of the filter 25 through the passages 26, 27, 28 to the orifice 29 immediately above the pilot valve element 21.This fluid is also in communication with the space 30 below the main valve element 10 and the space 31 above the main valve element.", "The main valve element 10 is slideably mounted on the structural parts of the assembly 32, 33, 34.It is to be noted that the effective operating areas, upon which a normally directed force component may cause the valve to move are the ring-shaped areas denoted as A1 and A2 in FIG.", "2.Area A1 is defined by the diameters shown as d1 and d2.Area A2 is defined by the diameters shown as d2 and d3 When fluid flows through the pulse generator, a small portion of the flow bypasses the main flow areas and passes through the filter 25 and the passageways 26, 27, 28 to a pilot valve orifice 29 (closable by movement of the pilot valve 27 under action of the actuator assembly 17, 13, 14, 15, 16).", "Passageway 27 forms a restriction controlling this pilot flow and ensuring that the pressure in passageway 28 is substantially less than the pressure P1.In this condition the pulse generator is inactive.", "The pressure in passageway 28 is communicated both to area A1 and area A2.The areas A1 and A2 are chosen so that the product (pressure in passageway 28)×(A2−A1) is insufficient to overcome the downwardly directed hydrodynamic force, caused by the main fluid flow, and the main valve element 10 remains in its rest position.", "To cause a pressure pulse to be generated in the main flow, the coil 13 is energised and the armature 15 moves upwards.", "This motion is transmitted to the shaft 17 and the pilot valve 21, which is carried upwards until it closes the pilot orifice 29.The closure of the pilot orifice stops the pilot flow and as a result the pressure throughout the set of passageways below the filter element 25 rises to the same value as the pressure at the exterior of the filter, the pressure P1.This pressure is applied to the areas A1 and A2, and since area A2 is substantially larger than A1 a net upwards force is applied to the main valve element 10.This force is sufficient to overcome the hydrodynamic resistance to movement and the valve element 10 moves upwards to increase the restriction offered to flow at the inlet area between it and the ring 9.Because the flow remains essentially constant, as described earlier, the pressure P1 now rises substantially.", "This change in pressure is detectable at the surface of the earth and forms the leading edge of a data pulse.", "When the coil 13 is de-energised, the forces provided by the pressure drop across the pilot valve and by the return spring 16 move the pilot valve back to its rest position.", "The net force on the main valve element is reversed in direction and the valve returns to the quiescent position described earlier.", "The excess pressure is relieved and the pressure change detected at surface forms the trailing edge of the data pulse.", "In the basic form described above the pulse generator operates generally according to the principles described in U.S. Pat.", "No.", "5,040,155.The present invention provides a much improved control of the amplitude of the pressure pulse generated in the wellbore when compared with the prior art, as will now be described, with reference to a preferred embodiment shown in FIG.", "3.This invention is equally applicable when it is used in conjunction with mechanism for improving performance and wear resistance in solids-bearing fluids described in our co-pending UK patent application No 0101802.7.It will be noted that in the basic form of the device described above, the operation of fully closing the pilot valve 21 causes the pressure acting on area A2 to remain the same as the pressure P1.The operation of the main valve causes pressure P1 to increase significantly, as described above, thus increasing the force tending to close the main valve 10.This positive feedback has the effect of largely offsetting the increase in drag forces experienced by the main valve element.", "Consequently the amplitude of the pressure change induced by the operation of the pulse generator tends to increase very substantially with flow rate.", "It is stated in U.S. Pat.", "No.", "5,040,155 that the main valve element can be configured in such a way that when the pulse generator is activated, the main valve element will come to rest in an intermediate position in which the main flow continues to pass through the reduced annular area between the ring 9 and the main valve element 10.This is indeed so, but that fact alone does not determine the final amplitude of the generated pulse.", "It is particularly desirable that a fluid pulse generator for use in MWD applications should provide stable pulsing characteristics over as wide as possible a range of drilling conditions and thus not act as any kind of constraint on the optimisation of such matters as flow rate and drilling fluid properties.", "It is well known in the field of drilling technology that there are many competing engineering factors that determine the choice of conditions for a particular part of a wellbore.", "The presence of instrumentation, such as MWD in the drill string, should have only a minimum effect on the freedom of choice drilling parameters.", "Although in any given drilling situation a certain minimum pulse amplitude is needed so that the pulse will be detectable at the earth's surface, it is unsatisfactory for the pulse to be made too large; the imposition of a succession of severe flow restrictions can stress or damage the drilling equipment and may cause the maximum pressure rating of the surface pumping equipment to be transiently exceeded.", "Furthermore, when mud pressure pulses are too large, significant pulse reflections occur at discontinuities in the process pipework.", "In particular a pulse can return to the lower end of the drillstring, be reflected, return to surface and be detected, incorrectly, as a data pulse.", "In order to keep pulse heights within acceptable limits, some types of pulse generator have to be physically adjusted to suit a particular combination of flow rate and type.", "This typically involves replacing parts of the downhole system, and is time consuming and expensive.", "There are cases too, in which for unexpected reasons, the planned flowrates for a particular well section have to be changed while the equipment is downhole.", "Removing the drilling equipment from the wellbore is generally very time consuming and expensive, and to do so solely to make a change in the operating characteristic of a part of the downhole system would be extremely inefficient.", "It is therefore very desirable to provide a single system which will operate satisfactorily over a wide range of drilling fluid flowrates.", "This makes for simplicity of the equipment and allows for flexibility in the drilling operation.", "In a basic, uncompensated, pulse generating system of the known general type described above, it could be expected that the pulse amplitude would be roughly proportional to the square of the flow rate but with an offsetting effect due to the increased drag force mentioned above.", "In a test of a pulse generator built as described above, the actual amplitude of the pressure pulse varied, to a reasonable approximation, according to (flow rate)1.75.It would not be unreasonable in a practical drilling operation to wish to use the same, unadjusted MWD equipment over a flow range of at least 3:1.With an uncompensated system this implies a pulse pressure range of almost 7:1.With a minimum detectable pulse amplitude of, say, 4 bar (a modest requirement in some deep wells) the amplitude of the generated pulse would become 28 bar at the higher flow rate.", "This is large enough to interfere seriously with the drilling operation and cause excessive wear on the pulse generating equipment.", "Alternatively a desirable pulse amplitude of 4 bar at the maximum flow rate would become 0.6 bar at the lower, which will be insufficient for detection in most circumstances.", "Returning now to the description of operation, the preferred embodiment of the present invention will be described in detail, with reference to FIG.", "3, and parts corresponding to those already described are given the same reference numerals.", "A control element in the form of a spring or other compliant device 20 is interposed between the actuator shaft 17 and the pilot valve head 21 i.e.", "there is no longer a solid link between the actuator and the pilot valve, as in FIG.", "2.Spring 20 is contained in housing 18 and acts against an increased diameter section of a rod 19 connected to the valve 21.Thus even when the coil 13 is energised and the armature 15 is in contact with the yoke 14, the valve 21 can take up an independent position intermediate between its rest position and full closure.", "Therefore, the spring 20 is one example of a resilient biassing means, (provided in an actuator link between the actuator (13, 14, 15) and the pilot valve 21), and which is effective to control the amplitude of the pressure signals produced by the generator as described later.", "When the coil 13 is energised to initiate a pulse, the valve 21 is forced against the seat 29 through the intermediary of the spring 20.The main valve element 10 starts to move upward as previously described, and as it does so the pressure communicated to the valve seat 29 steadily increases, also as previously described.", "This increases the force acting on the valve 21.When that force becomes sufficiently high, the valve 21 is forced off the seat 29 and some flow once again takes place through the valve seat 29 and the passages 26, 27 and 28.The pressure acting on area A2 of the main valve element 10 is now partially relieved, and the force acting on the main valve element 10 is stabilised.", "The valve 21 takes up an equilibrium position in which the forces acting on valve 21 are balanced, on one side by the spring 20 and on the other by the excess pressure created in region P1.This excess pressure is the amplitude of the generated pulse.", "Thus the pulse amplitude can be held essentially constant, and at the level desired for the application, over a wide range of flow rates.", "In practice the events described above occur almost simultaneously.", "The valve 21 does not necessarily close fully and then re-open partially, but may achieve an equilibrium position with only a slight overshoot of that position.", "Also there are cases to be considered in which the main flow rate is too low or too high to fall within the working range of the control system.", "If the flow is too low, the pressure drop (P2−P1) will remain below the control range even during the pulse and the valve 21 will remain completely closed.", "If the flow is too high, the force acting on valve 21 will be great enough to compress control spring 20 fully: no relative movement will take place between valve 21 and valve 29, and no pulse will be generated.", "When the pulse is to be terminated, the coil 13 is de-energised.", "The combined force, due to differential pressure, on pilot valve 21 and the return spring 16 causes the pilot valve 21 to retract.", "Full flow is re-established through the pilot valve and the pressure acting on area A2 falls.", "This restores the original force conditions on the main valve element 10, which now returns to its starting position, and the pressure pulse ends.", "Tests conducted with one embodiment of the invention show that the pulse amplitude is closely controlled over a flow range of at least 3:1.For example in a test running at flows between 150 US gallons per minute and 600 US gallons per minute (570 l/m−2270 l/m) the pulse amplitude variation is no more than 1.5:1 instead of the expected uncompensated range of 7:1, which would be quite unsuitable in practice.", "As an alternative to use of compression spring 20 (for pressure signal amplitude control), the flexible bellows 23 may be replaced by a floating piston assembly (not shown) through which the actuator shaft of the pilot valve extends.", "Although not shown in the drawings, by-pass ports may be provided in the restrictor ring 9 in order to provide a primary pressure drop.", "The by-pass may be used to increase the flow capability, without having to change the size of the main valve parts.", "This-may be important, because it means that the central part of the pulse generator can be exchanged across different pipe bores; only the mounting components have to be changed.", "The relative area of the by-pass ports may be of critical importance in a given flow situation.", "If the by-pass area is too large, there is insufficient initial pressure drop, the operation of the main valve becomes sluggish, and the pulse amplitude too low.", "If the by-pass area is too small, the flow velocity through the main valve becomes too great, causing rapid erosion.", "A number of circumferential by-pass ports, one of which is shown at 9a in FIG.", "4, may be provided and equipped with “lock-in” plugs that can easily be inserted or removed at the well site.", "By selecting the correct number of ports to remain open, the by-pass characteristics may be varied to suit the anticipated conditions." ] ]	Patent_10466984
[ [ "Human hyperpolarization-activated cyclic nucleotide-gated cation channel hcn1", "The present invention is directed to novel human DNA sequences encoding human HCN1 proteins, the protein encoded by the DNA sequences, vectors comprising the DNA sequences, host cells containing the vectors, and methods of identifying inhibitors and activators of cation channels containing the human HCN1 proteins." ], [ "1.An isolated DNA comprising a nucleotide sequence encoding human HCN1.2.The DNA of claim 1 comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, and 18.3.The DNA of claim 1 comprising a nucleotide sequence selected from the group consisting of: SEQ.ID.NO.", ":1, SEQ.ID.NO.", ":3, SEQ.ID.NO.", ":5, SEQ.ID.NO.", ":7, SEQ.ID.NO.", ":9, SEQ.ID.NO.", ":11, SEQ.ID.NO.", ":13, SEQ.ID.NO.", ":15, SEQ.ID.NO.", ":17, positions 26 to 2695 of SEQ.ID.NO.", ":1, positions 26 to 2695 of SEQ.ID.NO.", ":3, positions 26 to 2695 of SEQ.ID.NO.", ":5, positions 26 to 2695 of SEQ.ID.NO.", ":7, positions 26 to 2695 of SEQ.ID.NO.", ":9, positions 26 to 2695 of SEQ.ID.NO.", ":11, positions 26 to 2695 of SEQ.ID.NO.", ":13, positions 26 to 2695 of SEQ.ID.NO.", ":15, and positions 26 to 2695 of SEQ.ID.NO.", ":17.4.An isolated DNA that hybridizes under stringent conditions to the DNA of claim 3 and that encodes a protein having substantially the same biological activity as human HCN1.5.An expression vector comprising the DNA of claim 3.6.A recombinant host cell comprising the DNA of claim 3.7.DNA, substantially free of other nucleic acids, comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, and 18.8.DNA, substantially free of other nucleic acids, comprising a nucleotide sequence selected from the group consisting of: SEQ.ID.NO.", ":1, SEQ.ID.NO.", ":3, SEQ.ID.NO.", ":5, SEQ.ID.NO.", ":7, SEQ.ID.NO.", ":9, SEQ.ID.NO.", ":11, SEQ.ID.NO.", ":13, SEQ.ID.NO.", ":15, SEQ.ID.NO.", ":17, positions 26 to 2695 of SEQ.ID.NO.", ":1, positions 26 to 2695 of SEQ.ID.NO.", ":3, positions 26 to 2695 of SEQ.ID.NO.", ":5, positions 26 to 2695 of SEQ.ID.NO.", ":7, positions 26 to 2695 of SEQ.ID.NO.", ":9, positions 26 to 2695 of SEQ.ID.NO.", ":11, positions 26 to 2695 of SEQ.ID.NO.", ":13, positions 26 to 2695 of SEQ.ID.NO.", ":15, and positions 26 to 2695 of SEQ.ID.NO.", ":17.9.An isolated human HCN1 protein.", "10.The protein of claim 7 comprising an amino acid sequence selected from the group consisting of SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, and 18.11.The protein of claim 8 containing a single amino acid substitution.", "12.The protein of claim 8 containing two or more amino acid substitutions where the amino acid substitutions do not occur in conserved positions.", "13.A protein, substantially free of other proteins, comprising an amino acid sequence selected from the group consisting of SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, and 18.14.An antibody that binds specifically to a human HCN1 protein.", "15.A DNA or RNA oligonucleotide probe comprising at least 10 contiguous nucleotides from SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17.16.A method of identifying substances that bind to cation channels containing human HCN1 protein comprising: (a) providing cells expressing a cation channel containing human HCN1 protein; (b) exposing the cells to a substance that is not known to bind cation channels containing human HCN1 protein; (c) determining the amount of binding of the substance to the cells; (d) comparing the amount of binding in step (c) to the amount of binding of the substance to control cells where the control cells are substantially identical to the cells of step (a) except that the control cells do not express human HCN1 protein; where if the amount of binding in step (c) is greater than the amount of binding of the substance to control cells, then the substance binds to cation channels containing human HCN1 protein.", "17.A method of identifying substances that bind cation channels containing human HCN1 protein comprising: (a) providing cells expressing cation channels containing human HCN1 protein; (b) exposing the cells to a compound that is known to bind to the cation channels containing human HCN1 protein in the presence and in the absence of a substance not known to bind to cation channels containing human HCN1 protein; (c) determining the amount of binding of the compound to the cells in the presence and in the absence of the substance; where if the amount of binding of the compound in the presence of the substance differs from that in the absence of the substance, then the substance binds cation channels containing human HCN1 protein.", "18.A method of identifying activators or inhibitors of cation channels containing human HCN1 protein comprising: (a) recombinantly expressing human HCN1 protein in a host cell so that the recombinantly expressed human HCN1 protein forms cation channels either by itself or by forming heteromers with other cation channel subunit proteins; (b) measuring the biological activity of the cation channels formed in step (a) in the presence and in the absence of a substance not known to be an activator or an inhibitor of cation channels containing human HCN1 protein; where a change in the biological activity of the cation channels formed in step (a) in the presence as compared to the absence of the substance indicates that the substance is an activator or an inhibitor of cation channels containing human HCN1 protein." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The HCN genes encode a family of cation channels that are believed to carry a current known as I h or I q in neural tissue and I f in cardiac tissue.", "This current is activated by hyperpolarization beyond about −50 to −70 mV, does not inactivate, is carried by both Na + and K + , exhibits a small single channel conductance (about 1 pS), and has the effect of slowly depolarizing a cell toward the I h reversal potential of about −30 mV.", "The voltage dependence of I h can be modulated by cyclic nucleotides such as cAMP or cGMP.", "The I h current can contribute significantly to the total current at subthreshold membrane potentials, and thus can be an important factor in the regulation of neuronal firing and cardiac contraction.", "Three major roles for the I h current have been postulated in neurons: (a) I h contributes to the cell's resting membrane potential; (b) I h can modulate the summation of synaptic inputs into the neuron, e.g., by counteracting hyperpolarizing signals from inhibitory postsynaptic potentials; and (c) I h contributes to the generation of “pacemaker” or oscillatory activity (i.e., rhythmic, spontaneous firing of action potentials).", "In the heart, the I f current arises following repolarization of an action potential, which returns the cell to its hyperpolarized resting membrane potential.", "In pacemaker regions of the heart, such as the sinoatrial node, this hyperpolarization activates I f , which leads to a slow depolarization of the myocyte, eventually returning the membrane potential to the action potential threshold, and triggering another action potential.", "The larger the I f current, the more rapid the return to the action potential threshold and the faster the heart will beat.", "Agents that stimulate the heart by stimulating the β-adrenergic receptor act, in part, through the I f current.", "Such agents lead to an increase in intracellular cAMP which shifts the voltage dependence of the If current towards more positive (i.e., depolarized) levels, resulting in faster entry of this current into its role in moving the cell back toward the action potential threshold.", "For reviews of the I h /I f current, see Clapham, 1998, Neuron 21:5-7; Luthi & McCormick, 1998, Neuron 21:9-12; Pape, 1996, Ann.", "Rev.", "Physiol.", "58:299-327; DiFrancesco, 1993, Ann.", "Rev.", "Physiol.", "55:455-472.Certain HCN genes and their encoded protein products have been identified.", "The DNA and deduced amino acid sequences, as well as some electrophysiological properties, of human HCN2 and human HCN4 have been disclosed (Vaccari et al., 1999, Biochim.", "Biophys.", "Acta 1446:419425; Seifert et al., 1999, Proc.", "Natl.", "Acad.", "Sci.", "USA 96:9391-9396; Ludwig et al., 1999, EMBO J.", "18:2323-2329; GenBank accession nos.", "AF065164 and AJ012582 (HCN2); GenBank accession nos.", "AJ132429 and AJ238850 (HCN4)).", "GenBank accession no.", "AF064876 represents a partial, internal fragment of human HCN1, lacking 5′ and 3′ ends.", "GenBank accession no.", "AW054787 represents an EST containing only the carboxy terminal sequences of human HCN1.GenBank accession no.", "AC013384 represents human chromosome 2 genomic DNA sequences that encompass HCN1 but there is no indication of which portion of the disclosed sequence represents HCN1 coding sequence.", "Certain fragments of human HCN3 have appeared in certain databases (GenBank accession no.", "AI571225 is an amino terminal EST; AQ625620 is a partial genomic sequence).", "Full length mouse HCN1, HCN2, and HCN3 have been cloned as has a partial mouse cDNA encoding HCN4 (Santoro et al., 1998, Cell 93:717-729; Ludwig et al., 1998, Nature 393:587-591).", "Mouse (GenBank accession no.", "AJ225123), rat (GenBank accession no.", "AJ247450), and rabbit (GenBank accession no.", "AF168122) HCN1 sequences have been deposited in databases.", "Examination of the cDNAs encoding HCN channels revealed that the HCN proteins represent a family of ion channels having six putative transmembrane domains (S1-S6) and a cAMP binding domain.", "Functional expression of human HCN2 in a kidney cell line produced currents with properties similar to those of the heart I f current (Vaccari et al., 1999, Biochim.", "Biophys.", "Acta 1446:419-425).", "It is desirable to discover as wide a variety as possible of novel cation channels, especially those from humans and those exhibiting restricted tissue expression.", "Such novel cation channels would be attractive targets for drug discovery, useful in counterscreens for a variety of other drug targets, and would be valuable research tools for understanding more about ion channel biology." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed to a novel human DNA sequence encoding human HCN1, a hyperpolarization-activated cyclic nucleotide-gated cation channel.", "The present invention also includes certain polymorphic variants of human HCN1.The present invention includes DNA comprising the nucleotide sequences shown as SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, and 17 as well as DNA comprising the coding regions of SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, and 17.Also provided are proteins encoded by the novel DNA sequences.", "The human HCN1 proteins of the present invention comprise the amino acid sequences shown as SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, and 18 as well as fragments thereof.", "Methods of expressing the novel human HCN1 proteins in recombinant systems are provided.", "Also provided are methods of using human HCN1 as a drug target by identifying activators and inhibitors of cation channels comprising human HCN1 proteins.", "Also provided are methods of using the novel human HCN1 proteins and DNA encoding these HCN1 proteins in counterscreens for assays designed to identify activators and inhibitors of other drug targets." ], [ "CROSS-REFERENCE TO RELATED APPLICATIONS Not applicable.", "STATEMENT REGARDING FEDERALLY-SPONSORED R&D Not applicable.", "REFERENCE TO MICROFICHE APPENDIX Not applicable.", "FIELD OF THE INVENTION The present invention is directed to novel human DNA sequences encoding a hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1), proteins encoded by the DNA sequences, methods of expressing the proteins in recombinant cells, and methods of identifying activators and inhibitors of HCN1.BACKGROUND OF THE INVENTION The HCN genes encode a family of cation channels that are believed to carry a current known as Ih or Iq in neural tissue and If in cardiac tissue.", "This current is activated by hyperpolarization beyond about −50 to −70 mV, does not inactivate, is carried by both Na+ and K+, exhibits a small single channel conductance (about 1 pS), and has the effect of slowly depolarizing a cell toward the Ih reversal potential of about −30 mV.", "The voltage dependence of Ih can be modulated by cyclic nucleotides such as cAMP or cGMP.", "The Ih current can contribute significantly to the total current at subthreshold membrane potentials, and thus can be an important factor in the regulation of neuronal firing and cardiac contraction.", "Three major roles for the Ih current have been postulated in neurons: (a) Ih contributes to the cell's resting membrane potential; (b) Ih can modulate the summation of synaptic inputs into the neuron, e.g., by counteracting hyperpolarizing signals from inhibitory postsynaptic potentials; and (c) Ih contributes to the generation of “pacemaker” or oscillatory activity (i.e., rhythmic, spontaneous firing of action potentials).", "In the heart, the If current arises following repolarization of an action potential, which returns the cell to its hyperpolarized resting membrane potential.", "In pacemaker regions of the heart, such as the sinoatrial node, this hyperpolarization activates If, which leads to a slow depolarization of the myocyte, eventually returning the membrane potential to the action potential threshold, and triggering another action potential.", "The larger the If current, the more rapid the return to the action potential threshold and the faster the heart will beat.", "Agents that stimulate the heart by stimulating the β-adrenergic receptor act, in part, through the If current.", "Such agents lead to an increase in intracellular cAMP which shifts the voltage dependence of the If current towards more positive (i.e., depolarized) levels, resulting in faster entry of this current into its role in moving the cell back toward the action potential threshold.", "For reviews of the Ih/If current, see Clapham, 1998, Neuron 21:5-7; Luthi & McCormick, 1998, Neuron 21:9-12; Pape, 1996, Ann.", "Rev.", "Physiol.", "58:299-327; DiFrancesco, 1993, Ann.", "Rev.", "Physiol.", "55:455-472.Certain HCN genes and their encoded protein products have been identified.", "The DNA and deduced amino acid sequences, as well as some electrophysiological properties, of human HCN2 and human HCN4 have been disclosed (Vaccari et al., 1999, Biochim.", "Biophys.", "Acta 1446:419425; Seifert et al., 1999, Proc.", "Natl.", "Acad.", "Sci.", "USA 96:9391-9396; Ludwig et al., 1999, EMBO J.", "18:2323-2329; GenBank accession nos.", "AF065164 and AJ012582 (HCN2); GenBank accession nos.", "AJ132429 and AJ238850 (HCN4)).", "GenBank accession no.", "AF064876 represents a partial, internal fragment of human HCN1, lacking 5′ and 3′ ends.", "GenBank accession no.", "AW054787 represents an EST containing only the carboxy terminal sequences of human HCN1.GenBank accession no.", "AC013384 represents human chromosome 2 genomic DNA sequences that encompass HCN1 but there is no indication of which portion of the disclosed sequence represents HCN1 coding sequence.", "Certain fragments of human HCN3 have appeared in certain databases (GenBank accession no.", "AI571225 is an amino terminal EST; AQ625620 is a partial genomic sequence).", "Full length mouse HCN1, HCN2, and HCN3 have been cloned as has a partial mouse cDNA encoding HCN4 (Santoro et al., 1998, Cell 93:717-729; Ludwig et al., 1998, Nature 393:587-591).", "Mouse (GenBank accession no.", "AJ225123), rat (GenBank accession no.", "AJ247450), and rabbit (GenBank accession no.", "AF168122) HCN1 sequences have been deposited in databases.", "Examination of the cDNAs encoding HCN channels revealed that the HCN proteins represent a family of ion channels having six putative transmembrane domains (S1-S6) and a cAMP binding domain.", "Functional expression of human HCN2 in a kidney cell line produced currents with properties similar to those of the heart If current (Vaccari et al., 1999, Biochim.", "Biophys.", "Acta 1446:419-425).", "It is desirable to discover as wide a variety as possible of novel cation channels, especially those from humans and those exhibiting restricted tissue expression.", "Such novel cation channels would be attractive targets for drug discovery, useful in counterscreens for a variety of other drug targets, and would be valuable research tools for understanding more about ion channel biology.", "SUMMARY OF THE INVENTION The present invention is directed to a novel human DNA sequence encoding human HCN1, a hyperpolarization-activated cyclic nucleotide-gated cation channel.", "The present invention also includes certain polymorphic variants of human HCN1.The present invention includes DNA comprising the nucleotide sequences shown as SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, and 17 as well as DNA comprising the coding regions of SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, and 17.Also provided are proteins encoded by the novel DNA sequences.", "The human HCN1 proteins of the present invention comprise the amino acid sequences shown as SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, and 18 as well as fragments thereof.", "Methods of expressing the novel human HCN1 proteins in recombinant systems are provided.", "Also provided are methods of using human HCN1 as a drug target by identifying activators and inhibitors of cation channels comprising human HCN1 proteins.", "Also provided are methods of using the novel human HCN1 proteins and DNA encoding these HCN1 proteins in counterscreens for assays designed to identify activators and inhibitors of other drug targets.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1A shows a cDNA sequence encoding human HCN1 (SEQ.ID.NO.", ":1) and FIG.", "1B shows the corresponding amino acid sequence (SEQ.ID.NO.:2).", "The start ATG codon in FIG.", "1A is at position 26-28; the stop codon is at position 2696-2698.FIG.", "2A shows a cDNA sequence encoding human HCN1 with a single nucleotide polymorphism (SEQ.ID.NO.", ":3) as compared to SEQ.ID.NO.", ":1.Position 690 in SEQ.ID.NO.", ":3 is C rather than T as in SEQ.ID.NO.:1.FIG.", "2B shows the amino acid sequence (SEQ.ID.NO.", ":4) encoded by SEQ.ID.NO.:3.SEQ.ID.NO.", ":4 differs from SEQ.ID.NO.", ":2 in having an S rather than an F at position 222.FIG.", "3A shows a cDNA sequence encoding human HCN1 with a single nucleotide polymorphism (SEQ.ID.NO.", ":5) as compared to SEQ.ID.NO.", ":1.Position 1011 in SEQ.ID.NO.", ":5 is A rather than G as in SEQ.ID.NO.:1.FIG.", "3B shows the amino acid sequence (SEQ.ID.NO.", ":6) encoded by SEQ.ID.NO.:5.SEQ.ID.NO.", ":6 differs from SEQ.ID.NO.", ":2 in having a Y rather than a C at position 329.FIG.", "4A shows a cDNA sequence encoding human HCN1 with a single nucleotide polymorphism (SEQ.ID.NO.", ":7) as compared to SEQ.ID.NO.", ":1.Position 1401 in SEQ.ID.NO.", ":7 is G rather than A as in SEQ.ID.NO.:1.FIG.", "4B shows the amino acid sequence (SEQ.ID.NO.", ":8) encoded by SEQ.ID.NO.:7.SEQ.ID.NO.", ":8 differs from SEQ.ID.NO.", ":2 in having a G rather than an E at position 459.FIG.", "5A shows a cDNA sequence encoding human HCN1 with a single nucleotide polymorphism (SEQ.ID.NO.", ":9) as compared to SEQ.ID.NO.", ":1.Position 1532 in SEQ.ID.NO.", ":9 is G rather than A as in SEQ.ID.NO.:1.FIG.", "5B shows the amino acid sequence (SEQ.ID.NO.", ":10) encoded by SEQ.ID.NO.:9.SEQ.ID.NO.", ":10 differs from SEQ.ID.NO.", ":2 in having a V rather than an I at position 503.FIG.", "6A shows a cDNA sequence encoding human HCN1 with a single nucleotide polymorphism (SEQ.ID.NO.", ":11) as compared to SEQ.ID.NO.", ":1.Position 1743 in SEQ.ID.NO.", ":11 is C rather than T as in SEQ.ID.NO.:1.FIG.", "6B shows the amino acid sequence (SEQ.ID.NO.", ":12) encoded by SEQ.ID.NO.:11.SEQ.ID.NO.", ":12 differs from SEQ.ID.NO.", ":2 in having a P rather than an L at position 573.FIG.", "7A shows a cDNA sequence encoding human HCN1 with a single nucleotide polymorphism (SEQ.ID.NO.", ":13) as compared to SEQ.ID.NO.", ":1.Position 1973 in SEQ.ID.NO.", ":13 is G rather than A as in SEQ.ID.NO.:1.FIG.", "7B shows the amino acid sequence (SEQ.ID.NO.", ":14) encoded by SEQ.ID.NO.:13.SEQ.ID.NO.", ":14 differs from SEQ.ID.NO.", ":2 in having an A rather than a T at position 650.FIG.", "8A shows a cDNA sequence encoding human HCN1 with a single nucleotide polymorphism (SEQ.ID.NO.", ":15) as compared to SEQ.ID.NO.", ":1.Position 1997 in SEQ.ID.NO.", ":15 is A rather than T as in SEQ.ID.NO.:1.FIG.", "8B shows the amino acid sequence (SEQ.ID.NO.", ":16) encoded by SEQ.ID.NO.:15.SEQ.ID.NO.", ":16 differs from SEQ.ID.NO.", ":2 in having a T rather than an S at position 658.FIG.", "9A shows a cDNA sequence encoding human HCN1 with a single nucleotide polymorphism (SEQ.ID.NO.", ":17) as compared to SEQ.ID.NO.", ":1.Position 2417 in SEQ.ID.NO.", ":17 is C rather than T as in SEQ.ID.NO.:1.FIG.", "9B shows the amino acid sequence (SEQ.ID.NO.", ":18) encoded by SEQ.ID.NO.:17.SEQ.ID.NO.", ":18 differs from SEQ.ID.NO.", ":2 in having a P rather than an S at position 798.FIG.", "10A-B shows an amino acid sequence alignment of human HCN1 (SEQ.ID.NO.", ":2), rabbit HCN1 (SEQ.ID.NO.", ":21; GenBank accession no.", "AF168122), mouse HCN1 (SEQ.ID.NO.", ":19; GenBank accession no.", "AJ225123), and rat HCN1 (SEQ.ID.NO.", ":20; GenBank accession no.", "AJ247450).", "The consensus sequence is SEQ.ID.NO.", ":22.DETAILED DESCRIPTION OF THE INVENTION For the purposes of this invention: “Substantially free from other proteins” means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other proteins.", "Thus, a human HCN1 protein preparation that is substantially free from other proteins will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of proteins that are not human HCN1 proteins.", "Whether a given human HCN1 protein preparation is substantially free from other proteins can be determined by conventional techniques of assessing protein purity such as, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with appropriate detection methods, e.g., silver staining or immunoblotting.", "“Substantially free from other nucleic acids” means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other nucleic acids.", "Thus, a human HCN1 DNA preparation that is substantially free from other nucleic acids will contain, as a percent of its total nucleic acid, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of nucleic acids that are not human HCN1 nucleic acids.", "Whether a given human HCN1 DNA preparation is substantially free from other nucleic acids can be determined by conventional techniques of assessing nucleic acid purity such as, e.g., agarose gel electrophoresis combined with appropriate staining methods, e.g., ethidium bromide staining.", "A “conservative amino acid substitution” refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue.", "Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid); substitution of one aromatic amino acid (tryptophan, tyrosine, or phenylalanine) for another.", "A polypeptide has “substantially the same biological activity as human HCN1” if that polypeptide is able to either form a functional cation channel by itself, i.e., as a homomultimer, having properties similar to that of human HCN1 channels, or combine with at least one other cation channel subunit (e.g., HCN2, HCN3, or HCN4) so as to form a complex that constitutes a functional cation channel where the polypeptide confers upon the complex (as compared with the other subunit alone) altered electrophysiological or pharmacological properties that are similar to the electrophysiological or pharmacological properties that the human HCN1 protein having SEQ.ID.NO.", ":2 confers on the complex and where the polypeptide has an amino acid sequence that is at least about 50% identical, preferably at least about 80% identical, and even more preferably at least about 95% identical to SEQ.ID.NO.", ":2 when measured by such standard sequence comparison programs as BLAST or FASTA.", "See, e.g., Gish & States, 1993, Nature Genetics 3:266-272 and Altschul et al., 1990, J. Mol.", "Biol.", "215:403-410 for examples of sequence comparison programs.", "For the purposes of this definition, examples of electrophysiological or pharmacological properties are: cation selectivity, voltage dependence of activation and inactivation, activation kinetics, reversal potential, and modulation by cyclic nucleotides such as cAMP or cGMP.", "The present invention relates to the identification and cloning of DNA encoding the human HCN1 protein.", "Although cDNAs encoding mouse, rat, and rabbit HCN1 have been isolated, cDNA encoding the complete, correct human HCN1 protein has not previously been reported.", "A few ESTs, representing fragmentary sequences of human HCN1 (although not identified as HCN1 sequences) have been deposited in databanks.", "GenBank accession no.", "AF064876 represents a partial, internal fragment, lacking 5′ and 3′ ends; GenBank accession no.", "AW054787 represents an EST containing only the carboxy terminal sequences of human HCN1.GenBank accession no.", "AC013384 represents human chromosome 2 genomic DNA sequences that encompass HCN1 but there is no indication of which portion of the disclosed sequence represents HCN1 coding sequence.", "Other human HCN family members have been deposited.", "GenBank accession no.", "AI571225 is an amino terminal EST of HCN3; AQ625620 is a partial genomic sequence of HCN3.AF065164 and AJ012582 represent HCN2; AJ132429 and AJ238850 represent HCN4.Sequences from HCN family members of certain non-human species have been deposited in GenBank: AJ225123 (mouse HCN1); AJ247450 (rat HCN1); AF168122 (rabbit HCN1); AJ225122 (mouse HCN2); AJ225124 (mouse HCN3); AF247452 (rat HCN3); AF247453 (rat HCN4); AB022927 (rabbit HCN4).", "The present invention provides DNA encoding human HCN1 having SEQ.ID.NO.:1.SEQ.ID.NO.", ":1 encodes a human HCN1 protein having SEQ.ID.NO.", ":2.Other sequence variants of human HCN1 have also been identified.", "Eight single nucleotide polymorphisms (SNPs) were found in the cDNAs.", "They are highlighted and underlined below.", "In each case, more than one clone was found containing each sequence.", "The resulting amino acids from these polymorphisms are also highlighted and underlined 1.T (SEQ.ID.NO.", ":1) or C (SEQ.ID.NO.", ":3) at nucleotide position 690 ACCCCAAAGT GATCAAGATG AATTATTTAA AAAGCTGGT(T/C) TGTGGTTGAC (SEQ.ID.NO.", ":23) resulting in F (SEQ.ID.NO.", ":2) or S (SEQ.ID.NO.", ":4) at amino acid position 222 EDSSEILDP KVIKMNYLKS W(F/S)VVDFISSI PVDYIFLIVE KGMDSEVYKT (SEQ.ID.NO.", ":24) 2.G (SEQ.ID.NO.", ":1) or A (SEQ.ID.NO.", ":5) at nucleotide position 1011 1001 CCACCAGATT (G/A)CTGGGTGTC TTTAAATGAA ATGGTTAATG ATTCTTGGGG (SEQ.ID.NO.", ":25) resulting in C (SEQ.ID.NO.", ":2) or Y (SEQ.ID.NO.", ":6) at amino acid position 329 301 LIGMM LCH WDGCLQFLVP LLQDFPPD(C/Y)W VSLNEMVNDS WGKQYSYALF (SEQ.ID.NO.", ":26) 3.A (SEQ.ID.NO.", ":1) or G (SEQ.ID.NO.", ":7) at nucleotide position 1401 1401 (A/G)GGAGATAGT CAACTTCAAC TGTCGGAAAC TGGTGGCTAC AATGCCTTTA (SEQ.ID.NO.", ":27) resulting in E (SEQ.ID.NO.", ":2) or G (SEQ.ID.NO.", ":8) at amino acid position 459 451 NELNDPLR(E/G)E IVNFNCRKLV ATMPLFANAD PNFVTAMLSK LRFEVFQPGD (SEQ.ID.NO.", ":28) 4.A (SEQ.ID.NO.", ":1) or G (SEQ.ID.NO.", ":9) at nucleotide position 1532 1501 ATTTGAGGTG TTTCAACCTG GAGATTATAT C(A/G)TACGAGAA GGAGCCGTGG (SEQ.ID.NO.", ":29) resulting in I (SEQ.ID.NO.", ":2) or V (SEQ.ID.NO.", ":10) at amino acid position 503 501 YI(I/V)REGAVGK KMYFIQHGVA GVITKSSKEM KLTDGSYFGE ICLLTKGRRT (SEQ.ID.NO.", ":30) 5.T (SEQ.ID.NO.", ":1) or C (SEQ.ID.NO.", ":11) at nucleotide position 1743 1701 GTCGTCTTTA CTCACTTTCC GTGGACAATT TCAACGAGGT CC(T/C)GGAGGAA (SEQ.ID.NO.", ":31) resulting in L (SEQ.ID.NO.", ":2) or P (SEQ.ID.NO.", ":12) at amino acid position 573 551 ASVRADTYCR LYSLSVDNFN EV(L/P)EEYPM RAFETVA/DR LDRIGKKNSI (SEQ.ID.NO.", ":32) 6.A (SEQ.ID.NO.", ":1) or G (SEQ.ID.NO.", ":13) at nucleotide position 1973 1951 TCAAATGACA ACCCTGAATT CC(A/G)CATCGTC TACTACGACC CCGACCTCCC (SEQ.ID.NO.", ":33) resulting in T (SEQ.ID.NO.", ":2) or A (SEQ.ID.NO.", ":14) at amino acid position 650 601 LLQKFQKDLN TGVFNNQENE ILKQIVKHDR EMVQAIAPIN YPQMTTLNS(T/A) (SEQ.ID.NO.", ":34) 7.T (SEQ.ID.NO.", ":1) or A (SEQ.ID.NO.", ":15) at nucleotide position 1997 1951 TCAAATGACA ACCCTGAATT CCACATCGTC TACTACGACC CCGACC(T/A)CCC (SEQ.ID.NO.", ":35) resulting in S (SEQ.ID.NO.", ":2) or T (SEQ.ID.NO.", ":16) at amino acid position 658 651 SSTTTPT(S/T)RM RTQSPPVYTA TSLSHSNLHS PSPSTQTPQP SALSPCSYT (SEQ.ID.NO.", ":36) 8.T (SEQ.ID.NO.", ":1) or C (SEQ.ID.NO.", ":17) at nucleotide position 2417 2401 GCTGCCCCAT GAGGTG(T/C)CCA CTCTGATTTC CAGACCTCAT CCCACTGTGG (SEQ.ID.NO.", ":37) resulting in S (SEQ.ID.NO.", ":2) or P (SEQ.ID.NO.", ":18) at amino acid position 798 751 PSPQPQTPGS STPKNEVHKS TQALHNTNLT REVRPLSASQ PSLPHEV(S/P)TL (SEQ.ID.NO.", ":38) Northern blot analyses demonstrated expression of human HCN1 in a variety of tissues, including brain, heart, skeletal muscle, testes, liver, and pancreas.", "This pattern of expression suggests that the human HCN1 potassium channel subunit may have therapeutic relevance for the modulation of cellular excitability in the treatment of neurodegenerative diseases, cognitive and sensory disorders, pain, cardiac brady- and tachy-arrhythmias, ataxias, fertility disorders, hepatic dysfunction, pancreatic disorders (including diabetes), and diabetic neuropathy.", "The present invention provides nucleic acids encoding the human HCN1 hyperpolarization-activated and cyclic nucleotide-gated cation channel that are substantially free from other nucleic acids.", "The nucleic acids may be DNA or RNA.", "The present invention also provides isolated and/or recombinant DNA molecules encoding the human HCN1 cation channel.", "The present invention provides DNA molecules substantially free from other nucleic acids as well as isolated and/or recombinant DNA molecules comprising the nucleotide sequence shown in SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, and 17.The present invention includes isolated DNA molecules as well as DNA molecules that are substantially free from other nucleic acids comprising the coding region of SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, and 17.Accordingly, the present invention includes isolated DNA molecules and DNA molecules substantially free from other nucleic acids having a sequence comprising positions 26 to 2695 of SEQ.ID.NO.", ":1, 26 to 2695 of SEQ.ID.NO.", ":3, 26 to 2695 of SEQ.ID.NO.", ":5, 26 to 2695 of SEQ.ID.NO.", ":7, 26 to 2695 of SEQ.ID.NO.", ":9, 26 to 2695 of SEQ.ID.NO.", ":11, 26 to 2695 of SEQ.ID.NO.", ":13, 26 to 2695 of SEQ.ID.NO.", ":15, or 26 to 2695 of SEQ.ID.NO.", ":17.Also included are recombinant DNA molecules having a nucleotide sequence comprising positions 26 to 2695 of SEQ.ID.NO.", ":1, 26 to 2695 of SEQ.ID.NO.", ":3, 26 to 2695 of SEQ.ID.NO.", ":5, 26 to 2695 of SEQ.ID.NO.", ":7, 26 to 2695 of SEQ.ID.NO.", ":9, 26 to 2695 of SEQ.ID.NO.", ":11, 26 to 2695 of SEQ.ID.NO.", ":13, 26 to 2695 of SEQ.ID.NO.", ":15, or 26 to 2695 of SEQ.ID.NO.", ":17.The novel DNA sequences of the present invention encoding the human HCN1 protein, in whole or in part, can be linked with other DNA sequences, i.e., DNA sequences to which DNA encoding the human HCN1 protein is not naturally linked, to form “recombinant DNA molecules” encoding the human HCN1 protein.", "Such other sequences can include DNA sequences that control transcription or translation such as, e.g., translation initiation sequences, internal ribosome entry sites, promoters for RNA polymerase 11, transcription or translation termination sequences, enhancer sequences, sequences that control replication in microorganisms, sequences that confer antibiotic resistance, or sequences that encode a polypeptide “tag” such as, e.g., a polyhistidine tract, the FLAG epitope, or the myc epitope.", "The novel DNA sequences of the present invention can be inserted into vectors such as plasmids, cosmids, viral vectors, P1 artificial chromosomes, or yeast artificial chromosomes.", "Included in the present invention are DNA sequences that hybridize to the reverse complement of SEQ.ID.NO:1 under conditions of high stringency.", "Preferably, these sequences encode proteins that have substantially the same biological activity as human HCN1 protein having SEQ.ID.NO.", ":2 and that have at least about 50%, preferably at least about 75%, and even more preferably at least about 95% nucleotide sequence identity with SEQ.ID.NO.", ":1.By way of example, and not limitation, a procedure using conditions of high stringency is as follows: Prehybridization of filters containing DNA is carried out for 2 hr.", "to overnight at 65° C. in buffer composed of 6×SSC, 5× Denhardt's solution, and 100 μg/ml denatured salmon sperm DNA.", "Filters are hybridized for 12 to 48 hrs at 65° C. in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×106 cpm of 32P-labeled probe.", "Washing of filters is done at 37° C. for 1 hr in a solution containing 2×SSC, 0.1% SDS.", "This is followed by a wash in 0.1×SSC, 0.1% SDS at 50° C. for 45 min.", "before autoradiography.", "Other procedures using conditions of high stringency would include either a hybridization carried out in 5×SSC, 5× Denhardt's solution, 50% formamide at 42° C. for 12 to 48 hours or a washing step carried out in 0.2×SSPE, 0.2% SDS at 65° C. for 30 to 60 minutes.", "Reagents mentioned in the foregoing procedures for carrying out high stringency hybridization are well known in the art.", "Details of the composition of these reagents can be found in, e.g., Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press.", "In addition to the foregoing, other conditions of high stringency which may be used are well known in the art.", "The degeneracy of the genetic code is such that, for all but two amino acids, more than a single codon encodes a particular amino acid.", "This allows for the construction of synthetic DNA that encodes the human HCN1 protein where the nucleotide sequence of the synthetic DNA differs significantly from the nucleotide sequences of SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17 but still encodes the same human HCN1 protein as SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17.Such synthetic DNAs are intended to be within the scope of the present invention.", "Mutated forms of SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17 are intended to be within the scope of the present invention.", "In particular, mutated forms of SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17 encoding a protein that forms cation channels having altered voltage sensitivity, current carrying properties, or other properties as compared to cation channels formed by the proteins encoded by SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17, are within the scope of the present invention.", "Such mutant forms can differ from SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17 by having nucleotide deletions, substitutions, or additions.", "Also intended to be within the scope of the present invention are RNA molecules having sequences corresponding to SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17 or corresponding to the coding regions of SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17.The RNA molecules can be substantially free from other nucleic acids or can be isolated and/or recombinant RNA molecules.", "Antisense nucleotides, DNA or RNA, that are the reverse complements of SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17, or portions thereof, are also within the scope of the present invention.", "In addition, polynucleotides based on SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17 in which a small number of positions are substituted with non-natural or modified nucleotides such as inosine, methyl-cytosine, or deaza-guanosine are intended to be within the scope of the present invention.", "Polynucleotides of the present invention can also include sequences based on SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17 but in which non-natural linkages between the nucleotides are present.", "Such non-natural linkages can be, e.g., methylphosphonates, phosphorothioates, phosphorodithionates, phosphoroamidites, and phosphate esters.", "Polynucleotides of the present invention can also include sequences based on SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17 but having de-phospho linkages as bridges between nucleotides, e.g., siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, and thioether bridges.", "Other internucleotide linkages that can be present include N-vinyl, methacryloxyethyl, methacrylamide, or ethyleneimine linkages.", "Peptide nucleic acids based upon SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17 are also included in the present invention.", "Generally, such polynucleotides comprising non-natural or modified nucleotides and/or non-natural linkages between the nucleotides, as well as peptide nucleic acids, will encode the same, or highly similar, proteins as are encoded by SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17.Another aspect of the present invention includes host cells that have been engineered to contain and/or express DNA sequences encoding the human HCN1 protein.", "Such recombinant host cells can be cultured under suitable conditions to produce human HCN1 protein.", "An expression vector comprising DNA encoding human HCN1 protein can be used for the expression of human HCN1 protein in a recombinant host cell.", "Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, amphibian cells such as Xenopus oocytes, and insect cells including but not limited to Drosophila and silkworm derived cell lines (e.g., Spodoptera frugiperda).", "Cells and cell lines which are suitable for recombinant expression of human HCN1 protein and which are widely available, include but are not limited to, L cells L-M(TK−) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), HEK 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), CPAE (ATCC CCL 209), Saos-2 (ATCC HTB-85), ARPE-19 human retinal pigment epithelium (ATCC CRL-2302), Xenopus melanophores, and Xenopus oocytes.", "A variety of mammalian expression vectors can be used to express recombinant human HCN1 protein in mammalian cells.", "Commercially available mammalian expression vectors which are suitable include, but are not limited to, pMC1neo (Stratagene), pSG5 (Stratagene), pcDNAI and pcDNAIamp, pcDNA3, pcDNA3.1, pCR3.1 (Invitrogen), EBO-pSV2-neo (ATCC 37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pIZD35 (ATCC 37565), and pSV2-dhfr (ATCC 37146).", "Another suitable vector is the PT7TS oocyte expression vector.", "Following expression in recombinant cells, human HCN1 protein can be purified by conventional techniques to a level that is substantially free from other proteins.", "Techniques that can be used include ammonium sulfate precipitation, hydrophobic or hydrophilic interaction chromatography, ion exchange chromatography, affinity chromatography, phosphocellulose chromatography, size exclusion chromatography, preparative gel electrophoresis, and alcohol precipitation.", "In some cases, it may be advantageous to employ protein denaturing and/or refolding steps in addition to such techniques.", "Certain ion channel subunit proteins have been found to require the expression of other ion channel subunits in order to be properly expressed at high levels and inserted in membranes.", "For example, co-expression of KCNQ3 appears to enhance the expression of KCNQ2 in Xenopus oocytes (Wang et al., 1998, Science 282:1890-1893).", "Also, some voltage-gated potassium channel Kvα subunits require other related α subunits or Kvβ subunits (Shi et al., 1995, Neuron 16:843-852).", "Accordingly, the recombinant expression of human HCN1 proteins may under certain circumstances benefit from the co-expression of other ion channel proteins and such co-expression is intended to be within the scope of the present invention.", "Such co-expression can be effected by transfecting an expression vector encoding human HCN1 protein into a cell that naturally expresses another ion channel protein.", "Alternatively, an expression vector encoding human HCN1 protein can be transfected into a cell in which an expression vector encoding another ion channel protein has also been transfected.", "Preferably, such a cell does not naturally express human HCN1 subunit proteins or the other ion channel protein.", "Co-expression of human HCN1 with other HCN family proteins such as HCN2, HCN3, or HCN4 may be of benefit.", "In addition, since these cation channels are also modulated by cyclic nucleotides, co-expresion of HCN1 with other types of receptors, such as those that control levels of intracellular cyclic nucleotides (e.g., the beta adrenergic receptor) may also be of benefit and is also within the scope of the present invention.", "The present invention includes human HCN1 proteins substantially free from other proteins.", "The amino acid sequences of full-length human HCN1 subunit proteins are shown in SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, and 18.Thus, the present invention includes human HCN1 protein substantially free from other proteins comprising an amino acid sequence selected from the group consisting of SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, and 18.The present invention also includes isolated human HCN1 protein comprising an amino acid sequence selected from the group consisting of SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, and 18.Mutated forms of human HCN1 proteins are intended to be within the scope of the present invention.", "In particular, mutated forms of SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, or 18 that form cation channels having altered electrophysiological or pharmacological properties as compared to cation channels formed by SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, or 18 are within the scope of the present invention.", "As with many proteins, it may be possible to modify many of the amino acids of the human HCN1 protein and still retain substantially the same biological activity as for the original protein.", "Thus, the present invention includes modified human HCN1 proteins which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as naturally occurring human HCN1 proteins.", "It is generally accepted that single amino acid substitutions do not usually alter the biological activity of a protein (see, e.g., Molecular Biology of the Gene, Watson et al., 1987, Fourth Ed., The Benjamin/Cummings Publishing Co., Inc., page 226; and Cunningham & Wells, 1989, Science 244:1081-1085).", "Accordingly, the present invention includes polypeptides where one amino acid substitution has been made in SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, or 18 wherein the polypeptides still retain substantially the same biological activity as naturally occurring human HCN1 proteins.", "The present invention also includes polypeptides where two or more amino acid substitutions have been made in SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, or 18 wherein the polypeptides still retain substantially the same biological activity as naturally occurring human HCN1 proteins.", "In particular, the present invention includes embodiments where the above-described substitutions are conservative substitutions.", "In particular, the present invention includes embodiments where the above-described substitutions do not occur in conserved positions.", "Conserved positions are those positions in which the human HCN1 protein having SEQ.ID.NO:2, the mouse HCN1 protein (SEQ.ID.NO.", ":19), the rat HCN1 protein (SEQ.ID.NO.", ":20), and the rabbit HCN1 protein (SEQ.ID.NO.", ":21) share the same amino acid (see FIG.", "10).", "The human HCN1 proteins of the present invention may contain post-translational modifications, e.g., covalently linked carbohydrate, phosphorylation, myristoylation, palmytoylation.", "The present invention also includes chimeric human HCN1 proteins.", "Chimeric human HCN1 proteins consist of a contiguous polypeptide sequence of at least a portion of a human HCN1 protein fused to a polypeptide sequence that is not from a human HCN1 protein.", "The portion of the human HCN1 protein must include at least 10, preferably at least 25, and most preferably at least 50 contiguous amino acids from SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, or 18.The present invention also includes isolated human HCN1 protein and isolated DNA encoding human HCN1 protein.", "Use of the term “isolated” indicates that the human HCN1 protein or DNA has been removed from its normal cellular environment.", "Thus, an isolated human HCN1 protein may be in a cell-free solution or placed in a different cellular environment from that in which it occurs naturally.", "The term isolated does not necessarily imply that an isolated human HCN1 protein is the only, or predominant, protein present (although that is one of the meanings of isolated), but instead means that the isolated human HCN1 protein is at least 95% free of non-amino acid material (e.g., nucleic acids, lipids, carbohydrates) naturally associated with the human HCN1 protein.", "It is known that certain ion channel subunits can interact to form heteromeric complexes resulting in functional ion channels.", "For example, KCNQ2 and KCNQ3 can assemble to form a heteromeric functional potassium channel (Wang et al., 1998, Science 282:1890-1893).", "Accordingly, it is believed that the human HCN1 proteins of the present invention may also be able to form heteromeric structures with other proteins where such heteromeric structures form functional ion channels.", "Thus, the present invention includes such heteromers comprising human HCN1 protein.", "Preferred heteromers are those in which the human HCN1 protein forms heteromers with at least one other HCN family member, e.g., HCN2, HCN3, or HCN4.Preferably, the other HCN family member is a human HCN family member.", "DNA encoding human HCN1 proteins can be obtained by methods well known in the art.", "For example, a cDNA fragment encoding full-length human HCN1 protein can be isolated from human brain or heart cDNA by using the polymerase chain reaction (PCR) employing suitable primer pairs.", "Such primer pairs can be selected based upon the DNA sequences encoding the human HCN1 proteins shown in FIGS.", "1-9 as SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, and 17.Suitable primer pairs would be, e.g.", ": 5′ CCGTCGCCGGCCGCGTCCTCCGG 3′ (SEQ.ID.NO.", ":39) 5′ TAGTCTCAGTTTATGAGAG 3′ (SEQ.ID.NO.", ":40) The above primers are meant to be illustrative only; many acceptable primer pairs exist and one skilled in the art would readily be able to design other suitable primers based upon SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, and 17.Such primers could be produced by methods of oligonucleotide synthesis that are well known in the art.", "PCR reactions can be carried out with a variety of thermostable enzymes including but not limited to AmpliTaq, AmpliTaq Gold, or Vent polymerase.", "For AmpliTaq, reactions can be carried out in 10 mM Tris-Cl, pH 8.3, 2.0 mM MgCl2, 200 μM of each dNTP, 50 mM KCl, 0.2 μM of each primer, 10 ng of DNA template, 0.05 units/μl of AmpliTaq.", "The reactions are heated at 95° C. for 3 minutes and then cycled 35 times using the cycling parameters of 95° C., 20 seconds, 62° C., 20 seconds, 72° C., 3 minutes.", "In addition to these conditions, a variety of suitable PCR protocols can be found in PCR Primer, A Laboratory Manual, edited by C. W. Dieffenbach and G. S. Dveksler, 1995, Cold Spring Harbor Laboratory Press; or PCR Protocols: A Guide to Methods and Applications, Michael et al., eds., 1990, Academic Press.", "Since the human HCN1 proteins of the present invention are homologous to other cation channel subunit proteins, it is desirable to sequence the clones obtained by the herein-described methods, in order to verify that the desired human HCN1 protein has in fact been obtained.", "Sequencing is also advisable in order to ensure that one has obtained the desired cDNA from among SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, and 17.By these methods, cDNA clones encoding human HCN1 proteins can be obtained.", "These cDNA clones can be cloned into suitable cloning vectors or expression vectors, e.g., the mammalian expression vector pcDNA3.1 (Invitrogen, San Diego, Calif.).", "Human HCN1 protein can then be produced by transferring expression vectors encoding human HCN1 or portions thereof into suitable host cells and growing the host cells under appropriate conditions.", "Human HCN1 protein can then be isolated by methods well known in the art.", "As an alternative to the above-described PCR methods, cDNA clones encoding human HCN1 proteins can be isolated from cDNA libraries using as a probe oligonucleotides specific for human HCN1 and methods well known in the art for screening cDNA libraries with oligonucleotide probes.", "Such methods are described in, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Glover, D. M.", "(ed.", "), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K., Vol.", "I, II.", "Oligonucleotides that are specific for human HCN1 and that can be used to screen cDNA libraries can be readily designed based upon the DNA sequences shown in FIGS.", "1-9 (viz., SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, and 17) and can be synthesized by methods well-known in the art.", "Genomic clones containing the human HCN1 gene can be obtained from commercially available human PAC or BAC libraries from suppliers such as, e.g., Research Genetics, Huntsville, Ala. Alternatively, one may prepare genomic libraries, e.g., in P1 artificial chromosome vectors, from which genomic clones containing the human HCN1 gene can be isolated, using probes based upon the human HCN1 DNA sequences disclosed herein.", "Methods of preparing such libraries are known in the art (see, e.g., Ioannou et al., 1994, Nature Genet.", "6:84-89).", "The novel DNA sequences of the present invention can be used in various diagnostic methods.", "The present invention provides diagnostic methods for determining whether a patient carries a mutation or a polymorphism in the human HCN1 gene.", "In broad terms, such methods comprise determining the DNA sequence of a region in or near the human HCN1 gene from the patient and comparing that sequence to the sequence from the corresponding region of the human HCN1 gene from a non-affected person, i.e., a person who does not have the condition which is being diagnosed, where a difference in sequence between the DNA sequence of the gene from the patient and the DNA sequence of the gene from the non-affected person indicates that the patient has a mutation or a polymorphism in the human HCN1 gene.", "The present invention also provides oligonucleotide probes, based upon SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17 that can be used in diagnostic methods to identify patients having mutated or polymorphic forms of the human HCN1 gene, to determine the level of expression of RNA encoding human HCN1, or to isolate genes homologous to human HCN1 from other species.", "In particular, the present invention includes DNA oligonucleotides comprising at least about 10, 15, or 18 (but not more than 100) contiguous nucleotides of SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17 where the oligonucleotide probe comprises no stretch of contiguous nucleotides longer than 5 from SEQ.ID.NOs.", ":1, 3, 5, 7, 9, 11, 13, 15, or 17 other than the said at least about 10, 15, or 18 contiguous nucleotides.", "The oligonucleotides can be substantially free from other nucleic acids.", "Also provided by the present invention are corresponding RNA oligonucleotides.", "The DNA or RNA oligonucleotides can be packaged in kits.", "The present invention makes possible the recombinant expression of human HCN1 protein in various cell types.", "Such recombinant expression facilitates the study of this protein so that its biochemical activity and its possible role in various diseases such as neurodegenerative diseases, cognitive and sensory disorders, pain, cardiac brady- and tachy-arrhythmias, ataxias, fertility disorders, hepatic dysfunction, pancreatic disorders (including diabetes), and diabetic neuropathy can be elucidated.", "The present invention also makes possible the development of assays which measure the biological activity of cation channels containing human HCN1 protein.", "Assays using recombinantly expressed human HCN1 protein are especially of interest.", "Such assays can be used to screen libraries of compounds or other sources of compounds to identify compounds that are activators or inhibitors of the activity of cation channels containing human HCN1 protein.", "Such identified compounds can serve as “leads” for the development of pharmaceuticals that can be used to treat patients having diseases in which it is beneficial to enhance or suppress cation channel activity.", "In versions of the above-described assays, cation channels containing mutant human HCN1 proteins are used and inhibitors or activators of the activity of the mutant cation channels are identified.", "Preferred cell lines for recombinant expression of human HCN1 proteins are those which do not express endogenous cation channels.", "Cell lines expressing recombinant human HCN1 can be exposed to and loaded with 86Rb, an ion which can substitute for potassium in many ion channels.", "The efflux of 86Rb out of such cells can be assayed in the presence and absence of collections of substances (e.g., combinatorial libraries, natural products, analogues of lead compounds produced by medicinal chemistry), or members of such collections, and those substances that are able to alter 86Rb efflux thereby identified.", "Such substances are likely to be activators or inhibitors of cation channels containing human HCN1 protein.", "Activators and inhibitors of cation channels containing human HCN1 proteins are likely to be substances that are capable of binding to cation channels containing human HCN1 proteins.", "Thus, one type of assay determines whether one or more of a collection of substances is capable of such binding.", "Accordingly, the present invention provides a method of identifying substances that bind to cation channels containing human HCN1 protein comprising: (a) providing cells expressing a cation channel containing human HCN1 protein; (b) exposing the cells to a substance that is not known to bind cation channels containing human HCN1 protein; (c) determining the amount of binding of the substance to the cells; (d) comparing the amount of binding in step (c) to the amount of binding of the substance to control cells where the control cells are substantially identical to the cells of step (a) except that the control cells do not express human HCN1 protein; where if the amount of binding in step (c) is greater than the amount of binding of the substance to control cells, then the substance binds to cation channels containing human HCN1 protein.", "An example of control cells that are substantially identical to the cells of step (a) would be a parent cell line where the parent cell line is transfected with an expression vector encoding human HCN1 protein in order to produce the cells expressing a cation channel containing human HCN1 protein of step (a).", "Another version of this assay makes use of compounds that are known to bind to cation channels containing human HCN1 protein.", "Substances that are new binders are identified by virtue of their ability to augment or block the binding of these known compounds.", "This can be done if the known compound is used at a concentration that is far below saturation, in which case a substance that is a new binder is likely to be able to either augment or block the binding of the known compound.", "Substances that have this ability are likely themselves to be inhibitors or activators of cation channels containing human HCN1 protein.", "Accordingly, the present invention includes a method of identifying substances that bind cation channels containing human HCN1 protein and thus are likely to be inhibitors or activators of cation channels containing human HCN1 protein comprising: (a) providing cells expressing cation channels containing human HCN1 protein; (b) exposing the cells to a compound that is known to bind to the cation channels containing human HCN1 protein in the presence and in the absence of a substance not known to bind to cation channels containing human HCN1 protein; (c) determining the amount of binding of the compound to the cells in the presence and in the absence of the substance; where if the amount of binding of the compound in the presence of the substance differs from that in the absence of the substance, then the substance binds cation channels containing human HCN1 protein and is likely to be an inhibitor or activator of cation channels containing human HCN1 protein.", "Generally, the known compound is labeled (e.g., radioactively, enzymatically, fluorescently) in order to facilitate measuring its binding to the cation channels.", "Once a substance has been identified by the above-described methods, it can be assayed in functional tests, such as those described herein, in order to determine whether it is an inhibitor or an activator.", "In particular embodiments, the compound known to bind cation channels containing human HCN1 protein is selected from the group consisting of: ZD7288 and L-cis-diltiazem.", "The present invention includes a method of identifying activators or inhibitors of cation channels containing human HCN1 protein comprising: (a) recombinantly expressing human HCN1 protein in a host cell so that the recombinantly expressed human HCN1 protein forms cation channels either by itself or by forming heteromers with other cation channel subunit proteins; (b) measuring the biological activity of the cation channels formed in step (a) in the presence and in the absence of a substance not known to be an activator or an inhibitor of cation channels containing human HCN1 protein; where a change in the biological activity of the cation channels formed in step (a) in the presence as compared to the absence of the substance indicates that the substance is an activator or an inhibitor of cation channels containing human HCN1 protein.", "In particular embodiments of the methods described herein, the biological activity is the conduction of a mixed Na+/K+ current or the efflux of 86Rb.", "In particular embodiments, it may be advantageous to recombinantly express the other subunits of cation channels.", "Alternatively, it may be advantageous to use host cells that endogenously express such other subunits.", "Other subunits may be other HCN family members such as HCN2, HCN3, or HCN4, particularly other human HCN family members.", "In particular embodiments, a vector encoding human HCN1 protein is transferred into Xenopus oocytes in order to cause the expression of human HCN1 protein in the oocytes.", "Alternatively, RNA encoding human HCN1 protein can be prepared in vitro and injected into the oocytes, also resulting in the expression of human HCN1 protein in the oocytes.", "Following expression of the human HCN1 protein in the oocytes, and following the formation of cation channels containing human HCN1, membrane currents are measured after the transmembrane voltage is changed in steps.", "A change in membrane current is observed when the cation channels containing human HCN1 open or close, modulating sodium and potassium ion flow.", "Similar studies were reported for KCNQ2 and KCNQ3 potassium channels in Wang et al., 1998, Science 282:1890-1893 and for MinK channels by Goldstein & Miller, 1991, Neuron 7:403408.These references and references cited therein can be consulted for guidance as to how to carry out such studies.", "In such studies it may be advantageous to co-express other cation channel subunit proteins (e.g., HCN2, HCN3, or HCN4) in addition to human HCN1 in the oocytes.", "Inhibitors or activators of cation channels containing human HCN1 protein can be identified by exposing the oocytes to individual substances or collections of substances and determining whether the substances can block/diminish or enhance the membrane currents observed in the absence of the substance.", "Accordingly, the present invention provides a method of identifying inhibitors or activators of cation channels containing human HCN1 protein comprising: (a) expressing human HCN1 protein in cells such that cation channels containing human HCN1 protein are formed; (b) changing the transmembrane potential of the cells in step (a) from a potential where the cation channels containing human HCN1 protein are closed to a potential where cation channels containing human HCN1 protein are open in the presence and the absence of a substance not known to be an inhibitor or an activator of cation channels containing human HCN1 protein; (c) measuring mixed sodium/potassium currents following step (b); where if the mixed sodium/potassium currents measured in step (c) are less in the presence rather than in the absence of the substance, then the substance is an inhibitor of cation channels containing human HCN1 protein; where if the mixed sodium/potassium currents measured in step (c) are greater in the presence rather than in the absence of the substance, then the substance is an activator of cation channels containing human HCN1 protein.", "In general, for step (b), the potential where the cation channels containing human HCN1 protein are closed will be a depolarized potential and the potential where cation channels containing human HCN1 protein are open will be a hyperpolarized potential.", "The method described above can be practiced by the use of techniques that are well known in the art such as voltage clamp studies or patch clamp studies.", "Where the methods of the present invention involve measuring “mixed sodium/potassium currents” such measurements can be carried out by voltage clamp experiments.", "Alternatively, where the cells contain a β-adrenergic receptor as well as the HCN1 channel, instead of changing the membrane potential by voltage clamp to turn on the HCN1 current, the potential can be held steady and a β-adrenergic receptor agonist can be added to the cells.", "This should increase cAMP concentration and turn on the HCN1 channel.", "One could then assay for activators and inhibitors in the same way as above by looking at the currents plus/minus the compounds.", "The present invention also includes assays for the identification of activators and inhibitors of cation channels containing human HCN1 protein that are based upon fluorescence resonance energy transfer (FRET) between a first and a second fluorescent dye where the first dye is bound to one side of the plasma membrane of a cell expressing cation channels containing human HCN1 protein and the second dye is free to shuttle from one face of the membrane to the other face in response to changes in membrane potential.", "In certain embodiments, the first dye is impenetrable to the plasma membrane of the cells and is bound predominately to the extracellular surface of the plasma membrane.", "The second dye is trapped within the plasma membrane but is free to diffuse within the membrane.", "At polarized (i.e., negative) resting potentials of the membrane, the second dye is bound predominately to the inner surface of the extracellular face of the plasma membrane, thus placing the second dye in close proximity to the first dye.", "This close proximity allows for the generation of a large amount of FRET between the two dyes.", "At depolarized potentials, the second dye moves from the extracellular face of the membrane to the intracellular face, thus increasing the distance between the dyes.", "This increased distance results in a decrease in FRET, with a corresponding increase in fluorescent emission derived from the first dye and a corresponding decrease in the fluorescent emission from the second dye.", "In this way, the amount of FRET between the two dyes can be used to measure the polarization state of the membrane.", "For a description of this technique, see Gonzalez & Tsien, 1997, Chemistry & Biology 4:269-277.See also González & Tsien, 1995, Biophys.", "J.", "69:1272-1280 and U.S. Pat.", "No.", "5,661,035.In certain embodiments, the first dye is a fluorescent lectin or a fluorescent phospholipid that acts as the fluorescent donor.", "Examples of such a first dye are: a coumarin-labeled phosphatidylethanolamine (e.g., N-(6-chloro-7-hydroxy-2-oxo-2H—1-benzopyran-3-carboxamidoacetyl)-dimyristoylphosphatidylethanolamine) or N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dipalmitoylphosphatidylethanolamine); a fluorescently-labeled lectin (e.g., fluorescein-labeled wheat germ agglutinin).", "In certain embodiments, the second dye is an oxonol that acts as the fluorescent acceptor.", "Examples of such a second dye are: bis(1,3-dialkyl-2-thiobarbiturate)trimethineoxonols (e.g., bis(1,3-dihexyl-2-thiobarbiturate)trimethineoxonol) or pentamethineoxonol analogues (e.g., bis(1,3-dihexyl-2-thiobarbiturate)pentamethineoxonol; or bis(1,3-dibutyl-2-thiobarbiturate)pentamethineoxonol).", "See González & Tsien, 1997, Chemistry & Biology 4:269-277 for methods of synthesizing various dyes suitable for use in the present invention.", "In certain embodiments, the assay may comprise a natural carotenoid, e.g., astaxanthin, in order to reduce photodynamic damage due to singlet oxygen.", "The above described assays can be utilized to discover activators and inhibitors of cation channels containing human HCN1 protein.", "Such assays will generally utilize cells that express cation channels containing human HCN1 protein, e.g., by transfection with expression vectors encoding human HCN1 protein and, optionally, other cation channel subunits.", "The cellular membrane potential is determined by the balance between inward (depolarizing) and outward (repolarizing) ionic fluxes through various ion pumps and channels.", "FRET based assays could be developed by co-expressing HCN1 containing cation channels with an inward rectifier potassium channel.", "The inward rectifier will allow potassium efflux from the cell, which tends to stabilize the membrane potential near the potassium equilibrium potential, EK, (typically about −80 mV).", "When human HCN1 is expressed in cells having a resting membrane potential lower than about −30 mV, especially cells having resting membrane potentials lower than about −50 to −70 mV, the channels formed by human HCN1 will be open and will tend to pass a cation current into the cell, thus tending to depolarize the membrane potential.", "The presence of an inhibitor of a cation channel containing human HCN1 will prevent, or diminish, the ability of HCN1 to depolarize the membrane potential.", "Thus, membrane potential will remain negative (i.e., hyperpolarized) in the presence of human HCN1 inhibitors.", "Such changes in membrane potential that are caused by inhibitors of cation channels containing human HCN1 protein can be monitored by the assays using FRET described above.", "Accordingly, the present invention provides a method of identifying inhibitors of cation channels containing human HCN1 protein comprising: (a) providing cells comprising: (1) an expression vector that directs the expression of human HCN1 protein in the cells so that cation channels containing human HCN1 protein are formed in the cells and where the cells have a resting membrane potential lower than about −30 mV; (2) a first fluorescent dye, where the first dye is bound to one side of the plasma membrane of the cells; and (3) a second fluorescent dye, where the second fluorescent dye is free to distribute from one face of the plasma membrane of the cells to the other face in response to changes in membrane potential; (b) exposing the cells to a substance; (c) measuring the amount of fluorescence resonance energy transfer (FRET) in the cells in the presence and in the absence of the substance; (d) comparing the amount of FRET exhibited by the cells in the presence and in the absence of the substance; where if the amount of FRET exhibited by the cells in the presence of the substance is greater than the amount of FRET exhibited by the cells in the absence of the substance then the substance is an inhibitor of cation channels containing human HCN1 protein.", "If the cells are exposed to a substance that is an activator (rather than an inhibitor) of cation channels containing human HCN1 protein, then the HCN1 channels will pass more current into the cell, tending to move the membrane potential to a more positive (i.e., depolarized) level.", "This depolarization can also be monitored by the FRET assays described above.", "Accordingly, the present invention provides a method of identifying activators of cation channels containing human HCN1 protein comprising: (a) providing cells comprising: (1) an expression vector that directs the expression of human HCN1 protein in the cells so that cation channels containing human HCN1 protein are formed in the cells and where the cells have a resting membrane potential lower than about −30 mV; (2) a first fluorescent dye, where the first dye is bound to one side of the plasma membrane of the cells; and (3) a second fluorescent dye, where the second fluorescent dye is free to distribute from one face of the plasma membrane of the cells to the other face in response to changes in membrane potential; (b) exposing the cells to a substance; (c) measuring the amount of fluorescence resonance energy transfer (FRET) in the cells in the presence and in the absence of the substance; (d) comparing the amount of FRET exhibited by the cells in the presence and in the absence of the substance; where if the amount of FRET exhibited by the cells in the presence of the substance is less than the amount of FRET exhibited by the cells in the absence of the substance then the substance is an inhibitor of cation channels containing human HCN1 protein.", "As an alternative way of ensuring that the ion channels containing human HCN1 protein are turned on, one can utilize cells containing a β-adrenergic receptor and expose those cells to an agonist of the β-adrenergic receptor.", "This will cause an increase in cAMP concentration in the cells and thus open the ion channels containing human HCN1 protein.", "Further exposing such cells to substances that are inhibitors of ion channels containing human HCN1 protein will close those channels, leading to a hyperpolarization of the cells' membrane potentials.", "This hyperpolarization can be measured by FRET-based assays.", "Accordingly, the present invention includes a method of identifying inhibitors of ion channels containing human HCN1 protein comprising: (a) providing cells comprising: (1) an expression vector that directs the expression of human HCN1 protein in the cells so that ion channels containing human HCN1 protein are formed in the cells; (2) a β-adrenergic receptor; (3) a first fluorescent dye, where the first dye is bound to one side of the plasma membrane of the cells; and (4) a second fluorescent dye, where the second fluorescent dye is free to distribute from one face of the plasma membrane of the cells to the other face in response to changes in membrane potential; (b) exposing the cells to an agonist of the β-adrenergic receptor so that the cAMP concentration in the cells increases to a level such that the cation channels containing human HCN1 protein are open; (c) exposing the cells to a substance; (d) measuring the amount of fluorescence resonance energy transfer (FRET) in the cells in the presence and in the absence of the substance; (e) comparing the amount of FRET exhibited by the cells in the presence and in the absence of the substance; where if the amount of FRET exhibited by the cells in the presence of the substance is greater than the amount of FRET exhibited by the cells in the absence of the substance then the substance is an inhibitor of ion channels containing human HCN1 protein.", "In particular embodiments of the above-described methods, the cells also express an inward rectifier potassium channel, either endogenously (e.g., RBL cells) or recombinantly (e.g., as a result of having been transfected with an expression vector encoding the inward rectifier potassium channel).", "In such embodiments, it is desirable to perform control experiments to rule out the possibility that the substances identified are actually agonists of the inward rectifier potassium channel rather than inhibitors of cation channels containing human HCN1 protein.", "This can be done by expressing the HCN1 protein or the inward rectifier potassium channel individually in cells and testing the effect of the substances on the HCN1 protein and the inward rectifier potassium channel by patch clamp techniques.", "As another type of control experiment, in order to be sure that the effect of the substance in the above-described assays is arising through its action at cation channels containing human HCN1 protein, experiments can be run in which the cells are as above, except that they do not contain an expression vector that directs the expression of human HCN1 protein.", "In particular embodiments of the above-described methods, the expression vectors are transfected into the test cells.", "In particular embodiments of the above-described methods, the human HCN1 protein has an amino acid sequence selected from the group consisting of SEQ.ID.NOs.", ":2, 4, 6, 8, 10, 12, 14, 16, and 18.In particular embodiments of the above-described methods, the expression vector comprises positions 26 to 2695 of SEQ.ID.NO.", ":1, 26 to 2695 of SEQ.ID.NO.", ":3, 26 to 2695 of SEQ.ID.NO.", ":5, 26 to 2695 of SEQ.ID.NO.", ":7, 26 to 2695 of SEQ.ID.NO.", ":9, 26 to 2695 of SEQ.ID.NO.", ":11, 26 to 2695 of SEQ.ID.NO.", ":13, 26 to 2695 of SEQ.ID.NO.", ":15, or 26 to 2695 of SEQ.ID.NO.", ":17.In particular embodiments of the above-described methods, the first fluorescent dye is selected from the group consisting of: a fluorescent lectin; a fluorescent phospholipid; a coumarin-labeled phosphatidylethanolamine; N-(6-chloro-7-hydroxy-2-oxo-2H-1-benzopyran-3-carboxamidoacetyl)-dimyristoylphosphatidylethanolamine); N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dipalmitoylphosphatidylethanolamine); and fluorescein-labeled wheat germ agglutinin.", "In particular embodiments of the above-described methods, the second fluorescent dye is selected from the group consisting of: an oxonol that acts as the fluorescent acceptor; bis(1,3-dialkyl-2-thiobarbiturate)trimethineoxonols; bis(1,3-dihexyl-2-thiobarbiturate)trimethineoxonol; bis(1,3-dialkyl-2-thiobarbiturate) quatramethineoxonols; bis(1,3-dialkyl-2-thiobarbiturate)pentamethineoxonols; bis(1,3-dihexyl-2-thiobarbiturate)pentamethineoxonol; bis(1,3-dibutyl-2-thiobarbiturate)pentamethineoxonol); and bis(1,3-dialkyl-2-thiobarbiturate)hexamethineoxonols.", "In a particular embodiment of the above-described methods, the cells are eukaryotic cells.", "In another embodiment, the cells are mammalian cells, preferably human cells.", "In other embodiments, the cells are L cells L-M(TK−) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), HEK 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), or MRC-5 (ATCC CCL 171).", "In assays to identify activators or inhibitors of cation channels containing human HCN1 protein, it may be advantageous to co-express another cation channel subunit besides human HCN1.In particular, it may be advantageous to co-express another HCN family member subunit (e.g., HCN2, HCN3, or HCN4).", "Preferably, this is done by co-transfecting into the cells an expression vector encoding the other HCN family member subunit.", "The present invention also includes assays for the identification of inhibitors of cation channels containing human HCN1 protein that are based upon modulation of the growth phenotype of trk1Δtrk2Δ mutant yeast that also express cation channels containing human HCN1.The products of the yeast trk1 and trk2 genes are high affinity potassium transporters and their expression in wild type yeast allows growth under conditions in which the concentration of K+ in the medium is very low (e.g., <50 μM).", "Deletion, or inactivation, of these two genes abolishes high affinity K+ uptake and results in impaired growth in potassium limited (e.g, <7 mM) media.", "In addition, growth of trk1Δtrk2Δ yeast is also impaired by low (<3.0) pH even in the presence of otherwise permissive K+ concentrations (Nakamura & Gaber, 1999, Meth.", "Enzymol.", "293:89-104).", "Heterologous expression of a human HCN1 cation channel in trk1Δtrk2Δ yeast could rescue the mutant growth phenotype.", "That is, expression of such a channel could restore wild type growth to these cells in limiting K+ or low pH.", "Thus, inhibitors of human HCN1 cation channels will negate its effect in these mutant yeast and result in their reversion to the mutant growth phenotype (i.e., impaired growth in low K+ or low pH).", "Thus, the present invention includes a method of identifying inhibitors of cation channels containing human HCN1 protein comprising: (a) providing a yeast strain that has been engineered to (1) have inactivated trk1 and trk2 genes and (2) heterologously express a cation channel containing human HCN1 protein; (b) exposing the yeast to a substance; (c) measuring the growth rate of the yeast in the presence of the substance under either limiting K+ concentration or low pH and in the absence of the substance under either limiting K+ concentration or low pH; (d) comparing the growth rates measured in step (c) in the presence and in the absence of the substance; wherein if the growth rate in the presence of the substance is less than the growth rate in the absence of the substance then the substance is an inhibitor of cation channels containing human HCN1 protein.", "In certain embodiments, the yeast trk1 and trk2 genes have been inactivated by deletion or mutagenesis.", "Growth of the yeast is measured in media containing either 1) limiting K+ (e.g., <7 mM K+) or 2) permissive K+ and low pH (e.g., 100 mM K+ and pH <3.0).", "Growth rate may simply be measured as turbidity of the culture (e.g., as absorbance at 700 nm) as a function of time, or may be measured by other methods known in the art.", "Growth rate may also be measured in an all or none fashion by measuring the yeast's ability to form colonies in the presence or the absence of the substance.", "While the above-described methods are explicitly directed to testing whether “a”, substance is an activator or inhibitor of cation channels containing human HCN1 protein, it will be clear to one skilled in the art that such methods can be used to test collections of substances (e.g., combinatorial libraries, natural products extracts) to determine whether any members of such collections are activators or inhibitors of cation channels containing human HCN1 protein.", "Accordingly, the use of collections of substances, or individual members or subsets of such members of such collections, as the substance in the above-described methods is within the scope of the present invention.", "The present invention includes pharmaceutical compositions comprising activators or inhibitors of cation channels comprising human HCN1 protein that have been identified by the herein-described methods.", "The activators or inhibitors are generally combined with pharmaceutically acceptable carriers to form pharmaceutical compositions.", "Examples of such carriers and methods of formulation of pharmaceutical compositions containing activators or inhibitors and carriers can be found in Gennaro, ed., Remington's Pharmaceutical Sciences, 18th Edition, 1990, Mack Publishing Co., Easton, Pa. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain a therapeutically effective amount of the activators or inhibitors.", "Therapeutic or prophylactic compositions are administered to an individual in amounts sufficient to treat or prevent conditions where the activity of cation channels containing human HCN1 protein is abnormal.", "The effective amount can vary according to a variety of factors such as the individual's condition, weight, gender, and age.", "Other factors include the mode of administration.", "The appropriate amount can be determined by a skilled physician.", "Generally, an effective amount will be from about 0.01 to about 1,000, preferably from about 0.1 to about 250, and even more preferably from about 1 to about 50 mg per adult human per day.", "Compositions can be used alone at appropriate dosages.", "Alternatively, co-administration or sequential administration of other agents can be desirable.", "The compositions can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.", "For example, the compositions can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.", "Likewise, they can also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.", "Compositions can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three, four or more times daily.", "Furthermore, compositions can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.", "To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.", "The dosage regimen utilizing the compositions is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular composition thereof employed.", "A physician of ordinary skill can readily determine and prescribe the effective amount of the composition required to prevent, counter or arrest the progress of the condition.", "Optimal precision in achieving concentrations of composition within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the composition's availability to target sites.", "This involves a consideration of the distribution, equilibrium, and elimination of a composition.", "The inhibitors and activators of cation channels containing human HCN1 protein will be useful for treating a variety of diseases involving excessive or insufficient cation channel activity.", "Expression of human HCN1 in the human brain, heart, skeletal muscle, testes, liver, and pancreas was seen by Northern blot analysis.", "This suggests that inhibitors and activators of cation channels containing human HCN1 protein are likely to be useful for the treatment of neurodegenerative diseases, cognitive and sensory disorders, pain, cardiac brady- and tachy-arrhythmias, ataxias, fertility disorders, hepatic dysfunction, pancreatic disorders (including diabetes), and diabetic neuropathy.", "The human HCN1 nucleic acids and proteins of the present invention are useful in conjunction with screens designed to identify activators and inhibitors of other ion channels.", "When screening compounds in order to identify potential pharmaceuticals that specifically interact with a target ion channel, it is necessary to ensure that the compounds identified are as specific as possible for the target ion channel.", "To do this, it is necessary to screen the compounds against as wide an array as possible of ion channels that are similar to the target ion channel.", "Thus, in order to find compounds that are potential pharmaceuticals that interact with ion channel A, it is not enough to ensure that the compounds interact with ion channel A (the “plus target”) and produce the desired pharmacological effect through ion channel A.", "It is also necessary to determine that the compounds do not interact with ion channels B, C, D, etc.", "(the “minus targets”).", "The methods used to determine that a compound that is a drug candidate does not interact with minus targets are often referred to as “counterscreens.” In general, as part of a screening program, it is important to use as many minus targets in counterscreens as possible (see Hodgson, 1992, Bio/Technology 10:973-980, at 980).", "Human HCN1 protein, DNA encoding human HCN1 protein, and recombinant cells that have been engineered to express human HCN1 protein have utility in that they can be used as “minus targets” in screening programs designed to identify compounds that specifically interact with other ion channels.", "For example, Wang et al., 1998, Science 282:1890-1893 have shown that KCNQ2 and KCNQ3 form a heteromeric potassium ion channel know as the “M-channel.” The M-channel is an important target for drug discovery since mutations in KCNQ2 and KCNQ3 are responsible for causing epilepsy (Biervert et al., 1998, Science 279:403-406; Singh et al., 1998, Nature Genet.", "18:25-29; Schroeder et al., Nature 1998, 396:687-690).", "A screening program designed to identify activators or inhibitors of the M-channel would benefit greatly by the use of cation channels comprising human HCN1 protein as minus targets.", "Accordingly, the present invention includes methods for identifying drug candidates that modulate ion channels where the methods encompass using human HCN1 in a counterscreen.", "Such methods comprise: (a) determining that a compound is an activator or an inhibitor of an ion channel where the ion channel does not comprise human HCN1; and (b) determining that the compound is not an activator or an inhibitor of ion channels comprising human HCN1.Of course, human HCN1 may also be valuable in counterscreens where the primary drug target is not an ion channel.", "Thus, the present invention includes a method for determining that a drug candidate is not an activator or inhibitor of human HCN1 comprising: (a) selecting a drug target that is not human HCN1; (b) screening a collection of compounds to identify a compound that is an activator or an inhibitor of the drug target; and (c) determining that the compound identified in step (b) is not an activator or an inhibitor of human HCN1.The present invention also includes antibodies to the human HCN1 protein.", "Such antibodies may be polyclonal antibodies or monoclonal antibodies.", "The antibodies of the present invention can be raised against the entire human HCN1 protein or against suitable antigenic fragments that are coupled to suitable carriers, e.g., serum albumin or keyhole limpet hemocyanin, by methods well known in the art.", "Methods of identifying suitable antigenic fragments of a protein are known in the art.", "See, e.g., Hopp & Woods, 1981, Proc.", "Natl.", "Acad.", "Sci.", "USA 78:3824-3828; and Jameson & Wolf, 1988, CABIOS (Computer Applications in the Biosciences) 4:181-186.For the production of polyclonal antibodies, human HCN1 protein or antigenic fragments, coupled to a suitable carrier, are injected on a periodic basis into an appropriate non-human host animal such as, e.g., rabbits, sheep, goats, rats, mice.", "The animals are bled periodically and sera obtained are tested for the presence of antibodies to the injected human HCN1 protein or antigenic fragment.", "The injections can be intramuscular, intraperitoneal, subcutaneous, and the like, and can be accompanied with adjuvant.", "For the production of monoclonal antibodies, human HCN1 protein or antigenic fragments, coupled to a suitable carrier, are injected into an appropriate non-human host animal as above for the production of polyclonal antibodies.", "In the case of monoclonal antibodies, the animal is generally a mouse.", "The animal's spleen cells are then immortalized, often by fusion with a myeloma cell, as described in Kohler & Milstein, 1975, Nature 256:495-497.For a fuller description of the production of monoclonal antibodies, see Antibodies: A Laboratory Manual, Harlow & Lane, eds., Cold Spring Harbor Laboratory Press, 1988.Gene therapy may be used to introduce human HCN1 protein into the cells of target organs.", "Nucleotides encoding human HCN1 protein can be ligated into viral vectors, which mediate transfer of the nucleotides by infection of recipient cells.", "Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, lentivirus, and polio virus based vectors.", "Alternatively, nucleotides encoding human HCN1 protein can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted transfer using ligand-nucleotide conjugates, lipofection, membrane fusion, or direct microinjection.", "These procedures and variations thereof are suitable for ex vivo as well as in vivo gene therapy.", "Gene therapy with wild type human HCN1 proteins will be particularly useful for the treatment of diseases where it is beneficial to elevate cation channel activity.", "Gene therapy with a dominant negative mutant of human HCN1 protein will be particularly useful for the treatment of diseases where it is beneficial to decrease cation channel activity.", "The following non-limiting example is presented to better illustrate the invention.", "EXAMPLE Identification and Cloning of Human HCN1 cDNA The complete open reading frame of HCN1 was assembled from two overlapping cDNAs.", "These two cDNAs overlap in the region downstream (3′) of the putative S2 domain of the channel.", "Each cDNA was amplified from brain mRNA by PCR.", "For the cDNA encoding the 5′ sequence, PCR primers were derived from human genomic DNA sequence on chromosome 2 (GenBank accession no.", "AC013384) and from EST AF064876.The PCR primers used to amplify the 3′ region of the coding sequence were derived from ESTs AF064876 and AW054787.Three identical cDNAs, encoding the amino terminal sequence, were obtained by standard PCR techniques using the following primer pairs in nested PCR reactions.", "Primers with SEQ.ID.NOs.", ":41 and 43 are nested forward primers and those with SEQ.ID.NOs.", ":42 and 44 are nested reverse primers.", "5′ CCG GCG AGT CTG GAG CCC GCC 3′ (SEQ.ID.NO.", ":41) 5′ AAT AAT TCA TCT TGA TCA CTT (SEQ.ID.NO.", ":42) T 3′ 5′ CCGTCGCCGGCCGCGTCCTCC 3′ (SEQ.ID.NO.", ":43) 5′ TGT TGT TGT TTG CTC TGT 3′ (SEQ.ID.NO.", ":44) The cDNA encoding the 3′region was amplified in a similar manner.", "Primers with SEQ.ID.NOs.", ":45 and 47 represent the forward nested primers used in that amplification.", "SEQ.ID.NOs.", ":46 and 48 are the nested reverse primer pairs.", "5′ TGG AAT CAC ATT CTT TAC AGA GCA AAC A 3′ (SEQ.ID.NO.", ":45) 5′ TAG TCT CAG TTT ATG AGA GTA TTT CTT 3′ (SEQ.ID.NO.", ":46) 5′ GGACCCCAAAGTGATCAAGATGAAT 3′ (SEQ.ID.NO.", ":47) 5′ TCT GCT TTG ACA ATC AGC AGG 3′ (SEQ.ID.NO.", ":48) One 5′ cDNA (amplified using primer pair SEQ.ID.NO.", ":45 and SEQ.ID.NO.", ":46) and two 3′ cDNAs (amplified using primer pair SEQ.ID.NO.", ":47 and SEQ.ID.NO.", ":48) were isolated and sequenced.", "When all amino and carboxyl sequences were aligned and compared to the corresponding EST and genomic DNA sequences, eight putative single nucleotide polymorphisms were identified.", "The present invention is not to be limited in scope by the specific embodiments described herein.", "Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description.", "Such modifications are intended to fall within the scope of the appended claims.", "Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties." ] ]	Patent_10466992
[ [ "Transient eutectic phase process for ceramic-metal bonding metallization and compositing", "A method for directly joining ceramics (10) and metals (12).", "The method involves forming a structure having a ceramic component (10), a more refractory metallic component and a less refractory metallic-material-based interlayer (14) disposed between the ceramic component (10) and the metallic component (12); adding a eutectic forming reactant to the metallic interlayer (14); and heating the structure to approximately a eutectic melting temperature of the reactant and the interlayer to form a metallic-material-based eutectic liquid that interacts with the metallic component to form a bond that directly joins the ceramic and metallic components to one another." ], [ "1.A method for directly joining ceramics and metals, the method comprising: forming a structure having a ceramic component, a metallic component and a metallic interlayer disposed between the ceramic component and the metal metallic component, the metallic interlayer being less refractory than the metallic component; adding a eutectic liquid forming reactant to the metallic interlayer; and heating the structure to approximately a eutectic melting temperature of the reactant and the interlayer to form metallic-material-based eutectic liquid that interacts with the ceramic component and the metallic component to form a bond that directly joins the ceramic and metallic components to one another.", "2.The method according to claim 1, wherein the structure further includes a barrier layer that controls the interaction between the metallic interlayer and the metallic component.", "3.The method according to claim 1, wherein the adding step is performed prior to the heating step.", "4.The method according to claim 1, wherein the adding step is performed substantially concurrent with the heating step.", "5.The method according to claim 1, wherein the reactant comprises a gas.", "6.The method according to claim 5, wherein the gas comprises oxygen.", "7.The method according to claim 1, wherein the metallic interlayer comprises copper.", "8.The method according to claim 1, wherein the ceramic component comprises alumina.", "9.The method according to claim 1, wherein the metallic component comprises nickel.", "10.The method according to claim 1, wherein the reactant comprises oxygen, the metallic interlayer comprises copper, the ceramic component comprises alumina, and the metallic component comprises nickel.", "11.The method according to claim 1, wherein the ceramic component is selected from the group consisting of a ceramic layer, ceramic particles, ceramic fibers, ceramic fibrous structures, and combinations thereof; the metallic component is selected from the group consisting of a metal layer, a metal alloy layer, an intermetallic layer, metal particles, metal alloy particles, intermetallic particles, metal fibers, metal alloy fibers, intermetallic fibers, metal fibrous structures, metal alloy fibrous structures, intermetallic fibrous structures and combinations thereof; and the metallic interlayer is selected from the group consisting of a metal, a metal alloy, an intermetallic, and combinations thereof.", "12.A method for directly joining ceramics and metals, the method comprising: forming a structure having a ceramic component and a metallic component; and reacting a metallic-material-based eutectic liquid with the metallic component, which is more active than the eutectic liquid, such that active metal specie diffuse to the ceramic component thereby enhancing bonding between the ceramic component and the metallic component.", "13.The method according to claim 12, wherein the ceramic component is selected from the group consisting of a ceramic layer, ceramic particles, ceramic fibers, ceramic fibrous structures, and combinations thereof; the metallic component is selected from the group consisting of a metal layer, a metal alloy layer, an intermetallic layer, metal particles, metal alloy particles, intermetallic particles, metal fibers, metal alloy fibers, intermetallic fibers, metal fibrous structures, metal alloy fibrous structures, intermetallic fibrous structures and combinations thereof; and the metallic-material-based eutectic liquid is selected from the group consisting of a metal, a metal alloy, an intermetallic, and combinations thereof.", "14.A method for directly joining ceramics and metals, the method comprising: forming a structure having a ceramic component and a metallic component; and reacting a metallic-material-based eutectic liquid with the metallic component, which is more refractory than the eutectic liquid, to form a liquid composition that solidifies isothermally as a transient liquid phase joining the ceramic component and the metal component to one another.", "15.The method according to claim 14, wherein the ceramic component is selected from the group consisting of a ceramic layer, ceramic particles, ceramic fibers, ceramic fibrous structures, and combinations thereof; the metallic component is selected from the group consisting of a metal layer, a metal alloy layer, an intermetallic layer, metal particles, metal alloy particles, intermetallic particles, metal fibers, metal alloy fibers, intermetallic fibers, metal fibrous structures, metal alloy fibrous structures, intermetallic fibrous structures and combinations thereof; and the metallic-material-based eutectic liquid is selected from the group consisting of a metal, a metal alloy, an intermetallic, and combinations thereof.", "16.A method for directly joining ceramics and metals, the method comprising: forming a structure having a ceramic component and a metallic component; reacting the metallic component with a metallic-material-based eutectic liquid that transitions into a transient liquid phase that solidifies; and further reacting the solidified transient liquid phase with the metallic component, which is more refractory than the metallic component, at elevated temperature to form a solid metallic composition with a melting point that is greater than the solidified transient liquid phase.", "17.The method according to claim 16, wherein the ceramic component is selected from the group consisting of a ceramic layer, ceramic particles, ceramic fibers, ceramic fibrous structures, and combinations thereof; the metallic component is selected from the group consisting of a metal layer, a metal alloy layer, an intermetallic layer, metal particles, metal alloy particles, intermetallic particles, metal fibers, metal alloy fibers, intermetallic fibers, metal fibrous structures, metal alloy fibrous structures, intermetallic fibrous structures and combinations thereof; and the metallic-material-based eutectic liquid is selected from the group consisting of a metal, a metal alloy, an intermetallic, and combinations thereof.", "18.A method for directly joining ceramics and metals, the method comprising: forming a structure having a ceramic component and a metallic component; providing a metallic-material-based eutectic liquid that transitions into a transient liquid phase that solidifies; and reacting the solidified transient liquid phase with the metallic component, which is more refractory than the solidified transient liquid phase, at an elevated temperature to form a homogeneous metallically bonded material.", "19.The method according to claim 16, wherein the ceramic component is selected from the group consisting of a ceramic layer, ceramic particles, ceramic fibers, ceramic fibrous structures, and combinations thereof; the metallic component is selected from the group consisting of a metal layer, a metal alloy layer, an intermetallic layer, metal particles, metal alloy particles, intermetallic particles, metal fibers, metal alloy fibers, intermetallic fibers, metal fibrous structures, metal alloy fibrous structures, intermetallic fibrous structures and combinations thereof; and the metallic-material-based eutectic liquid is selected from the group consisting of a metal, a metal alloy, an intermetallic, and combinations thereof.", "20.A method of fabricating a composite structure or material, the method comprising: providing a ceramic component selected from the group consisting of ceramic particles, ceramic fibers, and ceramic fibrous structures and combinations thereof; providing a metallic component selected from the group consisting of metal particles, metal alloy particles, intermetallic particles, metal fibers, metal alloy fibers, intermetallic fibers, metal fibrous structures, metal alloy fibrous structures, intermetallic fibrous structures, and combinations thereof, the metallic component coated with a less refractory metallic interlayer selected from the group consisting of a metal, a metal alloy, an intermetallic, and combinations thereof; mixing the ceramic component with the metallic component, the metallic interlayer being disposed between the ceramic component and the metallic component; adding a eutectic liquid forming reactant to the metallic interlayer; and heating the structure to approximately a eutectic melting temperature of the reactant and the metallic interlayer to form a metallic-material-based eutectic liquid that interacts with the ceramic component and the metallic component to form a bond that directly joins the ceramic component and metallic component to one another.", "21.The method according to claim 20, wherein the eutectic liquid transitions into a transient liquid phase that solidifies; and reacting the solidified transient liquid phase with the metallic component, which is more refractory than the solidified transient liquid phase, at an elevated temperature to form a homogeneous metallically bonded material better." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>No single material is available today that possesses all of the material properties to meet the stringent demands of many traditional and advanced applications.", "Metals, although ductile with high thermal and electrical conductivity, often cannot withstand high temperatures or corrosion, and expand significantly with increasing temperature.", "An alternative to metals are ceramics, which are brittle insulators.", "Ceramics are refractory, hard, and wear-resistant, with excellent hot properties and relatively low thermal expansion.", "By joining ceramics and metals, composite components that may employ the desired properties of each material, can be manufactured to meet these increasing requirements.", "The technology required to bond these dissimilar materials effectively, reliably, and economically is in high demand.", "Several joining technologies, which utilize interfacial methods, have proven effective for bonding ceramics and metals, but their high processing costs limit their penetration of some potential markets.", "Direct joining or bonding requires few processing steps and therefore significantly reduces cost and eliminates interfacial joining material that may compromise properties.", "It can also provide property advantages such as hermeticity, stress transfer, stress reduction, continuity of strain, electrical response, interfacial properties, and mechanical interlocking.", "Therefore, an effective method of directly joining or bonding ceramics and metals is needed." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>A method is described herein for directly joining ceramics and metals.", "The method comprises forming a structure having a ceramic component, a metallic component and a metallic interlayer disposed between the ceramic component and the metallic component, the metallic interlayer being less refractory than the metallic component by means of a eutectic melt formed by adding a eutectic-forming reactant, such as a gas, an oxide of the metallic material of the interlayer or other compound, to the metallic interlayer (this is commonly termed gas-metal eutectic); and heating the structure to approximately a eutectic melting temperature of the eutectic-based interlayer system to form a metallic-material-based eutectic liquid that interacts with the metallic component to form a bond that directly joins the ceramic and metallic components to one another.", "The components may be bulk parts, metallization, ceramic coating layers, or compositing materials" ], [ "FIELD OF THE INVENTION The present invention relates to ceramic-metal bonding, ceramic metallization and ceramic-metal compositing and more particularly, to a method that utilizes a low temperature transient metallic-material-based eutectic liquid to directly bond ceramic bulk materials and coatings to metals and visa versa, metallize ceramics and produce ceramic-metal composites in a wide variety of configurations.", "BACKGROUND OF THE INVENTION No single material is available today that possesses all of the material properties to meet the stringent demands of many traditional and advanced applications.", "Metals, although ductile with high thermal and electrical conductivity, often cannot withstand high temperatures or corrosion, and expand significantly with increasing temperature.", "An alternative to metals are ceramics, which are brittle insulators.", "Ceramics are refractory, hard, and wear-resistant, with excellent hot properties and relatively low thermal expansion.", "By joining ceramics and metals, composite components that may employ the desired properties of each material, can be manufactured to meet these increasing requirements.", "The technology required to bond these dissimilar materials effectively, reliably, and economically is in high demand.", "Several joining technologies, which utilize interfacial methods, have proven effective for bonding ceramics and metals, but their high processing costs limit their penetration of some potential markets.", "Direct joining or bonding requires few processing steps and therefore significantly reduces cost and eliminates interfacial joining material that may compromise properties.", "It can also provide property advantages such as hermeticity, stress transfer, stress reduction, continuity of strain, electrical response, interfacial properties, and mechanical interlocking.", "Therefore, an effective method of directly joining or bonding ceramics and metals is needed.", "SUMMARY OF THE INVENTION A method is described herein for directly joining ceramics and metals.", "The method comprises forming a structure having a ceramic component, a metallic component and a metallic interlayer disposed between the ceramic component and the metallic component, the metallic interlayer being less refractory than the metallic component by means of a eutectic melt formed by adding a eutectic-forming reactant, such as a gas, an oxide of the metallic material of the interlayer or other compound, to the metallic interlayer (this is commonly termed gas-metal eutectic); and heating the structure to approximately a eutectic melting temperature of the eutectic-based interlayer system to form a metallic-material-based eutectic liquid that interacts with the metallic component to form a bond that directly joins the ceramic and metallic components to one another.", "The components may be bulk parts, metallization, ceramic coating layers, or compositing materials BRIEF DESCRIPTION OF THE DRAWINGS FIGS.", "1-4 are sectional views through a metal/metal eutectic-forming interlayer/ceramic system which illustrate the method of the present invention.", "DETAILED DESCRIPTION The present invention is a method of directly joining or bonding ceramics and metals (the terms “metal” and “metallic” being used herein to encompass metals, metal alloys, intermetallics, materials containing a substantial amount of metallically bonded materials, and any combination or combinations thereof) using a transient, low-temperature, metallic-material-based eutectic liquid or melt, i.e., where the metallic-material-based eutectic liquid “disappears” via solidification into a desired alloy or other metallically bonded material.", "To be consistent with current terminology and the illustrative embodiment to be described further on, the metallic-material-based eutectic liquid or melt will also be referred to hereinafter as “gas-metal eutectic.” It should be understood, however, that the eutectic constituents may also be provided by a liquid or solid in contact with an interlayer formed of a metallic material (metallic interlayer).", "The transient, low-temperature, metallic-material-based eutectic liquid is generated in the present invention by combining a eutectic-forming reactant, such as a gas, an oxide layer or another compound, with the metallic interlayer having a eutectic melting temperature which is lower than that of the metallic material of the interlayer of the subject ceramic-metal system.", "The method is useful for but not limited to: direct bonding ceramic coatings to metals, direct bonding metal coatings to ceramics, producing a metallic joint between two or more ceramic components for bonding ceramics to ceramics, metallizing ceramics and producing ceramic-metal composites.", "According to the principles of the present invention, the low temperature, metallic-material-based eutectic melt or liquid is made transient (by solidification) through interaction with a more refractory metallic component, that in some embodiments is more active than the metallic material of the eutectic liquid.", "It should be noted that the more active metallic component, which is nickel in the exemplary embodiment described herein, improves the bond and enhances the bond quality.", "The metallic-material-based eutectic liquid provides, when the metallic component is directly joined with a ceramic component, a ceramic-metal bond or joint having good wetting, high strength, a broad process window (relative to conventional gas-metal eutectic bonds), high thermal stability, and controlled thermo-elastic stress.", "The metallic-material-based eutectic liquid of the present invention also enables the transportation of a more active metal species to the ceramic interface to further improve adherence.", "For illustrative purposes only, the method of the present invention will now be described with application to a nickel/oxygen-copper/alumina system.", "One of ordinary skill in the art will of course recognize that the method of the invention is applicable to other metal/metallic-material-based eutectic/ceramic systems.", "The metallic-material-based eutectic interlayer is formed by interaction of the metallic interlayer with a gas, liquid or solid which promotes formation of a low-melting eutectic.", "Referring now to the drawings and initially to FIG.", "1, an exemplary embodiment of the method of the present invention may commence with the fabrication of a multilayer structure that includes a ceramic component layer 10, a metallic component layer 12, and a metallic interlayer 14.The metallic component layer 12 may be more active than the metallic interlayer 14 and is, therefore, referred to hereinafter as active metal component layer 12.The metallic interlayer 14, when combined with appropriate specie from a eutectic forming reactant, such as a gas, liquid or solid, has a lower melting temperature than the metallic interlayer 14 or the active metal component layer 12.In the exemplary metal/oxygen-copper/alumina system illustrated in FIGS.", "1-4, the ceramic component layer 10 comprises alumina, the active metal component layer 12 comprises nickel, and the metallic interlayer 14 comprises copper, preoxidized copper or copper containing dispersed copper oxide.", "Each metallic layer in the structure may be provided as a solid using one or more metal foils, a metal powder or a metal paste, or can be deposited in solution, i.e., plated, evaporated, or sputtered on either material surface prior to joining.", "Referring still to FIG.", "1, a barrier layer 18 is formed on the active metal component layer 12 to minimize competitive interaction of reactants (oxygen in the illustrated system) with the active metal component layer 12, which allows the gas-metal eutectic liquid to form and wet the ceramic prior to reaction with the active metal component layer 12.In the system illustrated in FIGS.", "1-4, the barrier layer 18 may comprise nickel oxide.", "The metallic interlayer 14 should be sufficiently thick, i.e., greater than 10 microns in the case of copper, to form an adequate amount of gas-metal eutectic liquid phase on heating.", "While a thinner metallic interlayer 14 (less than 10 microns in the case of copper) may be used, the heating rate required to achieve melting before the active metal component layer 12 and the metallic interlayer 14 form a metallic alloy or intermetallic may not be possible or practical.", "In addition, the small amount of gas-metal liquid formed from a thin metallic interlayer 14 requires polished surfaces and applied pressure to maintain intimate contact.", "Sufficiently thick metallic interlayers 14 do not require polished surfaces or pressure because the gas-metal eutectic liquid provides wetting, reaction, and adherence to the ceramic component layer 10.Accordingly, there is substantially no need for pressure and conformance fixturing beyond that required for holding parts together.", "Moreover, the gas-metal eutectic liquid penetrates roughness or keyholes for enhanced mechanical bonding.", "Additions of the gas are added to the metallic interlayer 14 of the multilayer structure.", "In the nickel/oxygen-copper/alumina system application, instead of using oxygen or oxygen containing gas mixtures, the oxygen additions may be accomplished by pre-oxidizing the metallic interlayer 14 prior to eutectic melting/bonding, thereby forming a copper oxide layer 16 on the copper interlayer 14.Alternatively, (or in addition to the copper oxide layer 16), gas additions may be accomplished in-situ, i.e., gas may be added during eutectic melting/bonding.", "Eutectic melting/bonding is achieved by heating the multilayer structure in a suitable oven to at least the eutectic melting temperature (copper-oxygen at 1065° C.) of the metallic interlayer 14 and the gas or other interlayer reactant, but below the melting point of the active metal layer 12 (nickel at 1452° C.), in an atmosphere of limited oxygen (reactant) fugacity (inert, controlled oxygen partial pressure, or vacuum).", "The level of oxygen (reactant) should be carefully controlled to facilitate formation of the gas-metal eutectic and to prevent the formation of an excessively thick (several microns thick) reaction layer to be described further on.", "As the temperature is raised above the eutectic melting point, the metal interlayer 14 uniformly forms a transient, low-temperature, gas-metal eutectic melt or liquid 20 at the interface between the active metal component layer 12 or active metal component barrier layer 18 and the ceramic component layer 10 as shown in FIG.", "2.There may be excess interlayer metal (copper) or reactant (oxygen) relative to the exact eutectic composition in which case the temperature must be raised above the liquidous temperature.", "The gas-metal (copper-oxygen) eutectic melt or liquid 20 wets the component layers 10 and 12 or 18, initiates contact therebetween, and begins to react with the ceramic component layer 10 to form a first reaction layer 22 thereon, which in the illustrated system comprises copper-aluminate.", "Because substantially the entire metallic interlayer 14 is melted by raising the temperature above the interlayer metal melting temperature (copper at 1083° C.), the processing window is wider than conventional gas-metal eutectic bonding processes in terms of temperature, atmosphere, and time.", "As the multilayer structure is held above the eutectic melting temperature, the gas-metal eutectic liquid 20 dissolves or consumes the optional barrier layer (nickel oxide) 18, and ultimately, part of the active metal component layer 12.The barrier layer 18, thus, controls the rate of dissolution or diffusion of the active metal component layer 12 into the gas-metal eutectic melt or liquid interlayer 20.The dissolved metallic material is transported toward the ceramic component layer interface where it reacts to form a second reaction layer or replacement reaction layer 26, which comprises nickel-aluminate (NiAl2O4) spinel in the illustrated system, superimposed on or replacing the previously formed first reaction layer 22, which together form a refractory bond phase joint 30 as shown in FIG.", "3.As the active metal (nickel) dissolves or diffuses into the interfacial gas-metal eutectic liquid 20 (copper-oxygen) at temperature, the interlayer liquid composition changes (copper-nickel-oxygen).", "Constant temperature isothermal solidification of the new interfacial gas-metal eutectic composition liquid 20 (copper-nickel-oxygen) occurs to form an interlayer of a solid metal alloy or other metallically bonded material by the transient liquid phase process as shown in FIG.", "4.Diffusional homogenization (blending of unlike elements) further increases the solidus temperature of the solid metallic interlayer portion 24 of the joint 30.An extended hold at the bond temperature or other elevated temperature causes interdiffusion of the active metal component layer 12 and the solid metallic interlayer 24, which results in a strong component layer 28 of metal alloy or other metallically bonded material, (a copper-nickel alloy in the illustrated system), that is bonded to the ceramic component layer 10 by a thin (micron-thick) interfacial compound formed by reaction layers 22 and 26.The interfacial metals of the reaction layers 22 and 26 may fully homogenize with the metallic component layer 28 (with a sufficient hold at elevated temperature thereby eliminating the metal-metal interface.", "Because the low-melting, liquid metal (copper in the illustrated system) of layer 20 incorporates the more refractory metal from the metallic component layer 12 to form the metal alloy layer 28, the resulting bond or joint 30 has a melting point significantly higher than the temperature at which the bond or joint 30 was formed.", "The thickness and composition of reaction layers 22 and 26 may be further modified by changing the oxygen fugacity during the extended hold at elevated temperature.", "The method of the present invention was evaluated using the illustrative nickel/copper-oxygen/alumina system.", "Multilayer bond structures were produced using both foils and plating.", "Oxygen additions were investigated using pre-oxidation of each metal and/or oxidation in-situ.", "The best bonds resulted from foils combining nickel pre-oxidation with a eutectic atmosphere.", "Adhesion was comparable to current technologies with a peel test strength of about 50 N/cm as compared to about 35 N/cm for nickel foil bonded by the direct bond copper method.", "The bond can exceed the ceramic strength as shown by occasional peel test failures in the ceramic rather than the bond interfaces.", "Typical peel failure occurred at the metal (the nickel/copper-nickel) interface.", "Residual thermo-elastic stress is reduced relative to conventional direct bond copper.", "A high-temperature peel test was developed to evaluate thermal stability.", "It showed that strength was maintained to 800° C., the apparatus limit.", "Long term exposure at 1000° C. did not deteriorate bond strength when interfacial oxidation was limited.", "The direct bond method of the present invention increases flexibility in processing temperature and atmosphere, reduces residual bond stresses, and significantly improves high temperature corrosion and mechanical properties at a reduced processing cost.", "Large parts, rough surfaces, and complex geometries can be accommodated in the method of the present invention.", "The method of the present invention may also be employed to produce metal matrix composites (MMC's) containing ceramic particles (cermets), fibers, fibrous structures (weaves and preforms) or combinations thereof.", "Similarly, it may be used to produce ceramic matrix composites.", "In order to fabricate a microscopic or macroscopic composite structure or material, metallic (such as nickel) particles, fibers, fibrous structures or combinations thereof are coated with a less refractory metallic interlayer (such as copper), thereby forming multilayer particles, fibers, or fibrous structures.", "The active metal particles, fibers, or fibrous structures may first be oxidized or otherwise coated with a barrier layer (such as nickel oxide) to prevent premature interaction between the interlayer and the active metal.", "These particles, fibers, or fibrous structures are then mixed with or infiltrated into the ceramic powder or preform.", "Alternately, an active metal preform or weave may be coated with a less refractory metal and infiltrated with ceramic.", "As in the case of the multilayer structure described above, by adding a eutectic forming reactant, such a gas, to the metallic interlayer; and heating the structure to approximately a eutectic melting temperature of the reactant and the interlayer, a eutectic gas-metal (in the case of a gas reactant) liquid may be formed.", "This eutectic liquid interacts with the ceramic component particles, fibers, or fibrous structure and the metallic component particles, fibers or fibrous structures to form a bond that directly joins the ceramic and metal composite particles, fibers, or fibrous structures to one another.", "The eutectic liquid to subsequently transforms to solid by the transient liquid phase method described above.", "The bond structure and further treatments are as described in the multilayer structure above, but possess a three-dimensional composite arrangement at microscopic and/or macroscopic scales.", "While the foregoing invention has been described with reference to the above embodiments, various modifications and changes can be made without departing from the spirit of the invention.", "Accordingly, all such modifications and changes are considered to be within the scope of the appended claims." ] ]	Patent_10467006
[ [ "Mutant sh-3 binding protein compositions and methods", "The invention provides mutant SH3-binding protein (SH3BP2) nucleic acids, polypeptides, and agents which selectively bind to the mutant SH3BP2 molecules and which do not bind to the wild type SH3BP2 molecules.", "Methods for selecting agents which inhibit mutant SH3BP2 expression, as well as diagnostic and therapeutic methods which utilize the mutant SH3BP2 molecules for diagnosing and treating disorders of bone homeostasis, also are provided." ], [ "1.An isolated nucleic acid molecule, comprising (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO: 25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9, (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to the degeneracy of the genetic code, and (c) complements of (a) or (b), provided that the nucleic acid molecule is not SEQ ID NO:1 (human “WT” SH3BP2 full length sequence) or SEQ ID NO:25 (human WT genomic).", "2.The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), and SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID Nos:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9.3.The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule codes for a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100.4.An isolated nucleic acid molecule selected from the group consisting of (a) a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, and other sequences capable of hybridizing to a mutant SH3BP2 nucleic acid and, thereby, useful in amplifying an exon 9 of a mutant SH3BP2 nucleic acid molecule, and (b) complements of (a).", "5.An expression vector comprising the isolated nucleic acid molecule of any of the foregoing claims, operably linked to a promoter.", "6.A host cell transformed or transfected with the expression vector of claim 5.7.A transgenic non-human animal having somatic and germ line cells that contain a SH3BP2 mutant nucleic acid molecule of claim 1, wherein expression of the SH3BP2 mutant nucleic acid molecule results in the animal having a bone homeostasis disorder.", "8.An isolated polypeptide encoded by the isolated nucleic acid molecule of claim 1.9.The isolated polypeptide of claim 8, wherein the isolated polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NOs:64-100.10.The isolated peptide of claim 8 or 9, wherein the peptide comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 contiguous amino acids having a sequence that is a unique fragment of a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100.11.A composition comprising an isolated binding agent that binds, selectively to a nucleic acid of claim A1 or to a polypeptide of claim 8.12.The composition of claim 11, wherein the isolated binding agent is a nucleic acid.", "13.The composition of claim 11, wherein the isolated binding agent is a peptide.", "14.The composition of claim 13, wherein the peptide is an antibody, or a fragment thereof.", "15.A method of identifying the presence of an SH3BP2 mutant molecule in a sample comprising analyzing the sample for the presence of a SH3BP2 mutant nucleic acid molecule or a SH3BP2 mutant polypeptide.", "16.The method of claim 15, further comprising contacting the sample with at least two nucleic acid amplification primers, wherein a first nucleic acid amplification primer and a second nucleic acid amplification primer are capable of hybridizing to a SH3BP2 mutant nucleic acid molecule and amplifying a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain), SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO: 19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9, amplifying a primed nucleic acid molecule which hybridizes to the first and the second nucleic acid amplification primers; and detecting the presence of an amplified nucleic acid molecule in the sample.", "17.The method of claim 15, further comprising contacting the sample with one or more nucleic acid probes, wherein the nucleic acid probe is capable of hybridizing to a nucleic acid sequence selected from the group consisting of: SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO: 19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9, and which does not hybridize to SEQ ID NO:1 (human “WT” SH3BP2 full length sequence), SEQ ID NO:3 (human WT domain), or SEQ ID NO:25 (genomic WT SH3BP2) and detecting the presence of a SH3BP2 mutant nucleic acid in the sample which hybridizes to the nucleic acid probe.", "18.The method of claim 15, further comprising contacting the sample with one or more binding agents, wherein the binding agent is capable of selectively binding to a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100 and detecting the presence of a SH3BP2 mutant polypeptide in the sample which binds the binding agent.", "19.The method of claim 18, wherein the binding agent is an antibody or a fragment thereof.", "20.A method for evaluating susceptibility of a human subject to a bone homeostasis disorder, comprising: obtaining a sample of DNA from the subject; and evaluating the sample of DNA for the presence of nucleotides encoding a mutant residue at an amino acid position encoded by exon 9 of the SH3BP2 gene product, wherein the presence of a mutant residue is indicative of increased susceptibility to a bone homeostasis disorder.", "21.The method of claim 20, further comprising the step of amplifying the DNA prior to evaluating the sample of DNA.", "22.A method of evaluating causation of a bone homeostasis disorder in a human subject, comprising obtaining a sample of DNA from the subject; and evaluating the sample of DNA for the presence of nucleotides encoding a mutant residue at an amino acid position encoded by exon 9 of the SH3BP2 gene product, wherein the presence of a mutant residue is indicative of SH3BP2 mutant causation of a bone homeostasis disorder.", "23.A method of evaluating the genetic predisposition of a human subject to have offspring with a bone homeostasis disorder, comprising obtaining a sample of DNA from the subject; and evaluating the sample of DNA for the presence of nucleotides encoding a mutant residue at an amino acid position encoded by exon 9 of the SH3BP2 gene product, wherein the presence of a mutant residue indicates that the human subject has a predisposition to having offspring with a bone homeostasis disorder.", "24.The method of claims 20, 21, 22, or 23, wherein evaluating comprises evaluating the sample of DNA for the presence of nucleotides encoding a mutant residue at an amino acid position selected from the group consisting of amino acid positions 415, 418, and 420 of the SH3BP2 gene product.", "25.A method of screening for an agent that inhibits the production of a SH3BP2 mutant polypeptide, comprising determining the level of a SH3BP2 mutant molecule (nucleic acid or polypeptide) in the absence of a putative inhibitor, determining the level of a SH3BP2 mutant molecule in the presence of the putative inhibitor, and comparing the level of the SH3BP2 mutant molecule in the presence and absence of the putative inhibitor, wherein a decrease in the level of the SH3BP2 mutant molecule in the presence of the putative inhibitor indicates that the putative inhibitor is an agent that inhibits the production of a SH3BP2 mutant polypeptide.", "26.The method of claim 25, wherein the agent inhibits transcription of a SH3BP2 mutant nucleic acid molecule.", "27.The method of claim 25, wherein the agent inhibits translation of a SH3BP2 mutant nucleic acid molecule.", "28.A method for treating a subject having a bone homeostasis disorder characterized by the presence of a SH3BP2 mutant nucleic acid molecule comprising administering a SH3BP2 mutant nucleic acid antisense molecule to a subject in need of such treatment in an amount effective to treat the subject, provided the subject is not otherwise in need of SH3BP2 mutant nucleic acid antisense molecule treatment.", "29.The method of claim 28, wherein the disorder is cherubism.", "30.The method of claim 28, wherein the disorder is a bone tumor.", "31.The method of claim 28, wherein the agent is locally administered.", "32.The method of claim 28, wherein the agent is locally administered to a periodontal pocket.", "33.The method of claim 28, wherein the agent is systemically administered.", "34.A method of reducing expression of a mutant SH3BP2 nucleic acid molecule in a cell having a mutant SH3BP2 nucleic acid molecule comprising: introducing a mutant SH3BP2 binding molecule into a cell, and allowing the mutant SH3BP2 binding molecule to hybridize to a sense mutant SH3BP2 nucleic acid molecule, thereby inhibiting expression of the sense mutant SH3BP2 nucleic acid molecule.", "35.The method of claim 34, wherein the mutant SH3BP2 binding molecule is an antisense molecule.", "36.The method of claim 34, wherein the mutant SH3BP2 binding molecule is a ribozyme." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The remodeling of bone is a dynamic process.", "Cells continuously lay down and resorb bone material.", "Bone homeostasis refers to the balance between bone formation and bone degradation.", "An imbalance in the activity of cells that lay down new bone (osteoblasts) and cells that resorb bone (osteoclasts) can result in serious, and sometimes fatal, disorders.", "Thus, imbalances in bone homeostasis are associated with a variety of adverse medical conditions.", "Osteoporosis is a term used for a number of diseases of diverse etiology, all involving a reduction in the mass of bone per unit volume.", "Osteoporosis is the most common of the metabolic bone diseases.", "Twenty-five million people in the United States and more than two hundred million people worldwide are affected by osteoporosis.", "Osteoporosis is frequent among post-menopausal women and is an important cause of morbidity in the elderly.", "It commonly results in bone fractures, and death can be a frequent occurrence in the months following fractures, particularly those of the hip in elderly individuals.", "Osteopetrosis is a disorder involving an increase in the mass of bone per unit volume.", "Its incidence is rare compared to osteoporosis, but it typically is life threatening.", "Despite having multiple causes, a defect in bone resorption is always the underlying mechanism.", "In many instances, the disorder is inherited as an autosomal recessive trait and involves abnormal osteoclast function.", "Bone marrow transplants from normal donors have been attempted to restore normal osteoclast precursor cells, but this therapy has shown only limited success.", "Present treatments for disorders of bone homeostasis are inadequate.", "In view of the foregoing, a need exists to develop new and improved methods and compositions for treating disorders of bone homeostasis.", "Preferably such methods and compositions are based upon inhibiting the particular interactions between cell components which mediate the abnormal physiological effect, thereby minimizing harmful side effects that may be due to non-specific therapeutic approaches." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention is based, in part, on our discovery of point mutations that cause amino acid substitutions in the SH3-binding protein SH3BP2.We have collected samples from 15 families, consisting of 66 individuals clinically diagnosed with cherubism (an exemplary bone homeostasis disorder), 4 obligate carriers, and 79 unaffected subjects.", "In 12 families, where mutations were found, the mutations co-segregate with the disease phenotype.", "We also cloned SH3BP2 cDNA from EBV-transformed patient lymphoblasts into TA-vectors and sequenced them.", "Half of the sequenced clones from each patient carried the mutation.", "We sequenced genomic DNAs from 100 unrelated and unaffected African-Brazilians and 100 Caucasian controls without detecting any of the sequence variants found in affected cherubism patients.", "The accumulation of co-segregating sequence variants in families with cherubism and their absence in unaffected controls provide compelling evidence that the mutations in SH3BP2 cause cherubism.", "In view of the foregoing discoveries, the mutant SH3BP2 molecules disclosed herein have a variety of uses including, for example, diagnostic applications (e.g., genetic testing including amniocentesis); screening methods to identify agents that are useful for treating bone homeostasis disorders such as cherubism, osteoporosis, osteopetrosis, and bone tumors (malignant or benign); and therapeutic applications that utilize the compositions of the invention (e.g., antibodies selective for mutant SH3P2 epitope, antisense) for treating bone homeostasis disorders.", "These and other aspects of the invention are summarized below.", "According to a first aspect of the invention, an isolated nucleic acid molecule is provided.", "The isolated nucleic acid molecule includes: (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO: 19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO: 25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9, (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to the degeneracy of the genetic code, and (c) complements of (a) or (b) (e.g., antisense to the mutant SH3BP2 domain SEQ ID NO:7), provided that the nucleic acid molecule is not SEQ ID NO: 1 (human “WT” SH3BP2 full length sequence) or SEQ ID NO: 25 (human WT genomic).", "In certain preferred embodiments, the nucleic acid molecule also is not SEQ ID NO:3 (human WT domain) or SEQ ID NO:102 (human WT exon 9).", "In these and/or other preferred embodiments, the isolated nucleic acid molecules of the invention do not hybridize under stringent conditions to a sequence selected from the group consisting of SEQ ID NO:1 (human “WT” SH3BP2 full length sequence), SEQ ID NO:3 (human WT domain), SEQ ID NO:25 (human WT genomic), and SEQ ID NO:101 (human WT exon 9).", "An abbreviated Sequence Listing which identifies the sequence and/or GenBank accession number information for the sequences relating to the invention is provided herewith as Table 1.In preferred embodiments, the isolated nucleic acid molecule of the invention has a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9.In these and other embodiments, the preferred isolated nucleic acid molecules of the invention code for a polypeptide having a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NOs:64-100.According to yet another aspect of the invention, primers for amplifying the above-identified nucleic acid sequences are provided.", "In this aspect, the preferred isolated nucleic acid molecules are selected from the group consisting of (a) a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO:23, 24 and other sequences which are capable of Is hybridizing to an SH3BP2 mutant molecule and, thereby, are useful in amplifying exon 9 in a mutant SH3BP3 nucleic acid molecule, and (b) complements of (a).", "The invention also embraces expression vectors containing the isolated nucleic acid molecules of the invention, operably linked to a promoter, as well as host cells transformed or transfected with such expression vectors.", "Pharmaceutical compositions comprising the compositions of the invention, as well as methods for making such medicaments also are provided.", "Transgenic non-human animals having somatic and germ line cells that contain any of the isolated SH3BP2 mutant nucleic acid molecules of the invention also are provided.", "Preferably, the expression of the SH3BP2 mutant nucleic acid molecule results in the animal having a bone homeostasis disorder.", "Suck animals are useful as models of human bone homeostasis disorders.", "According to another aspect of the invention, isolated polypeptides encoded by any of the isolated nucleic acid molecules of the invention are provided.", "Preferably, the isolated polypeptides of the invention include a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100.According to still another aspect of the invention, compositions containing an isolated binding agent that binds selectively to a nucleic acid molecule or to a polypeptide molecule of the invention also are provided.", "In certain embodiments, the isolated binding agent is a nucleic acid (e.g., an antisense, a ribozyme); in yet other embodiments, the isolated binding agent is a peptide (e.g., an antibody, or a fragment thereof).", "According to another aspect of the invention, a method of identifying the presence of an SH3BP2 mutant molecule in a sample is provided.", "In general, the method involves analyzing the sample for the presence of a SH3BP2 mutant nucleic acid molecule or a SH3BP2 mutant polypeptide.", "In certain embodiments, the method further involves the steps of contacting the sample with at least two nucleic acid amplification primers and amplifying DNA in the sample prior to analyzing the sample for the presence of a SH3BP2 mutant nucleic acid molecule.", "The method employs a first nucleic acid amplification primer and a second nucleic acid amplification primer that are capable of hybridizing to a SH3BP2 mutant nucleic acid molecule and, thereby, are useful in amplifying a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9.Following hybridization, the method involves amplifying a primed nucleic acid molecule which hybridizes to the first and the second nucleic acid amplification primers; and detecting the presence of an amplified nucleic acid molecule in the sample.", "In yet other embodiments, the method involves contacting the sample with one or more nucleic acid probes, wherein the nucleic acid probe is capable of hybridizing to a nucleic acid sequence selected from the group consisting of: nucleic acid sequences which hybridize to a sequence selected from one or more of the following sequences under stringent conditions: SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9 and, preferably, which does not hybridize to SEQ ID NO:1 (human “WT” SH3BP2 full length sequence), SEQ ID NO:3 (human WT domain), SEQ ID NO:25 (human WT genomic) or SEQ ID NO:101 (human WT exon 9), under stringent conditions, and detecting the presence of a SH3BP2 mutant nucleic acid in the sample which hybridizes to the nucleic acid probe.", "In yet other embodiments, the method involves contacting the sample with one or more binding agents, wherein the binding agent is capable of selectively binding to a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100; and detecting the presence of a SH3BP2 mutant polypeptide in the sample which binds the binding agent.", "Preferably, the binding agent is an antibody or a fragment thereof that selectively binds to the SH3BP2 mutant polypeptide in the sample and does not bind to a non-mutant SH3BP2 polypeptide that may be present in the sample.", "The diagnostic methods of the invention are particularly useful for: evaluating the susceptibility of a human subject to a disorder of bone homeostasis, evaluating causation of a bone homeostasis disorder in a human subject, or for evaluating the genetic predisposition of a human subject to have offspring with a bone homeostasis disorder.", "Such methods differ primarily in the selection of the human subject (e.g., the human subject exhibits a bone homeostasis disorder or is suspected of having a predisposition to developing the disorder or having offspring susceptible of developing the disorder).", "In general, such methods involve obtaining a sample of DNA from the subject; and evaluating the sample of DNA for the presence of nucleotides encoding a “mutant residue” at any one or more amino acid positions encoded by exon 9 of the SH3BP2 gene product.", "In certain preferred embodiments, the sample of DNA is evaluated for the presence of nucleotides encoding a “mutant residue” at any one or more amino acids encoded by exon 9, particularly at positions 415-420, inclusive, and, more particularly, at positions 415, 418, and 420, of the SH3BP2 gene product.", "The presence of a mutant residue is indicative of the condition for which the subject is being tested.", "Functional activity of a mutant SH3BP2 molecule can be determined, for example, by observing a detectable phenotype indicative of a bone homeostasis disorder as disclosed herein.", "Exemplary mutant residues that are diagnostic of cherubism are described in more detail in the Examples.", "According to yet another aspect of the invention, a method of screening for an agent that inhibits the production of a SH3BP2 mutant polypeptide is provided.", "The method involves: (a) determining the level of a SH3BP2 mutant molecule (e.g., nucleic acid, polypeptide) in the absence of a putative inhibitor, (b) determining the level of a SH3BP2 mutant molecule in the presence of the putative inhibitor, and (c) comparing the level of the SH3BP2 mutant molecule in the presence and absence of the putative inhibitor.", "A decrease in the level of the SH3BP2 mutant molecule in the presence of the putative inhibitor indicates that the putative inhibitor is an agent that inhibits the production of a SH3BP2 mutant polypeptide.", "In certain embodiments, the agent inhibits transcription of a SH3BP2 mutant nucleic acid molecule.", "In yet other embodiments, the agent inhibits translation of a SH3BP2 mutant nucleic acid molecule.", "According to a further aspect of the invention, a method for treating a subject having a bone homestasis disorder that is further characterized by the presence of a SH3BP2 mutant nucleic acid molecule is provided.", "The method involves administering a mutant binding molecule (such as an SH3BP2 mutant nucleic acid antisense molecule, or a ribozyme that selectively binds to and cleaves an SH3BP2 mutant nucleic acid) to a subject in need of such treatment in an amount effective to treat the subject, provided the subject is not otherwise in need of mutant SH3BP2 binding molecule treatment.", "In certain embodiments, the bone homeostasis disorder is selected from the group consisting of osteoporosis, osteopetrosis; bone tumors (malignant or benign); and cherubism.", "The agent may be administered in accordance with standard clinical practice (e.g., systemic); however, in certain embodiments, the agent is locally administered (e.g., to a periodontal pocket).", "The invention also embraces a method of reducing expression of an SH3BP2 mutant nucleic acid molecule in a cell or cell-free system containing a SH3BP2 mutant nucleic acid molecule.", "The method may be practiced in vivo or in vitro.", "In general, the method involves introducing a SH3BP2 mutant binding molecule (e.g., an antisense nucleic acid molecule, a ribozyme that selectively binds to and cleaves a SH3BP2 nucleic acid) into the system (e.g., a cell), and allowing the SH3BP2 mutant binding molecule to hybridize to a sense SH3BP2 mutant nucleic acid molecule, thereby inhibiting expression of the sense SH3BP2 mutant nucleic acid molecule.", "These and other objects of the invention will be described in further detail in connection with the detailed description of the invention." ], [ "GOVERNMENT SUPPORT This application was supported in part by grants numbers AR36819 and AR36820 from the National Institutes of Health.", "The government may have rights in the inventions.", "FIELD OF THE INVENTION This invention relates to nucleic acids and polypeptides derived from a mutant SH3-binding protein (SH3BP2) gene.", "The mutant gene differs from the wild type SH3BP2 gene in containing one or more point mutations.", "The nucleic acid molecules and encoded polypeptides are useful in, inter alia, research, diagnostic and therapeutic contexts, particularly for the development of antibodies and anti-sense molecules for treating disorders of bone homeostasis.", "BACKGROUND OF THE INVENTION The remodeling of bone is a dynamic process.", "Cells continuously lay down and resorb bone material.", "Bone homeostasis refers to the balance between bone formation and bone degradation.", "An imbalance in the activity of cells that lay down new bone (osteoblasts) and cells that resorb bone (osteoclasts) can result in serious, and sometimes fatal, disorders.", "Thus, imbalances in bone homeostasis are associated with a variety of adverse medical conditions.", "Osteoporosis is a term used for a number of diseases of diverse etiology, all involving a reduction in the mass of bone per unit volume.", "Osteoporosis is the most common of the metabolic bone diseases.", "Twenty-five million people in the United States and more than two hundred million people worldwide are affected by osteoporosis.", "Osteoporosis is frequent among post-menopausal women and is an important cause of morbidity in the elderly.", "It commonly results in bone fractures, and death can be a frequent occurrence in the months following fractures, particularly those of the hip in elderly individuals.", "Osteopetrosis is a disorder involving an increase in the mass of bone per unit volume.", "Its incidence is rare compared to osteoporosis, but it typically is life threatening.", "Despite having multiple causes, a defect in bone resorption is always the underlying mechanism.", "In many instances, the disorder is inherited as an autosomal recessive trait and involves abnormal osteoclast function.", "Bone marrow transplants from normal donors have been attempted to restore normal osteoclast precursor cells, but this therapy has shown only limited success.", "Present treatments for disorders of bone homeostasis are inadequate.", "In view of the foregoing, a need exists to develop new and improved methods and compositions for treating disorders of bone homeostasis.", "Preferably such methods and compositions are based upon inhibiting the particular interactions between cell components which mediate the abnormal physiological effect, thereby minimizing harmful side effects that may be due to non-specific therapeutic approaches.", "SUMMARY OF THE INVENTION The invention is based, in part, on our discovery of point mutations that cause amino acid substitutions in the SH3-binding protein SH3BP2.We have collected samples from 15 families, consisting of 66 individuals clinically diagnosed with cherubism (an exemplary bone homeostasis disorder), 4 obligate carriers, and 79 unaffected subjects.", "In 12 families, where mutations were found, the mutations co-segregate with the disease phenotype.", "We also cloned SH3BP2 cDNA from EBV-transformed patient lymphoblasts into TA-vectors and sequenced them.", "Half of the sequenced clones from each patient carried the mutation.", "We sequenced genomic DNAs from 100 unrelated and unaffected African-Brazilians and 100 Caucasian controls without detecting any of the sequence variants found in affected cherubism patients.", "The accumulation of co-segregating sequence variants in families with cherubism and their absence in unaffected controls provide compelling evidence that the mutations in SH3BP2 cause cherubism.", "In view of the foregoing discoveries, the mutant SH3BP2 molecules disclosed herein have a variety of uses including, for example, diagnostic applications (e.g., genetic testing including amniocentesis); screening methods to identify agents that are useful for treating bone homeostasis disorders such as cherubism, osteoporosis, osteopetrosis, and bone tumors (malignant or benign); and therapeutic applications that utilize the compositions of the invention (e.g., antibodies selective for mutant SH3P2 epitope, antisense) for treating bone homeostasis disorders.", "These and other aspects of the invention are summarized below.", "According to a first aspect of the invention, an isolated nucleic acid molecule is provided.", "The isolated nucleic acid molecule includes: (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO: 19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO: 25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9, (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to the degeneracy of the genetic code, and (c) complements of (a) or (b) (e.g., antisense to the mutant SH3BP2 domain SEQ ID NO:7), provided that the nucleic acid molecule is not SEQ ID NO: 1 (human “WT” SH3BP2 full length sequence) or SEQ ID NO: 25 (human WT genomic).", "In certain preferred embodiments, the nucleic acid molecule also is not SEQ ID NO:3 (human WT domain) or SEQ ID NO:102 (human WT exon 9).", "In these and/or other preferred embodiments, the isolated nucleic acid molecules of the invention do not hybridize under stringent conditions to a sequence selected from the group consisting of SEQ ID NO:1 (human “WT” SH3BP2 full length sequence), SEQ ID NO:3 (human WT domain), SEQ ID NO:25 (human WT genomic), and SEQ ID NO:101 (human WT exon 9).", "An abbreviated Sequence Listing which identifies the sequence and/or GenBank accession number information for the sequences relating to the invention is provided herewith as Table 1.In preferred embodiments, the isolated nucleic acid molecule of the invention has a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9.In these and other embodiments, the preferred isolated nucleic acid molecules of the invention code for a polypeptide having a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NOs:64-100.According to yet another aspect of the invention, primers for amplifying the above-identified nucleic acid sequences are provided.", "In this aspect, the preferred isolated nucleic acid molecules are selected from the group consisting of (a) a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO:23, 24 and other sequences which are capable of Is hybridizing to an SH3BP2 mutant molecule and, thereby, are useful in amplifying exon 9 in a mutant SH3BP3 nucleic acid molecule, and (b) complements of (a).", "The invention also embraces expression vectors containing the isolated nucleic acid molecules of the invention, operably linked to a promoter, as well as host cells transformed or transfected with such expression vectors.", "Pharmaceutical compositions comprising the compositions of the invention, as well as methods for making such medicaments also are provided.", "Transgenic non-human animals having somatic and germ line cells that contain any of the isolated SH3BP2 mutant nucleic acid molecules of the invention also are provided.", "Preferably, the expression of the SH3BP2 mutant nucleic acid molecule results in the animal having a bone homeostasis disorder.", "Suck animals are useful as models of human bone homeostasis disorders.", "According to another aspect of the invention, isolated polypeptides encoded by any of the isolated nucleic acid molecules of the invention are provided.", "Preferably, the isolated polypeptides of the invention include a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100.According to still another aspect of the invention, compositions containing an isolated binding agent that binds selectively to a nucleic acid molecule or to a polypeptide molecule of the invention also are provided.", "In certain embodiments, the isolated binding agent is a nucleic acid (e.g., an antisense, a ribozyme); in yet other embodiments, the isolated binding agent is a peptide (e.g., an antibody, or a fragment thereof).", "According to another aspect of the invention, a method of identifying the presence of an SH3BP2 mutant molecule in a sample is provided.", "In general, the method involves analyzing the sample for the presence of a SH3BP2 mutant nucleic acid molecule or a SH3BP2 mutant polypeptide.", "In certain embodiments, the method further involves the steps of contacting the sample with at least two nucleic acid amplification primers and amplifying DNA in the sample prior to analyzing the sample for the presence of a SH3BP2 mutant nucleic acid molecule.", "The method employs a first nucleic acid amplification primer and a second nucleic acid amplification primer that are capable of hybridizing to a SH3BP2 mutant nucleic acid molecule and, thereby, are useful in amplifying a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9.Following hybridization, the method involves amplifying a primed nucleic acid molecule which hybridizes to the first and the second nucleic acid amplification primers; and detecting the presence of an amplified nucleic acid molecule in the sample.", "In yet other embodiments, the method involves contacting the sample with one or more nucleic acid probes, wherein the nucleic acid probe is capable of hybridizing to a nucleic acid sequence selected from the group consisting of: nucleic acid sequences which hybridize to a sequence selected from one or more of the following sequences under stringent conditions: SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9 and, preferably, which does not hybridize to SEQ ID NO:1 (human “WT” SH3BP2 full length sequence), SEQ ID NO:3 (human WT domain), SEQ ID NO:25 (human WT genomic) or SEQ ID NO:101 (human WT exon 9), under stringent conditions, and detecting the presence of a SH3BP2 mutant nucleic acid in the sample which hybridizes to the nucleic acid probe.", "In yet other embodiments, the method involves contacting the sample with one or more binding agents, wherein the binding agent is capable of selectively binding to a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100; and detecting the presence of a SH3BP2 mutant polypeptide in the sample which binds the binding agent.", "Preferably, the binding agent is an antibody or a fragment thereof that selectively binds to the SH3BP2 mutant polypeptide in the sample and does not bind to a non-mutant SH3BP2 polypeptide that may be present in the sample.", "The diagnostic methods of the invention are particularly useful for: evaluating the susceptibility of a human subject to a disorder of bone homeostasis, evaluating causation of a bone homeostasis disorder in a human subject, or for evaluating the genetic predisposition of a human subject to have offspring with a bone homeostasis disorder.", "Such methods differ primarily in the selection of the human subject (e.g., the human subject exhibits a bone homeostasis disorder or is suspected of having a predisposition to developing the disorder or having offspring susceptible of developing the disorder).", "In general, such methods involve obtaining a sample of DNA from the subject; and evaluating the sample of DNA for the presence of nucleotides encoding a “mutant residue” at any one or more amino acid positions encoded by exon 9 of the SH3BP2 gene product.", "In certain preferred embodiments, the sample of DNA is evaluated for the presence of nucleotides encoding a “mutant residue” at any one or more amino acids encoded by exon 9, particularly at positions 415-420, inclusive, and, more particularly, at positions 415, 418, and 420, of the SH3BP2 gene product.", "The presence of a mutant residue is indicative of the condition for which the subject is being tested.", "Functional activity of a mutant SH3BP2 molecule can be determined, for example, by observing a detectable phenotype indicative of a bone homeostasis disorder as disclosed herein.", "Exemplary mutant residues that are diagnostic of cherubism are described in more detail in the Examples.", "According to yet another aspect of the invention, a method of screening for an agent that inhibits the production of a SH3BP2 mutant polypeptide is provided.", "The method involves: (a) determining the level of a SH3BP2 mutant molecule (e.g., nucleic acid, polypeptide) in the absence of a putative inhibitor, (b) determining the level of a SH3BP2 mutant molecule in the presence of the putative inhibitor, and (c) comparing the level of the SH3BP2 mutant molecule in the presence and absence of the putative inhibitor.", "A decrease in the level of the SH3BP2 mutant molecule in the presence of the putative inhibitor indicates that the putative inhibitor is an agent that inhibits the production of a SH3BP2 mutant polypeptide.", "In certain embodiments, the agent inhibits transcription of a SH3BP2 mutant nucleic acid molecule.", "In yet other embodiments, the agent inhibits translation of a SH3BP2 mutant nucleic acid molecule.", "According to a further aspect of the invention, a method for treating a subject having a bone homestasis disorder that is further characterized by the presence of a SH3BP2 mutant nucleic acid molecule is provided.", "The method involves administering a mutant binding molecule (such as an SH3BP2 mutant nucleic acid antisense molecule, or a ribozyme that selectively binds to and cleaves an SH3BP2 mutant nucleic acid) to a subject in need of such treatment in an amount effective to treat the subject, provided the subject is not otherwise in need of mutant SH3BP2 binding molecule treatment.", "In certain embodiments, the bone homeostasis disorder is selected from the group consisting of osteoporosis, osteopetrosis; bone tumors (malignant or benign); and cherubism.", "The agent may be administered in accordance with standard clinical practice (e.g., systemic); however, in certain embodiments, the agent is locally administered (e.g., to a periodontal pocket).", "The invention also embraces a method of reducing expression of an SH3BP2 mutant nucleic acid molecule in a cell or cell-free system containing a SH3BP2 mutant nucleic acid molecule.", "The method may be practiced in vivo or in vitro.", "In general, the method involves introducing a SH3BP2 mutant binding molecule (e.g., an antisense nucleic acid molecule, a ribozyme that selectively binds to and cleaves a SH3BP2 nucleic acid) into the system (e.g., a cell), and allowing the SH3BP2 mutant binding molecule to hybridize to a sense SH3BP2 mutant nucleic acid molecule, thereby inhibiting expression of the sense SH3BP2 mutant nucleic acid molecule.", "These and other objects of the invention will be described in further detail in connection with the detailed description of the invention.", "BRIEF DESCRIPTION OF THE FIGURES The figures, if present or referenced in this application, are illustrative only and are not required for enablement of the inventions disclosed herein.", "Example 1, FIG.", "1—Cherubism patient at age 12 (Family F).", "The facial swelling in this severe case affects the tipper and lower jaws.", "The computed tomography 3D image (right) shows excessive bone degradation and replacement by soft tissue masses.", "Excessive growth of the libro-osscous tissue results in widening of the mandible; it pushes the orbital floors upward and exposes the sclera.", "Several teeth are exfoliated due to the bone loss.", "Example 1, FIG.", "2—Gene structure and functional domains of SH3BP2.The gene consists of 13 exons extending over a 16.1 kb interval.", "The 561 amino acid (aa) residue-long protein consists of 3 modular domains (pleckstrin homology (PH) domain, SH3-binding domain, and SH2 domain).", "All the mutations found in the 12 families are located within a 6 residue-long sequence positioned 31 to 36 residues upstream of the SH2 domain.", "SEQ ID NO:105 contains a portion of the amino acid sequence of exon 9.Example 1, FIG.", "3—Electropherograms of partial sequences of exon 9 of the SH3BP2 gene showing the different heterozygous point mutations found in the cherubism families.", "Arrows (↓) indicate the different heterozygous point mutations.", "Capital letters next to the sequences indicate wild type sequence (WT) and list the families for which the respective mutation was detected.", "FIG.", "3A: Wild-type partial sequence of exon 9, SEQ ID NO:106.FIG.", "3B: Family H partial sequence of exon 9, SEQ ID NO:107.FIG.", "3C: Family K partial sequence of exon 9, SEQ ID NO:108.FIG.", "3D: Family A, B partial sequence of exon 9, SEQ ID NO:109.FIG.", "3E: Family C, F, J, M, O partial sequence of exon 9, SEQ ID NO:110.FIG.", "3F: Family L partial sequence of exon 9, SEQ ID NO:111.FIG.", "3G: Family G partial sequence of exon 9, SEQ ID NO:112.FIG.", "3H: Family N partial sequence of exon 9, SEQ ID NO:113.TABLE 1 ABBREVIATED SEQUENCE LISTING SEQ ID NO: DESCRIPTION OF NUCLEOTIDE OR AMINO ACID SEQUENCE 1 (full length wt Wild type SH3BP2 cDNA having GenBank Accession No.", "U56386.nucleic acid) 2 (full length wt Wild type SH3BP2 polypeptide having GenBank Accession No.", "AAB72034 (encoded polypeptide) by SEQ ID NO: 1).", "3 (wt domain) “Wild type nucleic acid domain” (CGATCACCCCCCGATGGGCAG) at nucleotide positions 1451-1471, inclusive, of SEQ ID NO: 1.4 (wt domain) “Wild type polypeptide domain” (RSPPDGQ) at amino acid positions 415-420, inclusive, of SEQ ID NO: 2, encoded by SEQ ID NO: 3.5 (full length Generic mutant SH3BP2 cDNA: Sequence is identical to SEQ ID NO: 1 but with “generic mutant” “mutant domain” (CXATCACCCCXCGATXXGCAG, SEQ ID NO: 7) in place of wild nucleic acid; type domain (CGATCACCCCCCGATGGGCAG, SEQ ID NO: 3) at nucleotide generic identifies positions 1451-1471, inclusive.", "all possible point mutations) 6 (full length Generic mutant SH3BP2 polypeptide: encoded by SEQ ID NO: 5.“generic mutant” polypeptide) 7 (generic mutant Generic mutant SH3BP2 nucleic acid “mutant domain”: domain) CXATCACCCCXCGATXXGCAG, wherein X is independently selected from the group consisting of S, R, Y, and M (i.e., X can be C, T, G, or A, provided that the mutant domain is not wild type).", "8 (generic mutant Generic mutant SH3BP2 polypeptide “mutant domain”: encoded by SEQ ID NO: 7.domain) 9 (Family H) Family H mutant SH3BP2 cDNA mutant domain: CSATCACCCCCCGATGGGCAG.", "10 Family H mutant SH3BP2 polypeptide mutant domain: encoded by SEQ ID NO: 9.11 (Family K) Family K mutant SH3BP2 cDNA mutant domain: CRATCACCCCCCGATGGGCAG.", "12 Family K mutant SH3BP2 polypeptide mutant domain: encoded by SEQ ID NO: 11.13 (Families Family A, B mutant SH3BP2 cDNA mutant domain: A, B) CGATCACCCCYCGATGGGCAG.", "14 Family A, B mutant SH3BP2 polypeptide mutant domain: encoded by SEQ ID NO: 13 15 (Families C, Family C, F, J, M, O mutant SH3BP2 cDNA mutant domain: F, J, M, and O) CGATCACCCCSCGATGGGCAG.", "16 Family C, F, J, M, O mutant SH3BP2 polypeptide mutant domain: encoded by SEQ ID NO: 15.17 (Family L) Family L mutant SH3BP2 cDNA mutant domain: CGATCACCCCMCGATGGGCAG.", "18 Family L mutant SH3BP2 polypeptide mutant domain: encoded by SEQ ID NO: 17 19 (Family G) Family G mutant SH3BP2 cDNA mutant domain: CGATCACCCCCCGATGRGCAG.", "20 Family G mutant SH3BP2 polypeptide mutant domain: encoded by SEQ ID NO: 19.21 (Family N) Family N mutant SH3BP2 cDNA mutant domain: CGATCACCCCCCGATSGGCAG.", "22 Family N mutant SH3BP2 polypeptide mutant domain: encoded by SEQ ID NO: 21.23 Primer #1 SH3BP29F: CTTGCCGTCCTCACACAGAG 24 Primer #2 SH3BP211R: TTAGGAACTGTGGAGTCCTG 25 Genomic WT GenBank Z68279 26 Genomic Genomic mutant SH3BP2 is identical to SEQ ID NO: 25 with the exception that mutant mutant exon 9 (SEQ ID NO: 27) is used in place of the wild type exon 9.27-63 Mutant SH3BP2 exon 9 nucleotide sequences.", "64-100 Mutant SH3BP2 exon 9 amino acid sequences.", "101 Human wild type exon 9 nucleotide sequence.", "102 Human wild type exon 9 amino acid sequence.", "103 PPAYPPPPVP 104 RSPPDG 105 HLQRSPPDGQSF 106 Wild-type partial sequence of exon 9.107 Family H partial sequence of exon 9.108 Family K partial sequence of exon 9.109 Family A, B partial sequence of exon 9.110 Family C, F, J, M, O partial sequence of exon 9.111 Family L partial sequence of exon 9.112 Family G partial sequence of exon 9.113 Family N partial sequence of exon 9.DETAILED DESCRIPTION OF THE INVENTION The invention is based, in part, on our discovery of point mutations that cause amino acid substitutions in the SH3-binding protein SH3BP2.We have collected samples from 15 families, consisting of 66 individuals clinically diagnosed with cherubism (an exemplary bone homeostasis disorder), 4 obligate carriers, and 79 unaffected subjects.", "In 12 families, where mutations were found, the mutations co-segregate with the disease phenotype.", "We also cloned SH3BP2 cDNA from EBV-transformed patient lymphoblasts into TA-vectors and sequenced them.", "Half of the sequenced clones from each patient carried the mutation.", "We sequenced genomic DNAs from 100 unrelated and unaffected African-Brazilians and 100 Caucasian controls without detecting any of the sequence variants found in affected cherubism patients.", "The accumulation of co-segregating sequence variants in families with cherubism and their absence in unaffected controls provide compelling evidence that the mutations in SH3BP2 cause cherubism.", "In view of the foregoing discoveries, the mutant SH3BP2 molecules disclosed herein have a variety of uses including, for example, diagnostic applications (e.g., genetic testing); screening methods to identify agents that are useful for treating bone homeostasis disorders such as cherubism, osteoporosis, osteopetrosis, and bone tumors; and therapeutic applications that utilize the compositions of the invention (e.g., antibodies selective for mutant SH3BP2 epitope, antisense, ribozymes) for treating bone homeostasis disorders.", "These and other aspects of the invention are summarized below.", "According to a first aspect of the invention, an isolated nucleic acid molecule is provided.", "The isolated nucleic acid molecule includes: (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO: 25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9, (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to the degeneracy of the genetic code, and (c) complements of (a) or (b) (e.g., antisense to the mutant SH3BP2 domain SEQ ID NO:7), provided that the nucleic acid molecule is not SEQ ID NO:1 (human “WT” SH3BP2 full length sequence) or SEQ ID NO:25 (human WT genomic).", "In certain preferred embodiments, the nucleic acid molecule also is not SEQ ID NO:3 (human WT domain) or SEQ ID NO:101 (human WT exon 9).", "In these and/or other preferred embodiments, the isolated nucleic acid molecules of the invention do not hybridize under stringent conditions to a sequence selected from the group consisting of SEQ ID NO:1 (human “WT” SH3BP2 full length sequence), SEQ ID NO:3 (human WT domain), SEQ ID NO:25 (human WT genomic), and SEQ ID NO:101 (human WT exon 9).", "An abbreviated Sequence Listing which identifies the sequence and/or GenBank accession number information for the sequences relating to the invention is provided herewith as Table 1.In preferred embodiments, the isolated nucleic acid molecule of the invention has a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9.In these and other embodiments, the preferred isolated nucleic acid molecules of the invention code for a polypeptide having a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100.As used herein, a mutant SH3BP2 nucleic acid molecule refers to a nucleic acid molecule which contains an SH3BP2-derived nucleotide sequence containing one or more point mutations in exon 9 (e.g., one or more point mutations in a “mutation domain”) and which results in a mutant SH3BP2 functional activity (e.g., an activity which can be measured in vitro or which can be determined by detecting a phenotype indicative of a bone homeostasis disorder, such as those disorders disclosed herein).", "As used herein, a “mutation domain” or a “mutant domain” refers to the nucleotide sequence between nucleotide positions 1451 to 1471, inclusive of the wild type SH3BP2 cDNA sequence (SEQ ID NO:1).", "The exact number of nucleotide changes in the mutant SH3BP2 compared to the wild type SH3BP2 nucleic acid can vary.", "In general, the nucleotide changes (one or more point mutations) in the mutant SH3BP2 nucleic acids of the invention are present in exon 9 (including nucleic acid positions 1451 to 1471, inclusive); however, the most common point mutations are those which occur at nucleotide positions 1452, 1461, 1466 and 1467.Table 2 illustrates the mutations in SH3BP2 causing cherubism in the families from which the mutant SH3BP2 sequence was identified.", "(See, also, FIG.", "3.)", "TABLE 2 MUTATIONS IN SH3BP2 CAUSING CHERUBISM Arg415 (CGA) to Gln (CAA) Family K Arg415 (CGA) to Pro (CCA) Family H Pro418 (CCC) to Leu (CTC) Family A, B Pro418 (CCC) to Arg (CGC) Family C, J, M, O, F Pro418 (CCC) to His (CAC) Family L Gly420 (GGG) to Glu (GAG) Family G Gly420 (GGG) to Arg (CGG) Family N The term “genomic mutant SH3BP2 nucleic acid molecule”, as used herein, refers to a mutant SH3BP2 nucleic acid molecule with a nucleic acid sequence identical to the genomic wild-type sequence of SEQ ID NO:25 with the exception that a mutant exon 9 with a sequence selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild-type exon 9 sequence, SEQ ID 101.The nucleotide changes in the mutant SH3BP2 exon 9 can occur at any nucleotide position within exon 9, provided that the change results in an amino acid change in the polypeptide encoded by exon 9 (1) which is distinct from the human wild type SH3BP2 exon 9 and (2) which exhibits a mutant SH3BP2 functional activity (e.g., a detectable phenotype indicative of a bone homeostasis disorder).", "Exemplary mutant SH3BP2 exon 9 nucleotide sequences (SEQ ID Nos: 27-63) are provided in Table 3.Exemplary mutant SH3BP2 exon 9 amino acid sequences (SEQ ID Nos:64-100) are provided in Table 4.For reference, Tables 5 and 6 show the wild type SH3BP2 exon positions (SH3BP2 is on the complementary strand of the published GenBank clone Z68279), and the wild type SH3BP2 exon 9 nucleotide and amino acid sequences, respectively.", "TABLE 3 MUTANT SH3BP2 EXON 9 NUCLEOTIDE SEQUENCES SEQ ID NO.", "SEQUENCE 27 5′-G 123 TCA CCC 123 GAT 123 GAG AGT TTC AGG AGC TTC TCC TTT GAA AAG CCC GGG CAA CCC TCA GAG GCT GAG ACT GGG GGG GAG GAG TGG GAG GAG GAG TAT GAG AAG-3′ 28 5′-G 123 TCA CCC CCC GAT GGG GAG AGT TTC AGG AGC TTG TGC TTT GAA AAG CCC GGG CAA CCC TCA GAG GGT GAG ACT GGG GGG GAG GAG TCG GAG GAG GAG TAT GAG AAG-3′ 29 5′-G GGA 123 CCC CCC GAT GGG GAG AGT TTG AGG AGG TTG TGG TTT GAA AAG CCC CGG CAA CCC TCA GAG GGT GAG ACT GCG GGG GAG GAG TGG GAG GAG GAG TAT GAG AAG-3′ 30 5′-G CGA TGA 123 CCC GAT GGG GAG AGT TTG AGG AGG TTC TCG TTT GAA AAG CCC GGG GAA CCC TCA GAG GGT GAG ACT GGC GGG GAG GAG TGG GAG GAG GAG TAT GAG AAG-3′ 31 5′-G GGA TGA CCC 123 GAT GGG GAG AGT TTC AGG AGC TTG TCG TTT GAA AAG CCC CGG CAA CCC TGA GAG GCT GAG ACT GGC GGG GAG GAG TGG GAG GAG GAG TAT GAG AAG-3′ 32 5′-G CGA TGA CCC CCC 123 GGG GAG AGT TTG AGG AGC TTG TCG TTT GAA AAG CCC GGG CAA CCC TGA GAG GGT GAG ACT GGG GGG GAG GAG TGG GAG GAG GAG TAT GAG AAG-3′ 33 5′-G GGA TCA CCC CCC GAT 123 GAG AGT TTG AGG AGG TTG TCG TTT GAA AAG CCC GGG GAA CCC TGA GAG GCT GAG ACT GGC GGG GAG GAG TCG GAG GAG GAG TAT GAG AAG-3′ 34 5′-G CGA TCA CCC CCC GAT GGG 123 AGT TTC AGG AGG TTG TCC TTT GAA AAG CCC CGG CAA CCC TGA GAG GGT GAG ACT GGG GGG GAG GAG TGG GAG GAG GAG TAT GAG AAG-3′ 35 5′-G CGA TCA CCC CCC GAT GGG GAG 123 TTG AGG AGC TTG TCC TTT GAA AAG CCC CGG CAA CCC TGA GAG GCT GAG ACT GGG GGG GAG GAG TGG GAG GAG GAG TAT GAG AAG-3′ 36 5′-G CGA TCA CCC CCC GAT GGG GAG AGT 123 AGG AGG TTG TGC TTT GAA AAG CCC GGG CAA CCC TGA GAG GGT GAG ACT GGG GGG GAG GAG TGG GAG GAG GAG TAT GAG AAG-3′ 37 5′-G GGA TCA CCC CCC GAT GGG GAG AGT TTC 123 AGG TTG TCG TTT GAA AAG CCC CGG CAA CCC TCA CAG GCT GAC ACT GGC GGG GAC GAC TCG GAC GAG GAC TAT GAG AAG-3′ 38 5′-C CGA TGA CCC CCC CAT GGG GAG AGT TTC AGG 123 TTC TGG TTT GAA AAG CCC CGG CAA CCC TCA GAG GCT GAC ACT GGC GGG GAC GAC TCG GAC GAG GAC TAT GAG AAG-3′ 39 5′-C CGA TCA CCC CCC GAT GGG GAG AGT TTG ACG AGC 123 TGC TTT GAA AAG CCC CGG CAA CCC TCA CAG GCT GAC ACT GGC GGG GAC GAC TCG GAC GAG GAC TAT GAG AAG-3′ 40 5′-G CGA TCA CCC CCC CAT GGG CAG AGT TTC AGG AGC TTC 123 TTT GAA AAG CCC CGG CAA CCC TCA CAG GCT GAC ACT CCC GGG GAC GAC TCG GAC GAG GAG TAT GAG AAG-3′ 41 5′-G CGA TCA CCC CCC GAT GGG CAG AGT TTC AGC AGC TTC TCC 123 GAA AAG CCC CGG CAA CCC TCA CAG GCT GAC ACT GGC GGG GAC GAC TCG GAG GAG GAC TAT GAG AAG-3′ 42 5′-G CGA TCA CCC CCC GAT GGG GAG ACT TTC AGG ACC TTC TCC TTT 123 AAG CCC CGG CAA CCC TCA GAG GCT GAG ACT CCC GGG GAG GAG TCG GAG GAG GAG TAT GAG AAG-3′ 43 5′-G CGA TGA CCC CCC GAT CGC GAG ACT TTC AGG ACC TTC TCC TTT GAA 123 CCC CGG CAA CCC TGA GAG GCT GAG ACT GGG GGG GAG GAG TCG GAG GAG GAG TAT GAG AAC-3′ 44 5′-G GGA TGA CCC CCC CAT CCC GAG ACT TTC AGG AGC TTG TCC TTT GAA AAG 123 CGG CAA CCC TGA GAG GCT GAG ACT GGC GGG GAG GAG TCG GAG GAG GAG TAT GAG AAG-3′ 45 5′-C CGA TCA CCC CCC CAT CCC GAG AGT TTG AGG AGC TTG TCG TTT GAA AAG CCC 123 CAA CCC TCA GAG GGT GAG ACT GGC CCC GAG GAG TCG GAG GAG GAG TAT GAG AAG-3′ 46 5′-G GCA TGA CCC CCC GAT CCC GAG ACT TTG AGG AGC TTG TCC TTT GAA AAG CCC CCC 123 CCC TCA GAG GCT GAG ACT CGC GCG GAC GAG TGG GAG GAG GAG TAT GAG AAG-3′ 47 5′-G CGA TCA CCC CCC CAT GCG GAG ACT TTC AGG AGG TTC TCC TTT GAA AAG CCC CGG CAA 123 TGA GAG GCT GAG ACT GGC GGG GAG GAG TCG GAG GAG GAG TAT GAG AAG-3′ 48 5′-G GGA TCA CCC CCC GAT GGG GAG AGT TTC AGG AGC TTC TCC TTT GAA AAG CGG CCC CAA CCC 123 GAG GGT GAG ACT CGG CGG GAG GAG TGG GAG GAG GAG TAT GAG AAC-3′ 49 5′-G CGA TCA CCC CCC CAT GGG GAG ACT TTC AGG AGC TTC TCC TTT GAA AAG CCC CCC GAA CCC TCA 123 GCT GAG ACT GGC GGG GAG GAG TCG GAG GAG GAG TAT GAG AAC-3′ 50 5′-C CGA TCA CCC CCC GAT CCG GAG AGT TTG AGG AGC TTC TCC TTT GAA AAG CCC CGG CAA CCC TGA GAG 123 GAG ACT GGC GGG GAG GAG TCG GAd GAG GAG TAT GAG AAG-3′ 51 5′-G CGA TCA CCC CCC CAT GGG GAG AGT TTG AGG AGC TTC TCG TTT GAA AAG CCC CGG GAA CCC TCA GAG GGT 123 ACT CCC GCG GAG GAG TCG GAG GAG GAG TAT GAG AAC-3′ 52 5′-C CCA TGA CCC CCC CAT CCC GAG ACT TTC AGG AGC TTC TGG TTT CAA AAG CCC CGG CAA CCC TCA GAG GGT GAG 123 CCC CCC GAG GAG TGG GAG GAG GAG TAT GAG AAG-3′ 53 5′-G CGA TCA CCC CCC CAT GCG GAG AGT TTC AGG ACC TTC TGC TTT GAA AAG CCC CCC GAA CCC TCA GAG GGT GAG ACT 123 GGG GAG GAG TGG GAG GAG GAG TAT GAG AAG-3′ 54 5′-G CGA TCA CCC CCC CAT CCG GAG ACT TTC AGC ACC TTC TCC TTT GAA AAG CCC CCC GAA CCC TCA GAG GCT GAG ACT CCC 123 GAG GAG TCC GAG GAG GAG TAT GAG AAG-3′ 55 5′-C GCA TCA CCC CCC CAT CCC GAG ACT TTC AGC ACG TTC TGG TTT GAA AAG CCC CGG CAA CCC TGA GAG GCT GAC ACT GGC GGG 123 GAC TCG GAG GAG GAG TAT GAG AAG-3′ 56 5′-G CGA TCA CCC CCC GAT GGG GAG AGT TTC AGG AGC TTC TGC TTT GAA AAG CCC CGG CAA CCC TCA GAG GCT GAG ACT GGG GGG GAG 123 TCG GAG GAG GAG TAT GAG AAG-3′ 57 5′-G CGA TGA CCC CCC GAT GGG GAG AGT TTC AGG AGG TTG TCC TTT GAA AAG CCC CGG CAA CCC TGA CAG GCT GAG ACT GGG GGG GAG GAC 123 GAG GAG GAG TAT GAG AAG-3′ 58 5′-G GGA TGA CCC CCC GAT GGG GAG AGT TTC AGG AGG TTC TGG TTT GAA AAG CCC GGG CAA CCC TGA GAG GCT GAG ACT GCG GGG GAG GAG TCG 123 GAG GAG TAT GAG AAG-3′ 59 5′-G CGA TCA CCC CCC GAT GGG GAG AGT TTG AGG AGG TTC TCC TTT GAA AAG CCC CGG CAA CCC TCA GAG GGT GAG ACT GGG GGG GAG GAG TCG GAG 123 GAG TAT GAG AAG-3′ 60 5′-G CGA TCA CCC CCC GAT GGG GAG AGT TTC AGG AGG TTG TGC TTT GAA AAG CCC GGG GAA CCC TCA GAG GGT GAG ACT GGG GGG GAG GAG TGG GAG GAG 123 TAT GAG AAG-3′ 61 5′-G CGA TCA CCC CCC GAT GGG GAG AGT TTC AGG AGC TTC TGG TTT GAA AAG CCC GGG CAA CCC TGA GAG GCT GAG ACT GGG GGG GAG GAG TCG GAG GAG GAG 123 GAG AAG-3′ 62 5′-G CGA TCA CCC CCC GAT GGG GAG AGT TTG AGG AGC TTC TCG TTT GAA AAG CCC CGG GAA CCC TCA GAG GGT GAG ACT GGG GGG GAG GAG TCG GAG GAG GAG TAT 123 AAG-3′ 63 5′-G GGA TGA CCC CCC GAT GGG GAG AGT TTC AGG AGC TTG TCC TTT GAA AAG CCC CGG CAA CCC TGA GAG GGT GAG ACT GGG GGG GAG GAG TCG GAG GAG GAG TAT GAG 123-3′ KEY: “123” refers to a coding triplet such that 1, 2, and 3 are independently selected from the group consisting of C,G,A, and T provided that the coding triplet does not encode the same amino acid as that located at the corresponding amino acid position in the wild type SH3BP2 exon 9 polypeptide (SEQ ID NO:102), and provided that en coded polypeptide has a mutant SH3BP2 functional activity.", "TABLE 4 MUTANT SH3BP2 EXON 9 AMINO ACID SEQUENCES SEQ ID NO.", "SEQUENCE 64 4 S P 5 D 6 Q S F R S F S F E K P R Q P S Q A D T G G D D S D E D Y E K 65 4 S P P D G Q S F R S F S F E K P R Q P S Q A D T G G D D S D E D Y E K 66 R 4 P P D G Q S F R S F S F E K P R Q P S Q A D T G G D D S D E D Y E K 67 R S 4 P D G Q S F R S F S F E K P R Q P S Q A D T G G D D S D E D Y E K 68 R S P 4 D G Q S F R S F S F E K P R Q P S Q A D T G G D D S D E D Y E K 69 R S P P 4 G Q S F R S F S F E K P R Q P S Q A D T G G D D S D E D Y E K 70 R S P P D 4 Q S F R S F S F E K P R Q P S Q A D T G G D D S D E D Y E K 71 R S P P D G 4 S F R S F S F E K P R Q P S Q A D T G G D D S D E D Y E K 72 R S P P D G Q 4 F R S F S F E K P R Q P S Q A D T G G D D S D E D Y E K 73 R S P P D G Q S 4 R S F S F E K P R Q P S Q A D T G G D D S D E D Y E K 74 R S P P D G Q S F 4 S F S F E K P R Q P S Q A D T G G D D S D E D Y E K 75 R S P P D G Q S F R 4 F S F E K P R Q P S Q A D T G G D D S D E D Y E K 76 R S P P D G Q S F R S 4 S F E K P R Q P S Q A D T G G D D S D E D Y E K 77 R S P P D G Q S F R S F 4 F E K P R Q P S Q A D T G G D D S D E D Y E K 78 R S P P D G Q S F R S F S 4 E K P R Q P S Q A D T G G D D S D E D Y E K 79 R S P P D G Q S F R S F S F 4 K P R Q P S Q A D T G G D D S D E D Y E K 80 R S P P D G Q S F R S F S F E 4 P R Q P S Q A D T G G D D S D E D Y E K 81 R S P P D G Q S F R S F S F E K 4 R Q P S Q A D T G G D D S D E D Y E K 82 R S P P D G Q S F R S F S F E K P 4 Q P S Q A D T G G D D S D E D Y E K 83 R S P P D G Q S F R S F S F E K P R 4 P S Q A D T G G D D S D E D Y E K 84 R S P P D G Q S F R S F S F E K P R Q 4 S Q A D T G G D D S D E D Y E K 85 R S P P D G Q S F R S F S F E K P R Q P 4 Q A D T G G D D S D E D Y E K 86 R S P P D G Q S F R S F S F E K P R Q P S 4 A D T G G D D S D E D Y E K 87 R S P P D G Q S F R S F S F E K P R Q P S Q 4 D T G G D D S D E D Y E K 88 R S P P D G Q S F R S F S F E K P R Q P S Q A 4 T G G D D S D E D Y E K 89 R S P P D G Q S F R S F S F E K P R Q P S Q A D 4 G G D D S D E D Y E K 90 R S P P D G Q S F R S F S F E K P R Q P S Q A D T 4 G D D S D E D Y E K 91 R S P P D G Q S F R S F S F E K P R Q P S Q A D T G 4 D D S D E D Y E K 92 R S P P D G Q S F R S F S F E K P R Q P S Q A D T G G 4 D S D E D Y E K 93 R S P P D G Q S F R S F S F E K P R Q P S Q A D T G G D 4 S D E D Y E K 94 R S P P D G Q S F R S F S F E K P R Q P S Q A D T G G D D 4 D E D Y E K 95 R S P P D G Q S F R S F S F E K P R Q P S Q A D T G G D D S 4 E D Y E K 96 R S P P D G Q S F R S F S F E K P R Q P S Q A D T G G D D S D 4 D Y E K 97 R S P P D G Q S F R S F S F E K P R Q P S Q A D T G G D D S D E 4 Y E K 98 R S P P D G Q S F R S F S F E K P R Q P S Q A D T G G D D S D E D 4 E K 99 R S P P D G Q S F R S F S F E K P R Q P S Q A D T G G D D S D E D Y 4 K 100 R S P P D G Q S F R S F S F E K P R Q P S Q A D T G G D D S D E D Y E 4 KEY: “4,” “5,” and “6\"” each refers to an amino acid, such that each 4, 5, and 6 is independently selected, provided that at least one 4, 5, 6 in each sequence is selected such that amino acid is not the same amino acid as that located at the corresponding amino acid position in the wild type SH3BP2 exon 9 polypeptide (SEQ ID:102), and provided that the encoded polypeptide has a mutant SH3BP2 functional activity.", "TABLE 5 SH3BPS EXONS IN GENBANK Z68279 3′-5′ EXON1 20503˜20707 EXON2 18142˜18281 EXON3 15858˜15960 EXON4 14163˜14280 EXON5 13697˜13767 EXON6 11602˜11690 EXON7 11246˜11314 EXON8 8774˜9427 EXON9 7242˜7350 EXON10 6943˜6998 EXON11 6509˜6590 EXON12 5872˜5931 EXON13 4631˜5224 TABLE 6 SH3BP2 EXON 9 NUCLEOTIDE AND AMINO ACID SEQUENCE 5′-G GGA TCA CCC CCC GAT GGG GAG AGT TTC AGG R S P P D G Q S F R AGC TTC TCC TTT GAA AAG CCC CGG CAA CCC TCA S F S F E K P R Q P S GAG GCT GAG ACT GGC GGG GAG GAG TCG GAG GAG Q A D T G G D D S D E GAG TAT GAG AAG-3′ D Y E K The presence of the mutant domain in the SH2BP2 sequence, identifies the mutant SH3BP2 nucleic acid molecule as a unique nucleic acid molecule derived as a point mutation(s) of the wild type SH3BP2 gene.", "The number of nucleotides contributed by the wild type SH3BP2 source gene on either side of the point mutation may differ and may be 1, 2, 3, 4 or more nucleotides, provided the mutant domain containing the point mutation is capable of uniquely identifying a mutant SH3BP2 nucleic acid molecule.", "Homologs and alleles of the mutant SH3BP2 nucleic acid molecules of the invention can be identified by conventional techniques.", "Thus, an aspect of the invention is those nucleotide sequences which code for mutant SH3BP2 polypeptides and which hybridize under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), and SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9, and which do not code for a wild type SH3BP2, such as that encoded by SEQ ID NO:1 (human “WT” SH3BP2 full length sequence).", "The term “stringent conditions” as used herein refers to parameters with which the art is familiar.", "Nucleic acid hybridization parameters may be found in references which compile such methods, e.g.", "Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.", "More specifically, stringent conditions, as used herein, refers, for example, to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4, pH 7, 0.5% SDS, 2 mM EDTA).", "SSC is 0.15M sodium chloride/0.15M sodium citrate, pH 7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetraacetic acid.", "After hybridization, the membrane upon which the DNA is transferred is washed at 2×SSC at room temperature and then at 0.1×SSC/0.1% SDS at temperatures up to 68° C. There are other conditions, reagents, and so forth which can be used, and would result in a similar degree of stringency.", "The skilled artisan will be familiar with such conditions, and thus they are not given here.", "It will be understood, however, that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of homologs and alleles of the mutant SH3BP2 nucleic acid molecules of the invention.", "The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid molecule and sequencing.", "In general, homologs and alleles typically will share at least 75% nucleotide identity to SEQ ID NO:5 (generic mutant—full length nucleic acid) and will include a sequence that shares at least 95% and, preferably, at least 96%, 97%, 98%, or 99% nucleotide sequence identity to a sequence selected from the group consisting of SEQ ID NO:7.", "(generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), and SEQ ID NO:21 (“N” family mutation).", "Similarly, the homologs and alleles typically will share at least 75% amino acid sequence identity to SEQ ID NO:6 (generic mutant—full length polypeptide) and will include a sequence that shares at least 85% and, preferably, at least 86% through 99% (and every integer therebetween) amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, SEQ.", "ID NO:18, SEQ ID NO:20 and SEQ ID NO:22.In some instances sequences will share at least 95% nucleotide identity and/or at least 90% amino acid identity and in still other instances sequences will share at least 95% nuclcotide identity and/or at least 95% or 99% amino acid identity.", "In certain embodiments, the homologs and alleles include an exon 9 encoded amino acid sequence that will share at least 98% and, preferably, at least 99% amino acid sequence identity with a sequence selected from the group consisting of SEQ ID Nos:64-100.Likewise, in certain embodiments, the homologs and alleles include an exon 9 nucleotide sequence that will share at least 85% and, more preferably, at least 90%- 95% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID Nos:27-63.The homology can be calculated using various, publicly available software tools developed by NCBI (Bethesda, Md.)", "that can be obtained through the internet at the NCBI/NIG website.", "Exemplary tools include the BLAST system available on the internet at the NCBI/NIH website.", "Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis can be obtained using the MacVector sequence analysis software (Oxford Molecular Group).", "Watson-Crick complements of the foregoing nucleic acid molecules also are embraced by the invention.", "In screening for mutant SH3BP2 related genes, such as homologs and alleles, a Southern blot may be performed using the foregoing conditions, together with a radioactive probe.", "After washing the membrane to which the DNA is finally transferred, the membrane can be placed against X-ray film or a phosphoimager plate to detect the radioactive signal.", "The invention also includes degenerate nucleic acid molecules which include alternative codons to those present in the native materials.", "For example, serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC.", "Each of the six codons is equivalent for the purposes of encoding a serine residue.", "Thus, it will be apparent to one of ordinary skill in the art that any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue into, for example, an elongating mutant SH3BP2 polypeptide.", "Similarly, nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons).", "Other amino acid residues may be encoded similarly by multiple nucleotide sequences.", "Thus, the invention embraces degenerate nucleic acid molecules that differ from the biologically isolated nucleic acid molecules in codon sequence due to the degeneracy of the genetic code.", "The invention also provides isolated unique fragments of a mutant SH3BP2 nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant full length nucleic acid); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO: 25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9.A unique fragment is one that is a ‘signature’ for the larger nucleic acid.", "For example, the unique fragment is long enough to assure that its precise sequence is not found in molecules within the human genome outside of the mutant SH3BP2 nucleic acid molecules defined above (and human alleles).", "Those of ordinary skill in the art may apply no more than routine procedures to determine if a fragment is unique within the human genome.", "The preferred unique fragments contain the mutant SH3BP2 exon 9 (SEQ ID Nos:27-63).", "Exemplary unique fragments are represented by SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO: 19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9 and smaller fragments of the foregoing nucleic acids which contain 4, 5, 6, 7, 8, 9, or 10 nucleotides, including the point mutation identified in each as distinctive of a mutant SH3BP2 mutant domain.", "Thus, according to one aspect of the invention, an isolated mutant SH3BP2 unique nucleic acid fragment is provided which is selected from the group consisting of: (a) a unique fragment of a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant full length nucleic acid); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and, a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9 (of sufficient length to represent a sequence unique within the human genome); and (b) complements of (a).", "The unique fragments of the invention include a sequence of contiguous nucleotides which excludes previously published sequences as of the date of invention or the filing date of this application or a priority document, (2) complements of (1), and optionally, (3) unique fragments of (1) and (2).", "In certain embodiments, the sequence of contiguous nucleotides is selected from the group consisting of (1) at least two contiguous nucleotides nonidentical to the sequence group (e.g., SEQ ID Nos: 1 or 25), (2) at least three contiguous nucleotides nonidentical to the sequence group, (3) at least four contiguous nucleotides nonidentical to the sequence group, (4) at least five contiguous nucleotides nonidentical to the sequence group, (5) at least six contiguous nucleotides nonidentical to the sequence group, (6) at least seven contiguous nucleotides nonidentical to the sequence group.", "In other embodiments, the unique fragment has a size selected from the group consisting of at least: 6 nucleotides, 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20, nucleotides, 22 nucleotides, 24 nucleotides, 26 nucleotides, 28 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 200 nucleotides, the number of nucleotides comprising the SH3BP2 gene, and every integer length therebetween.", "Unique fragments of mutant SH3BP2 nucleic acid molecules, however, exclude fragments completely composed of sequences of a wild type SH3BP2 nucleic acid (SEQ ID NO:1), wild type SH3BP2 fragment (SEQ ID NO:3), or wild type genomic SH3BP2 (SEQ ID NO:25) which do not contain a mutant domain.", "Unique fragments can be used as probes in Southern and Northern blot assays to identify such nucleic acid molecules, or can be used in amplification assays such as those employing PCR.", "As known to those skilled in the art, large probes such as 200, 250, 300 or more nucleotides are preferred for certain uses such as Southern and Northern blots, while smaller fragments will be preferred for use as PCR primers.", "Unique fragments also can be used to generate antibodies or to determine binding of a polypeptide fragment, or to generate immunoassay components.", "Likewise, unique fragments can be employed to produce nonfused fragments of the mutant SH3BP2, again useful, for example, in the preparation of antibodies, immunoassays or therapeutic applications.", "Unique fragments can be further used as antisense molecules to inhibit the expression of mutant SH3BP2 nucleic acid molecules and polypeptides, respectively.", "As will be recognized by those skilled in the art, the size of the unique fragment will depend upon its conservancy in the genetic code.", "Thus, some regions of SEQ ID NO:5 (generic mutant full length nucleic acid); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO: 25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9, and complements thereof will require longer segments to be unique while others will require only short segments, typically between 6 and 32 nucleotides long (e.g.", "6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 bases) or more, up to the entire length of the disclosed sequences.", "This disclosure intends to embrace each and every fragment of each mutant SH3BP2 nucleic acid molecule, including unique cDNA, mRNA, and genomic sequences.", "Those skilled in the art are well versed in methods for selecting such sequences, typically on the basis of the ability of the unique fragment to selectively distinguish the sequence of interest from other sequences in the human genome of the fragment to those on known databases typically is all that is necessary, although in vitro confirmatory hybridization and sequencing analysis may be performed.", "According to yet another aspect of the invention, expression vectors containing the isolated nucleic acid molecules of the invention, operably linked to a promoter, as well as host cells transformed or transfected with such expression vectors, are provided.", "In certain preferred embodiments, the host cells are eukaryotic cells.", "As used herein, a coding sequence and regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences.", "If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.", "Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.", "The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5′ non-transcribed and 5′ non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.", "Especially, such 5′ non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.", "Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.", "The vectors of the invention may optionally include 5′ leader or signal sequences.", "The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.", "Insertion of any of the nucleotide sequences described herein into an appropriate vector allows production of large quantities of such sequences.", "Indeed, vectors, methods for inserting nucleic acid-molecules into vectors, and use of such vectors for production of desired nucleic acid molecules, peptides and proteins are well known to those with skill in the art.", "Thus, the nucleotide sequences disclosed herein can also be inserted into cloning and/or expression vectors to produce peptides and proteins according to the present invention.", "Procedures and materials for preparation of replicable vectors, transformation of host cells with vectors, and host cell expression of polypeptides are described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor (1982) incorporated herein by reference.", "A replicable vector as used herein is a vector capable of being replicated and is thus useful for producing large quantities of a nucleic acid molecule of choice.", "Any replicable vector known to those with skill in the art may be used to clone or amplify mutant SH3BP2 nucleic acid molecules and/or to produce polypeptides encoded thereby.", "For example, suitable vectors include plasmids, phages, cosmids and artificial chromosomes.", "For example, bacteriophage lambda may be a useful cloning vector.", "This phage can accept pieces of foreign DNA up to about 20,000 base pairs in length.", "The lambda phage genome is a linear double stranded DNA molecule with single stranded complementary (cohesive) ends which can hybridize with each other when inside an infected host cell.", "The lambda DNA is cut with a restriction endonuclease and the foreign DNA, e.g., the DNA to be cloned, is ligated to the phage DNA fragments.", "The resulting recombinant molecule is then packaged into infective phage particles.", "Host cells are infected with the phage particles containing the recombinant DNA.", "The phage DNA replicates in the host cell to produce many copies of the desired DNA sequence.", "Cosmids are hybrid plasmid/bacteriophage vectors which can be used to clone DNA fragments of about 40,000 base pairs.", "Cosmids have one or more DNA sequences called “cos” sites derived from bacteriophage lambda for packaging lambda DNA into infective phage particles.", "Two cosmids are ligated to the DNA to be cloned.", "The resulting molecule is packaged into infective lambda phage particles and transfected into bacteria host cells.", "When the cosmids are inside the host cell they behave like plasmids and multiply under the control of a plasmid origin of replication.", "The origin of replication is a sequence of DNA which allows a plasmid to multiply within a host cell.", "Yeast artificial chromosome vectors (YAC) are similar to plasmids but allow for the incorporation of much larger DNA sequences of about 300 kb (kilobases) to 2 Mb (megabases), with an average of approximately 700 kb.", "The yeast artificial chromosomes contain sequences for replication in yeast.", "The yeast artificial chromosome containing the DNA to be cloned is transformed into yeast cells where it replicates thereby producing many copies of the desired DNA sequence.", "Where phage, cosmids or yeast artificial chromosomes are employed as cloning vectors, expression of the mutant SH3BP2 polypeptide may be obtained by culturing host cells that have been transfected or transformed with the cloning vector in a suitable culture medium.", "The bacterial artificial chromosomes used herein allow for the incorporation of 50-300 kb of DNA sequences.", "Suitable host/vector systems are available for propagation of nucleotide sequences and the expression of peptides and proteins.", "Replicable plasmids, viral vectors, and host cells such as CHO, COS, insect, yeast and bacterial are well-known for use in genetic engineering and can be used herein.", "As used herein with respect to nucleic acid molecules, the term “isolated” means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis.", "An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art.", "Thus, a nucleotide sequence contained in a vector in which 5′ and 3′ restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleotide sequence existing in its native state in its natural host is not.", "An isolated nucleic acid may be substantially purified, but need not be.", "For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides.", "Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.", "As used herein with respect to polypeptides (discussed below), the term “isolated” means separated from its native environment in sufficiently pure form so that it can be manipulated or used for any one of the purposes of the invention.", "Thus, isolated means sufficiently pure to be used (i) to raise and/or isolate antibodies, (ii) as a reagent in an assay, or (iii) for sequencing, etc.", "According to yet another aspect of the invention, antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding a mutant SH3BP2 polypeptide are provided.", "The antisense oligonucleotides are useful for decreasing mutant SH3BP2 expression (at a transcriptional and translational level) and functional activity.", "As used herein, the term “antisense oligonucleotide” or “anti sense” describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA.", "The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript.", "Antisepise oligonucleotides that selectively bind to mutant SH3BP2 nucleic acid molecules are particularly preferred.", "Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence.", "It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions.", "Based upon SEQ ID NO:5 (generic mutant full length nucleic acid); SEQ.", "ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9, or upon allelic or homologous genomic and/or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention.", "In order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least about 10 and, more preferably, at least about 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides.", "See Wagner et al., Nat.", "Med.", "1(11): 1116-1118, 1995.Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.", "Although oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5′ upstream sites such as translation initiation, transcription initiation or promoter sites.", "In addition, 3′-untranslated regions may be targeted by antisense oligonucleotides.", "Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs.", "In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol.", "Neurobiol.", "14(5):439-457, 1994) and at which proteins are not expected to bind.", "Finally, although SEQ ID NO:5 (generic mutant full length nucleic acid); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), and SEQ ID Nos:27-63, disclose cDNA sequences, one of ordinary skill in the art may easily derive the genomic DNA corresponding to each sequence.", "Thus, the present invention also provides for antisense oligonucleotides which are complementary to the genomic DNA corresponding to SEQ ID NO:5 (generic mutant full length nucleic acid); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), and SEQ ID Nos:27-63.Similarly, antisense to allelic or homologous mutant SH13BP2 and genomic DNAs are enabled without undue experimentation.", "In another aspect, ribozymes, which are catalytic RNA sequences that cleave specific RNA molecules, are used to disrupt translation of mutant SH3BP2 nucleic acid molecules.", "Several studies have demonstrated that ribozymes can be employed to inhibit oncogene expression, cell growth or induce apoptosis in tumor cell lines.", "U.S. Pat.", "No.", "5,635,385 to Leopold, et al., incorporated herein by reference, describes a therapeutic method for the treatment of a leukemia patient resulting from a chromosomal translocation (BCR/ABL) using a ribozyme that cleaves the oncogene mRNA and inhibits the expression of the polypeptide.", "A similar approach is employed according to the present invention using a synthetic ribozyme targeted to the mutant SH3BP2 mRNA molecules.", "In one set of embodiments, the antisense oligonucleotides of the invention may be composed of “natural” deoxyribonucleotides, ribonucleotides, or any combination thereof.", "That is, the 5′ end of one native nucleotide and the 3′ end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage.", "These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer.", "They also may be produced recombinantly by vectors.", "In preferred embodiments, however, the antisense oligonucleotides or ribozymes of the invention also may include “modified” oligonucleotides.", "That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.", "The term “modified oligonucleotide” as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5′ end of one nucleotide and the 3′ end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acid molecules has been covalently attached to the oligonucleotide.", "Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.", "The term “modified oligonucleotide” also encompasses oligonucleotides with a covalently modified base and/or sugar.", "For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3′ position and other than a phosphate group at the 5′ position.", "Thus modified oligonucleotides may include a 2′-O-alkylated ribose group.", "In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose.", "Thus, according to yet another aspect of the invention, a method of reducing expression of an SH3BP2 mutant nucleic acid molecule in a cell or cell-free system containing a SH3BP2 mutant nucleic acid molecule, is provided.", "The method may be practiced in vivo or in vitro.", "In general, the method involves introducing a SH3BP2 mutant antisense nucleic acid molecule or ribozyme that binds to and selectively cleaves a mutant SH3BP2 mRNA into the system (e.g., a cell), and allowing the SH3BP2 mutant antisense or ribozyme molecule to hybridize (and, if a ribozyme, cleave) to a sense SH3BP2 mutant nucleic acid molecule, thereby inhibiting expression of the sense SH3BP2 mutant nucleic acid molecule.", "The present invention also contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acid molecules encoding mutant SH3BP2 polypeptides, together with pharmaceutically acceptable carriers.", "Antisense oligonucleotides may be administered as part of a pharmaceutical composition.", "In this latter embodiment, it is preferable that localized or intravenous administration be used.", "Such a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art.", "The compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient.", "The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.", "The term “physiologically acceptable” refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.", "The characteristics of the carrier will depend on the route of administration.", "Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.", "Pharmaceutically acceptable carriers are discussed in more detail below in reference to the therapeutic methods of the invention.", "According to yet another aspect of the invention, transgenic non-human animals having somatic and germ line cells that contain any of the isolated SH3BP2 mutant nucleic acid molecules of the invention also are provided.", "Preferably, the expression of the SH3BP2 mutant nucleic acid molecule results in the animal having a bone homeostasis disorder.", "Such animals are useful as models of human bone homeostasis disorders and for studying transformation effects of mutant SH3BP2 nucleic acid molecules and polypeptides.", "Methods of creating transgenic animals are well known in the art.", "For example, U.S. Pat.", "No.", "4,873,191, incorporated herein by reference, describes genetic transformation of zygotes.", "Following such procedures, the mutant SH3BP2 nucleic acid molecule is microinjected into the nucleus of a zygote which is then allowed to undergo differentiation and development into a mature organism.", "Transgenic animals such as mice or pigs will have somatic and germ line cells containing the mutant SH3BP2 nucleic acid molecule.", "Such animals are useful as in vivo models for certain malignant syndromes and allow for the further development and testing of treatment modalities and screening assays for the identification of therapeutic agents.", "Other uses will be apparent to one of ordinary skill in the art.", "As used herein, “transgenic non-human animals” include non-human animals having one or more exogenous nucleic acid molecules incorporated in germ line cells and/or somatic cells.", "Thus transgenic animals include “knockout” and “knockin” animals having a homozygous or heterozygous gene disruption by homologous recombination, animals having episomal or chromosomally incorporated expression vectors, etc.", "Knockout and knockin animals can be prepared by homologous recombination using embryonic stem cells as is well known in the art.", "The recombination may be facilitated using, for example, the cre/lox system or other recombinase systems known to one of ordinary skill in the art.", "In certain embodiments, the recombinase system itself is expressed conditionally, for example, in certain tissues or cell types, at certain embryonic or post-embryonic developmental stages, inducibly by the addition of a compound which increases or decreases expression, and the like.", "In general, the conditional expression vectors used in such systems use a variety of promoters which confer the desired gene expression pattern (e.g., temporal or spatial).", "Conditional promoters also can be operably linked to, for example, mutant SH3BP2 nucleic acid molecules to increase expression of mutant SH3BP2 in a regulated or conditional manner.", "Trans-acting negative regulators of, for example, mutant SH3BP2 activity or expression also can be operably linked to a conditional promoter as described above.", "Such trans-acting regulators include antisense mutant SH3BP2 nucleic acid molecules, and the like.", "According to yet another aspect of the invention, primers for amplifying the above-identified nucleic acid sequences, including antisense sequences, are provided.", "In this aspect, the preferred isolated nucleic acid molecules are selected from the group consisting of a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO:23, 24 and other sequences which are capable of hybridizing to a mutant SH3BP2 nucleic acid molecule and, thereby useful in amplifying exon 9 in a mutant SH3BP2 nucleic acid molecule, and complements of the foregoing nucleic acids.", "The primers of the invention are useful for performing nucleic acid amplification techniques, such as PCR, and may be used to increase (i.e., amplify) mutant SH3BP2 nucleic acid molecules (either DNA or RNA in nature) or a fragment of such molecules.", "In some instances, the extent of amplification will depend upon the number of preexisting copies contained in the sample prior to manipulation.", "Preferably, the mutant SH3BP2 nucleic acid molecules encode all or portions of the mutant SH3BP2 polypeptide.", "The end result of such a PCR reaction may be a detectable amount of the amplified mutant SH3BP2 product, usually in the form of a nucleic acid molecule of a given length.", "In a variation of this approach, the PCR product (i.e., the amplified band) is visualized through hybridization with a labeled probe, as described earlier for Southern analysis.", "PCR techniques are well-known and described, for example, in Alberts et al., Molecular Biology of the Cell, 2nd ed., pp.", "269-276 (1989), incorporated herein by reference.", "Briefly, PCR is performed by heating the sample to separate complementary nucleic acid strands which are then annealed to complementary primer oligonucleotides which serve as primers for DNA synthesis catalyzed by polymerase enzymes between the primers.", "Multiple cycles of PCR provide multiple copies of the target sequence as long as the target sequence was originally present in the sample.", "Thus, the present invention provides a method for amplifying and detecting the presence of mutant SH3BP2 nucleic acid molecules in a sample by contacting the sample with at least two nucleic acid amplification primers, amplifying a primed nucleic acid molecule which hybridizes to the first and the second nucleic acid amplification primers; and detecting the presence of an amplified nucleic acid molecule in the sample.", "The first nucleic acid amplification primer and a second nucleic acid amplification primers are capable of hybridizing to a SH3BP2 mutant nucleic acid molecule and amplifying a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9 and, preferably, are not capable of hybridizing to and amplifying SEQ ID NO:1 (human “WT” SH3BP2 full length sequence), SEQ ID NO:3 (human WT domain), SEQ ID NO:15 (genomic WT SH3BP2), or SEQ ID NO:101 (human WT exon 9).", "Exemplary primers include SEQ ID Nos: 23 and 24.In theory, any combination of nucleic acid molecules each greater than approximately 10-20 base pairs in length which flank the point mutation(s) disclosed herein can serve as primers for PCR.", "The primers may be either DNA or RNA in nature although, usually DNA primers are preferred due to an increased stability.", "If the nucleic acid molecule to which the primers hybridize is RNA in nature, a first step of reverse transcription (RT) of the RNA molecule is required and the procedure is referred to as RT-PCR.", "RT-PCR methods are well known in the art.", "It should be understood that amplification primers may be derived from any region of the mutant SH3BP2 nucleic acid sequence which flank the point mutation(s), including intronic portions of genomic DNA.", "The target sequence for amplification can include genomic DNA or mRNA which encode all or unique fragments of the mutant SH3BP2 nucleotide sequences.", "It is apparent to those skilled in the art that other unique fragments derived from the mutant SH3BP2 nucleotide sequences or sequences complementary thereto can also be used as primers.", "The invention further provides nucleic acid detection techniques based on hybridization of labeled probes, e.g., fluorescent in-situ hybridization (FISH), which are capable of detecting small amounts of mutant SH3BP2 sequences and are extremely useful herein.", "Thus, in accordance with the present invention, the presence of a mutant SH3BP2 nucleic acid molecule containing a mutant SH3BP2 nucleic acid point mutation(s) in a sample can be detected by contacting the sample one or more nucleic acid probes which hybridize to a mutant SH3BP2 mutant domain, and detecting the presence of a nucleic acid molecule within the sample that hybridizes to the probe(s).", "Nucleic acid probes and primers for hybridization which are derived from mutant SH3BP2 can be synthesized on an oligonucleotide synthesizer such as those commercially available from Applied Biosystems (California).", "DNA or RNA probes can also be derived by PCR using two primers from the mutant SH3BP2 gene.", "As is well-known in the art, probes useful in detecting nucleic acid molecules can be labeled directly by attaching a label to the probe or indirectly by causing a labeled binding agent to couple to the probe after hybridization.", "Examples of labels include fluorochromes such as fluorescein, Texas Red® and green fluorescent protein, enzymes such as horse radish peroxidase and radioactive isotopes.", "Signal amplification systems may also be utilized herein, e.g., avidin, streptavidin and bidtin complexes or antibody hapten complexes.", "Such methods and systems are well known and are discussed generally, e.g., in Alberts et al., Molecular Biology of the Cell, 2nd ed., pp.", "174-193, incorporated herein by reference.", "Exemplary detectable labels include green fluorescent protein and Texas Red®, for use in assays which rely upon detecting fluorescence, microscopy, spectrophotometry, fluorescent plate readers and flow sorters.", "Additional compositions and methods for detecting the mutant SH3BP2 nucleic acid molecules and polypeptides utilize antibodies, fragments of antibodies (embraced within the definition of antibodies, herein) and labels, and signal amplification techniques involving antibodies.", "Antibodies which are immunoreactive to mutant SH3BP2 nucleic acid molecule or to the mutant SH3BP2 polypeptide or to unique fragments of these are generated by known techniques, e.g., by immunization of animals such as mice with mutant SH3BP2 nucleic acid or with mutant SH3BP2 polypeptide or unique fragments thereof which include the point mutation.", "Polyclonal and monoclonal antibodies may be generated using immortal cell lines for continuous production.", "Antibodies to mutant SH3BP2 nucleic acid or polypeptide or to unique fragments of each which include the point mutation are conjugated to labels such as those described above.", "Alternatively, if the so-called primary antibody is not labeled, it can be detected with a second labeled antibody which is immunoreactive with the first antibody.", "In addition to utilizing antibodies and fragments thereof, the foregoing detection methods can also be performed using other binding agents such as those described herein including peptide and non-peptide compounds produced in libraries.", "According to another aspect of the invention, isolated polypeptides encoded by any of the isolated mutant SH3BP2 nucleic acid molecules of the invention are provided.", "Preferably, the isolated polypeptides of the invention include a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100.As used herein, a mutant SH3BP2 refers to a polypeptide encoded by mutant SH3BP2 nucleic acid molecule of the invention and which contains a mutant SH3BP2 polypeptide domain.", "A mutant SH3BP2 polypeptide domain refers to the minimum amino acid sequence which identifies a polypeptide as a mutant SH3BP2 polypeptide.", "Typically, these mutations are present in the domain encoded by exon 9.", "(See, e.g., FIG.", "2).", "Mutant SH3BP2 polypeptides may be identified in a similar manner to the identification of unique polypeptide fragments, as described herein.", "The point mutations which result in the cherubism phenotype frequently result in changes in the amino acids at amino acid positions 415, 418, and 420 of SEQ ID NO:2 (see also Table 2); however, it is to be understood that the invention also embraces mutant SH3BP2 polypeptides having one or more amino acid mutations present in the polypeptide encoded by exon 9, in particular, mutations present at amino acid positions, 415, 416, 417, 418, 419 and/or 420 of SEQ ID NO:2.In the preferred embodiments, the isolated mutant SH3BP2 polypeptides are immunogenic and can be used to generate binding agents (e.g., antibodies and antibody fragments) for use in diagnostic and therapeutic applications.", "As diagnostic or prognostic indicators, such binding agents are useful for determining the presence or absence of a mutant SH3BP2 polypeptide, and/or for determining the level of such a polypeptide in a sample.", "Samples to be analyzed include but are not limited to biological samples such as biopsy samples.", "The mutant SH3BP2 polypeptides used for generating binding agents are unique polypeptides and, therefore, the binding agents so generated are those which selectively bind to a mutant SH3BP2 polypeptide and not to a wild type SH3BP3 polypeptide.", "A unique fragment of an mutant SH3BP2 polypeptide, in general, has the features and characteristics of unique fragments as discussed above in connection with nucleic acid molecules.", "As will be recognized by those skilled in the art, the size of the unique fragment will depend upon factors such as whether the fragment constitutes a portion of a conserved protein domain.", "Thus, some regions of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100 will require longer segments to be unique while others will require only short segments, typically between 4 and 12 amino acids (e.g.", "4, 5, 6, 7, 8, 9, 10, 11 and 12 amino acids long or more, including each integer up to the full length, </=561 amino acids long).", "Virtually any segment of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100, excluding the ones that share identity with it (e.g., the wild type SH3BP2 polypeptide, and fragments thereof, or other polypeptides published prior to the invention or application filing date) that is 9 or more amino acids in length will be unique.", "One important aspect of a unique fragment is its ability to act as a signature for identifying the polypeptide.", "Another is its ability to provide an immune response in an animal.", "Those skilled in the art are well versed in methods for selecting unique amino acid sequences, typically on the basis of the ability of the unique fragment to selectively distinguish the sequence of interest from unrelated proteins.", "A comparison of the sequence of the fragment to those on known databases typically is all that is necessary.", "The invention embraces variants of the mutant SH3BP2 polypeptides described above.", "As used herein, a “variant” of a mutant SH3BP2 polypeptide is a polypeptide which contains one or more modifications to the primary amino acid sequence of a mutant SH3BP2 polypeptide.", "Modifications which create a mutant SH3BP2 variant are typically made to the nucleic acid which encodes the mutant SH3BP2 polypeptide and can include deletions, point mutations, truncations, amino acid substitutions and addition of amino acids or non-amino acid moieties to: 1) reduce or eliminate a functional activity of the polypeptide, such as its ability to bind a ligand or to activate transcription of a particular genomic locus; 2) enhance a property of the polypeptide, such as protein stability in an expression system or the stability of protein-protein binding; 3) provide a novel activity or property to the polypeptide, such as addition of an antigenic epitope or addition of a detectable moiety; or 4) to provide equivalent or better binding to the polypeptide by another molecule, or to another molecule by the polypeptide.", "Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like.", "Modifications also embrace fusion proteins comprising all or part of the mutant SH3BP2 amino acid sequence.", "One of skill in the art will be familiar with methods for predicting the effect on protein conformation of a change in protein sequence, and can thus “design” a variant mutant SH3BP2 polypeptide according to known methods.", "One example of such a method is described by Dahiyat and Mayo in Science 278:82-87, 1997, whereby proteins can be designed de novo.", "The method can be applied to a known protein to vary only a portion of the polypeptide sequence.", "By applying the computational methods of Dahiyat and Mayo, specific variants of a mutant SH3BP2 polypeptide can be proposed and tested to determine whether the variant retains a desired conformation.", "Variants can include mutant SH3BP2 polypeptide which are modified specifically to alter a feature of the polypeptide unrelated to its physiological activity.", "For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages.", "Similarly, certain amino acids can be changed to enhance expression of a mutant SH3BP2 polypeptide by eliminating proteolysis by proteases in an expression system.", "Mutations of a nucleic acid molecule which encode a mutant SH3BP2 polypeptide preferably preserve the amino acid reading frame of the coding sequence and, preferably, do not create regions in the nucleic acid molecule which are likely to hybridize to form secondary structures, such a hairpins or loops, which can be deleterious to expression of the variant polypeptide.", "Mutations can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide.", "Variant polypeptides are then expressed and-tested for one or more activities to determine which mutation provides a variant polypeptide with the desired properties.", "Further mutations can be made to variants (or to non-variant mutant SH3BP2 polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host.", "Still other mutations can be made to the noncoding sequences of a mutant SH3BP2 gene or cDNA clone to enhance expression of the polypeptide.", "The skilled artisan will realize that conservative amino acid substitutions may be made in mutant SH3BP2 polypeptides to provide functionally equivalent variants of the foregoing polypeptides, i.e., the variants retain the functional capabilities of the mutant SH3BP2 polypeptides.", "As used herein, a “conservative amino acid substitution” refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.", "Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g.", "Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.", "Exemplary functionally equivalent variants of the mutant SH3BP2 polypeptides include conservative amino acid substitutions of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100.Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Thus functionally equivalent variants of mutant SH3BP2 polypeptides, i.e., variants of mutant SH3BP2 polypeptides which retain the function of the natural mutant SH3BP2 polypeptides, are contemplated by the invention.", "Conservative amino-acid substitutions in the amino acid sequence of mutant SH3BP2 polypeptides to produce functionally equivalent variants of mutant SH3BP2 polypeptides typically are made by alteration of the nucleic acid sequences encoding mutant SH3BP2 polypeptides.", "Such substitutions can be made by a variety of methods known to one of ordinary skill in the art.", "For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc.", "Nat.", "Acad.", "Sci.", "U.S.A. 82: 488-492,1985), or by chemical synthesis of a gene encoding a mutant SH3BP2 polypeptide.", "The activity of functionally equivalent fragments of mutant SH3BP2 polypeptides can be tested by cloning the gene encoding the altered mutant SH3BP2 polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered mutant SH3BP2 polypeptide, and testing for a functional capability of the mutant SH3BP2 polypeptides as disclosed herein.", "The mutant SH3BP2 polypeptides may be purified from cells which naturally produce the polypeptide by chromatographic means or immunological recognition.", "Alternatively, an expression vector may be introduced into cells to cause production of the polypeptide.", "In another method, mRNA transcripts may be microinjected or otherwise introduced into cells to cause production of the encoded polypeptide.", "Translation of mutant SH3BP2 mRNA in cell-free extracts such as the reticulocyte lysate system also may be used to produce mutant SH3BP2 polypeptides.", "Those skilled in the art also can readily follow known methods for isolating mutant SH3BP2 polypeptides.", "These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography and immune-affinity chromatography.", "According to another aspect of the invention, isolated mutant SH3BP2 binding agents (e.g., binding nucleic acid molecules such as probes or primers or ribozymes, and binding polypeptides such as antibodies) which selectively bind to a mutant SH3BP2 nucleic acid molecule or to a mutant SH3BP2 polypeptide encoded by the isolated nucleic acid molecules of the invention are provided.", "Preferably, the isolated binding agents selectively bind to a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:5 (generic mutant—full length); SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the lo wild type exon 9.In certain embodiments, the binding molecules do not hybridize to SEQ ID NO:1 (human “WT” SH3BP2 full length sequence) or SEQ ID NO:3 (human WT domain).", "Alternatively, the binding agents selectively bind to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID Nos:64-100, or to unique fragments of the foregoing nucleic acid molecules and polypeptides.", "In certain embodiments, the binding agents do not bind to SEQ ID NO:2 or to fragments thereof which do not include a mutant domain.", "In the preferred embodiments, the isolated binding polypeptides include antibodies and fragments of antibodies (e.g., Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region which binds selectively to a mutant SH3BP2 nucleic acid molecule or polypeptide).", "Preferably, the antibodies for human therapeutic applications are human antibodies.", "As is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I.", "(1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford).", "The pFc′ and Fe regions, for example; are effectors of the complement cascade but are not involved in antigen binding.", "An antibody from which the pFc′ region has been enzymatically cleaved, or which has been produced without the pFc′ region, designated an F(ab′)2 fragment, retains both of the antigen binding sites of an intact antibody.", "Similarly, an antibody from which the Fe region has been enzymatically cleaved, or which has been produced without the Fe region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule.", "Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.", "The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.", "Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991).", "In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3).", "The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.", "It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody.", "This is most clearly manifested in the development and use of “humanized” antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc′ regions to produce a functional antibody.", "Thus, for example, PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin.", "Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as “chimeric” antibodies.", "Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab′)2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab′)2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences.", "The present invention also includes so-called single chain antibodies.", "Thus, the invention involves binding agents in the form of binding polypeptides of numerous size and type that bind selectively to mutant SH3BP2 polypeptides, and complexes or fusion proteins containing mutant SH3BP2 polypeptides.", "These binding polypeptides also may be derived from sources other than antibody technology.", "For example, such polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form, as bacterial flagella peptide display libraries or as phage display libraries.", "Combinatorial libraries also can be synthesized of peptides containing one or more amino acids.", "Libraries further can be synthesized of peptides and non-peptide synthetic moieties.", "Phage display can be particularly effective in identifying binding peptides useful according to the invention.", "Briefly, one prepares a phage library (using e.g.", "m13, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures.", "The inserts may represent, for example, a completely degenerate or biased array.", "One then can select phage-bearing inserts which bind to the mutant SH3BP2 polypeptide or a complex containing a mutant SH3BP2 polypeptide, but not to a wild type SH3BP2 polypeptide.", "This process can be repeated through several cycles of reselection of phage that bind to the mutant SH3BP2 polypeptide or to a complex containing a mutant SH3BP2 polypeptide.", "Repeated rounds lead to enrichment of pliage bearing particular sequences.", "DNA sequence analysis can be conducted to identify the sequences of the expressed polypeptides.", "The minimal linear portion of the sequence that binds to the mutant SH3BP2 polypeptide or complex can be determined.", "One can repeat the procedure using a biased library containing inserts containing part or all of the minimal linear portion plus one or more additional degenerate residues upstream or downstream thereof.", "Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to the mutant SH3BP2 polypeptides.", "Thus, the mutant SH3BP2 polypeptides of the invention, or a unique fragment thereof, or complexes of mutant SH3BP2 can be used to screen peptide libraries, including phage display libraries, to identify and select peptide binding polypeptides that selectively bind to the mutant SH3BP2 polypeptides of the invention.", "Such molecules can be used, as described, for screening assays, for purification protocols, for interfering directly with the functioning of mutant SH3BP2 and for other purposes that will be apparent to those of ordinary skill in the art.", "In addition, such molecules can also be tested for their ability to inhibit mutant SH3BP2 polypeptide production.", "Such inhibition can result from interference with transcription and/or translation of mutant SH3BP2 nucleic acid molecules.", "Compounds and libraries can be so tested for these abilities using screening assays such as those described below.", "A mutant SH3BP2 polypeptide, or a unique fragment thereof, also can be used to isolate naturally occurring, polypeptide binding agents which may associate with the mutant SH3BP2 polypeptide in a cell.", "Isolation of binding agents may be performed according to well-known methods.", "For example, isolated mutant SH3BP2 polypeptides can be attached to a substrate, and then a solution suspected of containing an mutant SH3BP2 binding agent may be applied to the substrate.", "If the binding agent for mutant SH3BP2 polypeptides is present in the solution, then it will bind to the substrate-bound mutant SH3BP2 polypeptide.", "The binding agent then may be isolated.", "Other proteins which are binding agents for mutant SH3BP2 may be isolated by similar methods without undue experimentation.", "The isolated nucleic acid molecules disclosed herein have various utilities, including their use as probes and primers in diagnostic assays for identifying the presence of mutant SH3BP2 nucleic acid molecules in a sample.", "Additionally they may function as agents for generating mutant SH3BP2 polypeptides and mutant SH3BP2 binding agents that also can be used in diagnostic and therapeutic assays to determine the presence or absence of a mutant SH3BP2 molecule and/or to determine the level of a mutant SH3BP2 molecule in a sample.", "Thus, the foregoing mutant SH3BP2 nucleic acid molecules, polypeptides and binding agents can be used, inter alia, in the diagnosis or treatment of conditions characterized by the expression or presence of a mutant SH3BP2 nucleic acid molecule or polypeptide (See, for example, Examples).", "Since, as demonstrated herein, the mutant SH3BP2 nucleic acid molecule and polypeptide is found in tissue associated with a disorder of bone homeostasis, the role of mutant SH3BP2 molecules in mediating bone homeostasis is clear.", "Without wishing to be bound by any particular theory, there are several mechanisms by which, for example, the mutant SH3BP2 molecules may mediate a bone homeostasis disorder.", "SH3BP2 was initially identified by screening a phage expression library for proteins that can bind to the SH3 domain of the proto-oncogelle c-Abl, and it was shown to contain a proline-rich 10 amino acid residue-long sequence responsible for SH3 binding (Ren, R. et al., Science 259 (1993), 1157-1161).", "This SH3 ligand motif (PPAYPPPPVP, SEQ ID NO:103) is located 205 amino acid residues upstream of the mutated region Arg415-Gly420.The N-terminal pleckstrin homology (PH) domain belongs to a class of interaction domains that are found in a number of eukaryotic signaling proteins.", "They can bind to other proteins as well as to inositol phosphates, but the binding partners for the PH domain in SH3BP2 are still unknown.", "The C-terminal SH2 domain is likely to bind with high affinity to tyrosine-phosphorylated peptides (Songyang, Z. et al., Mol.", "Cell.", "Biol.", "14 (1994), 2777-2785;.Pawson, T. et al., Cell 71 (1992), 359-362).", "The combination of the three protein binding modules in SH3BP2 and its affinity for c-Abl via its SH3 (Ren, R. et al., Science 259 (1993), 1157-1161; Sparks, A.", "B. et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 93 (1996), 1540-1544) binding domain make it a possible adapter protein that could organize the formation of functional complexes involving c-Abl.", "SH3BP2 is also likely to interact with cytoplasmic signaling molecules playing a role in transcriptional activation in hematopoietic cells through its SH2 domain (Deckert, M., et al., Immunity 9 (1998), 595-605).", "Thus, it could function primarily as a regulator of c-Abl function in some cells, while controlling transcriptional regulation through interaction with other kinases and signaling complexes in other cells.", "Such a potential dual function may explain the cellular phenotype of the fibro-osseous lesions in cherubism.", "Not only are the lesions reminiscent of fibrous dysplasia of bone caused by mutations in GNSA1 resulting in deficient osteoblastic differentiation from mesenchymal percursors.", "(Weinstein, L S, et al., N. Engl.", "J. Med.", "325 (1991), 1688-1695; Bianco P. et al., J.", "Bone Miner Res.", "15 (2000), 120-128), but the accumulation of large numbers of osteoblast-like cells in cherubism suggests an abnormal formation of these monocyte-derived, multinucleated cells as well.", "The clustering of amino acid missense mutations in SH3BP2 suggest that they represent gain of function mutations.", "SH3BP2 lies within a region that is frequently deleted in Wolf-Hirschhorn Syndrome (WHS) (Hirschhorn, K. et al., Humangenetik 1 (1965), 479-482; Zollino, M., et al., Am.", "J. Med.", "Genet.", "18 (2000), 254-261) patients.", "Haploinsufficiency of SH3BP2 in these patients does not result in cherubism or cherubism-like characteristics, which supports our hypothesis that the mutations in SH3BP2 lead to a gain of function.", "Although not wishing to be bound to any particular theory or mechanism, we believe that SH3BP2 may enhance the ability of the protein to engage in complexes that are important for differentiation and activation of osteoblasts while they may affect in a negative manner the function of c-Abl in differentiating osteoblasts.", "Interestingly, osteoblastic differentiation is impaired in c-Abl null mice, resulting in osteoporosis (Li, B. et al., Nat.", "Genet.", "24.", "(2000), 304-308).", "The ability to shuttle between focal adhesions and the nucleus makes c-Abl a good candidate for being a mediator of integrin-regulated gene expression (Lewis, J. M. et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 93 (1996), 15174-15179) and cell cycle progression (Welch, P J, et al., Cell 75 (1993), 779-790).", "It is conceivable that at age 3 (frequently the age of onset of the disease) there are signals transmitted through the extracellular matrix of the mandible and maxilla that are unique to these bones and are triggered by the eruption of secondary teeth.", "Thus, the onset of the abnormalities of cherubism and their organ-restricted characteristics may be related to the dental developmental process.", "Thus, we believe that administration of agents which inhibit mutant SH3BP2 expression will alleviate the symptoms associated with various bone homeostasis disorders, including cherubism, bone tumors, osteoporosis and osteopetrosis.", "Similarly, we believe that the detection of the mutant SH3BP2 molecules of the invention will be useful for definitively diagnosing these and other bone homeostasis disorders.", "According to another aspect of the invention, a method of identifying the presence of an SH3BP2 mutant molecule in a sample is provided.", "In general, the method involves analyzing the sample for the presence of a SH3BP2 mutant nucleic acid molecule or a SH3BP2 mutant polypeptide.", "In certain embodiments, the method further involves the steps of contacting the sample with at least two nucleic-acid amplification primers and amplifying DNA in the sample prior to analyzing the sample for the presence of a SH3BP2 mutant nucleic acid molecule.", "The method employs a first nucleic acid amplification primer and a second nucleic acid amplification primer that are capable of hybridizing to a SH3BP2 mutant nucleic acid molecule and, thereby, useful in amplifying a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation), SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO:25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place of the wild type exon 9.Following hybridization, the method involves amplifying a primed nucleic acid molecule which hybridizes to the first and the second nucleic acid amplification primers; and detecting the presence of an amplified nucleic acid molecule in the sample.", "In yet other embodiments, the method involves contacting the sample with one or more nucleic acid probes, wherein the nucleic acid probe is capable of hybridizing to a nucleic acid sequence selected from the group consisting of: nucleic acid sequences which hybridize to a sequence selected from one or more of the following sequences under stringent conditions: SEQ ID NO:7 (generic mutant domain); SEQ ID NO:9 (“H” family mutation), SEQ ID NO:11 (“K” family mutation), SEQ ID NO:13 (“A,B” family mutation), SEQ ID NO:15 (“C,F,J,M,O” family mutation), SEQ ID NO:17 (“L” family mutation), SEQ ID NO:19 (“G” family mutation), SEQ ID NO:21 (“N” family mutation SEQ ID NO:26 (genomic mutant SH3BP2), SEQ ID NOs:27-63 (mutant SH3BP2 exon 9) and a genomic mutant SH3BP2 nucleic acid molecule having a sequence identical to SEQ ID NO: 25 with the exception that a mutant exon 9 selected from the group consisting of SEQ ID NOs: 27-63 is used in place-of the wild type exon 9 and, preferably, which does not hybridize to SEQ ID NO:1 (human “WT” SH3BP2 full length sequence) or SEQ ID NO:3 (human WT domain) or SEQ ID NO:25 (genomic WT SH3BP2) under stringent conditions, and detecting the presence of a SH3BP2 mutant nucleic acid in the sample which hybridizes to the nucleic acid probe.", "In yet other embodiments, the method involves contacting the sample with one or more binding agents, wherein the binding agent is capable of selectively binding to a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID Nos:64-100; and detecting the presence of a SH3BP2 mutant polypeptide in the sample which binds the binding agent.", "Preferably, the binding agent is an antibody or a fragment thereof that selectively binds to the SH3BP2 mutant polypeptide in the sample and does not bind to a non-mutant SH3BP2 polypeptide that may be present in the sample.", "The diagnostic methods of the invention are particularly useful for: evaluating the susceptibility of a human subject to a disorder of bone homeostasis, evaluating causation of a bone homeostasis disorder in a human subject, or for evaluating the genetic predisposition of a human subject to have offspring with a bone homeostasis disorder.", "Such methods differ primarily in the selection of the human subject (e.g., the human subject exhibits a bone homeostasis disorder or is suspected of having a predisposition to developing the disorder or having offspring susceptible of developing the disorder).", "In general, such methods involve obtaining a sample of DNA from the subject; and evaluating the sample of DNA for the presence of nucleotides encoding a “mutant residue” at any one or more amino acid positions encoded by exon 9 of the SH3BP2 gene product.", "In certain preferred embodiments, the sample of DNA is evaluated for the presence of nucleotides encoding a “mutant residue” in the polypeptide encoded by exon 9, particularly at positions 415-420 and, more particularly, at any one or more amino acids at positions 415, 418, and 420, of the SH3BP2 gene product.", "The presence of a mutant residue is indicative of the condition for which the subject is being tested.", "Exemplary mutant residues that are diagnostic of cherubism are described in more detail in the Examples.", "According to yet another aspect of the invention, a method of screening for an agent that inhibits the production of a SH3BP2 mutant polypeptide is provided.", "The method involves: (a) determining the level of a SH3BP2 mutant molecule (e.g., nucleic acid, polypeptide) in the absence of a putative inhibitor, (b) determining the level of a SH3BP2 mutant molecule in the presence of the putative inhibitor, and (c) comparing the level of the SH3BP2 mutant molecule in the presence and absence of the putative inhibitor.", "A decrease in the level of the SH3BP2 mutant molecule in the presence of the putative inhibitor indicates that the putative inhibitor is an agent that inhibits the production of a SH3BP2 mutant polypeptide.", "In certain embodiments, the agent inhibits transcription of a SH3BP2 mutant nucleic acid molecule.", "In yet other embodiments, the agent inhibits translation of a SH3BP2 mutant nucleic acid molecule.", "According to yet another aspect of the invention, methods for identifying agents which bind or otherwise interact with the SH3BP2 molecules of the invention are provided.", "The methods are useful for identifying agents which modulate SH3BP2 molecule activity at the level of its expression (defined herein as including replication, transcription or translation), or mutant SH3BP2 dependent cellular function (for example, binding to a nucleic acid or a ligand, mutant SH3BP2-mediated cellular signalling).", "Generally, the screening methods involve assaying for compounds which have a net result of inhibiting production of mutant SH3BP2 polypeptides or inhibiting the function of mutant SH3BP2 polypeptides.", "Such methods are adaptable to automated, high throughput screening of compounds.", "In a preferred embodiment, a method involves screening for an agent that inhibits the production of a mutant SH3BP2 molecule (nucleic acid or polypeptide).", "The method involves determining and comparing the level of, for example, a mutant SH3BP2 molecule in the absence and in the presence of a test compound.", "According to the method, a decrease in the level of a mutant SH3BP2 molecule in the presence of the compound is indicative of an agent that inhibits the production of a mutant SH3BP2 molecule.", "The compound can be synthesized or harvested from a variety of sources as described herein.", "The compound can be peptide or non-peptide in nature.", "As mentioned above, the agent may be one which inhibits transcription of a mutant SH3BP2 nucleic acid molecule.", "Alternatively, the agent may be one which inhibits translation of a mutant SH3BP2 nucleic acid molecule.", "The screening assay can be carried out in a cell, a cell-free system, a tissue or in an animal such as a transgenic, non-human animal.", "A wide variety of assays for screening pharmacological agents are provided, including, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays, cell-based assays such as two- or three-hybrid screens, expression assays, etc.", "For example, two-hybrid screens are used to rapidly examine the effect of transfected nucleic acid molecules on the intracellular binding of mutant SH3BP2 polypeptide or fragments thereof to intracellular targets.", "The transfected nucleic acid molecules can encode, for example, combinatorial peptide libraries or cDNA libraries.", "Convenient reagents for such assays, e.g., GAL4 fusion proteins, are known in the art.", "An exemplary cell-based assay involves transfecting a cell with a nucleic acid encoding, for example, a mutant SH3BP2 polypeptide fused to a GAL4 DNA binding domain and a nucleic acid encoding a reporter gene operably linked to a gene expression regulatory region, such as one or more GAL4 binding sites.", "Activation of reporter gene transcription occurs when the mutant SH3BP2 and reporter fusion polypeptides bind to each other such as to enable transcription of the reporter gene.", "Agents which modulate a mutant SH3BP2 polypeptide mediated cell function are then detected through a change in the expression of reporter gene.", "Methods for determining changes in the expression of a reporter gene are known in the art.", "In preferred embodiments, the agents so identified are also further screened to preclude those that also bind to the wild type SH3BP2 polypeptides.", "Mutant SH3BP2 polypeptide fragments used in the methods, when not produced by a transfected nucleic acid are added to an assay mixture as an isolated polypeptide.", "Mutant SH3BP2 polypeptides preferably are produced recombinantly, although such polypeptides may be isolated from biological extracts.", "Recombinantly produced mutant SH3BP2 polypeptides include chimeric proteins comprising a fusion of a mutant SH3BP2 polypeptide with another polypeptide, e.g., a polypeptide capable of providing or enhancing protein-protein binding, sequence specific nucleic acid binding (such as GAL4), enhancing stability of the mutant SH3BP2 polypeptide under assay conditions, or providing a detectable moiety, such as green fluorescent protein or Flag epitope.", "Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds.", "For example, numerous means,are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like.", "Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.", "Additionally, natural and synthetically produced libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means.", "Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc.", "to produce structural analogs of the agents.", "In connection with the screening assays and diagnostic methods, the invention also provides novel kits which are useful for detecting/measuring the levels of the nucleic acid molecules of the invention, expression products of the invention or anti-mutant SH3BP2 antibodies.", "In the case of nucleic acid detection, pairs of primers for amplifying mutant SH3BP2 nucleic acid molecules can be included.", "The preferred kits would include controls such as known amounts of nucleic acid probes, mutant SH3BP2 epitopes (such as mutant SH3BP2 expression products) or anti-mutant SH3BP2 antibodies, as well as instructions or other printed material.", "In certain embodiments the printed material can characterize the risk of developing a disorder that is characterized by mutant SH3BP2 nucleic acid or polypeptide expression based upon the outcome of the assay.", "The reagents may be packaged in containers and/or coated on wells in predetermined amounts, and the kits may include standard materials such as labeled immunological reagents (such as labeled anti-IgG antibodies) and the like.", "One kit is a packaged polystyrene microtiter plate coated with a mutant SH3BP2 polypeptide and a container containing labeled anti-human IgG antibodies.", "A well of the plate is contacted with, for example, a tissue lysate from a subject diagnosed as having a bone homeostasis disorder, washed and then contacted with the anti-IgG antibody.", "The label is then detected.", "A kit embodying features of the present invention is comprised of the following major elements: packaging an agent of the invention, a control agent, and instructions.", "Packaging is a box-like structure for holding a vial (or number of vials) containing an agent of the invention a vial (or number of vials) containing a control agent, and instructions.", "Individuals skilled in the art can readily modify packaging to suit individual needs.", "It should be understood that kits which include reagents that are used to detect mutant SH3BP2 nucleic acid molecules and polypeptides encoded thereby can also be assembled so as to provide convenient access and use in clinical settings.", "For example, a kit can include a container which holds one or more amplification primers, a container which holds enzymes used for amplification, a container which holds washing solution(s), a container which holds detection reagents, and a sample well.", "Alternatively, a kit can include a container which holds one or more antibodies directed to a mutant SH3BP2 polypeptide or a fragment thereof, a container which holds washing solution(s), a container which holds detection reagents, and a sample well.", "It is also contemplated that a kit may include a container having one or more labeled or unlabeled probes capable of hybridizing to the SH3BP2 nucleic acid molecule comprising nucleotides 1451-1471 of SEQ ID NO:5, or corresponding mRNA and, if the probe is unlabeled, a container having a labeled specific binding agent of the probe or to a recognition site on the probe, e.g., biotinylated probe, a container which holds washing solution(s), a container which holds detection reagents, and a sample well.", "Examples of detection reagents include radiolabeled probes, enzymatic labeled probes (horse radish peroxidase, alkaline phosphatase), and affinity labeled probes (biotin, avidin, or streptavidin).", "For antibodies, examples of detecting reagents include, but are not limited to, labeled secondary antibodies, or, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.", "The antibodies, primers and nucleic acid probes described herein can readily be incorporated into one of the established kit formats which are well known in the art.", "In some instances, the foregoing detection methods and kits may also comprise a control and/or comparison with a control.", "As used herein, a control can include a known amount of a mutant SH3BP2 nucleic acid molecule or a fragment thereof or a mutant SH3BP2 polypeptide or a fragment thereof (such as an antigenic fragment).", "In preferred embodiments the control is a similar tissue sample from a subject who does not express a mutant SH3BP2 nucleic acid molecule or polypeptide or similar normal tissue from the same subject.", "The methods described herein generally involve detection of a mutant SH3BP2 nucleic acid molecule or a polypeptide in a sample.", "Such a sample can be, but is not limited to, a tissue or a biological fluid, or any type of sample which typically is assessed to diagnose a disorder of bone homeostasis.", "SH3BP2 is expressed in blood leukocytes, bone marrow, and bone.", "Accordingly, exemplary samples that can be tested for the presence of a mutant SH3BP2 molecule include blood, bone marrow, skin biopsies, and iliac crest biopsies.", "Both invasive and non-invasive techniques can be used to obtain such samples and are well documented in the art.", "According to a yet another aspect of the invention, a method for treating a subject having a bone homestasis disorder which, preferably, is further characterized by the presence of a SH3BP2 mutant nucleic acid molecule is provided.", "The method of treatment involves administering a mutant SH3BP2 molecule binding agent (e.g., a mutant SH3BP2 antisense molecule) to a subject in need of such treatment in an amount effective to treat the subject, wherein the subject is not otherwise in need of mutant SH3BP2 binding molecule administration.", "In general, the therapeutic methods of the invention utilize the novel compositions of the invention (particularly antisense) to inhibit a mutant SH3BP2 function.", "All administration modes are possible, with localized (e.g., periodontal pockets)/systemic delivery preferred.", "Controlled release is also important.", "The agents of the invention can be administered alone or in combination with another therapeutic agent for treating the condition.", "Thus, as used herein, a therapeutic agent is an agent other than the mutant SH3BP2 nucleic acid molecules, polypeptides, binding agents of the invention which has been reported to possess therapeutic effectiveness toward the disorder being treated.", "A therapeutic agent is also different from those agents described herein which upon administration inhibit or downregulate the production of mutant SH3BP2 polypeptides.", "In some embodiments, the foregoing agents of the invention may be administered substantially simultaneously with the therapeutic agents.", "By substantially simultaneously, it is meant that the agent of the invention is administered to a subject close enough in time with the administration of the therapeutic agent so that the two compounds may exert an additive or even synergistic effect, (e.g., reducing a tumor mass).", "As used herein, a bone homeostasis disorder refers to a disorder characterized by an abnormal imbalance between bone formation and bone degradation.", "In certain embodiments, the bone homeostasis disorder is selected from the group consisting of osteoporosis, osteopetrosis; a bone tumor; and cherubism (MIM #118400).", "Additional disorders that can be tested in accordance with the methods of the invention to definitively diagnose whether the presented bone homeostasis disorder is mediated by a mutant SH3BP2 molecule include: Cassey disease (MIM #114000), Ramon syndrome (MIM #266270), Gingival fibromatosis (MIM #135300), Noonan-like multiple giant cell lesion syndrome (MIM #163955), McCune-albright syndrome (MIM #174800), giant cell granulomas, and giant cell tumors.", "The agent may be administered in accordance with standard clinical practice (e.g., systemic); however, in certain preferred embodiments relating to the treatment of cherubism, the agent is locally administered (e.g., to a periodontal pocket).", "As used herein, treatment of a subject includes the prophylactic treatment of a subject at risk of developing a bone homeostasis disorder (e.g., associated with mutant SH3BP2 expression), as well as treatment of subjects suspected of having a disorder or known to have a bone homeostasis disorder associated with, or characterized by, mutant SH3BP2 expression.", "In such embodiments, the amount effective to treat the subject is the amount which inhibits either the development or the progression of a disorder or decreases the rate of progression of a disorder associated with mutant SH3BP2 expression.", "In some instances, the disorder is a proliferative disorder, such as a bone tumor.", "Thus, alternatively an effective amount is that amount which inhibits the growth and/or proliferation of a cell expressing a mutant SH3BP2 molecule.", "Agents which are useful in this regard include but are not limited to agents which bind specifically to the mutant SH3BP2 nucleic acid molecule and thereby prevent replication, transcription and/or translation thereof, agents which bind specifically to the mutant SH3BP2 polypeptide and thereby interfere or, in other instances, inhibit the association of the polypeptide with other polypeptides or nucleic acid molecules in the cell.", "The prophylactic treatment methods of the invention optionally further comprise the selection of a subject at risk of developing a disorder prior to the administration of the agent.", "Such a subject may be identified using the diagnostic methods provided herein.", "A subject at risk may be one who exhibits symptoms of a bone homeostasis disorder, and/or one who expresses a mutant SH3BP2 nucleic acid molecule or a polypeptide.", "Other subjects at risk of developing such a disorder may be those with a family history of such disorders.", "Several such families are discussed in the Examples.", "Subjects with a family history of a bone homeostasis disorder, such as cherubism, may be considered subjects for prophylactic treatment.", "The invention further provides a method for treating a subject who has already developed a bone homeostasis disorder which, preferably, is characterized by the presence of a mutant SH3BP2.The method involves administering a mutant SH3BP2 binding agent (e.g., a mutant SH3BP2 antisense molecule) to the subject in need of such treatment in an amount effective to treat the subject.", "As used herein, an amount effective to treat the subject is that amount effective to cause a medically desirable affect.", "For example, an effective amount may be that amount necessary to prevent or halt the progression of the disorder.", "In the preferred therapeutic methods, a mutant SH3BP2 antisense molecule is administered to the subject to treat a bone homeostasis disorder.", "In an exemplary embodiment, DNA is introduced into cells producing mutant SH3BP2 polypeptide, the DNA being configured to produce antisense RNA that is complementary to mRNA that encodes mutant SH3BP2 polypeptide.", "In this latter example, the antisense mRNA hybridizes with the sense mRNA transcribed from the mutant SH3BP2 genomic locus thereby inhibiting synthesis of mutant SH3BP2 polypeptide.", "Methods of producing antisense mRNA and use thereof for inhibition of polypeptide production are well-known in the art.", "Expression vectors can be constructed to produce high levels of antisense RNA in transfected cells.", "This approach has led to reduced expression of oncogenes in exemplary instances whereby antisense oncogene constructs have reverted the growth properties of tumor cells to near normal, slowed their growth or induced apoptosis.", "See Watson et al., Recombinant DNA, 2d ed., 1992.For example, Philadelphia human chronic myelogenous leukemia (CML) cells that contain the BCR/ABL chromosomal translocation have been eradicated using antisense molecules targeted to this oncogene in clinical, pre-clinical, and laboratory settings.", "J. Nat'l.", "Cancer Inst.", "Vol.", "89, No.", "2, Jan. 15, 1997.A similar approach is provided herein directed to the treatment of disorders associated with the mutant SH3BP2 point mutations.", "For example, tumor cells harboring the mutant SH3BP2 genomic locus are treated in vivo or ex vivo with antisense molecules directed at the oncogene mRNA to induce an inhibition of cell responsiveness to tumor inducing factors, or an inhibition of factor-independent cell growth.", "The pharmaceutical preparations, as described above, are administered in effective amounts.", "The effective amount will depend upon the mode of administration, the particular condition being treated and the desired outcome.", "It will also depend upon, as discussed above, the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well known to the medical practitioner.", "For therapeutic applications, it is that amount sufficient to achieve a medically desirable result.", "In some cases this is a decrease in cell proliferation, a decrease in the size of a tumor, or an inhibition of tumor growth.", "The invention further provides a medicament and a method of making a medicament.", "The medicament comprises an agent and a pharmaceutically acceptable carrier.", "The method involves placing an agent in a pharmaceutically acceptable carrier.", "The agent may be but is not limited to a mutant SH3BP2 nucleic acid molecule or a fragment thereof, a mutant SH3BP2 antisense nucleic acid molecule, a mutant SH3BP2 polypeptide or fragment thereof, and a mutant SH3BP2 binding agent, as described herein.", "In a further embodiment, the invention provides a medicament and a method of making the same which includes a mutant SH3BP2 binding agent formulated for use in the treatment of a disorder characterized by expression of a mutant SH3BP2 nucleic acid molecule or a polypeptide.", "In one embodiment, the medicament is formulated in a dose and/or a delivery formulation particularly tailored to the treatment of cherubism and/or delivery to a periodontal pocket.", "Generally, doses of active compounds of the present invention would be from about 0.01 mg/kg per day to 1000 mg/kg per day.", "It is expected that doses ranging from 50-500 mg/kg will be suitable.", "A variety of administration routes are available.", "The methods of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.", "Such modes of administration include oral, rectal, topical, nasal, interdermal, or parenteral routes.", "In some embodiments of the invention, the mode of administration is direct injection into the tissue surrounding a bone suspected of abnormal homeostasis.", "The term “parenteral” includes subcutaneous, intravenous, intramuscular, or infusion.", "Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis.", "They could, however, be preferred in emergency situations.", "Oral administration will be preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule.", "When peptides are used therapeutically, in certain embodiments a desirable route of administration is by pulmonary aerosol.", "Techniques for preparing aerosol delivery systems containing peptides are well known to those of skill in the art.", "Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporated by reference).", "Those of skill in the art can readily determine the various parameters and conditions for producing antibody or peptide aerosols without resorting to undue experimentation.", "Compositions suitable for oral administration may be presented as discrete units, such as collagen matrices, floss, slow release capsules, tablets, lozenges, each containing a predetermined amount of the active agent.", "See, e.g., U.S. Pat.", "Nos.", "5,750,651; 5,171,574; 5,645,591; 6,120,789; 5,023,082; and 6,174,934 for exemplary controlled release matrices that are useful in accordance with practicing the methods of the invention.", "Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.", "Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.", "Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.", "Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.", "Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.", "Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.", "Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.", "Lower doses will result from other forms of administration, such as intravenous administration.", "In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.", "Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.", "The mutant SH3BP2 nucleic acid molecules, polypeptides or fragments thereof, as well as binding agents (including antisense molecules) may be combined, optionally, with a pharmaceutically-acceptable carrier.", "The term “pharmaceutically-acceptable carrier” as used herein means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human.", "The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.", "The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.", "When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptably compositions.", "Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.", "When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.", "Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.", "Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.", "Various techniques may be employed for introducing nucleic acid molecules of the invention into cells, depending on whether the nucleic acid molecules are introduced in vitro or in vivo in a host.", "Such techniques include transfection of nucleic acid-CaPO4 precipitates, transfection of nucleic acid molecules associated with DEAE, transfection with a retrovirus including the nucleic acid of interest, liposome mediated transfection, and the like.", "For certain uses, it is preferred to target the nucleic acid to particular cells.", "In such instances, a vehicle used for delivering a nucleic acid of the invention into a cell (e.g., a retrovirus, or other virus; a liposome) can have a targeting molecule attached thereto.", "For example, a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid delivery vehicle.", "For example, where liposomes are employed to deliver the nucleic acid molecules of the invention, proteins which bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake.", "Such proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like.", "Polymeric delivery systems also have been used successfully to deliver nucleic acid molecules into cells, as is known by those skilled in the art.", "Such systems even permit oral delivery of nucleic acid molecules.", "Other delivery system's can include time-release, delayed release or sustained release delivery systems.", "Such systems can avoid repeated administrations of the agents of the invention, increasing convenience to the subject and the physician.", "Many types of release delivery systems are available and known to those of ordinary skill in the art.", "They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides.", "Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat.", "No.", "5,075,109.Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.", "Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Pat.", "Nos.", "4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat.", "Nos.", "3,854,480, 5,133,974 and 5,407,686.In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.", "Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions.", "Long-term release, are used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.", "Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.", "The molecular characterization of mutant SH3BP2 nucleic acid molecules and the polypeptides encoded thereby also permits the production of therapeutic agents which selectively locate and/or destroy cells containing the mutant SH3BP2 nucleic acid molecule, or its corresponding polypeptide.", "For example, radiolabeled antibodies or fragments of antibodies which are formulated to penetrate the cell target and bind to the mutant SH3BP2 nucleic acid molecule, or corresponding polypeptide can be contacted with cells suspected of containing a mutant SH3BP2 nucleic acid molecule or polypeptide.", "Since the radiolabeled antibodies or antibody fragments collect in the area of cells having the nucleic acid molecule, or corresponding polypeptide, such cells may be detected and localized by observing the locus of radioactivity generated by the antibodies or fragments of antibodies.", "In one important embodiment, the cells are contacted with the radiolabeled antibodies or fragments thereof in vivo and correspondingly, the term “contacting” in these embodiments also encompasses “injecting into a subject”.", "Thus, in one embodiment, radiolabeled antibodies or fragments of antibodies which bind to the mutant SH3BP2 nucleic acid molecule, or corresponding polypeptide can be injected into a subject known to have or suspected of having a cell or a tumor containing mutant SH3BP2 nucleic acid molecules or corresponding polypeptides.", "Here, such cells or tumors may be detected and localized within a subject by observing the locus of radioactivity generated by the antibodies or fragments of antibodies.", "Methods of tumor localization using radiolabeled antibodies or fragments of antibodies (radioimmunodetection) are well-known in the art.", "See, e.g., U.S. Pat.", "No.", "4,348,376 incorporated herein by reference.", "In a method similar to that described herein, other binding agents can be used for locating or visualizing cells or tumors containing mutant SH3BP2 nucleic acid molecules or corresponding polypeptides.", "Also, detection labels other than radioactive labels may be conjugated to a binding agent and used to detect and localize cells or tumors containing mutant SH3BP2 nucleic acid molecules or corresponding polypeptides.", "The advantage of radiolabeled binding agents is the ability to deliver radioactivity to target cells and tissues, thereby providing toxic doses of radioactivity to such cell and tissues.", "Preferably, the radiolabeled binding agents are those which specifically bind to the mutant domain nucleic acid molecule or polypeptide but not the nucleic acid molecule or polypeptide of the wild type SH3BP2 molecule.", "Cells containing a mutant SH3BP2 nucleic acid molecule or polypeptide encoded thereby may be selectively destroyed by conjugating toxins to binding agents such as antibodies or fragments of antibodies which are capable of penetrating a cell target and binding to the nucleic acid molecule or polypeptide contained therein.", "Thus, as an example, by injecting a toxin/antibody or toxin/antibody fragment conjugate into a subject having mutant SH3BP2 nucleic acid molecule or polypeptide encoded thereby, wherein the antibody or antibody fragment is directed to a mutant SH3BP2 nucleic acid molecule or polypeptide, cells containing the nucleic acid molecule or polypeptide are preferentially destroyed by the toxin.", "Preferably, the binding agent in this latter embodiment is one which binds specifically to the mutant SH3BP2 nucleic acid molecule or polypeptide and not to the wild type molecules so as to reduce or prevent non-specific toxicity.", "In this manner, surgical resection of tumors may be avoided.", "Use of toxin conjugated antibodies or toxin conjugated antibody fragments is well-known in the art.", "See, e.g., U.S. Pat.", "No.", "4,671,958, incorporated herein by reference.", "Examples of suitable toxins include those derived from diphtheria toxin, ricin and the like.", "The following examples are included for purposes of illustration and are not intended to limit the scope of the invention.", "EXAMPLES Example 1 Cherubism Cherubism is an inherited syndrome characterized by excessive bone degradation of the upper and lower jaws.", "At around 3 years of age in affected individuals, multilocular cysts appear in mandibula and maxilla.", "The cysts fill with tumor-like tissue consisting of fibroblasts and osteoblast-like cells, causing a typical facial swelling (FIG.", "1).", "The swelling regresses after puberty, but the osseous defects can be radiologically detected even in old age.", "We have previously mapped cherubism to chromosome 4p16.32.Combining these published data with independent mapping results of Mangion et al.3 and haplotype analyses of 11 additional families, we have defined the cherubism locus to a 1.5 Mb interval.", "About 26 known or predicted genes are contained in this locus between D4S127 and D4S115, allowing systematic analyses of these genes as candidates for cherubism.", "By sequencing cDNA and/or genomic DNA from affected and unaffected members of several pedigrees, we have excluded 15 previously described genes4-6 as candidates, including GRK4, α-Adducin, WHSC2, RNF-4, WHSC1 LETM1, FGFR3 and TACC3.Finally, in 12 out of 15 families, we have detected point mutations that cause amino acid substitutions in the SH3-binding protein SH3BP27,8 (FIG.", "3).", "We have collected samples from 15 families, consisting of 66 individuals clinically diagnosed with cherubism, 4 obligate carries, and 79 unaffected subjects.", "Six of these families are of African-Brazilian, the others of Caucasian background.", "In 12 families, where mutations were found, the mutations co-segregate with the disease phenotype.", "We also cloned SH3BP2 cDNA from EBV-transformed patient lymphoblasts into TA-vectors and sequenced them.", "Half of the sequenced clones from each patient carried the mutation.", "We sequenced genomic DNAs from 100 unrelated and unaffected African-Brazilians and 100 Caucasian controls without detecting any of the sequence variants found in affected cherubism patients.", "The accumulation of co-segregating sequence variants in families with cherubism and their absence in unaffected controls provide compelling evidence that the mutations in SH3BP2 cause cherubism.", "Three families where no mutation in SH3BP2 could be detected were small, and linkage to the cherubism locus was inconclusive.", "Therefore, it is likely that cherubism in these families is caused by mutations in a gene other than SH3BP2.SH3BP2 contains three modular peptide recognition domains, an N-terminal pleckstrin homology domain, a 10 amino acid residue-long SH3 binding site, and a C-terminal SH2 domain7,8.All mutations identified to date are in exon 9 and affect three amino acid residues within a 6-residue long sequence, RSPPDG, SEQ ID NO: 104, located 31 to 36 residues upstream of the SH2 domain (FIG.", "2).", "As can be seen in Table 2, mutations in Pro418 (to Leu, Arg, or His) are the most common, seen in families A, B, C, F, J, L, M, and O.", "Other mutations result in Gly420 being replaced by Glu or Arg (in families G and N) and Arg415 being replaced by Pro or Glu (in families H and K).", "SH3BP2 was initially identified by screening a phage expression library for proteins that can bind to the SH3 domain of the proto-oncogene c-Abl, and it was shown to contain a proline-rich 10 amino acid residue-long sequence responsible for SH3 binding7.This SH3 ligand motif (PPAYPPPPVP, SEQ ID NO:103) is located 205 amino acid residues upstream of the mutated region Arg415-Gly420.The N-terminal pleckstrin homology (PH) domain belongs to a class of interaction domains that are found in a number of eukaryotic signaling proteins.", "They can bind to other proteins as well as to inositol phosphates, but the binding partners for the PH domain in SH3BP2 are still unknown.", "The C-terminal SH2 domain is likely to bind with high affinity to tyrosine-phosphorylated peptides9,10.The combination of the three protein binding modules in SH3BP2 and its affinity for c-Abl via its SH37,11 binding domain make it a possible adapter protein that could organize the formation of functional complexes involving c-Abl.", "SH3BP2 is also likely to interact with cytoplasmic signaling molecules playing a role in transcriptional activation in hematopoietic cells through its SH2 domain12.Thus, it could function primarily as a regulator of c-Abl function in some cells, while controlling transcriptional regulation through interaction with other kinases and signaling complexes in other cells.", "Such a potential dual function may explain the cellular phenotype of the fibro-osseous lesions in cherubism.", "Not only are the lesions reminiscent of fibrous dysplasia of bone caused by mutations in GNSA1 resulting in deficient osteoblastic differentiation from mesenchymal percursors13,14, but the accumulation of large numbers of osteoblast-like cells in cherubism suggests an abnormal formation of these monocyte-derived, multinucleated cells as well.", "The clustering of amino acid missense mutations in SH3BP2 suggest that they represent gain of function mutations.", "SH3BP2 lies within a region that is frequently deleted in Wolf-Hirschhorn Syndrome (WHS)15,16 patients.", "Haploinsufficiency of SH3BP2 in these patients does not result in cherubism or cherubism-like characteristics, which supports our hypothesis that the mutations in SH3BP2 lead to a gain of function.", "We speculate that they may enhance the ability of the protein to engage in complexes that are important for differentiation and activation of osteoblasts while they may affect in a negative manner the function of c-Abl in differentiating osteoblasts.", "Interestingly, osteoblastic differentiation is impaired in c-Abl null mice, resulting in osteoporosis17.The ability-to shuttle between focal adhesions and the nucleus makes c-Abl a good candidate for being a mediator of integrin-regulated gene expression18 and cell cycle progressions19.It is conceivable that at age 3 (frequently the age of onset of the disease) there are signals transmitted through the extracellular matrix of the mandible and maxilla that are unique to these bones and are triggered by the eruption of secondary teeth.", "Thus, the onset of the abnormalities of cherubism and their organ-restricted characteristics may be related to the dental developmental process.", "Example 1 Methods Cherubism Families.", "Probands were identified by clinical collaborators and detailed family histories were taken.", "The diagnosis of affected and unaffected subjects was radiographically confirmed when necessary and consent from all participating family members had been obtained.", "The study was approved by the Institutional Review Boards of the sponsoring institutions.", "Genetic analysis.", "Genomic DNA was prepared as previously described2.Information about published microsatellite markers for fine-mapping studies was obtained from the Genome Database (gdb).", "Additional markers were developed from Genbank sequences that were known to be located within the disease gene interval.", "Information about known genes within the locus were taken from publications.", "Searches for potential novel genes were performed with Gene Finder (http://searchlauncher.bcm.tmc.edu:9331/gene-finder/gf:html) and Genscan (http://genome.dkfz-heidelberg.de/cgi-bin/GENSCAN/genscan.cgi).", "Mutation screening.", "Exons of candidate genes were amplified with primers in the flanking intron sequences with genomic DNA of affected individuals from 6 families.", "Intronic sequences were either available in Genbank or were developed by sequencing PAC clone 184023 (BACPAC Resources, Oakland, Calif.) with exon-specific primers.", "PCR products were purified with a Qiagen PCR purification kit and the PAC clone with Qiagen Midiprep kit (Qiagen, Valencia, Calif.).", "PCR-generated templates were sequenced from both ends on an ABI 377 slab gel sequencer with ABI Big Dye chemistry.", "Alternatively, cDNA was reverse transcribed from RNA of EBV-transformed patient lymphoblasts and used for PCR amplification with exonic primers.", "These products were sequenced (manually or ABI 377) and searched for heterozygosity or splicing variants.", "PCR-amplified SH3BP2 cDNA was cloned into PCRII-TOPO TA cloning vectors (Invitrogen, Carlsbad, Calif.) and sequenced (ABI 377).", "Variations in patient sequences and wild type sequences were examined using Blast (NCBI).", "Electropherograms were visually examined for heterozygosities.", "Exhibit 1, References 1.Jones, W. A., Amer.", "J. of Cancer 17 (1933), 946-950.2.Tiziani, V. et al., Amer.", "J. of Human Genetics 65(1999),158-166.3.Mangion, J. et al., Amer.", "J. of Human Genetics 65(1999), 151-157.4.Pribill, I. et al., Somatic Cell & Molecular Genetics 23 (1997), 413-427.5.Hadano, S. et al., DNA Research 5 (1998), 177-186.6.Baxendale, S. et al., Nature Genetics 4 (1993), 181-186.7.Ren, R. et al., Science 259 (1993); 1157-1161.8.Bell, S. M., et al., Genomics 44 (1997), 163-170.9.Songyang, Z. et al., Mol.", "Cell.", "Biol.", "14 (1994), 2777-2785.10.Pawson, T. et al., Cell 71 (1992), 359-362.11.Sparks, A.", "B. et al., Proc.", "Natl.", "Acad Sci.", "USA 93 (1996), 1540-1544.12.Deckert, M., et al., Immunity 9 (1998), 595-605.13.Weinstein, L S, et al., N. Engl.", "J. Med.", "325 (1991), 1688-1695.14.Bianco P. et al., J.", "Bone Miner Res.", "15 (2000); 120-128.15.Hirschhorn, K. et al., Humangenetik 1 (1965), 479-482.16.Zollino, M., et al., Am.", "J. Med.", "Genet.", "18 (2000), 254-261.17.Li, B. et al., Nat.", "Genet.", "24 (2000), 304-308.18.Lewis, J. M. et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 93 (1996), 15174-15179.19.Welch, P J, et al., Cell 75 (1993), 779-790.Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.", "Such equivalents are intended to be encompassed by the following claims.", "All references disclosed herein are incorporated by reference in their entirety." ] ]	Patent_10467033
[ [ "Network selection in a mobile telecommunications system", "A mobile telephone (1) in a cellular telecommunications system uses data stored in a SIM cards (10) select a network (4) for registration and to perform optimise call routing to a call destination.", "If data relevant to networks which are currently available is not stored, a request message is sent to a control centre (14) and updating information received in a response message.", "The subscriber identity data stored in a memory file (200) of the SIM can also be replaced to enable a preferred subscriber identifier to be used with the currently registered network, selection being made using a look-up table (201) updated when required using request and response messages.", "Greater control of the network selection and cost saving by avoiding roaming agreements are thereby achievable." ], [ "1.A method of operating a mobile telecommunications apparatus in a telecommunications system wherein the apparatus comprises means for making telephone calls via the system, the method comprising: detecting a number of available networks; receiving network identification information from the available networks; selecting one of the available networks by comparing the network identification information with stored network information comprising at least one of a preferred network table and a barred network table; registering with the selected network; determining whether the stared information requires updating, and outputting a request message for receiving updating information for updating the stored network information if the stored information is determined to require updating.", "2.A method as claimed in claim 1, wherein the determining step comprises determining whether the network identification information indicates that the mobile telecommunications apparatus has moved to a country which is different from a previous country in which the apparatus was previously registered.", "3.A method as claimed in claim 1, wherein the determining step determines that the stored network information needs updating if the mobile telecommunications apparatus has registered with a network which is different from a network which the apparatus was last registered.", "4.A method as claimed in claim 1 including the step of receiving a response message comprising the requested updating information for updating the stored routing information.", "5.A method as claimed in claim 4 including the step of decrypting the received response message.", "6.A method as claimed in claim 4 including the step of updating the stored network information with the updating information.", "7.A method as claimed in claim 6, wherein the updating information is stored in a first portion of memory to constitute the updated stored network information, and including the further step of storing a duplicate of the updating information in a second portion of the memory to constitute a backup memory.", "8.A method as claimed in claim 7, wherein, when accessing the stored network information, the contents of the first and second portions of memory are compared and, if different, the contents of the backup memory in the second portion of the memory are copied to the first portion of memory to constitute the updated stored network information.", "9.A method as claimed in claim 7, wherein the stored network information is updated by an updating application which causes both first and second portions of memory to be updated and wherein other applications are prohibited from updating the second portion of memory.", "10.A method as claimed in claim 1, wherein updating the stored information comprises storing in a cache memory of the apparatus the information which is superseded by updating information.", "11.A mobile telecommunications apparatus for use in a telecommunications system, comprising: detecting means for detecting a number of available networks; receiving means for receiving network identification information from the available networks; selecting means for selecting one of the available networks by comparing the network identification information with stored network information comprising at least one of a preferred network table and a barred network table; and registering means for registering with the selected network; wherein the apparatus further comprises: determining means for determining whether the stored information requires updating, and output means for outputting a request message for receiving updating information for updating the stored information if the stored information is determined to require updating.", "12.Apparatus as claimed in claim 11, wherein the determining means is operable to determine whether the network identification information indicates that the mobile telecommunications apparatus has moved to a country which is different from a previous country in which the apparatus was previously registered.", "13.Apparatus as claimed in claim 11, wherein the determining means is operable to determine that the stored network information needs updating if the apparatus has registered with a network which is different from a network with which the apparatus was last registered.", "14.Apparatus as claimed in claim 11 including message receiving means for receiving a response message comprising the requested updating information for updating the stored information.", "15.Apparatus as claimed in claim 14 including decrypting means for decrypting the received response message.", "16.Apparatus as claimed in claim 14 including updating means for updating the stored routing information with the updating information.", "17.Apparatus as claimed in claims 16, wherein the updating means is operable to store the updating information in a first portion of the memory, and is further operable to store a duplicate of the updating information in a second portion of the memory to constitute a backup memory.", "18.Apparatus as claimed in claim 17, wherein the selecting means comprises accessing means for accessing the stored network information in the first portion of memory and means for comparing the contents of the first and second portions of memory and means for copying the contents of the backup memory in the second portion of the memory to the first portion of memory if the contents are different.", "19.Apparatus as claimed in claim 17, wherein the stored information is updated by an application which causes both first and second portions of memory to be updated and wherein an operating system prohibits other applications from updating the second portion of memory.", "20.Apparatus as claimed in claim 11 comprising a cache memory (146) for storing the stored network information which is superseded by updating information.", "21.A method of operating a mobile telecommunications apparatus in a telecommunications system wherein the apparatus comprises means for making outgoing telephone calls via the system, means for receiving a user generated input defining a call destination and routing means for selecting a preferred route to the call destination via the system for an outgoing telephone call by referring to routing information stored in the apparatus, the method comprising: registering with a network of the system which is local to the current location of the apparatus; receiving network identification information from the local network; determining from the network identification information whether the stored routing information requires updating, and outputting a request message for receiving updating information for updating the stored routing information if the stored routing information is determined to require updating.", "22.A method as claimed in claim 21, wherein the determining step comprises determining whether the stored routing information can be utilised in selecting a preferred route via the local network identified by the network identification information.", "23.A method as claimed in claim 21, wherein the determining step comprises determining whether the network identification information indicates that the mobile telecommunications apparatus has moved to a country which is different from a previous country in which the apparatus was previously registered.", "24.A method as claimed in claim 21, wherein the determining step comprises determining whether stored routing information relating to routing outgoing calls via the local network is valid by referring to expiry time information indicating a time at which the validity of the routing information expires.", "25.A method as claimed in claim 24, wherein the expiry information is stored in the apparatus in the form of a time at which the validity of the routing information expires and wherein the method comprises determining the current time with reference to a clock and comparing the time with the expiry information.", "26.A method as claimed in claim 24, wherein the determining step comprises determining whether a predetermined number of calls has been made by the apparatus since the last update of the routing information.", "27.A method as claimed in claim 24, wherein the determining step comprises counting the number of intervals of predetermined length for which the apparatus remains in operation and determining the validity of the routing information to have expired when a predetermined number of the intervals has been counted.", "28.A method as claimed in claim 24, wherein the determining step comprises outputting a request message to obtain the expiry information, receiving a response message including the expiry information and determining from the expiry information whether the validity of the routing information has expired.", "29.A method as claimed in claim 21 wherein the determining step comprises determining whether the network identification information indicates that the mobile telecommunications apparatus has registered with a network which is different from a network with which the apparatus was last registered.", "30.A method as claimed in claim 21 including the step of receiving a response message comprising the requested updating information for updating the stored routing information.", "31.A method as claimed in claim 30 including the step of decrypting the received response message.", "32.A method as claimed in claim 30 including the step of updating the stored routing information with the updating information.", "33.A method as claimed in claim 32, wherein the stored routing information comprises a routing table and comprising the step of updating the routing table.", "34.A method as claimed in claim 33, wherein the routing table is updated so as to include a field determining the time of expiry of the validity of the updated routing table.", "35.A method as claimed in claim 30 including the step of updating a preferred network table which defines a list of preferred networks in order of preference for use in the registering step when selecting a network from a plurality of available networks for registration.", "36.A method as claimed in claim 30 comprising updating a barred network table comprising a list of networks in respect of which the mobile telecommunications apparatus is barred from registration for use in the registering step when selecting a network from a plurality of available networks for registration.", "37.A method as claimed in claim 30, wherein the updating step comprises updating data stored in a SIM card of the mobile telecommunications apparatus.", "38.A method as claimed in claim 35 comprising the step of obtaining network identification information for a plurality of networks including the local network which are available for registration, comparing the network identification information for the available networks with the updated preferred network table, selecting a preferred network from the available networks and, if the selected network is not the local network, re-registering with the selected network.", "39.A method as claimed in claim 36 including the step of comparing the network identification information for the local network with the updated barred network table, and, if the local network is one of the barred networks, de-registering from the local network.", "40.A method as claimed in claim 39 including the step of registering with a further one of the available networks.", "41.A method as claimed in claim 1, wherein the telecommunications system is a GSM system.", "42.A method as claimed in claim 41, wherein the request message is output using the SMS protocol.", "43.A method as claimed in claim 41, wherein the request message is output using a USSD protocol.", "44.A method as claimed in claim 41, wherein the request message is output using a UMTS protocol.", "45.A method as claimed in claim 41, wherein a request message is output using a TCP/IP protocol.", "46.A method as claimed in claim 41, wherein the request message is output using a WAP protocol.", "47.A method as claimed in claim 1, wherein the apparatus comprises a mobile telephone.", "48.A method as claimed in claim 1, wherein the apparatus comprises a SIM card having a processor and wherein the determining step is performed by the processor of the SIM card.", "49.A method as claimed in claim 1 including the step of generating the request message to include call log information comprising accumulated data relating to use of the apparatus.", "50.A method as claimed in claim 21, wherein the updating information is stored in a first portion of memory, and including the further step of storing a duplicate of the information in a second portion of the memory to constitute a backup memory.", "51.A method as claimed in claim 50, wherein, when accessing the stored information, the contents of the first and second portions of memory are compared and, if different, the contents of the backup memory in the second portion of the memory are copied to the first portion of memory.", "52.A method as claimed in claim 50, wherein the stored information is updated by an application which causes both first and second portions of memory to be updated and wherein other applications are prohibited from updating the second portion of memory.", "53.A method as claimed in claim 22, wherein, if it is determined that the stored routing information cannot be utilised in selecting a preferred route via the local network, the apparatus is disabled from making outgoing telephone calls.", "54.A method as claimed in claim 21 for use with a prepaid subscriber service in which the subscriber makes prepayment to acquire credit for making calls, wherein the stored routing information provides routing of calls via a prepayment platform operable to allow connection if credit remains and provides for call disconnection if credit expires.", "55.A method as claimed in claim 54 including the step of outputting a validation request message to the prepaid platform and receiving a verification response therefrom before initiating the making of an outgoing call.", "56.A method as claimed in claim 21, wherein updating the stored information comprises storing in a cache memory the information which is superseded by updating information.", "57.A mobile telecommunications apparatus for use in a telecommunications system, comprising: registering means for registering with a network of the system which is a local network with respect to the current location of the apparatus; receiving means for receiving network identification information from the local network; storing means for storing routing information; routing means for determining a preferred route via the system for an outgoing call made by the apparatus based on a user input call number, the network identification information and the stored routing information; wherein the apparatus further comprises: determining means for determining whether the stored routing information requires updating, and output means for outputting a request message for receiving updating information for updating the stored routing information if the stored routing information is determined to require updating.", "58.Apparatus as claimed in claim 57, wherein the determining means is operable to determine whether the stored routing information can be utilised in selecting a preferred route via the local network identified by the network identification information.", "59.Apparatus as claimed in claim 57, wherein the determining means is operable to determine whether the network identification information indicates that the mobile telecommunications apparatus has moved to a country which is different from a previous country in which the apparatus was previously registered.", "60.Apparatus as claimed in claim 57, wherein the determining means is operable to determine whether stored routing information relating to routing outgoing calls via the local network is valid by referring to expiry time information indicating a time at which the validity of the routing information expires.", "61.Apparatus as claimed in claim 60, wherein the expiry information is stored in the apparatus in the form of a time at which the validity of the routing information expires and wherein the determining means is operable to determine the current time with reference to a clock and comparing the time with the expiry information.", "62.Apparatus as claimed in claim 60, wherein the determining means is operable to determine whether a predetermined number of calls has been made by the apparatus since the last update of the routing information.", "63.Apparatus as claimed in claim 60, wherein the determining means is operable to count the number of intervals of predetermined length for which the apparatus remains in operation and to determine the validity of the routing information to have expired when a predetermined number of the intervals has been counted.", "64.Apparatus as claimed in claim 60, wherein the determining means comprises means for outputting a request message to obtain the expiry information, means for receiving a response message including the expiry information, and wherein the determining means is operable to determine from the expiry information whether the validity of the routing information has expired.", "65.Apparatus as claimed in claim 57, wherein the determining means is operable to determine whether the network identification information indicates that the mobile telecommunications apparatus has registered with a network which is different from a network with which the apparatus was last registered.", "66.Apparatus as claimed in claim 57 including receiving means for receiving a response message comprising the requested updating information for updating the stored routing information.", "67.Apparatus as claimed in claim 66 including decrypting means for decrypting the received response message.", "68.Apparatus as claimed in claim 66 including updating means for updating the stored routing information with the updating information.", "69.Apparatus as claimed in claim 68, wherein the stored routing information comprises a routing table and wherein the updating means is operable to update the routing table.", "70.Apparatus as claimed in claim 69, wherein the updating means is operable to update the routing table so as to include a field determining the time of expiry of the validity of the updated routing table.", "71.Apparatus as claimed in claim 66 comprising a preferred network table which defines a list of preferred networks in order of preference for use in the registering step when selecting a network from a plurality of available networks for registration, and wherein the updating means is operable to update the preferred network table.", "72.Apparatus as claimed in claim 66 comprising a barred network table comprising a list of networks in respect of which the mobile telecommunications apparatus is barred from registration for use in the registering step when selecting a network from a plurality of available networks for registration, and wherein the updating means is operable to update the barred network table.", "73.Apparatus as claimed in claim 66 comprising a SIM card storing data which is updated by the updating means.", "74.Apparatus as claimed in claim 71, wherein the receiving means is operable to obtain network identification information for a plurality of networks including the local network which are available for registration, the apparatus further comprising means for comparing the network identification information for the available networks with the updated preferred network table, selecting a preferred network from the available networks and, if the selected network is not the local network, re-registering with the selected network.", "75.Apparatus as claimed in claim 72 including means for comparing the network identification information for the local network with the updated barred network table, and, if the local network is one of the barred networks, de-registering from the local network.", "76.Apparatus as claimed in claim 75 including means for registering with a further one of the available networks after de-registering from the local network.", "77.Apparatus as claimed in claim 11, wherein the telecommunications system is a GSM system.", "78.Apparatus as claimed in claim 77, wherein the output means is operable to output the request message using the SMS protocol.", "79.Apparatus as claimed in claim 77, wherein the output means is operable to output the request message using a USSR protocol.", "80.Apparatus as claimed in claim 77, wherein the output means is operable to output the request message using a UMTS protocol.", "81.Apparatus as claimed in claim 77, wherein the output means is operable to output the request message using a TCP/IP protocol.", "82.Apparatus as claimed in claim 41, wherein the output means is operable to output the request message using a WAP protocol.", "83.Apparatus as claimed in claim 11, wherein the apparatus comprises a mobile telephone.", "84.Apparatus as claimed in claim 11, wherein the apparatus comprises a SIM card having a processor and wherein the determining means is constituted by the processor of the SIM card.", "85.Apparatus as claimed in claim 11, wherein the output means generates the request message to include call log information comprising accumulated data relating to use of the apparatus.", "86.Apparatus as claimed in claim 76, wherein the updating means is operable to store the updating information in a first portion of memory, and to store a duplicate of the information in a second portion of the memory to constitute a backup memory.", "87.Apparatus as claimed in claim 86 comprising means for comparing the contents of the first and second portions of memory when and, if different, copying the contents of the backup memory to the first portion of memory.", "88.Apparatus as claimed in claim 86, wherein the updating means is operable to update the stored information using an application which causes both first and second portions of memory to be updated and to prohibit other applications from updating the second portion of memory.", "89.Apparatus as claimed in claim 58 comprising disabling means operable if it is determined that the stored routing information cannot be utilised in selecting a preferred route via the local network, to disable the apparatus from making outgoing telephone calls.", "90.Apparatus as claimed in claim 57 for use with a prepaid subscriber service in which the subscriber makes prepayment to acquire credit for making calls, wherein the stored routing information provides routing of calls via a prepayment platform operable to allow connection if credit remains and provides for call disconnection if credit expires.", "91.Apparatus as claimed in claim 90 comprising means for outputting a validation request message to the prepaid platform and means for initiating the making of an outgoing call which is conditional upon receiving a verification response therefrom.", "92.Apparatus as claimed in claim 57, wherein the updating means comprises a cache memory for storing the information which is superseded by updating information.", "93.A method of operating a mobile telecommunications apparatus in a telecommunications system wherein the apparatus comprises means for making telephone calls via the system and subscriber identity data stored in a first memory of the apparatus is used to associate telephone calls from the apparatus with a respective subscriber account, a set of subscriber identifiers being associated with the same account for use as the subscriber identity data, the method comprising: obtaining a network identifier from a network with which the apparatus is registered; determining which one of the set of subscriber identifiers is preferred for use as the subscriber identity data when the apparatus is registered to the network identified by the network identifier; and, if different from the subscriber identity data; replacing the subscriber identity data stored in the first memory with the preferred subscriber identifier.", "94.A method as claimed in claim 931 wherein the step of replacing the subscriber identity data comprises obtaining the preferred subscriber identifier by sending a request message via the network and receiving a response message containing the preferred subscriber identifier.", "95.A method as claimed in claim 93 further comprising storing in a second memory a look-up table of subscriber identifiers corresponding to respective network identifiers and wherein the determining step comprises accessing the look-up table.", "96.A method as claimed in claim 95 comprising the step of determining whether an entry exists in the look-up table for the network identifier, determining whether the entry remains valid and, if not, sending a request message via the network and receiving a response message containing updating information for populating the table and including an entry for the network identifier.", "97.A method as claimed in claim 93 comprising de-registering from the network when it is determined that the subscriber identity data does not correspond to the preferred subscriber identifier for the network identifier and subsequently re-registering after the subscriber identity data stored in the first memory has been replaced by the preferred subscriber identifier.", "98.A method as claimed in claim 93, wherein the system comprises a GSM system and wherein the subscriber identity data is an IMSI.", "99.A method as claimed in claim 93, wherein the first memory comprises a data file in a SIM card of the apparatus.", "100.A mobile telecommunications apparatus for use in a telecommunications system wherein the apparatus comprises means for making telephone calls via the system and subscriber identity data is stored in a first memory of the apparatus for associating telephone calls from the apparatus with a respective subscriber account, a set of subscriber identifiers being associated with the same account for use as the subscriber identity data, the apparatus comprising: registering means for obtaining a network identifier from a network with which the apparatus is registered; means for determining which one of the set of subscriber identifiers is preferred for use as the subscriber identity data when the apparatus is registered to the network identified by the network identifier; and replacing means operable, if different from the subscriber identity data, to replace the subscriber identity data stored in the first memory with the preferred subscriber identifier.", "101.Apparatus as claimed in claim 100, wherein the replacing means comprises sending means for sending a request message via the network and receiving means for receiving a response message containing the preferred subscriber identifier.", "102.Apparatus as claimed in claim 100 further comprising a second memory storing a look-up table of subscriber identifiers corresponding to respective network identifiers and wherein the determining means is operable to access the look-up table.", "103.Apparatus as claimed in claim 102 comprising means for determining whether an entry exists in the look-up table for the network identifier, means for determining whether the entry remains valid.", "104.Apparatus as claimed in claim 100 comprising registering means operable to de-register from the network when it is determined that the subscriber identity data does not correspond to the preferred subscriber identifier and further operable to subsequently re-register after the subscriber identity data stored in the first memory has been replaced by the preferred subscriber identifier.", "105.Apparatus as claimed in claim 100, wherein the system comprises a GSM system and wherein the subscriber identifier is an IMSI.", "106.Apparatus as claimed in claim 100, wherein the first memory comprises a data file in a SIM card of the apparatus.", "107.A telecommunications system for use in a method as claimed in claim 1, the system comprising a plurality of networks for providing communication with mobile telecommunications apparatus; and a control center for receiving request messages via one of said networks from mobile telecommunication apparatus registered with said network and for generating response messages containing updated data for storage in said apparatus.", "108.A control center for use in a method as claimed in claim 1 comprising: message receiving means for receiving request messages from mobile telecommunications apparatus; a response generator for generating a response message; a transmitter for transmitting the response message; and a database containing data for inclusion in the response message and comprising updating data for updating at least one of: preferred network data; forbidden network data; routing data; and preferred subscriber identifiers.", "109.A storage medium storing processor implementable instructions for instructing a processor to control a mobile telecommunications apparatus to perform all of the steps of claim 1.110.A signal comprising processor implementable instructions for instructing a processor to control a mobile telecommunications apparatus to perform all of the steps of claim 1.111.A SIM card comprising a processor and a memory 20 storing processor implementable instructions for operating the processor to control an apparatus to perform the steps of a method as claimed in claim 1." ], [ "This invention relates the operation of mobile stations in cellular telecommunications systems and in particular, but not exclusively, to the updating of information stored in memory within mobile telephones for the purpose of least cost routing.", "It is known from WO99/04578 and WO00/41486 to provide least cost routing of telephone calls made from mobile stations via a cellular network such that, when a user enters a destination call number, an application of the mobile station is called to refer to a lookup table to retrieve stored data which enables the mobile station to perform least cost routing.", "The stored data is updated by broadcasting of messages from a control centre when appropriate, for example to reflect changes in call charges made by different networks either in handling the cellular telephone link or subsequent landline telephone or internet connection to the call destination.", "The stored information within the mobile station, typically stored within a smart card referred to as a Subscriber Information Module (SIM) card, is generally relevant only to the territory in which normal usage of the mobile station is anticipated, as for example where the data covers the territory defined by the country in which the subscriber primarily uses the mobile station.", "A problem arises when the user moves to a different territory.", "Generally, agreements exist between operators in different countries which allow “international roaming” whereby the mobile station may register with a “roamed-to network” which will then communicate with the home network of the subscriber to establish authorisation and billing protocols for calls made by the subscriber using the roamed-to network.", "The least cost routing function of the mobile station is under these circumstances typically unusable because of the lack of available relevant data within its memory.", "A default mode of operation may thereby be utilised in which the routing of calls is determined by the roamed-to network and may therefore result in less than optimum call charges being rendered to the subscriber.", "A further problem arises from the usage in mobile stations of a preferred network table and forbidden network table, generally stored in the SIM card, which are referred to by the mobile station at the time of selecting a roamed-to network from a plurality of available networks at the time of registration.", "In the absence of stored data relevant to the currently available networks, the mobile station will typically register with whichever of the available networks presents the greatest signal strength.", "The present invention seeks to provide improved an apparatus and method for operating mobile stations in cooperation with mobile cellular networks.", "According to the present invention, a mobile station determines whether or not data stored in the mobile station needs to be updated.", "In a preferred embodiment, the mobile station registers with a mobile cellular network and makes the determination from network information received from the network.", "The mobile station may generate a request message sent to a control centre to enable the control centre to respond with appropriate data which is then stored in the mobile station for further use.", "Preferred embodiments of the present invention will now be described by way of example only and with reference to the accompanying drawings of which; FIG.", "1 is a schematic diagram illustrating registration of a mobile telephone with a local cellular network; FIG.", "2 is a schematic diagram of the components of the mobile telephone of FIG.", "1; FIG.", "3 is a schematic diagram of the components of the SIM card of the mobile telephone of FIG.", "2; FIG.", "4 is a diagram representing the data stored in the SIM card of the telephone of FIG.", "2; FIG.", "5 is a diagram illustrating the applications stored in the SIM card of FIG.", "2; FIG.", "6 is a flow chart of the network selection for registration; FIG.", "7 is a flow chart of the determination of whether to request updated tables; FIG.", "8 is a schematic diagram illustrating the call routing function; FIG.", "9 is a schematic diagram showing the storage and transmission of programs used in the embodiments; FIG.", "10 illustrates the functional elements of a generalized mobile station; FIG.", "11 illustrates the functional elements of the control centre; FIG.", "12 is a schematic diagram illustrating the monitoring for new networks and re-registration; FIG.", "13 is a schematic diagram illustrating memory management and backup files FIG.", "14 is a flowchart of the re-evaluation process; FIG.", "15 is a schematic diagram of an apparatus in which subscriber information is replaced; FIG.", "17 is a flowchart illustrating the updating of subscriber information; FIG.", "18 is a flowchart further illustrating the updating of subscriber information and expanding on the determining step of FIG.", "17; FIG.", "19 is a diagram illustrating networks using updated subscriber information; and FIG.", "20 illustrates the data structure of subscriber information.", "In the example of FIG.", "1, a mobile telecommunications apparatus is in the form of a mobile telephone 1 configured for use in a GSM (Global System for Mobile Communications) cellular telecommunications system.", "The GSM standard has been adopted by several countries including countries in the European community, agreements having been made to thereby allow the same mobile telephone 1 to operate in different countries.", "As illustrated in FIG.", "1, the mobile telephone 1 is currently located within a cell 2 served by a base station 3 of a local network 4, the term “local” here being used in the context that the local network is accessible via cellular broadcast to the mobile telephone 1 in its current location.", "Here the base station 3 represents the physical telemetric apparatus responsible for wireless communication with the mobile telephone 1 whereas the local network 4 represents a service provider utilising a base station controller apparatus to manage communications with a number of different base stations, including radio interface management and handover from one base station to another for mobility when the mobile telephone moves from one cell to another.", "The local network 4 maintains a database 5 which includes a Visitor Location Register (VLR) 6 containing information on mobile telephones currently registered with the local network, as in the case of mobile telephone 1.A home network 7 is operated by a home service provider with whom a subscriber using the mobile telephone 1 has in this example a contractual relationship.", "The home network 7 maintains a database 8 containing a Home Location Register (HLR) 9 which contains details of the subscriber's account.", "Subscriber information 40 is stored in a SIM card 10 within the mobile telephone 1 to providing a unique identifier allocated by the home service provider.", "The SIM (Subscriber Identity Module) card 10 is a smart card having the facilities of storing data and programs and having a moderate amount of processing power.", "The primary function of the SIM card 10 is to personalise the mobile telephone 1 to the subscriber, the main body of the mobile telephone 1 having its own microcomputer with greater processing power and memory, as described below in greater detail.", "In FIG.", "1, communication between the mobile telephone 1 and a call destination 11 such as a telephone receiver is represented by a generalised network 12 providing connection between the local network 4 and the call destination.", "This generalised representation signifies that any number of different connection paths could be defined by the generalised network 12 including connection via long distance carrier, public service telephone network, the internet, one or more cellular networks, or a combination thereof.", "The connection path utilised for any given call generally presents a number of available options depending for example on the interconnect facilities available to the local network 4.The mobile telephone 1 has a call routing facility which allows routing information to be accessed from the SIM card 10 and added to a user defined call number such that a setup procedure initiated via the local network 4 results in the connection path to the call destination 11 being determined in accordance with a preferred route.", "To achieve this routing function, the mobile telephone 1 has stored in its SIM card 10 a routing table 13 in the form of a look up table which allows routing information to be extracted rapidly and with minimal processing whenever a user initiates a call.", "The routing information is exchanged between the mobile telephone 1 and the local network via the base station 3 during the call setup procedure.", "The routing table 13 is populated with data provided to the mobile telephone 1 from a control centre 14 which maintains a database 15 of routing information obtained by monitoring parameters including call charges and network performance of the different components of the generalised network 12 and calculating therefrom the preferred routes corresponding to each call destination.", "When the mobile telephone 1 is turned on, it needs to register with the local network 4 so that subsequent calls can be rapidly set up by the local network.", "As part of the registration process, there is an exchange of information between the local network 4 and the home network 7 to establish the authenticity of the subscriber's proposed use of the network and to establish the manner in which calls are to be billed.", "To commence the registration process, the mobile telephone 1 will typically need to choose between a number of available networks including the local network 4 and additional networks 16, 17 and 18, any number of which may be detected as being available at the current location.", "For simplicity, FIG.", "1 shows three additional networks 16, 17, 18 sharing the common base station 3 with the local network 4.In selecting a network for registration purposes, the mobile telephone 1 refers to a preferred network table 19 which lists networks in order of preference.", "The mobile telephone 1 also refers to a forbidden network table 20 which lists those networks for which the subscriber does not have authorisation from the home network 7 to complete registration.", "Once registration is complete, the local network 4 includes details of the mobile telephone 1 in the Visitor Locator Register 6.The location of the mobile telephone 1 is therefore available to users of the networks in the GSM system, as for example in the case of routing calls to the mobile telephone 1.The above simplified scenario assumes that the current information stored in the SIM 10 is up to date and includes entries in the routing table 13, preferred network table 19 and forbidden network table 20 which are relevant to the currently available networks such as local network 4 and additional network 16 to 19.The routing table 13 for example comprises a number of sub-tables directed to different aspects of routing and some of these tables are network dependent in that they contain information which is specific to the identity of the network to which the mobile telephone is currently registered.", "Typically, in SIM cards of currently available memory size, the routing table 13 contains information for use with about 5 choices of local network 4.A situation may therefore arise in which the mobile telephone 1 is turned on at, or moves to, a new location at which the information stored in the SIM 10 is entirely inappropriate to the available networks for registration or is in some respect incomplete.", "Limitations on memory size and bandwidth for updating data to the SIM 10 prohibit the simultaneous maintenance of updated data covering all possible available networks throughout the world for the GSM system.", "The mobile telephone 1 of FIG.", "1 is therefore provided with means for determining whether the currently stored information requires updating and is further capable of outputting a request message in order to receive an update of whatever information is determined to be required.", "The components of the embodiment of FIG.", "1 will now be described in greater detail.", "The structure of the mobile telephone 1 is illustrated in schematic generalised form in FIG.", "2 and comprises a processor 21 with Read Only Memory (ROM) 22 and Random Access Memory (RAM) 23.A keypad 24 is provided for the input of user commands including dialled telephone numbers and a display 25 in the form of a liquid crystal display is operable to display dialled numbers and menu information.", "The SIM card 10 is removably inserted within the mobile telephone 1 so as to be electrically connected to a bus 26 which operatively connects the above components.", "A transmitting and receiving circuit 27 is also provided, being connected to an antenna 28, and a microphone and speaker unit 29 is coupled to an audio processor 30.The mobile telephone also includes an internal clock 31.FIG.", "3 illustrates schematically the contents of the SIM card 10 which comprises a processor 31 with a ROM 32 and RAM 33 formed of a flash memory so that the contents of the RAM persist when the SIM card is powered down.", "An interface 34 is also provided for external connection and communication with the bus 26 of the main body of the mobile telephone 1.An internal bus 35 interconnects the above components of the SIM card 10.The processor 21 of the mobile telephone initiates all communication with the SIM card 10, communication taking the form of a command passed from the processor 21 via the bus 26 and followed by a response returned from the SIM via the interface 34.Consequently, to ensure that the SIM card 10 is able to communicate with the processor 21, the processor 21 generates status commands at least every 30 seconds to the SIM card in order to give the SIM card the opportunity of establishing communication.", "The data stored in the SIM card 10 is illustrated schematically in FIG.", "4 and includes the routing table 13, subscriber information 40, preferred network table 19, and forbidden network table 20 as described above with reference to FIG.", "1.Additionally, the SIM card 10 stores encryption information 41 for decrypting incoming messages received from the control centre 14 via the local network 4.The SIM card also stores address information 42 to define the address to which a request message is to be output by the mobile telephone 1 when it is determined that updating information is required for the stored information, such as the routing table 13.Call log information 43 is also stored in order to provide a database of call information, including the duration of calls made and received by the mobile telephone 1 and the routing information used to setup the call.", "Table expiry information 44 is stored to identify the maximum usable life of data such as the routing table 13, each update having an associated expiry date beyond which it is not intended that the table should be used.", "The table expiry information 44 although illustrated as being separately stored is more conveniently stored as one of the fields of the data to which it relates.", "FIG.", "5 illustrates schematically the applications stored in the SIM 10.A data maintenance program 50 is an application for performing maintenance of data stored in the SIM 10, the data including primarily the routing table 13 which may require updating for a number of reasons.", "A key maintenance program 51 is provided to enable maintenance of decryption keys.", "A routing program 52 is provided to perform the call routing function of the mobile telephone 1 which is initiated when a user inputs a dialled number defining the call destination 11 and enabling the routing table 13 to be rapidly accessed to retrieve the required code which is to be transmitted to the local network 4 for use in call setup to the call destination 11.A key decryption program 53 is provided to perform the decryption of any new key which is transmitted to the mobile telephone 1 for use in decryption of data.", "A data decryption program 54 is provided for decrypting data received from a control centre 14, including updates to the routing table 13.At the time of registration, a registration procedure program operated by the processor 21 of the mobile telephone 1 determines the selection of the network in the case of more than one network being available for registration.", "After registration, the data maintenance program 50 is called to determine whether it is necessary to request an update of the stored routing information, including the routing table 13, preferred network table 19 and forbidden network table 20.FIG.", "6 illustrates the manner in which an initial part of the registration procedure is carried out.", "At step 60, the mobile telephone 1 analyses broadcast signals from the available networks in the cell 2 and obtains a list of the available networks together with network identification information.", "This information includes the mobile country code (MCC) and mobile network code (MNC) for each network.", "At step 61, the MNC for each network in the list is compared with the forbidden network table 20 and any networks which are excluded are removed from the list of available networks.", "At step 62, the list of available networks is compared with the preferred network table 19 which has entries for a number of mobile network codes in order of preference.", "If one or more of the MNCs in the available network list is found in the preferred network table 19, the preferred network is identified and at step 63 the preferred network is selected for registration.", "If, however, the preferred network table 19 does not include any of the networks in the list of available networks, a network is selected for registration at step 64 using the original list of available networks and selecting on the basis of maximum signal strength in the available broadcasts.", "At step 65, the registration process is initiated using the selected network from either step 63 or 64.During the registration procedure, the mobile telephone 1 and local network 4 exchange identifying signals.", "This allows the local network 4 to determine the identity of the subscribers home network 7 and home country.", "If the local network 4 is not the home network 7, the local network 4 exchanges messages with the home network 7 using one of the overhead data channels provided in the GSM system.", "This allows the subscriber's status to be authenticated and authority to be given by the home network 7 for calls by the mobile telephone 1 to be billed in an agreed manner.", "The local network 4 updates the visitor location register 6 in its database 5 with information obtained from the home network 7 and from the mobile telephone 1 such that other networks wishing to direct calls to the mobile telephone are able to locate its current location with reference to the stored information.", "The selection made in step 62 may be made in any one of a number of different ways, depending upon the capabilities of the apparatus.", "For example, the apparatus may be a mobile telephone having a SIM card in which an application is able to directly select the preferred network from the list of available networks using the command interaction between the main body of the apparatus and the SIM card.", "Alternatively, if the application is not capable of direct selection, the application may be able instead to modify some of the elementary files stored in the SIM card.", "As an example, the preferred and barred network, lists can be modified in a manner which forces registration to a network selected by the application.", "This therefore requires the steps of amending the elementary files and then initialising the network search procedure using a soft reset procedure (i.e.", "resetting without turning off and on the power to the apparatus).", "Where a soft reset facility is not available, the application must rely upon initiating re-registration when the user next turns off and on the power to the apparatus.", "FIG.", "7 illustrates schematically the process after registration in which it is determined whether new tables need to be requested from the control centre 14.At step 70, the processor 35 of the SIM card 10 receives details of the network with which the mobile telephone 1 is now registered and actuates the data maintenance program 50.At step 71, it is determined from the MCC of the registered network whether there has been a change in the country code indicating that the subscriber has moved to a location in a new country.", "The MCC may simply be compared with a country code field in a stored record of the last network with which the mobile telephone 1 registered.", "If it is determined that the country code is new, then it is determined that a request message must be generated in order to obtain new versions of the routing table 13, preferred network table 19 and the forbidden network table 20.A request message is generated at step 72 and transmitted as an SMS (Short Message Service) message via the local network 4 to the control centre 14.The control centre 14 responds by retrieving up-to-date tables from its database 15 and transmits a response message via the local network 4 to the mobile telephone 1.At step 73, the response message is received and the tables 13, 19 and 20 are updated with the received data.", "The MNC of the local network 4 with which the mobile telephone is now registered is then compared at step 74 with the updated preferred network table 19 and forbidden network table 20.It is determined at step 74 whether the registered network is the preferred network amongst the available network list according to the preferred network table 19.If it is determined that another one of the available networks has a higher level of preference in the list, a process of re-registration is initiated at step 75 in order to replace the currently registered network with the preferred network which then becomes the “local network” referred to above.", "The process then repeats from step 70.Similarly, it may be determined at step 74 that the currently registered network is listed in the forbidden network table 20, in which case step 75 similarly follows to force re-registration.", "If at step 74 it is determined that the currently registered network is in fact the network of most preference according to the preferred network table 19, the status of the mobile telephone is determined at step 76 to be ready to perform routing using the updated tables.", "If at step 71 the country code is determined not to be a new code, the contents of the routing table 13 are examined to determine whether routing information is available for use with the currently registered network.", "If this is the case, it is then determined at step 78 whether the routing table 13 is still valid by comparing current date and time information provided by clock 31 with an expiry date field included in the routing table.", "If the routing table 13 is determined to be still valid, the status of the mobile telephone 1 is then flagged at step 76 to be ready to perform routing.", "If however at step 78 it is determined that the routing table has expired, or at least that relevant parts of the routing table relating to the registered network have expired and are no longer valid, a request message is generated at step 79 for an update of the routing table 13.The request message is transmitted as an SMS message to the control centre 14 which responds by retrieving data from its database 15 and transmitting a response message in SMS format.", "At step 80, the response message is received and the routing table 13 is updated with the received data.", "The status of the mobile telephone is then flagged at step 76 to be ready to perform routing.", "The mobile telephone 1 then remains in standby mode until it is required to make or receive a telephone call.", "When the user initiates an outgoing call, the telephone number of call destination 11 is input using keypad 24 and buffered in RAM 23 of FIG.", "2.The processor 21 passes the dialled number to the SIM card 10 to actuate the routing program 52 which utilises the routing table 13 to extract routing information which is returned to the processor 21.The extraction of routing information is performed in the manner described in WO99/04578 and WO00/41486.The processor 21 initiates a call setup procedure with the local network 4 by outputting signals via the transmitter circuit 27 which include the call routing information.", "The local network 4 responds by completing the setup of the call for the preferred route.", "After “call connection”, the telephone call then proceeds until a “call disconnect” event is detected, the duration and other call parameters being logged by the SIM card and stored in the call information log 43.When outputting the call routing information, the mobile telephone 1 inserts carrier access dialling information in front of the dialled number generated by the user.", "This information is interpreted by the local network 4 to set up the call via the networks defined by the carrier access dialling information.", "The dialling information can be in the form of a 1XXX type access number or a free phone number such an International free phone number.", "The signalling of this information to the local network 4 is generally transparent to the user and utilises the conventional call setup signalling channel provided within the GSM system.", "If however the amount of information to be transmitted requires a string of digits which exceeds the normal field for such a signal, the final digits of the signal are output as DTMF (Dual Tone Multi Frequency) signals after the voice channel has been established.", "FIG.", "8 illustrates schematically the manner in which such routing via the generalised network 12 proceeds.", "The generalised network 12 is shown to comprise a number of constituent networks 80, each of which constitutes a node which is selected by the routing information to determine the route path to the call destination 11.Each of the nodes 80 could comprise a mobile network, long distance carrier, or any other private or public telecommunications network.", "In the example of FIG.", "8, the call destination 11 receives the call via the public service telephone network 81 which constitutes the last network (node) in the route.", "In other examples, however, the final network (node) could be a further mobile network, a long distance carrier network or the Internet.", "For a given local network 4, the available nodes of the generalised network 12 will depend upon the physical location of the connection to the local network and the interconnect facilities currently available, these being determined by commercial agreements and operational factors.", "The form of the request message and response message exchanged between the mobile telephone 1 and the control centre 14 has been referred above as being an SMS message.", "This exchange of SMS messages is transparent to the user of the mobile telephone 1 since a special type of SMS message is utilised which does not result in the SMS message being displayed in the display 25 of the mobile telephone.", "The response message is encrypted to preserve confidentiality of commercial information and to ensure subscriber authentication.", "Encryption keys are therefore required to be held in the SIM card 10 in the encryption information 41 shown in FIG.", "4, this information being periodically updated using encrypted messages from the control centre 14 and operation of the key maintenance program 51 of FIG.", "5.Decryption of the received keys is carried out using the key decryption program 53 and the received data is decrypted using data decryption program 54.The user part of the SMS messages is compressed in order to maximise use of available bandwidth for messaging, and thereby requires all received messages in the mobile telephone 1 to be decompressed using an algorithm available to the processor 21.When updating the routing table 13, memory provided by the SIM card 10 will be overwritten as required.", "Generally, the routing table 13 comprises a number of sub tables, for example containing information relating to a number of different selections of local network 4, and, depending upon the amount of available memory, it may not be necessary to overwrite all of the sub-tables currently stored when an update of routing information is received.", "The memory may therefore be managed to achieve caching of sub tables.", "Memory may be overwritten on the basis of retaining the most recently used information where possible so that it is available for reuse if necessary.", "This facility may for example by useful where the subscriber repeatedly crosses a territorial boundary between two adjacent territories so that caching the stored information reduces the number of updating messages required.", "In an alternative embodiment, the request and response messages may be transmitted using USSD (Unstructured Supplementary Service Data), an alternative protocol for data transmission within GSM.", "Embodiments using cellular telecommunications system other than GSM may similarly use whatever alternative message protocols are appropriate.", "The preferred embodiment has been described with reference to a mobile station in the form of a mobile telephone 1.Further embodiments are envisaged in which different forms of mobile station are used to make and receive telephone calls for voice, data or video communication and the above described method and apparatus may readily be adapted for such further embodiments.", "In particular, the mobile telephone may be a WAP telephone which makes calls to access data in the form of pages of WML (Wireless Mark-Up Language) or similar mark-up languages for retrieving documents or images.", "Handheld computer devices and facsimile devices may similarly utilise the above described call routing and routing table maintenance functions.", "FIG.", "10 illustrates schematically the functional elements of a mobile station in a generalised embodiment of the present invention.", "A registering means 100 interacts externally with the local network 4 of an air interface 101 and accesses internally the preferred network table 19 and forbidden network table 20 when selecting a network for registration from a number of available networks.", "The registering means 100 stores local network information in a buffer 102 which is accessed by a determining means 103 which determines whether the stored routing information requires updating.", "The determining means therefore also requires access to the routing table 13.If the routing table 13 requires updating, the determining means 103 commands a message generator 104 to generate and output an SMS message over the air interface 101 to the local network 4 for onward communication to the control centre 14.A received message is received by message receiver 105 which, after decompression and decryption by circuit 110, allows the updating information to be passed to an updating means 106 for updating the routing table 13 as well as the preferred network table 19 and the forbidden network table 20.A routing means 107 has access to the routing table 13 so that, when an outgoing call to a call destination 11 is initiated by a call number input 108, the routing means intercepts the call making process and provides a call output means 109 with routing information which will determine the route taken through the generalised network 12 between the local network 4 and the call destination 11.The call output means 109 outputs via the air interface 101 the call initiating signals used by the local network 4 in its call setup procedure.", "The call number input may simply be the keypad 24 of FIG.", "2 or may be constituted by a stored directory of telephone numbers selectively addressed by the user inputting an alpha-numeric string or a voice command interpreted by a speech recognition system.", "The mobile station 1 may optionally have an override for bypassing the operation of the routing means 107 or for imposing a user preference for routing the outgoing call.", "FIG.", "10 also illustrates a call log 111 used for storing the duration and call destination of calls generated by the mobile station 1.This data is periodically communicated to the control centre 14 by including call log information in the request message output by the message outputting means 104.The call log information may be utilised by the control centre 14 to verify billing information generated by networks within the generalised network 12 and by the local network 4.This information may also be useful in calculating preferred routes since it facilitates the collection of network usage data for a number of subscribers.", "FIG.", "11 illustrates the functional elements of the control centre 14.A receiver 115 receives SMS messages from mobile telephones 1 and a processor 116 extracts from the messages subscriber data, local network information and call log data which are stored in respective buffers 117, 118 and 119.The call log data is processed and transferred to database 15 as a call log database 120.The local network information in buffer 118 is input to a response generator 121 which refers to preferred network data 122, forbidden network data 123 and routing data 124 stored in the database 15, thereby extracting the relevant data for use with the local network with which the mobile telephone 1 is currently registered.", "Reference may also be made to a database of subscriber identifiers 129 when using a method described below with reference to FIG.", "17.An appropriate response is generated in which updating information is formulated for updating the routing table 13, preferred network table 19, and forbidden network table 20 of the mobile telephone 1 as illustrated in FIG.", "1.The response message is encrypted and compressed by circuit 125 before being output by a SMS message transmitter 126.The form of encryption used is controlled by an encryption manager 127 to ensure that an appropriate encryption key is utilised, depending upon the currently stored encryption information 41 in the SIM card 10 of the mobile telephone 1.Periodically, the encryption manager 127 requires the response generator 121 to include in the response additional updating information for updating the decryption key stored in the mobile telephone.", "As part of the encryption procedure, the SMS message received by the receiver 115 may include a random number challenge which is passed to the encryption manager 127 for use in the encryption performed by circuit 125.The described embodiment of FIG.", "1 provides optimum routing by means of routing information which is passed to the local network 4 to determine the choice of forward routing.", "The routing table 13 may include instructions for calls to certain call destinations 11 to be routed using a different protocol in which, instead of appending routing information to the outgoing call setup signals, an outgoing message, such as an SMS message is generated and forwarded to the control centre 14 to request routing information.", "The control centre 14 would then respond by returning the required routing information which the mobile telephone 1 would then add to the call routing information passed to the local network 4.The choice of optimum route will generally be made on the basis of least cost.", "Alternatively, the best route calculated by the control centre 14 may rely upon choice of best bandwidth available from the networks 80 of the generalised network 12 or other performance criteria, or to maximise use of commercial agreements for call routing made with certain networks or service providers.", "In the described embodiment of FIG.", "1, the control centre 14 calculates the preferred route before downloading data to the routing table 13 to enable the routing information to be accessed rapidly and with minimum computational effort.", "Alternative systems are envisaged in which the control centre 14 collates tariff information and downloads the raw data, or partially processed data, to the mobile telephone 1 for storage in the SIM card 10.The processor 21 of the mobile telephone body or the processor 35 of the SIM card 10 may then calculate the preferred route information based on the tariff data or other parameters defining the possible network connections available.", "This calculation may be performed at the time of call routing on demand and in response to the input of a destination call number or alternatively may be processed on receipt of the tariff data to populate the routing table 13 for use as a look-up table in the manner described above.", "Further aspects of the present invention have application to mobile stations such as mobile telephones in which the routing function is not employed.", "Such mobile stations may still include the preferred network table 19 and forbidden network table 20 of FIG.", "1 but without the routing table 13.For such mobile stations, the above described procedure for registration with reference to the preferred network table 19 and forbidden network table 20 may still be followed.", "After registration with a local network 4, a request message may be output using SMS or equivalent protocol to a control centre 14, requesting updating information for the tables 19 and 20.The mobile station 1 may in this embodiment determine whether such a request message is required by detecting whether the country code of the local network is different to the country code of the network in which the mobile station was previously registered or the tables may have an associated expiry date which may be compared with current time and date information to determine whether the stored information remains valid.", "Such mobile stations may accumulate call log data to be included periodically in the request message for analysis at the control centre 14.Also envisaged in accordance with the present invention are mobile stations in which the process of accumulating call log data and communicating the data to be control centre 14 occurs independently of any request messages for updating information.", "As such, in accordance with this aspect of the invention, the generation of request messages for updating information is optional.", "In the embodiment of FIG.", "1, the updating of stored information is initiated by the generation of a request message from the mobile telephone 1.The mobile telephone 1 may additionally be provided with means for responding to a control instructions from the control centre 14 such that the mobile telephone 1 generates a request message in response to receipt of the control instruction.", "The control centre 14 may issue such an instruction for example when major changes in the information stored in its database have been necessary, for example, to reflect a change in network costs or performance such as a failure of one of the networks 80 in the generalised network 12.Alternative embodiments are envisaged in which some or all of the data files of FIG.", "4 and related applications of FIG.", "5 are stored in the main body of the apparatus, as for example in ROM 22 and RAM 23 and operated using CPU 21 of FIG.", "2.In the above described method of FIG.", "7, the final step 76 places the apparatus ready to perform routing.", "In a preferred embodiment illustrated in FIG.", "12, the apparatus 1 continues to monitor at step 130 the availability of networks and, when a new network is detected at step 131 as becoming available, the network identity information for the new network is compared at step 132 with the stored, preferred and forbidden network tables.", "If the newly detected network is determined at step 133 to have a higher preference according to the preferred network table than the network with which the apparatus is currently registered, the apparatus re-registers at step 134 with the preferred network.", "If at step 135 it is determined that no routing table is available for the new network, a request message for updating tables is then output at step 136 and a subsequently received response message used to update the stored routing information at step 137.In FIG.", "4, the routing table 13, preferred network table 19 and forbidden network table 20 are stored in the SIM card in files which are accessible to any of the applications operable in the SIM card 10.This includes the facility for an external network such as the home network 7 to overwrite the information in these files using file maintenance facilities provided under the GSM system.", "A preferred embodiment of the present invention as illustrated in FIG.", "13 therefore additionally includes the facility of providing respective backup files 140, 141, 142 in a second portion 144 of memory and which contain all of the information in the routing table 13, preferred network table 19 and forbidden network table 20 in the form in which they are updated by response messages from the control centre 14.Access to these files is controlled by a memory management module 145 which restricts access to the back up files to applications such as the data maintenance program 50 and the routing program 52.Whenever accessing these files using the routing program 52, a comparison is first made between the stored files and the backup files and, if any disparity exists, the files are overwritten with the information contained in the backup files.", "This therefore negates the effect of any interference in the files from sources other than the control centre 14 which may for example occur when a network overwrites the preferred network table 19 or forbidden network table 20 with its own data.", "The memory management module 145 also controls access to storage in a cache memory 146 which is used to store a copy of any data deleted from memory so as to retain most recently deleted data.", "The cached data is then available for retrieval and may avoid the need for sending a repeat request message for data which has been recently overwritten.", "The memory management module 145 may be implemented by the processor 31 using program instructions stored in memory.", "The expiry of validity of stored data may simply be determined by storing with the data a time and date at which the validity expires, as described above.", "Alternatively, in those cases where the apparatus is not provided with an internal clock, the number of calls made by the apparatus may be counted and used as a parameter for determining when validity expires.", "This may be achieved for example by entering a count value when the data is updated and decrementing the count value at each call, validity being held to have expired when the count is reduced to zero.", "Alternatively, the prompt commands input to the SIM card 10 may be counted to effectively measure elapsed time during which the apparatus is in use.", "A count value may similarly be stored and decremented to determine when validity has expired.", "Alternatively, the apparatus may output a message to the control centre 14 periodically in order to be informed whether the validity of the data has expired.", "A particular problem associated with prepaid mobile telephone use is that they cannot generally be used in countries other than those in which the home network is located.", "An embodiment of the present invention illustrated in FIG.", "14 provides a solution to this problem by providing that all calls made by apparatus which is the subject of a prepaid contract are routed via a prepayment platform 150.This is achieved by including the network address of the platform in the routing information.", "The prepayment platform 150 then has the power to regulate whether the call is allowed to proceed and the ability to terminate the call based on the status of the subscriber's account, such as when credit runs out.", "Calls may thereby be made from any country since the prepayment platform 150 retains control of call making and call duration.", "Since the calls must in this embodiment be routed via the prepayment platform 150, the routing table 13 must always be used to ensure correct routing.", "The apparatus 1 therefore includes a lock out facility preventing the making of an outgoing call if no routing information is currently available for routing to the call destination 1 via the local network 4 and the prepayment platform 150.The routing means 107 of FIG.", "10 provides the lock out facility such that, for instance where the subscriber has a prepayment subscription, the routing means is operable to determine whether the currently available routing table 13 is valid in terms of expiry date or other relevant criteria and, if not valid, the routing means is operable to prevent the call output means 109 from initiating a telephone call and initiates instead the generation of an appropriate display message to the user.", "Various embodiments are herein disclosed for controlling the selection of network with which registration occurs, FIG.", "7 for example disclosing a method in which an initial registration can be replaced by a re-registration with a preferred network after an evaluation process in which reference is made to stored tables and those tables updated if necessary by sending a request message and receiving a response.", "Such control processes will naturally occur each time the apparatus is turned on.", "During continued operation of the apparatus, it is also appropriate to periodically re-evaluate the selection of network for registration since for example the user carrying the apparatus may move to a new location and may be continuously in motion so that it is not necessarily appropriate to remain registered with the same network.", "FIG.", "15 illustrates schematically an example of a re-evaluation process in which the apparatus re-evaluates the network selection and if necessary registers.", "At step 152, the apparatus begins operation, possibly at a different location from where the apparatus was previously in use.", "At step 153 the apparatus performs the steps described above with reference to FIG.", "7 including registering with an initial local network, evaluating the registration with reference to stored tables, updating the tables if necessary by sending and receiving messages, and if appropriate, re-registering with a new network.", "At step 154, the apparatus remains registered with the network arrived at as a result of the process of step 153 during a period of operation in which the apparatus awaits a trigger event for reevaluation.", "If at step 155 it is determined that a trigger event has occurred, the apparatus evaluates at step 156 whether the network with which the apparatus is currently registered is the optimum selection, the evaluation being made by referring to data stored in tables 19 and 20 for example.", "If at step 157 it is determined that the currently registered network is the most appropriate, no action is taken and the apparatus awaits a further trigger event at step 154.If however it is determined at step 157 that re-registration with a new network would be desirable, re-registration is proceeded with at step 158 and operation of the apparatus continues using the new network, and awaiting a further trigger event at step 154.A trigger event may be initiated periodically by the expiry of a timing interval, for example a thirty minute interval.", "A trigger event may be generated every time one of the tables is updated using new data received from the control centre 14.A trigger event may be generated whenever the apparatus receives a location updating message from the network 4 with which it is currently registered, such messages typically being generated at periods of about thirty minutes and contain current location information including country code, network code and an indication of the cell in which the apparatus is operating.", "A trigger event may be generated at each detection of movement of the apparatus from one cell to another.", "The apparatus may be configured such that step 155 is responsive to trigger events arising from any of the above occurrences.", "In each case, the decision making process will be carried out by the processor 31 of the SIM card in accordance with program instructions.", "The above process allows the reevaluation of network registration to be frequently implemented.", "This is advantageous in circumstances where for example the operator of the home network 7 wishes to have optimum control over network registration when a user is operating the apparatus abroad.", "The process may be used to maximise usage of a preferred foreign network, thereby allowing the operator of the home network 7 to enter into agreements with networks in other countries whereby cost benefit can be achieved in return for agreeing to provide subscribers apparatus with a preferred network list favouring a particular foreign network.", "An overall cost benefit to the user may thereby be achieved.", "A further example will now be described with reference to FIG.", "16 using corresponding reference numerals to those of preceding figures where appropriate for corresponding elements.", "In FIG.", "16 an apparatus such as a mobile telephone includes functional elements corresponding to those described above with reference to FIG.", "10, differing in that the data files accessed by the updating means 106 include the subscriber information 40.The subscriber information 40 is illustrated in FIG.", "20 and is stored in a data structure which includes a current subscriber identifier memory 200 containing subscriber identity data which in the GSM system is an IMSI (International Mobile Subscriber Identity).", "The memory 200 is here an elementary file which is generally referred to in GSM by the file name EF_IMSI.", "The IMSI comprises a fifteen digit number of which the first three digits define a MCC (Mobile Country Code), the next two digits define an MNC (Mobile Network Code) and the final ten digits define an MSIN (Mobile Subscriber Identity Number).", "The IMSI read from the memory 200 is communicated to the local network 4 which interprets this subscriber identity data as identifying the home network 7 of the subscriber and the country in which the home network 7 is located.", "The MSIN will be different for each subscriber of the home network 7 so that throughout the GSM system no two IMSI numbers are the same.", "In this embodiment however, the same subscriber account is allocated more than one possible subscriber identifier so that the subscriber is associated with a plurality of IMSI numbers.", "Each of these IMSI numbers is allocated by a respective network of a group of associated networks operating in different countries, one of the networks being the home network 7 which administers the subscriber account and has operating agreements with each of the associated networks of the group.", "The subscriber information 40 as shown in FIG.", "20 includes a data structure which includes a table in which, for each of a list of networks in various countries which the apparatus is likely to encounter, an IMSI number is indicated as being the preferred subscriber identifier.", "Each network is described in terms of a Mobile Country Code (MCC) and Mobile Network Code (MNC).", "The table including the preferred subscriber identifiers for each of a number of networks is written in the SIM card 10 when initially supplied to the apparatus.", "The subscriber identifiers can be updated for example in response to received messages from the control centre 14.Each subscriber identifier is provided with a validity field 202 from which the SIM card processor is able to determine whether the preferred subscriber identifier information is currently valid.", "The information may be time expired with reference to a system clock, or with reference to similar criteria as referred to above when describing tables 13, 19 and 20.At any given time, one of the subscriber identifiers will be stored in the current subscriber identifier memory 200.Depending upon the identity of the network with which the apparatus is currently registered, the contents of the file 200 may need to be updated to ensure that the preferred subscriber identifier for that network is stored in memory 200 and thereafter indicated as subscriber identity data to the local network 4.A process of determining whether the memory 200 contains the preferred subscriber information and of making updates when necessary is illustrated in the flowcharts of FIGS.", "17 and 18.At step 170 of FIG.", "17, the apparatus makes an initial registration with a local network 4 and at step 171 obtains details of the registered network, specifically determining a network identifier comprising a country code MCC for the network and its mobile network code MNC.", "At step 172 the apparatus determines the preferred subscriber identifier for use with this network, typically referring to a look-up table 201 as illustrated in FIG.", "20.The determining process 172 will be described in greater details with reference to the flowchart of FIG.", "18 below.", "The preferred subscriber identifier is compared at step 173 with the subscriber identity data currently stored in the memory 200, i.e.", "the elementary file EF_IMSI, and if they are the same, no change is made and the processing continues normally, for example by entering the procedure described above with reference to FIG.", "7.If however they are not the same, the contents of the current subscriber identifier memory 200 need to be updated.", "This is implemented in this example by de-registering from the local network 4 at step 174 and then overwriting the contents of the EF_IMSI file with the preferred subscriber identifier copied from the relevant entry in the table 201.At step 176, the registration process is restarted during this new registration process, the local network 4 is presented with signals from the apparatus 1 which contain new subscriber identity data 40 read from the memory 200 and therefore the local network assumes that the apparatus of a new customer has commenced operation in the cell from which the signals are received.", "The network will assume that the home network of the subscriber is the associated network as indicated in the subscriber identity data read from the memory 200 and will process the registration and any calls made via the network accordingly.", "Account information will then be passed to the actual home network 7 by the associated network.", "FIG.", "18 illustrates in greater detail the process referred to above at step 172 of determining the preferred subscriber identifier for use with the currently registered network.", "At step 180, the processor 31 of the SIM card 10 accesses the data structure to refer to the look-up table 201.For each row of the first column of the table 201, a network is identified by values of MCC and MNC.", "For a given network identifier, the preferred subscriber identifier is indicated in the second column at the same row, defining the IMSI number and defining each of its components, MCC, MNC and MNSI.", "In the same row, validity data 202 is provided in the third column for each of the preferred subscriber identifiers.", "At step 181, the processor 31 determines whether there is an existing entry in the table for the network identifier of the currently registered network and, if there is, determines at step 182 from the validity information 202 whether the entry is valid.", "If the entry is valid is indicated as being valid, the processor at step 186 takes the value of preferred subscriber identifier and proceeds with the process of FIG.", "17 at step 173.If however there is no entry in the table corresponding to the network identifier, or if the entry is indicated as not being valid, the processor at step 183 initiates the sending of a request message to the control centre 14, requesting updating information for updating the table 201 and providing a valid entry corresponding to the network identifier.", "A response message containing this information is received at step 184 and the table is updated at step 185.Step 186 then follows as described above, continuing the process of FIG.", "17 at step 173.As indicated above, step 175 results in the new value of subscriber identifier being written to the memory 200 so that the elementary file EF_IMSI now contains the preferred subscriber identifier corresponding to the network identifier for the currently registered network.", "The updating means 106 of FIG.", "16 updates the subscriber information 40 stored in the data files of the SIM card.", "The information previously stored in EF_IMSI is transferred to cache memory 169 for possible future use.", "The memory 200 will over time accumulate a table of IMSI numbers which are available for use with a range of networks in a number of countries.", "The usefulness of the ability to update the subscriber information is illustrated schematically in FIG.", "19 in which a network GB1 located in a first country, in this example the United Kingdom, has a roaming agreement with a network DE1 in a second country, in this case Germany, whereby subscribers whose home network is the network GB1 may operate their mobile telephones in Germany, but subject to each call incurring a roaming agreement surcharge paid by the subscriber.", "(The term “roaming” here is used to indicate use of a network in a country different from the home network).", "Subscribers of a further network GB2 in the United Kingdom do not have to pay the roaming agreement surcharge when using an associated German network DE2 because their mobile telephone apparatus utilises the features described above with reference to FIGS.", "16 to 20.The network GB2 has an arrangement with the associated network DE2 in Germany such that each subscriber of network GB2 is allocated a first IMSI by the network GB2 and a second IMSI by the network DE2.When operating in the United Kingdom, the mobile telephone apparatus uses the first IMSI and when operating in Germany and registered to network DE2 it uses the second IMSI.", "The subscriber may thereby make use of the network DE2 when operating in Germany without the requirement of a roaming agreement, the network DE2 treating calls to and from the mobile telephone apparatus in the same way as local calls from German customers having DE2 as home network.", "Since it is advantageous for the apparatus to register with network DE2 rather than DE1, the preferred network table 19 and forbidden network table 20 can be configured accordingly by the control centre 14 to make best use of this advantage.", "Typically the operators of network GB2 and network DE2 are the same or related companies who cooperate to minimise their own costs and the cost to the subscriber.", "This is particularly advantageous where the operator of network GB2 is a virtual network operator who does not own physical network infrastructure but is a service provider able to acquire an allocation of IMSI numbers from a network such as network GB1.The operator of network GB2 similarly obtains an allocation of IMSI numbers from network DE2 in order to allocate a second IMSI to each subscriber.", "The above allocation process can be extended to any number of cooperating network operators in any number of countries, thereby potentially extending the use of the method of updating subscriber information globally throughout the GSM system.", "Since conventional roaming agreements carry a high level of cost, the overall saving to the operator of network GB2 and ultimately to the subscriber can thereby be made substantial.", "In this example, the contents of the table 201 could be as follows.", "The first row would indicate network GB2 having a preferred subscriber identifier indicated as being the first IMSI.", "The second row would contain the network identifier for DE2 and would indicate the preferred subscriber identifier as being the second IMSI.", "The third row would contain the network identifier for GB1 and indicate the preferred subscriber identifier as being the first IMSI.", "Finally, the fourth row of data would indicate the network identifier of DE1 and the preferred subscriber identifier being the second IMSI.", "The result of this set of subscriber information 40 is that the apparatus is able to operate in both countries without incurring a roaming agreement surcharge.", "When for example registered with network DE1, calls made from the apparatus are treated as though the home network is DE2.Although network DE2 is required to pass accounting information to network GB2, the real home network, no roaming agreement charges are incurred.", "As a further option, the data entered in the table 201 may in a single row define a single preferred subscriber identifier for use with all networks in a specified country.", "This may be achieved by entering in the first column a value of MCC defining the country and a predetermined character, such as a string of zeros, in the field reserved for entry of the MNC.", "The application may then interpret this data as applying to all networks having the indicated value of MCC.", "For example, in the example of FIG.", "19, all networks in a third country such as France may be defined as using the preferred subscriber identifier for network DE1.This will enable the subscribers of home network GB2 to make use of a roaming agreement between DE1 and networks in France without network GB2 having entered into any separate roaming agreements.", "The routing of calls via preferred routes in a generalised network 12 as described above applies equally well to package switched networks in which calls are transmitted as packetised data and each packet is provided with the routing information necessary to enable the packet to reach its required destination.", "Such packetised routing can be configured for example to avoid specified nodes in the generalised network 12 or to always route via specific nodes such as the prepayment platform 150.When utilising the routing information, routing digits may be added to the routing data which is output to the local network.", "Alternatively, the number dialled may be replaced by an entirely different number determined by the routing information, provided that the resulting connection is to the same call destination.", "The above described embodiments refer to the possibility of the control centre 14 responding to request messages from apparatus 1 by providing response messages containing information for updating information stored and used by the apparatus.", "Embodiments are envisaged in which the updating information is provided for the routing table 13, preferred network table 19, forbidden network table 20 and subscriber information 40.Further embodiments are envisaged in which not all of these four items are updated by the use of messages from the control centre, as for example in the case where only the network tables are updated, or only the routing table is updated, or only the subscriber identifier is updated.", "The above embodiments may be implemented using hardware in which the various processors of the system are provided with computer programs which define processor implementable instructions for carrying out the methods disclosed above.", "A further aspect of the present invention is therefore constituted by such programs.", "The programs may be stored in storage media such as a disc 90 as illustrated in FIG.", "9 and may be communicated as a signal 91 over a network such as the internet.", "Further aspects of the present invention therefore comprise a storage medium storing such programs and an electronic signal embodying such programs.", "Implementation of the above embodiments generally requires that the SIM card is produced and supplied to manufacturers of mobile telecommunications apparatus such that the SIM card has an operating system and a set of data files which is customized to allow the above disclosed methods to be implemented.", "A further aspect of the present invention therefore comprises a SIM card having an operating system and data file system for carrying out the above disclosed methods.", "FIG.", "9 illustrates schematically that the programs stored on disc 90 or communicated as signals 91 may be provided to the control centre 14 from an originating station 92.Similarly, the programs required for operation of the SIM card 10 may be stored in a storage medium 90 and downloaded as electronic signals.", "Similarly, the programs required for operation of the processor 21 of the mobile telephone 1 may be stored in a storage medium 90 or downloaded as electronic signals." ] ]	Patent_10467055
[ [ "Network conduit for providing access to data services", "A web service conduit (3) receives data access requests from browsers (11) from hyperlinks on pages generated by web sites (4), converts the data access requests to web service access requests, and invokes the corresponding web services (40) with the web service access requests." ], [ "1.A method of providing access to a remote data service (40) over a network (14), comprising: a. receiving (S2; S11) from a remote terminal (11) a data access request, in a first format, identifying one or more parameters; b. converting the data access request into a second format; and c. forwarding (S5; S14) the data access request in the second format to the remote data service so as to perform a data access using the parameters; wherein the data access request is forwarded by the terminal (11) from a network service (4) remote from the terminal (11) and from the data service (40).", "2.The method of claim 1, wherein the data access request is a read access request, the method further comprising: d. receiving (S5) from the remote data service (40), in said second format, a data message containing parameter values corresponding to the parameters identified in said data access request; e. converting the data message from said second format to said first format; and f. forwarding (S6) the data message in the first format to the remote terminal (11).", "3.The method of claim 2, wherein the data access request is a write access request and contains parameter values corresponding to the parameters, and step c causes the parameter values to be written to the data service (40).", "4.A method of providing access to a web service (40) over the Internet (14), comprising: a. receiving (S2; S11) from a browser (11) a data access request, in a first format, identifying one or more parameters; b. converting the data access request into a second format; and c. forwarding (S5; S14) the data access request in the second format to the web service (40) so as to perform a data access using the parameters; wherein the data access request is forwarded by the browser (I 1) from a web site (4) remote from the web service (40).", "5.A method of providing access to a remote data service (40) over a network (14) using a link protocol, comprising sending to a remote terminal (11) a link to a network conduit (3), the link identifying one or more parameters and being formatted so as to cause the network conduit (3) to perform a data access at the remote data service (40) using the parameters when forwarded to the network conduit (3) by the remote terminal (11).", "6.The method of claim 5, wherein the link is formatted to provide a data read access at the remote data service (40) such that parameter values corresponding to the parameters are read (S5) from the data service (40) by the network conduit (3) and are forwarded to the remote terminal (11).", "7.The method of claim 6, further comprising sending to the remote terminal (11) a link to a program to cause the program to be loaded and executed by the remote terminal (11) so as to control the handling of the data read access by the terminal (11).", "8.The method of claim 7, further including the step of receiving the parameter values from the remote terminal (11).", "9.The method of claim 5, wherein the link is formatted to provide a data write access at the remote data service (40) and includes parameter values corresponding to the parameters, such that the parameter values are written to the data service (40) by the network conduit (3).", "10.A method of providing access to a web service (40) over the Internet (14), comprising sending to a browser (11) an HTML link to a conduit (3), the link identifying one or more parameters and being formatted so as to cause the network conduit (3) to perform a data access at the web service (40) using the parameters when forwarded to the conduit (3) by the browser (11).", "11.A method of performing a data access operation at a remote data service (40) over a network (14) from a terminal (11) using a link protocol, including: a. receiving (S1; S10) at the terminal (11) from a network service (4) a link directed to a network conduit (3) and identifying one or more parameters; and b. in response to user activation of the link, connecting (S2) the terminal (11) to the network conduit (3) over the network (14) and identifying the parameters to the network conduit (3), such that the parameters are forwarded by the network conduit (3) to the remote data service (40) so as to perform the data access operation.", "12.The method of claim 11, wherein the data access operation is a read access operation, the method further including receiving (S6) parameter values corresponding to the parameters from the network conduit (3).", "13.The method of claim 12, further including forwarding (S7) the parameter values to the network service (4).", "14.The method of any one of claims 11 to 13, including receiving a link to a program, the link causing the program to be loaded and executed by the terminal (11) so as to control the handling of the read access operation by the terminal (11).", "15.The method of claim 11, wherein the data access operation is a write access operation, and the link includes parameter values corresponding to the identified parameters, whereby the parameter values are written to the remote data service (40) by the network conduit (3).", "16.A method of performing a web service access operation over the Internet (14) from a browser (11), including: a. receiving (S1; S10) at the browser (11) from a web site (4) a hyperlink to a conduit (3), the hyperlink identifying one or more parameters; and b. in response to user activation of the hyperlink, connecting (S2) the browser (11) to the conduit (3) over the Internet (14) and identifying the parameters to the conduit (3), such that the parameters are forwarded by the conduit (3) to the web service (40) so as to perform the data access operation.", "17.A computer program for performing the method of any preceding claim.", "18.A carrier bearing a computer program according to claim 17.19.A system for providing access to a data service (40) over a network (14) using a link protocol, comprising: a. a network service (4) which provides a link to a conduit (3) including one or more parameters; b. a terminal (11) which receives said link and, in response to a user selection, sends the one or more parameters to the conduit (3); and c. said conduit (3) which receives said one or more parameters and sends a data access request including said one or more parameters to the data service (40) over the network (14)." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Web services are a class of computer program that runs on a server computer connected to the Internet.", "Instead of using protocols such as HTTP and FTP to communicate with a user, web services are invoked by other programs which may be running on clients or other servers connected to the Internet.", "Web services may use an XML-based protocol such as SOAP, with a transport protocol such as HTTP.", "In a conventional architecture shown in FIG.", "1 , a client browser 11 accesses web pages over the Internet 14 on a web site 4 , which invokes one or more web services 40 over the Internet 14 as part of the page generation process.", "Taking for example a web-based timetable lookup service, the user of the client browser 11 downloads a form page from the server 4 and fills in the lookup details of a timetable request.", "When the form is submitted, the client browser 11 sends a page request including the lookup details to the web site 4 using HTTP.", "The web site 4 invokes an underlying timetable lookup web service 40 using HTTP to get the data requested by the user, and formats the XML result into a form to be returned in a web page to the browser 11 .", "This architecture gives great flexibility, and allows the functionality of complex web sites to be distributed as underlying web services across geographic and commercial boundaries.", "However, there are certain technical requirements for the web site 4 to be able to invoke web services 40 : it must be able to make outgoing HTTP requests and to handle XML, SOAP and other protocols.", "These requirements can be a significant barrier to the use of web services.", "Furthermore, there is a great deal of freedom of data formats and protocols within web service standards such as HTTP, SOAP and XML, which makes the migration from one web service to another very difficult for a web site operator." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>Specific embodiments of the present invention will now be described with reference to the accompanying drawings, in which: FIG.", "1 is a diagram of a conventional web service architecture; FIG.", "2 is a diagram of a web service architecture in a general embodiment of the present invention; FIG.", "3 is a diagram of the steps in a read operation in an embodiment of the present invention; FIG.", "4 shows a web form as displayed on a browser for initiating the read operation; FIG.", "5 shows a log-in form as displayed by the browser during the read operation; FIG.", "6 shows a trusted web service check form as displayed by the browser during the read operation; FIG.", "7 is a diagram of the steps in a write operation in an embodiment of the present invention; and FIG.", "8 shows a web page displaying details of an event and a hyperlink to add the event to a web-based calendar.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention relates to a system for providing access to network data services.", "The present invention has particular but not exclusive applications to a method of facilitating access to web services by web sites.", "BACKGROUND OF THE INVENTION Web services are a class of computer program that runs on a server computer connected to the Internet.", "Instead of using protocols such as HTTP and FTP to communicate with a user, web services are invoked by other programs which may be running on clients or other servers connected to the Internet.", "Web services may use an XML-based protocol such as SOAP, with a transport protocol such as HTTP.", "In a conventional architecture shown in FIG.", "1, a client browser 11 accesses web pages over the Internet 14 on a web site 4, which invokes one or more web services 40 over the Internet 14 as part of the page generation process.", "Taking for example a web-based timetable lookup service, the user of the client browser 11 downloads a form page from the server 4 and fills in the lookup details of a timetable request.", "When the form is submitted, the client browser 11 sends a page request including the lookup details to the web site 4 using HTTP.", "The web site 4 invokes an underlying timetable lookup web service 40 using HTTP to get the data requested by the user, and formats the XML result into a form to be returned in a web page to the browser 11.This architecture gives great flexibility, and allows the functionality of complex web sites to be distributed as underlying web services across geographic and commercial boundaries.", "However, there are certain technical requirements for the web site 4 to be able to invoke web services 40: it must be able to make outgoing HTTP requests and to handle XML, SOAP and other protocols.", "These requirements can be a significant barrier to the use of web services.", "Furthermore, there is a great deal of freedom of data formats and protocols within web service standards such as HTTP, SOAP and XML, which makes the migration from one web service to another very difficult for a web site operator.", "STATEMENT OF THE INVENTION According to one aspect of the present invention, there is provided a web service conduit which receives data access requests from browsers on pages generated by web sites, converts the data access requests to web service access requests, and invokes the corresponding web services with the web service access requests.", "The web service access requests may be requests to read and/or write data.", "In the case of a read data request, the web service conduit receives data read from the web service, converts it into a browser format and sends it to the browser.", "An advantage of the invention is that web site authors can implement web services using a standard format required by the web service conduit, without the technical requirements of the different underlying web services.", "For example, there will be no need to enable outgoing HTTP at the web site, or to use XML or SOAP.", "BRIEF DESCRIPTION OF THE DRAWINGS Specific embodiments of the present invention will now be described with reference to the accompanying drawings, in which: FIG.", "1 is a diagram of a conventional web service architecture; FIG.", "2 is a diagram of a web service architecture in a general embodiment of the present invention; FIG.", "3 is a diagram of the steps in a read operation in an embodiment of the present invention; FIG.", "4 shows a web form as displayed on a browser for initiating the read operation; FIG.", "5 shows a log-in form as displayed by the browser during the read operation; FIG.", "6 shows a trusted web service check form as displayed by the browser during the read operation; FIG.", "7 is a diagram of the steps in a write operation in an embodiment of the present invention; and FIG.", "8 shows a web page displaying details of an event and a hyperlink to add the event to a web-based calendar.", "MODES FOR CARRYING OUT THE INVENTION GENERAL EMBODIMENT A general embodiment of the present invention is shown in FIG.", "2.In this architecture, web pages on the web site 4 include links to a conduit 3 connected to the Internet 14.The links cause the conduit 3 to invoke one or more underlying web services 40 over the Internet 14 to perform data read and/or write operations.", "In a read operation, the conduit 3 forwards data read from one of the web services to the browser 11.In a write operation, the data written to one of the web services 40 may be subsequently accessed by a read operation, or via another web server (not shown).", "As is well known, the web site 4 may be implemented by one or more web servers each comprising one or more computers connected to the Internet and running a web application on a web server platform.", "The web services 40 may be implemented by one or more application servers which comprise one or more computers connected to the Internet and running application server software serving as an interface or ‘middleware’ to one or more databases.", "The databases need not be collocated with the application servers but may instead be hosted at remote sites.", "However, the connections to the databases typically take place over high-bandwidth low-latency networks and not over the Internet.", "An example of a web service is the Microsoft .NET™ web services.", "The browser 11 may be implemented by a computer connected directly or indirectly to the Internet 14 and running browser software such as Microsoft™ Internet Explorer™ or Netscape™ Navigator versions 3 or above.", "The computer may be a desktop, laptop or palmtop computer or any other similar device which is capable of running browser software and connecting to the Internet.", "The computer may be connected to the Internet indirectly via a wireless circuit-switched or packet-switched network.", "The conduit 3 may be implemented by any suitable server configuration, but preferably by a secure, scalable, fault-tolerant server farm running a custom conduit application on suitable platform, such as Linux.", "The present invention is not limited to these specific configurations and may be implemented using other types of computer, device and/or network.", "Read Operation An example of a read operation in a first specific embodiment of the present invention will now be described with reference to FIGS.", "3 to 6.In this example, a form is populated with data retrieved from a user profile web service.", "At step S1, the user of the browser 11 requests a page from a web site 4, which returns a form with blank fields.", "For example, as shown in FIG.", "4, the page includes fields for the user's name and telephone number.", "The page includes a ‘Retrieve’ button which contains a link to the conduit 3 including parameters for passing to the conduit 3.The parameters include the identity of the web service 40, the identity of the web site 4, and the names of fields to be read from the web service 40.In this example, the user has already stored a full set of details, including name and address, on a web service 40.Rather than enter the details manually, the user clicks on the ‘Retrieve’ button.", "At step S2, the browser 11 is redirected to the conduit 3 and the parameters embedded in the link are passed to the conduit 3.The conduit 3 must authenticate the user and ensure that the user is authorized to access the requested web service.", "This may be done by verifying that the user is logged in to a user authentication service such as the Microsoft™ .NET Passport.", "Alternatively, the conduit 3 may use a separate authentication system from the web services 40.If the user is already authenticated, then the operation passes directly to step 4 and the user may read the authentication details stored on the user's computer, for example as an encrypted cookie.", "If the user is not logged in, at step S3 the conduit 3 sends a page 41 to the browser 11 requesting the user to log in to the conduit 3 and optionally the relevant web service 40, if this is required.", "An example of this page is shown in FIG.", "5.If it is the first visit of the user to the conduit 3 the user may be prompted for a choice of authentication mechanism for the desired category of the read/write web service access.", "For example, if the user selects .NET Passport to retrieve their profile, then the elected web service will be .NET Profile.", "This setting may be changed subsequently by the user.", "Preferably, the conduit 3 generates a separate pop-up window at the browser 11 for communication with the user, so as not to remove the page of the web site 4 from display.", "At step S4, the conduit 3 checks the identity of the indicated web service 40 against a list 42 of web services to which the user has granted the conduit 3 permission to access.", "If the indicated web service is not on this list, the conduit 3 sends a page to the browser 11 prompting the user to add the indicated web service 40 to the user's list 42, as shown for example in FIG.", "6.If the user agrees to add the indicated web service to the list 42 then the process continues.", "Otherwise, the process terminates and the conduit 3 sends a page to the browser 11, preferably in a pop-up window, indicating that the web service 40 cannot be used.", "At step S5, the conduit 3 sends a read request to the web service 40 using the parameters supplied in the link by the browser 11.The read request is formatted by the conduit 3 according to the protocols required by the web service.", "The conduit 3 receives the requested data from the web service 40.At step S6, the conduit 3 formats the received data and forwards it to the browser 11.At step S7, the browser 11 is redirected to the web site 4 with the received data encoded within the query string and the user submits the completed form to the web site 4.As an alternative to step S6, the conduit 3 may send the received data to the browser 11 as a form POST with the received data encoded in the HTTP request body.", "Web Site Implementation To implement a read operation link, three HTML fragments must be incorporated in the web site 4: a JavaScript HTML tag, an Onload event in the HTML body tag, and the hyperlink to the conduit 3.In one example, the JavaScript HTML tag is: <SCRIPT LANGUAGE=“javascript” SRC=http://conduitserver.com/scripts/conduit_read.js></SCRIPT> The file requested by this HTML tag contains a single generic function, for example WriteFormValues, to manage the form data requirements and any state data including any application-specific query string or hidden field data present on the web site 4.The JavaScript function first checks to see if there is a query string argument.", "If so, and none of the names within the query string name-value pairs correlate to form field names on the web page, then the query string text is appended to the end of the hyperlink to the conduit 3.Similarly, any hidden field data in the page is copied into the hyperlink to the conduit 3.When returning to the page, the JavaScript function initially associates form fields (either text box or application-defined hidden fields) with name-value pairs within the query string.", "Any query string information needed for proprietary purposes of the web site 4 is left unimpaired by the conduit 3 so as not to interfere with any web site server script which runs as a prelude to the delivery of the page.", "A sample script for WriteFormValues is given in Annex 1 below.", "This script is compatible with JavaScript DOM implementation in third-generation browsers (e.g.", "Microsoft™ Internet Explorer™ 3.00 and above or Netscape™ Navigator™ 3.00 and above).", "To copy the values from the page parameters to the associated fields, the function WriteFormValues is invoked using the OnLoad event in the HTML BODY tag, for example: OnLoad=“WriteFormValues( )” Finally, a hyperlink to the conduit 3 is included in the form page to allow the user to invoke the web service 40 via the conduit 3.The hyperlink may be associated with an icon allowing the user to identify a link to the conduit 3.For example, the hyperlink may be coded as: <A HREF = “http://conduitserver.com/conduitrequest.aspx ?name=con_profile_name&telno=con_profile_telno&con_pid=1234 > <IMG SRC=http://conduitserver.com/images/request.gif BORDER=“0”></A> The URL contains a list of parameters used by the conduit 3, where Name and Telno refer to the form text boxes with fieldnames Name and Telno respectively.", "PID is a partner identifier which identifies the web site 4 to the conduit 3.The parameter names are preferably those recognised by the web service 40, prefixed with ‘con_’ to represent the encoded version for the conduit 3.In some cases, additional parameters may need to be sent to the conduit 3 for supply to the web service 40.For example, the web service may require an email address of the user.", "This information is extracted by the JavaScript from either hidden field information or from a textbox on the form page, and the name-pair field is sent to the conduit 3.The conduit 3 recognises this field because of the ‘con_’ prefix before the actual parameter name recognised by the web service 40.Write Operation An example of a write operation in a second embodiment of the present invention will now be described with reference to FIGS.", "7 and 8.This embodiment uses the same general architecture as shown in FIG.", "2.In this example, the user adds the details of an event to a web calendar service.", "At step S10, the user of the browser 11 requests a page from a web site 4, which displays details of an event with a hyperlink to add that event to the user's calendar.", "For example, as shown in FIG.", "8, details of a concert are displayed with an ‘Add’ button alongside.", "At step S11, the user clicks on the ‘Add’ button and the browser is redirected to the conduit 3 by the underlying hyperlink.", "The hyperlink comprises the URL of the conduit 3 together with parameters which include the identity of the web service 40, the identity of the web site 4, and the names and values of the fields to be written to the web service 40.These parameters are passed to the conduit 3.If the user is not already authenticated, at step S12 the conduit 3 prompts the user to log in, as in the read operation.", "At step S13, the conduit 3 checks the identity of the indicated web service 40 against a list 42 of web services to which the user has granted the conduit 3 permission to access.", "If the indicated web service is not on this list, the conduit 3 sends a page to the browser 11 prompting the user to add the indicated web service 40 to the user's list 42.If the user agrees to add the indicated web service to the list 42 then the process continues.", "Otherwise, the process terminates and the conduit 3 sends a page to the browser 11 indicating that the web service 40 cannot be used.", "At step S14, the conduit 3 send parameter values derived from the parameter values in the hyperlink to the web service 40, and the web service responds by confirming that the values have been added to the user's calendar.", "At step S15, the conduit 3 displays a page, such as a pop-up window, confirming that the details of the event have been added to the user's calendar.", "Web Site Implementation To implement a write operation link, only a correctly formatted hyperlink is required in the displayed page at the web site 4.For example: <A HREF=“http://conduitserver.com/ ?con_pid=1234&con_eid=5678&con_day=08&con_month=04&con— year=2001&con_starttime=20.00&con_subject=beethoven@royal— albert_hall> <IMG SRC=http://conduitserver.com/images/add.gif BORDER=“0”></A> The hyperlink contains the names and values of parameters recognised by the web service, with the names prefixed by ‘con_’ so that the conduit 3 can identify parameters which it must forward to the web service 40.Alternatively, an address-based hyperlink may be used, such as: http://conduitserver.com/pid=1234/eid=5678/08/04/2001/20.00/beeth oven@royal_albert_hall> Conduit Implementation for Read/Write Operation The following description applies to both read and write operations and hence to both the first and second embodiments.", "The conduit 3 has access to a web service database identifying the format and protocol requirements of available web services 40, as well as their service types and field definitions.", "The conduit 3 is therefore able to translate a generic access request from the browser 11 to an access request to a specific web service 40.The conduit 3 also has access to a user database identifying, for each registered user, the web services 40 to which the user has granted permission to access, together with user logon details such as UserID and password.", "In each web service access request, the conduit 3 receives the parameters from the browser 11 and identifies those with the “con_” prefix as requiring processing.", "The conduit 3 identifies the requested web service type from the service type prefix of the parameters; for example, “profile_” indicates a user profile server type.", "Those parameters without a service type prefix, such as PID, are for internal processing by the conduit 3 and are not passed to a web service.", "The conduit 3 then identifies a specific web service 40 by searching the specified user record of the user database for a permitted web service of the specified web service type.", "The conduit reads the requirements of the specific web service 40 from the web service database, and formats and sends the parameters relevant to that web service 40 according to those requirements.", "The present invention is not limited to the general or specific embodiments described above.", "It is envisaged that various modifications and variations could be made without falling outside the scope of the present invention.", "Annex 1—Sample JavaScript Function function WriteFormValues( ) { var queryString = document.location.href; var twoQueryString = queryString.split(“?”); if(twoQueryString.length<=1) return; var eachFormElement = twoQueryString[1].split(“&”); var m_NameValue = new Array( ); //Search through hidden fields here //On the return trip the hidden fields will be pulled back out and //reloaded into form element values for(int a = 0; a < details.elements.length; i++) { if(details.elements[i].type==“hidden”) { m_NameValue[details.elements[i].name] = details.elements[i].", "value; } } //Do form handling here var details = document.forms[0]; var selected = false; for(i = 0; i < details.elements.length; i++) { if(details.elements[i].type==“text” ∥ details.elements[i].type== “hidden”) { for(var j = 0; j < eachFormElement.length; j++) { var tempString = eachFormElement[j].split(“=”); if(tempString[0] == details.elements[i].name) { details.elements[i].value=tempString[1]; selected = true; break; } } //Do the capturing of querystring information here - if the name from the //name-value pair doesn't correspond to a form element then the values will be //appended to the link querystring if(selected==false) { m_NameValue[tempString[0]] = tempString[1]; } selected = false; } } //Add to conduit link var addQuery = “&”; for(key in m_Name) { addQuery += (key + “=” + m_NameValue[key] + “&”); } document.all[“Link”].href += addQuery.substring(0, addQuery.length − 1); return true; } Annex 2—Glossary DOM: Document Object Model which allows JavaScript to interact with objects on an HTML page to change their behaviour.", "HTTP: HyperText Transport Protocol.", "The standard World Wide Web client-server protocol user for the exchange of information (such as HTML documents, and client requests for such documents) between a browser and a web server.", "HTML: HyperText Markup Language.", "A standard coding convention and set of codes for attaching presentation and linking attributes to informational content within web pages.", "During a web page authoring stage, the HTML codes are embedded within the informational content of the web page.", "When the web page is subsequently downloaded to a browser, the codes are interpreted by the browser and user to parse and display the web page.", "HTML codes are often used to create links to other web pages, commonly referred to as “hyperlinks”.", "JavaScript: A platform-independent scripting language which can interact with HTML to provide additional browser functionality.", ".NET: An operating system platform developed by Microsoft, which allows applications to be hosted on remote servers over the Internet.", "Passport: A standardized authentication system for NET which allows users to be authenticated to multiple different services using a single set of authentication details.", "SOAP: Simple Object Access Protocol.", "A platform-independent protocol for sending information over the Internet.", "SOAP uses an XML format with HTTP as a transport protocol, XML: Extensible Markup Language.", "A specification for designing customized tags (HTML codes), enabling the definition, transmission, validation and interpretation of data between applications and between organisations." ] ]	Patent_10467100
[ [ "Chemical vapor deposition devices and methods", "Apparatus is described for rapidly coating a large area, or for rapidly producing a powder.", "In one embodiment, a liquid having a coating chemical is pumped from a liquid reservoir to a distribution manifold.", "From the distribution manifold, the liquid is carried under pressure to a geometric array, e.g., linear, of atomization nozzles.", "Flow equalization means are provided for equalizing the flow of the liquid delivered to each nozzle, and, preferably, means are provided for equalizing the temperature of the liquid delivered to each nozzle.", "The liquid, upon exiting the nozzles with the attendant pressure drop atomizes.", "The atomized liquid coats a substrate either in non-reacted or reacted form, or forms a powder.", "In a preferred embodiment, a solution of precursor chemical is reacted in a geometric array of flames produced at the nozzles, and a coating material produced in the flame coats the substrate, or a powder is formed.", "In another embodiment, vaporized precursor and vaporized are fed to a burner chamber having a linear exit slit.", "The vapor exiting the slit is burned, and material produced in a flame reaction are deposited on a substrate, or the powder formed is collected." ], [ "1.Apparatus for producing a uniform flow aerosol of a liquid comprising: liquid reservoir means containing the liquid at a first temperature; means for pressurizing said liquid; liquid distribution manifold means, said pressurizing means delivering pressurized liquid to said liquid distribution manifold means, an array of thermally controlled nozzles, said nozzles each being configured such that the liquid reaches a second temperature in each of said nozzles and at the exit of each of the nozzles undergoes a pressure drop to a pressure below the liquid's boiling point relevant to the second temperature, and is thereby atomized, means to deliver the liquid from said manifold means to each of said nozzles, said delivery means having flow equalization means which use back pressure, whereby liquid flow is more uniformly delivered to and from each of said nozzles.", "2.Apparatus according to claim 1 having means to preheat the liquid delivered to each of said nozzles at an equalized temperature.", "3.Apparatus according to claim 1 having energy means associated with each of said nozzels to promote a chemical reaction of at least one component of said liquid so as to produce a powder or coating material.", "4.Apparatus according to claim 1 wherein each of said nozzles has associated means for delivering an oxidizing agent in the region of said nozzle, whereby at least one component of said atomized liquid is oxidized to produce a powder or coating material.", "5.Apparatus according to claim 1 wherein each of said nozzles has associated means for delivering a combustible agent in the region of said nozzle, whereby at least one component of said atomized liquid is combusted to produce a powder or coating material.", "6.Apparatus according to claim 1 wherein said flow equalization means comprises a plurality of conduits, a conduit from said manifold leading to each of said nozzles, the dimensions of each of said conduits being such that liquid of substantially equal flow is delivered to each of said nozzles.", "7.Apparatus according to claim 1 wherein said array comprises a linear array of said nozzles.", "8.Apparatus according to claim 1 wherein said array comprises a circular array of said nozzles.", "9.Apparatus according to claim 1 wherein said array comprises a curved array of said nozzles.", "10.Apparatus according to claim 1 wherein the pressure of the liquid in said manifold means is less than 5,000 psi.", "11.Apparatus according to claim 1 wherein the pressure of the liquid in said manifold means is less than 10,000 psi.", "12.Apparatus according to claim 1 wherein the pressure of the liquid in said manifold means is less than 20,000 psi.", "13.Apparatus according to claim 1 wherein the variation of liquid flow rate between any two of said nozzles is less than 10%.", "14.Apparatus according to claim 1 wherein the variation of liquid flow rate between any two of said nozzles is less than 5%.", "15.Apparatus according to claim 1 wherein the variation of liquid flow rate between any two of said nozzles is less than 2%.", "16.Apparatus for depositing a coating material comprising a burner unit comprising a liquid chamber and a liquid opening means exiting said liquid chamber wherein said opening means is elongated in at least one direction, means to provide to said liquid chamber at least one precursor chemical in liquid form and, means to ignite said liquid to produce said coating material from said precursor chemical.", "17.The apparatus of claim 16 further having means to provide an oxidizing agent to said chamber in gaseous form.", "18.The apparatus of claim 16 having pre-heating means to pre-heat at least one precursor chemical in liquid form fed to said gas chamber.", "19.The apparatus of claim 16 wherein said elongated opening means is linear.", "20.The apparatus of claim 16 wherein said opening means is an elongated slit 21.The apparatus of claim 16 wherein said opening means comprises a plurality of orifices.", "22.The apparatus of claim 21 having means to provide said precursor chemical to said chamber in pressurized liquid form and said orifices are sized sufficiently small such that pressurized liquid exiting said chamber atomizes via some boiling as it leaves said orifices.", "23.Apparatus for depositing a coating material comprising a burner unit comprising a fluid chamber and a fluid opening means exiting said fluid chamber wherein said opening is elongated in at least one direction, means to provide to said fluid chamber at least one precursor chemical in fluid form, and means to ignite said fluid to produce said coating material from said precursor chemical." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Vapor deposition is a well known method of producing coatings on substrates by exposing at least one surface of the substrate to a vapor phase of the deposition precursor.", "In CVD a chemical reaction of the precursor occurs on the surface of the substrate, or prior to deposition on the substrate to thereby form the coating on the substrate.", "The conventional methods of CVD require a chamber in which the substrate is held while the vaporized coating constituents are fed into the chamber.", "The portion of the vapor that does not deposit on the substrate to form the coating is exhausted out of the chamber where it must be collected for reuse or released into the atmosphere.", "In the more recently developed CCVD techniques, a combustion source (flame, plasma etc.)", "is used to promote the chemical reaction in the vicinity of the substrate.", "In this manner the coating species is formed in close proximity to the substrate such that a larger portion of the coating precursor is deposited on the substrate.", "This is due to the increased control of the deposition reactions and temperatures.", "Many coatings will only form at a specific deposition temperature, and below this temperature, the coating will not form on the substrate and is exhausted away.", "With CCVD, changes of this deposition temperature can be made much quicker, as the surface of the substrate is (in some cases) directly heated by the combustion source such that as the combustion source forms the coating species, and the majority of the precursor forms the coating with very little of the precursor material needing to be exhausted.", "This can allow open atmosphere depositions without the need for reclamation of the exhausted materials.", "These processes are detailed in U.S. Pat.", "Nos.", "5,652,021; 5,858,465; 5,863,604; 5,997,956; 6,013,318 and 6,132,653, and issued to Hunt et al.", "These patents, which are hereby incorporated by reference, disclose methods and apparatus for CCVD of films and coatings wherein a reagent and a carrier medium are mixed together to form a reagent mixture.", "The mixture, along with an oxidizing agent, is then ignited to create a flame or the mixture is fed to a plasma torch.", "The energy of the flame or torch vaporizes the reagent mixture and heats the substrate as well.", "These CCVD techniques have enabled a broad range of new applications and provided new types of coatings, with novel compositions and improved properties.", "In addition, these technologies are also useful in the formation of powders, as described in the above-referenced patents.", "A limitation of these previous CCVD processes is that the area coated by the combustion source is somewhat limited to the size of the combustion source, at least within reasonable time frames.", "The present invention is directed to overcoming this limitation by increasing the effective area coated by the combustion source, thereby increasing the overall rate of the deposition or powder production to a level appropriate for manufacturing processes.", "The apparatus of the invention is useful in any deposition process in which a liquid is atomized and the atomized liquid is used to form a coating.", "While a flame is one energy source that may be used to promote chemical reaction of a precursor chemical(s) in liquid form, other energy sources, such as heated gases, induction heaters, etc.", "may be used, particularly if a non-oxidizing reaction is to be promoted." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>To achieve the above objectives, the present invention provides for methods and apparatus that include at least one increased dimension of the combustion source, particularly for CCVD processes.", "It should be understood that the various embodiments of the present invention are useful for other methods of deposition such as pyrolytic spray or CVD, and the following detailed description is most specific to the CCVD method for simplicity only.", "Furthermore, the described devices are also useful in the production of powders when used in conjunction with well known powder collection apparatus.", "When used to deposit coatings, the present invention allows a greater area to be coated by a single pass of the CCVD apparatus.", "A first embodiment is an integrated discrete linear flame that is comprised of a plurality of CCVD nozzles aligned in a linear array.", "Each of the discrete nozzles must be precisely controlled in terms of pressure and temperature to insure a uniform deposition rate and composition across the width of the CCVD apparatus.", "A second embodiment of the apparatus is in the form of a continuous linear flame wherein vaporized coating material is fed into an extended tube with a flame slit extending along the length of the tube.", "The coating material ignites as it exits the slit, thereby forming a continuous linear flame that provides a uniform deposition rate and composition along the length of the flame.", "Both of the embodiments provide an efficient method of producing a large area uniform coating on large substrates from a single chemistry solution, thus increasing deposition rates to a level suitable for manufacturing purposes.", "In the first embodiment, several CCVD nozzles are arranged side-by-side.", "The typical deposition area or “footprint” of a single CCVD nozzle is dependent on several factors including but not limited to the material being deposited and distance from the substrate.", "In its simplest form, the multiple nozzle array consists of two deposition nozzles.", "The second nozzle may only increase the deposition area by a factor of 1.25, due to interaction effects between the two nozzles.", "The actual material throw rate, however, will typically increase by a factor close to 2.0.In order to maximize the coating uniformity, deposition area, and overall deposition material throw rate, the spacing between nozzles must be determined through experimentation for each particular application.", "Should the distance between nozzles for one of these factors interfere with another requirement, (such as maximizing deposition area at the expense of uniformity), two banks of nozzles may be arranged in succession, with the centerlines of the nozzles being offset to provide uniform coverage.", "To provide uniform deposition between the multiple nozzles, the temperature and flow of each nozzle must be held constant with reference to the other nozzles.", "To achieve this requirement, back-pressure regulators and a block heater are employed.", "Each of the nozzles is fed from a common, chemistry solution, distribution manifold.", "Between the manifold and each nozzle, a back-pressure regulator is provided.", "The back-pressure regulator may be a standard pressure regulator, a needle valve, or a coiled tubing.", "Each of the nozzles has an inherent pressure drop in the fluid as it flows from the common manifold and out the exit of the nozzle.", "If this pressure drop is for example 100 psi in one nozzle, and 200 psi in a second nozzle, then the flow rate differential between these nozzles would be 50% (assuming an equal cross sectional flow area).", "By providing back-pressure regulators that increase the pressure drop to around 1000 psi, this same pressure drop difference of 100 psi only causes a pressure drop variation of 900 to 100 psi or a flow rate differential of 10%.", "In the preferred embodiment, the back-pressure regulators are in the form of coiled, small inner diameter tubes, the lengths of which determine the pressure drop for each nozzle.", "Thus to adjust for equal pressure drops and resulting uniform deposition material flow, the relative length of each tube is adjusted accordingly.", "More specifically, since the effective flow rate of each orifice from a central manifold is inversely related to its back pressure, it is desired to maintain the back pressure to each line as closely as possible.", "Of particular importance to the present invention is the capability to maintain a uniform flow through multiple orifices from a single delivery system when the pressure drop across the orifices is not equal or as the pressure variation of an orifice varies over time (accumulation of material).", "Such a system exists, as per the present invention, when atomizing by releasing a thermally controlled liquid into a volume that is at a pressure below its boiling point relative to the controlled temperature of the liquid.", "Products to form the orifice are made with a large variation which results in different back pressures at the desired flow rate.", "Used alone, these orifices do not provide the desired control of the liquid through each nozzle.", "This is further complicated by the issue of heated liquids that can have variable amounts of material forming on the inside surfaces of the nozzles which causes a time variable back pressure at a constant flow rate.", "It is not desired to have a pump or liquid mass flow controllers (if one were found that works at the required pressures and flow rates) for each nozzle as the cost and maintenance becomes prohibitive.", "The current system works by have a precise pressure drop up stream from the nozzle to act in providing uniform flow to each nozzle.", "Downstream of this flow controlling pressure drop the liquid state would still be maintained so that material will not form over time on the walls of the flow controlling section.", "The pressure drop across this flow control region needs to be substantially higher than that of the orifice, so that any orifice pressure changes are minor in comparison to the constant pressure of the flow control section.", "Thus if orifice back pressure can vary from 100 psi to 300 psi, then the flow control pressure drop must be at least 2000 psi (10×) to maintain at least a 10% or better flow control through each nozzle.", "For even more uniform flow control, a 4000 psi (20×) pressure drop can be used (5% flow variation).", "For yet even more uniform flow control, a 10000 psi (50×) pressure drop can be used (2% flow variation).", "In some cases it will be desired to go to even a 100× factor (this would be a manifold pressure of 20,000 psi with a resultant pressure of 100 to 300 psi after the flow control section for the above example).", "Of course as higher pressures are needed the cost of the system increases, so added tolerance comes with a cost, complexity and size impact.", "This above example, without the flow control system of the present invention, would have a flow variation of about +/−50%.", "Available components (tubing, fittings, pumps, valves, etc.)", "for the present systems have significant price jumps at 5000, 10000, and 20000 psi.", "Thus there is desire to design a system with the exact level of flow variation required to produce the desired coating or powder, to avoid excessive costs.", "The flow rate of the deposition material is also dependent on other factors, such as density and viscosity, that are in turn dependent on the temperature of the fluid.", "In order to maintain a similar temperature between the nozzles, a block heater is used.", "Each of the nozzles and a thick walled tube leading to the nozzle from the coiled tube is encased in a block of material that is thermally conductive (such as a dense metal).", "Alternatively, precision-machined orifices may be drilled into the material to form the nozzles and passageway between the nozzles and the coiled tubes.", "Resistive element heaters are used to heat the block of material such that the fluid flowing through the nozzles is brought to a thermodynamically metastable state.", "A liquid is in a metastable state if its temperature at the exit of the nozzle is higher than its saturation temperature for a given pressure.", "By heating the fluid to this temperature, rapid expansion of the liquid is achieved which results in quick and uniform atomization of the precursor material.", "It should also be understood from the following description, that the apparatus and methods of the present invention can be used to form coatings using deposition techniques other than CCVD, as the use of a combustion source is not necessary for forming some materials.", "The linear deposition apparatus provides a uniform material deposition rate along its length, thereby forming a more uniform coating than could be previously achieved using prior art devices and techniques.", "This is due to the pressure and temperature regulation provided between the array of nozzles that form the integrated linear deposition apparatus.", "A second embodiment of the present invention provides a continuous linear deposition apparatus for applying coatings using precursor chemical-containing fluids." ], [ "GOVERNMENT CONTRACT The United States Government has rights in this invention pursuant to Contract No.", "70NANB8H4071 awarded by the Department of Commerce.", "FIELD OF THE INVENTION The present invention is directed to devices and methods for producing coatings with chemical vapor deposition (CVD).", "In particular, the invention is directed to increasing the rate of deposition in combustion chemical vapor deposition (CCVD) by extending the combustion and deposition zone, e.g., extending it in a linear direction.", "BACKGROUND OF THE INVENTION Vapor deposition is a well known method of producing coatings on substrates by exposing at least one surface of the substrate to a vapor phase of the deposition precursor.", "In CVD a chemical reaction of the precursor occurs on the surface of the substrate, or prior to deposition on the substrate to thereby form the coating on the substrate.", "The conventional methods of CVD require a chamber in which the substrate is held while the vaporized coating constituents are fed into the chamber.", "The portion of the vapor that does not deposit on the substrate to form the coating is exhausted out of the chamber where it must be collected for reuse or released into the atmosphere.", "In the more recently developed CCVD techniques, a combustion source (flame, plasma etc.)", "is used to promote the chemical reaction in the vicinity of the substrate.", "In this manner the coating species is formed in close proximity to the substrate such that a larger portion of the coating precursor is deposited on the substrate.", "This is due to the increased control of the deposition reactions and temperatures.", "Many coatings will only form at a specific deposition temperature, and below this temperature, the coating will not form on the substrate and is exhausted away.", "With CCVD, changes of this deposition temperature can be made much quicker, as the surface of the substrate is (in some cases) directly heated by the combustion source such that as the combustion source forms the coating species, and the majority of the precursor forms the coating with very little of the precursor material needing to be exhausted.", "This can allow open atmosphere depositions without the need for reclamation of the exhausted materials.", "These processes are detailed in U.S. Pat.", "Nos.", "5,652,021; 5,858,465; 5,863,604; 5,997,956; 6,013,318 and 6,132,653, and issued to Hunt et al.", "These patents, which are hereby incorporated by reference, disclose methods and apparatus for CCVD of films and coatings wherein a reagent and a carrier medium are mixed together to form a reagent mixture.", "The mixture, along with an oxidizing agent, is then ignited to create a flame or the mixture is fed to a plasma torch.", "The energy of the flame or torch vaporizes the reagent mixture and heats the substrate as well.", "These CCVD techniques have enabled a broad range of new applications and provided new types of coatings, with novel compositions and improved properties.", "In addition, these technologies are also useful in the formation of powders, as described in the above-referenced patents.", "A limitation of these previous CCVD processes is that the area coated by the combustion source is somewhat limited to the size of the combustion source, at least within reasonable time frames.", "The present invention is directed to overcoming this limitation by increasing the effective area coated by the combustion source, thereby increasing the overall rate of the deposition or powder production to a level appropriate for manufacturing processes.", "The apparatus of the invention is useful in any deposition process in which a liquid is atomized and the atomized liquid is used to form a coating.", "While a flame is one energy source that may be used to promote chemical reaction of a precursor chemical(s) in liquid form, other energy sources, such as heated gases, induction heaters, etc.", "may be used, particularly if a non-oxidizing reaction is to be promoted.", "SUMMARY OF THE INVENTION To achieve the above objectives, the present invention provides for methods and apparatus that include at least one increased dimension of the combustion source, particularly for CCVD processes.", "It should be understood that the various embodiments of the present invention are useful for other methods of deposition such as pyrolytic spray or CVD, and the following detailed description is most specific to the CCVD method for simplicity only.", "Furthermore, the described devices are also useful in the production of powders when used in conjunction with well known powder collection apparatus.", "When used to deposit coatings, the present invention allows a greater area to be coated by a single pass of the CCVD apparatus.", "A first embodiment is an integrated discrete linear flame that is comprised of a plurality of CCVD nozzles aligned in a linear array.", "Each of the discrete nozzles must be precisely controlled in terms of pressure and temperature to insure a uniform deposition rate and composition across the width of the CCVD apparatus.", "A second embodiment of the apparatus is in the form of a continuous linear flame wherein vaporized coating material is fed into an extended tube with a flame slit extending along the length of the tube.", "The coating material ignites as it exits the slit, thereby forming a continuous linear flame that provides a uniform deposition rate and composition along the length of the flame.", "Both of the embodiments provide an efficient method of producing a large area uniform coating on large substrates from a single chemistry solution, thus increasing deposition rates to a level suitable for manufacturing purposes.", "In the first embodiment, several CCVD nozzles are arranged side-by-side.", "The typical deposition area or “footprint” of a single CCVD nozzle is dependent on several factors including but not limited to the material being deposited and distance from the substrate.", "In its simplest form, the multiple nozzle array consists of two deposition nozzles.", "The second nozzle may only increase the deposition area by a factor of 1.25, due to interaction effects between the two nozzles.", "The actual material throw rate, however, will typically increase by a factor close to 2.0.In order to maximize the coating uniformity, deposition area, and overall deposition material throw rate, the spacing between nozzles must be determined through experimentation for each particular application.", "Should the distance between nozzles for one of these factors interfere with another requirement, (such as maximizing deposition area at the expense of uniformity), two banks of nozzles may be arranged in succession, with the centerlines of the nozzles being offset to provide uniform coverage.", "To provide uniform deposition between the multiple nozzles, the temperature and flow of each nozzle must be held constant with reference to the other nozzles.", "To achieve this requirement, back-pressure regulators and a block heater are employed.", "Each of the nozzles is fed from a common, chemistry solution, distribution manifold.", "Between the manifold and each nozzle, a back-pressure regulator is provided.", "The back-pressure regulator may be a standard pressure regulator, a needle valve, or a coiled tubing.", "Each of the nozzles has an inherent pressure drop in the fluid as it flows from the common manifold and out the exit of the nozzle.", "If this pressure drop is for example 100 psi in one nozzle, and 200 psi in a second nozzle, then the flow rate differential between these nozzles would be 50% (assuming an equal cross sectional flow area).", "By providing back-pressure regulators that increase the pressure drop to around 1000 psi, this same pressure drop difference of 100 psi only causes a pressure drop variation of 900 to 100 psi or a flow rate differential of 10%.", "In the preferred embodiment, the back-pressure regulators are in the form of coiled, small inner diameter tubes, the lengths of which determine the pressure drop for each nozzle.", "Thus to adjust for equal pressure drops and resulting uniform deposition material flow, the relative length of each tube is adjusted accordingly.", "More specifically, since the effective flow rate of each orifice from a central manifold is inversely related to its back pressure, it is desired to maintain the back pressure to each line as closely as possible.", "Of particular importance to the present invention is the capability to maintain a uniform flow through multiple orifices from a single delivery system when the pressure drop across the orifices is not equal or as the pressure variation of an orifice varies over time (accumulation of material).", "Such a system exists, as per the present invention, when atomizing by releasing a thermally controlled liquid into a volume that is at a pressure below its boiling point relative to the controlled temperature of the liquid.", "Products to form the orifice are made with a large variation which results in different back pressures at the desired flow rate.", "Used alone, these orifices do not provide the desired control of the liquid through each nozzle.", "This is further complicated by the issue of heated liquids that can have variable amounts of material forming on the inside surfaces of the nozzles which causes a time variable back pressure at a constant flow rate.", "It is not desired to have a pump or liquid mass flow controllers (if one were found that works at the required pressures and flow rates) for each nozzle as the cost and maintenance becomes prohibitive.", "The current system works by have a precise pressure drop up stream from the nozzle to act in providing uniform flow to each nozzle.", "Downstream of this flow controlling pressure drop the liquid state would still be maintained so that material will not form over time on the walls of the flow controlling section.", "The pressure drop across this flow control region needs to be substantially higher than that of the orifice, so that any orifice pressure changes are minor in comparison to the constant pressure of the flow control section.", "Thus if orifice back pressure can vary from 100 psi to 300 psi, then the flow control pressure drop must be at least 2000 psi (10×) to maintain at least a 10% or better flow control through each nozzle.", "For even more uniform flow control, a 4000 psi (20×) pressure drop can be used (5% flow variation).", "For yet even more uniform flow control, a 10000 psi (50×) pressure drop can be used (2% flow variation).", "In some cases it will be desired to go to even a 100× factor (this would be a manifold pressure of 20,000 psi with a resultant pressure of 100 to 300 psi after the flow control section for the above example).", "Of course as higher pressures are needed the cost of the system increases, so added tolerance comes with a cost, complexity and size impact.", "This above example, without the flow control system of the present invention, would have a flow variation of about +/−50%.", "Available components (tubing, fittings, pumps, valves, etc.)", "for the present systems have significant price jumps at 5000, 10000, and 20000 psi.", "Thus there is desire to design a system with the exact level of flow variation required to produce the desired coating or powder, to avoid excessive costs.", "The flow rate of the deposition material is also dependent on other factors, such as density and viscosity, that are in turn dependent on the temperature of the fluid.", "In order to maintain a similar temperature between the nozzles, a block heater is used.", "Each of the nozzles and a thick walled tube leading to the nozzle from the coiled tube is encased in a block of material that is thermally conductive (such as a dense metal).", "Alternatively, precision-machined orifices may be drilled into the material to form the nozzles and passageway between the nozzles and the coiled tubes.", "Resistive element heaters are used to heat the block of material such that the fluid flowing through the nozzles is brought to a thermodynamically metastable state.", "A liquid is in a metastable state if its temperature at the exit of the nozzle is higher than its saturation temperature for a given pressure.", "By heating the fluid to this temperature, rapid expansion of the liquid is achieved which results in quick and uniform atomization of the precursor material.", "It should also be understood from the following description, that the apparatus and methods of the present invention can be used to form coatings using deposition techniques other than CCVD, as the use of a combustion source is not necessary for forming some materials.", "The linear deposition apparatus provides a uniform material deposition rate along its length, thereby forming a more uniform coating than could be previously achieved using prior art devices and techniques.", "This is due to the pressure and temperature regulation provided between the array of nozzles that form the integrated linear deposition apparatus.", "A second embodiment of the present invention provides a continuous linear deposition apparatus for applying coatings using precursor chemical-containing fluids.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a top view of an integrated, discrete flame deposition apparatus of the present invention.", "FIG.", "2 is a side view of the deposition apparatus of FIG.", "1.FIG.", "3 is a top view of a continuous flame deposition apparatus of the present invention.", "FIG.", "4 a side view of the continuous flame deposition apparatus of FIG.", "3.FIG.", "5 is a top view of an alternative embodiment of the continuous flame deposition apparatus.", "FIG.", "6 is a top view of a further alternative embodiment of the continuous flame deposition apparatus.", "FIG.", "7 is a diagram showing a circular arrangement of nozzles.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The integrated nozzle embodiment 100 of the present invention is illustrated in FIGS.", "1 and 2.The integrated discrete flame deposition apparatus 100 includes a main body portion 116 having a plurality of discrete nozzles 102, such as those used for CCVD, linearly arranged across the width of main body portion 116.Each of the CCVD nozzles 102 includes a central atomizing liquid delivery tube 104 surrounded by a number of oxygen (or other deposition gas) delivery orifices 106.On both sides of each nozzle 102 a pilot flame nozzle 114 is located.", "Each of the pilot flame nozzles 114 includes an orifice 108 that provides a combustible gas (such as methane propane, hydrogen, etc.)", "that is ignited to form a pilot flame for each CCVD nozzle 102.While some precursor solutions are combustible enough to maintain a flame, others may require pilot flames to ensure constant and uniform burning of the precursor solution that exits orifice 104.Therefore pilot flame nozzles may not be required, or more than two pilot flame nozzles may be needed for each CCVD nozzle, or alternatively, the pilots may be in the form of a continuous flame provided from a slit-shaped nozzle.", "Each of the pilot flame nozzles 114 are rotatably mounted to main body portion 116 such that they can pivot about axis 200.This allows the spacing between each pilot flame nozzle 114 and CCVD nozzle 102 to be adjusted for optimum performance.", "Conduits 112 and 110 convey the appropriate gas to the tip oxygen orifices 106 and the pilot flame orifices 108, respectively.", "The conduits need to be of sufficient size to enable uniform flow through all orifices.", "To maintain a uniform precursor flow rate between the nozzles 102 (and therefore a more uniform coating), each liquid delivery tube 104 is attached to a liquid distribution manifold 208 via a flow equalization tube 206.Each of the flow equalization tubes includes a pressure regulating means 212, shown here as a loop of tubing, although other means may be used to equalize the flow between nozzles.", "For examples, the tubes could be of equal length, but different interior diameter, although it is most convenient to adjust tube length rather than diameter.", "Or the sizes of openings from manifold 208 could be of different, but precisely measured size.", "In order to increase the pressure drop between the liquid distribution manifold 208 and a liquid delivery tube 104, a longer length, larger diameter loop 216 is formed in the flow equalization tube 206.Conversely, in order to decrease the pressure drop between the liquid distribution manifold 208 and a liquid delivery tube 104, a smaller diameter loop 214 is formed in the flow equalization tube 206.By providing large pressure changes through the precise flow equalization tubes going into the liquid delivery tubes 104, equal flow rates of liquid can be realized between the nozzles 102 (assuming equal liquid temperatures).", "A liquid supply tube 210 delivers the liquid that is pumped by pump 213 from liquid reservoir 211 to a liquid distribution manifold 208.FIG.", "2 shows only one of flow equalization tube 206 exiting the manifold 208; however it is to be understand that all of the equalization tubes 206 that feed the nozzles 104 of the array exit the manifold 208.The pump 213 pressurizes the liquid so that the liquid exhibits a large pressure drop as it eventually exits the flow equalization tube 206 and another, smaller, pressure drop across the nozzle 104.Should active pressure regulation be required, pressure regulating valves and sensing means can be used in conjunction with or in place of the flow equalization tubes 206.The liquid may simply be a liquid material that forms a coating without reaction, e.g., a solution and/or suspension of a material that is to be deposited on a substrate.", "Controlled atomization will help to provide a uniform coating in such case.", "In the illustrated apparatus adapted for CCVD, the liquid in reservoir 211 is a solution of one or more precursor chemicals which, in conjunction with an oxidizing agent, particularly oxygen, undergoes a flame reaction that produces the material that is deposited as the coating.", "Energy sources other than flame may be used to react precursor chemicals in atomized liquids so as to produce coating materials.", "A very important advantage of the above-described apparatus is the ability to precisely control the flow rates through multiple outlets using back-pressure regulators for individual supply lines.", "This provides ability to induce controlled pressure drop in each supply line that is at least 5-10 times larger than the pressure drop in the discharge nozzle.", "The goal of the feed design it to minimize the effects of these pressure variations that can vary as nozzle orifices 104 are changed and as the nozzle ages.", "An important aspect of the present invention is the temperature of the precursor solution as it exits the orifices 104.One method to maintain a uniform temperature for the precursor solution is by heating the main body portion 116 using heating elements 202 that are embedded therein.", "The heating elements 202 are supplied electrical power via wires 204.Wires 204 may include two conductors for each element.", "Alternatively, the elements may be electrically grounded by the main body portion 116 if it is formed from electrically conductive material, in which case only a single conductor is needed to power each element 202.As the main body portion 116 is constructed of thermally conductive material, the elements heat the entire main body portion 116 as well as the liquid delivery tubes 104, and the liquid flowing therethrough.", "Of course it should be understood that the liquid delivery tubes 104 may be in the form of small orifices drilled or otherwise formed directly through the main body portion 116, without the need for separate tubes.", "In either case, by heating the main body portion, each nozzle delivers the liquid solution at the same temperature thereby greatly increasing the uniformity of the resulting coating.", "The degree of atomization, i.e., the droplet size, is determined in part by the temperature of the solution in the nozzle; the higher the temperature, the smaller the droplet size.", "Another important aspect to consider in constructing the integrated discrete flame deposition apparatus 100 of FIGS.", "1 and 2, is the distance A between the centers of adjacent nozzles 102.Considering the apparatus in its simplest embodiment (a two-nozzle array or integrated system) the effective deposition area is increased by at least a factor of 1.25 over a single nozzle system.", "While the effective area or deposition width is not necessarily increased by a factor of two due to possible interaction effects between the nozzles, the overall deposition material throw rate may increase by a factor close to two.", "Each nozzle, in its simplest form, typically has a radial bell distribution of coating thickness, the exact footprint being dependent upon the material being deposited and the exact processing parameters.", "Some experimentation is often needed to determine the optimum spacing A between the nozzles such that the coating uniformity, deposition area and overall deposition material throw rate are optimized.", "There may be inherent compromises between these factors that will be application dependent.", "Any limitations in optimizing this distance can be overcome by using additional arrays or nozzles with offset positioning of the centerlines of the nozzles between the two or more deposition apparatuses 100.The integrated nozzle apparatus described above is particularly suited to vapor deposition (such as CCVD) or spray deposition methods, such as pyrolytic spray onto large substrates, e.g., glass and sheet material.", "The deposition material throw rate and coating uniformity of the single-flame CCVD system were previously limited by the size of the deposition apparatus/nozzle.", "In a production environment, an array of flames has the potential to improve deposition material throw rate and uniformity due to increased flow precursor throw rates by using larger deposition zones.", "The integrated nozzle or discrete nozzle array provides uniform and efficient atomization and delivery of the liquid solution across a relatively large deposition area.", "It is important to note that the invention allows flexibility in designing the nozzle geometry (linear bank of flames, radial distribution of flames, or rastering arrangement), and almost unlimited expansion in the size.", "Because the individual supply tubes are connected (in parallel) to a common manifold, the additional lines do not result in increased pressure required by the pump.", "The manifold needs to be of sufficient size to enable little pressure variations along its length, so that the flow is better regulated.", "This feature of the present invention makes it particularly attractive for scale-up applications where high deposition rates and uniform large coverage areas are critical, using deposition equipment having a variable pressure drop component.", "The linear array of nozzles is shown in an array geometry particularly advantageous for many coating applications, such as for coating a continuously moving web of material.", "Other array geometries may be used for particular coating applications.", "For example, as shown in FIG.", "7, an array of nozzles 700 may be arranged in a circle for rapidly coating individual work pieces or for coating a strand 704 extending through center opening 702, or for coating the inner surface of a circular substrate 706.A partial circle (curve) geometry may also be used.", "Regardless of geometry of the array, equalization of temperature and flow promote uniform coating.", "The specific examples of form and shape are not to be deemed as limiting, as a wide range of forms and shapes are desired (as a many forms and shapes of substrates and powder collecting means can be used).", "In FIGS.", "3 and 4, a second embodiment of the deposition apparatus 300 of the present invention is illustrated.", "In this embodiment, a continuous linear flame is produced using a burner 302 having a central linear burner slit 304 extending longitudinally along one side of the tube.", "The illustrated burner 302 is a tube defining an interior gas chamber from which the gaseous or vaporized materials exit through the slit 302.The term “linear slit” in this context is intended to mean a slit that is at least 5 times the dimension in the linear dimension than its with, preferably at least 20 times, but often much longer than its width.", "A corrugated member or diffusion grating 303 within the slit 304 assists in maintaining a steady and uniform flame along the length of the slit 304.The grating 303 prevents fluttering of the flame along the burner slit 304.A gaseous precursor mixture is fed to the burner 302 from a mixing manifold 308 through an conical connector 306.In the illustrated embodiment, the mixing manifold 308 is fed by a conduit 310 that may contain a flammable gas that provides the main thermal energy and a conduit 312 that carries a gas-carried precursor solution.", "In the illustrated embodiment, the conduit 312 is fed by conduits 402 and 404.One of these conduits 402 may feed air that entrains precursor solution from a reservoir (not shown), such as a bubbler or sublimer, and the other conduit 404 may carry a fuel and/or oxygen to form a combustible mixture.", "Conduit 312 is shown passing through a heating unit 314, e.g., an inductive heater, to preheat the gases passing therethrough.", "Although not shown in the illustrated embodiment, a pre-heater could be used to heat the gas-carried precursor in conduit 310.For apparatus efficiency, however, generally only one of the gas streams is pre-heated, as sufficient heat may be provided to only one of the gas streams to provide the desired pre-heating energy for efficient operation.", "Such preheating of some or all of the gases may help to reduce the amount of fuel required for combustion and to more precisely control the vapors produced in the flame.", "At the end of the burner unit 302 is an overpressure relief device 316 that may simply be a rupture-able diaphragm 318 held between a pair of plates.", "Alternatively, a more elaborate relief valve may be used.", "While not required to form the coating, it may be beneficial to have exhaust gasses exit through exhaust plenums 320 and then through exhaust conduits 328.In a preferred mode of operation, exhaust conduits 328 are each connected to a vacuum (not shown).", "To more precisely control the exhaustion of gases, the illustrated plenums each have a rotating exhaust plate 324 that controls the size of the exhaust slots 322.The rotating plates in the illustrated embodiment are manipulated by set screws 326.By controlling the exhaust to each side of the burner unit, the depositing gasses can be evenly spread out as they exit the burner.", "Because of the large volume of space in the burner unit 302, the pressure through the burner slit 304 is generally equal from one end to the other.", "If greater equality of pressure is desired, various means may be used to more precisely equalize pressure along the length of the burner slot.", "For example, the burner slit 304 could be slightly wider at the downstream end than the upstream end.", "Or the burner unit 302 could be connected to the mixing manifold 308 from a central location behind the burner slit 304.The mixing manifold 308 should be of sufficient cross sectional size, that little variation in pressure exists along its length.", "It is also possible to have other configurations of the elongated burner slit, in which case the burner may be rectangular in cross section such that the top face of the burner is flat as shown by dotted line in FIG.", "4.For example, the slit could be of a wave configuration or a substantially circular configuration (with a center portion supported by struts).", "While the illustrated apparatus mixes oxidizing gas (oxygen) with the vaporized fuel/precursor mixture prior to introduction of the gaseous components into the burner unit 302, external oxygen in the air could be relied upon to maintain combustion of fuel/precursor exiting the slit 304.Illustrated in FIG.", "5 is an alternative embodiment of a linear spray apparatus 300′ apparatus of the present invention.", "In this embodiment, the linear slit 304 of FIG.", "3 is replaced by a linear array of orifices 305 which are spaced sufficiently close together so as to provide a generally continuous linear flame.", "In this embodiment, the fluid in the burner chamber 302 could be either in gaseous or liquid form.", "With the correct combination of orifices 305 of appropriately small size, temperature and liquid pressure, the liquid would atomize as it exited the chamber.", "Although the FIG.", "3-4 and FIG.", "5 embodiments are illustrated as linear, either the slit 304 (FIG.", "3-4) or linear array of orifices 305 (FIG.", "5) could be of other geometric arrangement, such as circular (as shown in FIG.", "7 with respect to the embodiment of FIGS.", "1 an 2), square or rectangular.", "To facilitate non-linear slit 304 or orifice array 305 configurations, the burner chamber might have a flat top face from which fluid exits the chamber through the slit 304 or orifice array 305.Illustrated in FIG.", "6 is a further alternative embodiment 300″ of the present invention.", "In this case, the burner face 352 is flat and rectangular, with the burner having a rectangular cross section (shown by dotted line in FIG.", "4).", "An array of very small orifices 305′, in this case in a rectangular configuration array, provide the fluid outlet and flame source.", "While gases could be burned in this burner, the FIG.", "6 embodiment is shown with a fluid conduit 348 leading to the connector 306; the conduit carries liquid pressurized by pump 350.While many possible arrays can be used, the linear arrays 305′ in FIG.", "6 are shown with offset centerlines to provide uniform coverage of the coating." ] ]	Patent_10467139
[ [ "Method of using a cyclooxygenase-2 inhibitor and sex steroids as a combination therapy for the treatment and prevention of dismenorrhea", "The present invention provides methods for the treatment and prevention of dysmenorrhea in a woman using a combination of a cyclooxygenase-2 inhibitor and sex steroids." ], [ "1.A therapeutic combination comprising an amount of a COX-2 inhibitor compound source and an amount of a sex steroid compound wherein the amount of a COX-2 inhibitor compound source and the amount of the sex steroid compound together comprises a dysmenorrhea-effective amount of the compounds.", "2.The combination of claim 1 wherein the COX-2 inhibitor source is a COX-2 inhibitor.", "3.The combination of claim 2 wherein the COX-2 inhibitor is a tricyclic COX-2 inhibitor.", "4.The combination of claim 3 wherein the tricyclic COX-2 inhibitor is selected from the group consisting of a pyrazole COX-2 inhibitor, a furanone COX-2 inhibitor, an isoxazole COX-2 inhibitor, a pyridine COX-2 inhibitor, and a pyridazinone COX-2 inhibitor.", "5.The combination of claim 4 wherein the tricyclic COX-2 inhibitor is a pyrazole COX-2 inhibitor.", "6.The combination of claim 5 wherein the tricyclic COX-2 inhibitor is celecoxib.", "7.The combination of claim 5 wherein the tricyclic COX-2 inhibitor is deracoxib.", "8.The combination of claim 4 wherein the tricyclic COX-2 inhibitor is a furanone COX-2 inhibitor.", "9.The combination of claim 8 wherein the tricyclic COX-2 inhibitor is rofecoxib.", "10.The combination of claim 4 wherein the tricyclic COX-2 inhibitor is an isoxazole COX-2 inhibitor.", "11.The combination of claim 10 wherein the tricyclic COX-2 inhibitor is valdecoxib.", "12.The combination of claim 4 wherein the tricyclic COX-2 inhibitor is a pyridine COX-2 inhibitor.", "13.The combination of claim 12 wherein the tricyclic COX-2 inhibitor is 5-chloro-6′-methyl-3-[4-(methylsulfonyl)phenyl]-2,3′-bipyridine.", "14.The combination of claim 4 wherein the tricyclic COX-2 inhibitor is a pyridazinone COX-2 inhibitor.", "15.The combination of claim 14 wherein the pyridazinone COX-2 inhibitor is 2-(3,4-difluorophenyl)-4-(3-hydroxy-3-methylbutoxy)-5-[4-(methylsulfonyl)phenyl]-3(2H)-pyridazinone.", "16.The combination of claim 2 wherein the COX-2 inhibitor is a benzopyran COX-2 inhibitor.", "17.The combination of claim 2 wherein the COX-2 inhibitor is a methane sulfonanilide COX-2 inhibitor.", "18.The combination of claim 17 wherein the methane sulfonanilide COX-2 inhibitor is N-(4-nitro-2-cyclohexyloxyphenyl)methanesulfonamide.", "19.The combination of claim 1 wherein the COX-2 inhibitor source is a prodrug of a COX-2 inhibitor.", "20.The combination of claim 19 wherein the prodrug of the COX-2 inhibitor is parecoxib.", "21.The combination of claim 1 wherein the sex steroid compound is a progestin sex steroid.", "22.The combination of claim 1 wherein the sex steroid compound is an estrogen sex steroid.", "23.The combination of claim 22 wherein the sex steroid compound further comprises a progestin sex steroid.", "24.The combination of claim 23 wherein the sex steroid compound comprises an amount of an estrogen sex steroid and an amount of a progestin sex steroid wherein the amount of the estrogen sex steroid and the amount of the progestin sex steroid together comprise a menstrual cycle controlling-effective amount of the compounds.", "25.The combination of claim 24 wherein the estrogen sex steroid is ethinyl estradiol.", "26.The combination of claim 24 wherein the progestin sex steroid is selected from the group consisting of levonorgestrel, norethindrone acetate, norgestimate, ethynodiol acetate, desogestrel, norgestrel and norethindrone.", "27.The combination of claim 26 wherein the progestin sex steroid is levonorgestrel.", "28.The combination of claim 26 wherein the progestin sex steroid is norethindrone acetate.", "29.The combination of claim 26 wherein the progestin sex steroid is norgestimate.", "30.The combination of claim 26 wherein the progestin sex steroid is ethynodiol acetate.", "31.The combination of claim 26 wherein the progestin sex steroid is desogestrel.", "32.The combination of claim 26 wherein the progestin sex steroid is norgestrel.", "33.The combination of claim 26 wherein the progestin sex steroid is norethindrone.", "34.The combination of claim 1 wherein the COX-2 inhibitor compound source and the sex steroid compound are present in a single composition.", "35.A combination therapy method for the treatment or prophylaxis of dysmenorrhea in a patient in need thereof, comprising: administering to the patient an amount of a COX-2 inhibitor compound source and administering to the patient an amount of a sex steroid compound wherein the amount of the COX-2 inhibitor compound source and the amount of the sex steroid compound together comprise a dysmenorrhea-effective amount of the compounds 36.The combination therapy method of claim 35 wherein the COX-2 inhibitor source is a COX-2 inhibitor.", "37.The combination therapy method of claim 36 wherein the COX-2 inhibitor compound is celecoxib.", "38.The combination therapy method of claim 36 wherein the COX-2 inhibitor compound is rofecoxib.", "39.The combination therapy method of claim 36 wherein the COX-2 inhibitor compound is valdecoxib.", "40.The combination therapy method of claim 36 wherein the COX-2 inhibitor compound is deracoxib.", "41.The combination therapy method of claim 36 wherein the COX-2 inhibitor compound is 5-chloro-6′-methyl-3-[4-(methylsulfonyl)phenyl]-2,3′-bipyridine.", "42.The combination therapy method of claim 36 wherein the COX-2 inhibitor compound is N-(4-nitro-2-phenoxyphenyl)methanesulfonamide.", "43.The combination therapy method of claim 36 wherein the COX-2 inhibitor compound is 2-(3,4-difluorophenyl)-4-(3-hydroxy-3-methylbutoxy)-5-[4-(methylsulfonyl)phenyl]-3(2H)-pyridazinone.", "44.The combination therapy method of claim 35 wherein the COX-2 inhibitor source is a prodrug of a COX-2 inhibitor.", "45.The combination therapy method of claim 44 wherein the prodrug of the COX-2 inhibitor is parecoxib.", "46.The combination therapy method of claim 35 wherein the sex steroid compound comprises an amount of an estrogen sex steroid and an amount of a progestin sex steroid wherein the amount of the estrogen sex steroid and the amount of the progestin sex steroid together comprise a menstrual cycle controlling-effective amount of the compounds.", "47.The combination therapy method of claim 46 wherein the estrogen sex steroid is ethinyl estradiol.", "48.The combination therapy method of claim 46 wherein the progestin sex steroid is selected from the group consisting of levonorgestrel, norethindrone acetate, norgestimate, ethynodiol acetate, desogestrel, norgestrel and norethindrone.", "49.The combination therapy method of claim 48 wherein the progestin sex steroid is levonorgestrel.", "50.The combination therapy method of claim 48 wherein the progestin sex steroid is norethindrone acetate.", "51.The combination therapy method of claim 48 wherein the progestin sex steroid is norgestimate.", "52.The combination therapy method of claim 48 wherein the progestin sex steroid is ethynodiol acetate.", "53.The combination therapy method of claim 48 wherein the progestin sex steroid is desogestrel.", "54.The combination therapy method of claim 48 wherein the progestin sex steroid is norgestrel.", "55.The combination therapy method of claim 48 wherein the progestin sex steroid is norethindrone." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to methods for the treatment and prevention of dysmenorrhea in a woman using a combination of a cyclooxygenase-2 inhibitor and sex steroids.", "2.Description of the Related Art In women, the menstrual cycle involves a complex series of hormonal changes.", "A consequence of these hormonal changes is the growth of the uterine lining (referred to as the endometrium).", "In the absence of pregnancy, the endometrium is shed in a process called menstruation.", "This process involves the release of prostaglandins, which cause contractions of the smooth muscle in the uterus.", "In some women, these contractions cause substantial pain, dysmenorrhea, which interferes with their daily activities.", "The time at which menstruation occurs varies in that it can not be predicted with certainty in any one woman.", "The variability in the onset of menstrual cycles is dependent upon many variables including the individual woman, her age and underlying medical and psychosocial conditions.", "This makes it difficult to predict the onset of menses.", "Non-steroidal anti-inflammatory agents (NSAIDs) that inhibit prostaglandin synthesis are effective in reducing dysmenorrhea (Lundstrom, V., et al.", "Acta Obstet.", "Gynecol.", "Scand.", "Suppl., 113, 83-85 (1983)).", "They are most effective when administered prior to the onset of menstrual pain by 24-48 hours.", "Since predicting the precise timing of menstruation is difficult, attempts to maximize efficacy by initiating treatment prior to menses may result in several days of unnecessary medication.", "The use of orally active contraceptives, composed of estrogen and progestin components, has been reported to reduce the intensity of the pain of dysmenorrhea (Nabrink, M. et al.", "Contraception, 42, 275-283 (1990)).", "The vast majority of oral contraceptives consist of a combination of a progestin sex steroid and an estrogen sex steroid.", "These sex steroids are administered concurrently for 21 days followed by either a 7 day pill free interval or by the administration of a placebo for 7 days in each 28 day cycle.", "Numerous regimens have been developed in which the progestin/estrogen combination is administered either as a fixed dosage combination (monophasic) or as a biphasic or a triphasic regimen in which the dosage of the combination is varied either once or twice throughout the menstrual cycle.", "Kuhl has reviewed the current state of hormonal contraception ( Handb.", "Exp.", "Pharmacol., 135/II, 363-407 (1999)).", "Various oral contraceptive combinations are listed in WO 98/04265.Most current oral contraceptives give good menstrual cycle control (Thorneycroft, I.", "Am.", "J. Obstet.", "Gynecol., 180 (2, Pt.", "2), S280-S287 (1999)).", "When good relief of dysmenorrhea is not obtained through the use of oral contraceptives, a nonsteroidal anti-inflammatory drug can be added as treatment (Deligeoroglou, E. Annals of the New York Academy of Science, 900, 237-244 (2000)).", "Prostaglandins play a major role in the inflammation process and the inhibition of prostaglandin production, especially production of PGG2, PGH2 and PGE2, has been a common target of anti-inflammatory drug discovery.", "However, common non-steroidal anti-inflammatory drugs (NSAIDs) that are active in reducing the prostaglandin-induced pain and swelling associated with the inflammation process are also active in affecting other prostaglandin-regulated processes not associated with the inflammation process.", "Thus, use of high doses of most common NSAIDs can produce severe side effects, including life-threatening ulcers, which limit their therapeutic potential.", "An alternative to NSAIDs is the use of corticosteroids, which have even more drastic side effects, especially when long-term therapy is involved.", "Previous NSAIDs have been found to prevent the production of prostaglandins by inhibiting enzymes in the human arachidonic acid/prostaglandin pathway, including the enzyme cyclooxygenase (COX).", "The recent discovery of an inducible enzyme associated with inflammation (named “cyclooxygenase II (COX II)” or “prostaglandin G/H synthase II”) provides a viable target of inhibition that more effectively reduces inflammation and produces fewer and less drastic side effects.", "U.S. Pat.", "No.", "5,466,823 discloses pyrazolyl cyclooxygenase-2 inhibitors useful in treating inflammation and inflammation-related disorders, including menstrual cramps.", "U.S. Pat.", "No.", "5,932,598 discloses prodrugs of cyclooxygenase-2 inhibitors useful in treating inflammation and inflammation-related disorders, including menstrual cramps.", "Morrison et al.", "describe a study where the cyclooxygenase-2 inhibitor, rofecoxib, is used to treat primary dysmenorrhea ( Obstet.", "Gynecol., 94(4), 504-508 (1999)).", "Compounds that selectively inhibit cyclooxygenase-2 and are useful in treating menstrual cramps have also been described in the following individual publications.", "U.S. Pat.", "No.", "5,521,207.U.S.", "Pat.", "No.", "5,633,272.The various classes of compounds that are selective inhibitors of cyclooxygenase-2 have been reviewed by J. Talley in Prog.", "Med.", "Chem., 36, 201-234 (1999).", "Compounds that selectively inhibit cyclooxygenase-2 have also been described in the following individual publications.", "U.S. Pat.", "No.", "5,380,738.U.S.", "Pat.", "No.", "5,344,991.U.S.", "Pat.", "No.", "5,393,790.U.S.", "Pat.", "No.", "5,434,178.U.S.", "Pat.", "No.", "5,474,995.U.S.", "Pat.", "No.", "5,510,368.WO 96/06840.WO 96/03388.WO 96/03387.WO 96/19469.WO 96/25405.WO 95/15316.WO 94/15932.WO 94/27980.WO 95/00501.WO 94/13635.WO 94/20480.WO 94/26731.The combination of NSAIDs and oral contraceptives has been used in cases where neither treatment alone was effective in treating primary dysmenorrhea (Coco, A., American Family Physician, 60(2), 489-496 (1999)).", "U.S. Pat.", "No.", "5,811,416 discloses the combination of an endothelin antagonist and/or an endothelin synthase inhibitor with at least one of a progestin, an estrogen, a combination of a progestin and estrogen, a cyclooxygenase inhibitor, a nitric oxide donor or a nitric oxide substrate for the treatment of menstrual disorders including dysmenorrhea.", "U.S. Pat.", "No.", "5,912,006 discloses the combination of an omega fatty acid and a cyclooxygenase inhibitor for the reduction or alleviation of uterine or vaginal pain associated with the onset of menstruation.", "However, a combination therapy method for the treatment and prevention of dysmenorrhea comprising a COX-2 inhibitor and sex steroids has not been previously described." ], [ "<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>To address the continuing need to find safe and effective agents for the prophylaxis and treatment of dysmenorrhea, combination therapies of therapeutic agents are now reported.", "Among its several embodiments, the present invention provides a therapeutic combination of a cyclooxygenase-2 inhibitor compound source and an amount of sex steroid compounds, wherein the compounds together comprise a dysmenorrhea-effective amount of the compounds.", "In another embodiment, the cyclooxygenase-2 inhibitor compound source is a cyclooxygenase-2 inhibitor compound.", "In yet another embodiment, the present invention provides a combination therapy method for the treatment or prophylaxis of dysmenorrhea in a patient in need thereof comprising the use of an amount of a cyclooxygenase-2 inhibitor compound and an amount of a sex steroid, wherein the amounts of the cyclooxygenase-2 inhibitor compound and the sex steroid compound together comprise a dysmenorrhea-effective amount of the compounds.", "The invention involves the preventive management of painful uterine cramps, dysmenorrhea, in women.", "A key improvement over existing technologies is that moderate to severe pain is not experienced prior to initiating treatment, but that it can be preempted, providing a much more satisfactory outcome.", "Another advantage is that by employing this regimen, lower doses of analgesic medication may be required.", "There should also be an advantage of a reduced blood loss compared with existing treatments.", "Further scope of the applicability of the present invention will become apparent from the detailed description provided below.", "However, it should be understood that the following detailed description and examples, while indicating preferred embodiments of the invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to methods for the treatment and prevention of dysmenorrhea in a woman using a combination of a cyclooxygenase-2 inhibitor and sex steroids.", "2.Description of the Related Art In women, the menstrual cycle involves a complex series of hormonal changes.", "A consequence of these hormonal changes is the growth of the uterine lining (referred to as the endometrium).", "In the absence of pregnancy, the endometrium is shed in a process called menstruation.", "This process involves the release of prostaglandins, which cause contractions of the smooth muscle in the uterus.", "In some women, these contractions cause substantial pain, dysmenorrhea, which interferes with their daily activities.", "The time at which menstruation occurs varies in that it can not be predicted with certainty in any one woman.", "The variability in the onset of menstrual cycles is dependent upon many variables including the individual woman, her age and underlying medical and psychosocial conditions.", "This makes it difficult to predict the onset of menses.", "Non-steroidal anti-inflammatory agents (NSAIDs) that inhibit prostaglandin synthesis are effective in reducing dysmenorrhea (Lundstrom, V., et al.", "Acta Obstet.", "Gynecol.", "Scand.", "Suppl., 113, 83-85 (1983)).", "They are most effective when administered prior to the onset of menstrual pain by 24-48 hours.", "Since predicting the precise timing of menstruation is difficult, attempts to maximize efficacy by initiating treatment prior to menses may result in several days of unnecessary medication.", "The use of orally active contraceptives, composed of estrogen and progestin components, has been reported to reduce the intensity of the pain of dysmenorrhea (Nabrink, M. et al.", "Contraception, 42, 275-283 (1990)).", "The vast majority of oral contraceptives consist of a combination of a progestin sex steroid and an estrogen sex steroid.", "These sex steroids are administered concurrently for 21 days followed by either a 7 day pill free interval or by the administration of a placebo for 7 days in each 28 day cycle.", "Numerous regimens have been developed in which the progestin/estrogen combination is administered either as a fixed dosage combination (monophasic) or as a biphasic or a triphasic regimen in which the dosage of the combination is varied either once or twice throughout the menstrual cycle.", "Kuhl has reviewed the current state of hormonal contraception (Handb.", "Exp.", "Pharmacol., 135/II, 363-407 (1999)).", "Various oral contraceptive combinations are listed in WO 98/04265.Most current oral contraceptives give good menstrual cycle control (Thorneycroft, I.", "Am.", "J. Obstet.", "Gynecol., 180 (2, Pt.", "2), S280-S287 (1999)).", "When good relief of dysmenorrhea is not obtained through the use of oral contraceptives, a nonsteroidal anti-inflammatory drug can be added as treatment (Deligeoroglou, E. Annals of the New York Academy of Science, 900, 237-244 (2000)).", "Prostaglandins play a major role in the inflammation process and the inhibition of prostaglandin production, especially production of PGG2, PGH2 and PGE2, has been a common target of anti-inflammatory drug discovery.", "However, common non-steroidal anti-inflammatory drugs (NSAIDs) that are active in reducing the prostaglandin-induced pain and swelling associated with the inflammation process are also active in affecting other prostaglandin-regulated processes not associated with the inflammation process.", "Thus, use of high doses of most common NSAIDs can produce severe side effects, including life-threatening ulcers, which limit their therapeutic potential.", "An alternative to NSAIDs is the use of corticosteroids, which have even more drastic side effects, especially when long-term therapy is involved.", "Previous NSAIDs have been found to prevent the production of prostaglandins by inhibiting enzymes in the human arachidonic acid/prostaglandin pathway, including the enzyme cyclooxygenase (COX).", "The recent discovery of an inducible enzyme associated with inflammation (named “cyclooxygenase II (COX II)” or “prostaglandin G/H synthase II”) provides a viable target of inhibition that more effectively reduces inflammation and produces fewer and less drastic side effects.", "U.S. Pat.", "No.", "5,466,823 discloses pyrazolyl cyclooxygenase-2 inhibitors useful in treating inflammation and inflammation-related disorders, including menstrual cramps.", "U.S. Pat.", "No.", "5,932,598 discloses prodrugs of cyclooxygenase-2 inhibitors useful in treating inflammation and inflammation-related disorders, including menstrual cramps.", "Morrison et al.", "describe a study where the cyclooxygenase-2 inhibitor, rofecoxib, is used to treat primary dysmenorrhea (Obstet.", "Gynecol., 94(4), 504-508 (1999)).", "Compounds that selectively inhibit cyclooxygenase-2 and are useful in treating menstrual cramps have also been described in the following individual publications.", "U.S. Pat.", "No.", "5,521,207.U.S.", "Pat.", "No.", "5,633,272.The various classes of compounds that are selective inhibitors of cyclooxygenase-2 have been reviewed by J. Talley in Prog.", "Med.", "Chem., 36, 201-234 (1999).", "Compounds that selectively inhibit cyclooxygenase-2 have also been described in the following individual publications.", "U.S. Pat.", "No.", "5,380,738.U.S.", "Pat.", "No.", "5,344,991.U.S.", "Pat.", "No.", "5,393,790.U.S.", "Pat.", "No.", "5,434,178.U.S.", "Pat.", "No.", "5,474,995.U.S.", "Pat.", "No.", "5,510,368.WO 96/06840.WO 96/03388.WO 96/03387.WO 96/19469.WO 96/25405.WO 95/15316.WO 94/15932.WO 94/27980.WO 95/00501.WO 94/13635.WO 94/20480.WO 94/26731.The combination of NSAIDs and oral contraceptives has been used in cases where neither treatment alone was effective in treating primary dysmenorrhea (Coco, A., American Family Physician, 60(2), 489-496 (1999)).", "U.S. Pat.", "No.", "5,811,416 discloses the combination of an endothelin antagonist and/or an endothelin synthase inhibitor with at least one of a progestin, an estrogen, a combination of a progestin and estrogen, a cyclooxygenase inhibitor, a nitric oxide donor or a nitric oxide substrate for the treatment of menstrual disorders including dysmenorrhea.", "U.S. Pat.", "No.", "5,912,006 discloses the combination of an omega fatty acid and a cyclooxygenase inhibitor for the reduction or alleviation of uterine or vaginal pain associated with the onset of menstruation.", "However, a combination therapy method for the treatment and prevention of dysmenorrhea comprising a COX-2 inhibitor and sex steroids has not been previously described.", "BRIEF SUMMARY OF THE INVENTION To address the continuing need to find safe and effective agents for the prophylaxis and treatment of dysmenorrhea, combination therapies of therapeutic agents are now reported.", "Among its several embodiments, the present invention provides a therapeutic combination of a cyclooxygenase-2 inhibitor compound source and an amount of sex steroid compounds, wherein the compounds together comprise a dysmenorrhea-effective amount of the compounds.", "In another embodiment, the cyclooxygenase-2 inhibitor compound source is a cyclooxygenase-2 inhibitor compound.", "In yet another embodiment, the present invention provides a combination therapy method for the treatment or prophylaxis of dysmenorrhea in a patient in need thereof comprising the use of an amount of a cyclooxygenase-2 inhibitor compound and an amount of a sex steroid, wherein the amounts of the cyclooxygenase-2 inhibitor compound and the sex steroid compound together comprise a dysmenorrhea-effective amount of the compounds.", "The invention involves the preventive management of painful uterine cramps, dysmenorrhea, in women.", "A key improvement over existing technologies is that moderate to severe pain is not experienced prior to initiating treatment, but that it can be preempted, providing a much more satisfactory outcome.", "Another advantage is that by employing this regimen, lower doses of analgesic medication may be required.", "There should also be an advantage of a reduced blood loss compared with existing treatments.", "Further scope of the applicability of the present invention will become apparent from the detailed description provided below.", "However, it should be understood that the following detailed description and examples, while indicating preferred embodiments of the invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.", "DETAILED DESCRIPTION OF THE INVENTION The following detailed description is provided to aid those skilled in the art in practicing the present invention.", "Even so, this detailed description should not be construed to unduly limit the present invention as modifications and variations in the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.", "The contents of each of the references cited herein, including the contents of the references cited within these primary references, are herein incorporated by reference in their entirety.", "Definitions The following definitions are provided in order to aid the reader in understanding the detailed description of the present invention.", "The phrase “cyclooxygenase-2 inhibitor” or “COX-2 inhibitor” or “cyclooxygenase-II inhibitor” includes agents that specifically inhibit a class of enzymes, cyclooxygenase-2, with less significant inhibition of cyclooxygenase-1.Preferably, it includes compounds that have a cyclooxygenase-2 IC50 of less than about 0.2 μM, and also have a selectivity ratio of cyclooxygenase-2 inhibition over cyclooxygenase-1 inhibition of at least 50, and more preferably of at least 100.Even more preferably, the compounds have a cyclooxygenase-1 IC50 of greater than about 1 μM, and more preferably of greater than 10 μM.", "The phrase “sex steroids” includes both estrogen and progestin steroid compounds.", "The phrase “combination therapy” (or “co-therapy”) embraces the administration of a cyclooxygenase-2 inhibitor and a sex steroid as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these therapeutic agents.", "The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents.", "Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).", "“Combination therapy” generally is not intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention.", "“Combination therapy” is intended to embrace administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner.", "Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents.", "Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues.", "The therapeutic agents can be administered by the same route or by different routes.", "For example, a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally.", "Alternatively, for example, all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection.", "The sequence in which the therapeutic agents are administered is not narrowly critical.", "“Combination therapy” also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies.", "The phrase “therapeutically effective” is intended to qualify the combined amount of inhibitors in the combination therapy.", "This combined amount will achieve the goal of reducing or eliminating dysmenorrhea.", "“Therapeutic compound” means a compound useful in the prophylaxis or treatment of dysmenorrhea.", "The term “comprising” means “including the following elements but not excluding others.” The term “hydrido” denotes a single hydrogen atom (H).", "This hydrido radical may be attached, for example, to an oxygen atom to form a hydroxyl radical or two hydrido radicals may be attached to a carbon atom to form a methylene (—CH2—) radical.", "Where used, either alone or within other terms such as “haloalkyl”, “alkylsulfonyl”, “alkoxyalkyl” and “hydroxyalkyl”, the term “alkyl” embraces linear or branched radicals having one to about twenty carbon atoms or, preferably, one to about twelve carbon atoms.", "More preferred alkyl radicals are “lower alkyl” radicals having one to about ten carbon atoms.", "Most preferred are lower alkyl radicals having one to about six carbon atoms.", "Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl and the like.", "The term “alkenyl” embraces linear or branched radicals having at least one carbon-carbon double bond of two to about twenty carbon atoms or, preferably, two to about twelve carbon atoms.", "More preferred alkenyl radicals are “lower alkenyl” radicals having two to about six carbon atoms.", "Examples of alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyl and 4-methylbutenyl.", "The term “alkynyl” denotes linear or branched radicals having two to about twenty carbon atoms or, preferably, two to about twelve carbon atoms.", "More preferred alkynyl radicals are “lower alkynyl” radicals having two to about ten carbon atoms.", "Most preferred are lower alkynyl radicals having two to about six carbon atoms.", "Examples of such radicals include propargyl, butynyl, and the like.", "The terms “alkenyl”, “lower alkenyl”, embrace radicals having “cis” and “trans” orientations, or alternatively, “E” and “Z” orientations.", "The term “cycloalkyl” embraces saturated carbocyclic radicals having three to twelve carbon atoms.", "More preferred cycloalkyl radicals are “lower cycloalkyl” radicals having three to about eight carbon atoms.", "Examples of such radicals include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.", "The term “cycloalkenyl” embraces partially unsaturated carbocyclic radicals having three to twelve carbon atoms.", "More preferred cycloalkenyl radicals are “lower cycloalkenyl” radicals having four to about eight carbon atoms.", "Examples of such radicals include cyclobutenyl, cyclopentenyl, cyclopentadienyl and cyclohexenyl.", "The term “halo” means halogens such as fluorine, chlorine, bromine or iodine.", "The term “haloalkyl” embraces radicals wherein any one or more of the alkyl carbon atoms is substituted with halo as defined above.", "Specifically embraced are monohaloalkyl, dihaloalkyl and polyhaloalkyl radicals.", "A monohaloalkyl radical, for one example, may have either an iodo, bromo, chloro or fluoro atom within the radical.", "Dihalo and polyhaloalkyl radicals may have two or more of the same halo atoms or a combination of different halo radicals.", "“Lower haloalkyl” embraces radicals having one to six carbon atoms.", "Examples of haloalkyl radicals include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl.", "The term “hydroxyalkyl” embraces linear or branched alkyl radicals having one to about ten carbon atoms any one of which may be substituted with one or more hydroxyl radicals.", "More preferred hydroxyalkyl radicals are “lower hydroxyalkyl” radicals having one to six carbon atoms and one or more hydroxyl radicals.", "Examples of such radicals include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl and hydroxyhexyl.", "The terms “alkoxy” and “alkyloxy” embrace linear or branched oxy-containing radicals each having alkyl portions of one to about ten carbon atoms.", "More preferred alkoxy radicals are “lower alkoxy” radicals having one to six carbon atoms.", "Examples of such radicals include methoxy, ethoxy, propoxy, butoxy and tert-butoxy.", "The term “alkoxyalkyl” embraces alkyl radicals having one or more alkoxy radicals attached to the alkyl radical, that is, to form monoalkoxyalkyl and dialkoxyalkyl radicals.", "The “alkoxy” radicals may be further substituted with one or more halo atoms, such as fluoro, chloro or bromo, to provide haloalkoxy radicals.", "More preferred haloalkoxy radicals are “lower haloalkoxy” radicals having one to six carbon atoms and one or more halo radicals.", "Examples of such radicals include fluoromethoxy, chloromethoxy, trifluoromethoxy, trifluoroethoxy, fluoroethoxy and fluoropropoxy.", "The term “aryl”, alone or in combination, means a carbocyclic aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused.", "The term “aryl” embraces aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl, indane and biphenyl.", "Aryl moieties may also be substituted at a substitutable position with one or more substituents selected independently from alkyl, alkoxyalkyl, alkylaminoalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, alkoxy, aralkoxy, hydroxyl, amino, halo, nitro, alkylamino, acyl, cyano, carboxy, aminocarbonyl, alkoxycarbonyl and aralkoxycarbonyl.", "The term “heterocyclo” embraces saturated, partially unsaturated and unsaturated heteroatom-containing ring-shaped radicals, where the heteroatoms may be selected from nitrogen, sulfur and oxygen.", "Examples of saturated heterocyclo radicals include saturated 3 to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms (e.g.", "pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl, etc.", "); saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g.", "morpholinyl, etc.", "); saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., thiazolidinyl, etc.).", "Examples of partially unsaturated heterocyclo radicals include dihydrothiophene, dihydropyran, dihydrofuran and dihydrothiazole.", "The term “heteroaryl” embraces unsaturated heterocyclo radicals.", "Examples of unsaturated heterocyclo radicals, also termed “heteroaryl” radicals include unsaturated 3 to 6 membered heteromonocyclic group containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g., 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.)", "tetrazolyl (e.g.", "1H-tetrazolyl, 2H-tetrazolyl, etc.", "), etc.", "; unsaturated condensed heterocyclo group containing 1 to 5 nitrogen atoms, for example, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl (e.g., tetrazolo[1,5-b]pyridazinyl, etc.", "), etc.", "; unsaturated 3 to 6-membered heteromonocyclic group containing an oxygen atom, for example, pyranyl, furyl, etc.", "; unsaturated 3 to 6-membered heteromonocyclic group containing a sulfur atom, for example, thienyl, etc.", "; unsaturated 3- to 6 membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, etc.)", "etc.", "; unsaturated condensed heterocyclo group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g.", "benzoxazolyl, benzoxadiazolyl, etc.", "); unsaturated 3 to 6-membered heteromonocyclic: group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for example, thiazolyl, thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.)", "etc.", "; unsaturated condensed heterocyclo group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., benzothiazolyl, benzothiadiazolyl, etc.)", "and the like.", "The term also embraces radicals where heterocyclo radicals are fused with aryl radicals.", "Examples of such fused bicyclic radicals include benzofuran, benzothiophene, benzopyran, and the like.", "The terms benzopyran and chromene are interchangeable.", "Said “heterocyclo group” may have 1 to 3 substituents such as alkyl, hydroxyl, halo, alkoxy, oxo, amino and alkylamino.", "The term “alkylthio” embraces radicals containing a linear or branched alkyl radical, of one to about ten carbon atoms attached to a divalent sulfur atom.", "More preferred alkylthio radicals are “lower alkylthio” radicals having alkyl radicals of one to six carbon atoms.", "Examples of such lower alkylthio radicals are methylthio, ethylthio, propylthio, butylthio and hexylthio.", "The term “alkylthioalkyl” embraces radicals containing an alkylthio radical attached through the divalent sulfur atom to an alkyl radical of one to about ten carbon atoms.", "More preferred alkylthioalkyl radicals are “lower alkylthioalkyl” radicals having alkyl radicals of one to six carbon atoms.", "Examples of such lower alkylthioalkyl radicals include methylthiomethyl.", "The term “alkylsulfinyl” embraces radicals containing a linear or branched alkyl radical, of one to ten carbon atoms, attached to a divalent —S(═O)-radical.", "More preferred alkylsulfinyl radicals are “lower alkylsulfinyl” radicals having alkyl radicals of one to six carbon atoms.", "Examples of such lower alkylsulfinyl radicals include methylsulfinyl, ethylsulfinyl, butylsulfinyl and hexylsulfinyl.", "The term “sulfonyl”, whether used alone or linked to other terms such as alkylsulfonyl, denotes respectively divalent radicals —SO2—.", "“Alkylsulfonyl” embraces alkyl radicals attached to a sulfonyl radical, where alkyl is defined as above.", "More preferred alkylsulfonyl radicals are “lower alkylsulfonyl” radicals having one to six carbon atoms.", "Examples of such lower alkylsulfonyl radicals include methylsulfonyl, ethylsulfonyl and propylsulfonyl.", "The “alkylsulfonyl” radicals may be further substituted with one or more halo atoms, such as fluoro, chloro or bromo, to provide haloalkylsulfonyl radicals.", "The terms “sulfamyl”, “aminosulfonyl” and “sulfonamidyl” denote NH2O2S—.", "The term “acyl” denotes a radical provided by the residue after removal of hydroxyl from an organic acid.", "Examples of such acyl radicals include alkanoyl and aroyl radicals.", "Examples of such lower alkanoyl radicals include formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, trifluoroacetyl.", "The term “carbonyl”, whether used alone or with other terms, such as “alkoxycarbonyl”, denotes —(C═O)—.", "The term “aroyl” embraces aryl radicals with a carbonyl radical as defined above.", "Examples of aroyl include benzoyl, naphthoyl, and the like and the aryl in said aroyl may be additionally substituted.", "The terms “carboxy” or “carboxyl”, whether used alone or with other terms, such as “carboxyalkyl”, denotes —CO2H.", "The term “carboxyalkyl” embraces alkyl radicals substituted with a carboxy radical.", "More preferred are “lower carboxyalkyl” which embrace lower alkyl radicals as defined above, and may be additionally substituted on the alkyl radical with halo.", "Examples of such lower carboxyalkyl radicals include carboxymethyl, carboxyethyl and carboxypropyl.", "The term “alkoxycarbonyl” means a radical containing an alkoxy radical, as defined above, attached via an oxygen atom to a carbonyl radical.", "More preferred are “lower alkoxycarbonyl” radicals with alkyl portions having 1 to 6 carbons.", "Examples of such lower alkoxycarbonyl (ester) radicals include substituted or unsubstituted methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl and hexyloxycarbonyl.", "The terms “alkylcarbonyl”, “arylcarbonyl” and “aralkylcarbonyl” include radicals having alkyl, aryl and aralkyl radicals, as defined above, attached to a carbonyl radical.", "Examples of such radicals include substituted or unsubstituted methylcarbonyl, ethylcarbonyl, phenylcarbonyl and benzylcarbonyl.", "The term “aralkyl” embraces aryl-substituted alkyl radicals such as benzyl, diphenylmethyl, triphenylmethyl, phenylethyl, and diphenylethyl.", "The aryl in said aralkyl may be additionally substituted with halo, alkyl, alkoxy, haloalkyl and haloalkoxy.", "The terms benzyl and phenylmethyl are interchangeable.", "The term “heterocycloalkyl” embraces saturated and partially unsaturated heterocyclo-substituted alkyl radicals, such as pyrrolidinylmethyl, and heteroarylsubstituted alkyl radicals, such as pyridylmethyl, quinolylmethyl, thienylmethyl, furylethyl, and quinolylethyl.", "The heteroaryl in said heteroaralkyl may be additionally substituted with halo, alkyl, alkoxy, halkoalkyl and haloalkoxy.", "The term “aralkoxy” embraces aralkyl radicals attached through an oxygen atom to other radicals.", "The term “aralkoxyalkyl” embraces aralkoxy radicals attached through an oxygen atom to an alkyl radical.", "The term “aralkylthio” embraces aralkyl radicals attached to a sulfur atom.", "The term “aralkylthioalkyl” embraces aralkylthio radicals attached through a sulfur atom to an alkyl radical.", "The term “aminoalkyl” embraces alkyl radicals substituted with one or more amino radicals.", "More preferred are “lower aminoalkyl” radicals.", "Examples of such radicals include aminomethyl, aminoethyl, and the like.", "The term “alkylamino” denotes amino groups that have been substituted with one or two alkyl radicals.", "Preferred are “lower N-alkylamino” radicals having alkyl portions having 1 to 6 carbon atoms.", "Suitable lower alkylamino may be mono or dialkylamino such as N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylamino or the like.", "The term “arylamino” denotes amino groups that have been substituted with one or two aryl radicals, such as N-phenylamino.", "The “arylamino” radicals may be further substituted on the aryl ring portion of the radical.", "The term “aralkylamino” embraces aralkyl radicals attached through an amino nitrogen atom to other radicals.", "The terms “N-arylaminoalkyl” and “N-aryl-N-alkylaminoalkyl” denote amino groups which have been substituted with one aryl radical or one aryl and one alkyl radical, respectively, and having the amino group attached to an alkyl radical.", "Examples of such radicals include N-phenylaminomethyl and N-phenyl-N-methylaminomethyl.", "The term “aminocarbonyl” denotes an amide group of the formula —C(═O)NH2.The term “alkylaminocarbonyl” denotes an aminocarbonyl group that has been substituted with one or two alkyl radicals on the amino nitrogen atom.", "Preferred are “N-alkylaminocarbonyl” and “N,N-dialkylaminocarbonyl” radicals.", "More preferred are “lower N-alkylaminocarbonyl” and “lower N,N-dialkylaminocarbonyl” radicals with lower alkyl portions as defined above.", "The term “aminocarbonylalkyl” denotes a carbonylalkyl group that has been substituted with an amino radical on the carbonyl carbon atom.", "The term “alkylaminoalkyl” embraces radicals having one or more alkyl radicals attached to an aminoalkyl radical.", "The term “aryloxyalkyl” embraces radicals having an aryl radical attached to an alkyl radical through a divalent oxygen atom.", "The term “arylthioalkyl” embraces radicals having an aryl radical attached to an alkyl radical through a divalent sulfur atom.", "Combinations The methods and combinations of the present invention provide one or more benefits.", "Combinations of COX-2 inhibitors with the compounds, compositions, agents and therapies of the present invention are useful in treating and preventing dysmenorrhea.", "Preferably, the COX-2 inhibitors and the compounds, compositions, agents and therapies of the present invention are administered in combination at a low dose, that is, at a dose lower than has been conventionally used in clinical situations.", "The combinations of the present invention will have a number of uses.", "For example, through dosage adjustment and medical monitoring, the individual dosages of the therapeutic compounds used in the combinations of the present invention will be lower than are typical for dosages of the therapeutic compounds when used in monotherapy.", "The dosage lowering will provide advantages including reduction of side effects of the individual therapeutic compounds when compared to the monotherapy.", "In addition, fewer side effects of the combination therapy compared with the monotherapies will lead to greater patient compliance with therapy regimens.", "Alternatively, the methods and combination of the present invention can also maximize the therapeutic effect at higher doses.", "When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.", "This new method of treatment for moderate to severe dysmenorrhea is superior to existing therapies, by reason of having the following characteristics.", "It inhibits the increased prostaglandin production induced by the complex series of hormonal changes characteristic of the menstrual cycle.", "The inhibition of prostaglandin synthesis occurs reproducibly 24-48 hours prior to initiation of menstruation.", "For safety reasons, it targets only the increased prostaglandin synthesis, which occurs immediately prior to menses, and not constitutive prostaglandin synthesis that may negatively impact other processes such as renal function.", "The COX-2 enzyme, which is responsible for prostaglandin synthesis, has been demonstrated in the endometrium and myometrium of the uterus in women.", "The tissue distribution of COX-2 is significantly different from COX-1 in the endometrium.", "Therefore one would expect differences in the effects of COX-2 inhibitors compared to COX-1 inhibitors.", "Among its several embodiments, the present invention provides a therapeutic combination of a cyclooxygenase-2 inhibitor compound source and a sex steroid compound, wherein the compounds together comprise a dysmenorrhea-effective amount of the compounds.", "In another embodiment, the cyclooxygenase-2 inhibitor compound source is a cyclooxygenase-2 inhibitor compound.", "In yet another embodiment, the cyclooxygenase-2 inhibitor compound source is a prodrug of a COX-2 inhibitor.", "Nonlimiting examples of COX-2 inhibitors that may be used in the present invention are identified in Table 1 below.", "TABLE NO.", "1 Cyclooxygenase-2 Inhibitors Trade/ Research Compound Name Reference Dosage 1,5-Diphenyl-3-substituted WO pyrazoles 97/13755 radicicol WO 96/25928.Kwon et al (Cancer Res (1992) 52 6296) GB- 02283745 TP-72 Cancer Res 1998 58 4 717-723 1-(4-chlorobenzoyl)-3-[4-(4- A-183827.0 fluoro-phenyl)thiazol-2- ylMethyl]-5-methoxy-2- methylindole GR-253035 4-(4-cyclohexyl-2- JTE-522 JP 9052882 methyloxazol-5-yl)-2- fluorobenzenesulfonamide 5-chloro-3-(4- (methylsulfonyl)phenyl)-2- (methyl-5-pyridinyl)-pyridine 2-(3,5-difluoro-phenyl)-3-4- (methylsulfonyl)-phenyl)-2- cyclopenten-1-one L-768277 L-783003 MK-966; U.S. Pat.", "No.", "12.5-100 VIOXX ® 5968974 mg po indomethacin-derived WO 200 indolalkanoic acid 96/374679 mg/kg/day 1-Methylsulfonyl-4-[1,1- WO dimethyl-4-(4- 95/30656.fluorophenyl)cyclopenta-2,4- WO dien-3-yl]benzene 95/30652.WO 96/38418.WO 96/38442.4,4-dimethyl-2-phenyl-3-[4- (methylsulfonyl)phenyl]cyclo- butenone 2-(4-methoxyphenyl)-4- EP 799823 methyl-1-(4- sulfamoylphenyl)-pyrrole N-[5-(4- RWJ-63556 fluoro)phenoxy]thiophene-2- methanesulfon-amide 5(E)-(3,5-di-tert-butyl-4- S-2474 EP 595546 hydroxy)benzylidene-2-ethyl- 1,2-isothiazolidine-1,1- dioxide 3-formylamino-7- T-614 DE methylsulfonylamino-6- 38/34204 phenoxy-4H-1-benzopyran-4- one Benzenesulfonamide, 4-(5-(4- celecoxib U.S. Pat.", "No.", "methylphenyl)-3- 5466823 (trifluoromethyl)-1H- pyrazol-1-yl)- CS 502 (Sankyo) MK 633 (Merck) meloxicam U.S. Pat.", "No.", "15-30 4233299 mg/day nimesulide U.S. Pat.", "No.", "3840597 The following references listed in Table No.", "2 below, hereby individually incorporated by reference, describe various COX-2 inhibitors suitable for use in the present invention described herein, and processes for their manufacture.", "TABLE NO.", "2 COX-2 Inhibitor References WO 99/30721 WO 99/30729 U.S. Pat.", "No.", "WO 98/15528 5760068 WO 99/25695 WO 99/24404 WO 99/23087 FR 27/71005 EP 921119 FR 27/70131 WO 99/18960 WO 99/15505 WO 99/15503 WO 99/14205 WO 99/14195 WO 99/14194 WO 99/13799 GB 23/30833 U.S. Pat.", "No.", "WO 99/12930 5859036 WO 99/11605 WO 99/10332 WO 99/10331 WO 99/09988 U.S. Pat.", "No.", "WO 99/05104 U.S. Pat.", "No.", "WO 98/47890 5869524 5859257 WO 98/47871 U.S. Pat.", "No.", "U.S. Pat.", "No.", "WO 98/45294 5830911 5824699 WO 98/43966 WO 98/41511 WO 98/41864 WO 98/41516 WO 98/37235 EP 86/3134 JP 10/175861 U.S. Pat.", "No.", "5776967 WO 98/29382 WO 98/25896 ZA 97/04806 EP 84/6,689 WO 98/21195 GB 23/19772 WO 98/11080 WO 98/06715 WO 98/06708 WO 98/07425 WO 98/04527 WO 98/03484 FR 27/51966 WO 97/38986 WO 97/46524 WO 97/44027 WO 97/34882 U.S. Pat.", "No.", "WO 97/37984 U.S. Pat.", "No.", "5681842 5686460 WO 97/36863 WO 97/40012 WO 97/36497 WO 97/29776 WO 97/29775 WO 97/29774 WO 97/28121 WO 97/28120 WO 97/27181 WO 95/11883 WO 97/14691 WO 97/13755 WO 97/13755 CA 21/80624 WO 97/11701 WO 96/41645 WO 96/41626 WO 96/41625 WO 96/38418 WO 96/37467 WO 96/37469 WO 96/36623 WO 96/36617 WO 96/31509 WO 96/25405 WO 96/24584 WO 96/23786 WO 96/19469 WO 96/16934 WO 96/13483 WO 96/03385 U.S. Pat.", "No.", "5510368 WO 96/09304 WO 96/06840 WO 96/06840 WO 96/03387 WO 95/21817 GB 22/83745 WO 94/27980 WO 94/26731 WO 94/20480 WO 94/13635 FR 27/70,131 U.S. Pat.", "No.", "5859036 WO 99/01131 WO 99/01455 WO 99/01452 WO 99/01130 WO 98/57966 WO 98/53814 WO 98/53818 WO 98/53817 WO 98/47890 U.S. Pat.", "No.", "U.S. Pat.", "No.", "WO 98/22101 5830911 5776967 DE 19/753463 WO 98/21195 WO 98/16227 U.S. Pat.", "No.", "5733909 WO 98/05639 WO 97/44028 WO 97/44027 WO 97/40012 WO 97/38986 U.S. Pat.", "No.", "WO 97/34882 WO 97/16435 5677318 WO 97/03678 WO 97/03667 WO 96/36623 WO 96/31509 WO 96/25928 WO 96/06840 WO 96/21667 WO 96/19469 U.S. Pat.", "No.", "WO 96/09304 GB 22/83745 WO 96/03392 5510368 WO 94/25431 WO 94/20480 WO 94/13635 JP 09052882 GB 22/94879 WO 95/15316 WO 95/15315 WO 96/03388 WO 96/24585 U.S. Pat.", "No.", "WO 95/00501 U.S. Pat.", "No.", "5344991 5968974 U.S. Pat.", "No.", "U.S. Pat.", "No.", "5945539 5994381 Three classes of cyclooxygenase-2 inhibitors are reviewed by J. Carter in Exp.", "Opin.", "Ther.", "Patents, 8(1), 21-29 (1997): methanesulfonanilides, tricyclics and structurally modified non-selective cyclooxygenase inhibitors.", "Methanesulfonanilides are a class of selective cyclooxygenase-2 inhibitors, of which NS-398, flosulide and nimesulide are example members.", "A preferred class of tricyclic cyclooxygenase-2 inhibitors comprises compounds of formula (1) wherein A is a substituent selected from partially unsaturated or unsaturated heterocyclyl and partially unsaturated or unsaturated carbocyclic rings; wherein n is 0 or 1; wherein X is O or S; wherein R1 is at least one substituent selected from heterocyclyl, cycloalkyl, cycloalkenyl and aryl, wherein R1 is optionally substituted at a substitutable position with one or more radicals selected from alkyl, haloalkyl, cyano, carboxyl, alkoxycarbonyl, hydroxyl, hydroxyalkyl, haloalkoxy, amino, alkylamino, arylamino, nitro, alkoxyalkyl, alkylsulfinyl, halo, alkoxy and alkylthio; wherein R2 is methyl, amino or aminocarbonylalkyl; and wherein R3 is one or more radicals selected from hydrido, halo, alkyl, alkenyl, alkynyl, oxo, cyano, carboxyl, cyanoalkyl, heterocyclyloxy, alkyloxy, alkylthio, alkylcarbonyl, cycloalkyl, aryl, haloalkyl, heterocyclyl, cycloalkenyl, aralkyl, heterocyclylalkyl, acyl, alkylthioalkyl, hydroxyalkyl, alkoxycarbonyl, arylcarbonyl, aralkylcarbonyl, aralkenyl, alkoxyalkyl, arylthioalkyl, aryloxyalkyl, aralkylthioalkyl, aralkoxyalkyl, alkoxyaralkoxyalkyl, alkoxycarbonylalkyl, aminocarbonyl, aminocarbonylalkyl, alkylaminocarbonyl, N-arylaminocarbonyl, N-alkyl-N-arylaminocarbonyl, alkylaminocarbonylalkyl, carboxyalkyl, alkylamino, N-arylamino, N-aralkylamino, N-alkyl-N-aralkylamino, N-alkyl-N-arylamino, aminoalkyl, alkylaminoalkyl, N-arylaminoalkyl, N-aralkylaminoalkyl, N-alkyl-N-aralkylaminoalkyl, N-alkyl-N-arylaminoalkyl, aryloxy, aralkoxy, arylthio, aralkylthio, alkylsulfinyl, alkylsulfonyl, aminosulfonyl, alkylaminosulfonyl, N-arylaminosulfonyl, arylsulfonyl and N-alkyl-N-arylaminosulfonyl, wherein R3 is optionally substituted at a substitutable position with one or more radicals selected from alkyl, haloalkyl, cyano, carboxyl, alkoxycarbonyl, hydroxyl, hydroxyalkyl, haloalkoxy, amino, alkylamino, arylamino, nitro, alkoxyalkyl, alkylsulfinyl, halo, alkoxy and alkylthio; or a pharmaceutically-acceptable salt thereof.", "Preferred COX-2 inhibitors are tricyclic COX-2 inhibitors wherein the A ring is selected from the heterocyclyl groups of pyrazolyl, furanonyl, isoxazolyl, pyridinyl and pyridazinonyl.", "More preferred COX-2 inhibitors that may be used in the present invention include, but are not limited to: JTE-522, 4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide; 5-chloro-3-(4-(methylsulfonyl)phenyl)-2-(2-methyl-5-pyridinyl)pyridine; 2-(3,5-difluorophenyl)-3-(4-(methylsulfonyl)phenyl)-2-cyclopenten-1-one; celecoxib, 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-benzenesulfonamide; rofecoxib, 4-(4-(methylsulfonyl)phenyl]-3-phenyl-2(5H)-furanone; valdecoxib, 4-(5-methyl-3-phenylisoxazol-4-yl)benzenesulfonamide; parecoxib, N-[[4-(5-methyl-3-phenylisoxazol-4-yl]phenyl]sulfonyl]propanamide; 4-[5-(4-chorophenyl)-3-(trifluoromethyl)-1H-pyrazole-1-yl]benzenesulfonamide; N-(2,3-dihydro-1,1-dioxido-6-phenoxy-1,2-benzisothiazol-5-yl)methanesulfonamide; 6-[[5-(4-chlorobenzoyl)-1,4-dimethyl-1H-pyrrol-2-yl]methyl]-3(2H)-pyridazinone; N-(4-nitro-2-phenoxyphenyl)methanesulfonamide; 3-(3,4-difluorophenoxy)-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]-2 (5H)-furanone; N-[6-[(2,4-difluorophenyl)thio]-2,3-dihydro-1-oxo-1H-inden-5-yl]methanesulfonamide; 3-(4-chlorophenyl)-4-[4-(methylsulfonyl)phenyl]-2 (3H)-oxazolone; 4-[3-(4-fluorophenyl)-2,3-dihydro-2-oxo-4-oxazolyl]benzenesulfonamide; 3-[4-(methylsulfonyl)phenyl]-2-phenyl-2-cyclopenten-1-one; 4-(2-methyl-4-phenyl-5-oxazolyl)benzenesulfonamide; 3-(4-fluorophenyl)-4-[4-(methylsulfonyl)phenyl]-2(3H)-oxazolone; 5-(4-fluorophenyl)-1-[4-(methylsulfonyl)phenyl]-3-(trifluoromethyl)-1H-pyrazole; 4-(5-phenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide; 4-[1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]benzenesulfonamide; 4-[5-(4-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide; NS-398, N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide; N-[6-(2,4-difluorophenoxy)-2,3-dihydro-1-oxo-1H-inden-5-yl]methanesulfonamide; 3-(4-chlorophenoxy)-4-[(methylsulfonyl)amino]benzenesulfonamide; 3-(4-fluorophenoxy)-4-[(methylsulfonyl)amino]benzenesulfonamide; 3-[(1-methyl-1H-imidazol-2-yl)thio]-4 [(methylsulfonyl)amino]benzenesulfonamide; 5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]-3-phenoxy-2(5H)-furanone; N-[6-[(4-ethyl-2-thiazolyl)thio)-1,3-dihydro-1-oxo-5-isobenzofuranyl]methanesulfonamide; 3-[(2,4-dichlorophenyl)thio]-4-[(methylsulfonyl)amino]benzenesulfonamide; 1-fluoro-4-[2-[4-(methylsulfonyl)phenyl]cyclopenten-1-yl]benzene; 4-[5-(4-chlorophenyl)-3-(difluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide; 3-[1-[4-(methylsulfonyl)phenyl]-4-(trifluoromethyl)-1H-imidazol-2-yl]pyridine; 4-[2-(3-pyridinyll)-4-(trifluoromethyl)-1H-imidazol-1-yl]benzenesulfonamide; 4-[5-(hydroxymethyl)-3-phenylisoxazol-4-yl]benzenesulfonamide; 4-[3-(4-chlorophenyl)-2,3-dihydro-2-oxo-4-oxazolyl]benzenesulfonamide; 4-[5-(difluoromethyl)-3-phenylisoxazol-4-yl]benzenesulfonamide; [1,1′:2′,1″-terphenyl]-4-sulfonamide; 4-(methylsulfonyl)-1,1′,2],1″-terphenyl; 4-(2-phenyl-3-pyridinyl)benzenesulfonamide; N-(2,3-dihydro-1,1-dioxido-6-phenoxy-1,2-benzisothiazol-5-yl)methanesulfonamide; and N-[3-(formylamino)-4-oxo-6-phenoxy-4H-1-benzopyran-7-yl]methanesulfonamide; 4-[4-methyl-1-[4-(methylthio)phenyl]-1H-pyrrol-2-yl]benzenesulfonamide; 4-[2-(4-ethoxyphenyl)-4-methyl-1H-pyrrol-1-yl]benzenesulfonamide; deracoxib, 4-[3-(difluoromethyl)-5-(3-fluoro-4-methoxyphenyl)-1H-pyrazol-1-yl]benzenesulfonamide; MK-663, etoricoxib, 5-chloro-6′-methyl-3-[4-(methylsulfonyl)phenyl]-2,3′-bipyridine; DuP 697, 5-bromo-2-(4-fluorophenyl)-3-[4-(methylsulfonyl)phenyl]thiophene; ABT-963, 2-(3,4-difluorophenyl)-4-(3-hydroxy-3-methylbutoxy)-5-[4-(methylsulfonyl)phenyl]-3(2H)-pyridazinone; 6-nitro-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid; 6-chloro-8-methyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid; (2S)-6-chloro-7-(1,1-dimethylethyl)-2-(trifluoromethyl)-2H-1-benzopyran-3-carboxylic acid; (2S)-6,8-dichloro-2-(trifluoromethyl)-2H-1-benzopyran-3-carboxylic acid; 2-trifluoromethyl-2H-naphtho[2,3-b]pyran-3-carboxylic acid; 6-chloro-7-(4-nitrophenoxy)-2-(trifluoromethyl)-2H-1-benzopyran-3-carboxylic acid; (2S)-6,8-dichloro-2-(trifluoromethyl)-2H-1-benzopyran-3-carboxylic acid, ethyl ester; 6-chloro-2-(trifluoromethyl)-4-phenyl-2H-1-benzopyran-3-carboxylic acid; 6-(4-hydroxybenzoyl)-2-(trifluoromethyl)-2H-1-benzopyran-3-carboxylic acid; 2-(trifluoromethyl)-6-[(trifluoromethyl)thio]-2H-1-benzothiopyran-3-carboxylic acid; (2S)-6,8-dichloro-2-(trifluoromethyl)-2H-1-benzopyran-3-carboxylic acid, sodium salt; 6,8-dichloro-2-trifluoromethyl-2H-1-benzothiopyran-3-carboxylic acid; 6-(1,1-dimethylethyl)-2-(trifluoromethyl)-2H-1-benzothiopyran-3-carboxylic acid; (2S)-6,8-dichloro-2-(trifluoromethyl)-2H-1-benzopyran-3-carboxamide; 6,7-difluoro-1,2-dihydro-2-(trifluoromethyl)-3-quinolinecarboxylic acid; 6-chloro-1,2-dihydro-1-methyl-2-(trifluoromethyl)-3-quinolinecarboxylic acid; 6-chloro-2-(trifluoromethyl)-1,2-dihydro[1,8]naphthyridine-3-carboxylic acid; 6,8-dichloro-7-methyl-2-(trifluoromethyl)-2H-1-benzopyran-3-carboxylic acid, ethyl ester; (2S)-6-chloro-1,2-dihydro-2-(trifluoromethyl)-3-quinolinecarboxylic acid.", "In a further preferred embodiment of the invention the cyclooxygenase inhibitor can be selected from the class of phenylacetic acid derivative cyclooxygenase-2 selective inhibitors represented by the general structure of Formula V: wherein R16 is methyl or ethyl; R17 is chloro or fluoro; R1 is hydrogen or fluoro R19 is hydrogen, fluoro, chloro, methyl, ethyl, methoxy, ethoxy or hydroxy; R20 is hydrogen or fluoro; and R21 is chloro, fluoro, trifluoromethyl or methyl, provided that R17, R18, R19 and R20 are not all fluoro when R16 is ethyl and R19 is H. A particularly preferred phenylacetic acid derivative cyclooxygenase-2 selective inhibitor that is described in WO 99/11605 is a compound that has the designation of COX189 (CAS RN 346670-74-4), and that has the structure shown in Formula V, wherein R16 is ethyl; R17 and R19 are chloro; R19 and R20 are hydrogen; and and R21 is methyl.", "Other preferred cyclooxygenase-2 selective inhibitors that can be used in the present invention have the general structure shown in formula VI, where the J group is a carbocycle or a heterocycle.", "Particularly preferred embodiments have the structure: where: X is O; J is 1-phenyl; R21 is 2-NHSO2CH3; R22 is 4-NO2; and there is no R23 group, (nimesulide), and X is O; J is 1-oxo-inden-5-yl; R2, is 2-F; R22 is 4-F; and R23 is 6-NHSO2CH3, (flosulide); and X is O; J is cyclohexyl; R21 is 2-NHSO2CH3; R22 is 5-NO2; and there is no R23 group, (NS-398); and X is S; J is 1-oxo-inden-5-yl; R21 is 2-F; R22 is 4-F; and R23 is 6-N−SO2CH3.Na+, (L-745337); and X is S; J is thiophen-2-yl; R21 is 4-F; there is no R22 group; and R23 is 5-NHSO2CH3, (RWJ-63556); and X is O; J is 2-oxo-5(R)-methyl-5-(2,2,2-trifluoroethyl)furan-(5H)-3-yl; R2, is 3-F; R22 is 4-F; and R23 is 4-(p-SO2CH3)C6H4, (L-784512).", "Further information on the applications of N-(2-cyclohexyloxynitrophenyl)methane sulfonamide (NS-398, CAS RN 123653-11-2), having a structure as shown in formula B-26, have been described by, for example, Yoshimi, N. et al., in Japanese J.", "Cancer Res., 90(4): 406-412 (1999); Falgueyret, J.-P. et al., in Science Spectra, available at: http://www.gbhap.com/Science_Spectra/20-1-article.htm (Jun.", "6, 2001); and Iwata, K. et al., in Jpn.", "J.", "Pharmacol., 75(2): 191-194 (1997).", "An evaluation of the antiinflammatory activity of the cyclooxygenase-2 selective inhibitor, RWJ 63556, in a canine model of inflammation, was described by Kirchner et al., in J Pharmacol Exp Ther 282, 1094-1101 (1997).", "Other compounds useful as the cyclooxygenase-2 selective inhibitor in the present invention include diarylmethylidenefuran derivatives such as those described in U.S. Pat.", "No.", "6,180,651.Such diarylmethylidenefuran derivatives have the general formula shown below in formula VII: wherein: the rings T and M independently are: a phenyl radical, a naphthyl radical, a radical derived from a heterocycle comprising 5 to 6 members and possessing from 1 to 4 heteroatoms, or a radical derived from a saturated hydrocarbon ring having from 3 to 7 carbon atoms; at least one of the substituents Q1, Q2, L1 or L2 is: an —S(O)n—R group, in which n is an integer equal to 0, 1 or 2 and R is a lower alkyl radical having 1 to 6 carbon atoms or a lower haloalkyl radical having 1 to 6 carbon atoms, or an —SO2NH2 group; and is located in the para position, the others independently being: a hydrogen atom, a halogen atom, a lower alkyl radical having 1 to 6 carbon atoms, a trifluoromethyl radical, or a lower O-alkyl radical having 1 to 6 carbon atoms, or Q1 and Q2 or L1 and L2 are a methylenedioxy group; and R24, R25, R26 and R27 independently are: a hydrogen atom, a halogen atom, a lower alkyl radical having 1 to 6 carbon atoms, a lower haloalkyl radical having 1 to 6 carbon atoms, or an aromatic radical selected from the group consisting of phenyl, naphthyl, thienyl, furyl and pyridyl; or, R24, R25 or R26, R27 are an oxygen atom, or R24, R25 or R26, R27, together with the carbon atom to which they are attached, form a saturated hydrocarbon ring having from 3 to 7 carbon atoms; or an isomer or prodrug thereof.", "Particular materials that are included in this family of compounds, and which can serve as the cyclooxygenase-2 selective inhibitor in the present invention, include N-(2-cyclohexyloxynitrophenyl)methane sulfonamide, and (E)-4-[(4-methylphenyl)(tetrahydro-2-oxo-3-furanylidene) methyl] benzenesulfonamide.", "Preferred cyclooxygenase-2 selective inhibitors that are useful in the present invention include the following individual compounds; darbufelone (Pfizer), CS-502 (Sankyo), LAS 34475 (Almirall Profesfarma), LAS 34555 (Almirall Profesfarma), S-33516 (Servier), SD 8381 (Pharmacia, described in U.S. Pat.", "No.", "6,034,256), BMS-347070 (Bristol Myers Squibb, described in U.S. Pat.", "No.", "6,180,651), MK-966 (Merck), L-783003 (Merck), T-614 (Toyama), D-1367 (Chiroscience), L-748731 (Merck), CT3 (Atlantic Pharmaceutical), CGP-28238 (Novartis), BF-389 (Biofor/Scherer), GR-253035 (Glaxo Wellcome), 6-dioxo-9H-purin-8-yl-cinnamic acid (Glaxo Wellcome), and S-2474 (Shionogi).", "In another preferred embodiment of the invention, the compound BMS-347070 having the formula: Information about S-33516, mentioned above, can be found in Current Drugs Headline News, at http://www.current-drugs.com/NEWS/Inflaml.htm, Oct. 4, 2001, where it was reported that S-33516 is a tetrahydroisoinde derivative which has IC50 values of 0.1 and 0.001 mM against cyclooxygenase-1 and cyclooxygenase-2, respectively.", "In human whole blood, S-33516 was reported to have an ED50=0.39 mg/kg.", "The CAS reference numbers for nonlimiting examples of COX-2 inhibitors are identified in Table 3 below.", "TABLE NO.", "3 COX-2 Inhibitors Compound Number CAS Reference Number C1 180200-68-4 C2 202409-33-4 C3 212126-32-4 C4 169590-42-5 C5 162011-90-7 C6 181695-72-7 C7 198470-84-7 C8 170569-86-5 C9 187845-71-2 C10 179382-91-3 C11 51803-78-2 C12 189954-13-0 C13 158205-05-1 C14 197239-99-9 C15 197240-09-8 C16 226703-01-1 C17 93014-16-5 C18 197239-97-7 C19 162054-19-5 C20 170569-87-6 C21 279221-13-5 C22 170572-13-1 C23 123653-11-2 C24 80937-31-1 C25 279221-14-6 C26 279221-15-7 C27 187846-16-8 C28 189954-16-3 C29 181485-41-6 C30 187845-80-3 C31 158959-32-1 C32 170570-29-3 C33 177660-77-4 C34 177660-95-6 C35 181695-81-8 C36 197240-14-5 C37 181696-33-3 C38 178816-94-9 C39 178816-61-0 C40 279221-17-9 C41 187845-71-2 C42 123663-49-0 C43 197905-01-4 C44 197904-84-0 C45 169590-41-4 C46 202409-33-4 C47 88149-94-4 C48 266320-83-6 C49 215122-43-3 C50 215122-44-4 C51 215122-74-0 C52 215123-80-1 C53 215122-70-6 C54 264878-87-7 C55 279221-12-4 C56 215123-48-1 C57 215123-03-8 C58 215123-60-7 C59 279221-18-0 C60 215123-61-8 C61 215123-52-7 C62 279221-19-1 C63 215123-64-1 C64 215123-70-9 C65 215123-79-8 C66 215123-91-4 C67 215123-77-6 More preferably, the COX-2 inhibitors that may be used in the present invention include, but are not limited to celecoxib, valdecoxib, parecoxib, rofecoxib, NS-398, deracoxib, Merck MK-663 and ABT-963.Various classes of cyclooxygenase-2 inhibitors can be prepared as follows.", "Pyrazoles can be prepared by methods described in WO 95/15316.Pyrazoles can further be prepared by methods described in WO 95/15315.Pyrazoles can also be prepared by methods described in WO 96/03385.Thiophene analogs can be prepared by methods described in WO 95/00501.Preparation of thiophene analogs is also described in WO 94/15932.Oxazoles can be prepared by the methods described in WO 95/00501.Preparation of oxazoles is also described in WO 94/27980.Isoxazoles can be prepared by the methods described in WO 96/25405.Imidazoles can be prepared by the methods described in WO 96/03388.Preparation of imidazoles is also described in WO 96/03387.Cyclopentene cyclooxygenase-2 inhibitors can be prepared by the methods described in U.S. Pat.", "No.", "5,344,991.Preparation of cyclopentene COX-2 inhibitors is also described in WO 95/00501.Terphenyl compounds can be prepared by the methods described in WO 96/16934.Thiazole compounds can be prepared by the methods described in WO 96/03,392.Pyridine compounds can be prepared by the methods described in WO 96/03392.Preparation of pyridine compounds is also described in WO 96/24,585.Benzopyranopyrazolyl compounds can be prepared by the methods described in WO 96/09304.Benzopyran compounds can be prepared by the methods described in WO 98/47890.Preparation of benzopyran compounds is also described in WO 00/23433.Benzopyran compounds can further be prepared by the methods described in U.S. Pat.", "No.", "6,077,850.Preparation of benzopyran compounds is further described in U.S. Pat.", "No.", "6,034,256.Arylpyridazinones can be prepared by the methods described in WO 00/24719.The celecoxib used in the therapeutic combinations of the present invention can be prepared in the manner set forth in U.S. Pat.", "No.", "5,466,823.The valdecoxib used in the therapeutic combinations of the present invention can be prepared in the manner set forth in U.S. Pat.", "No.", "5,633,272.The parecoxib used in the therapeutic combinations of the present invention can be prepared in the manner set forth in U.S. Pat.", "No.", "5,932,598.The rofecoxib used in the therapeutic combinations of the present invention can be prepared in the manner set forth in U.S. Pat.", "No.", "5,474,995.The deracoxib used in the therapeutic combinations of the present invention can be prepared in the manner set forth in U.S. Pat.", "No.", "5,521,207.The compound MK-663 used in the therapeutic combinations of the present invention can be prepared in the manner set forth in WO 98/03484.The compound NS-398 used in the therapeutic combinations of the present invention can be prepared in the manner set forth in U.S. Pat.", "No.", "4,885,367.The compound ABT-963 used in the therapeutic combinations of the present invention can be prepared in the manner set forth in WO 00/24719.The estrogen sex steroid is preferably selected from, but is not limited to, the group consisting of ethinyl estradiol, 17β-estradiol and mestranol.", "Still more preferably the estrogen sex steroid is ethinyl estradiol.", "The progestin sex steroid is preferably selected from, but is not limited to, the group consisting of levonorgestrel, norethindrone acetate, norgestimate, ethynodiol acetate, desogestrel, norgestrel, gestodene, 3-ketodesogestrel, Org 30659, dienogest, trimegestone and norethindrone.", "More preferably the progestin sex steroid is selected from the group consisting of levonorgestrel, norethindrone acetate, norgestimate, ethynodiol acetate, desogestrel, norgestrel and norethindrone.", "Even more preferably, the progestin sex steroid is selected from the group consisting of levonorgestrel, norethindrone acetate and norgestimate.", "The structures and CAS registry numbers of preferred estrogen and progestin sex steroids are listed in Table No.", "4 below.", "TABLE NO.", "4 Sex Steroid Structures CAS Registry Name Number Structure Ethinyl estradiol 57-63-6 17β-Estradiol 50-28-2 Mestranol 72-33-3 Levonorgestrel 797-63-7 Norethindrone acetate 51-98-9 Norgestimate 35189-28-7 Ethynodiol diacetate 297-76-7 Desogestrel 54024-22-5 Norgestrel 6533-00-2 Norethindrone 68-22-4 3-Ketodesogestrel 54048-10-1 Gestodene 60282-87-3 Org 30659 110072-15-6 Trimegestone 74513-62-5 Dienogest 65928-58-7 The following references listed in Table No.", "5 below, hereby individually incorporated by reference, describe various sex steroids suitable for use in the present invention described herein, and processes for their manufacture.", "TABLE NO.", "5 Sex Steroid References Sex Steroid Reference Ethinyl estradiol U.S. Pat.", "No.", "3,759,961 17β-Estradiol U.S. Pat.", "No.", "3,274,182 Mestranol U.S. Pat.", "No.", "3,759,961 Levonorgestrel U.S. Pat.", "No.", "3,759,961 Norethindrone acetate U.S. Pat.", "No.", "3,408,371 Norgestimate U.S. Pat.", "No.", "4,027,019 Ethynodiol diacetate U.S. Pat.", "No.", "3,383,384 Desogestrel U.S. Pat.", "No.", "3,927,046 Norgestrel U.S. Pat.", "No.", "3,892,779 Norethindrone U.S. Pat.", "No.", "3,383,384 3-Ketodesogestrel U.S. Pat.", "No.", "4,371,529 Gestodene U.S. Pat.", "No.", "4,081,537 Org 30659 U.S. Pat.", "No.", "5,236,913 Trimegestone U.S. Pat.", "No.", "4,273,771 Dienogest U.S. Pat.", "No.", "4,167,517 The compounds useful in the present invention can have no asymmetric carbon atoms, or, alternatively, the useful compounds can have one or more asymmetric carbon atoms.", "When the useful compounds have one or more asymmetric carbon atoms, they therefore include racemates and stereoisomers, such as diastereomers and enantiomers, in both pure form and in admixture.", "Such stereoisomers can be prepared using conventional techniques, either by reacting enantiomeric starting materials, or by separating isomers of compounds of the present invention.", "Isomers may include geometric isomers, for example cis-isomers or trans-isomers across a double bond.", "All such isomers are contemplated among the compounds useful in the present invention.", "The compounds useful in the present invention also include tautomers.", "The compounds useful in the present invention also include their salts, solvates and prodrugs.", "Dosages, Formulations and Routes of Administration For the prophylaxis or treatment of the conditions referred to above, the compounds useful in the combinations and methods of the present invention can be used as the compound per se.", "Pharmaceutically acceptable salts are particularly suitable for medical applications because of their greater aqueous solubility relative to the parent compound.", "Such salts must clearly have a pharmaceutically acceptable anion or cation.", "Suitable pharmaceutically acceptable acid addition salts of the compounds of the present invention when possible include those derived from inorganic acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, sulfonic, and sulfuric acids, and organic acids such as formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, stearic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, cyclohexylaminosulfonic, algenic, b-hydroxybutyric, galactaric and galacturonic acids.", "Suitable pharmaceutically-acceptable base addition salts of compounds of the present invention include metallic ion salts and organic ion salts.", "More preferred metallic ion salts include, but are not limited to appropriate alkali metal (group Ia) salts, alkaline earth metal (group IIa) salts and other physiological acceptable metal ions.", "Such salts can be made from the ions of aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.", "Preferred organic salts can be made from tertiary amines and quaternary ammonium salts, including in part, trimethylamine, diethylamine, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.", "All of the above salts can be prepared by those skilled in the art by conventional means from the corresponding compound of the present invention.", "The compounds useful in the present invention can be presented with an acceptable carrier in the form of a pharmaceutical composition.", "The carrier must, of course, be acceptable in the sense of being compatible with the other ingredients of the composition and must not be deleterious to the recipient.", "The carrier can be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose composition, for example, a tablet, which can contain from 0.05% to 95% by weight of the active compound.", "Other pharmacologically active substances can also be present, including other compounds of the present invention.", "The pharmaceutical compositions of the invention can be prepared by any of the well-known techniques of pharmacy, consisting essentially of admixing the components.", "Optionally, the combination of the present invention can comprise a composition comprising a cyclooxygenase-2 inhibiting compound and a sex steroid compound.", "In such a composition, the cyclooxygenase-2 inhibiting compound and the sex steroid can be present in a single dosage form, for example a pill, a capsule, or a liquid that contains both of the compounds.", "These compounds can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic compounds or as a combination of therapeutic compounds.", "The amount of compound which is required to achieve the desired biological effect will, of course, depend on a number of factors such as the specific compound chosen, the use for which it is intended, the mode of administration, and the clinical condition of the recipient.", "Dosages Dosage levels of COX-2 inhibitors on the order of about 0.1 mg to about 10,000 mg of the active ingredient compound are useful in the treatment of the above conditions, with preferred levels of about 1.0 mg to about 1,000 mg and even more preferred levels of about 5 mg to about 500 mg.", "The amount of active ingredient will vary depending upon the host treated and the particular mode of administration.", "It is understood, however, that a specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated and form of administration.", "Treatment dosages generally may be titrated to optimize safety and efficacy.", "Typically, dosage-effect relationships from in vitro initially can provide useful guidance on the proper doses for patient administration.", "Studies in animal models also generally may be used for guidance regarding effective dosages for treatment of cancers in accordance with the present invention.", "In terms of treatment protocols, it should be appreciated that the dosage to be administered will depend on several factors, including the particular agent that is administered, the route administered, the condition of the particular patient, etc.", "Generally speaking, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro.", "Thus, where a compound is found to demonstrate in vitro activity at, e.g., 10 μM, one will desire to administer an amount of the drug that is effective to provide about a 10 μM concentration in vivo.", "Determination of these parameters is well within the skill of the art.", "These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.", "An estrogen sex steroid at a daily dosage equivalent in estrogenic activity to about 5-75 ug ethinyl estradiol is useful in the treatment of the above conditions, with preferred levels of about 10 ug to about 50 ug and even more preferred levels of about 15 ug to about 35 ug.", "Actual dosage levels for other estrogen sex steroids may vary relative to the levels listed for ethinyl estradiol.", "A progestin sex steroid at a daily dosage equivalent in progestinic activity to about 10-600 ug levonorgestrel is useful in the treatment of the above conditions, with preferred levels of about 25 ug to about 400 ug and even more preferred levels of about 50 ug to about 200 ug.", "Actual dosage levels for other progestin sex steroids may vary relative to the levels listed for levonorgestrel.", "The compounds of the present invention can be formulated as a pharmaceutical composition.", "Such a composition can then be administered orally, parenterally, by inhalation-spray, rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.", "Formulation of drugs is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975.Another discussion of drug formulations can be found in Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980.Solid dosage forms for oral administration can include capsules, tablets, pills, powders, and granules.", "In such solid dosage forms, the compounds of this invention are ordinarily combined with one or more adjuvants appropriate to the indicated route of administration.", "If administered per os, a contemplated inhibitor compound can be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration.", "Such capsules or tablets can contain a controlled-release formulation as can be provided in a dispersion of active compound in hydroxypropylmethyl cellulose.", "In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents such as sodium citrate, magnesium or calcium carbonate or bicarbonate.", "Tablets and pills can additionally be prepared with enteric coatings.", "Liquid dosage forms for oral administration can include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water.", "Such compositions can also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.", "The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.", "Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.", "The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.", "Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution.", "In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.", "For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides.", "In addition, fatty acids such as oleic acid find use in the preparation of injectables.", "Dimethyl acetamide, surfactants including ionic and non-ionic detergents, polyethylene glycols can be used.", "Mixtures of solvents and wetting agents such as those discussed above are also useful.", "For therapeutic purposes, formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions.", "These solutions and suspensions can be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration.", "A contemplated therapeutic compound can be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers.", "Other adjuvants and modes of administration are well and widely known in the pharmaceutical art.", "Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter, synthetic mono- di- or triglycerides, fatty acids and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.", "Topical administration can also involve the use of transdermal administration such as transdermal patches or iontophoresis devices.", "The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the mammalian host treated and the particular mode of administration.", "Treatment Regimen The dosage regimen to prevent, give relief from, or ameliorate a disease condition having dysmenorrhea as an element of the disease or to protect against or treat a further dysmenorrhea related disorder with the compounds and/or compositions of the present invention is selected in accordance with a variety of factors.", "These include the type, age, weight, diet, and medical condition of the patient, the severity of the disease, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetics and toxicology profiles of the particular compound employed, whether a drug delivery system is utilized, and whether the compound is administered as part of a drug combination.", "Thus, the dosage regimen actually employed may vary widely and therefore deviate from the preferred dosage regimen set forth above.", "In order to create a reproducible time of menses, specific combinations of daily administration of orally active sex steroids will be used to pharmacologically regulate the onset of menses within a small (24-48 hour) window of time.", "These steroids will include an estrogenic component and a progestagenic component with the effects of the latter predominating.", "The use of such a regimen should also result in less growth of the endometrial lining resulting in a reduced blood loss at the time of menses.", "The use of daily orally active sex steroids to regulate endometrial growth will upon their discontinuation result in menses within 48-72 hours.", "The addition of a cyclooxygenase-2 inhibitor, such as celecoxib, starting 24 hours following discontinuation of the sex steroids will synchronize events such that the cyclooxygenase-2 inhibitor will be reproducibly administered at the time of initiation of increased prostaglandin synthesis triggered by the withdrawal of the steroid hormones.", "The cyclooxgenase-2 inhibitor can be administered until the end of menses with a variety of regimens.", "For example, the cyclooxygenase-2 inhibitor can be administered daily (od), twice a day (bid) or three times a day (tid).", "Thus the invention refers to the sequential administration of daily orally active sex steroids followed by a selective COX-2 inhibitor.", "This would be administered in a regular schedule (every 28 days) with the sex steroids being administered for 21 days followed by 2-7 days of a cyclooxygenase-2 inhibitor.", "More preferably, the sex steroids would be administered for 21 days followed by 4-7 days of a cyclooxygenase-2 inhibitor.", "Patients undergoing treatment with the compounds or compositions disclosed herein can be routinely monitored to determine the effectiveness of the combination therapy.", "Continuous analysis of such data permits modification of the treatment regimen during therapy so that optimal effective amounts of each type of therapeutic compound are administered at any point in time, and so that the duration of treatment can be determined as well.", "In this way, the treatment regimen/dosing schedule can be rationally modified over the course of therapy so that the lowest amount of the therapeutic compounds which together exhibit satisfactory effectiveness is administered, and so that administration is continued only so long as is necessary to successfully treat the dysmenorrhea related condition.", "A potential advantage of the combination therapy disclosed herein may be reduced dosage amount of any individual therapeutic compound, or all therapeutic compounds, effective in treating dysmenorrhea related conditions.", "The dosage lowering will provide advantages including reduction of side effects of the individual therapeutic compounds when compared to the monotherapy.", "One of the several embodiments of the present invention provides a combination therapy comprising the use of a first amount of a COX-2 inhibitor and a second amount of sex steroids useful in the prophylaxis or treatment of dysmenorrhea, wherein said first and second amounts together comprise an dysmenorrhea-effective amount of said compounds.", "For example one of the many embodiments of the present invention is a combination therapy regimen comprising therapeutic dosages of a pyrazole COX-2 inhibitor, ethinyl estradiol and levonorgestrel.", "The following non-limiting examples serve to illustrate various aspects of the present invention.", "EXAMPLES Table 6 illustrates examples of some combinations of the present invention wherein the combination comprises a first amount of a COX-2 inhibitor source, a second amount of a estrogen sex steroid and a third amount of a progestin sex steroid wherein the amounts together comprise an dysmenorrhea-effective amount of the compounds.", "TABLE NO.", "6 Combination Examples Example COX-2 Number Inhibitor Estrogen Sex Steroid Progestin Sex Steroid 1 C1 Ethinyl estradiol Levonorgestrel 2 C2 Ethinyl estradiol Levonorgestrel 3 C3 Ethinyl estradiol Levonorgestrel 4 C4 Ethinyl estradiol Levonorgestrel 5 C5 Ethinyl estradiol Levonorgestrel 6 C6 Ethinyl estradiol Levonorgestrel 7 C7 Ethinyl estradiol Levonorgestrel 8 C8 Ethinyl estradiol Levonorgestrel 9 C9 Ethinyl estradiol Levonorgestrel 10 C10 Ethinyl estradiol Levonorgestrel 11 C11 Ethinyl estradiol Levonorgestrel 12 C12 Ethinyl estradiol Levonorgestrel 13 C13 Ethinyl estradiol Levonorgestrel 14 C14 Ethinyl estradiol Levonorgestrel 15 C15 Ethinyl estradiol Levonorgestrel 16 C16 Ethinyl estradiol Levonorgestrel 17 C17 Ethinyl estradiol Levonorgestrel 18 C18 Ethinyl estradiol Levonorgestrel 19 C19 Ethinyl estradiol Levonorgestrel 20 C20 Ethinyl estradiol Levonorgestrel 21 C21 Ethinyl estradiol Levonorgestrel 22 C22 Ethinyl estradiol Levonorgestrel 23 C23 Ethinyl estradiol Levonorgestrel 24 C24 Ethinyl estradiol Levonorgestrel 25 C25 Ethinyl estradiol Levonorgestrel 26 C26 Ethinyl estradiol Levonorgestrel 27 C27 Ethinyl estradiol Levonorgestrel 28 C28 Ethinyl estradiol Levonorgestrel 29 C29 Ethinyl estradiol Levonorgestrel 30 C30 Ethinyl estradiol Levonorgestrel 31 C31 Ethinyl estradiol Levonorgestrel 32 C32 Ethinyl estradiol Levonorgestrel 33 C33 Ethinyl estradiol Levonorgestrel 34 C34 Ethinyl estradiol Levonorgestrel 35 C35 Ethinyl estradiol Levonorgestrel 36 C36 Ethinyl estradiol Levonorgestrel 37 C37 Ethinyl estradiol Levonorgestrel 38 C38 Ethinyl estradiol Levonorgestrel 39 C39 Ethinyl estradiol Levonorgestrel 40 C40 Ethinyl estradiol Levonorgestrel 41 C41 Ethinyl estradiol Levonorgestrel 42 C42 Ethinyl estradiol Levonorgestrel 43 C43 Ethinyl estradiol Levonorgestrel 44 C44 Ethinyl estradiol Levonorgestrel 45 C45 Ethinyl estradiol Levonorgestrel 46 C46 Ethinyl estradiol Levonorgestrel 47 C47 Ethinyl estradiol Levonorgestrel 48 C48 Ethinyl estradiol Levonorgestrel 49 C49 Ethinyl estradiol Levonorgestrel 50 C50 Ethinyl estradiol Levonorgestrel 51 C51 Ethinyl estradiol Levonorgestrel 52 C52 Ethinyl estradiol Levonorgestrel 53 C53 Ethinyl estradiol Levonorgestrel 54 C54 Ethinyl estradiol Levonorgestrel 55 C55 Ethinyl estradiol Levonorgestrel 56 C56 Ethinyl estradiol Levonorgestrel 57 C57 Ethinyl estradiol Levonorgestrel 58 C58 Ethinyl estradiol Levonorgestrel 59 C59 Ethinyl estradiol Levonorgestrel 60 C60 Ethinyl estradiol Levonorgestrel 61 C61 Ethinyl estradiol Levonorgestrel 62 C62 Ethinyl estradiol Levonorgestrel 63 C63 Ethinyl estradiol Levonorgestrel 64 C64 Ethinyl estradiol Levonorgestrel 65 C65 Ethinyl estradiol Levonorgestrel 66 C66 Ethinyl estradiol Levonorgestrel 67 C67 Ethinyl estradiol Levonorgestrel 68 C1 Ethinyl estradiol Norethindrone acetate 69 C2 Ethinyl estradiol Norethindrone acetate 70 C3 Ethinyl estradiol Norethindrone acetate 71 C4 Ethinyl estradiol Norethindrone acetate 72 C5 Ethinyl estradiol Norethindrone acetate 73 C6 Ethinyl estradiol Norethindrone acetate 74 C7 Ethinyl estradiol Norethindrone acetate 75 C8 Ethinyl estradiol Norethindrone acetate 76 C9 Ethinyl estradiol Norethindrone acetate 77 C10 Ethinyl estradiol Norethindrone acetate 78 C11 Ethinyl estradiol Norethindrone acetate 79 C12 Ethinyl estradiol Norethindrone acetate 80 C13 Ethinyl estradiol Norethindrone acetate 81 C14 Ethinyl estradiol Norethindrone acetate 82 C15 Ethinyl estradiol Norethindrone acetate 83 C16 Ethinyl estradiol Norethindrone acetate 84 C17 Ethinyl estradiol Norethindrone acetate 85 C18 Ethinyl estradiol Norethindrone acetate 86 C19 Ethinyl estradiol Norethindrone acetate 87 C20 Ethinyl estradiol Norethindrone acetate 88 C21 Ethinyl estradiol Norethindrone acetate 89 C22 Ethinyl estradiol Norethindrone acetate 90 C23 Ethinyl estradiol Norethindrone acetate 91 C24 Ethinyl estradiol Norethindrone acetate 92 C25 Ethinyl estradiol Norethindrone acetate 93 C26 Ethinyl estradiol Norethindrone acetate 94 C27 Ethinyl estradiol Norethindrone acetate 95 C28 Ethinyl estradiol Norethindrone acetate 96 C29 Ethinyl estradiol Norethindrone acetate 97 C30 Ethinyl estradiol Norethindrone acetate 98 C31 Ethinyl estradiol Norethindrone acetate 99 C32 Ethinyl estradiol Norethindrone acetate 100 C33 Ethinyl estradiol Norethindrone acetate 101 C34 Ethinyl estradiol Norethindrone acetate 102 C35 Ethinyl estradiol Norethindrone acetate 103 C36 Ethinyl estradiol Norethindrone acetate 104 C37 Ethinyl estradiol Norethindrone acetate 105 C38 Ethinyl estradiol Norethindrone acetate 106 C39 Ethinyl estradiol Norethindrone acetate 107 C40 Ethinyl estradiol Norethindrone acetate 108 C41 Ethinyl estradiol Norethindrone acetate 109 C42 Ethinyl estradiol Norethindrone acetate 110 C43 Ethinyl estradiol Norethindrone acetate 111 C44 Ethinyl estradiol Norethindrone acetate 112 C45 Ethinyl estradiol Norethindrone acetate 113 C46 Ethinyl estradiol Norethindrone acetate 114 C47 Ethinyl estradiol Norethindrone acetate 115 C48 Ethinyl estradiol Norethindrone acetate 116 C49 Ethinyl estradiol Norethindrone acetate 117 C50 Ethinyl estradiol Norethindrone acetate 118 C51 Ethinyl estradiol Norethindrone acetate 119 C52 Ethinyl estradiol Norethindrone acetate 120 C53 Ethinyl estradiol Norethindrone acetate 121 C54 Ethinyl estradiol Norethindrone acetate 122 C55 Ethinyl estradiol Norethindrone acetate 123 C56 Ethinyl estradiol Norethindrone acetate 124 C57 Ethinyl estradiol Norethindrone acetate 125 C58 Ethinyl estradiol Norethindrone acetate 126 C59 Ethinyl estradiol Norethindrone acetate 127 C60 Ethinyl estradiol Norethindrone acetate 128 C61 Ethinyl estradiol Norethindrone acetate 129 C62 Ethinyl estradiol Norethindrone acetate 130 C63 Ethinyl estradiol Norethindrone acetate 131 C64 Ethinyl estradiol Norethindrone acetate 132 C65 Ethinyl estradiol Norethindrone acetate 133 C66 Ethinyl estradiol Norethindrone acetate 134 C67 Ethinyl estradiol Norethindrone acetate 135 C1 Ethinyl estradiol Norgestimate 136 C2 Ethinyl estradiol Norgestimate 137 C3 Ethinyl estradiol Norgestimate 138 C4 Ethinyl estradiol Norgestimate 139 C5 Ethinyl estradiol Norgestimate 140 C6 Ethinyl estradiol Norgestimate 141 C7 Ethinyl estradiol Norgestimate 142 C8 Ethinyl estradiol Norgestimate 143 C9 Ethinyl estradiol Norgestimate 144 C10 Ethinyl estradiol Norgestimate 145 C11 Ethinyl estradiol Norgestimate 146 C12 Ethinyl estradiol Norgestimate 147 C13 Ethinyl estradiol Norgestimate 148 C14 Ethinyl estradiol Norgestimate 149 C15 Ethinyl estradiol Norgestimate 150 C16 Ethinyl estradiol Norgestimate 151 C17 Ethinyl estradiol Norgestimate 152 C18 Ethinyl estradiol Norgestimate 153 C19 Ethinyl estradiol Norgestimate 154 C20 Ethinyl estradiol Norgestimate 155 C21 Ethinyl estradiol Norgestimate 156 C22 Ethinyl estradiol Norgestimate 157 C23 Ethinyl estradiol Norgestimate 158 C24 Ethinyl estradiol Norgestimate 159 C25 Ethinyl estradiol Norgestimate 160 C26 Ethinyl estradiol Norgestimate 161 C27 Ethinyl estradiol Norgestimate 162 C28 Ethinyl estradiol Norgestimate 163 C29 Ethinyl estradiol Norgestimate 164 C30 Ethinyl estradiol Norgestimate 165 C31 Ethinyl estradiol Norgestimate 166 C32 Ethinyl estradiol Norgestimate 167 C33 Ethinyl estradiol Norgestimate 168 C34 Ethinyl estradiol Norgestimate 169 C35 Ethinyl estradiol Norgestimate 170 C36 Ethinyl estradiol Norgestimate 171 C37 Ethinyl estradiol Norgestimate 172 C38 Ethinyl estradiol Norgestimate 173 C39 Ethinyl estradiol Norgestimate 174 C40 Ethinyl estradiol Norgestimate 175 C41 Ethinyl estradiol Norgestimate 176 C42 Ethinyl estradiol Norgestimate 177 C43 Ethinyl estradiol Norgestimate 178 C44 Ethinyl estradiol Norgestimate 179 C45 Ethinyl estradiol Norgestimate 180 C46 Ethinyl estradiol Norgestimate 181 C47 Ethinyl estradiol Norgestimate 182 C48 Ethinyl estradiol Norgestimate 183 C49 Ethinyl estradiol Norgestimate 184 C50 Ethinyl estradiol Norgestimate 185 C51 Ethinyl estradiol Norgestimate 186 C52 Ethinyl estradiol Norgestimate 187 C53 Ethinyl estradiol Norgestimate 188 C54 Ethinyl estradiol Norgestimate 189 C55 Ethinyl estradiol Norgestimate 190 C56 Ethinyl estradiol Norgestimate 191 C57 Ethinyl estradiol Norgestimate 192 C58 Ethinyl estradiol Norgestimate 193 C59 Ethinyl estradiol Norgestimate 194 C60 Ethinyl estradiol Norgestimate 195 C61 Ethinyl estradiol Norgestimate 196 C62 Ethinyl estradiol Norgestimate 197 C63 Ethinyl estradiol Norgestimate 198 C64 Ethinyl estradiol Norgestimate 199 C65 Ethinyl estradiol Norgestimate 200 C66 Ethinyl estradiol Norgestimate 201 C67 Ethinyl estradiol Norgestimate 202 C1 Ethinyl estradiol Ethynodiol diacetate 203 C2 Ethinyl estradiol Ethynodiol diacetate 204 C3 Ethinyl estradiol Ethynodiol diacetate 205 C4 Ethinyl estradiol Ethynodiol diacetate 206 C5 Ethinyl estradiol Ethynodiol diacetate 207 C6 Ethinyl estradiol Ethynodiol diacetate 208 C7 Ethinyl estradiol Ethynodiol diacetate 209 C8 Ethinyl estradiol Ethynodiol diacetate 210 C9 Ethinyl estradiol Ethynodiol diacetate 211 C10 Ethinyl estradiol Ethynodiol diacetate 212 C11 Ethinyl estradiol Ethynodiol diacetate 213 C12 Ethinyl estradiol Ethynodiol diacetate 214 C13 Ethinyl estradiol Ethynodiol diacetate 215 C14 Ethinyl estradiol Ethynodiol diacetate 216 C15 Ethinyl estradiol Ethynodiol diacetate 217 C16 Ethinyl estradiol Ethynodiol diacetate 218 C17 Ethinyl estradiol Ethynodiol diacetate 219 C18 Ethinyl estradiol Ethynodiol diacetate 220 C19 Ethinyl estradiol Ethynodiol diacetate 221 C20 Ethinyl estradiol Ethynodiol diacetate 222 C21 Ethinyl estradiol Ethynodiol diacetate 223 C22 Ethinyl estradiol Ethynodiol diacetate 224 C23 Ethinyl estradiol Ethynodiol diacetate 225 C24 Ethinyl estradiol Ethynodiol diacetate 226 C25 Ethinyl estradiol Ethynodiol diacetate 227 C26 Ethinyl estradiol Ethynodiol diacetate 228 C27 Ethinyl estradiol Ethynodiol diacetate 229 C28 Ethinyl estradiol Ethynodiol diacetate 230 C29 Ethinyl estradiol Ethynodiol diacetate 231 C30 Ethinyl estradiol Ethynodiol diacetate 232 C31 Ethinyl estradiol Ethynodiol diacetate 233 C32 Ethinyl estradiol Ethynodiol diacetate 234 C33 Ethinyl estradiol Ethynodiol diacetate 235 C34 Ethinyl estradiol Ethynodiol diacetate 236 C35 Ethinyl estradiol Ethynodiol diacetate 237 C36 Ethinyl estradiol Ethynodiol diacetate 238 C37 Ethinyl estradiol Ethynodiol diacetate 239 C38 Ethinyl estradiol Ethynodiol diacetate 240 C39 Ethinyl estradiol Ethynodiol diacetate 241 C40 Ethinyl estradiol Ethynodiol diacetate 242 C41 Ethinyl estradiol Ethynodiol diacetate 243 C42 Ethinyl estradiol Ethynodiol diacetate 244 C43 Ethinyl estradiol Ethynodiol diacetate 245 C44 Ethinyl estradiol Ethynodiol diacetate 246 C45 Ethinyl estradiol Ethynodiol diacetate 247 C46 Ethinyl estradiol Ethynodiol diacetate 248 C47 Ethinyl estradiol Ethynodiol diacetate 249 C48 Ethinyl estradiol Ethynodiol diacetate 250 C49 Ethinyl estradiol Ethynodiol diacetate 251 C50 Ethinyl estradiol Ethynodiol diacetate 252 C51 Ethinyl estradiol Ethynodiol diacetate 253 C52 Ethinyl estradiol Ethynodiol diacetate 254 C53 Ethinyl estradiol Ethynodiol diacetate 255 C54 Ethinyl estradiol Ethynodiol diacetate 256 C55 Ethinyl estradiol Ethynodiol diacetate 257 C56 Ethinyl estradiol Ethynodiol diacetate 258 C57 Ethinyl estradiol Ethynodiol diacetate 259 C58 Ethinyl estradiol Ethynodiol diacetate 260 C59 Ethinyl estradiol Ethynodiol diacetate 261 C60 Ethinyl estradiol Ethynodiol diacetate 262 C61 Ethinyl estradiol Ethynodiol diacetate 263 C62 Ethinyl estradiol Ethynodiol diacetate 264 C63 Ethinyl estradiol Ethynodiol diacetate 265 C64 Ethinyl estradiol Ethynodiol diacetate 266 C65 Ethinyl estradiol Ethynodiol diacetate 267 C66 Ethinyl estradiol Ethynodiol diacetate 268 C67 Ethinyl estradiol Ethynodiol diacetate 269 C1 Ethinyl estradiol Desogestrel 270 C2 Ethinyl estradiol Desogestrel 271 C3 Ethinyl estradiol Desogestrel 272 C4 Ethinyl estradiol Desogestrel 273 C5 Ethinyl estradiol Desogestrel 274 C6 Ethinyl estradiol Desogestrel 275 C7 Ethinyl estradiol Desogestrel 276 C8 Ethinyl estradiol Desogestrel 277 C9 Ethinyl estradiol Desogestrel 278 C10 Ethinyl estradiol Desogestrel 279 C11 Ethinyl estradiol Desogestrel 280 C12 Ethinyl estradiol Desogestrel 281 C13 Ethinyl estradiol Desogestrel 282 C14 Ethinyl estradiol Desogestrel 283 C15 Ethinyl estradiol Desogestrel 284 C16 Ethinyl estradiol Desogestrel 285 C17 Ethinyl estradiol Desogestrel 286 C18 Ethinyl estradiol Desogestrel 287 C19 Ethinyl estradiol Desogestrel 288 C20 Ethinyl estradiol Desogestrel 289 C21 Ethinyl estradiol Desogestrel 290 C22 Ethinyl estradiol Desogestrel 291 C23 Ethinyl estradiol Desogestrel 292 C24 Ethinyl estradiol Desogestrel 293 C25 Ethinyl estradiol Desogestrel 294 C26 Ethinyl estradiol Desogestrel 295 C27 Ethinyl estradiol Desogestrel 296 C28 Ethinyl estradiol Desogestrel 297 C29 Ethinyl estradiol Desogestrel 298 C30 Ethinyl estradiol Desogestrel 299 C31 Ethinyl estradiol Desogestrel 300 C32 Ethinyl estradiol Desogestrel 301 C33 Ethinyl estradiol Desogestrel 302 C34 Ethinyl estradiol Desogestrel 303 C35 Ethinyl estradiol Desogestrel 304 C36 Ethinyl estradiol Desogestrel 305 C37 Ethinyl estradiol Desogestrel 306 C38 Ethinyl estradiol Desogestrel 307 C39 Ethinyl estradiol Desogestrel 308 C40 Ethinyl estradiol Desogestrel 309 C41 Ethinyl estradiol Desogestrel 310 C42 Ethinyl estradiol Desogestrel 311 C43 Ethinyl estradiol Desogestrel 312 C44 Ethinyl estradiol Desogestrel 313 C45 Ethinyl estradiol Desogestrel 314 C46 Ethinyl estradiol Desogestrel 315 C47 Ethinyl estradiol Desogestrel 316 C48 Ethinyl estradiol Desogestrel 317 C49 Ethinyl estradiol Desogestrel 318 C50 Ethinyl estradiol Desogestrel 319 C51 Ethinyl estradiol Desogestrel 320 C52 Ethinyl estradiol Desogestrel 321 C53 Ethinyl estradiol Desogestrel 322 C54 Ethinyl estradiol Desogestrel 323 C55 Ethinyl estradiol Desogestrel 324 C56 Ethinyl estradiol Desogestrel 325 C57 Ethinyl estradiol Desogestrel 326 C58 Ethinyl estradiol Desogestrel 327 C59 Ethinyl estradiol Desogestrel 328 C60 Ethinyl estradiol Desogestrel 329 C61 Ethinyl estradiol Desogestrel 330 C62 Ethinyl estradiol Desogestrel 331 C63 Ethinyl estradiol Desogestrel 332 C64 Ethinyl estradiol Desogestrel 333 C65 Ethinyl estradiol Desogestrel 334 C66 Ethinyl estradiol Desogestrel 335 C67 Ethinyl estradiol Desogestrel 336 C1 Ethinyl estradiol Norgestrel 337 C2 Ethinyl estradiol Norgestrel 338 C3 Ethinyl estradiol Norgestrel 339 C4 Ethinyl estradiol Norgestrel 340 C5 Ethinyl estradiol Norgestrel 341 C6 Ethinyl estradiol Norgestrel 342 C7 Ethinyl estradiol Norgestrel 343 C8 Ethinyl estradiol Norgestrel 344 C9 Ethinyl estradiol Norgestrel 345 C10 Ethinyl estradiol Norgestrel 346 C11 Ethinyl estradiol Norgestrel 347 C12 Ethinyl estradiol Norgestrel 348 C13 Ethinyl estradiol Norgestrel 349 C14 Ethinyl estradiol Norgestrel 350 C15 Ethinyl estradiol Norgestrel 351 C16 Ethinyl estradiol Norgestrel 352 C17 Ethinyl estradiol Norgestrel 353 C18 Ethinyl estradiol Norgestrel 354 C19 Ethinyl estradiol Norgestrel 355 C20 Ethinyl estradiol Norgestrel 356 C21 Ethinyl estradiol Norgestrel 357 C22 Ethinyl estradiol Norgestrel 358 C23 Ethinyl estradiol Norgestrel 359 C24 Ethinyl estradiol Norgestrel 360 C25 Ethinyl estradiol Norgestrel 361 C26 Ethinyl estradiol Norgestrel 362 C27 Ethinyl estradiol Norgestrel 363 C28 Ethinyl estradiol Norgestrel 364 C29 Ethinyl estradiol Norgestrel 365 C30 Ethinyl estradiol Norgestrel 366 C31 Ethinyl estradiol Norgestrel 367 C32 Ethinyl estradiol Norgestrel 368 C33 Ethinyl estradiol Norgestrel 369 C34 Ethinyl estradiol Norgestrel 370 C35 Ethinyl estradiol Norgestrel 371 C36 Ethinyl estradiol Norgestrel 372 C37 Ethinyl estradiol Norgestrel 373 C38 Ethinyl estradiol Norgestrel 374 C39 Ethinyl estradiol Norgestrel 375 C40 Ethinyl estradiol Norgestrel 376 C41 Ethinyl estradiol Norgestrel 377 C42 Ethinyl estradiol Norgestrel 378 C43 Ethinyl estradiol Norgestrel 379 C44 Ethinyl estradiol Norgestrel 380 C45 Ethinyl estradiol Norgestrel 381 C46 Ethinyl estradiol Norgestrel 382 C47 Ethinyl estradiol Norgestrel 383 C48 Ethinyl estradiol Norgestrel 384 C49 Ethinyl estradiol Norgestrel 385 C50 Ethinyl estradiol Norgestrel 386 C51 Ethinyl estradiol Norgestrel 387 C52 Ethinyl estradiol Norgestrel 388 C53 Ethinyl estradiol Norgestrel 389 C54 Ethinyl estradiol Norgestrel 390 C55 Ethinyl estradiol Norgestrel 391 C56 Ethinyl estradiol Norgestrel 392 C57 Ethinyl estradiol Norgestrel 393 C58 Ethinyl estradiol Norgestrel 394 C59 Ethinyl estradiol Norgestrel 395 C60 Ethinyl estradiol Norgestrel 396 C61 Ethinyl estradiol Norgestrel 397 C62 Ethinyl estradiol Norgestrel 398 C63 Ethinyl estradiol Norgestrel 399 C64 Ethinyl estradiol Norgestrel 400 C65 Ethinyl estradiol Norgestrel 401 C66 Ethinyl estradiol Norgestrel 402 C67 Ethinyl estradiol Norgestrel 403 C1 Ethinyl estradiol Norethindrone 404 C2 Ethinyl estradiol Norethindrone 405 C3 Ethinyl estradiol Norethindrone 406 C4 Ethinyl estradiol Norethindrone 407 C5 Ethinyl estradiol Norethindrone 408 C6 Ethinyl estradiol Norethindrone 409 C7 Ethinyl estradiol Norethindrone 410 C8 Ethinyl estradiol Norethindrone 411 C9 Ethinyl estradiol Norethindrone 412 C10 Ethinyl estradiol Norethindrone 413 C11 Ethinyl estradiol Norethindrone 414 C12 Ethinyl estradiol Norethindrone 415 C13 Ethinyl estradiol Norethindrone 416 C14 Ethinyl estradiol Norethindrone 417 C15 Ethinyl estradiol Norethindrone 418 C16 Ethinyl estradiol Norethindrone 419 C17 Ethinyl estradiol Norethindrone 420 C18 Ethinyl estradiol Norethindrone 421 C19 Ethinyl estradiol Norethindrone 422 C20 Ethinyl estradiol Norethindrone 423 C21 Ethinyl estradiol Norethindrone 424 C22 Ethinyl estradiol Norethindrone 425 C23 Ethinyl estradiol Norethindrone 426 C24 Ethinyl estradiol Norethindrone 427 C25 Ethinyl estradiol Norethindrone 428 C26 Ethinyl estradiol Norethindrone 429 C27 Ethinyl estradiol Norethindrone 430 C28 Ethinyl estradiol Norethindrone 431 C29 Ethinyl estradiol Norethindrone 432 C30 Ethinyl estradiol Norethindrone 433 C31 Ethinyl estradiol Norethindrone 434 C32 Ethinyl estradiol Norethindrone 435 C33 Ethinyl estradiol Norethindrone 436 C34 Ethinyl estradiol Norethindrone 437 C35 Ethinyl estradiol Norethindrone 438 C36 Ethinyl estradiol Norethindrone 439 C37 Ethinyl estradiol Norethindrone 440 C38 Ethinyl estradiol Norethindrone 441 C39 Ethinyl estradiol Norethindrone 442 C40 Ethinyl estradiol Norethindrone 443 C41 Ethinyl estradiol Norethindrone 444 C42 Ethinyl estradiol Norethindrone 445 C43 Ethinyl estradiol Norethindrone 446 C44 Ethinyl estradiol Norethindrone 447 C45 Ethinyl estradiol Norethindrone 448 C46 Ethinyl estradiol Norethindrone 449 C47 Ethinyl estradiol Norethindrone 450 C48 Ethinyl estradiol Norethindrone 451 C49 Ethinyl estradiol Norethindrone 452 C50 Ethinyl estradiol Norethindrone 453 C51 Ethinyl estradiol Norethindrone 454 C52 Ethinyl estradiol Norethindrone 455 C53 Ethinyl estradiol Norethindrone 456 C54 Ethinyl estradiol Norethindrone 457 C55 Ethinyl estradiol Norethindrone 458 C56 Ethinyl estradiol Norethindrone 459 C57 Ethinyl estradiol Norethindrone 460 C58 Ethinyl estradiol Norethindrone 461 C59 Ethinyl estradiol Norethindrone 462 C60 Ethinyl estradiol Norethindrone 463 C61 Ethinyl estradiol Norethindrone 464 C62 Ethinyl estradiol Norethindrone 465 C63 Ethinyl estradiol Norethindrone 466 C64 Ethinyl estradiol Norethindrone 467 C65 Ethinyl estradiol Norethindrone 468 C66 Ethinyl estradiol Norethindrone 469 C67 Ethinyl estradiol Norethindrone 470 C1 Ethinyl estradiol 3-Ketodesogestrel 471 C2 Ethinyl estradiol 3-Ketodesogestrel 472 C3 Ethinyl estradiol 3-Ketodesogestrel 473 C4 Ethinyl estradiol 3-Ketodesogestrel 474 C5 Ethinyl estradiol 3-Ketodesogestrel 475 C6 Ethinyl estradiol 3-Ketodesogestrel 476 C7 Ethinyl estradiol 3-Ketodesogestrel 477 C8 Ethinyl estradiol 3-Ketodesogestrel 478 C9 Ethinyl estradiol 3-Ketodesogestrel 479 C10 Ethinyl estradiol 3-Ketodesogestrel 480 C11 Ethinyl estradiol 3-Ketodesogestrel 481 C12 Ethinyl estradiol 3-Ketodesogestrel 482 C13 Ethinyl estradiol 3-Ketodesogestrel 483 C14 Ethinyl estradiol 3-Ketodesogestrel 484 C15 Ethinyl estradiol 3-Ketodesogestrel 485 C16 Ethinyl estradiol 3-Ketodesogestrel 486 C17 Ethinyl estradiol 3-Ketodesogestrel 487 C18 Ethinyl estradiol 3-Ketodesogestrel 488 C19 Ethinyl estradiol 3-Ketodesogestrel 489 C20 Ethinyl estradiol 3-Ketodesogestrel 490 C21 Ethinyl estradiol 3-Ketodesogestrel 491 C22 Ethinyl estradiol 3-Ketodesogestrel 492 C23 Ethinyl estradiol 3-Ketodesogestrel 493 C24 Ethinyl estradiol 3-Ketodesogestrel 494 C25 Ethinyl estradiol 3-Ketodesogestrel 495 C26 Ethinyl estradiol 3-Ketodesogestrel 496 C27 Ethinyl estradiol 3-Ketodesogestrel 497 C28 Ethinyl estradiol 3-Ketodesogestrel 498 C29 Ethinyl estradiol 3-Ketodesogestrel 499 C30 Ethinyl estradiol 3-Ketodesogestrel 500 C31 Ethinyl estradiol 3-Ketodesogestrel 501 C32 Ethinyl estradiol 3-Ketodesogestrel 502 C33 Ethinyl estradiol 3-Ketodesogestrel 503 C34 Ethinyl estradiol 3-Ketodesogestrel 504 C35 Ethinyl estradiol 3-Ketodesogestrel 505 C36 Ethinyl estradiol 3-Ketodesogestrel 506 C37 Ethinyl estradiol 3-Ketodesogestrel 507 C38 Ethinyl estradiol 3-Ketodesogestrel 508 C39 Ethinyl estradiol 3-Ketodesogestrel 509 C40 Ethinyl estradiol 3-Ketodesogestrel 510 C41 Ethinyl estradiol 3-Ketodesogestrel 511 C42 Ethinyl estradiol 3-Ketodesogestrel 512 C43 Ethinyl estradiol 3-Ketodesogestrel 513 C44 Ethinyl estradiol 3-Ketodesogestrel 514 C45 Ethinyl estradiol 3-Ketodesogestrel 515 C46 Ethinyl estradiol 3-Ketodesogestrel 516 C47 Ethinyl estradiol 3-Ketodesogestrel 517 C48 Ethinyl estradiol 3-Ketodesogestrel 518 C49 Ethinyl estradiol 3-Ketodesogestrel 519 C50 Ethinyl estradiol 3-Ketodesogestrel 520 C51 Ethinyl estradiol 3-Ketodesogestrel 521 C52 Ethinyl estradiol 3-Ketodesogestrel 522 C53 Ethinyl estradiol 3-Ketodesogestrel 523 C54 Ethinyl estradiol 3-Ketodesogestrel 524 C55 Ethinyl estradiol 3-Ketodesogestrel 525 C56 Ethinyl estradiol 3-Ketodesogestrel 526 C57 Ethinyl estradiol 3-Ketodesogestrel 527 C58 Ethinyl estradiol 3-Ketodesogestrel 528 C59 Ethinyl estradiol 3-Ketodesogestrel 529 C60 Ethinyl estradiol 3-Ketodesogestrel 530 C61 Ethinyl estradiol 3-Ketodesogestrel 531 C62 Ethinyl estradiol 3-Ketodesogestrel 532 C63 Ethinyl estradiol 3-Ketodesogestrel 533 C64 Ethinyl estradiol 3-Ketodesogestrel 534 C65 Ethinyl estradiol 3-Ketodesogestrel 535 C66 Ethinyl estradiol 3-Ketodesogestrel 536 C67 Ethinyl estradiol 3-Ketodesogestrel 537 C1 Ethinyl estradiol Gestodene 538 C2 Ethinyl estradiol Gestodene 539 C3 Ethinyl estradiol Gestodene 540 C4 Ethinyl estradiol Gestodene 541 C5 Ethinyl estradiol Gestodene 542 C6 Ethinyl estradiol Gestodene 543 C7 Ethinyl estradiol Gestodene 544 C8 Ethinyl estradiol Gestodene 545 C9 Ethinyl estradiol Gestodene 546 C10 Ethinyl estradiol Gestodene 547 C11 Ethinyl estradiol Gestodene 548 C12 Ethinyl estradiol Gestodene 549 C13 Ethinyl estradiol Gestodene 550 C14 Ethinyl estradiol Gestodene 551 C15 Ethinyl estradiol Gestodene 552 C16 Ethinyl estradiol Gestodene 553 C17 Ethinyl estradiol Gestodene 554 C18 Ethinyl estradiol Gestodene 555 C19 Ethinyl estradiol Gestodene 556 C20 Ethinyl estradiol Gestodene 557 C21 Ethinyl estradiol Gestodene 558 C22 Ethinyl estradiol Gestodene 559 C23 Ethinyl estradiol Gestodene 560 C24 Ethinyl estradiol Gestodene 561 C25 Ethinyl estradiol Gestodene 562 C26 Ethinyl estradiol Gestodene 563 C27 Ethinyl estradiol Gestodene 564 C28 Ethinyl estradiol Gestodene 565 C29 Ethinyl estradiol Gestodene 566 C30 Ethinyl estradiol Gestodene 567 C31 Ethinyl estradiol Gestodene 568 C32 Ethinyl estradiol Gestodene 569 C33 Ethinyl estradiol Gestodene 570 C34 Ethinyl estradiol Gestodene 571 C35 Ethinyl estradiol Gestodene 572 C36 Ethinyl estradiol Gestodene 573 C37 Ethinyl estradiol Gestodene 574 C38 Ethinyl estradiol Gestodene 575 C39 Ethinyl estradiol Gestodene 576 C40 Ethinyl estradiol Gestodene 577 C41 Ethinyl estradiol Gestodene 578 C42 Ethinyl estradiol Gestodene 579 C43 Ethinyl estradiol Gestodene 580 C44 Ethinyl estradiol Gestodene 581 C45 Ethinyl estradiol Gestodene 582 C46 Ethinyl estradiol Gestodene 583 C47 Ethinyl estradiol Gestodene 584 C48 Ethinyl estradiol Gestodene 585 C49 Ethinyl estradiol Gestodene 586 C50 Ethinyl estradiol Gestodene 587 C51 Ethinyl estradiol Gestodene 588 C52 Ethinyl estradiol Gestodene 589 C53 Ethinyl estradiol Gestodene 590 C54 Ethinyl estradiol Gestodene 591 C55 Ethinyl estradiol Gestodene 592 C56 Ethinyl estradiol Gestodene 593 C57 Ethinyl estradiol Gestodene 594 C58 Ethinyl estradiol Gestodene 595 C59 Ethinyl estradiol Gestodene 596 C60 Ethinyl estradiol Gestodene 597 C61 Ethinyl estradiol Gestodene 598 C62 Ethinyl estradiol Gestodene 599 C63 Ethinyl estradiol Gestodene 600 C64 Ethinyl estradiol Gestodene 601 C65 Ethinyl estradiol Gestodene 602 C66 Ethinyl estradiol Gestodene 603 C67 Ethinyl estradiol Gestodene 604 C1 Ethinyl estradiol Org 30659 605 C2 Ethinyl estradiol Org 30659 606 C3 Ethinyl estradiol Org 30659 607 C4 Ethinyl estradiol Org 30659 608 C5 Ethinyl estradiol Org 30659 609 C6 Ethinyl estradiol Org 30659 610 C7 Ethinyl estradiol Org 30659 611 C8 Ethinyl estradiol Org 30659 612 C9 Ethinyl estradiol Org 30659 613 C10 Ethinyl estradiol Org 30659 614 C11 Ethinyl estradiol Org 30659 615 C12 Ethinyl estradiol Org 30659 616 C13 Ethinyl estradiol Org 30659 617 C14 Ethinyl estradiol Org 30659 618 C15 Ethinyl estradiol Org 30659 619 C16 Ethinyl estradiol Org 30659 620 C17 Ethinyl estradiol Org 30659 621 C18 Ethinyl estradiol Org 30659 622 C19 Ethinyl estradiol Org 30659 623 C20 Ethinyl estradiol Org 30659 624 C21 Ethinyl estradiol Org 30659 625 C22 Ethinyl estradiol Org 30659 626 C23 Ethinyl estradiol Org 30659 627 C24 Ethinyl estradiol Org 30659 628 C25 Ethinyl estradiol Org 30659 629 C26 Ethinyl estradiol Org 30659 630 C27 Ethinyl estradiol Org 30659 631 C28 Ethinyl estradiol Org 30659 632 C29 Ethinyl estradiol Org 30659 633 C30 Ethinyl estradiol Org 30659 634 C31 Ethinyl estradiol Org 30659 635 C32 Ethinyl estradiol Org 30659 636 C33 Ethinyl estradiol Org 30659 637 C34 Ethinyl estradiol Org 30659 638 C35 Ethinyl estradiol Org 30659 639 C36 Ethinyl estradiol Org 30659 640 C37 Ethinyl estradiol Org 30659 641 C38 Ethinyl estradiol Org 30659 642 C39 Ethinyl estradiol Org 30659 643 C40 Ethinyl estradiol Org 30659 644 C41 Ethinyl estradiol Org 30659 645 C42 Ethinyl estradiol Org 30659 646 C43 Ethinyl estradiol Org 30659 647 C44 Ethinyl estradiol Org 30659 648 C45 Ethinyl estradiol Org 30659 649 C46 Ethinyl estradiol Org 30659 650 C47 Ethinyl estradiol Org 30659 651 C48 Ethinyl estradiol Org 30659 652 C49 Ethinyl estradiol Org 30659 653 C50 Ethinyl estradiol Org 30659 654 C51 Ethinyl estradiol Org 30659 655 C52 Ethinyl estradiol Org 30659 656 C53 Ethinyl estradiol Org 30659 657 C54 Ethinyl estradiol Org 30659 658 C55 Ethinyl estradiol Org 30659 659 C56 Ethinyl estradiol Org 30659 660 C57 Ethinyl estradiol Org 30659 661 C58 Ethinyl estradiol Org 30659 662 C59 Ethinyl estradiol Org 30659 663 C60 Ethinyl estradiol Org 30659 664 C61 Ethinyl estradiol Org 30659 665 C62 Ethinyl estradiol Org 30659 666 C63 Ethinyl estradiol Org 30659 667 C64 Ethinyl estradiol Org 30659 668 C65 Ethinyl estradiol Org 30659 669 C66 Ethinyl estradiol Org 30659 670 C67 Ethinyl estradiol Org 30659 671 C1 Ethinyl estradiol Trimagestone 672 C2 Ethinyl estradiol Trimegestone 673 C3 Ethinyl estradiol Trimegestone 674 C4 Ethinyl estradiol Trimegestone 675 C5 Ethinyl estradiol Trimegestone 676 C6 Ethinyl estradiol Trimegestone 677 C7 Ethinyl estradiol Trimegestone 678 C8 Ethinyl estradiol Trimegestone 679 C9 Ethinyl estradiol Trimegestone 680 C10 Ethinyl estradiol Trimegestone 681 C11 Ethinyl estradiol Trimegestone 682 C12 Ethinyl estradiol Trimegestone 683 C13 Ethinyl estradiol Trimegestone 684 C14 Ethinyl estradiol Trimegestone 685 C15 Ethinyl estradiol Trimegestone 686 C16 Ethinyl estradiol Trimegestone 687 C17 Ethinyl estradiol Trimegestone 688 C18 Ethinyl estradiol Trimegestone 689 C19 Ethinyl estradiol Trimegestone 690 C20 Ethinyl estradiol Trimegestone 691 C21 Ethinyl estradiol Trimegestone 692 C22 Ethinyl estradiol Trimegestone 693 C23 Ethinyl estradiol Trimegestone 694 C24 Ethinyl estradiol Trimegestone 695 C25 Ethinyl estradiol Trimegestone 696 C26 Ethinyl estradiol Trimegestone 697 C27 Ethinyl estradiol Trimegestone 698 C28 Ethinyl estradiol Trimegestone 699 C29 Ethinyl estradiol Trimegestone 700 C30 Ethinyl estradiol Trimegestone 701 C31 Ethinyl estradiol Trimegestone 702 C32 Ethinyl estradiol Trimegestone 703 C33 Ethinyl estradiol Trimegestone 704 C34 Ethinyl estradiol Trimegestone 705 C35 Ethinyl estradiol Trimegestone 706 C36 Ethinyl estradiol Trimegestone 707 C37 Ethinyl estradiol Trimegestone 708 C38 Ethinyl estradiol Trimegestone 709 C39 Ethinyl estradiol Trimegestone 710 C40 Ethinyl estradiol Trimegestone 711 C41 Ethinyl estradiol Trimegestone 712 C42 Ethinyl estradiol Trimegestone 713 C43 Ethinyl estradiol Trimegestone 714 C44 Ethinyl estradiol Trimegestone 715 C45 Ethinyl estradiol Trimegestone 716 C46 Ethinyl estradiol Trimegestone 717 C47 Ethinyl estradiol Trimegestone 718 C48 Ethinyl estradiol Trimegestone 719 C49 Ethinyl estradiol Trimegestone 720 C50 Ethinyl estradiol Trimegestone 721 C51 Ethinyl estradiol Trimegestone 722 C52 Ethinyl estradiol Trimegestone 723 C53 Ethinyl estradiol Trimegestone 724 C54 Ethinyl estradiol Trimegestone 725 C55 Ethinyl estradiol Trimegestone 726 C56 Ethinyl estradiol Trimegestone 727 C57 Ethinyl estradiol Trimegestone 728 C58 Ethinyl estradiol Trimegestone 729 C59 Ethinyl estradiol Trimegestone 730 C60 Ethinyl estradiol Trimegestone 731 C61 Ethinyl estradiol Trimegestone 732 C62 Ethinyl estradiol Trimegestone 733 C63 Ethinyl estradiol Trimegestone 734 C64 Ethinyl estradiol Trimegestone 735 C65 Ethinyl estradiol Trimegestone 736 C66 Ethinyl estradiol Trimegestone 737 C67 Ethinyl estradiol Trimegestone 738 C1 Ethinyl estradiol Dienogest 739 C2 Ethinyl estradiol Dienogest 740 C3 Ethinyl estradiol Dienogest 741 C4 Ethinyl estradiol Dienogest 742 C5 Ethinyl estradiol Dienogest 743 C6 Ethinyl estradiol Dienogest 744 C7 Ethinyl estradiol Dienogest 745 C8 Ethinyl estradiol Dienogest 746 C9 Ethinyl estradiol Dienogest 747 C10 Ethinyl estradiol Dienogest 748 C11 Ethinyl estradiol Dienogest 749 C12 Ethinyl estradiol Dienogest 750 C13 Ethinyl estradiol Dienogest 751 C14 Ethinyl estradiol Dienogest 752 C15 Ethinyl estradiol Dienogest 753 C16 Ethinyl estradiol Dienogest 754 C17 Ethinyl estradiol Dienogest 755 C18 Ethinyl estradiol Dienogest 756 C19 Ethinyl estradiol Dienogest 757 C20 Ethinyl estradiol Dienogest 758 C21 Ethinyl estradiol Dienogest 759 C22 Ethinyl estradiol Dienogest 760 C23 Ethinyl estradiol Dienogest 761 C24 Ethinyl estradiol Dienogest 762 C25 Ethinyl estradiol Dienogest 763 C26 Ethinyl estradiol Dienogest 764 C27 Ethinyl estradiol Dienogest 765 C28 Ethinyl estradiol Dienogest 766 C29 Ethinyl estradiol Dienogest 767 C30 Ethinyl estradiol Dienogest 768 C31 Ethinyl estradiol Dienogest 769 C32 Ethinyl estradiol Dienogest 770 C33 Ethinyl estradiol Dienogest 771 C34 Ethinyl estradiol Dienogest 772 C35 Ethinyl estradiol Dienogest 773 C36 Ethinyl estradiol Dienogest 774 C37 Ethinyl estradiol Dienogest 775 C38 Ethinyl estradiol Dienogest 776 C39 Ethinyl estradiol Dienogest 777 C40 Ethinyl estradiol Dienogest 778 C41 Ethinyl estradiol Dienogest 779 C42 Ethinyl estradiol Dienogest 780 C43 Ethinyl estradiol Dienogest 781 C44 Ethinyl estradiol Dienogest 782 C45 Ethinyl estradiol Dienogest 783 C46 Ethinyl estradiol Dienogest 784 C47 Ethinyl estradiol Dienogest 785 C48 Ethinyl estradiol Dienogest 786 C49 Ethinyl estradiol Dienogest 787 C50 Ethinyl estradiol Dienogest 788 C51 Ethinyl estradiol Dienogest 789 C52 Ethinyl estradiol Dienogest 790 C53 Ethinyl estradiol Dienogest 791 C54 Ethinyl estradiol Dienogest 792 C55 Ethinyl estradiol Dienogest 793 C56 Ethinyl estradiol Dienogest 794 C57 Ethinyl estradiol Dienogest 795 C58 Ethinyl estradiol Dienogest 796 C59 Ethinyl estradiol Dienogest 797 C60 Ethinyl estradiol Dienogest 798 C61 Ethinyl estradiol Dienogest 799 C62 Ethinyl estradiol Dienogest 800 C63 Ethinyl estradiol Dienogest 801 C64 Ethinyl estradiol Dienogest 802 C65 Ethinyl estradiol Dienogest 803 C66 Ethinyl estradiol Dienogest 804 C67 Ethinyl estradiol Dienogest Biological Assays The utility of the combinations of the present invention can be shown by the following assays.", "These assays are performed in vitro and in animal models essentially using procedures recognized to show the utility of the present invention.", "Rat Carrageenan Foot Pad Edema Test The carrageenan foot edema test is performed with materials, reagents and procedures essentially as described by Winter, et al., (Proc.", "Soc.", "Exp.", "Biol.", "Med., 111, 544 (1962)).", "Male Sprague-Dawley rats are selected in each group so that the average body weight is as close as possible.", "Rats are fasted with free access to water for over sixteen hours prior to the test.", "The rats are dosed orally (1 mL) with compounds suspended in vehicle containing 0.5% methylcellulose and 0.025% surfactant, or with vehicle alone.", "One hour later a subplantar injection of 0.1 mL of 1% solution of carrageenan/sterile 0.9% saline is administered and the volume of the injected foot is measured with a displacement plethysmometer connected to a pressure transducer with a digital indicator.", "Three hours after the injection of the carrageenan, the volume of the foot is again measured.", "The average foot swelling in a group of drug-treated animals is compared with that of a group of placebo-treated animals and the percentage inhibition of edema is determined (Otterness and Bliven, Laboratory Models for Testing NSAIDS, in Non-steroidal Anti-Inflammatory Drugs, (J. Lombardino, ed.", "1985)).", "The % inhibition shows the % decrease from control paw volume determined in this procedure.", "Rat Carrageenan-Induced Analgesia Test The analgesia test using rat carrageenan is performed with materials, reagents and procedures essentially as described by Hargreaves, et al., (Pain, 32, 77 (1988)).", "Male Sprague-Dawley rats are treated as previously described for the Carrageenan Foot Pad Edema test.", "Three hours after the injection of the carrageenan, the rats are placed in a special plexiglass container with a transparent floor having a high intensity lamp as a radiant heat source, positionable under the floor.", "After an initial twenty minute period, thermal stimulation is begun on either the injected foot or on the contralateral uninjected foot.", "A photoelectric cell turns off the lamp and timer when light is interrupted by paw withdrawal.", "The time until the rat withdraws its foot is then measured.", "The withdrawal latency in seconds is determined for the control and drug-treated groups, and percent inhibition of the hyperalgesic foot withdrawal determined.", "Evaluation of COX-1 and COX-2 Activity In Vitro The compounds of this invention exhibit inhibition in vitro of COX-2.The COX-2 inhibition activity of the compounds of this invention illustrated in the Examples is determined by the following methods.", "a.", "Preparation of Recombinant COX Baculoviruses A 2.0 kb fragment containing the coding region of either human or murine COX-1 or human or murine COX-2 is cloned into a BamH1 site of the baculovirus transfer vector pVL1393 (Invitrogen) to generate the baculovirus transfer vectors for COX-1 and COX-2 in a manner similar to the method of D. R. O'Reilly et al (Baculovirus Expression Vectors: A Laboratory Manual (1992)).", "Recombinant baculoviruses are isolated by transfecting 4 μg of baculovirus transfer vector DNA into SF9 insect cells (2×10 eB) along with 200 ng of linearized baculovirus plasmid DNA by the calcium phosphate method.", "See M. D. Summers and G. E. Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agric.", "Exp.", "Station Bull.", "1555 (1987).", "Recombinant viruses are purified by three rounds of plaque purification and high titer (10E7-10E8 pfu/ml) stocks of virus are prepared.", "For large scale production, SF9 insect cells are infected in 10 liter fermentors (0.5×106/ml) with the recombinant baculovirus stock such that the multiplicity of infection is 0.1.After 72 hours the cells are centrifuged and the cell pellet homogenized in Tris/Sucrose (50 mM: 25%, pH 8.0) containing 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).", "The homogenate is centrifuged at 10,000×G for 30 minutes, and the resultant supernatant is stored at −80° C. before being assayed for COX activity.", "b. Assay for COX-1 and COX-2 Activity COX activity is assayed as PGE2 formed/μg protein/time using an ELISA to detect the prostaglandin released.", "CHAPS-solubilized insect cell membranes containing the appropriate COX enzyme are incubated in a potassium phosphate buffer (50 mM, pH 8.0) containing epinephrine, phenol, and heme with the addition of arachidonic acid (10 μM).", "Compounds are pre-incubated with the enzyme for 10-20 minutes prior to the addition of arachidonic acid.", "Any reaction between the arachidonic acid and the enzyme is stopped after ten minutes at 37° C./room temperature by transferring 40 μl of reaction mix into 160 μl ELISA buffer and 25 μM indomethacin.", "The PGE2 formed is measured by standard ELISA technology (Cayman Chemical).", "The examples herein can be performed by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.", "The invention being thus described, it is apparent that the same can be varied in many ways.", "Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications and equivalents as would be obvious to one skilled in the art are intended to be included within the scope of the following claims." ] ]	Patent_10467146
[ [ "Gel emulsions in the form of o/w emulsions having a hydrocolloid content", "The invention relates to cosmetic preparations in the form of gel emulsions on preparations of the O/W emulsion type having a hydrocolloid content.", "Said hydrocolloids are selected from the group of preparations based on (i) hydroxyethylcellulose, (ii) xanthan gum, (iii) carbomer.", "Said preparations further contain (iv) one or more lipids, (v) one or more non-ionic and/or anionic emulsifiers with HLB values between 8 and 16, the overall content of the emulsifiers not exceeding 1.5% by weight." ], [ "1-9.", "(canceled) 10.A composition which is present as a gel emulsion, wherein the composition is based on an O/W emulsion and comprises the following components: (i) hydroxyethylcellulose; (ii) xanthan gum; (iii) carbomer; (iv) one or more lipids; and (v) one or more emulsifiers having HLB values of from 8 to 16, said emulsifiers comprising at least one of a nonionic and an anionic emulsifier; wherein a ratio of (i):(ii):(iii) is a:b:c and a, b and c independently are numbers of from 1 to 5 and b may additionally be equal to 0; components (i), (ii) and (iii) are present in a total concentration of from 0.25% to 1.5% by weight, component (iv) is present in a concentration of from 3.0% to 20.0% by weight, and component (v) is present in a concentration not exceeding 1.5% by weight, all percentages being based on a total weight of the composition.", "11.The composition of claim 10, wherein a, b and c independently are numbers of from 1 to 3.12.The composition of claim 11, wherein components (i), (ii) and (iii) are present in a total concentration of from 0.5% to 1.0% by weight.", "13.The composition of claim 10, wherein component (iv) is present in a concentration of from 7.5% to 15% by weight.", "14.The composition of claim 10, wherein the one or more emulsifiers (v) comprise one or more emulsifiers having HLB values of from 10 to 12.15.The composition of claim 10, wherein component (v) is present in a concentration of from 0.5% to 1.0% by weight.", "16.The composition of claim 15, wherein component (v) comprises glyceryl stearate citrate.", "17.The composition of claim 10, wherein the composition further comprises (vi) one or more fatty alcohols.", "18.The composition of claim 17, wherein component (vi) is present in a concentration of up to 10% by weight, based on the total weight of the composition.", "19.The composition of claim 17, wherein component (vi) is present in a concentration of up to 5.0% by weight.", "20.The composition of claim 17, wherein component (vi) is present in a concentration of up to 3.0% by weight.", "21.The composition of claim 13, wherein component (iv) comprises less than about 30% by weight of polar lipids.", "22.The composition of claim 13, wherein component (iv) comprises at least one lipid which has an interfacial tension with water of from about 20 to about 30 mN/m.", "23.The composition of claim 10, wherein the composition comprises an emulsion having an oil phase which comprises at least 50% by weight of at least one of petrolatum, paraffin oil and polyolefins.", "24.The composition of claim 23, wherein the oil phase comprises at least 75% by weight of at least one of petrolatum, paraffin oil and polyolefins.", "25.The composition of claim 10, wherein the composition comprises an emulsion having an oil phase which comprises a silicone oil.", "26.The composition of claim 10, wherein the composition further comprises at least one antioxidant.", "27.The composition of claim 26, wherein the at least one antioxidant is present in a concentration of from 0.001% to 30% by weight, based on the total weight of the composition.", "28.The composition of claim 12, wherein the composition further comprises at least one antioxidant in a concentration of from 0.05% to 20% by weight, based on the total weight of the composition.", "29.The composition of claim 10, wherein the composition further comprises at least one antioxidant in a concentration of from 1% to 10% by weight, based on the total weight of the composition.", "30.The composition of claim 27, wherein the at least one antioxidant comprises an oil-soluble antioxidant.", "31.The composition of claim 26, wherein the at least one antioxidant comprises at least one of vitamin A and derivatives thereof.", "32.The composition of claim 31, wherein the at least one of vitamin A and derivatives thereof is present in a concentration of from 0.001% to 10% by weight, based on the total weight of the composition.", "33.The composition of claim 26, wherein the at least one antioxidant comprises at least one of vitamin E and derivatives thereof.", "34.The composition of claim 33, wherein the at least one of vitamin E and derivatives thereof is present in a concentration of from 0.001% to 10% by weight, based on the total weight of the composition.", "35.The composition of claim 10, wherein the composition further comprises at least one UV filter substance which is at least one of a UV-A filter substance, a UV-B filter substance and an inorganic pigment.", "36.The composition of claim 35, wherein the at least one UV filter substance is present in a total concentration of from 0.1% to 30% by weight, based on the total weight of the composition.", "37.A composition which is present as a gel emulsion, wherein the composition is based on an O/W emulsion and comprises the following components: (i) hydroxyethylcellulose; (ii) xanthan gum; (iii) carbomer; (iv) one or more lipids; (v) one or more emulsifiers having HLB values of from 10 to 12, said emulsifiers comprising at least one of a nonionic and an anionic emulsifier; and (vi) one or more fatty alcohols; wherein a ratio of (i):(ii):(iii) is a:b:c and a, b and c independently are numbers of from 1 to 3 and b may additionally be equal to 0; components (i), (ii) and (iii) are present in a total concentration of from 0.5% to 1.0% by weight, component (iv) is present in a concentration of from 7.5% to 15% by weight, component (v) is present in a concentration of from 0.5% to 1.0% by weight, and component (vi) is present in a concentration of up to 3.0% by weight, all percentages being based on a total weight of the composition.", "38.The composition of claim 37, wherein component (v) comprises glyceryl stearate citrate.", "39.The composition of claim 37, wherein component (iv) comprises less than about 30% by weight of polar lipids.", "40.The composition of claim 37, wherein the composition further comprises at least one antioxidant in a concentration of from 1% to 10% by weight, based on the total weight of the composition.", "41.The composition of claim 40, wherein the at least one antioxidant comprises at least one of vitamin A, vitamin E and derivatives thereof.", "42.The composition of claim 37, wherein the composition further comprises at least at least one of a UV-A filter substance, a UV-B filter substance and an inorganic pigment in a total concentration of 1.0% to 6.0% by weight, based on the total weight of the composition.", "43.A cosmetic preparation which comprises the composition of claim 10.44.The cosmetic preparation of claim 43, which comprises one of a skin protection cream, a cleansing cream, a cleansing milk, a sunscreen lotion, a nourishing cream, a day cream and a night cream.", "45.A dermatological preparation which comprises the composition of claim 10.46.A sunscreen preparation which comprises the composition of claim 35.47.A lip care product which comprises the composition of claim 10.48.A hair care product which comprises the composition of claim 10.49.An aerosol container which comprises the composition of claim 10 and a propellant.", "50.The aerosol container of claim 49, wherein the propellant comprises a liquefied hydrocarbon.", "51.A roll-on device which comprises the composition of claim 10.52.A method of treating and protecting skin, wherein the method comprises an application onto the skin of a composition which is present as a gel emulsion, is based on an O/W emulsion and comprises the following components: (i) hydroxyethylcellulose; (ii) xanthan gum; (iii) carbomer; (iv) one or more lipids; and (v) one or more emulsifiers having HLB values of from 10 to 12, said emulsifiers comprising at least one of a nonionic and an anionic emulsifier; wherein a ratio of (i):(ii):(iii) is a:b:c and a, b and c independently are numbers of from 1 to 3 and b may additionally be equal to 0; components (i), (ii) and (iii) are present in a total concentration of from 0.25% to 1.5% by weight, component (iv) is present in a concentration of from 3.0% to 20.0% by weight, and component (v) is present in a concentration not exceeding 1.5% by weight, all percentages being based on a total weight of the composition." ], [ "The present invention relates to cosmetic and dermatological preparations, in particular those of the oil-in-water type, to processes for their preparation and to their use for cosmetic and medicinal purposes.", "The human skin is the largest human organ and performs numerous vital functions.", "Having an average area of about 2 m2 in adults, it has a prominent role as a protective and sensory organ.", "The purpose of this organ is to transmit and avert mechanical, thermal, actinic, chemical and biological stimuli.", "In addition, it has an important role as a regulatory and target organ in human metabolism.", "The main aim of skincare in the cosmetics sense is to strengthen or restore the skin's natural function as a barrier against environmental influences (e.g.", "dirt, chemicals, microorganisms) and against the loss of endogenous substances (e.g.", "water, natural fats, electrolytes), and also to assist its horny layer in its natural regeneration ability in cases of existing damage.", "If the barrier properties of the skin are impaired, increased resorption of toxic or allergenic substances or attack by microorganisms may result, leading to toxic or allergic skin reactions.", "Another aim of skincare is to compensate for the loss by the skin of sebum and water caused by daily washing.", "This is particularly important if the natural regeneration ability is inadequate.", "Furthermore, skincare products should protect against environmental influences, in particular against sun and wind, and delay skin aging.", "Medicinal topical compositions usually comprise one or more medicaments in an effective concentration.", "For the sake of simplicity, in order to clearly distinguish between cosmetic and medicinal use and corresponding products, reference is made to the legal provisions in the Federal Republic of Germany (e.g.", "Cosmetics Directive, Foods and Drugs Act).", "Emulsions are generally understood as meaning heterogeneous systems which consist of two liquids, which are usually referred to as phases, and which are immiscible or miscible with one another only to a limited extent.", "In an emulsion, one of the two liquids is dispersed in the form of very fine droplets in the other liquid.", "If the two liquids are water and oil and oil droplets are very finely dispersed in water, this is an oil-in-water emulsion (O/W emulsion, e.g.", "milk).", "The basic character of an O/W emulsion is determined by the water.", "In the case of a water-in-oil emulsion (W/O emulsion, e.g.", "butter), the principle is reversed, the basic character being determined here by the oil.", "Gel creams are particularly light products with a low emulsifier and lipid content.", "They are characterized by the fact that they can be readily distributed on the skin and impart a fresh feel.", "Following product application, no or only a little residue should remain on the skin.", "Gel creams usually comprise a relatively high content of hydrophilic thickeners (e.g.", "carbopols, xanthan gum, hydroxyethylcellulose) for thickening and stabilizing the systems.", "Since the thickener or the thickener system is present in the external phase, it has a significant influence on the sensory properties of the product.", "Current thickener systems can either not be distributed readily, do not produce a fresh feeling or leave behind an excessively greasy residue on the fingers and/or a harsh, sticky feel on the skin after the product has been distributed on the skin.", "The aim was to overcome these disadvantages.", "Surprisingly, it has been found that cosmetic preparations in the form of gel emulsions, namely preparations based on preparations of the O/W emulsion type with a content of hydrocolloids chosen from the group (i) hydroxyethylcellulose (ii) xanthan gum (iii) carbomer, where the ratio of (i):(ii):(iii) is chosen as a:b:c, where a, b and c, independently of one another are rational numbers from 1 to 5, preferably from 1 to 3, and where in particular b can also assume the value zero, where (i), (ii) and (iii) are present in the preparations in total concentrations (a+b+c) of 0.25-1.5% by weight, based on the total weight of the preparation, preferably in total concentrations of 0.5-1.0% by weight, where these preparations further comprise (iv) one or more lipids, where the total content of the lipids is chosen from the concentration range from 3.0 to 20.0% by weight, based on the total weight of the preparations, preferably in total concentrations of from 7.5 to 15% by weight, where these preparations further comprise (v) one or more nonionic and/or anionic emulsifiers with HLB values between 8 and 16, preferably between 10 and 12 where the total content of the emulsifiers does not exceed 1.5% by weight and is preferably chosen from the concentration range from 0.5 to 1.0% by weight, based on the total weight of the preparations, achieve these objects.", "It has not been foreseen by the person skilled in the art that the preparations according to the invention are more effective moisture-donating preparations, are easier to formulate, better promote skin smoothing, are characterized by better care action, are better vehicles for cosmetic and medicinal-dermatological active ingredients, would have-better sensory properties, such as, for example, ability to be spread on the skin or the absorption capacity into the skin, have greater stability against decomposition in oil and water phases and would be characterized by better biocompatibility than the prior art preparations.", "The preparations according to the invention therefore represent an enrichment of the prior art.", "Xanthan gum (CASE No.", "11138-66-2), also called xanthan, represents an anionic heteropolysaccharide which is usually formed by fermentation from corn sugar and is isolated as the potassium salt.", "It is produced by Xanthomonas campestris and a number of other species under aerobic conditions with a molecular weight of 2×106 to 24×106.Xanthan gum is formed from a chain having β-1,4-bonded glucose (cellulose) with side chains.", "The structure of the subgroups consists of glucose, mannose, glucuronic acid, acetate and pyruvate.", "Xanthan gum is the name of the first microbial anionic heteropolysaccharide.", "It is produced from Xanthomonas campestris and a number of other species under aerobic conditions with a molecular weight of 2-15 106.Xanthan gum is formed from a chain having β-1,4-bonded glucose (cellulose) with side chains.", "The structure of the subgroups consists of glucose, mannose, glucuronic acid, acetate and pyruvate.", "The number of pyruvate units determines the viscosity of the xanthan gum.", "Xanthan gum is produced in two-day batch cultures with a yield of 70-90%, based on carbohydrate used.", "Here, yields of 25-30 g/l are achieved.", "After the culture has been killed; work-up is carried out by precipitation with, for example, 2-propanol.", "Xanthan gum is then dried and ground.", "Within the scope of the present disclosure, the expression “lipids” is sometimes used as the generic term for fats, oils, waxes and the like, as is entirely familiar to the person skilled in the art.", "The terms “oil phase” and “lipid phase” are also used synonymously.", "Advantageously, the lipid or lipids are chosen from the group of moderately polar to nonpolar llipids.", "It is preferred to make the proportion by weight of polar lipids in the lipid phase less than about 30%.", "Oils and fats differ from one another, inter alia, in their polarity, which is difficult to define.", "It has already been proposed to adopt the interfacial tension toward water as a measure of the polarity index of an oil or of an oil phase.", "This means that the lower interfacial tension between this oil phase and water, the greater the polarity of the oil phase in question.", "According to the invention, the interfacial tension is regarded as one possible measure of the polarity of a given oil component.", "The interfacial tension is the force which acts on an imaginary line of one meter in length in the interface between two phases.", "The physical unit for this interfacial tension is conventionally calculated from the force/length relationship and is usually expressed in mN/m (millinewtons divided by meter).", "It has a positive sign if it endeavors to reduce the interface.", "In the converse case, it has a negative sign.", "Table 1 below lists moderately polar lipids which are advantageous according to the invention as individual substances or else as mixtures with one another.", "The relevant interfacial tensions toward water are given in the last column.", "It is also advantageous in some circumstances to use mixtures of higher and lower polarity and the like, particularly if the overall polarity of the oil phase corresponds to of the moderate or low polarity.", "TABLE 1 Trade name INCI name (mN/m) Isofol ® 14 T Butyl Decanol + 27.6 Hexyl Decanol + Hexyl Octanol + Butyl Octanol Isofol ® 16 Hexyl Decanol 24.3 Eutanol ® G Octyldodecanol 24.8 Cetiol ® OE Dicaprylyl Ether 22.1 Miglyol ® 812 Caprylic/Capric Triglyceride 21.3 Cegesoft ® C24 Octyl Palmitate 23.1 Isopropylstearate Isopropyl Stearate 21.9 Estol ® 1540 Octyl Octanoate 30.0 EHC Finsolv ® TN C12-15 Alkyl Benzoate 21.8 Cetiol ® SN Cetearyl Isonoanoate 28.6 Dermofeel ® Butylene Glycol Caprylate/Caprate 21.5 BGC Trivent ® OCG Tricaprylin 20.2 MOD Octyldodeceyl Myristate 22.1 Cosmacol ® ETI Di-C12-13Alkyl Tartrate 29.4 Miglyol ® 829 Caprylic/Capric Diglyceryl Succinate 29.5 Prisorine ® Octyl Isostearate 29.7 2036 Tegosoft ® SH Stearyl Heptanoate 28.7 Abil ® Wax Cetyl Dimethicone 25.1 9840 Cetiol ® LC Coco-Caprylate/Caprate 24.8 IPP Isopropyl Palmitate 22.5 Luvitol ® EHO Cetearyl Octanoate 28.6 Cetiol ® 868 Octyl Stearate 28.4 For the purposes of the present invention, the oil phase can also advantageously comprise substances chosen from the group of esters of saturated and/or unsaturated, branched and/or unbranched alkanecarboxylic acids having a chain length of from 3 to 30 carbon atoms, and saturated and/or unsaturated, branched and/or unbranched alcohols having a chain length of from 3 to 30 carbon atoms and from the group of esters of aromatic carboxylic acids and saturated and/or unsaturated, branched and/or unbranched alcohols having a chain length of from 3 to 30 carbon atoms.", "Such ester oils can then advantageously be chosen from the group consisting of isopropyl myristate, isopropyl palmitate, isopropyl stearate, isopropyl oleate, n-butyl stearate, n-hexyl laurate, n-decyl oleate, isooctyl stearate, isononyl stearate, isononyl isononanoate, 2-ethylhexyl palmitate, 2-ethylhexyl laurate, 2-hexyldecyl stearate, 2-octyidodecyl palmitate, oleyl oleate, oleyl erucate, erucyl oleate, erucyl erucate and also synthetic, semisynthetic and natural mixtures of such esters, such as, for example, jojoba oil.", "The oil phase can also be chosen advantageously from the group of branched and unbranched hydrocarbons and hydrocarbon waxes, silicone oils, dialkyl ethers, the group of saturated or unsaturated, branched or unbranched alcohols, and fatty acid triglycerides, namely the triglycerol esters of saturated and/or unsaturated, branched and/or unbranched alkanecarboxylic acids having a chain length of from 8 to 24 carbon atoms, in particular 12-18 carbon atoms.", "The fatty acid triglycerides can, for example, be advantageously chosen from the group of synthetic, semisynthetic and natural oils, e.g.", "olive oil, sunflower oil, soya oil, peanut oil, rapeseed oil, almond oil, palm oil, coconut oil, palm kernel oil and the like.", "If desired, fatty and/or wax components which are to be used in the oil phase—as secondary constituents in a minor amount—can be chosen from the group of vegetable waxes, animal waxes, mineral waxes and petrochemical waxes.", "Examples which are favorable according to the invention are candelilla wax, carnauba wax, japan wax, esparto grass wax, cork wax, guaruma wax, rice germ oil wax, sugarcane wax, berry wax, ouricury wax, montan wax, jojoba wax, shea butter, beeswax, shellac wax, spermaceti, lanolin (wool wax), uropygial grease, ceresin, ozocerite (earth wax), paraffin waxes and microcrystalline waxes.", "Other advantageous fatty and/or wax components are chemically modified waxes and synthetic waxes such as, for example, those obtainable under the trade names Syncrowax HRC (glyceryl tribehenate), Syncrowax HGLC (C16-36-fatty acid triglyceride) and Syncrowax AW 1C (C18-36-fatty acid) from CRODA GmbH, and also montan ester waxes, Sasol waxes, hydrogenated jojoba waxes, synthetic or modified beeswaxes (e.g.", "dimethicone copolyol beeswax and/or C30-50-alkyl beeswax), polyalkylene waxes, polyethylene glycol waxes, but also chemically modified fats, such as, for example, hydrogenated vegetable oils (for example hydrogenated castor oil and/or hydrogenated coconut fatty glycerides), triglycerides, such as, for example, trihydroxystearin, fatty acids, fatty acid esters and glycol esters, such as, for example, C20-40-alkyl stearate, C20-40-alkylhydroxystearoyl stearate and/or glycol montanate.", "Also advantageous are certain organosilicon compounds, which have similar physical properties to the specified fatty and/or wax components, such as, for example, stearoxytrimethylsilane.", "If desired, the fatty and/or wax components can be present either individually or as a mixture.", "Any desired mixtures of such oil and wax components can also be used advantageously for the purposes of the present invention.", "In some instances, it can also be advantageous to use waxes, for example cetyl palmitate, as the lipid component of the oil phase.", "Of the hydrocarbons, paraffin oil, hydrogenated polyolefins (e.g.", "hydrogenated polyisobutene), squalane and squalene can be used advantageously for the purposes of the present invention.", "According to the invention, emulsions which are particularly advantageous are those which are characterized in that the oil phase consists of at least 50% by weight, preferably of more than 75% by weight, of at least one substance chosen from the group consisting of Vaseline (petrolatum), paraffin oil and polyolefins, and of the latter, preference is given to polydecenes.", "The oil phase can advantageously additionally have a content of cyclic or linear silicone oils or consist entirely of such oils, although it is preferable to use an additional content of other oil phase components apart from the silicone oil or the silicone oils.", "Cyclomethicone (octamethylcyclotetrasiloxane, cyclopentasiloxane and cyclohexasiloxane) can be used advantageously.", "However, other silicone oils can also be used advantageously for the purposes of the present invention, for example hexamethylcyclotrisiloxane, polydimethylsiloxane and poly(methylphenylsiloxane).", "The aqueous phase of the preparations according to the invention in some instances advantageously comprises alcohols, diols or polyols of low carbon number, and ethers thereof, preferably ethanol, isopropanol, propylene glycol, glycerol, ethylene glycol, ethylene glycol monoethyl or monobutyl ether, propylene glycol monomethyl, monoethyl or monobutyl ethers, diethylene glycol monomethyl or monoethyl ethers and analogous products, and also alcohols of low carbon number, e.g.", "ethanol, isopropanol, 1,2-propanediol and glycerol, and, in particular, one or more thickeners which may advantageously be chosen from the group consisting of silicon dioxide, aluminum silicates.", "According to the invention, the carbopol or carbopols can advantageously be chosen, for example, from the carbopol grades from Goodrich (Carbopol 980, 981, 1382, 5984, 2984, EDT 2001 or Pemulen TR2).", "The anionic or nonionic emulsifier or emulsifiers can advantageously be chosen from the group of a) partial fatty acid esters and fatty acid esters of polyhydric alcohols and ethoxylated derivatives thereof (e.g.", "glyceryl monostearate, sorbitan stearate, glyceryl stearyl citrate, sucrose stearate) b) ethoxylated fatty alcohols and fatty acids c) ethoxylated fatty amines, fatty acid amides, fatty acid alkanolamides d) alkylphenol polyglycol ethers (e.g.", "Triton X) A preferred emulsifier according to the invention is glyceryl stearate citrate.", "This is available, for example, under the product name “IMWITOR® 370” from Hüls AG.", "Preference is given to preparations which are characterized in that they additionally comprise (vi) one or more fatty alcohols where the total content of the fatty alcohol is advantageously chosen from the concentration range from 0 to 10% by weight, preferably from the range from 0 to 5.0% by weight, particularly preferably 0 to 3.0% by weight, in each case based on the total weight of the preparations.", "Particularly advantageous preparations are also obtained if antioxidants are used as additives or active ingredients.", "According to the invention, the preparations advantageously comprise one or more antioxidants.", "Favorable, but nevertheless optional, antioxidants which may be used are all antioxidants customary or suitable for cosmetic and/or dermatological applications.", "It is also advantageous to add antioxidants to the preparations according to the invention.", "The antioxidants are advantageously selected from the group consisting of amino acids (e.g.", "glycine, histidine, tyrosine, tryptophan) and their derivatives, imidazoles, (e.g.", "urocanic acid) and their derivatives, peptides, such as D,L-carnosine, D-carnosine, L-carnosine and their derivatives (e.g.", "anserine), carotenoids, carotenes (e.g.", "α-carotene, β-carotene, lycopene) and their derivatives, chlorogenic acid and derivatives thereof, lipoic acid and its derivatives (e.g.", "dihydrolipoic acid), aurothioglucose, propylthiouracil and other thiols (e.g.", "thioredoxin, glutathione, cysteine, cystine, cystamine and their glycosyl, N-acetyl, methyl, ethyl, propyl, amyl, butyl and lauryl, palmitoyl, oleyl, γ-linoleyl, cholesteryl and glyceryl esters) and their salts, dilauryl thiodipropionate, distearyl thiodipropionate, thiodipropionic acid and its derivatives (esters, ethers, peptides, lipids, nucleotides, nucleosides and salts) and sulfoximine compounds (e.g.", "buthionine sulfoximines, homocysteine sulfoximine, buthionine sulfones, penta-, hexa-, heptathionine sulfoximine) in very low tolerated doses (e.g.", "pmol to μmol/kg), and also (metal) chelating agents (e.g.", "α-hydroxy fatty acids, palmitic acid, phytic acid, lactoferrin), α-hydroxy acids (e.g.", "citric acid, lactic acid, malic acid), humic acid, bile acid, bile extracts, bilirubin, biliverdin, EDTA, EGTA and their derivatives, unsaturated fatty acids and their derivatives (e.g.", "γ-linolenic acid, linoleic acid, oleic acid), folic acid and its derivatives, ubiquinone and ubiquinol and their derivatives, vitamin C and derivatives (e.g.", "ascorbyl palmitate, Mg ascorbyl phosphate, ascorbyl acetate), tocopherols and derivatives (e.g.", "vitamin E acetate), vitamin A and derivatives (vitamin A palmitate) and coniferyl benzoate of benzoin resin, rutinic acid and its derivatives, α-glucosylrutin, ferulic acid, furfurylideneglucitol, carnosine, butylhydroxytoluene, butylhydroxyanisole, nordihydroguaiacic acid, nordihydroguaiaretic acid, trihydroxybutyrophenone, uric acid and its derivatives, mannose and its derivatives, zinc and its derivatives (e.g.", "ZnO, ZnSO4), selenium and its derivatives (e.g.", "selenomethionine), stilbenes and their derivatives (e.g.", "stilbene oxide, trans-stilbene oxide), and the derivatives (salts, esters, ethers, sugars, nucleotides, nucleosides, peptides and lipids) of said active ingredients which are suitable according to the invention.", "For the purposes of the present invention, oil-soluble antioxidants can be used particularly advantageously.", "A surprising property of the present invention is that preparations according to the invention are very good vehicles for cosmetic or dermatological active ingredients into the skin, preferred active ingredients being antioxidants which are able to protect the skin against oxidative stress.", "Preferred antioxidants are vitamin E and its derivatives and vitamin A and its derivatives.", "The amount of antioxidants (one or more compounds) in the preparations is preferably from 0.001 to 30% by weight, particularly preferably 0.05-20% by weight, in particular 1-10% by weight, based on the total weight of the preparation.", "If vitamin E and/or its derivatives are used as the antioxidant or antioxidants, their respective concentrations are advantageously chosen from the range of 0.001-10% by weight, based on the total weight of the formulation.", "If vitamin A or vitamin A derivatives or carotenes or their derivatives are used as the antioxidant or antioxidants, their respective concentrations are advantageously chosen from the range of 0.001-10% by weight, based on the total weight of the formulation.", "The person skilled in the art is of course aware that cosmetic preparations are in most cases inconceivable without the customary auxiliaries and additives.", "The cosmetic and dermatological preparations according to the invention can, accordingly, also comprise cosmetic auxiliaries, as are customarily used in such preparations, for example bodying agents, stabilizers, fillers, preservatives, perfumes, antifoams, dyes, pigments which have a coloring action, thickeners, surface-active substances, emulsifiers, emollients, moisturizers and/or humectants, anti-inflammatory substances, additional active ingredients such as vitamins or proteins, sunscreens, insect repellants, bactericides, virucides, water, salts, antimicrobial, proteolytic or keratolytic substances, medicaments or other customary constituents of a cosmetic or dermatological formulation such as alcohols, polyols, polymers, foam stabilizers, organic solvents or also electrolytes.", "The latter can be chosen, for example, from the group of salts containing the following anions: chlorides, also inorganic oxo element anions, of these, in particular sulfates, carbonates, phosphates, borates and aluminates.", "Electrolytes based on organic anions are also advantageous, e.g.", "lactates, acetates, benzoates, propionates, tartrates, citrates, amino acids, ethylenediaminetetraacetic acid and salts thereof and others.", "Preferred cations of the salts are ammonium, alkylammonium, alkali metal, alkaline earth metal, magnesium, iron or zinc ions.", "It does not need to be mentioned that only physiologically acceptable electrolytes should be used in cosmetics.", "Particular preference is given to potassium chloride, sodium chloride, magnesium sulfate, zinc sulfate and mixtures thereof.", "Corresponding requirements apply mutatis mutandis to the formulation of medicinal preparations.", "The gel creams according to the invention can be used as a basis for cosmetic or dermatological formulations.", "The latter can have the customary composition and be used, for example, for the treatment and care of the skin and/or the hair, as lip care product, as deodorant product and as make-up or make-up remover product in decorative cosmetics or as a sunscreen preparation.", "For use, the cosmetic and dermatological preparations according to the invention are applied to the skin and/or the hair in a sufficient amount in a manner customary for cosmetics or dermatological compositions.", "Accordingly, for the purposes of the present invention, cosmetic or topical dermatological compositions can, depending on their composition, be used, for example, as a skin protection cream, cleansing milk, sunscreen lotion, nourishing cream, day or night cream, etc.", "In some circumstances it is possible and advantageous to use the compositions according to the invention as a base for pharmaceutical formulations.", "The cosmetic or dermatological compositions according to the invention can, for example, be in the form of preparations which can be sprayed from aerosol containers, squeezable bottles or by means of a pump device, or in the form of a liquid composition which can be applied by means of roll-on devices, but also in the form of an emulsion which can be applied from normal bottles and containers.", "Suitable propellants for cosmetic or dermatological preparations which can be sprayed from aerosol containers for the purposes of the present invention are the customary known readily volatile, liquefied propellants, for example hydrocarbons (propane, butane, isobutane), Which can be used alone or in a mixture with one another.", "Compressed air is also used advantageously.", "The person skilled in the art is of course aware that there are propellants which are nontoxic per se which would be suitable in principle for realizing the present invention in the form of aerosol preparations, but which must nevertheless be avoided because of their unacceptable impact on the environment or other accompanying circumstances, in particular fluorocarbons and chlorofluorocarbons (CFCs).", "Also favorable are cosmetic and dermatological preparations which are in the form of a sunscreen.", "As well as the active ingredient combinations according to the invention, these preferably additionally comprise at least one UV-A filter substance and/or at least one UV-B filter substance and/or at least one inorganic pigment.", "For the purposes of the present invention, however, it is also advantageous to provide cosmetic and dermatological preparations whose main purpose is not protection against sunlight, but which nevertheless have a content of UV protectants.", "Thus, for example, UV-A or UV-B filter substances are usually incorporated into day creams.", "UV protectants, like antioxidants and, if desired, preservatives, also effectively protect the preparations themselves against decay.", "Preparations according to the invention can advantageously comprise further substances which absorb UV radiation in the UV-B range, the total amount of filter substances being, for example, from 0.1% by weight to 30% by weight, preferably from 0.5 to 10% by weight, in particular from 1.0 to 6.0% by weight, based on the total weight of the preparations, in order to provide cosmetic preparations which protect the hair and/or the skin from the whole region of ultraviolet radiation.", "They can also be used as sunscreens for the hair or the skin.", "If the emulsions according to the invention contain UV-B filter substances, the latter may be oil-soluble or water-soluble.", "Examples of oil-soluble UV-B filters which are advantageous according to the invention are: 3-benzylidenecamphor derivatives, preferably 3-(4-methylbenzylidene)camphor, 3-benzylidenecamphor; 4-aminobenzoic acid derivatives, preferably 2-ethylhexyl 4-(dimethylamino)benzoate, amyl 4-(dimethylamino)benzoate; esters of cinnamic acid, preferably 2-ethylhexyl 4-methoxycinnamate, isopentyl 4-methoxycinnamate; esters of salicylic acid, preferably 2-ethylhexyl salicylate, 4-isopropylbenzyl salicylate, homomenthyl salicylate; derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4′-methylbenzophenone, 2,2′-dihydroxy-4-methoxy-benzophenone; esters of benzalmalonic acid, preferably di(2-ethylhexyl) 4-methoxybenzalmalonate; derivatives of 1,3,5-triazine, preferably 2,4,6-trianilino(p-carbo-2′-ethyl-1′-hexyloxy)-1,3,5-triazine.", "The list of said UV-B filters, which may be used in combination with the active ingredient combinations according to the invention is of course not intended to be limiting.", "It can also be advantageous to formulate lipodispersions according to the invention with UV-A filters which have hitherto been customarily present in cosmetic preparations.", "These substances are preferably derivatives of dibenzoylmethane, in particular 1-(4′-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione and 1-phenyl-3-(4′-isopropylphenyl)propane-1,3-dione.", "Cosmetic and dermatological preparations according to the invention can also comprise inorganic pigments which are customarily used in cosmetics for protecting the skin against UV rays.", "These are oxides of titanium, zinc, iron, zirconium, silicon, manganese, aluminum, cerium and mixtures thereof, and modifications in which the oxides are the active agents.", "Particular preference is given to pigments based on titanium dioxide.", "Further constituents which can be used are: fats, waxes and other natural and synthetic fatty substances, preferably esters of fatty acids with alcohols of low carbon number, e.g.", "with isopropanol, propylene glycol or glycerol, or esters of fatty alcohols with alkanoic acids of low carbon number or with fatty acids; alcohols, diols or polyols of low carbon number, and their ethers, preferably ethanol, isopropanol, propylene glycol, glycerol, ethylene glycol, ethylene glycol monoethyl or monobutyl ethers, propylene glycol monomethyl, monoethyl or monobutyl ethers, diethylene glycol monomethyl or monoethyl ethers and analogous products.", "The examples below are intended to illustrate the present invention without limiting it.", "EXAMPLES % by wt.", "(1) Gel cream Glyceryl stearate citrate 1.00 Hydrogenated coconut fatty acid glycerides 1.00 Dicaprylyl carbonate 3.00 Hydroxyethylcellulose 0.375 Xanthan gum 0.125 Carbomer 0.125 Dimethicone 3.00 NaOH 0.31 Hydrogenated polyisobutene 3.00 Glycerol 5.00 Perfume qs Preservative qs Water 100.00 (2) Gel lotion Glyceryl stearate citrate 1.00 Hydrogenated coconut fatty acid glycerides 0.50 Dicaprylyl carbonate 1.00 Hydroxyethylcellulose 0.25 Carbomer 0.15 Cyclomethicone 5.00 NaOH 0.10 Hydrogenated polyisobutene 5.00 Glycerol 12.00 Perfume qs Preservative qs Water 100.00" ] ]	Patent_10467176
[ [ "Antisense modulation of wrn espression", "Antisense compounds, compositions and methods are provided for modulating the expression of WRN.", "The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding WRN.", "Methods of using these compounds for modulation of WRN expression and for treatment of diseases associated with expression of WRN are provided." ], [ "1.A compound 8 to 50 nucleobases in length targeted to a nucleic acid molecule encoding WRN, wherein said compound specifically hybridizes with and inhibits the expression of WRN.", "2.The compound of claim 1 which is an antisense oligonucleotide.", "3.The compound of claim 2 wherein the antisense oligonucleotide has a sequence comprising SEQ ID NO: 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 85, 86, 87, 89 or 90.4.The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage.", "5.The compound of claim 4 wherein the modified internucleoside linkage is a phosphorothioate linkage.", "6.The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.", "7.The compound of claim 6 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.", "8.The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified nucleobase.", "9.The compound of claim 8 wherein the modified nucleobase is a 5-methylcytosine.", "10.The compound of claim 2 wherein the antisense oligonucleotide is a chimeric oligonucleotide.", "11.A compound 8 to 50 nucleobases in length which specifically hybridizes with at least an 8-nucleobase portion of an active site on a nucleic acid molecule encoding WRN.", "12.A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent.", "13.The composition of claim 12 further comprising a colloidal dispersion system.", "14.The composition of claim 12 wherein the compound is an antisense oligonucleotide.", "15.A method of inhibiting the expression of WRN in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of WRN is inhibited.", "16.A method of treating an animal having a disease or condition associated with WRN comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of WRN is inhibited.", "17.The method of claim 16 wherein the disease or condition is a hyperproliferative disorder.", "18.The method of claim 17 wherein the hyperproliferative disorder is cancer.", "19.The method of claim 16 wherein the disease or condition involves premature aging.", "20.The method of claim 16 wherein the disease or condition is viral infection." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Genomic integrity is critical to the health and survival of any organisms and cells have evolved multiple pathways for the repair of DNA damage.", "One class of enzymes involved in the maintenance of genomic integrity and stability are DNA helicases.", "These proteins play important roles in DNA replication, repair, recombination and transcription by unwinding duplex genomic strands allowing the repair machinery access to damaged or mispaired DNA.", "For example, the RecQ family of helicases has been shown to be important players in linking cell cycle checkpoint responses to recombination repair (Chakraverty and Hickson, BioEssays, 1999, 21, 286-294; Frei and Gasser, J.", "Cell Sci., 2000, 113, 2641-2646; Wu et al., Curr.", "Biol., 1999, 9, R518-520).", "More recently, these helicases have been implicated in the process of posttranscriptional gene silencing (PTGS) (Cogoni and Macino, Science, 1999, 286, 2342-2344).", "In this process, the helicase is required to separate the double-stranded DNA (dsDNA) before any hybridization and silencing mechanism could be initiated.", "The RecQ family consists of five members and can be divided into two distinct groups according to whether they contain an additional carboxy- or amino-terminus group.", "One class containing the longest members of the family include genes known to be defective in several syndromes including the BLM gene in Bloom's syndrome, the WRN gene in Werner's syndrome and the RECQ4 gene in Rothmund-Thompson syndrome.", "Mutations in these genes lead to an increase in the incidence of cancer as well as other physiologic abnormalities (Karow et al., Curr.", "Opin.", "Genet.", "Dev., 2000, 10, 32-38; Kawabe et al., Oncogene, 2000, 19, 4764-4772).", "The second class contains the RECQL gene and the RECQ5 gene which encode little more than the central helicase domain and have not been associated with any human disease.", "WRN (also known as RECQL3) was originally identifed by positional cloning as the gene responsible for Werner's syndrome and localized to chromosome 8p12 (Yu et al., Science, 1996, 272, 258-262.).", "Werner's syndrome is a rare autosomal recessive disorder characterized by symptoms similar to premature aging including atherosclerosis, osteoporosis, type II diabetes, cataracts and cancers (Goto, Clin.", "Exp.", "Rheumatol., 2000, 18, 760-766; Oshima, BioEssays, 2000, 22, 894-901; Shen and Loeb, Trends Genet., 2000, 16, 213-220).", "In an effort to better define the role of the WRN gene, Marciniak et al.", "determined the subcellular localization of the protein using indirect immunofluorescence and a polyclonal antibody.", "These studies revealed a predominant nuclear localization and no difference in localization was detected in normal compared to transformed human cell lines (Marciniak et al., Proc.", "Natl.", "Acad.", "Sci.", "U.S.A., 1998, 95, 6887-6892).", "In addition to having helicase activity, WRN also contains intrinsic exonuclease activity and has been shown to bind single-stranded DNA with higher affinity than double-stranded DNA (Orren et al., Nucleic Acids Res., 1999, 27, 3557-3566; Shen et al., J. Biol.", "Chem., 1998, 273, 34139-34144).", "Disclosed in U.S. Pat.", "No.", "6,090,620 are the nucleic acid molecules encoding the WRN gene as well as WRN gene products, expression vectors, viral vectors and host cells suitable for expressing the WRN gene products (Fu et al., 2000).", "Disclosed and claimed in European Patent EP 1135502 is a nucleotide sequence encoding a protein characterized in having a silencing activity comprising a recQ helicase domain, or a functional portion of, wherein the domain is at least 30% homologous with the amino acid sequence of the recQ helicase domain of the product of the qde-3 gene from Neurospora crassa, or its complementary sequence, an expression vector comprising said nucleotide sequence in the sense or antisense orientation for expression of said sequence in bacteria, fungi, plants, and animals, organisms transformed said vector, and use of the nucleotide sequence to modulate the gene silencing in plants, animals and fungi.", "The human WRN protein is also generally disclosed.", "Disclosed and claimed in U.S. Pat.", "No.", "6,228,583 are methods of identifying an agent which inhibits the replication or accumulation of ribosomal DNA circles in a yeast cell or in a mammalian cell, wherein the agent inhibits the formation of ribosomal DNA circles in a yeast cell with a mutation in the SGS1 gene, and wherein the agent extends the lifespan of the yeast or mammalian cell.", "The yeast SGS1 gene is the homolog of human WRN.", "Further disclosed is the homologous mouse gene, mWRN, and in one embodiment, the invention relates to a method of screening a compound for the ability to alter life span comprising administering the compound to a mouse with a suppressed level of mWRN expression and assaying the mouse for altered life span, wherein antisense nucleic acids are generally disclosed as a means of reducing gene expression.", "Currently, there are no known therapeutic agents which effectively inhibit the synthesis of WRN.", "There are reports of DNA minor groove-binding drugs which inhibit the helicase activity of WRN (Brosh et al., Nucleic Acids Res., 2000, 28, 2420-2430).", "WRN-deficient mice display reduced embryonic survival while live-born mice otherwise appear normal during the first year of life (Lebel and Leder, Proc.", "Natl.", "Acad.", "Sci.", "U.S.A., 1998, 95, 13097-13102).", "While mutations and targeted disruptions resulting in altered protein expression in the WRN gene are responsible for Werner's syndrome, the normal function of the WRN gene product and its regulation are still unclear.", "It is, however, believed to be involved in DNA metabolism and is therefore a potential therapeutic target in conditions involving the production of aberrant DNA products, including the recognition of foreign DNA products as is the case upon viral infection.", "Consequently, there remains a long felt need for agents capable of effectively inhibiting and/or modulating WRN function.", "Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of WRN expression.", "The present invention provides compositions and methods for modulating WRN expression." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding WRN, and which modulate the expression of WRN.", "Pharmaceutical and other compositions comprising the compounds of the invention are also provided.", "Further provided are methods of modulating the expression of WRN in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention.", "Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of WRN by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention provides compositions and methods for modulating the expression of WRN.", "In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding WRN.", "Such compounds have been shown to modulate the expression of WRN.", "BACKGROUND OF THE INVENTION Genomic integrity is critical to the health and survival of any organisms and cells have evolved multiple pathways for the repair of DNA damage.", "One class of enzymes involved in the maintenance of genomic integrity and stability are DNA helicases.", "These proteins play important roles in DNA replication, repair, recombination and transcription by unwinding duplex genomic strands allowing the repair machinery access to damaged or mispaired DNA.", "For example, the RecQ family of helicases has been shown to be important players in linking cell cycle checkpoint responses to recombination repair (Chakraverty and Hickson, BioEssays, 1999, 21, 286-294; Frei and Gasser, J.", "Cell Sci., 2000, 113, 2641-2646; Wu et al., Curr.", "Biol., 1999, 9, R518-520).", "More recently, these helicases have been implicated in the process of posttranscriptional gene silencing (PTGS) (Cogoni and Macino, Science, 1999, 286, 2342-2344).", "In this process, the helicase is required to separate the double-stranded DNA (dsDNA) before any hybridization and silencing mechanism could be initiated.", "The RecQ family consists of five members and can be divided into two distinct groups according to whether they contain an additional carboxy- or amino-terminus group.", "One class containing the longest members of the family include genes known to be defective in several syndromes including the BLM gene in Bloom's syndrome, the WRN gene in Werner's syndrome and the RECQ4 gene in Rothmund-Thompson syndrome.", "Mutations in these genes lead to an increase in the incidence of cancer as well as other physiologic abnormalities (Karow et al., Curr.", "Opin.", "Genet.", "Dev., 2000, 10, 32-38; Kawabe et al., Oncogene, 2000, 19, 4764-4772).", "The second class contains the RECQL gene and the RECQ5 gene which encode little more than the central helicase domain and have not been associated with any human disease.", "WRN (also known as RECQL3) was originally identifed by positional cloning as the gene responsible for Werner's syndrome and localized to chromosome 8p12 (Yu et al., Science, 1996, 272, 258-262.).", "Werner's syndrome is a rare autosomal recessive disorder characterized by symptoms similar to premature aging including atherosclerosis, osteoporosis, type II diabetes, cataracts and cancers (Goto, Clin.", "Exp.", "Rheumatol., 2000, 18, 760-766; Oshima, BioEssays, 2000, 22, 894-901; Shen and Loeb, Trends Genet., 2000, 16, 213-220).", "In an effort to better define the role of the WRN gene, Marciniak et al.", "determined the subcellular localization of the protein using indirect immunofluorescence and a polyclonal antibody.", "These studies revealed a predominant nuclear localization and no difference in localization was detected in normal compared to transformed human cell lines (Marciniak et al., Proc.", "Natl.", "Acad.", "Sci.", "U.S.A., 1998, 95, 6887-6892).", "In addition to having helicase activity, WRN also contains intrinsic exonuclease activity and has been shown to bind single-stranded DNA with higher affinity than double-stranded DNA (Orren et al., Nucleic Acids Res., 1999, 27, 3557-3566; Shen et al., J. Biol.", "Chem., 1998, 273, 34139-34144).", "Disclosed in U.S. Pat.", "No.", "6,090,620 are the nucleic acid molecules encoding the WRN gene as well as WRN gene products, expression vectors, viral vectors and host cells suitable for expressing the WRN gene products (Fu et al., 2000).", "Disclosed and claimed in European Patent EP 1135502 is a nucleotide sequence encoding a protein characterized in having a silencing activity comprising a recQ helicase domain, or a functional portion of, wherein the domain is at least 30% homologous with the amino acid sequence of the recQ helicase domain of the product of the qde-3 gene from Neurospora crassa, or its complementary sequence, an expression vector comprising said nucleotide sequence in the sense or antisense orientation for expression of said sequence in bacteria, fungi, plants, and animals, organisms transformed said vector, and use of the nucleotide sequence to modulate the gene silencing in plants, animals and fungi.", "The human WRN protein is also generally disclosed.", "Disclosed and claimed in U.S. Pat.", "No.", "6,228,583 are methods of identifying an agent which inhibits the replication or accumulation of ribosomal DNA circles in a yeast cell or in a mammalian cell, wherein the agent inhibits the formation of ribosomal DNA circles in a yeast cell with a mutation in the SGS1 gene, and wherein the agent extends the lifespan of the yeast or mammalian cell.", "The yeast SGS1 gene is the homolog of human WRN.", "Further disclosed is the homologous mouse gene, mWRN, and in one embodiment, the invention relates to a method of screening a compound for the ability to alter life span comprising administering the compound to a mouse with a suppressed level of mWRN expression and assaying the mouse for altered life span, wherein antisense nucleic acids are generally disclosed as a means of reducing gene expression.", "Currently, there are no known therapeutic agents which effectively inhibit the synthesis of WRN.", "There are reports of DNA minor groove-binding drugs which inhibit the helicase activity of WRN (Brosh et al., Nucleic Acids Res., 2000, 28, 2420-2430).", "WRN-deficient mice display reduced embryonic survival while live-born mice otherwise appear normal during the first year of life (Lebel and Leder, Proc.", "Natl.", "Acad.", "Sci.", "U.S.A., 1998, 95, 13097-13102).", "While mutations and targeted disruptions resulting in altered protein expression in the WRN gene are responsible for Werner's syndrome, the normal function of the WRN gene product and its regulation are still unclear.", "It is, however, believed to be involved in DNA metabolism and is therefore a potential therapeutic target in conditions involving the production of aberrant DNA products, including the recognition of foreign DNA products as is the case upon viral infection.", "Consequently, there remains a long felt need for agents capable of effectively inhibiting and/or modulating WRN function.", "Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of WRN expression.", "The present invention provides compositions and methods for modulating WRN expression.", "SUMMARY OF THE INVENTION The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding WRN, and which modulate the expression of WRN.", "Pharmaceutical and other compositions comprising the compounds of the invention are also provided.", "Further provided are methods of modulating the expression of WRN in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention.", "Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of WRN by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.", "DETAILED DESCRIPTION OF THE INVENTION The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding WRN, ultimately modulating the amount of WRN produced.", "This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding WRN.", "As used herein, the terms “target nucleic acid” and “nucleic acid encoding WRN” encompass DNA encoding WRN, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA.", "The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid.", "This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”.", "The functions of DNA to be interfered with include replication and transcription.", "The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA.", "The overall effect of such interference with target nucleic acid function is modulation of the expression of WRN.", "In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.", "In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.", "It is preferred to target specific nucleic acids for antisense.", "“Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process.", "The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated.", "This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent.", "In the present invention, the target is a nucleic acid molecule encoding WRN.", "The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e-.g., detection or modulation of expression of the protein, will result.", "Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene.", "Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”.", "A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo.", "Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes).", "It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions.", "In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding WRN, regardless of the sequence(s) of such codons.", "It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).", "The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon.", "Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon.", "The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively.", "Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene.", "The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage.", "The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap.", "The 5′ cap region may also be a preferred target region.", "Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated.", "The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence.", "mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease.", "Aberrant fusion junctions due to rearrangements or deletions are also preferred targets.", "It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.", "Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.", "In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.", "For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.", "“Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides.", "For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position.", "The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.", "Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target.", "It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.", "An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.", "Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target are identified through experimentation, and the sequences of these compounds are hereinbelow identified as preferred embodiments of the invention.", "The target sites to which these preferred sequences are complementary are hereinbelow referred to as “active sites” and are therefore preferred sites for targeting.", "Therefore another embodiment of the invention encompasses compounds which hybridize to these active sites.", "Antisense compounds are commonly used as research reagents and diagnostics.", "For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes.", "Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway.", "Antisense modulation has, therefore, been harnessed for research use.", "For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.", "Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined.", "These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.", "Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov.", "Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc.", "Natl.", "Acad.", "Sci.", "U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J.", "Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.", "Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr.", "Opin.", "Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J.", "Cell Biochem.", "Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur.", "J.", "Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in (To, Comb.", "Chem.", "High Throughput Screen, 2000, 3, 235-41).", "The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses.", "Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man.", "Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway.", "It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.", "In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.", "This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly.", "Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.", "While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below.", "The antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e.", "from about 8 to about 50 linked nucleosides).", "Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases.", "Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.", "As is known in the art, a nucleoside is a base-sugar combination.", "The base portion of the nucleoside is normally a heterocyclic base.", "The two most common classes of such heterocyclic bases are the purines and the pyrimidines.", "Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.", "For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar.", "In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.", "In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred.", "Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.", "The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.", "Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages.", "As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.", "For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.", "Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.", "Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e.", "a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).", "Various salts, mixed salts and free acid forms are also included.", "Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat.", "Nos.", "3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.", "Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.", "These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.", "Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat.", "Nos.", "5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.", "In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.", "The base units are maintained for hybridization with an appropriate nucleic acid target compound.", "One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).", "In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.", "The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.", "Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat.", "Nos.", "5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference.", "Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene(methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —O—N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above referenced U.S. Pat.", "No.", "5,489,677, and the amide backbones of the above referenced U.S. Pat.", "No.", "5,602,240.Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat.", "No.", "5,034,506.Modified oligonucleotides may also contain one or more substituted sugar moieties.", "Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl.", "Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.", "A preferred modification includes 2′ -methoxyethoxy (2′—O—CH2CH2OCH3, also known as 2′—O—(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.", "Chim.", "Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group.", "A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylamino-ethoxyethoxy (also known in the art as 2′-O-dimethylamino ethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2, also described in examples hereinbelow.", "A further prefered modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety.", "The linkage is preferably a methelyne(—CH2—), group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2.LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.Other preferred modifications include 2′-methoxy (2′-O—CH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2), 2′-allyl (2′-CH2—CH═CH2), 2′-O-allyl (2′-O—CH2—CH═CH2) and 2′-fluoro (2′-F).", "The 2′-modification may be in the arabino (up) position or ribo (down) position.", "A preferred 2′-arabino modification is 2′-F.", "Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide.", "Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.", "Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat.", "Nos.", "4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.", "Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.", "As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).", "Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.", "Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.", "9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine(2H-pyrimido[4,5-b)indol-2-one), pyridoindole cytidine(H-pyrido[3′, 2′: 4,5]pyrrolo[2,3-d]pyrimidin-2-one).", "Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.", "Further nucleobases include those disclosed in U.S. Pat.", "No.", "3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.", "John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.", "ed., CRC Press, 1993.Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.", "These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyl-adenine, 5-propynyluracil and 5-propynylcytosine.", "5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.", "276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.", "Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat.", "No.", "3,687,808, as well as U.S. Pat.", "Nos.", "4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat.", "No.", "5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.", "Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.", "The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups.", "Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.", "Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.", "Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA.", "Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion.", "Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference.", "Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg.", "Med.", "Chem.", "Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann.", "N.Y. Acad.", "Sci., 1992, 660, 306-309; Manoharan et al., Bioorg.", "Med.", "Chem.", "Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl.", "Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl.", "Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.", "Biophys.", "Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.", "Exp.", "Ther., 1996, 277, 923-937.Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.", "Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser.", "No.", "09/334,130 (filed Jun.", "15, 1999) which is incorporated herein by reference in its entirety.", "Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat.", "Nos.", "4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.", "It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.", "The present invention also includes antisense compounds which are chimeric compounds.", "“Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.", "These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.", "An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.", "By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.", "Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression.", "Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region.", "Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.", "Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above.", "Such compounds have also been referred to in the art as hybrids or gapmers.", "Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat.", "Nos.", "5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.", "The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.", "Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.).", "Any other means for such synthesis known in the art may additionally or alternatively be employed.", "It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.", "The antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules.", "The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.", "Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat.", "Nos.", "5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.", "The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.", "Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.", "The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.", "In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat.", "No.", "5,770,713 to Imbach et al.", "The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.", "Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.", "Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like.", "Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66, 1-19).", "The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.", "The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.", "The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.", "As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention.", "These include organic or inorganic acid salts of the amines.", "Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates.", "Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid.", "Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation.", "Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations.", "Carbonates or hydrogen carbonates are also possible.", "For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.", "; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.", "The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits.", "For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of WRN is treated by administering antisense compounds in accordance with this invention.", "The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier.", "Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.", "The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding WRN, enabling sandwich and other assays to easily be constructed to exploit this fact.", "Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding WRN can be detected by means known in the art.", "Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means.", "Kits using such detection means for detecting the level of WRN in a sample may also be prepared.", "The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention.", "The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.", "Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral.", "Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.", "Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.", "Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.", "Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.", "Coated condoms, gloves and the like may also be useful.", "Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.", "Preferred lipids and liposomes include neutral (e.g.", "dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g.", "dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.", "dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).", "Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes.", "Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids.", "Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-10 alkyl ester (e.g.", "isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.", "Topical formulations are described in detail in U.S. patent application Ser.", "No.", "09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.", "Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets.", "Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.", "Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators.", "Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.", "Prefered bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate.", "Prefered fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g.", "sodium).", "Also prefered are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts.", "A particularly prefered combination is the sodium salt of lauric acid, capric acid and UDCA.", "Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.", "Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles.", "Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches.", "Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.", "p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG).", "Oral formulations for oligonucleotides and their preparation are described in detail in U.S. applications Ser.", "No.", "08/886,829 (filed Jul.", "1, 1997), Ser.", "No.", "09/108,673 (filed Jul.", "1, 1998), Ser.", "No.", "09/256,515 (filed Feb. 23, 1999), Ser.", "No.", "09/082,624 (filed May 21, 1998) and Ser.", "No.", "09/315,298 (filed May 20, 1999) each of which is incorporated herein by reference in their entirety.", "Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.", "Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations.", "These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.", "The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry.", "Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s).", "In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.", "The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.", "The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.", "Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.", "The suspension may also contain stabilizers.", "In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams.", "Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes.", "While basically similar in nature these formulations vary in the components and the consistency of the final product.", "The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.", "Emulsions The compositions of the present invention may be prepared and formulated as emulsions.", "Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter.", "(Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301).", "Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other.", "In general, emulsions may be either water-in-oil (w/o) or of the oil-in-water (o/w) variety.", "When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase the resulting composition is called a water-in-oil (w/o) emulsion.", "Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion.", "Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase.", "Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed.", "Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.", "Such complex formulations often provide certain advantages that simple binary emulsions do not.", "Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion.", "Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.", "Emulsions are characterized by little or no thermodynamic stability.", "Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation.", "Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams.", "Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion.", "Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).", "Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).", "Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion.", "The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations.", "Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).", "Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia.", "Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum.", "Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations.", "These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.", "A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions.", "These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).", "Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers).", "These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.", "Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives.", "Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.", "Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.", "Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.", "The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).", "Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint.", "(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).", "Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.", "In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions.", "A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).", "Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system.", "Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).", "Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte.", "Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).", "The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335).", "Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.", "Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants.", "The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.", "Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art.", "The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol.", "The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.", "Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs.", "Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth.", "Find.", "Exp.", "Clin.", "Pharmacol., 1993, 13, 205).", "Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.", "Sci., 1996, 85, 138-143).", "Often microemulsions may form spontaneously when their components are brought together at ambient temperature.", "This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides.", "Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications.", "It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.", "Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention.", "Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92).", "Each of these classes has been discussed above.", "Liposomes There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs.", "These include monolayers, micelles, bilayers and vesicles.", "Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the is standpoint of drug delivery.", "As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.", "Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior.", "The aqueous portion contains the composition to be delivered.", "Cationic liposomes possess the advantage of being able to fuse to the cell wall.", "Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.", "In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient.", "Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.", "Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.", "), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).", "Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.", "Liposomes are useful for the transfer and delivery of active ingredients to the site of action.", "Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes.", "As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.", "Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs.", "There is growing evidence that for topical administration, liposomes present several advantages over other formulations.", "Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.", "Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin.", "Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin.", "The majority of applications resulted in the targeting of the upper epidermis.", "Liposomes fall into two broad classes.", "Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex.", "The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome.", "Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem.", "Biophys.", "Res.", "Commun., 1987, 147, 980-985).", "Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it.", "Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs.", "Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes.", "pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture.", "Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).", "One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine.", "Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).", "Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE).", "Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.", "Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.", "Several studies have assessed the topical delivery of liposomal drug formulations to the skin.", "Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g.", "as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410).", "Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).", "Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol.", "Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin.", "Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al.", "S.T.P.", "Pharma.", "Sci., 1994, 4, 6, 466).", "Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.", "Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.", "While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).", "Various liposomes comprising one or more glycolipids are known in the art.", "Papahadjopoulos et al.", "(Ann.", "N.Y. Acad.", "Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes.", "These findings were expounded upon by Gabizon et al.", "(Proc.", "Natl.", "Acad.", "Sci.", "U.S.A., 1988, 85, 6949).", "U.S. Pat.", "No.", "4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester.", "U.S. Pat.", "No.", "5,543,152 (Webb et al.)", "discloses liposomes comprising sphingomyelin.", "Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).", "Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art.", "Sunamoto et al.", "(Bull.", "Chem.", "Soc.", "Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety.", "Illum et al.", "(FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives.", "Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat.", "Nos.", "4,426,330 and 4,534,899).", "Klibanov et al.", "(FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives.", "Blume et al.", "(Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG.", "Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No.", "EP 0 445 131 B1 and WO 90/04384 to Fisher.", "Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al.", "(U.S. Pat.", "Nos.", "5,013,556 and 5,356,633) and Martin et al.", "(U.S. Pat.", "No.", "5,213,804 and European Patent No.", "EP 0 496 813 B1).", "Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat.", "No.", "5,225,212 (both to Martin et al.)", "and in WO 94/20073 (Zalipsky et al.)", "Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).", "U.S. Pat.", "No.", "5,540,935 (Miyazaki et al.)", "and U.S. Pat.", "No.", "5,556,948 (Tagawa et al.)", "describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.", "A limited number of liposomes comprising nucleic acids are known in the art.", "WO 96/40062 to Thierry et al.", "discloses methods for encapsulating high molecular weight nucleic acids in liposomes.", "U.S. Pat.", "No.", "5,264,221 to Tagawa et al.", "discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA.", "U.S. Pat.", "No.", "5,665,710 to Rahman et al.", "describes certain methods of encapsulating oligodeoxynucleotides in liposomes.", "WO 97/04787 to Love et al.", "discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.", "Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles.", "Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet.", "Transfersomes are adaptable to the environment in which they are used, e.g.", "they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading.", "To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition.", "Transfersomes have been used to deliver serum albumin to the skin.", "The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.", "Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes.", "The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB).", "The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).", "If the surfactant molecule is not ionized, it is classified as a nonionic surfactant.", "Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values.", "In general their HLB values range from 2 to about 18 depending on their structure.", "Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters.", "Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.", "The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.", "If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic.", "Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.", "The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.", "If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic.", "Cationic surfactants include quaternary ammonium salts and ethoxylated amines.", "The quaternary ammonium salts are the most used members of this class.", "If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric.", "Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.", "The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).", "Penetration Enhancers In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals.", "Most drugs are present in solution in both ionized and nonionized forms.", "However, usually only lipid soluble or lipophilic drugs readily cross cell membranes.", "It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer.", "In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.", "Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92).", "Each of the above mentioned classes of penetration enhancers are described below in greater detail.", "Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced.", "In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43.Takahashi et al., J. Pharm.", "Pharmacol., 1988, 40, 252).", "Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)", "(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm.", "Pharmacol., 1992, 44, 651-654).", "Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.", "Eds., McGraw-Hill, New York, 1996, pp.", "934-935).", "Various natural bile salts, and their synthetic derivatives, act as penetration enhancers.", "Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.", "The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm.", "Exp.", "Ther., 1992, 263, 25; Yamashita et al., J. Pharm.", "Sci., 1990, 79, 579-583).", "Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced.", "With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J.", "Chromatogr., 1993, 618, 315-339).", "Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J.", "Control Rel., 1990, 14, 43-51).", "Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33).", "This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm.", "Pharmacol., 1987, 39, 621-626).", "Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention.", "For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat.", "No.", "5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.", "Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.", "Carriers Certain compositions of the present invention also incorporate carrier compounds in the formulation.", "As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.", "The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.", "For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res.", "Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl.", "Acid Drug Dev., 1996, 6, 177-183).", "Excipients In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal.", "The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.", "Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.", "); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.", "); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.", "); disintegrants (e.g., starch, sodium starch glycolate, etc.", "); and wetting agents (e.g., sodium lauryl sulphate, etc.).", "Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention.", "Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.", "Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.", "The solutions may also contain buffers, diluents and other suitable additives.", "Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.", "Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.", "Other Components The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.", "Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.", "However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.", "The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.", "Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.", "The suspension may also contain stabilizers.", "Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism.", "Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES).", "See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed.", "1987, pp.", "1206-1228, Berkow et al., eds., Rahway, N.J.", "When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide).", "Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention.", "See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively).", "Other non-antisense chemotherapeutic agents are also within the scope of this invention.", "Two or more combined compounds may be used together or sequentially.", "In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target.", "Numerous examples of antisense compounds are known in the art.", "Two or more combined compounds may be used together or sequentially.", "The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art.", "Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.", "Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient.", "Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.", "Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models.", "In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years.", "Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.", "Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.", "While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.", "EXAMPLES Example 1 Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy amidites 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g.", "Chemgenes, Needham Mass.", "or Glen Research, Inc. Sterling Va.).", "Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat.", "No.", "5,506,351, herein incorporated by reference.", "For oligonucleotides synthesized using 2′-alkoxy amidites, the standard cycle for unmodified oligonucleotides was utilized, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds.", "Oligonucleotides containing 5-methyl-2′-deoxycytidine(5-Me-C)nucleotides were synthesized according to published methods [Sanghvi, et.", "al., Nucleic Acids Research, 1993, 21, 3197-3203] using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).", "2′-Fluoro amidites 2′-Fluorodeoxyadenosine amidites 2′-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et.", "al., J. Med.", "Chem., 1993, 36, 831-841) and U.S. Pat.", "No.", "5,670,633, herein incorporated by reference.", "Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2′-alpha-fluoro atom is introduced by a SN2-displacement of a 2′-beta-trityl group.", "Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′, 5′-ditetrahydropyranyl (THP) intermediate.", "Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.", "2′-Fluorodeoxyguanosine The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyryl-arabinofuranosylguanosine.", "Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine.", "Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups.", "Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.", "2′-Fluorouridine Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a literature procedure in which 2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine.", "Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.", "2′-Fluorodeoxycytidine 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine.", "Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.", "2′-O-(2-Methoxyethyl) modified amidites 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared as follows, or alternatively, as per the methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine] 5-Methyluridine(ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL).", "The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner.", "After 1 hour, the slightly darkened solution was concentrated under reduced pressure.", "The resulting syrup was poured into diethylether (2.5 L), with stirring.", "The product formed a gum.", "The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca.", "400 mL).", "The solution was poured into fresh ether (2.5 L) to yield a stiff gum.", "The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid that was crushed to a light tan powder (57 g, 85% crude yield).", "The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca.", "5%).", "The material was used as is for further reactions (or it can be purified further by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4° C.).", "2′-O-Methoxyethyl-5-methyluridine 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL).", "The residue was suspended in hot acetone (1 L).", "The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated.", "The residue (280 g) was dissolved in CH3CN (600 mL) and evaporated.", "A silica gel column (3 kg) was packed in CH2Cl2/acetone/MeOH (20:5:3) containing 0.5% Et3NH.", "The residue was dissolved in CH2Cl2 (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column.", "The product was eluted with the packing solvent to give 160 g (63%) of product.", "Additional material was obtained by reworking impure fractions.", "2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L).", "A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour.", "A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour.", "Methanol (170 mL) was then added to stop the reaction.", "HPLC showed the presence of approximately 70% product.", "The solvent was evaporated and triturated with CH3CN (200 mL).", "The residue was dissolved in CHCl3 (1.5 L) and extracted with 2×500 mL of saturated NaHCO3 and 2×500 mL of saturated NaCl.", "The organic phase was dried over Na2SO4, filtered and evaporated.", "275 g of residue was obtained.", "The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et3NH.", "The pure fractions were evaporated to give 164 g of product.", "Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).", "3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours.", "The reaction was monitored by TLC by first quenching the TLC sample with the addition of MeOH.", "Upon completion of the reaction, as judged by TLC, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl3 (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl.", "The water layers were back extracted with 200 mL of CHCl3.The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx.", "90% product).", "The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/hexane(4:1).", "Pure product fractions were evaporated to yield 96 g (84%).", "An additional 1.5 g was recovered from later fractions.", "3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH3CN (700 mL) and set aside.", "Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH3CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer.", "POCl3 was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours.", "The first solution was added dropwise, over a 45 minute period, to the latter solution.", "The resulting reaction mixture was stored overnight in a cold room.", "Salts were filtered from the reaction mixture and the solution was evaporated.", "The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration.", "The filtrate was washed with 1×300 mL of NaHCO3 and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated.", "The residue was triturated with EtOAc to give the title compound.", "2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH4OH (30 mL) was stirred at room temperature for 2 hours.", "The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL).", "The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel.", "MeOH (400 mL) saturated with NH3 gas was added and the vessel heated to 100° C. for 2 hours (TLC showed complete conversion).", "The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL).", "The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.", "N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring.", "After stirring for 3 hours, TLC showed the reaction to be approximately 95% complete.", "The solvent was evaporated and the residue azeotroped with MeOH (200 mL).", "The residue was dissolved in CHCl3 (700 mL) and extracted with saturated NaHCO3 (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO4 and evaporated to give a residue (96 g).", "The residue was chromatographed on a 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et3NH as the eluting solvent.", "The pure product fractions were evaporated to give 90 g (90%) of the title compound.", "N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH2Cl2 (1 L).", "Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere.", "The resulting mixture was stirred for 20 hours at room temperature (TLC showed the reaction to be 95% complete).", "The reaction mixture was extracted with saturated NaHCO3 (1×300 mL) and saturated NaCl (3×300 mL).", "The aqueous washes were back-extracted with CH2Cl2 (300 mL), and the extracts were combined, dried over MgSO4 and concentrated.", "The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) as the eluting solvent.", "The pure fractions were combined to give 90.6 g (87%) of the title compound.", "2′-O-(Aminooxyethyl)nucleoside amidites and 2′-O-(dimethylaminooxyethyl)nucleoside amidites 2′-(Dimethylaminooxyethoxy)nucleoside amidites 2′-(Dimethylaminooxyethoxy)nucleoside amidites [also known in the art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs.", "Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.", "5′-O-tert-Butyldiphenylsilyl-O2-2′-anhydro-5-methyluridine O2-2′-anhydro-5-methyluridine (Pro.", "Bio.", "Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring.", "tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion.", "The reaction was stirred for 16 h at ambient temperature.", "TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.", "The solution was concentrated under reduced pressure to a thick oil.", "This was partitioned between dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L) and brine is (1 L).", "The organic layer was dried over sodium sulfate and concentrated under reduced pressure to a thick oil.", "The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) and the solution was cooled to −10° C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid.", "TLC and NMR were consistent with pure product.", "5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine In a 2 L stainless steel, unstirred pressure reactor was added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL).", "In the fume hood and with manual stirring, ethylene glycol (350 mL, excess) was added cautiously at first until the evolution of hydrogen gas subsided.", "5′-O-tert-Butyldiphenylsilyl-O2-2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring.", "The reactor was sealed and heated in an oil bath until an internal temperature of 160° C. was reached and then maintained for 16 h (pressure <100 psig).", "The reaction vessel was cooled to ambient and opened.", "TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T side product, ethyl acetate) indicated about 70% conversion to the product.", "In order to avoid additional side product formation, the reaction was stopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol.", "[Alternatively, once the low boiling solvent is gone, the remaining solution can be partitioned between ethyl acetate and water.", "The product will be in the organic phase.]", "The residue was purified by column chromatography (2 kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1).", "The appropriate fractions were combined, stripped and dried to product as a white crisp foam (84 g, 50%), contaminated starting material (17.4 g) and pure reusable starting material 20 g. The yield based on starting material less pure recovered starting material was 58%.", "TLC and NMR were consistent with 99% pure product.", "2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with 25 triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol).", "It was then dried over P2O5 under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear solution.", "Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture.", "The rate of addition is maintained such that resulting deep red coloration is just discharged before adding the next drop.", "After the addition was complete, the reaction was stirred for 4 hrs.", "By that time TLC showed the completion of the reaction (ethylacetate:hexane, 60:40).", "The solvent was evaporated in vacuum.", "Residue obtained was placed on a flash column and eluted with ethyl acetate:hexane (60:40), to get 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%).", "5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine 2′-O-([2-phthalimidoxy)ethyl)-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH2Cl2 (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate was washed with ice cold CH2Cl2 and the combined organic phase was washed with water, brine and dried over anhydrous Na2SO4.The solution was concentrated to get 2′-O-(aminooxyethyl)thymidine, which was then dissolved in MeOH (67.5 mL).", "To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.)", "was added and the resulting mixture was strirred for 1 h. Solvent was removed under vacuum; residue chromatographed to get 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).", "5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL).", "Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at 10° C. under inert atmosphere.", "The reaction mixture was stirred for 10 minutes at 10° C. After that the reaction vessel was removed from the ice bath and stirred at room temperature for 2 h, the reaction monitored by TLC (5% MeOH in CH2Cl2).", "Aqueous NaHCO3 solution (5%, 10 mL) was added and extracted with ethyl acetate (2×20 mL).", "Ethyl acetate phase was dried over anhydrous Na2SO4, evaporated to dryness.", "Residue was dissolved in a solution of 1M PPTS in MeOH (30.6 mL).", "Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes.", "Reaction mixture cooled to 10° C. in an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction mixture stirred at 10° C. for 10 minutes.", "After 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature for 2 hrs.", "To the reaction mixture 5% NaHCO3 (25 mL) solution was added and extracted with ethyl acetate (2×25 mL).", "Ethyl acetate layer was dried over anhydrous Na2SO4 and evaporated to dryness The residue obtained was purified by flash column chromatography and eluted with 5% MeOH in CH2Cl2 to get 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%).", "2′-O-(dimethylaminooxyethyl)-5-methyluridine Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).", "This mixture of triethylamine-2HF was then added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs.", "Reaction was monitored by TLC (5% MeOH in CH2Cl2).", "Solvent was removed under vacuum and the residue placed on a flash column and eluted with 10% MeOH in CH2Cl2 to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).", "5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P2O5 under high vacuum overnight at 40° C. It was then co-evaporated with anhydrous pyridine (20 mL).", "The residue obtained was dissolved in pyridine (11 mL) under argon atmosphere.", "4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the mixture and the reaction mixture was stirred at room temperature until all of the starting material disappeared.", "Pyridine was removed under vacuum and the residue chromatographed and eluted with 10% MeOH in CH2Cl2 (containing a few drops of pyridine) to get 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).", "5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL).", "To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and dried over P2O5 under high vacuum overnight at 40° C. Then the reaction mixture was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N1,N1-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added.", "The reaction mixture was stirred at ambient temperature for 4 hrs under inert atmosphere.", "The progress of the reaction was monitored by TLC (hexane:ethyl acetate 1:1).", "The solvent was evaporated, then the residue was dissolved in ethyl acetate (70 mL) and washed with 5% aqueous NaHCO3 (40 mL).", "Ethyl acetate layer was dried over anhydrous Na2SO4 and concentrated.", "Residue obtained was chromatographed (ethyl acetate as eluent) to get 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%).", "2′-(Aminooxyethoxy)nucleoside amidites 2′-(Aminooxyethoxy)nucleoside amidites [also known in the art as 2′-O-(aminooxyethyl)nucleoside amidites] are prepared as described in the following paragraphs.", "Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.", "N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside.", "Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl)diaminopurine riboside along with a minor amount of the 3′-O-isomer.", "2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.", "(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)", "Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.", "As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].", "2′-dimethylaminoethoxyethoxy(2′-DMAEOE)nucleoside amidites 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH2—O—CH2—N(CH2)2, or 2′-DMAEOE nucleoside amidites) are prepared as follows.", "Other nucleoside amidites are prepared similarly.", "2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb.", "Hydrogen gas evolves as the solid dissolves.", "O2—, 2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oil bath and heated to 155° C. for 26 hours.", "The bomb is cooled to room temperature and opened.", "The crude solution is concentrated and the residue partitioned between water (200 mL) and hexanes (200 mL).", "The excess phenol is extracted into the hexane layer.", "The aqueous layer is extracted with ethyl acetate (3×200 mL) and the combined organic layers are washed once with water, dried over anhydrous sodium sulfate and concentrated.", "The residue is columned on silica gel using methanol/methylene chloride 1:20 (which has 2% triethylamine) as the eluent.", "As the column fractions are concentrated a colorless solid forms which is collected to give the title compound as a white solid.", "5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.)", "are added and stirred for 1 hour.", "The reaction mixture is poured into water (200 mL) and extracted with CH2Cl2 (2×200 mL).", "The combined CH2Cl2 layers are washed with saturated NaHCO3 solution, followed by saturated NaCl solution and dried over anhydrous sodium sulfate.", "Evaporation of the solvent followed by silica gel chromatography using MeOH:CH2Cl2:Et3N (20:1, v/v, with 1% triethylamine) gives the title compound.", "5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.)", "are added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH2Cl2 (20 mL) under an atmosphere of argon.", "The reaction mixture is stirred overnight and the solvent evaporated.", "The resulting residue is purified by silica gel flash column chromatography with ethyl acetate as the eluent to give the title compound.", "Example 2 Oligonucleotide Synthesis Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine.", "Phosphorothioates (P═S) are synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages.", "The thiation wait step was increased to 68 sec and was followed by the capping step.", "After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (18 h), the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution.", "Phosphinate oligonucleotides are prepared as described in U.S. Pat.", "No.", "5,508,270, herein incorporated by reference.", "Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat.", "No.", "4,469,863, herein incorporated by reference.", "3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat.", "No.", "5,610,289 or U.S. Pat.", "No.", "5,625,050, herein incorporated by reference.", "Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.", "No., 5,256,775 or U.S. Pat.", "No.", "5,366,878, herein incorporated by reference.", "Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.", "3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat.", "No.", "5,476,925, herein incorporated by reference.", "Phosphotriester oligonucleotides are prepared as described in U.S. Pat.", "No.", "5,023,243, herein incorporated by reference.", "Borano phosphate oligonucleotides are prepared as described in U.S. Pat.", "Nos.", "5,130,302 and 5,177,198, both herein incorporated by reference.", "Example 3 Oligonucleoside Synthesis Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedi-methylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat.", "Nos.", "5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.", "Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat.", "Nos.", "5,264,562 and 5,264,564, herein incorporated by reference.", "Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat.", "No.", "5,223,618, herein incorporated by reference.", "Example 4 PNA Synthesis Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23.They may also be prepared in accordance with U.S. Pat.", "Nos.", "5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.", "Example 5 Synthesis of Chimeric Oligonucleotides Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types.", "These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound.", "Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides.", "Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.", "[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 380B, as above.", "Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.", "The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2′-O-methyl.", "The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 ammonia/ethanol at room temperature overnight then lyophilized to dryness.", "Treatment in methanolic ammonia for 24 hrs at room temperature is then done to deprotect all bases and sample was again lyophilized to dryness.", "The pellet is resuspended in 1M TBAF in THF for 24 hrs at room temperature to deprotect the 2′ positions.", "The reaction is then quenched with 1M TEAA and the sample is then reduced to ½ volume by rotovac before being desalted on a G25 size exclusion column.", "The oligo recovered is then analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.", "[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)]Chimeric Phosphorothioate Oligonucleotides [2′-O-(2-methoxyethyl)]-[2′-deoxy)-[-2′-O-(methoxy-ethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites.", "[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester]Chimeric Oligonucleotides [2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxy phosphorothioate]-[2′-O-(methoxyethyl)phosphodiester]chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidization with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.", "Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat.", "No.", "5,623,065, herein incorporated by reference.", "Example 6 Oligonucleotide Isolation After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.", "Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material.", "The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by 31p nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol.", "Chem.", "1991, 266, 18162-18171.Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.", "Example 7 Oligonucleotide Synthesis—96 Well Plate Format Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 96 well format.", "Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine.", "Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile.", "Standard base-protected beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial vendors (e.g.", "PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.).", "Non-standard nucleosides are synthesized as per known literature or patented methods.", "They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.", "Oligonucleotides were cleaved from support and deprotected with concentrated NH40H at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo.", "The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.", "Example 8 Oligonucleotide Analysis—96 Well Plate Format The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy.", "The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270).", "Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy.", "All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors.", "Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.", "Example 9 Cell Culture and Oligonucleotide Treatment The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels.", "This can be routinely determined using, for example, PCR or Northern blot analysis.", "The following 4 cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen.", "This can be readily determined by methods routine in the art, for example Northern blot analysis, Ribonuclease protection assays, or RT-PCR.", "T-24 cells: The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.).", "T-24 cells were routinely cultured in complete McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, Md.)", "supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.", "), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.).", "Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.", "Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.", "For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.", "A549 Cells: The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.).", "A549 cells were routinely cultured in DMEM basal media (Gibco/Life Technologies, Gaithersburg, Md.)", "supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.", "), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.).", "Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.", "NHDF Cells: Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville Md.).", "NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)", "supplemented as recommended by the supplier.", "Cells were maintained for up to 10 passages as recommended by the supplier.", "HEK Cells: Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville Md.).", "HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)", "formulated as recommended by the supplier.", "Cells were routinely maintained for up to 10 passages as recommended by the supplier.", "Treatment with Antisense Compounds: When cells reached 80% confluency, they were treated with oligonucleotide.", "For cells grown in 96-well plates, wells were washed once with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Gibco BRL) and the desired concentration of oligonucleotide.", "After 4-7 hours of treatment, the medium was replaced with fresh medium.", "Cells were harvested 16-24 hours after oligonucleotide treatment.", "The concentration of oligonucleotide used varies from cell line to cell line.", "To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations.", "For human cells the positive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H-ras.", "For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf.", "The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line.", "If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line.", "If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments.", "Example 10 Analysis of Oligonucleotide Inhibition of WRN Expression Antisense modulation of WRN expression can be assayed in a variety of ways known in the art.", "For example, WRN mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR).", "Real-time quantitative PCR is presently preferred.", "RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.", "Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp.", "4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp.", "4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.", "Protein levels of WRN can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS).", "Antibodies directed to WRN can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods.", "Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp.", "11.12.1-11.12.9, John Wiley & Sons, Inc., 1997.Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp.", "11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp.", "10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp.", "10.8.1-10.8.21, John Wiley & Sons, Inc., 1997.Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp.", "11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.Example 11 Poly(A)+ mRNA Isolation Poly(A)+ mRNA was isolated according to Miura et al., Clin.", "Chem., 1996, 42, 1758-1764.Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp.", "4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS.", "60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes.", "55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.).", "Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl).", "After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes.", "60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C. was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.", "Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.", "Example 12 Total RNA Isolation Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia Calif.) following the manufacturer's recommended procedures.", "Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS.", "100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds.", "100 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down.", "The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source.", "Vacuum was applied for 15 seconds.", "1 mL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum again applied for 15 seconds.", "1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 15 seconds.", "The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes.", "The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels.", "The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes.", "RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds.", "The elution step was repeated with an additional 60 μL water.", "The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.).", "Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.", "Example 13 Real-time Quantitative PCR Analysis of WRN mRNA Levels Quantitation of WRN mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions.", "This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time.", "As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate.", "This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes.", "A reporter dye (e.g., JOE, FAM, or VIC, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end of the probe.", "When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye.", "During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase.", "During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated.", "With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System.", "In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.", "Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction.", "In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample.", "In this analysis, mRNA isolated from untreated cells is serially diluted.", "Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing).", "Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples.", "If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable.", "Other methods of PCR are also known in the art.", "PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl2, 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL total RNA solution.", "The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).", "Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.).", "GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately.", "Total RNA is quantified using RiboGreen™ RNA quantification reagent from Molecular Probes.", "Methods of RNA quantification by RiboGreen™ are taught in Jones, L.J., et al, Analytical Biochemistry, 1998, 265, 368-374.In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 25 uL purified, cellular RNA.", "The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.", "Probes and primers to human WRN were designed to hybridize to a human WRN sequence, using published sequence information (GenBank accession number AF181897, incorporated herein as SEQ ID NO:3).", "For human WRN the PCR primers were: forward primer: ATCCCAAGCGGTGAAAGCT (SEQ ID NO: 4) reverse primer: GGTTTCGGATAACATCAGCAATAA (SEQ ID NO: 5) and the PCR probe was: FAM-CCCCCTTGATTTGGAGCGAGCA-TAMRA (SEQ ID NO: 6) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.", "For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCCX-TAMRA 3′ (SEQ ID NO: 9) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.", "Example 14 Northern Blot Analysis of WRN mRNA Levels Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B”Inc., Friendswood, Tex.).", "Total RNA was prepared following manufacturer's recommended protocols.", "Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio).", "RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.).", "RNA transfer was confirmed by UV visualization.", "Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.", "To detect human WRN, a human WRN specific probe was prepared by PCR using the forward primer ATCCCAAGCGGTGAAAGCT (SEQ ID NO: 4) and the reverse primer GGTTTCGGATAACATCAGCAATAA (SEQ ID NO: 5).", "To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).", "Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.).", "Data was normalized to GAPDH levels in untreated controls.", "Example 15 Antisense Inhibition of Human WRN Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human WRN gene, using published sequences (GenBank accession number AF181897 characterized as genomic sequence encoding exons 21-35 of the human WRN gene, incorporated herein as SEQ ID NO: 3; GenBank accession number AF181896 characterized as genomic sequence encoding exons 1-20 of the human WRN gene, incorporated herein as SEQ ID NO: 10; GenBank accession number AF091214 encoding the complete cds, incorporated herein as SEQ ID NO: 11; and GenBank accession number AA249288 which extends the 3′UTR region of the human WRN gene, incorporated herein as SEQ ID NO: 12).", "The oligonucleotides are shown in Table 1.“Target site” indicates the first (5′-most)nucleotide number on the particular target sequence to which the oligonucleotide binds.", "All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.", "The wings are composed of 2′-methoxyethyl(2′-MOE)nucleotides.", "The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide.", "All cytidine residues are 5-methylcytidines.", "The compounds were analyzed for their effect on human WRN mRNA levels by quantitative real-time PCR as described in other examples herein.", "Data are averages from two experiments.", "If present, “N.D.” indicates “no data”.", "TABLE 1 Inhibition of human WRN mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB NO 137442 Intron 10 37091 tctcacggtttgggactcaa 91 13 137443 Intron 10 38448 agataatagctcttctatat 50 14 137444 Intron 10 54755 tcagtagagcaaagctgctt 91 15 137445 Intron 10 55220 ggtaattacgtggcaaaacc 24 16 137446 Intron 10 81922 caaactttaggttttcaatg 76 17 137447 Intron 10 94616 tcacctaagatctgtagaaa 69 18 137448 Intron 3 9993 gtcagaaaacactttctata 71 19 137449 Intron 3 12240 cacggtttgccaatgaggca 68 20 137450 Intron 3 17702 taaaggaatcatattccctt 93 21 137451 Intron 3 18784 cagaggttcaaagatgttaa 79 22 137452 Intron 3 64718 atgtgtggctgactgctgag 47 23 137453 Intron 3 80767 tgcttcaacaagtaattaca 0 24 137454 5′UTR 11 32 aaactttattcccgcgctgc 91 25 137455 5′UTR 11 174 tcttcatgggtaaatacaaa 70 26 137456 Start 11 222 tttcactcatctttgaaatg 88 27 Codon 137457 coding 11 318 gaacacatgcctttctttct 60 28 137458 Coding 11 390 tagcatcgtaactatacaca 90 29 137459 Coding 11 431 tagactcatgctaatatctt 80 30 137460 Coding 11 460 atgtcaaatcccaccacatc 91 31 137461 Coding 11 822 gaaatttactccaattgcta 14 32 137462 Coding 11 844 agtttctggtcctcagtgag 67 33 137463 Coding 11 850 gcatacagtttctggtcctc 76 34 137464 Coding 11 856 gtggctgcatacagtttctg 59 35 137465 Coding 11 862 gcatcagtggctgcatacag 63 36 137466 Coding 11 1249 gtttcatcttcaacgtgaat 13 37 137467 Coding 11 1262 tgttgggtcccatgtttcat 75 38 137468 Coding 11 1282 tgtttagctaaatgatcaag 69 39 137469 Coding 11 1427 caaaatttggagttcatgtt 82 40 137470 Coding 11 1491 gagataaatgctcagtagat 26 41 137471 Coding 11 1496 attgggagataaatgctcag 76 42 137472 Coding 11 1572 gagataaatgcttaagcatc 29 43 137473 Coding 11 1580 atcattgggagataaatgct 85 44 137474 Coding 11 1653 tttctaaagacttaagcatc 83 45 137475 Coding 11 1684 tgagttggttctaccgtgcc 84 46 137476 Coding 11 1847 gccaaagtacatcttgaggc 67 47 137477 Coding 11 1874 ccactgaactggtttaaaac 77 48 137478 Coding 11 1938 catatccagttgccatgaca 77 49 137479 Coding 11 2051 gttggacattttaagctgta 0 50 137480 Coding 11 2153 tgaacagtattctggagtta 51 51 137481 Coding 11 2221 gcctcatccacagcaatgag 64 52 137482 Coding 11 2227 cagtgagcctcatccacagc 95 53 137483 Coding 11 2495 ccagtgggaacttgttttga 63 54 137484 Coding 11 2511 ttggaccttcaaattcccag 95 55 137485 Coding 11 2665 acacactgaatttcatctct 56 56 137486 Coding 11 2670 ctatgacacactgaatttca 20 57 137487 Coding 11 2696 aatgcccattccaaaagcta 71 58 137488 Coding 11 2702 tttattaatgcccattccaa 91 59 137489 Coding 11 2709 tgtcagctttattaatgccc 89 60 137490 Coding 11 2715 ggcgaatgtcagctttatta 47 61 137491 Coding 11 2769 caatctcctgataatatgat 88 62 137492 Coding 11 2852 aagaaggtgcctatttaagt 60 63 137493 Coding 11 2954 caagatgatttgtctcctac 72 64 137494 Coding 11 3016 catttttcagttcccataat 77 65 137495 Coding 11 3024 tatcacagcatttttcagtt 85 66 137496 Coding 11 3030 tgcaattatcacagcatttt 82 67 137497 Coding 11 3181 ttagatcctcggagaaataa 80 68 137498 Coding 11 3187 tgagaattagatcctcggag 70 69 137499 Coding 11 3225 caaataaactgtgcctgcga 93 70 137500 Coding 11 3265 aaagccttccaccaactctc 92 71 137501 Coding 11 3455 agttttcgaactaggcagaa 59 72 137502 Coding 11 3531 ccaagttagacttcttctct 88 73 137503 Coding 11 3681 ataacacaatctgagtctcc 84 74 137504 Coding 11 3830 ttcagaaacaccatcaatcc 90 75 137505 Coding 11 3877 aaatgtttgatgacttccaa 88 76 137506 Coding 11 3930 cttgaggttttgtacttgaa 80 77 137507 Coding 11 3999 atgtgatggccatagactgt 71 78 137508 Coding 11 4234 tcaggaactaacattctgat 91 79 137509 Coding 11 4240 atgttttcaggaactaacat 77 80 137510 Coding 11 4274 gatctcaattgccatgtgga 74 81 137511 Stop 11 4519 ttgccagcttaactaaaaag 83 82 Codon 137512 3′UTR 11 4540 gaaacataattgttctggta 83 83 137513 3′UTR 11 4589 tccttactcttcagaaataa 0 84 137514 3′UTR 11 4605 taagccaaaatactactcct 79 85 137515 3′UTR 11 4642 ttcaataaacagtgaacttt 88 86 137516 3′UTR 11 4980 acgtatttaagaacttcttc 86 87 137517 3′UTR 11 5156 aaaaacattgttttattact 10 88 137518 3′UTR 12 261 gtcacatgtgctacataaga 72 89 137519 3′UTR 12 299 ttacccaactctgtgtcaca 90 90 As shown in Table 1, SEQ ID NOs 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 85, 86, 87, 89 and 90 demonstrated at least 40% inhibitition of human WRN expression in this assay and are therefore preferred.", "The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.", "Example 16 Western Blot Analysis of WRN Protein Levels Western blot analysis (immunoblot analysis) is carried out using standard methods.", "Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel.", "Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting.", "Appropriate primary antibody directed to WRN is used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species.", "Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.)." ] ]	Patent_10467182
[ [ "Method and Device for Carrying Out the Functional Check and Functional Checking of a Technical Unit", "The invention relates to a method for carrying out the functional check and a functional checking of a technical unit, comprising the following steps, creation of a check program, by selection and storage of control commands from a number of control commands held in a data bank, depending upon the unit for checking, input of the check program which defines a number of control commands and the sequence thereof into a controller, output of the control commands to the unit in the defined sequence and receiving and displaying of returned information from the unit.", "The invention further relates to a corresponding device." ], [ "1-47.", "(canceled) 48.A method for performing a functionality test of a technical unit including: providing a plurality of control commands stored in a data base; selecting control commands from said plurality of control commands as a function of a technical unit to be tested; storing said selected control commands; preparing a testing program for the technical unit using said stored, selected control commands; providing a control unit; transferring said testing program to said control unit; connecting said control unit with the technical unit to be tested; outputting said selected control commands to the technical unit in a defined sequence; and receiving and displaying acknowledgment information from the technical unit.", "49.A method of performing a functionality test of a technical unit including: providing a plurality of control commands stored in a data base; selecting control commands from said plurality of control commands as a function of a technical unit to be tested; storing said selected control commands; preparing a testing program for the technical unit using said stored, selected control commands; providing a control unit; transferring said testing program to said control unit; providing a bus identical to a bus usable with said technical unit for communicating with other components of a finished printing press; outputting said selected control commands to the technical unit using said bus in a defined sequence; and receiving and displaying acknowledgment information from the technical unit.", "50.The method of claim 48 further including preparing a recording of said acknowledgment information received from the technical unit.", "51.The method of claim 49 further including preparing a recording of said acknowledgment information received from the technical unit.", "52.The method of claim 50 further including all of said selected control commands in said recording.", "53.The method of claim 51 further including all of said selected control commands in said recording.", "54.The method of claim 48 further including represented said control commands in one of text form and graphic symbols.", "55.The method of claim 49 further including represented said control commands in one of text form and graphic symbols.", "56.The method of claim 48 further including displaying said control commands during performance of said testing program.", "57.The method of claim 49 further including displaying said control commands during performance of said testing program.", "58.The method of claim 48 further including providing a bus and using said bus for transmitting said control commands of said testing program to said technical unit.", "59.The method of claim 48 further including providing said testing program with only control commands intended for said technical unit.", "60.The method of claim 49 further including providing said testing program with only control commands intended for said technical unit.", "61.The method of claim 48 including testing at least one of the functions of the technical unit selected from the functions including: engaging and disengaging inking rollers, engaging and disengaging inking ductors, engaging and disengaging ink aspirators, engaging and disengaging dampening ductors, operating drive units of inking rollers, operating drive units of inking ductors, operating drive units of ink aspirators, operating drive units of dampening ductors, operating drive units of cylinders, operating drive units for circumferential registrations of cylinders, operating drive units for diagonal registrations of cylinders, operating drive units for axial registrations of cylinders, operating locking devices of dressings on cylinders, operating dampening water control, and the addition of printing plates.", "63.A device for functionality testing of a technical unit comprising: at least one displaying screen for displaying input masks; at least one memory unit; and a control unit with a testing program, said input masks representing a plurality of control commands in one of text form and graphic symbols, said memory unit being adapted to store said control commands selected by said input masks, as a function of a unit to be tested, as a testing program, said testing program being prepared using one of a program different from said testing program and a development computer.", "64.The device of claim 63 further including a first interface adapted to transmit control commands to said technical unit and to secure acknowledgment information from said technical unit, said first interface containing a second interface, said second interface being adapted for receiving said testing program, said testing program defining a plurality of control commands and their sequence, said second interface being adapted to output said control commands defined in said testing sequence via said first interface in a defined sequence.", "65.The device of claim 64 further including a third interface adapted for outputting acknowledgment information received from said technical unit.", "66.The device of claim 65 further wherein said third interface is adapted to output all of said control commands issued by said first interface.", "67.The device of claim 66 further including a writing device for a data carrier, not able to be overwritten, connected to said third interface.", "68.The device of claim 63 including a second display screen adapted to display control commands and parameters of control commands.", "69.The device of claim 63 wherein said memory unit stores said input masks.", "70.The device of claim 63 further including a buffer memory adapted for the buffer memory adapted for the intermediate storage of not yet executed memory commands.", "71.The device of claim 63 wherein said technical unit is a part of a printing press.", "72.The device of claim 63 wherein said technical unit is a printing unit.", "73.The device of claim 63 wherein said technical unit is a folding unit." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>At present, so-called test control consoles are employed for the functionality testing of various units, such as, for example, printing presses or parts thereof, which test control consoles make it possible for an operator, identified as consumer, to control the individual functions of the unit to be tested and to check whether the unit reacts in the intended manner to the settings input at the test control console.", "Customarily the settings required for a complete functionality test are listed in a test protocol, in which test protocol the consumer can enter, if desired, the reactions of the unit to the settings performed and reported by the test control console in order to document, in this way, the correct or incorrect functioning of the unit.", "This procedure is lengthy, time-consuming and contains uncertainties which never completely eliminate the possibility that the consumer does not make erroneous settings, does not correctly enter results in the protocol, or omits test steps.", "DE 197 25 916 A1 describes a diagnostic device for electrically controlled systems, in which additional libraries are added to a diagnostic program.", "The automatic running of several functions and the input of plain language are not disclosed.", "DE 195 22 937 C2 discloses a diagnostic system for a motor vehicle with a “fixed” diagnostic program.", "No plain language input is provided.", "A testing device for an electro-medical apparatus is known from DE 36 02 171 A1.Access to a data bank for preparing a test program is not possible.", "U.S. Pat.", "No.", "5,588,109 discloses a method and a device for the remote diagnosis of units already placed in an installation by the customer.", "A unit to be diagnosed can be selected from a predetermined number.", "Parameters are assigned to the unit, whose measured values are then detected by the device by use of a remote diagnosis and are shown on a display.", "A user can either make notes in a “memo field”, or can select further parameters to be measured from a predetermiend number of parameters and display them.", "By use of a further tool, the user can select a testing program from a predetermined number, which has a fixed sequence of steps." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The object of the present invention is directed to providing a method and a device for performing a functionality test, as well as to a functionality test of a technical unit.", "In accordance with the present invention, this object is attained by the provision of a method for performing a functionality test of a technical unit.", "A testing program is prepared using control commands selected from a larger group of control commands which are stored in a data bank.", "The testing program is prepared as a function of the unit to be tested.", "The testing program can be transferred to a control unit.", "The technical unit can be connected to the control unit and can be tested.", "A bus may be used for transmitting the control commands, with the bus being the same as the type of bus which the technical unit uses to communicate with other components of a finished printing press.", "A display screen is used to display input masks that represent control commands in text form, or as graphic symbols.", "A memory element stores the control commands selected by the input masks, as a function of the unit to be tested, as a testing program.", "The advantages to be gained by the present invention lie, in particular, in that it assures a complete and correct performance of the functionality test with only little outlay for time and work.", "Since the control unit includes a complete testing program, the accidental omission of individual test steps is not possible.", "A time savings results, inter alia, from the fact that the control unit can immediately issue a command, which is already known to the control unit from the testing program, to the unit to be tested as soon as the previous test step is completed.", "It is thus not necessary to wait until an operator has made the settings required for performing the next steps.", "In order to be able to generate a complete and a dependable protocol regarding the results of the individual test steps, it is useful for the control unit to have a third interface for issuing acknowledgement information which it has received from the unit, in response to an issued control command.", "The totality of the acknowledgement information can be considered to be a testing protocol, by the use of which, the correct performance of the functionality test can be verified at any desired later time.", "To make this later test easier, it is useful not only if the acknowledgement information can be issued via this third interface, but moreover also the issuance of all control commands issued to the unit via the first interface, which have resulted in the respective acknowledgements.", "In this way, it is possible to provide proof, beyond all doubt, regarding the performance of every individual step of the test program.", "For creating the protocol, a writing device for a data carrier, which cannot be overwritten, is preferably connected to the third interface.", "In the simplest embodiment, this writing device can be a printer.", "For storing extensive protocols in a space-saving manner, the employment of a CD burner can also be considered.", "To make it possible for a user to follow the course of the testing program, the control unit can be equipped with a display screen for use in displaying control commands issued to the unit, if desired also inclusive of the parameters of such control commands.", "In the simplest configuration this control unit can be realized as a computer, and in particular as an inexpensive workplace computer, which computer communicates with the technical unit to be tested via a bus.", "In order to prevent any tampering with the progress of the testing program by the user, the control unit may be configured so that it does not have any interface for use for entering control commands by a user.", "This can be realized, in the case of a control unit in the form of a workplace computer, if the conversion of the testing program into commands, which commands can be performed by the unit, and the issuance of such commands, is performed under the control of a control program, which control program does not accept any commands from a user during the running of the testing program.", "It is also possible to provide the user the possibility, within reason, of affecting the control commands sent to the unit, in particular if these control commands are also entered into the protocol to be prepared.", "To make the fixing of such control commands, or their parameters, easier for the user, it is possible to provide a memory element for storing input masks, each of which input masks contains input fields for specifying a control command, or the parameters of a control command specified by the user." ], [ "FIELD OF THE INVENTION The present invention is directed to a method and to a device for performing a functionality test, as well as to the functionality test of a technical unit of a printing press.", "A testing program is prepared by selecting control commands from a data bank.", "A display panel can display the control commands.", "BACKGROUND OF THE INVENTION At present, so-called test control consoles are employed for the functionality testing of various units, such as, for example, printing presses or parts thereof, which test control consoles make it possible for an operator, identified as consumer, to control the individual functions of the unit to be tested and to check whether the unit reacts in the intended manner to the settings input at the test control console.", "Customarily the settings required for a complete functionality test are listed in a test protocol, in which test protocol the consumer can enter, if desired, the reactions of the unit to the settings performed and reported by the test control console in order to document, in this way, the correct or incorrect functioning of the unit.", "This procedure is lengthy, time-consuming and contains uncertainties which never completely eliminate the possibility that the consumer does not make erroneous settings, does not correctly enter results in the protocol, or omits test steps.", "DE 197 25 916 A1 describes a diagnostic device for electrically controlled systems, in which additional libraries are added to a diagnostic program.", "The automatic running of several functions and the input of plain language are not disclosed.", "DE 195 22 937 C2 discloses a diagnostic system for a motor vehicle with a “fixed” diagnostic program.", "No plain language input is provided.", "A testing device for an electro-medical apparatus is known from DE 36 02 171 A1.Access to a data bank for preparing a test program is not possible.", "U.S. Pat.", "No.", "5,588,109 discloses a method and a device for the remote diagnosis of units already placed in an installation by the customer.", "A unit to be diagnosed can be selected from a predetermined number.", "Parameters are assigned to the unit, whose measured values are then detected by the device by use of a remote diagnosis and are shown on a display.", "A user can either make notes in a “memo field”, or can select further parameters to be measured from a predetermiend number of parameters and display them.", "By use of a further tool, the user can select a testing program from a predetermined number, which has a fixed sequence of steps.", "SUMMARY OF THE INVENTION The object of the present invention is directed to providing a method and a device for performing a functionality test, as well as to a functionality test of a technical unit.", "In accordance with the present invention, this object is attained by the provision of a method for performing a functionality test of a technical unit.", "A testing program is prepared using control commands selected from a larger group of control commands which are stored in a data bank.", "The testing program is prepared as a function of the unit to be tested.", "The testing program can be transferred to a control unit.", "The technical unit can be connected to the control unit and can be tested.", "A bus may be used for transmitting the control commands, with the bus being the same as the type of bus which the technical unit uses to communicate with other components of a finished printing press.", "A display screen is used to display input masks that represent control commands in text form, or as graphic symbols.", "A memory element stores the control commands selected by the input masks, as a function of the unit to be tested, as a testing program.", "The advantages to be gained by the present invention lie, in particular, in that it assures a complete and correct performance of the functionality test with only little outlay for time and work.", "Since the control unit includes a complete testing program, the accidental omission of individual test steps is not possible.", "A time savings results, inter alia, from the fact that the control unit can immediately issue a command, which is already known to the control unit from the testing program, to the unit to be tested as soon as the previous test step is completed.", "It is thus not necessary to wait until an operator has made the settings required for performing the next steps.", "In order to be able to generate a complete and a dependable protocol regarding the results of the individual test steps, it is useful for the control unit to have a third interface for issuing acknowledgement information which it has received from the unit, in response to an issued control command.", "The totality of the acknowledgement information can be considered to be a testing protocol, by the use of which, the correct performance of the functionality test can be verified at any desired later time.", "To make this later test easier, it is useful not only if the acknowledgement information can be issued via this third interface, but moreover also the issuance of all control commands issued to the unit via the first interface, which have resulted in the respective acknowledgements.", "In this way, it is possible to provide proof, beyond all doubt, regarding the performance of every individual step of the test program.", "For creating the protocol, a writing device for a data carrier, which cannot be overwritten, is preferably connected to the third interface.", "In the simplest embodiment, this writing device can be a printer.", "For storing extensive protocols in a space-saving manner, the employment of a CD burner can also be considered.", "To make it possible for a user to follow the course of the testing program, the control unit can be equipped with a display screen for use in displaying control commands issued to the unit, if desired also inclusive of the parameters of such control commands.", "In the simplest configuration this control unit can be realized as a computer, and in particular as an inexpensive workplace computer, which computer communicates with the technical unit to be tested via a bus.", "In order to prevent any tampering with the progress of the testing program by the user, the control unit may be configured so that it does not have any interface for use for entering control commands by a user.", "This can be realized, in the case of a control unit in the form of a workplace computer, if the conversion of the testing program into commands, which commands can be performed by the unit, and the issuance of such commands, is performed under the control of a control program, which control program does not accept any commands from a user during the running of the testing program.", "It is also possible to provide the user the possibility, within reason, of affecting the control commands sent to the unit, in particular if these control commands are also entered into the protocol to be prepared.", "To make the fixing of such control commands, or their parameters, easier for the user, it is possible to provide a memory element for storing input masks, each of which input masks contains input fields for specifying a control command, or the parameters of a control command specified by the user.", "BRIEF DESCRIPTION OF THE DRAWINGS A preferred embodiment of the present invention is represented in the drawings and will be described in greater detail in what follows.", "Shown are in: FIG.", "1, a block diagram of an integrated system for developing a testing program and for performing it on a unit to be tested, in accordance with the present invention, in FIG.", "2, an example of a graphic representation of a command which can be performed by the unit, and in FIG.", "3, a graphic representation of the steps of a testing program which makes it possible for a user to follow the performance of the testing program by the use of the device in accordance with the present invention.", "DESCRIPTION OF THE PREFERRED EMBODIMENT The development and testing system represented in FIG.", "1, in accordance with the present invention, contains a first computer, identified as a development computer 01, which is connected to a data bank 02, a second computer identified as a test computer 03, which is connected to a testing program memory 04 and to a protocol printer 06, and a unit 07 to be tested.", "The unit 07 to be tested, is part of a printing press, and can be, for example, a printing unit, a folding apparatus, a roll changer, a draw-in device, a cooling group, a dryer, a superstructure with guide rollers and turning bars, a sheet feeder, a sheet delivery device, a sheet conveying installation, a turning installation, or the like, or can be a component of a unit 07 to be tested, for example an inking unit, a dampening unit, and the like.", "The development computer 01, the test computer 03 and the unit 07 to be tested are each equipped with suitable interfaces in order to be able to communicate with each other via a common bus 08.The common bus 08 is of the same type as a suitable bus, not shown, through the one via which the unit 07, if it is a part of a printing press would, a finished printing press, communicate with other components of the finished printing press, for example for receiving control commands from a central control unit and for providing acknowledgements regarding the execution of a control command, of a set parameters, and the like to the control unit or to other components.", "The data bank 02 contains a data set whose elements each correspond to one of a plurality of control commands which the unit 07, or which other components of the printing press are able to process.", "A programmer, during the development of a testing program for a given unit 07, sequentially selects elements of the data set which are intended to form control commands of the testing program and which are to be sequentially executed.", "Each element of the data set consists of a plurality of data fields, as well as of information which fixes the type of the display of the individual data fields on a display screen of the development computer 01.An example for such a display, which display is shown to the programmer, after the selection of an element of the data set, is represented in FIG.", "2.Consecutive numbers, in this example the number 17, is assigned to each one of the elements of the data set.", "An identification of the command, in text form, in this case “operation of the color zone modules”, the designation 12 of a component of the printing press which is the sender of the report, and the designation 13 of a receiver of the control command, are shown in a first partial window 11.It is also within the scope of the preset invention to represent the control commands as graphic symbols.", "Depending on the importance of a particular control command, it is possible to assign various parameters to it, which can assume various values.", "A second partial window 14 is divided into a plurality of fields 16, each of which fields 16 is assigned to one of these parameters, contains information regarding the importance of the assigned parameter, and on which field a programmer can write a selected value of the parameter.", "In the example of a command for the control of the color zone module of an inking system, which is represented in FIG.", "2, a total of 10 parameters, each of a width of one bit, is defined, each of which parameters can assume values of 0 or 1 as set or not set.", "In this case, the parameters are distributed over the bits LB0 to LB7 and HB6 to HB7 of two bytes.", "Here, a group of “n” parameters each is assigned to an operating property of the unit 07 to be tested, which can assume “n” values.", "For example, the bits LB0, LB1 are used for activating or deactivating a testing mode of the ink color modules.", "The testing mode is distinguished in that the module cyclically transmits a plurality of internal measuring values, such as temperature, potentiometer voltages, and the like, which transmission would not be the case during normal operations of the module.", "The bits LB3, LB4 are used for switching the color zone module back and forth between a normal operational state and a stand-by state.", "By transmitting a command “operation of the color zone modules” with the bit LB5 set, the module can be switched into the stand-by state, in which state control commands addressed to it are not evaluated, with the exception of the inverse command “operation of the color zone modules” with the bit LB4 set.", "In this case, n=4 different states are defined for the color zones of the module, to each of which different states a parameter bit is assigned: all color zones 100% open (LB7), all color zones closed (LB6), setting of all color zones to a nominal value determined in a previous control command (HB7), and setting all color zones to pre-inking (HB6).", "Of the parameter bits, which are all a part of a group of alternative parameters, such as for example LB6, LB7, HB6, HB7, only one can be selected at one time.", "If none of the parameters of a group has been set, the property of the module, which can be affected by these parameters, remains unchanged in the course of performing the control command by the module.", "For example, to get the color zone module ready, by use of a single control command, to process control commands for releasing the positioning of the actuators and to set all color zones to the nominal value, a programmer therefore can select the fields LB4, LB2, HB7 in the partial window 14, whereupon the development computer 01 generates a testing program step with the control command “operation of the color zone modules” and an associated 16 bit parameter word of the hexadecimal value 8014, or the binary values 1000 0000 0001 0100.The setting of other control commands of the testing program for other components of a printing press to be tested, can also take place in the same way as described above.", "A testing program developed in this way as a sequence of control commands can now be transferred via the bus 08 to the testing program memory 04 of the testing computer 03.The testing program can have the form of a data set which contains nothing more than the compilation of control commands to be issued by the testing computer 03 to the unit 07 to be tested, as well as their parameters and, if required, auxiliary information, whose function will be addressed at a later time.", "The conversion of this data set into commands transmitted to the unit is performed by a system program of the testing computer 03.In general, the testing program can also be in the form of a program which is performed directly by the testing computer 03 for issuing the fixed control commands.", "However, the first mentioned case is assumed in the continued description, namely that the testing program substantially only includes the control commands intended for the unit and that it is performed under control of a system program of the testing computer 03.After the testing program has been transferred to the testing program memory 04, the development computer 01 is no longer needed for further testing of the unit 07.The development computer 01 and the unit 07 to be tested do not communicate directly with each other via the bus 08.It is therefore possible and also is useful that, at an initial time, the testing computer 03 is only connected with the development computer 01 for transferring the testing program into the memory 04, and that, at a later time, the testing computer 03 is connected via a bus 08 of the same type with the unit 07 to be tested.", "The testing computer 03 can usefully be a portable computer, such as a laptop, for example, so that the testing program in a first network can be transferred to the memory 04 via the bus 08, and that actual testing of the unit 07 can be performed at any other arbitrary location in a second network via the bus 08.It is, of course, possible to transfer the testing program in any arbitrary other way into the testing program memory 04, for example by writing the testing program on a data carrier at the development computer 01 and by reading the data carrier out at the testing computer 03.It is also conceivable for the development computer 01 and for the testing computer 03 to be the same device, which single device merely executes different programs at different times.", "FIG.", "3 shows the structure of an image appearing on a display screen, which is not represented, of the testing computer 03 in the course of processing the testing program entered into the testing program memory 04.The testing program is divided into a plurality of sections, each of which sections has been assigned a title, here “initialize SPS”, “test color zones”, “operate main drive unit”, which sections are arranged in a tree structure in the partial window 18.These titles, as well as information regarding their hierarchical structuring required for generating the tree structure, are contained in the above mentioned auxiliary information.", "At the stage of the performance of the testing program represented in FIG.", "3, the testing computer 03 is in the process of performing the testing program “test color zones”, to which end the individual control commands, or “jobs”, of which the partial program is composed, are listed in the partial window 18.Only the titles of presently not performed program sections, such as “initialize SPS” and “operate main drive unit”, are represented.", "The control command being executed at this time is represented inverted, i.e.", "as white print on a black background.", "A partial window 19 can be used for displaying comments or instructions in text form, which can also be contained in the auxiliary information, synchronized with the running of the testing program, to a user of the testing program.", "In a plurality of named fields, a further partial window 21 shows a sequence of numerical values which, when transmitted in a fixed sequence on the bus 08 to the unit 07, constitute the control command represented inverted in the window 18.Parameters of the control command, here the decimal value 32788, for example, which corresponds to the above hexadecimal value 8014 in the above mentioned example, are represented in a partial window 22.Thus a user which lets the test computer 03 perform the control program can obtain a complete overview regarding the progress of the testing program by utilization of the display represented in FIG.", "3.By the use of a plurality of buttons 23, 24, 26, 27, the user has the ability to stop the testing program run, by using button 23, to manually trigger the transmission of a control command to the unit to be tested 07, by using button 24, or to switch the display of the status shown in FIG.", "3, in which the sent commands are displayed, into a state wherein the acknowledgements received from the unit 07 are displayed, and vice versa, by using buttons 26, 27.An addition or removal of control commands, or a manipulation of their parameters, is not provided.", "Acknowledgements received by the testing computer 03 from the unit to be tested 7, as a response to a transmitted control command, are output by the system program on a protocol printer 06 in the sequence of their arrival.", "On the basis of this testing program, and with a knowledge of the performed control program, it is easily and assuredly possible to prove, at a later time, that the functionality test has taken place with all required steps and correct parameters, and what results it has provided.", "To make such a later test even more easily possible, it can be provided that not only the acknowledgement information sent by the unit 07 is output on the protocol printer 06, but also the control commands sent by the testing computer 03 to the unit 07, which had triggered the recorded acknowledgements are also output on the protocol printer 06.If the system program completely records the control commands sent to the unit 07 on the printer 06, it is also possible, with this system program, to provide a possibility for a mask, of the type represented in FIG.", "2, to be displayed on the testing computer 03, so that a user has the option, in this way, to insert additional testing commands, or to change the parameters of testing commands if the result delivered by the testing program make this desirable.", "Since these additional or modified commands, and the acknowledgements sent as a reaction thereto by the unit 07, are recorded on the printer 06, the reproducibility of the test process and the possibility of documenting its correct and complete performance is assured.", "It is, of course, possible to employ any arbitrary other data recording device in place of the printer, by the use of which a recording, safe against later falsification, of the progress of the testing program, can be installed.", "For example, it is possible to employ a CD burner for recording the protocol on a CD-ROM which can only be written on once.", "Also conceivable is a recordation on any arbitrary data carriers which, per se, can be overwritten, for example any arbitrary magnetic data carrier which is protected by program-technological means, for example by a so-called digital signature, against subsequent falsification.", "The functionality test of a printing unit can include the following steps, for example: turning the printing unit on and off, drive units of inking rollers, inking ductor, ink aspirator, dampening unit, dampening ductor, drive units of cylinders, circumferential, diagonal and axial registration of the rollers, locking devices for dressings on the cylinders, print-on and print-off of the cylinders, washing function of the inking unit and the cylinders, dampening fluid level control, adding of printing plates.", "A device in accordance with the present invention may be characterized in that the unit to be tested 07 is embodied as a printing unit or as a folding apparatus or as a draw-in device or as a cooling group or as a dryer, or as a superstructure with guide rollers and turning bars, or a sheet feeder, or a sheet delivery device, or a sheet conveying installation, or a turning installation.", "While preferred embodiments of a method and device for carrying out the functional check and functional checking of a technical unit, in accordance with the present invention, have been set forth fully and completely hereinabove, it will be apparent to one of skill in the art that a number of changes in, for example, the structure of the technical unit to be tested, the specific computer or computers used to conduct the tests, and the like, could be made without departing from the true spirit and scope of the present invention, which is accordingly to be limited only by the following claims." ] ]	Patent_10467332
[ [ "Androgenic 7-substituted 11-halogen steroids", "The invention relates to 11β-halogen steroids with general formula (I), whereby R11 is halogen, X—Y-Z represents a group with one of the two structures CH═C—C or CH2—C═C and the other radicals have the meaning that is indicated in the claims, also the production and use of these compounds for the production of pharmaceutical agents as well as pharmaceutical preparations that contain 11β-halogen steroids." ], [ "1.11β-Halogen steroids with the general formula I: in which X—Y-Z represents a group with one of the two structures CH═C—C or CH2—C═C R1 can be in α- or β-position, and stands for hydrogen, R or P-Q-R that is bonded via P to the basic ring structure, whereby P and Q represent straight-chain or branched-chain, optionally partially or completely fluorinated C1 to C8 alkylene, -alkenylene, -alkinylene groups and can be the same or different, and R represents a CH3 radical or CF3 radical, provided that no substituent R1 is present at Z if X—Y-Z represents the group CH2—C═C, R6 is a hydrogen atom or can have the meanings that are indicated under R7, R7 stands for R or P-Q-R bonded via P to the basic ring structure, whereby these groups have the above-mentioned meanings, R11 represents a halogen, R13 is methyl or ethyl, and R17 is hydrogen or stands for C(O)—R18, whereby R18 is a straight-chain or branched-chain C1 to C18 alkyl, -alkenyl, -alkinyl radical or an aryl radical, or stands for T-U—V bonded via T to the C(O) group, whereby T and U represent straight-chain or branched-chain C, to C18 alkylene, -alkenylene, -alkinylene groups, alicyclic C3 to C12 groups or aryl groups and are the same or different, and V is a straight-chain or branched-chain C, to C18 alkyl, -alkenyl or -alkinyl radical or an aryl radical, or R18 has one of the above-mentioned meanings and in addition is substituted with one or more groups NR19R20 or one or more groups SOxR21, whereby x=0, 1 or 2, and R19, R20 and R21 in each case are hydrogen or T-U—V bonded via T to N, S with the above-mentioned meaning, provided that in addition, the physiologically compatible addition salts with in organic and organic acids are included.", "2.11β-Halogen steroids according to claim 2, characterized in that R11 is fluorine.", "3.11β-Haologen steroids according to claim 2, wherein R6 is hydrogen, methyl, ethyl or fluoromethyl.", "4.11β-Halogen steroids according to claim 2, wherein R7 is methyl, ethyl or fluoromethyl.", "5.11β-Halogen steroids according to claim 2, wherein R1 is hydrogen.", "6.11β-Halogen steroids according to claim 2, wherein R13 is methyl.", "7.11β-Halogen steroids according to claim 2, wherein R17 is hydrogen or C(O)—R18, whereby R18 is a straight-chain or branched-chain C1 to C18 alkyl radical.", "8.11β-Halogen steroids of general formula I, namely 11β-Fluoro 17β-hydroxy-7α-methyl-estr-4-en-3-one, 11β-Chloro-17β-hydroxy-7α-methyl-ester-4-en-3-one, 11β-Bromo-17β-hydroxy-7α-methyl-estr-4-en-3-one, 17β-Hydroxy-11β-iodo-7α-methyl-estr-4-en-3-one, 7α-Ethyl-11β-fluoro-17β-hydroxy-estr-4-en-3-one, 11β-Fluoro-7α-(fluoromethyl)-17β-hydroxy-estr-4-en-3-one, 11β-Fluoro-17β-heptanoyloxy-7α-methyl-estr-4-en-3-one, 11β-Fluoro-7α-methyl-17β-undecanoyloxy-estr-4-ene-3-one, 11β-Fluoro-17β-hydroxy-7α-methyl-estr-5(10)-en-3-one.", "9.Use of the structural portion of general formula as a component of compounds with androgenic action, in which X—Y-Z represents a group with one of the two structures CH═C—C or CH2—C═C, the other bonds are saturated in ring A, R7 and R13 are not hydrogen, and R11 is a halogen.", "10.Process for the production of 11β-halogen steroids of the general formula I according to claim 1, in which, starting from a compound with general formula B, D whereby R11 means either a hydroxyl group, a halogen or a nucleophilic leaving group, and R13 has the above-mentioned meaning, a compound with general formula B′, E is first formed by 1,6-addition of a metallated alkyl with general formula R7-MX or R7-M, whereby M is an alkali metal, M′ is an alkaline-earth metal, X is a halogen atom, and R7 has the above-mentioned meaning: and this compound then is reduced either selectively to a compound with general formula I or, if R11 is not halogen, is nucleophilically substituted in 11-position with a halogenating agent and then is reduced selectively to the compound with general formula I: 11.Use of the 11β-halogen steroids according to claim 1 for the production of pharmaceutical agents.", "12.Pharmaceutical preparations that contain at least one 11β-halogen steroid according to claim 1 as well as at least one pharmaceutically compatible vehicle." ], [ "DESCRIPTION The invention relates to 11β-halogen steroids, their production and use for the production of pharmaceutical agents, pharmaceutical preparations that contain 11β-halogen steroids as well as the use of certain 11β-halogen steroid derivatives as a component of compounds with androgenic action.", "For therapy of male menopause and for development of male sex organs as well as for male birth control, androgens, especially testosterone, are used.", "In addition, these hormones also have partial anabolic active components that promote, i.a., muscle growth.", "Male menopause is characterized by an age-related reduction in endogenous androgen production, so that hormone replacement (HRT: hormone replacement therapy) is performed for treatment thereof.", "In addition to a reduction of spermatogenesis, the LH-RH administration for male birth control also results in the release of LH and in reducing testosterone levels and libido, which are compensated for by administration of testosterone pharmaceutical agents (D. E. Cummings et al., “Prostate-Sparing Effects of the Potent Androgen 7α-Methyl-19-Nortestosterone: A Potential Alternative to Testosterone for Androgen Replacement and Male Contraception,” Journal of Clinical Endocrinology and Metabolism, Vol.", "83, No.", "12, pages 4212-4219 (1998)).", "Combinations including an androgen and a progestogen can be used to control male fertility (c.f.", "for instance WO 01/60376 and the references cited therein).", "In the case of treatment with testosterone, it has been shown that side effects occur, especially an enlargement of the prostate owing to an increase in the number of cells and glands of the stroma (BPH: benign prostate hyperplasia).", "In the metabolism of testosterone that is mediated by 5α-reductase, dihydrotestosterone (DHT) that can result, i.a., in the occurrence of BPH is produced (Cummings et al., ibid.", "; WO 99/13883 A1).", "The inhibition of the 5α-reductase is therefore used for treating BPH in clinical practice (finasterides).", "The quick metabolism of the androgenic steroid testosterone in the human body further results not only in the formation of undesirable DHT, but also in that an oral administration of higher doses is necessary to reach the desired effect level of testosterone.", "Alternative forms for dispensing, such as i.m.-injections or large patches, are therefore necessary.", "To replace testosterone in the above-mentioned indication areas, 7α-methyl-19-nortestosterone (MeNT) was proposed which has, on the one hand, a higher biological effectiveness as testosterone, since it has a higher binding affinity to the androgen receptors.", "On the other hand, because of a steric inhibition by the 7α-methyl group, it presumably resists metabolization by 5α-reductase (Cummings et al., ibid., WO 99/13883 A1, WO 99/13812 A1, U.S. Pat.", "No.", "5,342,834).", "During metabolism of testosterone, a smaller portion of this compound is also reacted by aromatization of ring A of the steroid system to form estradiol, especially in the brain, in the liver and in the fatty tissue.", "With respect to the total action of the testosterone and its metabolites, estradiol is substantially responsible for sex-specific behavior and gonadotrophin regulation.", "Therefore, its action just like that of testosterone for the adult male is regarded as advantageous (Cummings et al., ibid.).", "It has been shown, however, that the pharmacokinetics of testosterone is not satisfactory.", "In particular in the case of oral dispensing, testosterone is quickly excreted again, so that the effectiveness and the duration of action of the thus produced pharmaceutical agents is unsatisfactory.", "Other testosterone derivatives were therefore also synthesized.", "Such derivatives are described in, i.a., U.S. Pat.", "No.", "5,952,319, in particular 7α-,11β-dimethyl derivatives of 19-nortestosterone, namely 7α,11β-dimethyl-17β-hydroxyestr-4-en-3-one, 7α,11β-dimethyl-17β-heptanoyloxyestr-4-en-3-one, 7α,11β-dimethyl-17β-[[(2-cyclopentylethyl)-carbonyl]-oxy]-estr-4-en-3-one, 7α,11β-dimethyl-17β-(phenylacetyloxy]-estr-4-en-3-one and 7α,11β-dimethyl-17β-[[(trans-4-[n-butyl]cyclohexyl)-carbonyl]-oxy]-estr-4-en-3-one.", "The above-mentioned 7α,11β-dimethyl-derivatives have the above-mentioned advantages, like MeNT, including an improved pharmacokinetics, i.e., its effectiveness and duration of action are improved relative to testosterone.", "These derivatives, however, can be produced only via an expensive synthesis method.", "The problem on which the invention is based is therefore to find derivatives of testosterone that are not sensitive relative to a reduction by means of 5α-reductase, that also have an improved pharmacokinetics compared to 7α-methyl-19-nortestosterone and that can be produced easier than the 7α,11β-dimethyl-derivatives at the same time.", "The problem on which this invention is based is solved by 11β-halogen steroids according to claim 1, by the use of 11-β-halogen steroids as a component of compounds with androgenic action according to claim 9, also by a process for the production of these compounds according to claim 10, a use of these steroids for the production of pharmaceutical agents according to claim 11 as well as by pharmaceutical preparations according to claim 12.Preferred embodiments of the invention are indicated in the subclaims.", "As components of compounds with androgenic action, the 11β-halogen steroids according to the invention have the following basic structure: whereby X—Y-Z represents a group with one of the two structures CH═C—C or CH2—C═C, the other bonds are saturated in ring A, R7 and R13 are not hydrogen, and R11 is a halogen.", "In the other positions on the ring skeleton, any other substituents including hydrogen consequently can be present.", "The invention thus basically relates to the following two basic structures, namely 11β-halogen-estr-5(10)-en-3-one: as well as 11β-halogen-estr-4-en-3-one: In particular, the androgenic steroids according to the invention have the following general formula I: in which X—Y-Z represents a group with one of the two structures CH═C—C or CH2—C═C R1 can be in α- or β-position, and stands for hydrogen, R or P-Q-R that is bonded via P to the basic ring structure, whereby P and Q represent straight-chain or branched-chain, optionally partially or completely fluorinated C1 to C8 alkylene, -alkenylene, -alkinylene groups and can be the same or different, and R represents a CH3 radical or CF3 radical, provided that no substituent R1 is present at Z if X—Y-Z represents the group CH2—C═C, R6 is a hydrogen atom or can also have the meanings that are indicated under R7, R7 stands for R or P-Q-R bonded via P to the basic ring structure, whereby these groups have the above-mentioned meanings, R11 represents a halogen, R13 is a methyl or ethyl group, and R17 is hydrogen or stands for C(O)—R18, whereby R18 is a straight-chain or branched-chain C1 to C18 alkyl, -alkenyl, -alkinyl radical or an aryl radical, or stands for T-U—V bonded via T to the C(O) group, whereby T and U represent straight-chain or branched-chain C1 to C18 alkylene, -alkenylene, -alkinylene groups, alicyclic C3 to C12 groups or aryl groups and are the same or different, and V is a straight-chain or branched-chain C1 to C18 alkyl, -alkenyl or -alkinyl radical or an aryl radical, or R18 has one of the above-mentioned meanings and in addition is substituted with one or more groups NR19R20 or one or more groups SOxR21, whereby x=0, 1 or 2, and R19, R20 and R21 in each case are hydrogen or T-U—V bonded via T to N, S with the above-mentioned meaning, provided that in addition, the physiologically compatible addition salts with inorganic and organic acids are included.", "The R11 group is preferably fluorine.", "As an alternative, in this case, it can also be chlorine, bromine or iodine, however.", "In particular, the R6 group can be hydrogen, methyl, ethyl or fluoromethyl.", "R6 preferably stands for hydrogen.", "In particular, the R7 group can be methyl, ethyl or fluoromethyl.", "R7 is preferably methyl.", "It is especially advantageous if R1 is hydrogen, so that this is a 19-nortestosterone derivative.", "Basically, however, R1 can also be methyl, ethyl, propyl or butyl or a fluorinated, especially perfluorinated derivative of these radicals.", "R13 can be methyl or ethyl.", "In particular, R13 is methyl.", "R17 is preferably hydrogen or C(O)—R18, whereby R17 is a straight-chain or branched-chain alkyl radical.", "If substituent R18 in the grouping C(O)—R18 for R17 is an alkyl, in this connection this can be in particular methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl and octadecyl, whereby both the unbranched and the branched derivatives, thus in particular isopropyl, isobutyl, tert-butyl, isopentyl, tert-pentyl and neopentyl, are suitable.", "Within the aforementioned alkyl residues hexyl, heptyl, octyl, nonyl and decyl are especially preferred.", "If R18 is an alkenyl, this can be in particular ethenyl as well as 1- or 2-propenyl.", "If R18 is alkinyl, this can be in particular ethinyl as well as 1- or 2-propinyl.", "If R18 is represented by grouping T-U—V, in this case this can be in first line methylenecyclohexyl that is bonded via the methylene group to the CO group or ethylene cyclohexyl that is bonded via the ethylene group in 1- or 2-position to the CO group.", "The upper limit of carbon atoms in the straight of P-Q-R and T-U—V shall be 22.As aryl radicals in the C(O)—R18 substituent, in particular phenyl and 1-naphthyl or 2-naphthyl can be suitable.", "In the same manner, aralkyl radicals T-U—V can be in particular benzyl, phenylethyl, phenylpropyl, naphthylmethyl and naphthylethyl.", "Aryl in the C(O)—R18 substituent can likewise mean heteroaryl.", "Examples of heteroaryl radicals are in particular 2-, 3- and 4-pyridinyl, 2- and 3-furyl, 2- and 3-thienyl, 2- and 3-pyrrolyl, 2-, 4- and 5-imidazolyl, pyridazinyl, 2-, 4- and 5-pyrimidinyl as well as 2- and 4-pyridazinyl.", "If R18 in addition is substituted with a group NR19R20, in this connection this can be a methylamino, dimethylamino, ethylamino, diethylamino, cyclohexylamino, dicyclohexylamino, phenylamino, diphenylamino, benzylamino or dibenzylamino group.", "In this case, the corresponding physiologically compatible addition salts with inorganic and organic acids are also included.", "As physiologically compatible addition salts with inorganic acids, in particular the hydrochlorides, hydrobromides, acetates, citrates, oxalates, tartrates and methanesulfonates are suitable.", "In particular, the following 11β-halogen steroids are preferred: 11β-Fluoro-17β-hydroxy-7α-methyl-estr-4-en-3-one, 11β-Chloro-17β-hydroxy-7α-methyl-estr-4-en-3-one, 11β-Bromo-17β-hydroxy-7α-methyl-estr-4-en-3-one, 17β-Hydroxy-11β-iodo-7α-methyl-estr-4-en-3-one, 7α-Ethyl-11β-fluoro-17β-hydroxy-estr-4-en-3-one, 11β-Fluoro-7α-(fluoromethyl)-17β-hydroxy-estr-4-en-3-one, 11β-Fluoro-17β-heptanoyloxy-7α-methyl-estr-4-en-3-one, 11β-Fluoro-7α-methyl-17β-undecanoyloxy-estr-4-ene-3-one, 11β-Fluoro-17β-hydroxy-7α-methyl-estr-5(10)-en-3-one.", "The above-mentioned 11β-halogen steroids with general formula I are especially suitable for the production of pharmaceutical agents.", "In addition, the invention relates to pharmaceutical preparations that contain at least one pharmaceutically compatible vehicle in addition to at least one of the above-mentioned 11β-halogen steroids.", "The 11β-halogen steroids of general formula I according to the invention are compounds with strong androgenic action without the above-mentioned side effects as for instance stimulation of the prostate (in particular no benign prostate hyperplasia).", "They can be easily synthesized.", "It has been shown that the 11β-halogen steroids can be used not only for male HRT but are also suitable without additional administration of additional active ingredients as effective male contraceptive agents if a sufficient dosage is made to drop the blood level of LH, of testosterone that is produced in the body as well as FSH (follicle stimulating hormone) sufficiently.", "This depends on the 11β-halogen steroids according to the invention inhibiting the release of LH and FSH.", "LH stimulates the Leydig cells, so that testosterone is secreted.", "If the blood level of the LH is kept low, the endogenous testosterone release also drops.", "Testosterone is required for spermatogenesis, while FSH stimulates the germ cells.", "Sufficiently high FSH and LH blood levels are therefore necessary for an effective spermatogenesis, whereby a sufficiently high LH blood level results in the testosterone release that is necessary for spermatogenesis.", "Since treatment with just the 11β-halogen steroids without additional active ingredients for sterilization can provide for effective male contraception, the administration of a pharmaceutical agent that is suitable for this purpose can be significantly simplified, and the costs of the treatment are considerably decreased.", "The 11β-halogen steroids of this invention can also be used in combination with a progestogen to control male fertility.", "Moreover, the 11β-halogen steroids according to the invention effectively inhibit 5α-reductase and the steroid-11-hydroxylase [CYP11B (P450c11), G. Zhang, W. L. Miller, Journal of Clinical Endocrinology and Metabolism, Vol.", "81, pages 3254-3256 (1996)], so that, for example, the stimulation of the prostate is avoided in a selective manner, and these compounds exhibit improved pharmacokinetics.", "The inhibition of the 11-hydroxylase results in a reduced deactivation of the androgenic compounds and in their reduced excretion from the human body.", "As a result, the effectiveness and the duration of action of these compounds compared to known compounds are improved especially after oral administration.", "For the reasons above, these compounds are especially suitable for use in male birth control as well as for androgen replacement therapy with a reduced tendency toward 5α-reduction with simultaneously obtained aromatizability to form estrogenic steroids and advantageous influence on serum lipids and the central nervous system.", "The androgenic action and the observation that the above-mentioned side effects do not occur were determined “in vivo” in rats: Anabolic and Androgenic Activity in the Juvenile Male Rat The test by Hershberger et al.", "(1953) is used as a screening test method for detecting androgenic properties: Principle.", "The function and size of the accessory reproductive glands (seminal vesicles, prostate) and of the muscle (e.g.", "M. levator ani) are androgen dependent.", "Castration leads to the atrophy of these organs, the substitution with androgenic compounds stimulates growth and function in a dose dependent manner.", "Test description.", "In the juvenile male castrated rat, pharmacologically active agonists of androgens stimulate the growth of the prostate, seminal vesicles (androgenic property) and Musculus levator ani (anabolic property).", "The treatment for seven days of juvenile castrated male rats with daily administration of compound A: 11β-Fluoro-17β-hydroxy-7α-methyl-estr-4-en-3-one,(different doses), compound B: 7α-methyl-19-nortestosterone (different doses), testosterone propionate (reference) and benzylbenzoate/ricinus oil (solvent and control), respectively, was used to determine the androgenic and anabolic potency of eF-MENT.", "Analyses: relative weights of prostate, seminal vesicles and M. levator ani.", "(Hershberger, LG, Shipley, EG, Meyer, RK (1953).", "Myotrophic activity of 19-nortestosterone and other steroids determined by modified levator ani muscle method.", "Proc.", "Soc.", "Exp.", "Biol.", "Med.", "83: 175-180.)", "Comparison of Compound A: 11β-Fluoro-17β-hydroxy-7α-methyl-estr-4-en-3-one, and compound B: 7α-methyl-19-nortestosterone Prostate: Compound A Dose rel.", "Compound B Stimu- mg * kg−1 * Weight Stimulation rel.", "Weight lation Day−1 [mg/100 g] [mean] [mg/100 g] [mean] 0.005 8.9 4.3 0.015 19.7/18.5 30.0 14.6/20.0 24.0 0.03 25.2 40.1 0.05 34.1 76.6 24.7/31.7 51.3 0.15 44.9/47.5/ 93.8 40.6 90.2 41.7 0.3 59.5 125.1 1.5 64.6 137.3 Animals: ORX, juvenile rats, duration of treatment: 7 days.", "Seminal Vesicles: Compound A Compound B Dose rel.", "rel.", "mg * kg−1 * Weight Stimulation Weight Stimulation Day−1 [mg/100 g] [mean] [mg/100 g] [mean] 0.005 9.2 4.6 0.015 18.6/15.5 18.6 13.6/19.2 17.0 0.03 26.8 35.6 0.05 43.6 59.2 36.8/39.0 51.8 0.15 57.9/64.4/ 85.5 65.5 87.8 49.3 0.3 93.4 144.8 1.5 120.0 189.3 Animals: ORX, juvenile rats, duration of treatment: 7 days.", "M. Levator Ani Compound A Compound B Dose rel.", "rel.", "mg * kg−1 * Weight Stimulation Weight Stimulation Day−1 [mg/100 g] [mean] [mg/100 g] [mean] 0.005 36.6 71.3 0.015 54.9/50.8 79.5 38.1/53.7 92.6 0.03 56.5 93.3 0.05 59.4 118.0 48.1/59.4 150.7 0.15 64.7/63.67 154.5 59.4 283.4 1.7 0.3 69.8 180.4 1.5 69.9 181.0 Animals: ORX, juvenile rats, duration of treatment: 7 days.", "In these tests the compound A as one representative of the compounds according to the invention shows an activity profile comparable to the prior art compound B demonstrating that the compounds of the invention can be used analogously to the known compounds.", "Stability in Liver Microsomes The metabolic stability of the compounds has been tested in liver microsomes of various species (commercially available).", "It is possible to make predictions as to the first-pass-metabolism in human by using human test material.", "A rapid degradation of the test material gives hints at low oral bio-availability of the substance tested.", "Isolated microsomes from various species are incubated in the two different periods of time at comparable metabolic activity (cytochrome P 450 enzymes).", "The points in time correspond with the ones in which degradation of reference compounds can be demonstrated.", "The amount of the remaining compound is quantified by comparison with the amount remaining after 0 mins.", "Bei entsprechender Gestaltung können Hinweise auf Phase-I-Metaboliten und auf beteiligte CYP Isoenzyme erhalten werden.", "Werden Lebermikrosomen mit Zusatzstoffen supplementiert, können neben Phase-I-Reaktionen auch Glucuronidierungen (Phase-II-Reaktion) beobachtet werden.", "Species Specific Stability of Test Compounds in Liver at a Concentration of 3 μM Compound B Compound A (%) (%) left unchanged left unchanged Species after 30 min after 60 min after 30 min after 60 min Human 37 19 59 41 Mouse 4 4 20 11 Rat 4 6 5 7 The metabolic stability of compound A representing compounds according to this invention has proved higher than that of compounds of the prior art.", "The compounds according to the invention or the pharmaceutical preparations according to the invention that contain these compounds are extremely well suited for treating non-sterile male patients as well as basically also male mammals.", "A treatment for contraception results in that the male patients to be treated are only temporarily sterile.", "After the treatment with the active ingredients according to the invention or the pharmaceutical preparations is completed, the original state is produced again, so that the previously treated male patient is no longer sterile, and the spermatogenesis takes place again to the original extent.", "To keep the condition of temporary sterility constant over a desired period, the administration of the active ingredient or the preparation is to be performed continuously, whereby the administration, depending on the form of administration, is to be repeated either daily, at a shorter interval or else periodically at a longer interval.", "After the one-time or repeated administration of the active ingredient or the preparation is completed, the non-sterile condition of the male patient optionally is not immediately restored but rather only slowly restored, whereby the time span that is necessary for this purpose depends on various factors, for example the dosage, the body constitution of the treated patients and the parallel administration of other pharmaceutical agents.", "If the purpose of treatment in the contraception exists, the dosage of the 11β-halogen steroids must be set high so that the blood levels of LH and FSH in each case are at most 2.5 I.E./ml (I.E.", ": International Units), especially at most 1.0 I.E./ml, and the blood level of testosterone is at most 10 nmol/l, especially at most 3 nmol/l.", "If the compounds according to the invention are to be used for HRT without a contraception being achieved, the dosage is set lower.", "For this case, an attempt is made to achieve effect levels that make possible the blood levels for LH and FSH of respectively more than 2.5 I.E./ml and for testosterone of more than 10 nmol/l.", "The dosages of the 11β-halogen steroids according to the invention that are required to set the blood level of LH, FSH and testosterone depend on a number of factors and must therefore be determined in an application-specific manner.", "First, the dosage is naturally dependent on the type of therapy.", "If the compounds for male contraception are to be used, significantly higher doses must be given than in the case of a use for HRT.", "The dosage also depends on the type of 11β-halogen steroid and its bio-availability.", "The type of use is also essential for the amount to be administered.", "Finally, the dosage also depends on the body constitution of the patient to be treated and other factors, for example the state of whether other pharmaceutical agents are provided in parallel.", "The compounds can be administered orally and parenterally, for example i.p.", "(intraperitoneally), i.v.", "(intravenously), i.m.", "(intramuscularly) or percutaneously.", "The compounds can also be implanted in the tissue.", "The amount of the compounds to be administered can fluctuate within a wide range if an effective amount is administered.", "Based on the condition to be treated or based on the effect to be achieved and the type of dispensing, the amount of administered compound can vary within a wide range.", "In humans, the daily dose is in the range of 0.1 to 100 mg.", "The preferred daily dosage in humans is 0.1 to 10 mg.", "The duration of treatment depends on the purpose to be achieved.", "Capsules, pills, tablets, coated tablets, creams, ointments, lotions, liquids, such as syrups, gels, injectable liquids, for example for i.p., i.v., i.m.", "or percutaneous injection, etc., are suitable for use, whereby the individual forms for dispensing release the compounds according to the invention to the body gradually or in the entire amount within a short time depending on the type thereof.", "For oral administration, capsules, pills, tablets, coated tablets and liquids or other known oral forms for dispensing are used as pharmaceutical preparations.", "In this case, the pharmaceutical agents can be formulated in such a way that they release the active ingredients either in a short time and deliver them to the body or they have a depot action, so that a prolonged, slow feed of active ingredient to the body is achieved.", "In addition to the 11β-halogen steroid, the dosage units can contain one or more pharmaceutically compatible vehicles, for example substances for adjusting the rheology of the pharmaceutical agent, surfactants, solubilizers, microcapsules, microparticles, granulates, diluents, binders, such as starch, sugar, sorbitol and gelatin, also fillers, such as silicic acid and talc, lubricants, dyes, perfumes and other substances.", "In particular, the 11β-halogen steroids according to the invention can also be formulated in the form of a solution that is intended for oral administration and that in addition to the active 11β-halogen steroid contains as the following components: a pharmaceutically compatible oil and/or a pharmaceutically compatible lipophilic surfactant and/or a pharmaceutically compatible hydrophilic surfactant and/or a pharmaceutically compatible water-miscible solvent.", "In this respect, reference is also made to WO-A-97/21440.To achieve better bio-availability of the steroid, the compounds can also be formulated as cyclodextrin clathrates.", "For this purpose, the compounds are reacted with α-, β- or γ-cyclodextrin or derivatives thereof (PCT/EP95/02656).", "If creams, ointments, lotions and liquids that can be applied topically are to be used, the latter must be constituted in such a way that the compounds according to the invention are fed to the body in a sufficient amount.", "In these forms for dispensing, adjuvants are contained, for example substances for adjusting the rheology of pharmaceutical agents, surfactants, preservatives, solubilizers, diluents, substances for increasing the permeability of the steroids according to the invention through the skin, dyes, perfumes and skin protection agents, such as conditioners and moisturizers.", "Together with the steroids according to the invention, other active ingredients can also be contained in the pharmaceutical agent.", "For parenteral administration, the active ingredients can be dissolved or suspended in a physiologically compatible diluent.", "As diluents, very often oils with or without the addition of a solubilizer, a surfactant, a suspending agent or emulsifier are used.", "Examples of oils that are used are olive oil, peanut oil, cottonseed oil, soybean oil, castor oil and sesame oil.", "To formulate an injectable preparation, any liquid vehicle can be used in which the compounds according to the invention are dissolved or emulsified.", "These liquids frequently also contain substances to regulate viscosity, surfactants, preservatives, solubilizers, diluents and other additives, with which the solution is set to isotonic.", "Other active ingredients can also be administered together with the 11β-halogen steroids.", "The 11β-halogen steroids can be administered in the form of a depot injection or an implant preparation, for example subcutaneously, which can be formulated in such a way that a delayed release of active ingredients is made possible.", "To this end, known techniques can be used, for example depots that dissolve or that operate with a membrane.", "Implants can contain as inert materials, for example, biodegradable polymers or synthetic silicones, for example silicone gum.", "The 11β-halogen steroids can also be incorporated in, for example, a patch, for percutaneous administration.", "There are various methods for synthesis of the 11β-halogen steroids according to the invention.", "Below, two of these methods are shown by way of example: Starting from a compound with general formula B, D whereby R11 can represent either a hydroxyl group, a halogen or a nucleophilic leaving group, and R13 has the above-indicated meaning, a compound with general formula B′, E is first formed by 1,6-addition of a metallated alkyl with general formula R7-M or R7-M′X, whereby M is an alkali metal, M′ is an alkaline-earth metal, X is a halogen atom, and R7 has the above-mentioned meaning: and this compound then is reduced either selectively to a compound with general formula I or, if R11 is not halogen, is nucleophilically substituted in 11-position with a halogenating agent and then is reduced selectively to the compound with general formula I: For synthesis, the process starts from a first embodiment of compound A, for example 11β-hydroxy-19-norandrost-4-ene-3,17-dione (J. de Flines et al., Recl.", "Trav.", "Chim.", "Pays-Bas, Vol.", "82, pages 129 ff.", "(1963)): For 11α-hydroxy-19-norandrost-4-ene-3,17-dione, R11=OH and R13=methyl.", "Compound A (with R11=OH) is reacted in a first reaction with a halodehydroxylating reagent, for example hydrofluoric acid, hydrochloric acid, hydrobromic acid or hydroiodic acid, thionyl chloride or thionyl bromide, phosphorus pentachloride, phosphorus oxychloride, N-chlorosuccinimide, triphenylphosphine/carbon tetrachloride, HF/pyridine or diethylaminosulfur trifluoride or preferably nonaflyl fluoride/1,5-diazabicyclo[5.4.0]undecene to form halo-norandrostenedione.", "The 11β-halogen-norandrost-4-ene-3,17-dione is formed according to general formula A′ above (R11=halogen).", "Then, a Δ6-double bond is introduced, for example by the 3-keto-Δ4 system first being converted under proton catalysis into the 3,5-dien-3-ol-ether A″: whereby Hal=F, Cl, Br, I and compound A″ then is converted, for example, by bromination/dehydrobromination, especially with N-bromosuccinimide or 1,3-dibromo-5,5-dimethylhydantoin and pyridine treatment or lithium bromide/lithium carbonate treatment, into the Δ4,6-ketone with general formula B.", "By copper-catalyzed 1,6-addition of a metallated alkyl, for example methylmagnesium bromide or methyllithium, B is then alkylated in 7-position to compound B′: In the next step, the 7α-alkyl-11β-halogen-norandrost-4-ene-3,17-dione B′ that is produced is treated at low temperatures, for example in THF or diethyl ether with sodium borohydride, whereby the 17-ketone is reduced selectively.", "In this case, 7α-alkyl-11β-halogen-17β-hydroxy-norandrost-4-en-3-one I according to the invention is produced: The hydroxy group is then optionally esterified in 17-position with acyl anhydrides or acyl chlorides, for example with acetyl chloride, butanoyl chloride or enantoyl chloride, in the presence of suitable bases, such as, e.g., pyridine.", "In this case, the corresponding esters I′ according to the invention are produced: For the production of the alternative 11β-halogen steroids I″, namely the 7α-alkyl-11β-halogen-17β-hydroxy-norandrost-5(10)-en-3-ones, the Δ4-3-ketone group of compound I is first ketalized, whereby mixtures of the Δ5- and the Δ5(10)-double-bond-isomeric ketals C are produced: These mixtures are carefully deketalized, and the isomers are separated, whereby 7α-alkyl-11β-halogen-17β-hydroxy-norandrost-5(10)-en-3-ones I″ according to the invention are isolated: In an alternative synthesis method, the compounds of formula I, starting from 11α-acetoxy-19-norandrosta-4,6-diene-3,17-dione D (CAS: 228570-21-6): whereby R13=methyl, can be reacted by copper-catalyzed 1,6-addition of a metal alkyl, such as, e.g., methylmagnesium bromide or methyllithium, for example in THF or diethyl ether, at low temperatures to form compounds with general formula E: Then, compounds E are saponified, for example with a methanolic KOH solution, whereby 11β-hydroxy derivatives F are produced: The latter are then reacted with halodehydroxylating reagents, for example hydrofluoric acid, hydrochloric acid, hydrobromic acid or hydroiodic acid, thionyl chloride or thionyl bromide, phosphorus pentachloride, phosphorous oxychloride, N-chlorosuccinimide, triphenylphosphine/carbon tetrachloride, HF/pyridine or diethylaminosulfur trifluoride or preferably nonaflyl chloride/1,5-diazabicyclo[5.4.0]undecene to form 7α-alkyl-11β-halogen-17β-hydroxyandrost-4-ene-3,17-dione, which then is reduced at low temperatures, for example in THF or diethyl ether, selectively with sodium borohydride in 17-position to 7α-alkyl-11β-halogen-17β-hydroxy-androst-4-en-3-one I.", "The invention is explained in more detail with the examples below: EXAMPLE 1 Production of 11β-Fluoro-17β-hydroxy-7α-methylestr-4-en-3-one (I) (Diagram A, FIG.", "1): a) 11β-Fluoro-estr-4-ene-3,17-dione (A′): 4.6 ml of perfluorobutane-1-sulfonic acid fluoride was added in drops at 0° C. to 5.0 g of 11β-hydroxy-estr-4-ene-3,17-dione (A) [J. de Flines et al., Recl.", "Trav.", "Chim.", "Pays-Bas, Vol.", "82, pages 129 ff., (1963)) in 100 ml of toluene and 7.3 ml of 1,8-diazabicyclo[5.4.0]undec-7-ene.", "After 30 minutes, the solution was diluted with ethyl acetate, washed with saturated sodium chloride solution and dried.", "Then, it was concentrated by evaporation in a vacuum.", "After the crude product was chromatographed on silica gel with a hexane-ethyl acetate gradient, 3.8 g of 11β-fluoro-estr-4-ene-3,17-dione (A′) with a melting point of 173-174° C. was obtained.", "b) 11β-Fluoro-3-methoxy-estra-3,5-dien-17-one (A″): 7.8 g of 11β-fluoro-estr-4-ene-3,17-dione (A′) was stirred in 40 ml of 2,2-dimethoxypropane with 780 mg of pyridinium-toluene-4-sulfonate for 5 hours at 80° C. Then, 1.5 ml of triethylamine was added.", "Then, it was diluted with ethyl acetate and washed with saturated sodium chloride solution.", "After crystallization from methanol, 5.3 g of 11β-fluoro-3-methoxy-estra-3,5-dien-17-one (A″) with a melting point of 173° C. was obtained.", "c) 11β-Fluoro-estra-4,6-diene-3,17-dione (B): 5 ml of a 10% by weight sodium acetate solution and, in portions, 2.5 g of 1,3-dibromo-5,5-dimethylhydantoin were added in succession at 0° C. to 5.0 g of 11β-fluoro-3-methoxy-estra-3,5-dien-17-one (A″) in 50 ml of DMF.", "After 30 minutes, 2.3 g of sodium sulfite and then 2.5 g of lithium bromide and 2.0 g of lithium carbonate were added and stirred for 2 hours at 100° C. The reaction mixture was stirred into ice water.", "The precipitated product was suctioned off, dissolved in ethyl acetate, washed with water, dried and concentrated by evaporation in a vacuum.", "After recrystallization from ethyl acetate, 3.6 g of 11β-fluoro-estra-4,6-diene-3,17-dione (B) with a melting point of 198° C. was obtained.", "d) 11β-Fluoro-7α-methylestr-4-ene-3,17-dione (B′): 68.7 mg of copper(I) chloride was added at room temperature to a solution of 2 g of 11β-fluoro-estra-4,6-diene-3,17-dione (B) in 36 ml of tetrahydrofuran.", "The solution was then stirred for 10 minutes, before the reaction mixture was cooled to −15° C. Then, it was mixed with 427 mg of aluminum chloride, stirred for 30 minutes at this temperature, mixed drop by drop with 4.63 ml of methylmagnesium bromide solution and stirred for another 5 hours at −10° C. For working-up, the reaction mixture was mixed at −10° C. with 4N hydrochloric acid, stirred for 1.5 hours at room temperature, added to water, extracted three times with ethyl acetate, dried on sodium sulfate, concentrated by evaporation in a vacuum and chromatographed on silica gel with hexane/ethyl acetate.", "1.0 g of pure 11β-fluoro-7α-methylestr-4-ene-3,17-dione (B′) with a melting point of 101.4° C. was obtained ([α]D20=+135.8° (c=0.525% in chloroform)).", "From a later fraction, 0.17 g of the isomeric 11β-fluoro-7β-methylestr-4-ene-3,17-dione with a melting point of 84.8° C. was obtained ([α]D20=+93.7° (c=0.525% in chloroform)).", "e) 11β-Fluoro-17β-hydroxy-7α-methylestr-4-en-3-one (I): A solution of 500 mg of 11β-fluoro-7α-methylestr-4-ene-3,17-dione (B′) in 10 ml of tetrahydrofuran, 5.8 ml of ethanol and 1.15 ml of water was stirred at −55° C. with 175.6 mg of sodium borohydride for 2.5 hours.", "After another 351.2 mg of sodium borohydride was added, it was stirred for another 1.5 hours at −55° C., then added to ice water, extracted three times with ethyl acetate, washed neutral, dried on sodium sulfate and concentrated by evaporation in a vacuum.", "Then, it was chromatographed on silica gel with hexane/ethyl acetate.", "230 mg of pure 11β-fluoro-17β-hydroxy-7α-methylestr-4-en-3-one (I) with a melting point of 126.7° C. was obtained ([α]D20=+79.9° (c=0.53% in chloroform)).", "The synthesis above can also be modified to introduce higher homologs of the 7α-methyl group and derivatives thereof into the steroid skeleton by the corresponding alkylmagnesium bromide solution being used in synthesis step d) instead of methylmagnesium bromide solution or the corresponding derivatives thereof.", "Thus, for example, 7α-ethyl-11β-fluoro-17β-hydroxy-estr-4-ene-3-one and 11β-fluoro-7α-(fluoromethyl)-17β-hydroxy-estr-4-en-3-one can also be produced.", "EXAMPLE 2 Production of 11β-Fluoro-17β-heptanoyloxy-7α-methylestr-4-en-3-one (I′) (Diagram B, FIG.", "2): A solution of 220 mg of 11β-fluoro-17β-hydroxy-7α-methylestr-4-ene-3-one (I) in 5 ml of tetrahydrofuran and 1.09 ml of pyridine was mixed drop by drop at 0° C. with 1.09 ml of heptanoyl chloride and stirred for 1 hour.", "Then, it was mixed with sodium bicarbonate solution, extracted three times with ethyl acetate, washed neutral, dried on sodium sulfate, concentrated by evaporation in a vacuum and chromatographed on silica gel with hexane/ethyl acetate.", "210 mg of pure 11β-fluoro-17β-heptanoyloxy-7α-methylestr-4-en-3-one (I′) was obtained as a foam ([α]D20=+40.8° (c=0.56% in chloroform)).", "The synthesis above can also be modified to introduce higher homologs of the 17β-heptanoyl group by the corresponding alkanoyl chloride being used instead of heptanoyl chloride.", "Thus, for example, 11β-fluoro-7α-methyl-17β-undecanoyloxy-estr-4-en 3-one can also be produced.", "EXAMPLE 3 Production of 11β-Fluoro-17β-hydroxy-7α-methylestr-5(10)-en-3-one (I″) (Diagram C, FIG.", "3): a) 3-Ethylenedioxy-11β-fluoro-7α-methylestr-5(10)-en-17β-ol (C) 3.63 ml of ethylene glycol and 1.82 ml of trimethyl orthoformate and 23.6 ml of para-toluenesulfonic acid hydrate were added to a solution of 500 mg of 11β-fluoro-17β-hydroxy-7α-methyl-estr-4-en-3-one (I) in 6.35 ml of dichloromethane.", "The reaction mixture was stirred for 20 hours at room temperature.", "Then, it was mixed with sodium carbonate solution, diluted with ethyl acetate, washed neutral, dried on sodium sulfate, concentrated by evaporation in a vacuum and chromatographed on silica gel with hexane/ethyl acetate.", "170 mg of 3-ethylenedioxy-11β-fluoro-7α-methylestr-5(10)-en-17β-ol (C), which contained portions of double-bond-isomeric 3-ethylenedioxy-11β-fluoro-7α-methylestr-5-en-17β-ol (C), was obtained.", "b) 11β-Fluoro-17β-hydroxy-7α-methyl-estr-5(10)-en-3-one (I″): A solution of 170 mg of product (C), obtained under 3a), in 22.5 ml of methanol was stirred with 300 mg of oxalic acid and 3 ml of water for 24 hours at room temperature.", "Then, it was mixed with sodium bicarbonate solution, extracted three times with ethyl acetate, washed neutral, dried on sodium sulfate, concentrated by evaporation in a vacuum and chromatographed on silica gel with hexane/ethyl acetate.", "62.7 mg of pure 11β-fluoro-17β-hydroxy-7α-methyl-estr-5(10)-en-3-one (I″) was obtained.", "Crystallization from hexane yielded 47.6 mg of compound I″ with a melting point of 169.9° C. ([α]D20=+134.4° (c=0.56% chloroform)).", "EXAMPLE 4 Production of 11β-Chloro-17β-hydroxy-7α-methyl-estr-4-en-3-one (I) (Diagram D, FIG.", "4): a) 11α-Acetoxy-7α-methyl-estr-4-ene-3,17-dione (E): A solution of 20 g of 11α-acetoxy-estra-4,6-diene-3,17-dione (D) in 500 ml of tetrahydrofuran, 20 ml of 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone and 20 ml of trimethylchlorosilane were added in drops within 1 hour at −30° C. in a prepared solution of 20 g of copper(I) iodide in 200 ml of tetrahydrofuran and 70 ml of methylmagnesium bromide.", "It was stirred for 1 hour at −20° C. For working-up, the reaction mixture was then mixed at −20° C. with 20 ml of glacial acetic acid, stirred for 1 hour at room temperature, added to water, extracted three times with ethyl acetate, dried on sodium sulfate, concentrated by evaporation in a vacuum and chromatographed on silica gel with hexane/ethyl acetate.", "12.8 g of pure 11α-acetoxy-7α-methylestr-4-ene-3,17-dione (E) with a melting point of 182.7° C. was obtained ([α]D20=+24.70 (c=0.525% in chloroform)).", "b) 11α-Hydroxy-7α-methyl-estr-4-ene-3,17-dione (F): A solution of 1 g of 11α-acetoxy-7α-methyl-estr-4-ene-3,17-dione (E) in 20 ml of a 0.2 molar methanolic potassium hydroxide solution was stirred at room temperature for 3 hours.", "Then, it was added to water, extracted three times with ethyl acetate, washed neutral, dried on sodium sulfate, concentrated by evaporation in a vacuum and recrystallized from diethyl ether/acetone.", "878 mg of pure 11α-hydroxy-7α-methyl-estr-4-ene-3,17-dione (F) with a melting point of 202.3° C. was obtained ([α]D20=+44.80 (c=0.5% in chloroform)).", "c) 11β-Chloro-7α-methyl-estr-4-ene-3,17-dione (F′); A solution of 800 mg of 11α-hydroxy-7α-methylestr-4-ene-3,17-dione (F) in 8 ml of dichloromethane was mixed at 0° C. with 1.21 ml of hexachloroacetone and 764 mg of triphenylphosphine and stirred at room temperature for 4 hours.", "Then, it was diluted with dichloromethane, washed neutral, dried on sodium sulfate, concentrated by evaporation in a vacuum and chromatographed on silica gel with hexane/ethyl acetate.", "430 mg of pure 11β-chloro-7α-methyl-estr-4-ene-3,17-dione (F′) with a melting point of 215.1° C. was obtained ([α]D20=+182.7° (c=0.5% in chloroform)).", "d) 11β-Chloro-17β-hydroxy-7α-methyl-estr-4-en-3-one (I): A solution of 380 mg of 11β-chloro-7α-methyl-estr-4-ene-3,17-dione (F′) in 7.6 ml of tetrahydrofuran, 4.4 ml of ethanol and 1.15 ml of water was mixed at −55° C. in portions with 596 mg of sodium borohydride and stirred for 7 hours at −55° C. Then, the mixture was added to ice water, extracted three times with ethyl acetate, washed neutral, dried on sodium sulfate, concentrated by evaporation in a vacuum and chromatographed on silica gel with hexane/ethyl acetate.", "130 mg of pure 11β-chloro-17β-hydroxy-7α-methyl-estr-4-en-3-one (I) was obtained ([α]D20=+136.9° (in chloroform), FD 183.2° C.).", "The synthesis above can also be modified to produce the 11β-bromine and 11β-iodine derivatives, by a brominating or iodizing agent being used.", "Thus, for example, 11β-bromo-17β-hydroxy-7α-methyl-estr-4-en-3-one and 17β-hydroxy-11β-iodo-7α-methyl-estr-4-en-3-one can also be produced.", "In analogy to Example 17α-Ethyl-11β-fluoro-17β-hydroxy-estr-4-en-3-one, using ethylmagnesium bromide instead of methylmagnesium bromide, is obtained; Fp.", "115,1° C., [α]D20=+62,1°(in chloroform).", "The intermediate 7α-Ethyl-11β-fluoro-estr-4-en-3,17-dione shows Fp.=174,9° C. and [α]D20=+107,1° (chloroform).", "Further compounds according to the invention are accesible by performing procedures in analogy to the mentioned prior art." ] ]	Patent_10467352
[ [ "Normalizing circuit with reduced error voltage", "A circuit for calibrating a voltage signal.", "The circuit includes a first voltage-to-current converter receiving the signal to be calibrated and a reference voltage, and providing a current proportional to the voltage difference between the voltage of the signal to be calibrated and the reference voltage, a first current amplifier driven by the first voltage-to-current converter, a second voltage-to-current converter receiving an adjustment voltage and the reference voltage and providing a current proportional to the voltage difference between the adjustment voltage and the reference voltage, a second current amplifier driven by the second voltage-to-current converter, and a means for providing a calibrated voltage signal based on the difference between the currents provided by the first and second current amplifiers." ], [ "1.A circuit for calibrating a voltage signal, including: a first voltage-to-current converter receiving a voltage signal to be calibrated and a reference voltage, and providing a current proportional to the voltage difference between the voltage of the signal to be calibrated and the reference voltage, a first current amplifier driven by the first voltage-to-current converter, a second voltage-to-current converter receiving an adjustment voltage and the reference voltage and providing a current proportional to the voltage difference between the adjustment voltage and the reference voltage, a second current amplifier driven by the second voltage-to-current converter, and means for providing a calibrated voltage signal based on the difference between the currents provided by the first and second current amplifiers.", "2.The circuit of claim 1, wherein said means for providing a calibrated voltage signal includes: a current conversion means driven by the second current amplifier and providing a current of same amplitude and of sign opposite to that of the current that it receives, and a current-to-voltage converter driven by the first current amplifier and by the current conversion means and providing the calibrated voltage signal.", "3.The circuit of claim 1, wherein the first and second current amplifiers, as well as the first and second voltage-to-current converters, have an identical structure.", "4.The circuit of claim 3, wherein the first and second current amplifiers have a variable gain and their gain is identically controlled.", "5.The circuit of claim 1, wherein each of the first and second current amplifiers includes several amplifier units.", "6.The circuit of claim 5, wherein each of the first and second current amplifiers includes a first unit and a second unit, the second unit ensuring a current gain equal to 1 or to 9.7.The circuit of claim 6, wherein the first unit includes: an input node coupled to ground via a diode-connected transistor, 16 identical branches, each formed of a transistor, and 31 identical branches, each including a transistor in series with a switch, each of the transistors coupled to the input node being flown, when on, by a same current, and an output node coupled to ground via 16 identical branches, each formed of a transistor, and 31 identical branches, each including a transistor in series with a switch, each of the transistors coupled with the output node being flown, when on, by a current of same value as any each transistor coupled with the input node, in the on state.", "8.The circuit of claim 1, wherein the adjustment voltage is adjustable and is used to set the minimum value of the calibrated signal.", "9.The circuit of claim 1, including a first current source arranged between the output of the first voltage-to-current converter and a fixed voltage, and a second current source arranged between the output of the second voltage-to-current converter and said fixed voltage.", "10.", "(canceled) 11.A circuit for calibrating a voltage signal, including: a first voltage-to-current converter receiving a voltage signal to be calibrated and a reference voltage, and providing a current proportional to the voltage difference between the voltage of the signal to be calibrated and the reference voltage, a first current amplifier driven by the first voltage-to-current converter, a second voltage-to-current converter receiving an adjustment voltage and the reference voltage and providing a current proportional to the voltage difference between the adjustment voltage and the reference voltage, a second current amplifier driven by the second voltage-to-current converter, and means for providing a calibrated voltage signal based on the difference between the currents provided by the first and second current amplifiers, each of the first and second current amplifiers includes a first unit and a second unit, the second unit configured to ensure a current gain equal to a range of 1-9, the first unit including: an input node coupled to ground via a diode-connected transistor, 16 identical branches, each formed of a transistor, and 31 identical branches, each including a transistor in series with a switch, each of the transistors coupled to the input node being flown, when on, by a same current, and an output node coupled to ground via 16 identical branches, each formed of a transistor, and 31 identical branches, each including a transistor in series with a switch, each of the transistors coupled with the output node being flown, when on, by a current of same value as any each transistor coupled with the input node, in the on state.", "12.The circuit of claim 11, further comprising a first current source arranged between the output of the first voltage-to-current converter and a fixed 13.A circuit for calibrating a voltage signal, including: a first voltage-to-current converter receiving a voltage signal to be calibrated and a reference voltage, and providing a current proportional to the voltage difference between the voltage of the signal to be calibrated and the reference voltage, a first current amplifier driven by the first voltage-to-current converter, a second voltage-to-current converter receiving an adjustment voltage and the reference voltage and providing a current proportional to the voltage difference between the adjustment voltage and the reference voltage, a second current amplifier driven by the second voltage-to-current converter, and means for providing a calibrated voltage signal based on the difference between the currents provided by the first and second current amplifiers; and a first current source arranged between the output of the first voltage-to-current converter and a fixed voltage, and a second current source arranged between the output of the second voltage-to-current converter and the fixed voltage.", "14.The circuit of claim 13, wherein each of the first and second current amplifiers includes a first unit and second unit, the first unit including: an input node coupled to ground via a diode-connected transistor, 16 identical branches, each formed of a transistor, and 31 identical branches, each including a transistor in series with a switch, each of the transistors coupled to the input node being flown, when on, by a same current, and an output node coupled to ground via 16 identical branches, each formed of a transistor, and 31 identical branches, each including a transistor in series with a switch, each of the transistors coupled with the output node being flown, when on, by a current of same value as any, each transistor coupled with the input node, in the on state.", "15.A calibration circuit, comprising: first and second current amplifiers receiving as input first and second proportional currents from first and second voltage-to-current converters, respectively; a current converter driven by the second current amplifier and configured to provide a current of the same amplitude and of a sign that is opposite to that of the current that it receives; and a current-to-voltage converter driven by the first current amplifier and by the current conversion circuit and configured to provide on an output thereof a calibrated voltage signal.", "16.A calibration circuit, comprising: first and second current amplifiers receiving as input first and second proportional currents from first and second voltage-to-current converters, respectively; a current converter driven by the second current amplifier and configured to provide a current of the same amplitude and of a sign that is opposite to that of the current that it receives; and a current-to-voltage converter driven by the first current amplifier and by the current conversion circuit and configured to provide on an output thereof a calibrated voltage signal; and a first current source arranged between the output of the first voltage-to-current converter and a fixed voltage, and a second current source arranged between the output of the second voltage-to-current converter and a fixed voltage.", "17.The circuit of claim 16, wherein each of the first and second current amplifiers includes a first unit and a second unit, the first unit comprising: an input node coupled to ground via a diode-connected transistor, 16 identical branches that are each formed of a transistor, and 31 identical branches that are each comprising a transistor in series with a switch, each of the transistors coupled to an input node supplied by a same current, and an output node coupled to ground via 16 identical branches that are each formed of a transistor, and 31 identical branches that are each comprising a transistor in series with a switch, each of the transistors coupled with the output node being supplied by a current of same value as any of each of the transistors coupled with the input node when in an on state." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to a circuit for calibrating a signal, that is, for providing, based on an input signal, a signal included in a predetermined range.", "2.Description of the Related Art There are various applications for calibration circuits.", "For example, calibration circuits are used in optical disk read systems.", "In such systems, a calibration circuit is arranged between a photodetector and an analog-to-digital converter to adapt the signal from the photodetector to the analog-to-digital converter input range.", "The integrated circuit “L6300” of STMicroelectronics is an example of a calibration circuit used in such a system.", "FIG.", "1 illustrates the shape, along time, of a voltage V at the input of a calibration circuit.", "Voltage V, shown as a sinusoid, oscillates between a minimum value Vmin and a maximum value Vmax.", "In the mentioned application, voltage V coming from a photodetector oscillates above a reference level Vmin approximately equal to 2.5 volts.", "Peak-to-peak amplitude Vmax-Vmin of voltage V can vary within a large range from 25 to 500 millivolts, according to whether the photodetector receives all or part of the light emitted by the laser.", "FIG.", "2 illustrates the shape, along time, of voltage VOUT at the output of the calibration circuit.", "Voltage VOUT has the same frequency as voltage V, but its amplitude is constant and the signal oscillates between a minimum value Vbot and a maximum value Vtop corresponding to desired limiting values.", "Typical values of Vbot and Vtop, in the application mentioned hereabove, are respectively around 125 and 875 millivolts.", "Generally, the forming of a calibration circuit must take into account error or offset voltages likely to affect the limiting values of output voltage VOUT.", "For example, in the mentioned application, an error voltage which can reach ±70 millivolts systematically affects the reference level Vmin.", "Further, each element of the calibration circuit introduces an error voltage specific to it.", "This effect is particularly substantial if the circuit includes MOS transistors, since these transistors generate greater error voltages than bipolar transistors.", "Because of this, many calibration circuits, like circuit “L6300” mentioned hereabove, are formed by means of bipolar transistors.", "Further, since the signal processing circuits that follow the calibration circuit are generally formed by means of CMOS transistors, the calibration circuit can hardly be formed together with the circuits that follow it on a same silicon wafer to form an integrated circuit.", "This results in high manufacturing, testing, and interconnection costs.", "Further, known calibration circuits are powered by relatively high supply voltages, greater than those supplying CMOS circuits." ], [ "<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>The disclosed embodiments of the present invention provide a calibration circuit in which the effect of error voltages is decreased.", "The calibration circuit of the present invention can be easily be made on a same integrated circuit as a digital signal processing circuit, and it can be powered under a reduced voltage.", "The present invention provides a circuit for calibrating a voltage signal that includes: a first voltage-to-current converter receiving a voltage signal to be calibrated and a reference voltage, and providing a current proportional to the voltage difference between the voltage of the signal to be calibrated and the reference voltage, a first current amplifier driven by the first voltage-to-current converter, a second voltage-to-current converter receiving an adjustment voltage and the reference voltage and providing a current proportional to the voltage difference between the adjustment voltage and the reference voltage, a second current amplifier driven by the second voltage-to-current converter, and a means for providing a calibrated voltage signal based on the difference between the currents provided by the first and second current amplifiers.", "According to an embodiment of the present invention, the means for providing the calibrated voltage signal includes: a current conversion means driven by the second current amplifier and providing a current of same amplitude and of a direction opposite to that of the current that it receives, and a current-to-voltage converter driven by the first current amplifier and by the current conversion means and providing the calibrated voltage signal.", "According to an embodiment of the present invention, the first and second current amplifiers, as well as the first and second voltage-to-current converters, have an identical structure.", "According to an embodiment of the present invention, the first and second current amplifiers have a variable gain and their gain is identically controlled.", "According to an embodiment of the present invention, each of the first and second current amplifiers includes several amplifier units.", "According to an embodiment of the present invention, each of the first and second current amplifiers includes a first unit and a second unit, the second unit ensuring a current gain equal to 1 or to 9.According to an embodiment of the present invention, the first unit includes: an input node coupled to ground via a diode-connected transistor, 16 identical branches, each formed of a transistor, and 31 identical branches, each including a transistor in series with a switch, each of the transistors coupled to the input node being run through, when on, by a same current, and an output node coupled to ground via 16 identical branches, each formed of a transistor, and 31 identical branches, each including a transistor in series with a switch, each of the transistors coupled with the output node being run through, when on, by a current of same value as any one, in the on state, of the transistors coupled with the input node.", "According to an embodiment of the present invention, the adjustment voltage is adjustable and is used to set the minimum value of the calibrated signal.", "According to an embodiment of the present invention, the circuit includes a first current source arranged between the output of the first voltage-to-current converter and a fixed voltage, and a second current source arranged between the output of the second voltage-to-current converter and said fixed voltage.", "The present invention also provides a method for calibrating a signal by means of a circuit including a first branch receiving the signal to be calibrated and a second branch receiving an adjustment voltage, the first branch including a first variable-gain current amplifier and the second branch including a second variable-gain current amplifier, the first and second current amplifiers being of identical structure and their gain being adjusted in the same way.", "The method includes the adjustment steps of: a) the gain of the first and second current amplifiers being set to its maximum value, injecting into the circuit a voltage equal to the minimum value of the voltage to be calibrated and adjusting the adjustment voltage so that the minimum level of the signal at the circuit output correspond to the desired minimum level, then b) injecting the signal to be calibrated and adjusting the gain of the first and second current amplifiers so that the maximum level of the signal at the circuit output corresponds to the desired maximum level." ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to a circuit for calibrating a signal, that is, for providing, based on an input signal, a signal included in a predetermined range.", "2.Description of the Related Art There are various applications for calibration circuits.", "For example, calibration circuits are used in optical disk read systems.", "In such systems, a calibration circuit is arranged between a photodetector and an analog-to-digital converter to adapt the signal from the photodetector to the analog-to-digital converter input range.", "The integrated circuit “L6300” of STMicroelectronics is an example of a calibration circuit used in such a system.", "FIG.", "1 illustrates the shape, along time, of a voltage V at the input of a calibration circuit.", "Voltage V, shown as a sinusoid, oscillates between a minimum value Vmin and a maximum value Vmax.", "In the mentioned application, voltage V coming from a photodetector oscillates above a reference level Vmin approximately equal to 2.5 volts.", "Peak-to-peak amplitude Vmax-Vmin of voltage V can vary within a large range from 25 to 500 millivolts, according to whether the photodetector receives all or part of the light emitted by the laser.", "FIG.", "2 illustrates the shape, along time, of voltage VOUT at the output of the calibration circuit.", "Voltage VOUT has the same frequency as voltage V, but its amplitude is constant and the signal oscillates between a minimum value Vbot and a maximum value Vtop corresponding to desired limiting values.", "Typical values of Vbot and Vtop, in the application mentioned hereabove, are respectively around 125 and 875 millivolts.", "Generally, the forming of a calibration circuit must take into account error or offset voltages likely to affect the limiting values of output voltage VOUT.", "For example, in the mentioned application, an error voltage which can reach ±70 millivolts systematically affects the reference level Vmin.", "Further, each element of the calibration circuit introduces an error voltage specific to it.", "This effect is particularly substantial if the circuit includes MOS transistors, since these transistors generate greater error voltages than bipolar transistors.", "Because of this, many calibration circuits, like circuit “L6300” mentioned hereabove, are formed by means of bipolar transistors.", "Further, since the signal processing circuits that follow the calibration circuit are generally formed by means of CMOS transistors, the calibration circuit can hardly be formed together with the circuits that follow it on a same silicon wafer to form an integrated circuit.", "This results in high manufacturing, testing, and interconnection costs.", "Further, known calibration circuits are powered by relatively high supply voltages, greater than those supplying CMOS circuits.", "BRIEF SUMMARY OF THE INVENTION The disclosed embodiments of the present invention provide a calibration circuit in which the effect of error voltages is decreased.", "The calibration circuit of the present invention can be easily be made on a same integrated circuit as a digital signal processing circuit, and it can be powered under a reduced voltage.", "The present invention provides a circuit for calibrating a voltage signal that includes: a first voltage-to-current converter receiving a voltage signal to be calibrated and a reference voltage, and providing a current proportional to the voltage difference between the voltage of the signal to be calibrated and the reference voltage, a first current amplifier driven by the first voltage-to-current converter, a second voltage-to-current converter receiving an adjustment voltage and the reference voltage and providing a current proportional to the voltage difference between the adjustment voltage and the reference voltage, a second current amplifier driven by the second voltage-to-current converter, and a means for providing a calibrated voltage signal based on the difference between the currents provided by the first and second current amplifiers.", "According to an embodiment of the present invention, the means for providing the calibrated voltage signal includes: a current conversion means driven by the second current amplifier and providing a current of same amplitude and of a direction opposite to that of the current that it receives, and a current-to-voltage converter driven by the first current amplifier and by the current conversion means and providing the calibrated voltage signal.", "According to an embodiment of the present invention, the first and second current amplifiers, as well as the first and second voltage-to-current converters, have an identical structure.", "According to an embodiment of the present invention, the first and second current amplifiers have a variable gain and their gain is identically controlled.", "According to an embodiment of the present invention, each of the first and second current amplifiers includes several amplifier units.", "According to an embodiment of the present invention, each of the first and second current amplifiers includes a first unit and a second unit, the second unit ensuring a current gain equal to 1 or to 9.According to an embodiment of the present invention, the first unit includes: an input node coupled to ground via a diode-connected transistor, 16 identical branches, each formed of a transistor, and 31 identical branches, each including a transistor in series with a switch, each of the transistors coupled to the input node being run through, when on, by a same current, and an output node coupled to ground via 16 identical branches, each formed of a transistor, and 31 identical branches, each including a transistor in series with a switch, each of the transistors coupled with the output node being run through, when on, by a current of same value as any one, in the on state, of the transistors coupled with the input node.", "According to an embodiment of the present invention, the adjustment voltage is adjustable and is used to set the minimum value of the calibrated signal.", "According to an embodiment of the present invention, the circuit includes a first current source arranged between the output of the first voltage-to-current converter and a fixed voltage, and a second current source arranged between the output of the second voltage-to-current converter and said fixed voltage.", "The present invention also provides a method for calibrating a signal by means of a circuit including a first branch receiving the signal to be calibrated and a second branch receiving an adjustment voltage, the first branch including a first variable-gain current amplifier and the second branch including a second variable-gain current amplifier, the first and second current amplifiers being of identical structure and their gain being adjusted in the same way.", "The method includes the adjustment steps of: a) the gain of the first and second current amplifiers being set to its maximum value, injecting into the circuit a voltage equal to the minimum value of the voltage to be calibrated and adjusting the adjustment voltage so that the minimum level of the signal at the circuit output correspond to the desired minimum level, then b) injecting the signal to be calibrated and adjusting the gain of the first and second current amplifiers so that the maximum level of the signal at the circuit output corresponds to the desired maximum level.", "BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS The foregoing features and advantages of the present invention will be discussed in detail in the following non-limiting description of specific embodiments in connection with the accompanying drawings, wherein.", "FIG.", "1 illustrates the shape of an input voltage of a calibration circuit; FIG.", "2 illustrates the shape of the output voltage of a calibration circuit; FIG.", "3 shows, in the form of blocks, a calibration circuit according to the present invention; FIG.", "4 shows an embodiment of an element of the circuit of FIG.", "3; FIG.", "5 shows the current-vs.-voltage graph of an element of the circuit of FIG.", "3; FIG.", "6 shows the current-vs.-voltage graph of an element of the circuit of FIG.", "3; FIGS.", "7, 8, and 9 show examples of forming of elements of the circuit of FIG.", "3.DETAILED DESCRIPTION OF THE INVENTION In FIG.", "3, a voltage V to be calibrated drives a voltage-to-current converter 1.Voltage-to-current converter 1 also receives a reference voltage V0, and it provides a current I1 which varies proportionally to difference V−V0.As will be seen hereafter, the general expression of current I1 is I1=Im+k1.", "(V−V0), Im and k1 being constants.", "A current source 3 is connected at the output of voltage-to-current converter 1 and pulls a constant current I0 to the circuit ground.", "The output of voltage-to-current converter 1 is connected to a current amplifier 5.Current amplifier 5 is a variable-gain amplifier and its gain G is controlled by a control terminal, also designated as G. Current amplifier 5 provides a current I2 equal to G×(I1−I0).", "Elements 1, 3, and 5 form a first branch of the calibration circuit.", "The calibration circuit includes a second branch having the same structure as the first one.", "Thus, the second branch includes a voltage-to-current converter 1′, a current source 3′, and a current amplifier 5′.", "Voltage-to-current converter 1′ receives an adjustment voltage Vreg and reference voltage V0.Voltage-to-current converter 1′ provides a current I′1 expressed as I′1=I′m+k′1.", "(Vreg−V0), I′m and k′1 being constants.", "Part of current I′1 is pulled to ground by current source 3′, run through by a current I′0.Voltage-to-current converter 1′ drives current amplifier 5′, which provides a current I′2.Current amplifier 5′ is a variable-gain current amplifier.", "Its gain G′ is controlled by a control terminal connected, in FIG.", "3, to control terminal G of amplifier 5.Current I′2 is equal to G′×(I′1−I′0).", "The output of current amplifier 5′ drives a current conversion unit 7.Current conversion unit 7 outputs a current I′2 of same amplitude as the current I′2 that it receives, but of opposite direction.", "Unit 7 may be formed by means of a current mirror, including for example two MOS transistors T1 and T2 connected in parallel, one of which is diode-mounted, as illustrated in FIG.", "4.The output of current amplifier 5 and the output of unit 7 are connected together to the input of a current-to-voltage converter 9.Current-to-voltage converter 9 receives a current i equal to the difference between currents I2−I′2.Current-to-voltage converter 9 provides a voltage VOUT corresponding to input voltage V, but the limiting values Vbot and Vtop of which correspond to the desired values.", "Current sources 3 and 3′ are optional.", "They are used to pull to ground part of the D.C. component of currents I1 and I′1.This enables current amplifier 5 to act on lower currents.", "Amplifier 5 thus is easier to form.", "It can be formed by means of smaller components and consumes less.", "Advantageously, elements 1′, 3′, 5′ of the second branch of the circuit have a structure identical to that of elements 1, 3, 5, respectively, of the first circuit branch.", "This-enables easy forming of the calibration circuit and compensation for various drifts, as will be seen hereafter.", "FIG.", "5 illustrates the current-vs.-voltage characteristics of voltage-to-current converter 1.Current I1 is on the on the Y-axis and voltage V is on the X-axis.", "Preferably, voltage V0 is selected to be as close as possible to minimum value Vmin of the voltage V to be calibrated.", "Lower value Vmin of voltage V thus is equal to V0+ΔV, ΔV being a positive or negative error voltage.", "As mentioned, current I1 is provided by expression I1=Im+k1 (V−V0).", "When the input voltage is equal to V0, output current I1 is equal to current Im.", "Current Im is chosen to obtain good noise performances of the calibration circuit.", "In an example of practical implementation, current Im has been chosen to be equal to 500 μA.", "FIG.", "6 illustrates the voltage-vs.-current characteristic of current-to-voltage converter 9.This characteristic is such that, when current i entering converter 9 is equal to 0, output voltage VOUT is equal to Vbot.", "When current i is maximum (imax), output voltage VOUT is equal to Vtop.", "VOUT=Vbot+k2.i, k2 being a coefficient specific to converter 9.The operation of the calibration circuit of FIG.", "3 will now be explained.", "Voltage V corresponds to the graph of FIG.", "1, and voltage VOUT provided by the calibration circuit must range between levels Vbot and Vtop of FIG.", "2.The adjustment of the limiting values of the voltage to be provided is performed in two steps.", "First, the minimum level of voltage VOUT is adjusted.", "For this purpose, a voltage equal to Vmin is applied to the circuit input.", "Since voltage V0 is substantially equal to voltage Vmin, voltage V corresponds to V0 plus a positive or negative offset voltage, ΔV, which is small as compared to V0.Gain G is set to its maximum.", "The value of voltage VOUT at the output of the calibration circuit is then observed, and adjustment voltage Vreg is adjusted to obtain Vbot as an output.", "The adjustment of Vreg may be performed in any appropriate manner.", "For example, the adjustment of Vreg will be automatically done by means of a unit coupled to output VOUT.", "Assuming that G=Gmax and that elements 1′, 3′, and 5′ are respectively identical to elements 1, 3, and 5 (Im=I′m, I0=I′0, k1=k′1, k2=k′2): I1=Im+k1.ΔV I2=Gmax.(I1−I0)=Gmax.", "(Im−I0+k1.ΔV)+Ioffset, Ioffset being an error current caused by the elements of the first circuit branch.", "One also has: I ′ ⁢ 1 = ⁢ I ′ ⁢ m + k ′ ⁢ 1.⁢ ( Vreg - V 0 ) I ′ ⁢ 2 = ⁢ Gmax .", "( I ′ ⁢ 1 - I ′ ⁢ 0 ) = ⁢ Gmax .", "( I ′ ⁢ m - I ′ ⁢ 0 + k ⁢ ⁢ 1.⁡ [ Vreg - V 0 ] ) + I ′ ⁢ offset , I′offset being an error current due to the elements of the second circuit branch.", "The expression of VOUT is: VOUT=Vbot+k2.i=Vbot+k2.", "(I2−I2′) that is, with Im=I′m, I0=I′0, k1=k′1, and k2=k′2: VOUT=Vbot+k1.k2.Gmax[ΔV−(Vreg−V0)]+k2(Ioffset−I′offset).", "Since Vreg is adjusted so that VOUT is equal to Vbot, one has: k1.k2.Gmax.", "(ΔV+V0−Vreg)+k2(Ioffset−I′offset)=0, that is, Vreg = V 0 + Δ ⁢ ⁢ V + ( I offset - I offset ′ ) K 1 ⁢ G max .", "When the adjustment of voltage Vreg is performed, it is not modified until a new voltage V is to be calibrated.", "The maximum level of output voltage VOUT is then adjusted.", "For this purpose, voltage Vmax is applied to the input of the calibration circuit.", "By acting upon control terminal G, the gain of amplifiers 5 and 5′ is adjusted so that the maximum value of signal VOUT is equal to Vtop.", "Gain G will be adjusted by any appropriate means.", "For example, an adequate digital unit coupled to the circuit output will drive terminal G and will automatically adjusted the gain.", "After the adjustment of gain G, the adjustment of the limits of VOUT is ended and the adjustments will no longer be modified until a new voltage exhibiting values different from Vmin and/or Vmax is desired to be calibrated.", "In normal operation, one has: I1=Im+k1 (V−V0) I2=G.(I1−I0)=G.(Im−I0+k1.V−k1.V0)+Ioffset.", "One also has: I′1=I′m+k′1 (Vreg−V0) I′2=G.(I′1−I′0)=G.(I′m−I′0+k′1.[Vreg−V0])+I′offset.", "Further, VOUT=Vbot+k2.(I2−I′2).", "Since Im=I′m, k1=k′1, and I0=I′0, one has: VOUT=Vbot+k1.k2.G(V−Vreg)+k2(Ioffset−I′offset).", "Since Vreg = V 0 + Δ ⁢ ⁢ V + ( I offset - I offset ′ ) K 1 ⁢ G max , one has: VOUT=Vbot+k1.k2.G(V−V0−ΔV−[Ioffset−I′offset]/k1.Gmax)+k2(Ioffset−I′offset).", "(V−V0−ΔV) represents the component of voltage V above voltage Vmin.", "Call it Ve.", "One thus has: VOUT=Vbot+k1.k2.G.Ve+k2(Ioffset−I′offset)(1−G/Gmax).", "If there was no error voltage in output voltage VOUT, VOUT would only be equal to the sum of the first two terms, that is, Vbot+k1.k2.G.Ve.", "The expression of VOUT clearly indicates that the structure of the calibration circuit according to the present invention is such that the influence of the error voltages (currents) introduced by the structure is very limited.", "Indeed, in the expression of the output voltage, the error currents are multiplied by a factor 1−G/Gmax, this factor ranging between 0 and 1, whatever gain G. This is particularly important in a technology using MOS transistors, since this type of transistor generates significant error voltages.", "It should be noted that the adjustment of the limiting values of the voltage to be calibrated is particularly simple, even in the case where the circuit is made with MOS transistors.", "Further, since the two branches of the circuit are identical, if one of currents I1, I2 varies for any reason, for example after a temperature or supply voltage variation, the other one of currents I1, I2 undergoes the same variation and this variation does not affect current i since currents I1 and I2 are subtracted.", "It should be noted that, since the input voltage is converted into current and the amplification is a current amplification, the calibration circuit of the present invention does not clip, even under a low supply voltage.", "For example, if the maximum value of V is on the order of 3 volts, the calibration circuit of the present invention can be supplied with a supply voltage as low as 3.3 volts.", "As a comparison, circuit “L6300” mentioned hereabove is supplied with 5 volts.", "The calibration circuit of the present invention can thus be formed with small transistors, it occupies a reduced surface area and consumes little.", "Further, the calibration circuit of the present invention can easily be formed in CMOS technology, and thus easily be formed on a same integrated circuit together with other circuit using MOS transistors.", "This results in gains in manufacturing time, testing, interconnection, etc.", "whereby a generally reduced cost for the circuit and a more extensive integration are obtained.", "An integrated circuit including the calibration circuit according to the present invention and downstream digital circuits may further be supplied with various supply voltages, for example, 3.3 volts for the signal calibration portion, and 1.8 volts for the digital portion.", "The various elements of the circuit of FIG.", "3 will easily be formed by those skilled in the art.", "As a non-limiting example, FIGS.", "7, 8, and 9 respectively show examples of forming of elements 1, 9, and 5 of FIG.", "3.In FIG.", "7, voltage-to-current converter 1 includes two MOS transistors T3 and T4 forming a differential pair.", "The respective gates of transistors T3 and T4 are driven by voltages V and V0.The sources of these transistors are connected together via resistors R and their junction point is coupled to ground via a current source run through by current Im.", "The drain of transistor T3 is connected to a positive supply voltage source VDD and the drain of transistor T4 is connected to a diode-mounted transistor T5.Transistor T5 forms, with a transistor T6, a current mirror.", "Current I1 at the output of voltage-to-current converter 1 is provided by the source of transistor T6.FIG.", "7 further illustrates current source I0 connecting the output of converter 1 to ground.", "The example of FIG.", "7 is an example only and its operation will not be explained any further.", "FIG.", "8 illustrates a non-limiting example of forming of current-to-voltage converter 9.An operational amplifier 11 receives current i on its inverting input (−) and a voltage equal to Vtop on its non-inverting input (+).", "The inverting input of amplifier 11 is coupled to the output of amplifier 11 via a resistor R1.Voltage Vi present between the output of amplifier 11 and the ground is equal to Vtop−R1.i.", "Current-to-voltage converter 9 includes a second stage driven by voltage Vi.", "This second stage includes an operational amplifier 12 having its inverting input (−) connected to the output of amplifier 11 via a resistor R2.The non-inverting input (+) of amplifier 12 receives a voltage Vmid equal to (Vtop+Vbot)/2.The inverting input of amplifier 12 is coupled to the output of amplifier 12 via a resistor R′2 equal to R2.Output voltage VOUT is equal to Vmid−R2.i2, i2 being the current flowing through resistor R′2.Since Vi is equal to R2.i2+Vmid, VOUT=2Vmid−Vi, that is, VOUT=Vbot+R1.i, which corresponds to the characteristic of FIG.", "7, with a coefficient k2 equal to R1.FIG.", "9 shows an embodiment of current amplifier 5 enabling easy variation of the amplifier gain in large proportions.", "In FIG.", "9, amplifier 5 is formed of two units 15 and 16.Unit 15 includes an input node N receiving current I to be amplified (equal to I1−I0).", "A diode-connected MOS transistor, T, couples node N to ground and is run through by a current Iref.", "16 branches Ai, i ranging from 1 to 16, connect node N to ground.", "Each branch Ai is formed of a MOS transistor TAi.", "The gate of each transistor TAi is connected to the gate of transistor T. Each transistor TAi forms a current mirror with transistor T and is run through by a current equal to Iref.", "Node N is also grounded by thirty-one identical branches Bj, j ranging from 1 to 31.Each branch Bj includes a transistor TBj in series with a switch SBj.", "The gate of each transistor TBj is connected to the gate of transistor T and, when switch SBj is on, the corresponding transistor TBj is run through by a current equal to Iref.", "Switches SBj are controllable independently from one another and, if a number n of switches SBj is on, current Iref is equal to I/(17+n).", "When all switches SBj are off, current Iref is equal to I/17; and, when all switches SBj are on, current Iref is equal to I/48.A node P forms the output of unit 15.Sixteen branches Ci connected in parallel connect node P to ground.", "Each branch Ci is formed of a MOS transistor TCi.", "Each transistor TCi has its gate connected to the gate of transistor T and is run through by a current equal to Iref.", "Node P is also coupled to ground by thirty-one identical branches Dj connected in parallel.", "Each branch Dj includes a MOS transistor TDj in series with a switch SDj.", "The gate of each transistor TDj is connected to the gate of transistor T. When a switch SDj is on, the corresponding transistor TDj is run through by a current equal to Iref.", "Node P is run through by a current I15 equal to Iref×(16+m), m being the number of on switches SDj.", "Switches SBj and SDj are controlled so that if a switch SBj is off, the corresponding switch SDj is on, and vice versa.", "Accordingly, the number m of on switches SDj is equal to (31−n), n being the number of on switches SBj.", "Gain G1 of unit 15 can vary between ⅓ and 47/17.Indeed, if all switches SBj are on, current Iref is equal to 1/48.All switches SDj are then off and current I15 is equal to Iref×16.G1 is then equal to I15/I, that is 16/48 (⅓).", "If all switches SBj are off, current Iref is equal to I/17.All switches SDj are on and current I15 is equal to Iref×47.G1 is then equal to I15/I, that is, 47/17 (approximately 2.7).", "Unit 16 has its input connected to node P. Unit 16 includes three MOS transistors T8, T9, and T10.Transistor T8 is diode-connected.", "It has its source connected to node P and its drain connected to supply voltage VDD.", "Transistor T9 has its drain connected to supply voltage VDD and its source connected to a node S forming the output of amplifier 5.The gate of transistor T9 is connected to the gate of transistor T8.Transistors T8 and T9 form together a current mirror.", "The geometry of transistors T8 and T9 is chosen so that the current running through transistor T9 is equal to current I15 running through transistor T8.Transistor T10 has its drain connected to supply voltage VDD.", "The gate of transistor T10 is connected to the gate of transistor T8.The source of transistor T10 is connected to the output via a switch S10.Transistors T8 and T10 form a current mirror.", "The geometry of transistors T8 and T10 is chosen so that, when on, transistor T10 is run through by a current equal to eight time current I15 running through transistor T8.Thus, when switch S10 is on, current I1 provided at the output of amplifier 15 is equal to 9 times current I15.By playing on the turning-off or on of switches SBj, SDj, and S10, the gain of amplifier 5 can vary from ⅓ to 47× 9/17, that is, approximately 24.8.Current amplifier 5 of FIG.", "9 is particularly advantageous.", "On the one hand, it is made in the form of two units, which enables easily obtaining a more significant gain.", "Further, switches SBj, SDj, and S10 can be easily made in the form of MOS transistors controlled to be turned off or to be turned on by logic signals 0 or 1.Unit 15 is then controlled by a digital number N coded over five bits, providing values from 0 to 31.Switches SBj will for example be controlled by number N and switches SDj by the inverse of number N. The control signal applied on input G of amplifier 5 then is a number coded over six bits, the five least significant bits (N) controlling unit 15 and the most significant bit controlling switch S10 of unit 16.Since the gain of the second unit has been chosen to be equal to 1 or to 9, the gain of amplifier 5 varies monotonously when the binary control number varies from 011111 to 100000.Such a structure, which is said to be linear in decibels, is particular advantageous since the gain is easily adjusted by a digital procedure.", "Of course, the present invention is likely to have various alterations, modifications, and improvements which will readily occur to those skilled in the art.", "In particular, it should be noted that branches 1, 2, 5 and 1′, 2′, 5′ are not necessarily identical, in a circuit where specifications in terms of error voltage are not critical.", "Further, although current amplifiers 5 and 5′ have been described as having an adjustable gain, they may have a fixed gain, if the A.C. component of voltage V to be calibrated does not vary excessively.", "Moreover, it should be noted that voltage V0 is not necessarily chosen to be substantially equal to the minimum level of the voltage to be calibrated.", "Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and the scope of the present invention.", "Accordingly, the foregoing description is by way of example only and is not intended to be limiting.", "The present invention is limited only as defined in the following claims and the equivalents thereto." ] ]	Patent_10467378
[ [ "Process for the polymerization of olefins", "The present invention relates to a process for homopolymerization of ethylene or copolymerization of ethylene with alpha-olefins by contacting ethylene or ethylene and alpha-olefin with a catalyst composition comprising: (a) a solid catalyst precursor comprising at least one vanadium compound, at least one magnesium compound and a polymeric material or a solid catalyst precursor comprising at least one vanadium compound, at least one further transition metal compound and/or at least one alcohol, at least one magnesium compound and a polymeric material; and (b) a cocatalyst comprising an aluminum compound." ], [ "1.A process for homopolymerization of ethylene or copolymerization of ethylene with alpha-olefins comprising contacting ethylene or ethylene and alpha-olefin with a catalyst composition comprising: (i) a solid catalyst precursor comprising at least one vanadium compound represented by the general formulas V(OR1)nX4-n, V(R2)nX4-n, VX3 and VOX3 wherein R1 and R2 represent an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms, and X represents a halogen and n represents a number satisfying 0≦n≦4, at least one Grignard compound represented by the general formula R6MgX wherein R6 is a hydrocarbon group having 1 to 20 carbon atoms and X is a halogen atom or a dialkyl magnesium compound represented by the general formula R7R8Mg, wherein R7 and R8 are each a hydrocarbon group having 1 to 20 carbon atoms, and a polymeric support; and (ii) a cocatalyst comprising an aluminum compound represented by the general formula R9nAlX3-n wherein R9 represents a hydrocarbon group having 1 to 10 carbon atoms; X represents a halogen and n represents a number satisfying 0≦n≦3, or by the general formula R10R11Al—O—AlR12R13, wherein R10, R11, R12 and R13 are either the same or different linear, branched or cyclic alkyl group having 1 to 12 carbons, or is an aluminoxane.", "2.The process according to claim 1 wherein said solid catalyst precursor further comprises at least transition metal compound represented by the general formulas Tm(OR3)nX4-n, TmOX3 and Tm(R4)nX4-n, wherein Tm is titanium or vanadium, R3 and R4 represent an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms, X represents a halogen atom and n represents a number satisfying 0≦n≦4 or at least one alcohol represented by the general formula R5OH, wherein R5 is an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms.", "3.", "(canceled) 4.", "(canceled) 5.The process according to claim 1, wherein said vanadium compound is selected from the group comprising vanadium tetraethoxide, vanadium tetrapropoxide, vanadium tetrabutoxide, vanadium trichloride, vanadium tetrachloride, vanadium oxytrichloride, and vanadium dichlorodiethoxide.", "6.The process according to claim 5, wherein said vanadium compound comprises vanadium tetrachloride or vanadium oxytrichloride.", "7.", "(canceled) 8.The process according to claim 2, wherein the transition metal compound is selected from the group comprising titanium trichloromethoxide, titanium dichlorodimethoxide, titanium tetramethoxide, titanium trichloroethoxide, titanium dichlorodiethoxide, titanium tetraethoxide, titanium trichloropropoxide, titanium dichlorodipropoxide, titanium chlorotripropoxide, titanium tetrapropoxide, titanium trichlorobutoxide, titanium dichlorodibutoxide, titanium tetrabutoxide, vanadium tetrachloride, vanadium tetraethoxide, vanadium tetrapropoxide, vanadium tetrabutoxide, vanadium oxytrichloride, and vanadium dichlorodiethoxide.", "9.The process according to claim 8, wherein the transition metal compound comprises titanium tetraethoxide, titanium tetrapropoxide or titanium tetrabutoxide.", "10.", "(canceled) 11.The process according to claim 2, wherein the alcohol is selected from the group comprising methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, cyclohexanol, phenol, methylphenol, and ethylphenol.", "12.", "(canceled) 13.The process according to claim 5, wherein the magnesium compound comprises diethylmagnesium, di-n-propylmagnesium, di-isopropylmagnesium, di-n-butylmagnesium, di-isobutylmagnesium butylethylmagnesium, dihexylmagnesium, dioctylmagnesium, butyloctylmagnesium, ethylmagnesium chloride, butylmagnesium chloride, or hexylmagnesium chloride.", "14.The process according to claim 5, wherein the polymeric support is in the form of particles having a mean particle diameter of about 5 to 1000 microns, a pore volume of at least about 0.05 cm3/g, a pore diameter of about 20 to 10000 angstroms and a surface area of about 0.1 to 100 m2/g.", "15.The process according to claim 14, wherein said polymeric support has a pore diameter from about 500 to 10000 angstroms and a surface area from about 0.2 to 30 m2/g.", "16.The process according to claim 15, wherein the polymeric support is comprised of polyvinylchloride, polyvinylalcohol, polyketone, hydrolyzed polyketone, ethylene-vinylalcohol copolymer, or polycarbonate.", "17.(canceled).", "18.The process according to claim 15, wherein the polymeric support is comprised of polyvinylchloride having a molecular weight in the range of about 5000 to 500000 g/mole.", "19.The process according to claim 18, wherein the Grignard compound is used in the range of about 0.05 to 20 mmol per gram of polymeric support.", "20.The process according to claim 1, wherein the molar ratio of vanadium to magnesium in the catalyst precursor is in the range of about 0.01 to 50.21.The process according to claim 2, wherein the molar ratio of vanadium to transition metal in said transition compound in the catalyst precursor is of about 0.01 to 50.22.The process according to claim 2, wherein said catalyst precursor comprises an alcohol and the molar ratio of vanadium to OH groups in said alcohol in the catalyst precursor is in the range of about 0.01 to 50.23.", "(canceled) 24.The process according to claim 6, wherein the aluminum compound is trimethylaluminum, triethylaluminum, tri-isobutylaluminum or tri-n-hexylaluminum.", "25.", "(canceled) 26.The process according to claim 5, wherein the aluminum compound comprises methyl aluminoxane (MAO) or modified methyl aluminoxane (MMAO).", "27.The process according to claim 5, wherein the aluminum compound comprises a mixture of an alkylaluminum and an aluminoxane.", "28.The process according to claim 2, wherein the ratio of moles of aluminum in said cocatalyst to the moles of transition metal in said catalyst precursor is about 10 to 5000.29.", "(canceled) 30.", "(canceled) 31.", "(canceled)" ], [ "<SOH> FIELD OF THE INVENTION <EOH>The present invention relates to a process for the polymerization of olefins." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a process for homopolymerization of ethylene or copolymerization of ethylene with alpha-olefins by contacting ethylene or ethylene and alpha-olefin with a catalyst composition comprising: (a) a solid catalyst precursor comprising at least one vanadium compound, at least one magnesium compound and a polymeric material; and (b) a cocatalyst comprising at least one aluminum compound.", "Most preferably, the component (a) used in the catalyst composition for the process of the present invention further comprises at least one further transition metal compound and/or at least one alcohol.", "As a result of the present invention polyolefin and especially polyethylenes and copolymers of ethylene and alpha-olefins are provided which have a density of about 0.88 to 0.98 g/cm 3 and weight average molecular weight of about 500 to 900 000 g/mole and molecular weight distribution range of about 2 to about 30.The products have a uniform spherical particle morphology, very low level of fines and catalyst residues, improved thermal stability, excellent optical and improved environmental stress cracking resistance (ESCR).", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention relates to a process for the polymerization of olefins.", "DESCRIPTION OF THE PRIOR ART Several publications are referenced in this application.", "These references describe the state of the art to which this invention pertains, and are incorporated herein by reference.", "The application of vanadium based Ziegler-Natta catalysts for industrial use has been limited to vanadium trichloride and vanadium oxytrichloride as homogenous catalysts in solution polymerization for the production of ethylene-propylene copolymers.", "To attain suitable activity for polymerization these catalysts require the use of halogenated organic molecules such as chloroform and trichlorofluoromethane as a promoter.", "In the absence of the promoter the catalyst activity is low.", "Further, the kinetic behavior of these catalysts during polymerization display high initial rates of polymerization followed by a sharp decrease with time, decay type rate-time kinetics, and as a result produce resin of poor morphology.", "The application of vanadium based catalysts for ethylene polymerization, high density and linear low density polyethylene production, has been comparatively very limited.", "Attempts have been made to support vanadium based catalysts on silica or magnesium chloride to produce catalysts for ethylene polymerization.", "U.S. Pat.", "No.", "4,508,842 describes a catalyst preparation in which a complex of VCl3 and tetrahydrofuran (THF) is treated with silica, the solvent is then removed from the solid by distillation and a boron trihalide or alkylaluminum halide modifier is added to the solid.", "In addition, chloroform is used as a promoter with the catalyst for ethylene polymerization.", "However the productivity and stability of this catalyst system is relatively poor.", "U.S. Pat.", "Nos.", "5,534,472 and 5,670,439 describe a silica supported vanadium catalyst prepared by the prior contacting of silica with an organomagnesium compound and a trialkylaluminum compound.", "The catalysts are suitable for the production of ethylene-hexene copolymer.", "However, the polymerizations are conducted with trichlorofluoromethane or dibromomethane promoters.", "Further, procedures typically used for the preparation of suitable magnesium chloride and silica supports such as spray drying or re-crystallization processes are complicated and expensive.", "Also, the inorganic supports remain in the product, which can affect the product properties, such as optical properties, or processing.", "It is an object of the present invention to provide a process for polymerization of olefins which overcomes the drawbacks of the prior art, especially providing a process which is cheap in carrying out and which produces a polymer with improved productivity and activity without using halogenated organic molecules as a promoter.", "SUMMARY OF THE INVENTION The present invention provides a process for homopolymerization of ethylene or copolymerization of ethylene with alpha-olefins by contacting ethylene or ethylene and alpha-olefin with a catalyst composition comprising: (a) a solid catalyst precursor comprising at least one vanadium compound, at least one magnesium compound and a polymeric material; and (b) a cocatalyst comprising at least one aluminum compound.", "Most preferably, the component (a) used in the catalyst composition for the process of the present invention further comprises at least one further transition metal compound and/or at least one alcohol.", "As a result of the present invention polyolefin and especially polyethylenes and copolymers of ethylene and alpha-olefins are provided which have a density of about 0.88 to 0.98 g/cm3 and weight average molecular weight of about 500 to 900 000 g/mole and molecular weight distribution range of about 2 to about 30.The products have a uniform spherical particle morphology, very low level of fines and catalyst residues, improved thermal stability, excellent optical and improved environmental stress cracking resistance (ESCR).", "DETAILED DESCRIPTION OF THE INVENTION Further advantages and features of the present invention will become apparent by the following detailed description in connection with the accompanying drawing.", "In the drawing the Figure is a polymerization kinetic rate-time profile for ethylene polymerization using the catalyst composition of the present invention described in Example 1 below and a process of the present invention described in Example 5 below.", "The profile shows a rate build up followed by a stable rate-time profile resulting in product polymer of spherical morphology and uniform particle size distribution.", "The ethylene homopolymers and copolymers which may be prepared by the process of the present invention can have a wide density range of about 0.88 to 0.98 g/cm3.The process of the present invention provides polyolefins, and preferably high density polyethylene and linear low density polyethylene.", "The density of the polymer at a given melt index can be regulated by the amount of the alpha-olefin comonomer used.", "The amount of alpha-olefin comonomer needed to achieve the same density is varied according to the type of comonomer used.", "These alpha-olefins have 4 to 10 carbon atoms and include 1-butene, 1-pentene, 4-methyl-1-pentene, 1-hexene, 1-heptene, 1-octene and/or mixtures thereof.", "The weight average molecular weight (Mw) of the polymers produced by the process of the present invention is in the range of about 500 to 900 000 g/mole or higher, preferably from about 10 000 to 750 000 g/mole; depending on the amount of hydrogen used, the polymerization temperature and the polymer density attained.", "The homopolymers and copolymers of the present invention have a melt index (MI) range of more than 0 and up to 100, preferably between 0.3 to 50.The polymers of such wide range of MI are capable of being used in film and molding applications.", "The molecular weight distribution (MWD) of the polymers produced by the process of the present invention expressed as weight average molecular weight/number average molecular weight (Mw/Mn), is in the range of about 2 to about 30.The polymers produced by the process of the present invention are granular materials, uniform and spherical particles with an average particle size of about 0.1 to 4 mm in diameter and contain a very low level of fines.", "The solid catalyst precursor used in the present invention comprises a polymeric material in the form of particles having a mean particle diameter of about 5 to 1 000 microns and a pore volume of at least about 0.05 cm3/g and a pore diameter of about 20 to 10 000 angstroms, preferably from about 500 to 10 000 angstroms, and a surface area of about 0.1 to 100 m2/g, preferably from about 0.2 to 30 m2/g.", "The vanadium compound used for the catalyst composition for the process of the present invention is represented by the general formulas V(OR1)nX4-n, V(R2)nX4-n, VX3 and VOX3, wherein R1 and R2 represent an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms, X represents a halogen and n represents a number satisfying 0≦n≦4.Examples of R1 and R2 include alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like.", "Preferred examples of the above mentioned vanadium compounds are selected from the group comprising vanadium tetraethoxide, vanadium tetrapropoxide, vanadium tetrabutoxide, vanadium trichloride, vanadium tetrachloride, vanadium oxytrichloride, vanadium dichlorodiethoxide and/or the like.", "Vanadium tetrachloride and/or vanadium oxytrichloride are most preferred.", "The transition metal compound which may be used for the catalyst composition for the process of the present invention is represented by the general formulas Tm(OR3)nX4-n, TmOX3 and Tm(R4)nX4-n, wherein Tm represents a transition metal of Group IVB, VB, or VIB, wherein R3 and R4 represent an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms, X represents a halogen atom and n represents a number satisfying 0≦n≦4.Non-limiting examples of the transition metal are titanium and vanadium, examples of R3 and R4 include alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like.", "Preferred examples of the above mentioned transition metal compounds are selected from the group comprising titanium trichloromethoxide, titanium dichlorodimethoxide, titanium tetramethoxide, titanium trichloroethoxide, titanium dichlorodiethoxide, titanium tetraethoxide, titanium trichloropropoxide, titanium dichlorodipropoxide, titanium chlorotripropoxide, titanium tetrapropoxide, titanium trichlorobutoxide, titanium dichlorodibutoxide, titanium tetrabutoxide, vanadium tetrachloride, vanadium tetraethoxide, vanadium tetrapropoxide, vanadium tetrabutoxide, vanadium oxytrichloride, vanadium dichlorodiethoxide and/or the like.", "Titanium tetraethoxide, titanium tetrapropoxide and/or titanium tetrabutoxide are most preferred.", "The alcohol compound which may be used for the catalyst composition for the process of the present invention includes compounds represented by the general formula R5OH, wherein R5 is an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms.", "Examples of R5 include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclohexyl, phenyl, methylphenyl, ethylphenyl and the like.", "Preferred examples of the above mentioned alcohols are selected from the group comprising methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, cyclohexanol, phenol, methylphenol, ethylphenol and/or mixtures thereof.", "The magnesium compound used for the catalyst composition for the process of the present invention include Grignard compounds represented by the general formula R6MgX, wherein R6 is a hydrocarbon group having 1 to 20 carbon atoms and X is a halogen atom, preferably chlorine.", "Other preferred magnesium compounds are represented by the general formula R7R8Mg, wherein R7 and R8 are each a hydrocarbon group having 1 to 20 carbon atoms.", "Preferred examples of the above mentioned magnesium compounds include dialkylmagnesium such as diethylmagnesium, di-n-propylmagnesium, di-isopropylmagnesium, di-n-butylmagnesium, di-isobutylmagnesium butylethylmagnesium, dihexylmagnesium, dioctylmagnesium, butyloctylmagnesium; alkylmagnesium chloride such as ethylmagnesium chloride, butylmagnesium chloride, hexylmagnesium chloride and/or mixtures thereof.", "The polymer particles used for the catalyst composition for the process of the present invention have a spherical shape with a mean particle diameter of about 5 to 1 000 microns and a pore volume of at least about 0.05 cm3/g and a pore diameter of about 20 to 10 000 angstroms, preferably from about 500 to 10 000 angstroms, and a surface area of about 0.1 to 100 m2/g, preferably from about 0.2 to 30 m2/g.", "Examples of the above polymeric supports used for the catalyst composition for the process of the present invention are selected from the group comprising polymer particles of polyvinylchloride, polyvinylalcohol, polyketone, hydrolyzed polyketone, ethylene-vinylalcohol copolymer, polycarbonate and the like.", "Among these polymeric materials polyvinylchloride is more preferred and non-crosslinked polyvinylchloride particles are most preferred.", "Polyvinylchloride having a molecular weight in the range of about 5 000 to 500 000 g/mole is most preferred.", "The use of the polymer particles mentioned in the present invention, in catalyst preparation offers significant advantages over traditional olefin polymerization catalysts using supports such as silica or magnesium chloride.", "In comparison to the silica supported catalyst, the polymer particles described in catalyst preparation of the present invention do not require high temperature and prolonged dehydration steps prior to their use in catalyst synthesis, thereby simplifying the synthesis process and thus reducing the overall cost of catalyst preparation.", "Further, the cost of the polymeric support used in the present invention is substantially cheaper than silica or magnesium chloride supports.", "In addition, the catalyst of the present invention uses significantly lower levels of catalyst components for catalyst preparation than silica or magnesium chloride supported catalysts.", "Also, the process of the present invention exhibits a higher activity than processes with conventional silica or magnesium chloride supported catalysts.", "According to one embodiment of the present invention, a polyvinylchloride support is used in the process.", "The synthesis of the solid catalyst precursor of the present invention involves introducing the polymeric material described above into a vessel and then adding a diluent.", "Suitable diluents include isopentane, hexane, cyclohexane, heptane, isooctane and pentamethylheptane.", "The polymeric material is then treated with a magnesium compound described above at a temperature in the range of about 10° C. to 130° C. The ratio of magnesium compound to the polymer support may be in the range of about 0.05 to 20 mmol per gram of polymer, preferably 0.1 to 10 mmol per gram polymer.", "The solvent is then vaporized using a nitrogen purge at a temperature in the range of about 20° C. to 100° C. The resulting free-flowing solid product is then slurried.", "Suitable solvents for slurring include isopentane, hexane, cyclohexane, heptane, isooctane and pentamethylheptane.", "The magnesium modified polymeric material is then treated with a transition metal compound and/or an alcohol described above at a temperature in the range of about 10° C. to 130° C. According to the present invention titanium tetramethoxide, titanium tetraethoxide, titanium tetrapropoxide, titanium tetrabutoxide are preferred transition metal compounds and methanol, ethanol, n-propanol, isopropanol, n-butanol are preferred alcohols.", "The resulting material is then treated with a vanadium compound described above at a temperature in the range of about 10° C. to 130° C. According to the present invention vanadium tetrachloride and/or vanadium oxytrichloride are preferred vanadium compounds.", "The produced solid catalyst precursor is then washed several times with a suitable solvent such as isopentane, hexane, cyclohexane, heptane, or isooctane.", "The solid catalyst precursor is then dried using a nitrogen purge at a temperature in the range of 20° C. to 100° C. In the final dried solid catalyst precursor, the molar ratio of vanadium to magnesium is in the range of about 0.01 to 50.In the case where the transition metal compound is used in catalyst preparation the molar ratio of vanadium to transition metal in the catalyst precursor is in the range of about 0.01 to 50, and in the case where alcohol is used in catalyst preparation the molar ratio of vanadium to OH groups in the catalyst precursor is in the range of about 0.01 to 50.The catalyst compositions used for the process of the present invention are not subjected to halogenation treatments, for example chlorination treatments.", "The thus-formed catalyst precursor of the present invention is suitably activated by aluminum compounds, also known as cocatalysts.", "The activation process can be a one step in which the catalyst is fully activated in the reactor, or two steps, in which the catalyst is partially activated outside the reactor and the complete activation occurs inside the reactor.", "Unlike catalyst compositions described in the prior art the catalyst compositions of the present invention do not require promoters such as chloroform, trichlorofluoromethane or dibromomethane during polymerization.", "The aluminum compounds, also known as cocatalyst, used in the process of the present invention are represented by the general formula R9nAlX3-n, wherein R9 represents a hydrocarbon group having 1 to 10 carbon atoms, X represents a halogen and represents a number satisfying 0≦n≦3.Illustrative but not limiting examples include trialkylaluminums such as trimethylaluminum, triethylaluminum, tri-isobutylaluminum or tri-n-hexylaluminum; dialkylaluminum chloride such as dimethylaluminum chloride, diethylaluminum chloride.", "The preferred activators of the above general formula are trimethylaluminum, triethylaluminum, tri-isobutylaluminum and tri-n-hexylaluminum.", "Examples of other suialuminum compounds of the present invention are represented by the general formula R10R11Al—O—AlR12R13, wherein R10, R11, R12 and R13 are either the same or different linear, branched or cyclic alkyl group having 1 to 12 carbon atoms, such as methyl, ethyl, propyl or isobutyl.", "The preferred examples are methylaluminoxane (MAO) and modified methylaluminoxane (MMAO).", "Further, mixtures of alkylaluminum compounds and aluminoxane compounds described above can also be conveniently used in the present invention.", "The cocatalyst in the present invention can be added to the catalyst precursor before and/or during the polymerization reaction.", "The cocatalyst in the present invention can be used in an amount of about 10 to 5 000 in terms moles of aluminum in the cocatalyst to moles of transition metal in the catalyst precursor, and is preferably in the range of 20 to 3 000.The process of the present invention can be carried out in slurry, solution and gas phase conditions.", "Gas phase polymerization can be carried out in stirred bed reactors and in fluidized bed reactors.", "Suitable ethylene pressures are in the range of about 3 to 40 bar, more preferably 5 to 30 bar and suitable polymerization temperatures are in the range of about 30° C. to 110° C., preferably 50° C. to 95° C. In addition to polyethylene homopolymer, ethylene copolymers with alpha-olefins having 4 to 10 carbon atoms are readily prepared by the present invention.", "Particular examples include ethylene/1-butene, ethylene/1-pentene, ethylene/1-heptene, ethylene/1-hexene, ethylene/1-octene and ethylene/4-methyl 1-pentene.", "The density of the polymer at a given melt index can be regulated by the amount and nature of the alpha-olefin comonomer used.", "Hydrogen can be very conveniently used during polymerization using the catalyst described in the present invention to regulate the molecular weight of the polymer product.", "Catalysts described in the prior art display undesirable decay type polymerization rate-time profiles, however the catalysts described in the present invention display build up followed by stable polymerization rate-time profiles, as seen in the Figure, such profiles are very suitable in gas, slurry and solution phase polymerization processes and provide polymer of good morphology and high resin bulk density.", "EXAMPLES The following examples are intended to be illustrative of this invention.", "They are, of course, not be taken in any way limiting on the scope of this invention.", "Numerous changes and modifications can be made with respect to the invention.", "Test Methods The properties of the polymers produced in the following examples were determined by the following test methods: Mw, Mn, and MWD were measured at 135° C. by Gel Permeation Chromatography (GPC) using mixed mode columns and trichlorobenzene as solvent.", "Melting point (Mp) was determined by Differential Scanning Calorimetry (DSC).", "Example 1 Synthesis of Solid Catalyst Precursor (A) To a three-necked round bottom flask, equipped with a condenser and a magnetic stirrer, was placed 10.0 g of polyvinylchloride spheres with an average particle size of 36 microns.", "The flask containing the polyvinylchloride was heated up to 70° C. using an oil bath and then evacuated at 30 mm Hg pressure for 30 minutes.", "The flask and its contents were then purged with dried nitrogen and the polyvinylchloride was slurried using 30 cm3 of isopentane.", "Then 1.5 cm3 of butylmagnesium chloride (2.0 molar in diethylether) was added to the slurry and the resultant mixture was stirred for 60 minutes at an oil bath temperature of 35° C., under reflux conditions.", "The isopentane was then evaporated to obtain a free-flowing powder by using a nitrogen purge at 35° C. Then the magnesium-modified polyvinylchloride was slurried using 30 cm3 of isopentane and 2.0 cm3 of titanium tetraethoxide (1.0 molar in n-hexane) was added to the slurry and the resulting mixture was stirred at 35° C. for 40 minutes.", "A dark green/brown colored solid was produced.", "Then 8.0 cm3 of vanadium tetrachloride (1.0 molar in n-hexane) was added to contents of the flask and the resulting mixture was stirred at 35° C. for 20 minutes.", "The supernatant liquid was decanted and the resulting solid product was washed by stirring with 80 cm3 of isopentane and then removing the isopentane, then washed again twice with 80 cm3 of isopentane in each wash.", "Finally, the solid catalyst was dried using a nitrogen purge at 35° C. to yield a free-flowing brown colored solid product.", "The solid catalyst precursor was analyzed by atomic adsorption spectroscopy and was found to contain 0.57% by weight magnesium, 0.54% by weight titanium and 1.77% by weight vanadium.", "Example 2 Ethylene Polymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.", "After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.", "The reactor was then pressurized to 3 barg hydrogen pressure.", "Then 5.0 cm3 of tri-isobutylaluminum, TIBAL (1.0 molar in n-hexane) was injected into the reactor.", "Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.", "This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.", "Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.", "390 grams of polyethylene were recovered with a catalyst productivity of 3900 gPE/g catalyst, a resin bulk density of 0.290 g/cm3, a weight average molecular weight of 94 050 g/mole, a number average molecular weight of 28 350, a molecular weight distribution of 3.32 and a melting point of 135° C. Examples 3,4 Ethylene Polymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.", "After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.", "The reactor was then pressurised to 3 barg hydrogen pressure.", "Then, the desired quantity of triethylaluminum, TEAL (1.0 molar in n-hexane), described in Table 1 below, was injected into the reactor.", "Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.", "This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.", "Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.", "Catalyst performance and product polymer properties are described in Table 1.TABLE 1 Influence of TEAL Concentration Productivity/ Bulk TEAL/ Yield/ (g PE/g Density/ Mw/ Mn/ DSC/ Example (mmol) (g PE) catalyst) (g/cm3) (1000 g/mol) (1000) MWD (Mp/° C.) 3 4 500 5000 0.310 135 36.4 3.71 138 4 6 455 4550 0.303 109 35.2 3.10 137 Examples 5,6 Ethylene Polymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.", "After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.", "The reactor was then pressurised to the desired hydrogen pressure, described in Table 2 below.", "Then 4.0 cm3 of triethylaluminum, TEAL (1.0 molar in n-hexane) was injected into the reactor.", "Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.", "This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.", "Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.", "Catalyst performance and product polymer properties are described in Table 2.TABLE 2 Influence of Hydrogen Pressure Hydrogen Productivity/ Bulk Pressure/ Yield/ (g PE/g Density/ Mw/ Mn/ DSC/ Example (barg) (g PE) catalyst) (g/cm3) (1000 g/mol) (1000) MWD (Mp/° C.) 5 2 490 4900 0.290 151 43.3 3.48 139 3 3 500 5000 0.310 135 36.4 3.71 13 6 4 305 3050 0.260 114 33.7 3.37 137 Example 7 Ethylene-Butene Copolymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.", "After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.", "Then the desired quantity of butene, described in Table 3 below, was added to the reactor.", "The reactor was then pressurized to 3 barg hydrogen pressure.", "Then 4.0 cm3 of triethylaluminum, TEAL (1.0 molar in n-hexane) was injected into the reactor.", "Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.", "This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.", "Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.", "Catalyst performance and product polymer properties are described in Table 3.TABLE 3 Influence of Butene Productivity/ Bulk Butene/ Yield/ (g PE/g Density/ Mw/ Mn/ DSC/ Example (cm3) (g PE) catalyst) (g/cm3) (1000 g/mol) (1000) MWD (Mp/° C.) 3 0 500 5000 0.310 135 36.4 3.71 138 7 10 502 5020 0.300 135 35.7 3.77 138 Example 8 Ethylene-Hexene Copolymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.", "After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.", "Then the desired quantity of hexene, described in Table 4 below, was added to the reactor.", "The reactor was then pressurized to 3 barg hydrogen pressure.", "Then 4.0 cm3 of triethylaluminum, TEAL (1.0 molar in n-hexane) was injected into to the reactor.", "Ethylene was introduced to the reactor such as to raise the: reactor pressure to 15 barg.", "This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.", "Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.", "Catalyst performance and product polymer properties are described in Table 4.TABLE 4 Influence of Hexene Productivity/ Bulk Hexene/ Yield/ (g PE/g Density/ Mw/ Mn/ DSC/ Example (cm3) (g PE) catalyst) (g/cm3) (1000 g/mol) (1000) MWD (Mp/° C.) 3 0 500 5000 0.310 135 36.4 3.71 138 8 10 476 4760 0.302 134 41.8 3.21 137 Example 9 Synthesis of Solid Catalyst Precursor (B) To a three-necked round bottom flask, equipped with a condenser and a magnetic stirrer, was placed 10.0 g of polyvinylchloride spheres with an average particle size of 36 microns.", "The flask containing the polyvinylchloride was heated up to 70° C. using an oil bath and then evacuated at 30 mm Hg pressure for 30 minutes.", "The flask and its contents were then purged with dried nitrogen and the polyvinylchloride was slurried using 30 cm3 of isopentane.", "Then 1.5 cm3 of butylmagnesium chloride (2.0 molar in diethylether) was added to the slurry and the resultant mixture was stirred for 60 minutes at an oil bath temperature of 35° C., under reflux conditions.", "The isopentane was then evaporated to obtain a free-flowing powder by using a nitrogen purge at 35° C. Then the magnesium-modified polyvinylchloride was slurried using 30 cm3 of isopentane and 2.0 cm3 of titanium tetraethoxide (1.0 molar in n-hexane) was added to the slurry and the resulting mixture was stirred at 35° C. for 40 minutes.", "A dark green/brown colored solid was produced.", "Then 8.0 cm3 of vanadium oxytrichloride (1.0 molar in n-hexane) was added to contents of the flask and the resulting mixture was stirred at 35° C. for 20 minutes.", "The supernatant liquid was decanted and the resulting solid product was washed by stirring with 80 cm3 of isopentane and then removing the isopentane, then washed again twice with 80 cm3 of isopentane in each wash.", "Finally, the solid catalyst was dried using a nitrogen purge at 35° C. to yield a free-flowing brown colored solid product.", "The solid catalyst precursor was analyzed by atomic adsorption spectroscopy and was found to contain 0.74% by weight magnesium, 0.56% by weight titanium and 1.50% by weight vanadium.", "Example 10 Ethylene Polymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.", "After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.", "The reactor was then pressurised to 3 barg hydrogen pressure.", "Then, 4.0 cm3 of triethylaluminum, TEAL (1.0 molar in n-hexane) was injected into the reactor.", "Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.", "This was followed by injection of 0.1 g of the solid catalyst precursor “B” described in Example 9 after being slurried in 20 cm3 of n-hexane solvent.", "Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.", "200 grams of polyethylene were recovered with a catalyst productivity of 2000 gPE/g catalyst, a resin bulk density of 0.260 g/cm3, a weight average molecular weight of 157 000 g/mole, a number average molecular weight of 44 950, a molecular weight distribution of 3.49 and a melting point of 138° C. The features disclosed in the foregoing description, in the claims and/or in the drawing may, both separately and in any combination thereof, be material for realising the invention in diverse forms thereof." ] ]	Patent_10467389
[ [ "Modulating apparatus and method, and dsv control bit producing method", "The present invention relates to a modulation apparatus and method and a DSV-control-bit generating method for suppressing an increase in circuit size of the modulation apparatus.", "When an input data stream is supplied to a DSV control bit determination unit 31, the DSV control bit determination unit 31 determines a DSV control bit to be inserted into the input data stream.", "Upon supplying the input data stream to the DSV control bit determination unit 31, the input data stream is simultaneously supplied to a delay processor 32.", "The input data stream is delayed for a predetermined delay time and supplied to a determined-DSV-control-bit insertion unit 33.", "The determined-DSV-control-bit insertion unit 33 inserts the DSV control bit determined by the DSV control bit determination unit 3.1 into a predetermined position of the input data stream supplied by the delay means and supplies the input data stream containing the DSV control bit to a modulator 34.", "The modulator 34 modulates the input data stream containing the DSV control bit into a code string in accordance with a predetermined coding rule (for example, 1,7PP modulation)." ], [ "1.A modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string, comprising: DSV-control-bit generating means for generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; timing adjusting means for adjusting transmission timing for transmitting the input bit string; DSV-control-bit-inserted bit string generating means for generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated by the DSV-control-bit generating means into a predetermined position of the input bit string whose transmission timing is adjusted by the timing adjusting means; and first modulation means for modulating the DSV-control-bit-inserted bit string generated by the DSV-control-bit-inserted bit string generating means-into the channel bit string on the basis of a conversion rule (d, k; m, n; r).", "2.The modulation apparatus according to claim 1, further comprising NRZI means for performing NRZI modulation of the channel bit string generated by modulation by the first modulation means to generate the recording code string or the transmission code string.", "3.The modulation apparatus according to claim 1, wherein the conversion rule states that the remainder of the number of “1s” in a predetermined block of the input bit string or the DSV-control-bit-inserted bit string divided by two equals the remainder of the number of “1s” in the corresponding block of the channel bit string divided by two.", "4.The modulation apparatus according to claim 1, wherein the conversion rule states that the number of consecutive minimum run length d of the channel bit string is limited to a predetermined number or less.", "5.The modulation apparatus according to claim 1, wherein the conversion rule states a variable-length code (d, k; m, n; r), where the minimum run length d=1, the maximum run length k=7, the length of basic data prior to conversion m=2, and the length of basic channel bits subsequent to conversion n=3.6.The modulation apparatus according to claim 1, wherein data of m length, which is the length of basic data, is input for a time period in which the channel bit string of n length, which is the length of the basic channel bits, is output.", "7.The modulation apparatus according to claim 1, wherein the DSV-control-bit generating mean includes: first-candidate-bit-inserted bit string generating means for generating a first-candidate-bit-inserted bit string, which is a candidate for the DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into the predetermined position of the input bit string; second-candidate-bit-inserted bit string generating means for generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; second modulation means for modulating, on the basis of the same conversion rule as the conversion rule used by the first modulation means, the first-candidate-bit-inserted bit string generated by the first-candidate-bit-inserted bit string generating means into a first candidate channel bit string, which is a candidate for the channel bit string, and for modulating the second-candidate-bit-inserted bit string generated by the second-candidate-bit-inserted bit string generating means into a second candidate channel bit string, which is another candidate for the channel bit string; DSV calculating means for calculating a DSV of each of the first and second candidate channel bit strings generated by modulation by the second modulation means; and DSV control bit determining means for determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated by the DSV calculating means.", "8.The modulation apparatus according to claim 7, wherein the DSV calculating means includes: section DSV calculating means for calculating a section DSV of a current DSV control section of each of the first and second candidate channel bit strings; cumulative DSV calculating means for calculating a cumulative DSV on the basis of the determination result by the DSV control bit determining means; and adding means for calculating the DSV by adding each section DSV calculated by the section DSV calculating means and the cumulative DSV immediately before the current DSV control section, the cumulative DSV being calculated by the cumulative DSV calculating means.", "9.The modulation apparatus according to claim 7, wherein the first and second modulation means each have a minimum number of registers required to perform modulation based on the coding rule.", "10.The modulation apparatus according to claim 1, further comprising: first sync signal inserting means for inserting a sync pattern including a preset unique pattern into the channel bit string, wherein the DSV-control-bit generating means includes second sync signal inserting means for inserting the same sync pattern as the sync pattern inserted by the first sync signal inserting means into each of the first and second candidate channel bit strings generated by modulating the first- and second-candidate-bit-inserted bit strings generated by inserting the first and second candidate bits, respectively, into the input bit string, and wherein the DSV calculating means calculates the DSV on the basis of each of the first and second candidate channel bit strings, each of which includes the sync pattern inserted by the second sync signal inserting means.", "11.The modulation apparatus according to claim 1, wherein the timing adjusting means adjusts the transmission timing by adding a delay time to the input bit string.", "12.The modulation apparatus according to claim 1, wherein the timing adjusting means inserts a temporary value prior to determination of the DSV control bit into the input bit string at predetermined intervals.", "13.The modulation apparatus according to claim 1, further comprising checking information generating means for calculating a final cumulative DSV of the recording code string or the transmission code string, determining whether or not the calculated final cumulative DSV is within a predetermined range, and generating checking information on the basis of the determination result, wherein the DSV-control-bit generating means generates the DSV control bit on the basis of the checking information generated by the checking information generating means.", "14.The modulation apparatus according to claim 13, wherein, when it is determined that the final cumulative DSV is out of the predetermined range, the checking information generating means resets the final cumulative DSV to zero and generates an error signal serving as the checking information, and wherein the DSV-control-bit generating means internally calculates a cumulative DSV for generating the DSV control bit and, when the error signal is generated by the checking information generating means, resets the cumulative DSV to zero.", "15.A modulation method for a modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string, comprising: a DSV-control-bit generating step of generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into the channel bit string on the basis of a conversion rule (d, k; m, n; r).", "16.A recording medium providing a computer-readable computer program for controlling a modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string, the program comprising: a DSV-control-bit generating step of generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into the channel bit string on the basis of a conversion rule (d, k; m, n; r).", "17.A program for causing a computer that controls a modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string to perform a process comprising: a DSV-control-bit generating step of generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into the channel bit string on the basis of a conversion rule (d, k; m, n; r).", "18.A DSV-control-bit generating method for generating a DSV control bit to be inserted into an input bit string, comprising: a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into a predetermined position of the input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string generated from the input bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.", "19.A recording medium providing a computer-readable program for generating a DSV control bit to be inserted into an input bit string, the program comprising: a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into a predetermined position of the input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string generated from the input bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.", "20.A program for generating a DSV control bit to be inserted into an input bit string, the program causing a computer to perform a process comprising: a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into a predetermined position of the input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string generated from the input bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step." ], [ "<SOH> BACKGROUND ART <EOH>In transferring data to a predetermined transmission line or in recording data onto a recording medium, such as a magnetic disk, an optical disk, or a magneto-optical disk, the data is modulated to suit the transfer or recording.", "One known modulation method is block coding.", "Block coding is to divide a data stream into blocks of m×i bits (hereinafter the blocks are referred to as data words), and each data word is converted into a codeword of n×i bits in accordance with an appropriate coding rule.", "When i=1, the resultant code is a fixed-length code.", "When i can be of multiple values, that is, when i is selected from a range of 1 to i max (maximum i) and conversion is performed with the selected i, the resultant code is a variable-length code.", "The code generated by block coding is defined as a variable-length code (d, k; m, n; r).", "In the above description, i denotes the constraint length, and i max is r (maximum constraint length); d denotes the minimum number of consecutive “0s” between consecutive “1s”, e.g., the minimum run length of “0”; and k denotes the maximum number of consecutive “0s” between consecutive “1s”, e.g., the maximum run length of “0”.", "In recording the code generated as described above onto an optical disk, a magneto-optical disk, or the like, such as a compact disk (CD) or mini disk (MD), the variable-length code is subjected to NRZI (Non Return to Zero Inverted) modulation in which “1” is inverted whereas “0” is not inverted, and recording is done on the basis of the NRZI-modulated variable-length code (hereinafter referred to as the recording code string).", "There is a system, such as that on a magneto-optical disk in an initial ISO format having a not-so-high recording density, which records a modulated recording bit string that has not been subjected to NRZI modulation.", "The minimum inversion interval of the recording code string is denoted by Tmin, and the maximum inversion interval of the recording code string is denoted by Tmax.", "Higher recording density in the direction of linear velocity is realized when the minimum inversion interval Tmin is longer, that is, when the minimum run length d is greater.", "On the other hand, in terms of clock reading, it is preferable to have a shorter maximum inversion interval Tmax, that is, a smaller maximum run length k. Various modulations methods have been proposed.", "Specifically, modulation systems proposed or actually used in, for example, optical disks, magnetic disks, magneto-optical disks, and the like are described as follows.", "For example, RLL codes (Run Length Limited Code) with the minimum run length d=2 include an EFM (Eight to Fourteen Modulation) code (may also be represented as (2, 10: 8, 17; 1)) used in CD, MD, and the like; an 8-16 code (may also be represented as (2, 10: 8, 16; 1)) used in DVD (Digital Video Disk); an RLL (2-7) (may also be represented as (2, 7; 1, 2; r)) used in PD (Phase Change Disk); and the like.", "RLL codes with the minimum run length d=1 include a fixed-length RLL (1-7) (may also be represented as (1, 7; 2, 3; 1)) used in an ISO-format MO disk (Magnetic-Optical Disk); and a variable-length RLL (1-7) (may also be represented as (1, 7; 2, 3; r)) used in a disk drive for a high-density optical disk, magneto-optical disk, or the like.", "A conversion table for the variable-length RLL (1-7) is as follows: TABLE 1 RLL (1, 7, 2, 3, 2) Data word Codeword i = 1 11 00x 10 010 01 10x i = 2 0011 000 00x 0010 000 010 0001 100 00x 0000 100 010 The symbol x in the conversion table corresponds to 1 when the subsequent codeword is 0 and corresponds to 0 when the subsequent codeword is 1.The maximum constraint length r is 2.The parameters of the variable-length RLL (1-7) are (1, 7; 2, 3; 2).", "When the bit interval of the recording code string is denoted by T, the minimum inversion interval Tmin expressed as (d+1) is 2(=1+1)T. When the bit interval of the data stream is denoted by Tdata, the minimum inversion interval Tmin expressed as (m/n)×2 is 1.33(=(⅔)×2)Tdata.", "In the above description, m/n denotes conversion at the ratio m:n. For example, ⅔ denotes conversion at 2:3 (conversion of a data word of 2×i bits into a codeword of 3×i bits).", "The maximum inversion interval Tmax expressed as (k+1)T is 8(=7+1)T ((=(⅔)×8Tdata=5.33Tdata).", "The detection window margin Tw is expressed as (m/n)×Tdata and is 0.67(=⅔)Tdata.", "In a code string (channel bit string) generated by RLL (1-7) modulation in Table 1, 2T which is Tmin occurs most frequently, which is followed by 3T, 4T, and so forth.", "The fact that edge information, such as 2T or 3T, occurs many times with a short cycle is advantageous in reading clock.", "In contrast, when the recording linear density becomes higher, the minimum run length causes a problem.", "Specifically, when 2T (the minimum run length) occurs consecutively, the recording waveform is easily distorted because the waveform output of 2T is smaller than the others and is influenced more easily by defocus, tangential tilt, or the like.", "In recording at high linear density, recording with consecutive minimum marks is influenced more easily by disturbance, such as noise, and this may easily lead to a data read error.", "In such a case in which a data read error occurs, the error often resides in shift of start-edge and end-edge of the consecutive minimum marks.", "In other words, the generated bit error length becomes longer.", "In order to solve this problem, the consecutive minimum run lengths are controlled to better suit high linear density.", "In contrast, in recording onto a recording medium or in data transfer, code modulation based on each medium (transfer) is done.", "When a modulated code contains a DC component, fluctuation or jitter may be caused in various error signals, such as for a tracking error in servo control of a disk drive.", "It is preferable for the modulated code not to contain a DC component.", "In order to solve this problem, DSV (Digital Sum Value) control is proposed.", "DSV is the sum of bits of an NRZI-modulated (level-coded) bit string (channel bit string) in which “1” corresponds to +1 and “0” corresponds to −1.DSV serves as a reference for a DC component in the code string.", "Minimizing the absolute value of DSV, that is, performing DSV control, enables suppression of a DC component in the code string.", "In the code modulated according to the variable-length RLL (1-7) table shown in Table 1, no DSV control is performed.", "In such a case, DSV control is performed by calculating DSV of the modulated channel bit string at a predetermined interval and inserting predetermined DSV control bits into the code string.", "Basically, the DSV control bits are redundant bits.", "In view of the efficiency of code conversion, the fewer the DSV control bits, the better.", "It is preferable that the minimum run length d and the maximum run length k remain unchanged by the inserted DSV control bits.", "Changes of (d, k) influence the reading and writing characteristics.", "In order to satisfy the above requirements, DSV control must be performed in as efficiently as possible.", "While the actual RLL code must satisfy the minimum run length requirement, the maximum run length requirement need not be satisfied.", "There is a format that uses a pattern exceeding the maximum run length for a sync signal.", "For example, although EFM plus for DVD has a maximum run length of 11T, EFM plus for DVD allows for 14T as a matter of convenience of the format.", "By exceeding the maximum run length, for example, the capability of detecting a sync signal or the like is enhanced greatly.", "In the RLL (1-7) format with improved conversion efficiency, it is important to “control the consecutive minimum run lengths so as to better suit the high linear density” and to “perform DSV control as efficiently as possible” in association with an increase in linear density.", "Accordingly, the assignee of the present invention et al.", "discloses, in Japanese Patent Application No.", "10-150280, a conversion table including, as a conversion code, a basic code where d=1, k=7, m=2, and n=3; a coding rule that the remainder of the number of “1s” in each element of a data stream divided by two must be one or zero and equal the remainder of the number of “1s” in a converted channel bit string divided by two; a first replacement code for limiting the consecutive minimum run lengths d to a predetermined number or less; and a second replacement code for satisfying the run length limitation.", "Specifically, when a disk drive with high linear density reads/writes an RLL code, a pattern with consecutive minimum run lengths often causes a long error.", "When DSV control is performed on an RLL code, such as the RLL (1-7) code, DSV control bits need to be inserted into a code string (channel bit string) at arbitrary intervals.", "As described above, since the DSV control bits are redundant bits, it is preferable to have fewer DSV control bits.", "In order to maintain the minimum run length or the maximum run length, the DSV control bits of at least 2 bits or greater are necessary.", "The assignee of the present invention et al.", "discloses, in Japanese Patent Application No.", "10-150280, an RLL code with the minimum run length d=1 (d, k; m, n) and a conversion table, which is shown in Table 2, for limiting the number of consecutive minimum run lengths and for performing complete DSV control using efficient control bits while maintaining the minimum run length and the maximum run length (hereinafter referred to as a 1,7PP table; and a code according to 1,7PP table is referred to as a 1,7PP code): TABLE 2 1, 7PP (d, k, m, n, r) = (1, 7, 2, 3, 4) Data word Codeword 11 0 10 001 01 010 0011 010 100 0010 010 000 0001 000 100 000011 000 100 100 000010 000 100 000 000001 010 100 100 000000 010 100 000 “110111 001 000 000 (next010) 00001000 000 100 100 100 00000000 010 100 100 100 if xx1 then 0 = 000 xx0 then 0 = 101 Termination table 00 000 0000 010 100 “110111 001 000 000 (next010) When next channel bits are ‘010’ convert ‘11 01 11’ to ‘001 000 000’ after using main table and termination table.", "As an example of a modulation apparatus using the 1,7PP table, the assignee of the present invention discloses, in Japanese Patent Application No.", "10-150280, a modulation apparatus 1 shown in FIG.", "1 .", "The modulation apparatus 1 includes a DSV control bit determination and insertion unit 11 for determining “1” or “0” serving as a DSV control bit and inserting the DSV control bit into an input data stream at arbitrary intervals; a modulator 12 for modulating the data stream containing the DSV control bits; and an NRZI unit 13 for converting the output of the modulator 12 into a recording code string.", "Although not shown in the diagram, the modulation apparatus 1 includes a timing management unit for generating a timing signal, supplying the timing signal to the above components, and managing timing.", "In Japanese Patent Application No.", "09-342416, the assignee of the present invention et al.", "discloses a specific example of another modulation apparatus, namely, a modulation apparatus 2 shown in FIG.", "2 .", "The modulation apparatus includes a DSV control bit insertion unit 21 for inserting “1” or “0” serving as a DSV control bit into a data stream at arbitrary intervals.", "At this time, there is a data stream into which the DSV control bit “ 1 ” is inserted and another data stream into which the DSV control bit “ 0 ” is inserted.", "The modulation apparatus further includes a modulator 22 for modulating the data stream containing the DSV control bits and a DSV controller 23 for NRZI-modulating the modulated code string into level data, calculating DSV of the level data, and consequently outputting a DSV-controlled recording code string.", "As described above, the 1,7PP code is advantageous in solving the above problems.", "In contrast, compared with a modulation apparatus using a known method or a technique for performing DSV control on the RLL (1,7) code, the configuration of the known modulation apparatus using the 1,7PP code is complicated, and the circuit size is increased.", "For example, in the modulation apparatus 2 shown in FIG.", "2 , the register configuration in the modulator 22 is shown in FIG.", "3 .", "Specifically, the modulator 22 has a modulation (1,7PP modulation) portion and a delay portion corresponding to a DSV control interval (DSV section) in one integrated unit in order to transfer data corresponding to the DSV control interval to the DSV controller 23 at a subsequent stage.", "As a result, the modulator 22 needs two registers, that is, an input register 22 a (register 22 a for the data stream) and an output register 22 b (register 22 b for the channel bit string).", "The number of registers required corresponds to the DSV control interval.", "Two pairs of registers (registers 22 a and 22 b ) are necessary for the DSV control bit “0” and the DSV control bit “1”." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 is a block diagram showing an example of the configuration of a known modulation apparatus.", "FIG.", "2 is a block diagram showing an example of the configuration of another known modulation apparatus.", "FIG.", "3 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG.", "2 .", "FIG.", "4 is a block diagram showing an example of the configuration of a modulation apparatus according to the present invention.", "FIG.", "5 is a block diagram showing details of the configuration of the modulation apparatus shown in FIG.", "4 .", "FIG.", "6 is a flowchart for describing the operation of the modulation apparatus shown in FIG.", "4 .", "FIG.", "7 is a diagram for describing the data format at each stage of a data stream modulated by the modulation apparatus shown in FIG.", "4 .", "FIG.", "8 is a chart for describing timing of data to be input to the modulation apparatus shown in FIG.", "4 .", "FIG.", "9 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG.", "4 .", "FIG.", "10 is a block diagram showing an example of the configuration of another modulation apparatus according to the present invention.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to modulation apparatuses and methods and DSV-control-bit generating methods, and more particularly relates to a modulation apparatus and method and a DSV-control-bit generating method for suppressing an increase in circuit size.", "BACKGROUND ART In transferring data to a predetermined transmission line or in recording data onto a recording medium, such as a magnetic disk, an optical disk, or a magneto-optical disk, the data is modulated to suit the transfer or recording.", "One known modulation method is block coding.", "Block coding is to divide a data stream into blocks of m×i bits (hereinafter the blocks are referred to as data words), and each data word is converted into a codeword of n×i bits in accordance with an appropriate coding rule.", "When i=1, the resultant code is a fixed-length code.", "When i can be of multiple values, that is, when i is selected from a range of 1 to i max (maximum i) and conversion is performed with the selected i, the resultant code is a variable-length code.", "The code generated by block coding is defined as a variable-length code (d, k; m, n; r).", "In the above description, i denotes the constraint length, and i max is r (maximum constraint length); d denotes the minimum number of consecutive “0s” between consecutive “1s”, e.g., the minimum run length of “0”; and k denotes the maximum number of consecutive “0s” between consecutive “1s”, e.g., the maximum run length of “0”.", "In recording the code generated as described above onto an optical disk, a magneto-optical disk, or the like, such as a compact disk (CD) or mini disk (MD), the variable-length code is subjected to NRZI (Non Return to Zero Inverted) modulation in which “1” is inverted whereas “0” is not inverted, and recording is done on the basis of the NRZI-modulated variable-length code (hereinafter referred to as the recording code string).", "There is a system, such as that on a magneto-optical disk in an initial ISO format having a not-so-high recording density, which records a modulated recording bit string that has not been subjected to NRZI modulation.", "The minimum inversion interval of the recording code string is denoted by Tmin, and the maximum inversion interval of the recording code string is denoted by Tmax.", "Higher recording density in the direction of linear velocity is realized when the minimum inversion interval Tmin is longer, that is, when the minimum run length d is greater.", "On the other hand, in terms of clock reading, it is preferable to have a shorter maximum inversion interval Tmax, that is, a smaller maximum run length k. Various modulations methods have been proposed.", "Specifically, modulation systems proposed or actually used in, for example, optical disks, magnetic disks, magneto-optical disks, and the like are described as follows.", "For example, RLL codes (Run Length Limited Code) with the minimum run length d=2 include an EFM (Eight to Fourteen Modulation) code (may also be represented as (2, 10: 8, 17; 1)) used in CD, MD, and the like; an 8-16 code (may also be represented as (2, 10: 8, 16; 1)) used in DVD (Digital Video Disk); an RLL (2-7) (may also be represented as (2, 7; 1, 2; r)) used in PD (Phase Change Disk); and the like.", "RLL codes with the minimum run length d=1 include a fixed-length RLL (1-7) (may also be represented as (1, 7; 2, 3; 1)) used in an ISO-format MO disk (Magnetic-Optical Disk); and a variable-length RLL (1-7) (may also be represented as (1, 7; 2, 3; r)) used in a disk drive for a high-density optical disk, magneto-optical disk, or the like.", "A conversion table for the variable-length RLL (1-7) is as follows: TABLE 1 RLL (1, 7, 2, 3, 2) Data word Codeword i = 1 11 00x 10 010 01 10x i = 2 0011 000 00x 0010 000 010 0001 100 00x 0000 100 010 The symbol x in the conversion table corresponds to 1 when the subsequent codeword is 0 and corresponds to 0 when the subsequent codeword is 1.The maximum constraint length r is 2.The parameters of the variable-length RLL (1-7) are (1, 7; 2, 3; 2).", "When the bit interval of the recording code string is denoted by T, the minimum inversion interval Tmin expressed as (d+1) is 2(=1+1)T. When the bit interval of the data stream is denoted by Tdata, the minimum inversion interval Tmin expressed as (m/n)×2 is 1.33(=(⅔)×2)Tdata.", "In the above description, m/n denotes conversion at the ratio m:n. For example, ⅔ denotes conversion at 2:3 (conversion of a data word of 2×i bits into a codeword of 3×i bits).", "The maximum inversion interval Tmax expressed as (k+1)T is 8(=7+1)T ((=(⅔)×8Tdata=5.33Tdata).", "The detection window margin Tw is expressed as (m/n)×Tdata and is 0.67(=⅔)Tdata.", "In a code string (channel bit string) generated by RLL (1-7) modulation in Table 1, 2T which is Tmin occurs most frequently, which is followed by 3T, 4T, and so forth.", "The fact that edge information, such as 2T or 3T, occurs many times with a short cycle is advantageous in reading clock.", "In contrast, when the recording linear density becomes higher, the minimum run length causes a problem.", "Specifically, when 2T (the minimum run length) occurs consecutively, the recording waveform is easily distorted because the waveform output of 2T is smaller than the others and is influenced more easily by defocus, tangential tilt, or the like.", "In recording at high linear density, recording with consecutive minimum marks is influenced more easily by disturbance, such as noise, and this may easily lead to a data read error.", "In such a case in which a data read error occurs, the error often resides in shift of start-edge and end-edge of the consecutive minimum marks.", "In other words, the generated bit error length becomes longer.", "In order to solve this problem, the consecutive minimum run lengths are controlled to better suit high linear density.", "In contrast, in recording onto a recording medium or in data transfer, code modulation based on each medium (transfer) is done.", "When a modulated code contains a DC component, fluctuation or jitter may be caused in various error signals, such as for a tracking error in servo control of a disk drive.", "It is preferable for the modulated code not to contain a DC component.", "In order to solve this problem, DSV (Digital Sum Value) control is proposed.", "DSV is the sum of bits of an NRZI-modulated (level-coded) bit string (channel bit string) in which “1” corresponds to +1 and “0” corresponds to −1.DSV serves as a reference for a DC component in the code string.", "Minimizing the absolute value of DSV, that is, performing DSV control, enables suppression of a DC component in the code string.", "In the code modulated according to the variable-length RLL (1-7) table shown in Table 1, no DSV control is performed.", "In such a case, DSV control is performed by calculating DSV of the modulated channel bit string at a predetermined interval and inserting predetermined DSV control bits into the code string.", "Basically, the DSV control bits are redundant bits.", "In view of the efficiency of code conversion, the fewer the DSV control bits, the better.", "It is preferable that the minimum run length d and the maximum run length k remain unchanged by the inserted DSV control bits.", "Changes of (d, k) influence the reading and writing characteristics.", "In order to satisfy the above requirements, DSV control must be performed in as efficiently as possible.", "While the actual RLL code must satisfy the minimum run length requirement, the maximum run length requirement need not be satisfied.", "There is a format that uses a pattern exceeding the maximum run length for a sync signal.", "For example, although EFM plus for DVD has a maximum run length of 11T, EFM plus for DVD allows for 14T as a matter of convenience of the format.", "By exceeding the maximum run length, for example, the capability of detecting a sync signal or the like is enhanced greatly.", "In the RLL (1-7) format with improved conversion efficiency, it is important to “control the consecutive minimum run lengths so as to better suit the high linear density” and to “perform DSV control as efficiently as possible” in association with an increase in linear density.", "Accordingly, the assignee of the present invention et al.", "discloses, in Japanese Patent Application No.", "10-150280, a conversion table including, as a conversion code, a basic code where d=1, k=7, m=2, and n=3; a coding rule that the remainder of the number of “1s” in each element of a data stream divided by two must be one or zero and equal the remainder of the number of “1s” in a converted channel bit string divided by two; a first replacement code for limiting the consecutive minimum run lengths d to a predetermined number or less; and a second replacement code for satisfying the run length limitation.", "Specifically, when a disk drive with high linear density reads/writes an RLL code, a pattern with consecutive minimum run lengths often causes a long error.", "When DSV control is performed on an RLL code, such as the RLL (1-7) code, DSV control bits need to be inserted into a code string (channel bit string) at arbitrary intervals.", "As described above, since the DSV control bits are redundant bits, it is preferable to have fewer DSV control bits.", "In order to maintain the minimum run length or the maximum run length, the DSV control bits of at least 2 bits or greater are necessary.", "The assignee of the present invention et al.", "discloses, in Japanese Patent Application No.", "10-150280, an RLL code with the minimum run length d=1 (d, k; m, n) and a conversion table, which is shown in Table 2, for limiting the number of consecutive minimum run lengths and for performing complete DSV control using efficient control bits while maintaining the minimum run length and the maximum run length (hereinafter referred to as a 1,7PP table; and a code according to 1,7PP table is referred to as a 1,7PP code): TABLE 2 1, 7PP (d, k, m, n, r) = (1, 7, 2, 3, 4) Data word Codeword 11 0 10 001 01 010 0011 010 100 0010 010 000 0001 000 100 000011 000 100 100 000010 000 100 000 000001 010 100 100 000000 010 100 000 “110111 001 000 000 (next010) 00001000 000 100 100 100 00000000 010 100 100 100 if xx1 then 0 = 000 xx0 then 0 = 101 Termination table 00 000 0000 010 100 “110111 001 000 000 (next010) When next channel bits are ‘010’ convert ‘11 01 11’ to ‘001 000 000’ after using main table and termination table.", "As an example of a modulation apparatus using the 1,7PP table, the assignee of the present invention discloses, in Japanese Patent Application No.", "10-150280, a modulation apparatus 1 shown in FIG.", "1.The modulation apparatus 1 includes a DSV control bit determination and insertion unit 11 for determining “1” or “0” serving as a DSV control bit and inserting the DSV control bit into an input data stream at arbitrary intervals; a modulator 12 for modulating the data stream containing the DSV control bits; and an NRZI unit 13 for converting the output of the modulator 12 into a recording code string.", "Although not shown in the diagram, the modulation apparatus 1 includes a timing management unit for generating a timing signal, supplying the timing signal to the above components, and managing timing.", "In Japanese Patent Application No.", "09-342416, the assignee of the present invention et al.", "discloses a specific example of another modulation apparatus, namely, a modulation apparatus 2 shown in FIG.", "2.The modulation apparatus includes a DSV control bit insertion unit 21 for inserting “1” or “0” serving as a DSV control bit into a data stream at arbitrary intervals.", "At this time, there is a data stream into which the DSV control bit “1” is inserted and another data stream into which the DSV control bit “0” is inserted.", "The modulation apparatus further includes a modulator 22 for modulating the data stream containing the DSV control bits and a DSV controller 23 for NRZI-modulating the modulated code string into level data, calculating DSV of the level data, and consequently outputting a DSV-controlled recording code string.", "As described above, the 1,7PP code is advantageous in solving the above problems.", "In contrast, compared with a modulation apparatus using a known method or a technique for performing DSV control on the RLL (1,7) code, the configuration of the known modulation apparatus using the 1,7PP code is complicated, and the circuit size is increased.", "For example, in the modulation apparatus 2 shown in FIG.", "2, the register configuration in the modulator 22 is shown in FIG.", "3.Specifically, the modulator 22 has a modulation (1,7PP modulation) portion and a delay portion corresponding to a DSV control interval (DSV section) in one integrated unit in order to transfer data corresponding to the DSV control interval to the DSV controller 23 at a subsequent stage.", "As a result, the modulator 22 needs two registers, that is, an input register 22a (register 22a for the data stream) and an output register 22b (register 22b for the channel bit string).", "The number of registers required corresponds to the DSV control interval.", "Two pairs of registers (registers 22a and 22b) are necessary for the DSV control bit “0” and the DSV control bit “1”.", "DISCLOSURE OF INVENTION In view of these circumstances, it is an object of the present invention to suppress an increase in circuit size of a modulation apparatus.", "A modulation apparatus of the present invention includes DSV-control-bit generating means for generating a DSV control bit to be inserted into an input bit string in order to control a DSV of a recording code string or a transmission code string; timing adjusting means for adjusting transmission timing for transmitting the input bit string; DSV-control-bit-inserted bit string generating means for generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated by the DSV-control-bit generating means into a predetermined position of the input bit string whose transmission timing is adjusted by the timing adjusting means; and first modulation means for modulating the DSV-control-bit-inserted bit string generated by the DSV-control-bit-inserted bit string generating means into a channel bit string on the basis of a conversion rule (d, k; m, n; r).", "The modulation apparatus may further include NRZI means for performing NRZI modulation of the channel bit string generated by modulation by the first modulation means to generate the recording code string or the transmission code string.", "The conversion rule may state that the remainder of the number of “1s” in a predetermined block of the input bit string or the DSV-control-bit-inserted bit string divided by two equals the remainder of the number of “1s” in the corresponding block of the channel bit string divided by two.", "The conversion rule may state that the number of consecutive minimum run length d of the channel bit string is limited to a predetermined number or less.", "The conversion rule may state a variable-length code (d, k; m, n; r) where the minimum run length d=1, the maximum run length k=7, the length of basic data prior to conversion m=2, and the length of basic channel bits subsequent to conversion n=3.Data of m length, which is the length of basic data, may be input for a time period in which the channel bit string of n length, which is the length of the basic channel bits, is output.", "The DSV-control-bit generating mean may include first-candidate-bit-inserted bit string generating means for generating a first-candidate-bit-inserted bit string, which is a candidate for the DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into the predetermined position of the input bit string; second-candidate-bit-inserted bit string generating means for generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; second modulation means for modulating, on the basis of the same conversion rule as the conversion rule used by the first modulation means, the first-candidate-bit-inserted bit string generated by the first-candidate-bit-inserted bit string generating means into a first candidate channel bit string, which is a candidate for the channel bit string, and for modulating the second-candidate-bit-inserted bit string generated by the second-candidate-bit-inserted bit string generating means into a second candidate channel bit string, which is another candidate for the channel bit string; DSV calculating means for calculating a DSV of each of the first and second candidate channel bit strings generated by modulation by the second modulation means; and DSV control bit determining means for determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated by the DSV calculating means.", "The DSV calculating means may include section DSV calculating means for calculating a section DSV of a current DSV control section of each of the first and second candidate channel bit strings; cumulative DSV calculating means for calculating a cumulative DSV on the basis of the determination result by the DSV control bit determining means; and adding means for calculating the DSV by adding each section DSV calculated by the section DSV calculating means and the cumulative DSV immediately before the current DSV control section, the cumulative DSV being calculated by the cumulative DSV calculating means.", "The first and second modulation means each may have a minimum number of registers required to perform modulation based on the coding rule.", "The modulation apparatus may further include first sync signal inserting means for inserting a sync pattern including a preset unique pattern into the channel bit string.", "The DSV-control-bit generating means may include second sync signal inserting means for inserting the same sync pattern as the sync pattern inserted by the first sync signal inserting means into each of the first and second candidate channel bit strings generated by modulating the first- and second-candidate-bit-inserted bit strings generated by inserting the first and second candidate bits, respectively, into the input bit string.", "The DSV calculating means may calculate the DSV on the basis of each of the first and second candidate channel bit strings, each of which includes the sync pattern inserted by the second sync signal inserting means.", "The timing adjusting means may adjust the transmission timing by adding a delay time to the input bit string.", "The timing adjusting means may insert a temporary value prior to determination of the DSV control bit into the input bit string at predetermined intervals.", "The modulation apparatus may further include checking information generating means for calculating a final cumulative DSV of the recording code string or the transmission code string, determining whether or not the calculated final cumulative DSV is within a predetermined range, and generating checking information on the basis of the determination result.", "The DSV-control-bit generating means may generate the DSV control bit on the basis of the checking information generated by the checking information generating means.", "When it is determined that the final cumulative DSV is out of the predetermined range, the checking information generating means may reset the final cumulative DSV to zero and generate an error signal serving as the checking information.", "The DSV-control-bit generating means may internally calculate a cumulative DSV for generating the DSV control bit and, when the error signal is generated by the checking information generating means, may reset the cumulative DSV to zero.", "A modulation method of the present invention includes a DSV-control-bit generating step of generating a DSV control bit to be inserted into an input bit string in order to control a DSV of a recording code string or a transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into a channel bit string on the basis of a conversion rule (d, k; m, n; r).", "A program on a recording medium of the present invention includes a DSV-control-bit generating step of generating a DSV control bit to be inserted into an input bit string in order to control a DSV of a recording code string or a transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into a channel bit string on the basis of a conversion rule (d, k; m, n; r).", "A program of the present invention includes a DSV-control-bit generating step of generating a DSV control bit to be inserted into an input bit string in order to control a DSV of a recording code string or a transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into a channel bit string on the basis of a conversion rule (d, k; m, n; r).", "According to a modulation apparatus and method, a recording medium, and a program of the present invention, a DSV control bit to be inserted into an input bit string is generated in order to control a DSV of a recording code string or a transmission code string.", "Transmission timing for transmitting the input bit string is adjusted.", "The generated DSV control bit is inserted into a predetermined position of the input bit string, whose transmission timing is adjusted, to generate a DSV-control-bit-inserted bit string.", "The generated DSV-control-bit-inserted bit string is modulated into a channel bit string on the basis of a conversion rule (d, k; m, n; r).", "A DSV-control-bit generating method of the present invention includes a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for a DSV control bit into a predetermined position of an input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.", "A program on a recording medium of the present invention includes a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for a DSV control bit into a predetermined position of an input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.", "A program of the present invention includes a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for a DSV control bit into a predetermined position of an input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.", "According to a DSV-control-bit generating method, a recording medium, and a program of the present invention, a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, is generated by inserting a first candidate bit for a DSV control bit into a predetermined position of an input bit string.", "A second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, is generated by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string.", "On the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the generated first-candidate-bit-inserted bit string is modulated into a first candidate channel bit string, which is a candidate for channel bits; and the generated second-candidate-bit-inserted bit string is modulated into a second candidate channel bit string, which is another candidate for the channel bits.", "A DSV of each of the first and second candidate channel bit strings is calculated.", "One of the first and second candidate bits is determined as the DSV control bit on the basis of the calculated DSVs.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a block diagram showing an example of the configuration of a known modulation apparatus.", "FIG.", "2 is a block diagram showing an example of the configuration of another known modulation apparatus.", "FIG.", "3 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG.", "2.FIG.", "4 is a block diagram showing an example of the configuration of a modulation apparatus according to the present invention.", "FIG.", "5 is a block diagram showing details of the configuration of the modulation apparatus shown in FIG.", "4.FIG.", "6 is a flowchart for describing the operation of the modulation apparatus shown in FIG.", "4.FIG.", "7 is a diagram for describing the data format at each stage of a data stream modulated by the modulation apparatus shown in FIG.", "4.FIG.", "8 is a chart for describing timing of data to be input to the modulation apparatus shown in FIG.", "4.FIG.", "9 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG.", "4.FIG.", "10 is a block diagram showing an example of the configuration of another modulation apparatus according to the present invention.", "BEST MODE FOR CARRYING OUT THE INVENTION FIG.", "4 shows an example of the configuration of a modulation apparatus 3 according to the present invention.", "The modulation method employed by the modulation apparatus 3 is not limited.", "In this example, the modulation apparatus 3 converts a data stream into, for example, a variable-length code (d, k; m, n; r)=(1, 7; 2, 3; 4).", "An input unit 38 receives an input data stream supplied from the outside and supplies the input data stream to a DSV control bit determination unit 31 and a delay processor 32.The DSV control bit determination unit 31 executes a predetermined arithmetic operation to determine the DSV control bit “1” or “0” to be inserted into the supplied input data stream and supplies the determination result to a determined-DSV-control-bit insertion unit 33.The delay processor 32 delays the supplied input data stream for a predetermined delay time and supplies the delayed input data stream to the determined-DSV-control-bit insertion unit 33.Specifically, the delay processor 32 adjusts the transmission timing to supply the input data stream to the determined-DSV-control-bit insertion unit 32.The predetermined delay time is set on the basis of the timing to insert, by the determined-DSV-control-bit insertion unit 33 described below, the DSV control bit determined by the DSV control bit determination unit 31 into a predetermined position of the input data stream output by the delay processor 32.When the determined-DSV-control-bit insertion unit 33 receives, at a predetermined time, the input data stream supplied by the delay processor 32 and the DSV-control-bit determination result (the result indicating whether the DSV control bit is “0” or “1”) supplied at a predetermined time by the DSV control bit determination unit 31, the determined-DSV-control-bit insertion unit 33 inserts a DSV control bit corresponding to the received determination result into the predetermined position (hereinafter referred to as the DSV position) of the received input data stream to generate a new data stream (hereinafter referred to as a DSV-control-bit-inserted bit string in order to be distinguished from other data streams) and supplies the DSV-control-bit-inserted bit string to a modulator 34.Specifically, the predetermined delay time of the delay processor 32 is set so that a bit corresponding to the DSV position of the input data stream will be input to the determined-DSV-control-bit insertion unit 33 at the above-described predetermined time.", "The modulator 34 modulates the DSV-control-bit-inserted bit string into a code string (channel bit string) in accordance with a predetermined conversion rule (e.g., 1,7PP table shown in Table 2) and supplies the code string to a sync signal insertion unit 35.The sync signal insertion unit 35 inputs a prepared sync signal to a predetermined position of the code string supplied by the modulator 34 at a predetermined time (differing from that of the determined-DSV-control-bit insertion unit 33) and supplies the resultant code string to an NRZI unit 36.The sync signal consists of a predetermined pattern containing a predetermined number of channel bits and is referred to as Frame Sync hereinafter.", "The NRZI unit 36 performs NRZI modulation of the code string supplied by the sync signal insertion unit 35 to generate a recording code string and outputs the recording code string to the outside and to a cumulative DSV checking unit 37.As described above, bit-string permutation by inverting 1 and not inverting 0 of a code string is referred to as NRZI modulation.", "In other words, the code string prior to being NRZI-modulated is a bit string indicating the edge position, whereas the NRZI-modulated recording code string corresponds to a bit string indicating the H/L (High/Low) level of recording data.", "The cumulative DSV checking unit 37 receives the recording code string supplied by the NRZI unit 36, calculates the cumulative DSV so far (hereinafter referred to as the final cumulative DSV in order to be distinguished from other DSVs), determines whether or not the calculated final cumulative DSV is within a predetermined range, and generates checking information based on the determination result.", "Specifically, when the cumulative DSV checking unit 37 determines that the final cumulative DSV is out of the predetermined range, the cumulative DSV checking unit 37 resets the final cumulative DSV to “0” or sets the final cumulative DSV to a predetermined initial value, generates an error signal serving as the checking information, and supplies the error signal to the DSV control bit determination unit 31.Specifically, the cumulative DSV checking unit 37 determines whether or not the final cumulative DSV at each moment exceeds the predetermined range (e.g., a range from −128 to +127 or a range from 0 to 255 when represented in sign and magnitude notation).", "When it is determined that the final cumulative DSV exceeds the predetermined range, the error signal is supplied to a cumulative DSV calculator 55, which will be described later, in the DSV control bit determination unit 31 shown in FIG.", "5.The cumulative DSV calculator 55 resets the calculated cumulative DSV so far to “0” or sets the calculated cumulative DSV so far to the predetermined initial value.", "In this example, when the cumulative DSV checking unit 37 determines that the final cumulative DSV is within the predetermined range, the cumulative DSV checking unit 37 generates no checking information.", "Alternatively, the cumulative DSV checking unit 37 may generate a signal corresponding to “normal” or the like serving as the checking information.", "Referring to FIG.", "5, the modulation apparatus 3 will be described in more detail.", "Specifically, FIG.", "5 shows an example of the detailed configuration of the modulation apparatus 3.In FIG.", "5, the symbol “+” in the square represents a portion that processes a data stream and functions as an insertion unit or a selector.", "In order to simplify the description, the symbol is simply referred to as an adder.", "Referring to FIG.", "5, the input data stream received by the input unit 38 is supplied to the DSV control bit determination unit 31 and the delay processor 32 at the same time.", "In the DSV control bit determination unit 31, an adder 42 inserts “0” serving as a first candidate bit for the DSV control bit into the predetermined position (DSV position) of the received input data stream to generate a data stream serving as a first candidate for the DSV-control-bit-inserted bit string (hereinafter referred to as a first-candidate-bit-inserted bit string) and supplies the first-candidate-bit-inserted bit string to a 1,7PP modulator 45.The 1,7PP modulator 45 modulates the first-candidate-bit-inserted bit string into a code string (hereinafter referred to as a first candidate code string in order to be distinguished from other code strings) in accordance with, for example, the above-described 1,7PP table shown in Table 2 and supplies the first candidate code string to an adder 48.The adder 48 inserts, at a predetermined time, Frame Sync (sync signal) into a predetermined position of the first candidate code string supplied by the 1,7PP modulator 45 and supplies the resultant code string to an NRZI unit 51.As described above, for example, the 1,7PP table shown in Table 2 is used in this example.", "The 1,7PP table contains a termination table for inserting Frame Sync.", "The 1,7PP modulator 45 performs termination based on the termination table.", "Specifically, termination is to set, on a data stream, a break point at a position immediately before a position at which Frame Sync is to be inserted and to terminate table-based conversion (modulation) at the break point.", "Since the 1,7PP table has a variable-length structure, the table-based conversion has a variable end position.", "The modulation apparatus 3 uses the above-described termination table if necessary to terminate the table-based conversion at an arbitrary position on the data stream in units of two.", "The adder 48 inserts Frame Sync containing the predetermined number of channel bits into a position immediately after the end position of the table-based conversion at the time the table-based conversion is terminated.", "As described above, Frame Sync contains a predetermined pattern distinguishable from other code strings (a unique pattern not included in conversion codes in the conversion table).", "In this example, the same Frame Sync is inserted by the above-described adder 48, an adder 49, and an adder 65, which will be described later.", "The NRZI unit 51 performs NRZI modulation of the first candidate code string supplied by the adder 48 to generate a recording code string (hereinafter referred to as a first candidate symbol code string in order to be distinguished from other recording code strings) and supplies the first candidate recording code string to a section DSV calculator 53.The section DSV calculator 53 calculates a DSV in a predetermined DSV section (hereinafter referred to as a section DSV in order to be distinguished from other DSVs) on the basis of the first candidate recording code string and supplies the section DSV to an adder 56.The adder 56 adds the section DSV of the first candidate recording code string and the cumulative DSV so far supplied by the cumulative DSV calculator 55 described below and supplies the sum to a comparator 58.In contrast, an adder 43 inserts “1” serving as a second candidate bit for the DSV control bit into the predetermined position (DSV position) of the received input data stream to generate a data stream serving as a second candidate for the DSV-control-bit-inserted bit string (hereinafter referred to as a second-candidate-bit-inserted bit string) and supplies the second-candidate-bit-inserted bit string to a 1,7PP modulator 46.The second-candidate-bit-inserted bit string supplied to the 1,7PP modulator 46 is modulated, as in the above-described first-candidate-bit-inserted bit string, by the 1,7PP modulator 46 into a code string (hereinafter referred to as a second candidate code string to be distinguished from other code strings).", "The adder 49 inserts Frame Sync into the second candidate code string at a predetermined time (predetermined position).", "An NRZI unit 52 performs NRZI modulation of the second candidate code string containing Frame Sync to generate a recording code string (hereinafter referred to as a second candidate recording code string to be distinguished from other recording code strings).", "The second candidate recording code string is supplied to a section DSV calculator 54.The section DSV calculator 54 calculates a section DSV in a predetermined DSV section on the basis of the second candidate recording code string and supplies the section DSV to an adder 57.The adder 57 adds the section DSV of the second candidate recording code string and the cumulative DSV so far supplied by the cumulative DSV calculator 55 described below and supplies the sum to the comparator 58.Accordingly, the cumulative DSV of the data stream generated by inserting “0” serving as a candidate for the DSV control bit into the input data stream (the first-candidate-bit-inserted bit string) and the cumulative DSV of the data stream generated by inserting “1” serving as another candidate for the DSV control bit into the input data stream (the second-candidate-bit-inserted bit string) are supplied to the comparator 58.The comparator 58 compares the absolute values of the two cumulative DSVs, selects the data stream having the cumulative DSV whose absolute value is smaller (first- or second-candidate-bit-inserted bit string), and determines, as the DSV control bit to be actually inserted into the input data stream, the first or second candidate DSV control bit included in the selected data stream (“0” when the first-candidate-bit-inserted bit string is selected and “1” when the second-candidate-bit-inserted bit string is selected).", "Specifically, the comparator 58 supplies a DSV control bit selection signal (signal indicating “1” or “0”) corresponding to the determined DSV control bit to an AND operator 62.The comparator 58 supplies the cumulative DSV of the selected data stream to the cumulative DSV calculator 55.The cumulative DSV calculator 55 receives the cumulative DSV supplied by the comparator 58 and determines the received cumulative DSV as the cumulative DSV.", "When the subsequent section DSV of the first or second candidate recording code string is supplied to the adder 56 or the adder 57, the cumulative DSV calculator 55 supplies the cumulative DSV determined immediately before the supply of the section DSV to the adder 56 or the adder 57.As described above, when the final cumulative DSV corresponding to the currently output recording code string exceeds the predetermined range, the cumulative DSV checking unit 37 supplies the error signal to the cumulative DSV calculator 55.The cumulative DSV calculator 55 receives the error signal and resets the currently determined cumulative DSV to 0 or sets the currently determined cumulative DSV to the predetermined initial value.", "In the delay processor 32, an adder 60 inserts “0” serving as a temporary value prior to determination of the DSV control bit into the predetermined position (DSV position) of the input data stream supplied by the input unit 38 to generate a new data stream (hereinafter referred to as a temporary-DSV-control-bit-inserted bit string to be distinguished from other data streams) and supplies the temporary-DSV-control-bit-inserted bit string to a DSV section delay shift register 61.Specifically, the temporary-DSV-control-bit-inserted bit string is the same data stream as the first-candidate-bit-inserted bit string generated by the above-described adder 42.In this example, “0” is inserted as the temporary value prior to determination of the DSV control bit.", "When a combination of logic circuits in the determined-DSV-control-bit insertion unit 33 described below changes, “1” may be inserted.", "A temporary-DSV-control-bit-inserted bit string in this case is the same data stream as the second-candidate-bit-inserted bit string generated by the above-described adder 43.The DSV section delay shift register 61 delays the temporary-DSV-control-bit-inserted bit string for a predetermined delay time and supplies the delayed temporary-DSV-control-bit-inserted bit string to the determined-DSV-control-bit insertion unit 33.The DSV section delay shift register 61 contains registers, the number of which corresponds to a delay of x bits corresponding to the DSV control section, and, if necessary, a delay of α bits corresponding to a circuit delay (delay corresponding to the circuit delay α shown in FIG.", "5).", "The order of the adder 60 and the shift register 61 of the delay processor 32 may be reversed.", "Specifically, the adder 60 may insert “0” serving as the temporary value prior to determination of the DSV control bit into the input data stream delayed by the shift register 61 to generate a temporary-DSV-control-bit-inserted bit string and may supply the temporary-DSV-control-bit-inserted bit string to the determined-DSV-control-bit insertion unit 33.In the determined-DSV-control-bit insertion unit 33, the AND operator 62 performs an AND operation (logical AND) of “1” supplied at a predetermined time by a DSV control bit position gate 64 and the DSV control bit selection signal indicating “0” or “1” supplied by the above-described comparator 58 and supplies the logical operation result to an OR operator 63.The OR operator 63 performs an OR operation (logical OR) of the operation result (“1” or “0”) supplied by the AND operator 62 and predetermined bit data of the temporary-DSV-control-bit-inserted bit string supplied by the DSV section delay shift register 61 and supplies the logical operation result to the 1,7PP modulator 34.When the delay processor 32 (DSV section delay shift register 61) supplies bit data corresponding to the DSV position of the temporary-DSV-control-bit-inserted bit string (“0” inserted by the adder 60 and serving as the temporary value prior to determination of the DSV control bit) to the OR operator 63, the above-described DSV control bit position gate 64 supplies “1” to the AND operator 62 at that time.", "As described above, when “1” serving as the DSV control bit selection signal is supplied to the AND operator 62 and “1” is supplied by the DSV control bit position gate 64 to the AND operator 62 (when a bit corresponding to the DSV position is supplied to the OR operator 63), the AND operator 62 supplies “1” serving as the logical operation result to the OR operator 63.Specifically, the OR operator 63 receives “1” supplied by the AND operator 62 and bit data that is supplied by the delay processor 32 and that corresponds to the DSV position, that is, “0” inserted as the temporary DSV control bit by the adder 60, performs an OR operation of the received “1” and “0”, and supplies “1” serving as the logical operation result to the 1,7PP modulator 34.In other words, when the DSV control bit determined by the DSV control bit determination unit 31 is “1”, the determined-DSV-control-bit insertion unit 33 converts “0 (temporary value prior to determination of the DSV control bit)” inserted into the DSV position by the adder 60 into “1 (DSV control bit determined by the DSV control bit determination unit 31)”.", "In contrast, when the DSV control bit determined by the DSV control bit determination unit 31 is “0”, the determined-DSV-control-bit insertion unit 33 uses “0 (temporary value prior to determination of the DSV control bit)” inserted into the DSV position by the adder 60, unchanged, as the DSV control bit (performs no conversion).", "Accordingly, the determined-DSV-control-bit insertion unit 33 inserts the DSV control bit determined by the DSV control bit determination unit 31 into the DSV position of the temporary-DSV-control-bit-inserted bit string (position at which the temporary value prior to determination of the DSV control bit is inserted) to generate a DSV-control-bit-inserted bit string and supplies the DSV-control-bit-inserted bit string to the 1,7PP modulator 34.Since the 1,7PP modulator 34 has the same configuration as the above-described 1,7PP modulator 45 and the 1,7PP modulator 46, and since the adder 65 of the sync signal insertion unit 35 has the same configuration as the above-described adder 48 and adder 49, descriptions of the 1,7PP modulator 34 and the adder 65 are omitted.", "Since the NRZI unit 36 and the cumulative DSV checking unit 37 have already been described, descriptions of the NRZI unit 36 and the cumulative DSV checking unit 37 are omitted.", "The modulator 34 in FIG.", "4 and the 1,7PP modulator 34 in FIG.", "5 are the same modulator.", "In order to emphasize that the modulator performs 1,7PP modulation, in FIG.", "5, the modulator is expressed as the 1,7PP modulator 34.With reference to the flowchart of FIG.", "6, the operation of the modulation apparatus 3 will now be described.", "An input data stream 71 shown in FIG.", "7 is supplied to the modulation apparatus 3.In step S11, the modulation apparatus 3 receives the input data stream 71.The time at which the input data stream 71 is input is as shown in FIG.", "8.Specifically, a channel bit string (code string) 74 output by the 1,7PP modulator 34 is output as a serial recording code string in synchronization with a predetermined clock 75.In other words, one codeword is output every clock cycle.", "In contrast, the input data stream 71 is input in accordance with the conversion ratio m/n of the 1,7PP modulator 34.Specifically, in this example, the conversion ratio is ⅔.", "The amount of data of a codeword is three, whereas the amount of data of a data word of the input data stream 71 is two.", "As shown in FIG.", "8, the modulation apparatus 3 receives, of the input data stream 71, predetermined two data words within two clock cycles and stops receiving the input data stream 71 for one clock cycle.", "Accordingly, a mismatch in the conversion ratio between the input data and the output code is adjusted.", "Referring to FIG.", "7, a DSV section of the input data stream 71 has x bits.", "A 1-bit DSV control bit is inserted at the end of each x-bit data.", "In order to be distinguished from unit data, i.e., a data word, to be modulated by the 1,7PP modulator 34, the x-bit data is referred to as data Dk (k is an integer).", "In other words, the DSV position of data Dk is immediately after the end of the data Dk.", "Data D1 into which Frame Sync is inserted has a short DSV section.", "Specifically, Data D1 consists of x-Fsx(⅔) bits (Fs is the number of bits of Frame Sync).", "Referring back to FIG.", "6, in step S12, the modulation apparatus 3 determines the DSV control bit and inserts the DSV control bit into the predetermined position of the input data stream 71 to generate a DSV-control-bit-inserted bit string 72 shown in FIG.", "7.Specifically, when each data word of the input data stream 71 is supplied to the DSV control bit determination unit 31 in the order shown in FIG.", "8, the DSV control bit determination unit 31 receives each data word and determines the DSV control bit to be inserted into the DSV position of x-bit data received, that is, data Dk.", "At the same time, the input data stream 71 is also supplied to the delay processor 32 in the order shown in FIG.", "8 and delayed for a predetermined delay time, and the delayed input data stream 71 is supplied to the determined-DSV-control-bit insertion unit 33.The determined-DSV-control-bit insertion unit 33 inserts the DSV control bit (bit “0” or “1”) of the data Dk, which is determined by the DSV control bit determination unit 31, into the DSV position of the data Dk at the time the bit at the DSV position of the data Dk is supplied by the delay processor 32 to generate the DSV-control-bit-inserted bit string 72 and supplies the DSV-control-bit-inserted bit string 72 to the 1,7PP modulator 34.In step S13, the modulation apparatus 3 performs 1,7PP modulation of the DSV-control-bit-inserted bit string 72 to generate a predetermined code string.", "Specifically as described above (as shown in FIG.", "8), since the input data is received in units of two data words (stopped for one clock cycle), the 1,7PP modulator 34 modulates the DSV-control-bit-inserted bit string 72 in units of two data words.", "In other words, the 1,7PP modulator 34 processes the data in units of three clock cycles (three channel bits).", "The timing is generated by a counter or the like (not shown).", "The register configuration in the 1,7PP modulator 34 is as shown in FIG.", "9.Also, the register configuration in each of the 1,7PP modulator 45 and the 1,7PP modulator 46 is as shown in FIG.", "9.Specifically, an input register 81 of the 1,7PP modulator 34 (register 81 associated with the DSV-control-bit-inserted bit string 72 supplied to the 1,7PP modulator 34) and an output register 82 (register 82 associated with a code string 73 output by the 1,7PP modulator 34) have minimum number of registers required to modulate each data word in accordance with the 1,7PP table shown in Table 2.Specifically, the input register 81 is provided with a number of registers which are 12 bits.", "The output register 82 is provided with a number of registers which are 18 bits.", "The 1,7PP modulator 34 includes a timing control register (not shown).", "The number of registers required by the 1,7PP modulator 34 is the minimum number of registers required to modulate each data word and does not depend on the DSV interval of the format (in this example, the x-bit section).", "In other words, the registers required by the 1,7PP modulator 34 are provided without taking into consideration a delay corresponding to a DSV control interval portion.", "When the input data word consists of two bits (constraint length i=1), the 1,7PP modulator 34 places the bits into [0, 1] of the input register 81 shown in FIG.", "9.Referring to [0,1] replaced with the corresponding bits, the 1,7PP modulator 34 modulates the bits when a predetermined condition is satisfied and places the channel bits into [0, 1, 2] of the output register 82.Similarly, when the input data word consists of four bits (constraint length i=2), the 1,7PP modulator 34 refers to [0, 1, 2, 3] of the input register 81 containing the corresponding bits, modulates the bits when a predetermined condition is satisfied, and places the channel bits into [0, 1, 2, 3, 4, 5] of the output register 82.When the input data word consists of six bits (constraint length i=3), the 1,7PP modulator 34 refers to [0, 1, 2, 3, 4, 5] of the input register 81 containing the corresponding bits, modulates the bits when a predetermined condition is satisfied, and places the channel bits into [0, 1, 2, 3, 4, 5, 6, 7, 8] of the output register 82.In the case of the maximum constraint length, that is, when the input data word consists of eight bits (constraint length i=4), the 1,7PP modulator 34 refers to [0, 1, 2, 3, 4, 5, 6, 7] of the input register 81 containing the corresponding bits, modulates the bits when a predetermined condition is satisfied, and places the channel bits into [0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] of the output register 82.When processing the Prohibit rmtr portion (110111-next_cbit:010), the 1,7PP modulator 34 refers to [0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] of the input register 81 and, when a predetermined condition is satisfied, replaces predetermined positions of the output register 82 with the channel bits.", "Accordingly, the 1,7PP modulator 34 uses the input register 81 and the output register 82 to perform 1,7PP modulation of the DSV-control-bit-inserted bit string 72 to generate a channel bit string (code string) and supplies the channel bit string (code string) to the sync signal insertion unit 35.In step S14, the modulation apparatus 3 inserts Frame Sync into a predetermined position of the code string output by the 1,7PP modulator 34 to generate the code string (channel bit string) 73 shown in FIG.", "7.Specifically, when the 1,7PP modulator 34 performs the above-described termination, the sync signal insertion unit 35 inserts Frame Sync (sync signal) at the head of the immediate data Dk (data D1 in this example) to generate the code string (channel bit string) 73 shown in FIG.", "7 and supplies the code string 73 to the NRZI unit 36.Subsequent to the insertion of Frame Sync into the code string 73, DSV control bits are inserted into the code string 73 at equal intervals (span 1=span 2=span 3), thus achieving appropriate DSV control.", "More specifically, when the 1,7PP modulator 34 performs termination, the 1,7PP modulator 34 stops outputting data.", "At the same time, the adder 65 of the sync signal insertion unit 35 changes the selector and adds Frame Sync of a predetermined length.", "When Frame Sync is added, the adder 65 changes the selector (to the original state), and the 1,7PP modulator 34 resumes data output (the code string 74 is supplied to the sync signal insertion unit 65).", "Although the above method is described as an example of a method of inserting Frame Sync, the method is not limited to the above-described method.", "For example, the 1,7PP modulator 34 may perform termination and then supply a temporary code string of the same length as predetermined Frame Sync to the sync signal insertion unit 35, and the sync signal insertion unit 35 may replace the temporary code string with the predetermined Frame Sync.", "In step S15, the modulation apparatus 3 performs NRZI modulation of the code string 73 to generate a recording code string and outputs the recording code string to the outside.", "Specifically, the NRZI unit 36 performs NRZI modulation of the code string 73 supplied by the sync signal insertion unit 35 to generate a recording code string and outputs the recording code string to the outside and to the cumulative DSV checking unit 37.The cumulative DSV checking unit 37 receives the recording code string supplied by the NRZI unit 36, calculates the final cumulative DSV, and determines whether or not the calculated final cumulative DSV is within a predetermined range.", "When it is determined that the final cumulative DSV is out of the predetermined range, the cumulative DSV checking unit 37 supplies the determination result to the cumulative DSV calculator 55 of the DSV control bit determination unit 31.The cumulative DSV calculator 55 resets the calculated cumulative DSV so far to 0 or sets the calculated cumulative DSV so far to a predetermined initial value.", "The cumulative DSV supplied to the cumulative DSV calculator 55 of the DSV control bit determination unit 31 may be a value supplied by the comparator 58 shown in FIG.", "5 or may be, for example, the final cumulative DSV calculated by the above-described cumulative DSV checking unit 37.Specifically, supplying the final cumulative DSV calculated by the cumulative DSV checking unit 37 to the cumulative DSV calculator 55 at a predetermined time enables the cumulative DSV calculator 55 to operate in a manner similar to the above-described operation.", "The code string output by the NRZI unit 36 is a recording code string in this example.", "In the case of transmission of the output result, the NRZI unit 36 outputs a transmission code string.", "In this case, the operation of the solid-state imaging device 3 remains unchanged.", "As described above, according to the modulation apparatus 3 of the present invention, as shown in FIG.", "9, the number of registers (the input register 81 and the output register 82) in each of the 1,7PP modulator 34, the 1,7PP modulator 45, and the 1,7PP modulator 46 is the minimum number of registers required to perform 1,7PP modulation of each data word and does not depend on the DSV control interval since the portion that performs 1,7PP modulation has an independent structure.", "On the other hand, registers in a known modulating apparatus (e.g., the modulation apparatus 2 shown in FIG.", "2) must be of sufficient number corresponding to the DSV control interval, as shown in FIG.", "3.In the delay processor 32, only the DSV section delay shift register 61) is required, which consists of registers, the number of which corresponds to the total of the channel bit string corresponding to the DSV control interval and the circuit delay α (only one shift register is necessary).", "Accordingly, the registers required in the modulation apparatus 3 of the present invention are more compact than those in a known modulation apparatus.", "As a result, the manufacturer can make circuits of the modulation apparatus 3 more compact.", "A reduction in the number of registers reduces, for example, the power consumption.", "In particular, when the DSV control interval is increased or the conversion table for converting a data word into a codeword becomes smaller, the advantage of using the modulation apparatus 3 becomes more apparent.", "A series of the above-described processes may be executed by hardware or software.", "In the latter case, for example, the modulation apparatus 4 includes a personal computer shown in FIG.", "10.Referring to FIG.", "10, a CPU 101 performs various processes in accordance with a program stored in a ROM 102 or a program loaded from a storage unit 108 into a RAM 103.If necessary, the RAM 103 stores data necessary for the CPU 101 to perform the various processes.", "The CPU 101, the ROM 102, and the RAM 103 are interconnected with one another via a bus 104.An input/output interface 105 is connected to the bus 104.An input unit 106 including a keyboard and a mouse, an output unit 107 including a display, the storage unit 108 including a hard disk, a communication unit 109 including a modem and a terminal adapter are connected to the input/output interface 105.The communication unit 109 performs communication via a network including the Internet.", "If necessary, a drive 110 is connected to the input/output interface 105.A magnetic disk 121, an optical disk 122, a magneto-optical disk 123, or a semiconductor memory 124 is appropriately placed on the drive 111, and a computer program read from the placed medium is installed into the storage unit 108 if necessary.", "When a series of processes is to be performed using software, a program providing the software is installed via a network or a recording medium onto a computer included in dedicated hardware or, for example, a general personal computer capable of performing various functions by installing therein various programs.", "As shown in FIG.", "10, the recording medium includes a packaged medium including the magnetic disk 121 (including a floppy disk), the optical disk 122 (including a CD-ROM (Compact Disk-Read Only Memory) and DVD (Digital Versatile Disk)), the magneto-optical disk 123 (including MD (Mini-Disk)), or the semiconductor memory 124, all of which have recorded thereon the program and which are distributed, separately from the apparatus, to supply the program to the user.", "Also, the recording medium includes the ROM 102 or the hard disk included in the storage unit 108, which have recorded thereon the program and which are included in advance in the apparatus to be supplied to the user.", "In this specification, steps for writing the program stored in the recording medium not only include time-series processing performed in accordance with the described order but also include parallel or individual processing, which may not necessarily be performed in time series.", "INDUSTRIAL APPLICABILITY As described above, according to a modulation apparatus and method and to a DSV-control-bit generating method of the present invention, an increase in circuit size of the modulation apparatus is suppressed." ] ]	Patent_10467399
[ [ "Systems and methods for automatic carburetor enrichment during cold start", "An automatic carburetor (20) enrichment system that controls the air-fuel mixture during cold start of an engine (10) having a carburetor including a fuel bowl (22) and an induction passage (23), includes a sensor (50) that provides a signal indicative of an engine temperature at engine start, a fuel line (30) connected between the fuel bowl (22) and the induction passage, a solenoid valve (40) disposed in the fuel line, and a controller (10) that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture at engine start.", "The automatic carburetor enrichment system reduces cranking time during cold start, eliminates the need for driver input during cold start, prevents engine stalling without assistance from the operator during the warm up phase, provides a simpler, more cost effective and reliable carburetor enrichment, provides self-drowning protection without the use of an electronic idle switch and eliminates the risk of engine drowning when the engine is cranked with the choke ON and the ignition switches OFF." ], [ "1.A carburetor enrichment system that controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor, that is supplied fuel from a fuel reservoir, and includes an induction passage, the carburetor enrichment system comprising: a sensor that provides a signal indicative of an engine temperature; a fuel line connected between the fuel reservoir and the induction passage; a solenoid valve disposed in the fuel line; and a controller that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture.", "2.A carburetor enrichment system according to claim 1, wherein the controller sets the duty cycle of the solenoid valve at one of a) an engine start time and b) a boost duration time associated with the engine temperature.", "3.A carburetor enrichment system according to claim 2, wherein the engine start time is determined when the engine reaches a predetermined idle threshold speed.", "4.A carburetor enrichment system according to claim 2, wherein the controller stores a plurality of engine temperatures and associated boost duration times.", "5.A carburetor enrichment system according to claim 1, wherein the controller stores a plurality of engine temperatures and associated duty cycles.", "6.A carburetor enrichment system according to claim 2, wherein the controller reduces the duty cycle of the solenoid valve at a predetermined rate after one of a) the engine start time and b) the boost duration time.", "7.A carburetor enrichment system according to claim 6, wherein the predetermined rate is constant.", "8.A carburetor enrichment system according to claim 2, wherein the duty cycle of the solenoid valve is a maximum duty cycle prior to one of a) the engine start time and b) the boost duration time.", "9.A carburetor enrichment system that controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start the engine having a carburetor, that is supplied fuel from a fuel reservoir, and includes an induction passage, the carburetor enrichment system comprising: a sensor that provides a signal indicative of an engine temperature; a fuel line connected between the fuel reservoir and the induction passage; a solenoid valve disposed in the fuel line; and a controller that sets a duty cycle of the solenoid valve to a maximum duty cycle, receives the signal, reduces the duty cycle from the maximum duty cycle to a determined duty cycle associated with the engine temperature, and further reduces the duty cycle at a predetermined rate after one of a) engine start and b) a boost duration time associated with the engine temperature.", "10.A carburetor enrichment system according to claim 9, wherein the engine start time is determined when the engine reaches a predetermined idle threshold speed.", "11.A method of controlling an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor including an induction passage that is supplied fuel by a fuel line connected between a fuel reservoir and the induction passage, the fuel line having a solenoid valve disposed therein, the method comprising: determining an engine temperature; and setting a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture.", "12.A method according to claim 11, wherein setting the duty cycle occurs at one of a) an engine start time and b) a boost duration time associated with the engine temperature.", "13.A method according to claim 12, wherein the engine start time is determined when the engine reaches a predetermined idle threshold speed.", "14.A method according to claim 12, wherein a plurality of engine temperatures are associated with a plurality of boost duration times.", "15.A method according to claim 11, wherein a plurality of engine temperatures ar associated with a plurality of duty cycles.", "16.A method according to claim 12, further comprising reducing the duty cycle of the solenoid valve at a predetermined rate after one of a) the engine start time and b) the boost duration time.", "17.A method according to claim 16, wherein the predetermined rate is constant.", "18.A method according to claim 12, wherein the duty cycle of the solenoid valve is a maximum duty cycle prior to one of a) the engine start time and b) the boost duration time.", "19.A method for controlling an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start the engine having a carburetor including an induction passage that is supplied by a fuel line connected between a fuel reservoir and the induction passage, the fuel line having a solenoid valve disposed therein, the method comprising: determining an engine temperature; setting a duty cycle of the solenoid valve to a maximum duty cycle; reducing the duty cycle from the maximum duty cycle to a determined duty cycle associated with the engine temperature; and further reducing the duty cycle at a predetermined rate after one of a) engine start and b) a boost duration time associated with the engine temperature.", "20.A method according to claim 19, wherein the engine start time is determined when the engine reached a predetermined idle threshold speed." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to systems and methods for automatic carburetor enrichment during cold start of an engine.", "2.Description of the Related Art In internal combustion engines having carburetor controlled fuel supplies, for example, engines used in vehicles such as snowmobiles, personal watercraft, and all terrain vehicles, the rate of fuel flow in a fixed or variable venturi carburetor is dependent on the pressure differential between the venturi and the fuel bowl, also known as the float bowl or float chamber.", "In a conventional float bowl carburetor the pressure differential is measured between the pressure in the fluid float chamber, which is normally atmospheric, and the pressure at the discharge orifice of the fuel metering system which is normally located in or adjacent the venturi in the induction passage.", "For optimum combustion, the relationship between the mass air flow and the mass fuel flow delivered to the engine by the carburetor should be kept to a controllable rate.", "A fixed or variable venturi may be used to provide a constant relationship between the mass air flow and the mass fuel flow.", "As the air velocity in the induction passage increases, a pressure reduction, or vacuum, is created in the venturi.", "The pressure reduction creates a pressure differential between the induction passage and the fuel in the float chamber causing fuel to be drawn into the induction passage at a flow rate that is proportional to the pressure differential.", "The pressure reduction is mainly a function of the air velocity through the induction passage.", "However, at a given velocity, the mass air flow rate is dependent on the air density which in turn is dependent on the barometric pressure and temperature.", "For a given air velocity, the induction passage delivers a reduced mass air flow at higher altitudes due to the reduced barometric pressure and correspondingly reduced air density.", "Operating the engine at higher altitudes thus causes the engine to be supplied with an over rich air-fuel mixture.", "Conversely, for a given air velocity, the induction passage delivers an increased mass air flow at lower temperatures due to the increased air density.", "Operating the engine at lower temperatures thus causes the engine to be supplied with an over lean air-fuel mixture.", "U.S. Pat.", "No.", "5,021,198 to Bostelmann, the entire contents of which are herein incorporated by reference, discloses a carburetor with a high altitude compensator that includes a pressure splitter connected with the lower pressure of the venturi throat in the area where the fuel delivery line opens out and with the induction pressure in the area of the inlet end of the air flow passage.", "The pressure splitter includes a pressure line with two chokes that are connected in series.", "The fuel bowl is connected to the pressure line between the two chokes and one or both of the chokes is controlled as a function of the specific air density.", "The control system of U.S. Pat.", "No.", "5,021,198 cannot provide an enriched air-fuel mixture during cold start When the engine is cold it is difficult to start because it is difficult to create a sufficient amount of fuel vapor in the combustion chamber because atomization and vaporization are less effective at lower temperatures.", "It is therefore necessary to increase the amount of fuel in order to compensate for the lack of atomization.", "It has been known to increase the amount of fuel by using a manual primer or an air pump enrichment system at the carburetor.", "U.S. application Ser.", "No.", "08/948,064, the entire contents of which are hereby incorporated by reference, discloses an electronic compensation system for an internal combustion engine including a manifold connected to the float chamber and the venturi of each carburetor.", "A barometric pressure sensor and an engine temperature sensor provide signals to an electronic control unit that controls first and second solenoids connected to the manifold.", "During a cold start, the first solenoid is controlled to provide pressurized gas, provided by a pressure line in communication with the crankcase interior, from the manifold to the float chambers of the carburetors to increase the fuel flow and enrich the air-fuel mixture.", "As the engine temperature increases, the electronic control unit responds to signals from the engine temperature sensor to reduce the duty cycle of the first solenoid and thus reduce the supply of pressurized gas to the float chambers until the normal engine operating temperature is reached.", "The second solenoid is controlled during normal engine operation to apply the underpressure, or vacuum, from the venturis of the carburetors to the manifold to reduce the float chamber pressure to decrease the fuel flow when the air density is reduced, for example, at increased altitudes.", "The electronic compensation system of U.S. application Ser.", "No.", "08/948,064 requires the use of an air pump when the rotation speed of the engine c corresponds to idling or higher.", "While completely reliable during operation, condensation of fuel, oil, and water in the air pump decreases the reliability of the air pump.", "The electronic compensation system of U.S. application Ser.", "No.", "08/948,064 also requires an expensive idle switch to signal the position of the throttle during idling to the electronic control unit and to limit fuel distribution to avoid flooding the engine.", "In addition, the electronic compensation system of U.S. application Ser.", "No.", "08/948,064 requires the insertion of an impulse line from the interior of the crankcase to the air pump to activate a diaphragm of the air pump." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>There exists a need for an engine control system that automatically provides an enriched air-fuel mixture during cold start and that does not require a manifold and an air pump for providing pressurized gas to the float chamber and an impulse line for activating the diaphragm of the air pump.", "There also exists a need for an engine control system that automatically reduces the enriched air-fuel mixture as the engine operating temperature approaches a normal engine operating temperature and that does not require an idle switch for limiting the fuel distribution to the engine when in off-idle mode.", "A system for automatic carburetor enrichment during cold start according to the present invention controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor and being supplied fuel from a fuel reservoir, for example a fuel bowl, and an induction passage, and includes a sensor that provides a signal indicative of an engine temperature at engine start, a fuel line connected between the fuel bowl and the induction passage, a solenoid valve disposed in the fuel line, and a controller that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture at engine start A method according to the present invention for controlling the air-fuel ratio of an air fuel mixture supplied to a carburetor of an internal combustion engine during cold start, the engine having a carburetor including a fuel bowl, a fuel line between the fuel reservoir and an induction passage of the carburetor, and a solenoid valve disposed in the fuel line, includes determining a temperature of the engine, determining a duty cycle of the solenoid valve associated with the determined temperature, setting the duty cycle of the solenoid valve to the associated duty cycle at an engine start time to increase the air-fuel ratio of the air-fuel mixture at engine start." ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to systems and methods for automatic carburetor enrichment during cold start of an engine.", "2.Description of the Related Art In internal combustion engines having carburetor controlled fuel supplies, for example, engines used in vehicles such as snowmobiles, personal watercraft, and all terrain vehicles, the rate of fuel flow in a fixed or variable venturi carburetor is dependent on the pressure differential between the venturi and the fuel bowl, also known as the float bowl or float chamber.", "In a conventional float bowl carburetor the pressure differential is measured between the pressure in the fluid float chamber, which is normally atmospheric, and the pressure at the discharge orifice of the fuel metering system which is normally located in or adjacent the venturi in the induction passage.", "For optimum combustion, the relationship between the mass air flow and the mass fuel flow delivered to the engine by the carburetor should be kept to a controllable rate.", "A fixed or variable venturi may be used to provide a constant relationship between the mass air flow and the mass fuel flow.", "As the air velocity in the induction passage increases, a pressure reduction, or vacuum, is created in the venturi.", "The pressure reduction creates a pressure differential between the induction passage and the fuel in the float chamber causing fuel to be drawn into the induction passage at a flow rate that is proportional to the pressure differential.", "The pressure reduction is mainly a function of the air velocity through the induction passage.", "However, at a given velocity, the mass air flow rate is dependent on the air density which in turn is dependent on the barometric pressure and temperature.", "For a given air velocity, the induction passage delivers a reduced mass air flow at higher altitudes due to the reduced barometric pressure and correspondingly reduced air density.", "Operating the engine at higher altitudes thus causes the engine to be supplied with an over rich air-fuel mixture.", "Conversely, for a given air velocity, the induction passage delivers an increased mass air flow at lower temperatures due to the increased air density.", "Operating the engine at lower temperatures thus causes the engine to be supplied with an over lean air-fuel mixture.", "U.S. Pat.", "No.", "5,021,198 to Bostelmann, the entire contents of which are herein incorporated by reference, discloses a carburetor with a high altitude compensator that includes a pressure splitter connected with the lower pressure of the venturi throat in the area where the fuel delivery line opens out and with the induction pressure in the area of the inlet end of the air flow passage.", "The pressure splitter includes a pressure line with two chokes that are connected in series.", "The fuel bowl is connected to the pressure line between the two chokes and one or both of the chokes is controlled as a function of the specific air density.", "The control system of U.S. Pat.", "No.", "5,021,198 cannot provide an enriched air-fuel mixture during cold start When the engine is cold it is difficult to start because it is difficult to create a sufficient amount of fuel vapor in the combustion chamber because atomization and vaporization are less effective at lower temperatures.", "It is therefore necessary to increase the amount of fuel in order to compensate for the lack of atomization.", "It has been known to increase the amount of fuel by using a manual primer or an air pump enrichment system at the carburetor.", "U.S. application Ser.", "No.", "08/948,064, the entire contents of which are hereby incorporated by reference, discloses an electronic compensation system for an internal combustion engine including a manifold connected to the float chamber and the venturi of each carburetor.", "A barometric pressure sensor and an engine temperature sensor provide signals to an electronic control unit that controls first and second solenoids connected to the manifold.", "During a cold start, the first solenoid is controlled to provide pressurized gas, provided by a pressure line in communication with the crankcase interior, from the manifold to the float chambers of the carburetors to increase the fuel flow and enrich the air-fuel mixture.", "As the engine temperature increases, the electronic control unit responds to signals from the engine temperature sensor to reduce the duty cycle of the first solenoid and thus reduce the supply of pressurized gas to the float chambers until the normal engine operating temperature is reached.", "The second solenoid is controlled during normal engine operation to apply the underpressure, or vacuum, from the venturis of the carburetors to the manifold to reduce the float chamber pressure to decrease the fuel flow when the air density is reduced, for example, at increased altitudes.", "The electronic compensation system of U.S. application Ser.", "No.", "08/948,064 requires the use of an air pump when the rotation speed of the engine c corresponds to idling or higher.", "While completely reliable during operation, condensation of fuel, oil, and water in the air pump decreases the reliability of the air pump.", "The electronic compensation system of U.S. application Ser.", "No.", "08/948,064 also requires an expensive idle switch to signal the position of the throttle during idling to the electronic control unit and to limit fuel distribution to avoid flooding the engine.", "In addition, the electronic compensation system of U.S. application Ser.", "No.", "08/948,064 requires the insertion of an impulse line from the interior of the crankcase to the air pump to activate a diaphragm of the air pump.", "SUMMARY OF THE INVENTION There exists a need for an engine control system that automatically provides an enriched air-fuel mixture during cold start and that does not require a manifold and an air pump for providing pressurized gas to the float chamber and an impulse line for activating the diaphragm of the air pump.", "There also exists a need for an engine control system that automatically reduces the enriched air-fuel mixture as the engine operating temperature approaches a normal engine operating temperature and that does not require an idle switch for limiting the fuel distribution to the engine when in off-idle mode.", "A system for automatic carburetor enrichment during cold start according to the present invention controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor and being supplied fuel from a fuel reservoir, for example a fuel bowl, and an induction passage, and includes a sensor that provides a signal indicative of an engine temperature at engine start, a fuel line connected between the fuel bowl and the induction passage, a solenoid valve disposed in the fuel line, and a controller that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture at engine start A method according to the present invention for controlling the air-fuel ratio of an air fuel mixture supplied to a carburetor of an internal combustion engine during cold start, the engine having a carburetor including a fuel bowl, a fuel line between the fuel reservoir and an induction passage of the carburetor, and a solenoid valve disposed in the fuel line, includes determining a temperature of the engine, determining a duty cycle of the solenoid valve associated with the determined temperature, setting the duty cycle of the solenoid valve to the associated duty cycle at an engine start time to increase the air-fuel ratio of the air-fuel mixture at engine start.", "BRIEF DESCRIPTION OF THE DRAWINGS The present invention will further be described with reference to the accompanying drawings wherein: FIG.", "1 is a schematic illustration of an exemplary automatic carburetor enrichment system according to the present invention; FIG.", "2 is a graphical representation of an exemplary relationship between a solenoid duty cycle and an engine start time according to the present invention; FIG.", "3 is a graphical representation of a table including duty cycles values at various engine starting temperatures; FIG.", "4 is a graphical representation of a table including enrichment mode durations at various engine starting temperatures; and FIG.", "5 is a graphical representation of a value of the duty cycle slope at an idle threshold speed.", "DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS Various exemplary embodiments of the present invention will now be described in detail with reference to the drawings.", "Referring to FIG.", "1, an automatic carburetor enrichment system for an internal combustion engine 10 includes a carburetor 20 having a throttle valve 21.It should be appreciated that a carburetor having an air inlet of fixed size may also be used.", "The carburetor 20 also includes a float chamber 22 configured in the usual manner to ensure a constant fuel level within the chamber 22.It should be appreciated however that fuel may be supplied to the carburetor from a fuel reservoir separate from the carburetor.", "A fuel delivery line 30 extends from the float chamber 22 into the induction passage 23 of the carburetor 20.A solenoid valve 40 controls the flow of fuel from the float chamber 22 to the induction passage 23.A check valve 60 provides a balanced distribution of fuel to the cylinders of the engine 10 and prevents fuel from flowing back from the induction passage 23 to the float chamber 22 through the fuel delivery line 30.An electronic control unit 70 receives a signal indicative of the engine temperature from an engine temperature sensor 50.The engine temperature sensor 50 provides a signal indicative of the engine temperature by sensing, for example, the temperature of the engine coolant.", "The electronic control unit 70 controls the duty cycle of the solenoid valve 40 based on the temperature sensed by the temperature sensor 50.The duty cycle of the solenoid valve 40 is the percentage of the opening time of the solenoid valve in relation to its fixed cycle time.", "The solenoid valve 40 has a fixed frequency of, for example, 10 Hz and the electronic control unit 70 controls the duty cycle of the solenoid valve 40 to increase the amount of fuel delivered to the induction passage 23 through the fuel delivery line 30 during cold start.", "The electronic control unit 70 reduces the duty cycle of the solenoid valve 40 at a constant rate until it reaches zero.", "This prevents an over enrichment of fuel mixture once the engine has started, which may otherwise damage the ignition components (spark plugs) or reduce the engine performance.", "Referring to FIG.", "2, the duty cycle at the beginning of engine cranking is set at 100%, regardless of the engine temperature, to minimize the crank time before engine start-up.", "Immediately upon engine start, defined as the electronic control unit 70 detecting an engine speed corresponding to an idle threshold speed, indicated in FIG.", "5, the electronic control unit 70 reduces the duty cycle of the solenoid valve 40 to a value associated with the temperature determined by the sensor 50 that is stored in a table, such as the table shown in FIG.", "3.Although the duty cycle is shown in FIG.", "2 as being changed from 100% to the associated value in zero time, it should be appreciated that the change in duty cycle does require some time, albeit a very short time.", "The table may be stored, for example, in a memory of the electronic control unit 70.The A/D value in FIG.", "3 is an analog to digital parameter readable by the electronic control unit 70.The idle threshold speed shown in FIG.", "5 is empirically determined.", "The rate at which the duty cycle will decrease after engine start will be determined by dividing the duty cycle pitch by the time pitch, shown in FIG.", "5.For example, the duty cycle will be reduced 1% every two seconds.", "The duty cycle pitch and the time pitch are empirically determined for the vehicle and are not dependent on the temperature.", "The duty cycle/motor temp table shown in FIG.", "3 includes duty cycle values at four temperatures, ranging from 40° C. to −40° C. The duty cycle values at the four temperatures are empirically determined.", "Although the table shown in FIG.", "3 includes duty cycle values at four temperatures, it should be appreciated that the table may include duty cycle values at any number of temperatures.", "If the signal from the temperature sensor 50 indicates a temperature that is not stored in the table shown in FIG.", "3, the electronic control unit 70 extrapolates a duty cycle value from the table based on the signal provided by the temperature sensor 50.Referring again to FIG.", "2, if the engine does not start before a predetermined boost duration time To, such as the boost duration time shown in the table of FIG.", "4, the electronic control unit 70, at time To, will reduce the duty cycle of the solenoid valve 40 to that corresponding to the engine temperature at the beginning of cranking, found in FIG.", "3, and continue to reduce it at the constant rate determined by the duty cycle pitch and the time pitch of FIG.", "5.The boost duration time To is also empirically determined and is temperature dependent Again, assuming that the engine temperature, as determined by the signal from the temperature sensor 50, is not stored in the tables of FIGS.", "3 and 4, the electronic control unit 70 extrapolates a value of the temperature and the boost duration time To.", "Thereafter, regardless of the start time of the engine after the boost duration time To, the duty cycle of the solenoid valve 40 continues to decrease at the constant rate until it reaches zero.", "The automatic carburetor enrichment system according to the present invention reduces cranking time during cold start and eliminates the need for driver input during cold start.", "The automatic carburetor enrichment system according to the present invention also prevents engine stalling without assistance from the operator during the warm up phase and provides a simpler, more cost effective and reliable carburetor enrichment than current systems, including those that use an air pump.", "The automatic carburetor enrichment system according to the present invention provides self-drowning protection without the use of an electronic idle switch and eliminates the risk of engine drowning when the engine is cranked with the choke ON and the ignition switches OFF.", "Although the present invention has been described with reference to the exemplary embodiments described above, it should be appreciated that various modifications would be within the level of ordinary skill in the art.", "For example, a manual choke may be included as a backup to the automatic carburetor enrichment system.", "As another example, although the electronic control unit has been described as determining the engine temperature at the beginning of engine start, it should be appreciated that the electronic control unit may monitor the engine temperature a plurality of times, or even continuously, during engine start and control the solenoid valve duty cycle based on the plural signals or the continuous signal from the temperature sensor.", "Additionally, although the duty cycle has been described as reduced at a constant linear rate, it should be appreciated that the duty cycle may be reduced at an exponential or logarithmic rate.", "Accordingly, the scope of the protection sought by the grant of Letters Patent is defined by the Claims appended hereto." ] ]	Patent_10467451
[ [ "Sequence of the photorhabdus luminescens strain tt01 genome and uses", "The present invention relates to the genomic sequence and to nucleotide sequences encoding polypeptides of Photorhabdus luminescens.", "The present invention further relates to polypeptides involved in operons involved in the biosynthesis of antibiotics or toxins, as well as polypeptides with activity of the antibiotic or toxin.", "Uses of the aforementioned polypeptides in pesticides, bactericides, or fungicides is provides.", "In addition, the present invention provides vectors, cells, or animals containing the sequences of the present invention." ], [ "1-56.", "(canceled) 57.An isolated nucleotide sequence derived from the Photorhabdus luminescens genome, comprising a sequence selected from the group consisting of SEQ ID No.", "1 to SEQ ID No.", "41 and SEQ ID No.", "5826 to SEQ ID No.", "5834.58.An isolated nucleotide sequence derived from the Photorhabdus luminescens genome, selected from the group consisting of: a) a nucleotide sequence comprising at least 75% identity with a sequence chosen selected from the group consisting of SEQ ID No.", "1 to SEQ ID No.", "41 and SEQ ID No.", "5826 to SEQ ID No.", "5834; b) a nucleotide sequence comprising a fragment of a sequence selected from the group consisting of SEQ ID No.", "1 to SEQ ID No.", "41 and SEQ ID No.", "5826 to SEQ ID No.", "5834; c) a nucleotide sequence complementary to a nucleotide sequence as defined in a) or b); d) a nucleotide sequence of the RNA corresponding to one of the sequences as defined in a), b) or c); and e) a nucleotide sequence as defined in a), b), c) or d), which has been modified.", "59.The nucleotide sequence as claimed in claim 58, wherein the nucleotide sequence is a fragment of a nucleotide sequence selected from the group consisting of SEQ ID No.", "1 to SEQ ID No.", "41 and SEQ ID No.", "5826 to SEQ ID No.", "5834, and wherein said fragment encodes a polypeptide selected from the group consisting of SEQ ID No.", "42 to SEQ ID No.", "3855 or a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.", "5835 to SEQ ID No.", "10784.60.The nucleotide sequence as claimed in claim 59, wherein: a) said nucleotide sequence encodes a polypeptide selected from the group consisting of SEQ ID No.", "61, SEQ ID No.", "62, SEQ ID No.", "67, SEQ ID No.", "171, SEQ ID No.", "221, SEQ ID No.", "268, SEQ ID No.", "288, SEQ ID No.", "380, SEQ ID No.", "426, SEQ ID No.", "438, SEQ ID No.", "448, SEQ ID No.", "453, SEQ ID No.", "455, SEQ ID No.", "456, SEQ ID No.", "458, SEQ ID No.", "501, SEQ ID No.", "516, SEQ ID No.", "530, SEQ ID No.", "542, SEQ ID No.", "551, SEQ ID No.", "720, SEQ ID No.", "761, SEQ ID No.", "762, SEQ ID No.", "814, SEQ ID No.", "859, SEQ ID No.", "860, SEQ ID No.", "861, SEQ ID No.", "862, SEQ ID No.", "869, SEQ ID No.", "1079, SEQ ID No.", "1168, SEQ ID No.", "1174, SEQ ID No.", "1176, SEQ ID No.", "1413, SEQ ID No.", "1414, SEQ ID No.", "1415, SEQ ID No.", "1416, SEQ ID No.", "1417, SEQ ID No.", "1457, SEQ ID No.", "1651, SEQ ID No.", "1856, SEQ ID No.", "1869, SEQ ID No.", "2021, SEQ ID No.", "2080, SEQ ID No.", "2152, SEQ ID No.", "2162, SEQ ID No.", "2173, SEQ ID No.", "2251, SEQ ID No.", "2295, SEQ ID No.", "2306, SEQ ID No.", "2317, SEQ ID No.", "2328, SEQ ID No.", "2340, SEQ ID No.", "2342, SEQ ID No.", "2351, SEQ ID No.", "2500, SEQ ID No.", "3228, SEQ ID No.", "3230, SEQ ID No.", "3311, SEQ ID No.", "3317, SEQ ID No.", "3318, SEQ ID No.", "3319, SEQ ID No.", "3320, SEQ ID No.", "3322, SEQ ID No.", "3323, SEQ ID No.", "3326, SEQ ID No.", "3327, SEQ ID No.", "3328, SEQ ID No.", "3375, SEQ ID No.", "3376, SEQ ID No.", "3377, SEQ ID No.", "3378, SEQ ID No.", "3422, SEQ ID No.", "3489, SEQ ID No.", "3503, SEQ ID No.", "3609, SEQ ID No.", "3623, SEQ ID No.", "3624, SEQ ID No.", "3772, SEQ ID No.", "3783, SEQ ID No.", "3788 and SEQ ID No.", "3794; or b) said nucleotide sequence is selected from the group consisting of SEQ ID No.", "5835 to SEQ ID No.", "10784.61.A nucleotide sequence, comprising a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence as claimed in claim 59; b) a nucleotide sequence comprising at least 75% identity with a nucleotide sequence as claimed in claim 59; c) a complementary or RNA nucleotide sequence corresponding to a sequence as defined in a) or b); d) a nucleotide sequence of a representative fragment of a sequence as defined in a) or c); and e) a sequence as defined in a) or c), which has been modified.", "62.A polypeptide encoded by a nucleotide sequence as claimed in claim 58.63.A polypeptide as claimed in claim 62, wherein: a) said polypeptide is selected from the group consisting of SEQ ID No.", "61, SEQ ID No.", "62, SEQ ID No.", "67, SEQ ID No.", "171, SEQ ID No.", "221, SEQ ID No.", "268, SEQ ID No.", "288, SEQ ID No.", "380, SEQ ID No.", "426, SEQ ID No.", "438, SEQ ID No.", "448, SEQ ID No.", "453, SEQ ID No.", "455, SEQ ID No.", "456, SEQ ID No.", "458, SEQ ID No.", "501, SEQ ID No.", "516, SEQ ID No.", "530, SEQ ID No.", "542, SEQ ID No.", "551, SEQ ID No.", "720, SEQ ID No.", "761, SEQ ID No.", "762, SEQ ID No.", "814, SEQ ID No.", "859, SEQ ID No.", "860, SEQ ID No.", "861, SEQ ID No.", "862, SEQ ID No.", "869, SEQ ID No.", "1079, SEQ ID No.", "1168, SEQ ID No.", "1174, SEQ ID No.", "1176, SEQ ID No.", "1413, SEQ ID No.", "1414, SEQ ID No.", "1415, SEQ ID No.", "1416, SEQ ID No.", "1417, SEQ ID No.", "1457, SEQ ID No.", "1651, SEQ ID No.", "1856, SEQ ID No.", "1869, SEQ ID No.", "2021, SEQ ID No.", "2080, SEQ ID No.", "2152, SEQ ID No.", "2162, SEQ ID No.", "2173, SEQ ID No.", "2251, SEQ ID No.", "2295, SEQ ID No.", "2306, SEQ ID No.", "2317, SEQ ID No.", "2328, SEQ ID No.", "2340, SEQ ID No.", "2342, SEQ ID No.", "2351, SEQ ID No.", "2500, SEQ ID No.", "3228, SEQ ID No.", "3230, SEQ ID No.", "3311, SEQ ID No.", "3317, SEQ ID No.", "3318, SEQ ID No.", "3319, SEQ ID No.", "3320, SEQ ID No.", "3322, SEQ ID No.", "3323, SEQ ID No.", "3326, SEQ ID No.", "3327, SEQ ID No.", "3328, SEQ ID No.", "3375, SEQ ID No.", "3376, SEQ ID No.", "3377, SEQ ID No.", "3378, SEQ ID No.", "3422, SEQ ID No.", "3489, SEQ ID No.", "3503, SEQ ID No.", "3609, SEQ ID No.", "3623, SEQ ID No.", "3624, SEQ ID No.", "3772, SEQ ID No.", "3783, SEQ ID No.", "3788 and SEQ ID No.", "3794; or b) said polypeptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID No.", "5835 to SEQ ID No.", "10784.64.A polypeptide, comprising a polypeptide selected from the group consisting of: a) a polypeptide as claimed in claim 62; b) a polypeptide exhibiting at least 80% identity with a polypeptide of sequence selected from the group consisting of SEQ ID No.", "42 to SEQ ID No.", "3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.", "5835 to SEQ ID No.", "10784; c) a fragment of at least 5 amino acids of a polypeptide of sequence selected from the group consisting of SEQ ID No.", "42 to SEQ ID No.", "3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.", "5835 to SEQ ID No.", "10784; d) a biologically active fragment of a polypeptide of sequence selected from the group consisting of SEQ ID No.", "42 to SEQ ID No.", "3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.", "5835 to SEQ ID No.", "10784; and e) a polypeptide of sequence selected from the group consisting of SEQ ID No.", "42 to SEQ ID No.", "3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.", "5835 to SEQ ID No.", "10784, or as defined in c) or d), which has been modified.", "65.A nucleotide sequence encoding a polypeptide as claimed in claim 62.66.The nucleotide sequence as claimed in claim 58, wherein said nucleotide sequence encodes a polypeptide of P. luminescens having toxin activity or antibiotic activity, or is involved in the synthesis of toxins or antibiotics.", "67.The polypeptide as claimed in claim 62, wherein said polypeptide is a polypeptide of P. luminescens having toxin activity or antibiotic activity, or is involved in the synthesis of toxins or antibiotics, or a fragment thereof.", "68.A recording medium, comprising one or more nucleotide sequences as claimed in claim 57.69.The recording medium as claimed in claim 68, wherein said recording medium is selected from the group consisting of a CD-ROM, a computer disk and a computer server.", "70.A method of identifying primers or probes for determining genes in strains related to P. luminescens comprising inspecting the sequences recorded on a recording medium as claimed in claim 68 and identifying a primer pair or probe corresponding to a desired nucleotide or to a nucleotide sequence encoding a polypeptide with a desired function.", "71.A method of studying the genetic polymorphism of strains related to P. luminescens comprising obtaining a nucleotide sequence from a strain related to P. luminescens sequencing the nucleotide sequence comparing the nucleotide sequence to the nucleotide sequences recorded on a recording medium as claimed in claim 68.72.A method for the automatic annotation of genes originating from a genome other than P. luminescens comprising obtaining genomic DNA from a strain other than P. luminescens sequencing the genomic DNA comparing the nucleotide sequence of the genomic DNA to the nucleotide sequences recorded on a recording medium as claimed in claim 68 to assign putative function associated therewith.", "73.A recording medium, comprising one or more polypeptides sequences as claimed in claim 62.74.The recording medium as claimed in claim 73, wherein said recording medium is selected from the group consisting of a CD-ROM, a computer disk and a computer server.", "75.A method of identifying primers or probes for determining genes in strains related to P. luminescens comprising inspecting the sequences recorded on a recording medium as claimed in claim 73 and identifying a primer pair or probe corresponding to a desired nucleotide or to a nucleotide sequence encoding a polypeptide with a desired function.", "76.A method of studying the genetic polymorphism of strains related to P. luminescens comprising obtaining a nucleotide sequence from a strain related to P. luminescens sequencing the nucleotide sequence comparing the nucleotide sequence to the nucleotide sequences encoding the polypeptide sequences recorded on a recording medium as claimed in claim 73.77.A method for the automatic annotation of genes originating from a genome other than P. luminescens comprising obtaining genomic DNA from a strain other than P. luminescens sequencing the genomic DNA comparing the nucleotide sequence of the genomic DNA to the nucleotide sequences encoding the polypeptide sequences recorded on a recording medium as claimed in claim 73 to assign putative function associated therewith 78.A primer or a probe, comprising one or more sequences as claimed in claim 57.79.The nucleotide sequence as claimed in claim 78, wherein said primer or probe is labeled with a radioactive compound or with a nonradioactive compound.", "80.The nucleotide sequence as claimed in claim 78, wherein said primer or probe is immobilized on a support by a covalent or noncovalent interaction.", "81.The nucleotide sequence as claimed in claim 80, wherein said support is a high density filter or a DNA chip.", "82.A DNA chip or a filter, comprising one or more nucleotide sequences as claimed in claim 78.83.The DNA chip or the filter as claimed in claim 82, further comprising one or more nucleotide sequences from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said nucleotide sequences are immobilized on the support of said chip.", "84.The DNA chip or the filter as claimed in claim 83, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens.", "85.A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a DNA chip or a filter as claimed in claim 82.86.A cloning and/or expression vector, comprising a nucleotide sequence as claimed in claim 57.87.The cloning and/or expression vector as claimed in claim 86, comprising a nucleotide sequence selected from the group consisting of SEQ ID No.", "3856 to SEQ ID No.", "5825 and SEQ ID No.", "5835 to SEQ ID No.", "10784, or fragments thereof.", "88.A host cell, transformed with a vector as claimed in claim 86.89.A plant or an animal, except a human, comprising a transformed cell as claimed in claim 88.90.A method for preparing a polypeptide, comprising culturing a cell transformed with a vector as claimed in claim 88 under conditions which allow the expression of said polypeptide, and recovering the resultant recombinant polypeptide.", "91.A recombinant polypeptide obtained by the method as claimed in claim 90.92.A method for preparing a polypeptide as claimed in claim 62, comprising chemical synthesizing said polypeptide and isolating said polypeptide.", "93.An antibody selected from the group consisting of a monoclonal antibody, a polyclonal antibody, and a chimeric antibody, or a fragment thereof, wherein said antibody specifically recognizes a polypeptide as claimed in claim 62.94.The antibody as claimed in claim 93, wherein said antibody is a labeled antibody.", "95.A method for detecting and/or identifying bacteria belonging to the species P. luminescens, in a biological sample, comprising a) contacting said biological sample with an antibody as claimed in claim 93; b) detecting the formation of an antigen-antibody complex.", "96.A method for detecting the expression of a gene of P. luminescens, comprising contacting a strain of P. luminescens with an antibody as claimed in claim 93, and detecting the formation of an antigen-antibody complex.", "97.A kit for performing the method as claimed in claim 95, comprising the following elements: a) said antibody; and at least one of either b) reagents for constituting the medium suitable for an immunoreaction; or c) reagents for detecting the antigen-antibody complexes resulting from the immunoreaction.", "98.A kit for performing the method as claimed in claim 96, comprising the following elements: a) said antibody; b) reagents for constituting the medium suitable for an immunoreaction; and c) reagents for detecting the antigen-antibody complexes resulting from the immunoreaction.", "99.The polypeptide as claimed in claim 62, wherein said polypeptide is immobilized on a support 100.The polypeptide as claimed in claim 99, wherein said support a protein chip.", "101.The polypeptide as claimed in claim 91, wherein said polypeptide is immobilized on a support 102.The polypeptide as claimed in claim 101, wherein said support a protein chip.", "103.The antibody as claimed in claim 93, wherein said polypeptide is immobilized on a support 104.The antibody as claimed in claim 103, wherein said support a protein chip.", "105.A protein chip comprising one or more polypeptide as claimed in claim 62 immobilized on the support of said chip.", "106.The protein chip as claimed in claim 105, further comprising one or more polypeptides from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said polypeptides are immobilized on the support of said chip.", "107.The protein chip as claimed in claim 106, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens.", "108.A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a protein chip as claimed in claim 105.109.A protein chip comprising one or more polypeptide as claimed in claim 91 immobilized on the support of said chip.", "110.The protein chip as claimed in claim 109, further comprising one or more polypeptides from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said polypeptides are immobilized on the support of said chip.", "111.The protein chip as claimed in claim 110, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens.", "112.A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a protein chip as claimed in claim 109.113.A protein chip comprising one or more antibodies as claimed in claim 93 immobilized on the support of said chip.", "114.The protein chip as claimed in claim 113, further comprising one or more polypeptides from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said polypeptides are immobilized on the support of said chip.", "115.The protein chip as claimed in claim 114, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens.", "116.A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a protein chip as claimed in claim 113.117.A method for detecting and/or identifying bacteria belonging to the species P. luminescens, in a biological sample, comprising: a) isolating the DNA, or cDNA from the RNA, from the biological sample; b) specifically amplifying the DNA of bacteria belonging to the species P. luminescens using at least one primer as claimed in claim 78; c) identifying the amplification products.", "118.A kit or set for detecting and/or identifying bacteria belonging to the species P. luminescens, comprising: a) a nucleotide probe and/or primer as claimed in claim 78; and at least one of b) reagents required for carrying out a hybridization reaction; or c) reagents for a DNA amplification reaction.", "119.A composition comprising one or more nucleotide sequences as claimed in claim 57.120.A pharmaceutical composition comprising the composition as claimed in claim 119 and a pharmaceutically acceptable vehicle.", "121.A biopesticidal composition comprising the composition as claimed in claim 119.122.A composition comprising one or more polypeptides as claimed in claim 62.123.A pharmaceutical composition comprising the composition as claimed in claim 122 and a pharmaceutically acceptable vehicle.", "124.A biopesticidal composition comprising the composition as claimed in claim 122.125.A composition comprising a vector as claimed in claim 86.126.A pharmaceutical composition comprising the composition as claimed in claim 125 and a pharmaceutically acceptable vehicle.", "127.A biopesticidal composition comprising the composition as claimed in claim 125.128.A composition comprising one or more antibodies as claimed in claim 93.129.A pharmaceutical composition comprising the composition as claimed in claim 128 and a pharmaceutically acceptable vehicle.", "130.A biopesticidal composition comprising the composition as claimed in claim 128.131.A method of preparing a toxin or an antibiotic comprising culturing a cell as claimed in claim 88, and expressing a polypeptide involved in production of a toxin or an antibiotic.", "132.A genomic library of a bacterium of the genus Photorhabdus.", "133.The genomic DNA library of a bacterium of the genus Photorhabdus as claimed in claim 132, wherein said DNA library is cloned into a plasmid.", "134.The genomic DNA library as claimed in claim 132, wherein said bacterium is P. luminescens or P. luminescens strain TT01.135.The genomic DNA library as claimed in claim 132, wherein said genomic DNA library is the genomic DNA library deposited with the CNCM on May 12, 2000, under accession No.", "I-2478.136.A method for identifying at least one nucleotide sequence of P. luminescens not present in the genome of another species of bacterium or for identifying at least one nucleotide sequence of a genome of a bacterium of a species other than P. luminescens and not present in the P. luminescens genome, comprising: a) aligning the genomic sequences of the other bacterial species with the nucleotide sequences of P. luminescens as claimed in claim 57; and b) analyzing the data obtained by said aligning to identify and isolate said sequences only present in one or the other genome.", "137.A method for identifying at least one nucleotide sequence of P. luminescens not present in the genome of another species of bacterium or for identifying at least one nucleotide sequence of a genome of a bacterium of a species other than P. luminescens and not present in the P. luminescens genome, comprising: a) aligning the genomic sequences of the other bacterial species with the genomic DNA library as claimed in claim 134; and b) analyzing the data obtained by said aligning to identify and isolate said sequences only present in one or the other genome." ], [ "The invention relates to the genomic sequence and to nucleotide sequences encoding polypeptides of Photorhabdus luminescens, such as polypeptides involved in operons for biosynthesis of antibiotics, or of toxins, or polypeptides with activity of the toxin or antibiotic type which can be used as a pesticide, bactericide or fungicide, and also to vectors which include said sequences, and to cells or animals transformed with these vectors.", "Photorhabdus luminescens is an entomopathogenic, commensal intestinal bacterium of a nematode and insect parasite.", "This bacterium is both a model for studying host-parasite interactions and a bacterium which has many industrial applications because of its ability to synthesize numerous toxins (insecticides, bactericides and fungicides) and to secrete numerous enzymes.", "In order to obtain an overall understanding of the genetic determinants involved in these processes, sequencing of the Photorhabdus luminescens genome was carried out.", "The choice of the TT01 strain of Photorhabdus luminescens, subspecies laumondii, the sequencing of whose genome was carried out in the present invention, is very important since this strain has several advantages: its genome is stable; it can be cultured on a Petri dish; it is central in the phylogenetic tree, and therefore representative of the species; and its associated nematode is known and cultured (Heterorhabditis bacteriophora HP88, Trinidad).", "The present invention thus relates to the nucleotide and polypeptide sequences of Photorhabdus luminescens strain TT01.Thus, an object of the present invention is to disclose the sequence of the genome of Photorhabdus luminescens strain TT01, contained in the genomic library prepared from the genome of this strain and deposited with the CNCM [French National Collection of Cultures and Micro-organisms] on May 12, 2000, under the number I-2478, and of all the genes and noncoding regulatory sequences contained in said genome.", "Photorhabdus luminescens strain TT01 is also identified in the present application by Photorhabdus luminescens , in an interchangeable manner.", "The invention also relates to novel tools for typing Photorhabdus strains.", "These tools might be of the DNA “chip” type or of another type.", "The novel characteristics of these typing tools will be as follows: rapidity and simplicity of use; high capacity for discriminating between strains; and possibility of providing information on the genomic content of the strain analyzed.", "The present invention therefore relates to an isolated nucleotide sequence derived from the Photorhabdus luminescens genome, characterized in that it comprises a sequence chosen from the sequences SEQ ID No.", "1 to SEQ ID No.", "41 and the sequences SEQ ID No.", "5826 to SEQ ID No.", "5834.The sequences SEQ ID No.", "1 to SEQ ID No.", "41 represent the sequences of 41 contigs which altogether cover the genomic sequence of Photorhabdus luminescens TT01.It has been possible to reassemble these sequences SEQ ID No.", "1 to SEQ ID No.", "41 and to smooth them back out into 9 new contigs which altogether also cover the genomic sequence of Photorhabdus luminescens TT01.The sequences SEQ ID No.", "5826 to SEQ ID No.", "5834 represent the sequences of these 9 contigs.", "The nucleotide sequences SEQ ID No.", "1 to SEQ ID No.", "41 and SEQ ID No.", "5826 to SEQ ID No.", "5834 were obtained by sequencing the Photorhabdus luminescens TT01 genome using the “shotgun” technique (cf.", "examples).", "Despite the great precision of these sequences SEQ ID No.", "1 to SEQ ID No.", "41 or SEQ ID No.", "5826 to SEQ ID No.", "5834, it is possible that these sequences do not give a 100% perfect representation, after assembly, of the nucleotide sequence of the Photorhabdus luminescens TT01 genome, and that some rare sequencing errors or indeterminations remain in these sequences.", "In the present invention, the presence of an indetermination of an amino acid is denoted by “Xaa” and that of a nucleotide is denoted by “N” or “n” in the sequence listing hereinafter.", "These few rare errors or indeterminations may be easily demonstrated and corrected by those skilled in the art using the whole chromosome and/or its representative fragments according to the invention, and standard methods of amplification, cloning and sequencing, it being possible for the sequences obtained to be easily compared, in particular by means of computer software, and using computer-readable media for recording the sequences according to the invention, as described, for example, below.", "After correction of these possible rare errors or indeterminations, the corrected nucleotide sequence obtained would still comprise at least 97%, preferably at least 98%, 98.5%, 99% or 99.9%, identity with the genomic sequence obtained after assembly of these nucleotide sequences SEQ ID No.", "1 to SEQ ID No.", "41 or SEQ ID No.", "5826 to SEQ ID No.", "5834.The present invention also relates to an isolated nucleotide sequence derived from the Photorhabdus luminescens genome, characterized in that it is chosen from: a) a nucleotide sequence comprising at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with a sequence chosen from the sequences SEQ ID No.", "1 to SEQ ID No.", "41 or SEQ ID No.", "5826 to SEQ ID No.", "5834; b) a nucleotide sequence comprising a representative fragment of a sequence chosen from the sequences SEQ ID No.", "1 to SEQ ID No.", "41 or SEQ ID No.", "5826 to SEQ ID No.", "5834; c) a nucleotide sequence complementary to a nucleotide sequence as defined in a) or b); d) a nucleotide sequence of the RNA corresponding to one of the sequences as defined in a), b) or c); e) a nucleotide sequence as defined in a), b), c) or d), which has been modified; f) a nucleotide sequence which hybridizes, under high stringency conditions, with a sequence chosen from SEQ ID No.", "1 to SEQ ID No.", "41 or SEQ ID No.", "5826 to SEQ ID No.", "5834, and which comprises at least 20 nucleotides, preferably at least 25, 30, 50, 75, 100, 150, 200, 250, 500, 750, 1 000, 1 500, 2 000 or 2 500 nucleotides.", "More particularly, a subject of the present invention is also a nucleotide sequence included in one of the sequences SEQ ID No.", "1 to SEQ ID No.", "41 or one of the sequences SEQ ID No.", "5826 to SEQ ID No.", "5834, and in that it encodes a polypeptide chosen from the polypeptides of sequence SEQ ID No.", "42 to SEQ ID No.", "3855 or from the polypeptides encoded by a sequence SEQ ID No.", "5835 to SEQ ID No.", "10784.Preferably, the polypeptides encoded by one of the sequences SEQ ID No.", "5835 to SEQ ID No.", "10784 are the polypeptides for which the sequence of at least 5 amino acids is obtained by taking as reading frame the first nucleotide of the sequences SEQ ID No.", "5835 to SEQ ID No.", "10784.Very preferably, a subject of the invention is also a nucleotide sequence, characterized in that it encodes a polypeptide whose function annotated in table I hereinafter, final column, or in table II hereinafter, penultimate column, corresponds to an activity of the toxin and/or antibiotic type, or to an operon involved in the synthesis of a toxin and/or of an antibiotic, which polypeptide is preferably chosen from: a) the polypeptides of sequences SEQ ID No.", "61, SEQ ID No.", "62, SEQ ID No.", "67, SEQ ID No.", "171, SEQ ID No.", "221, SEQ ID No.", "268, SEQ ID No.", "288, SEQ ID No.", "380, SEQ ID No.", "426, SEQ ID No.", "438, SEQ ID No.", "448, SEQ ID No.", "453, SEQ ID No.", "455, SEQ ID No.", "456, SEQ ID No.", "458, SEQ ID No.", "501, SEQ ID No.", "516, SEQ ID No.", "530, SEQ ID No.", "542, SEQ ID No.", "551, SEQ ID No.", "720, SEQ ID No.", "761, SEQ ID No.", "762, SEQ ID No.", "814, SEQ ID No.", "859, SEQ ID No.", "860, SEQ ID No.", "861, SEQ ID No.", "862, SEQ ID No.", "869, SEQ ID No.", "1079, SEQ ID No.", "1168, SEQ ID No.", "1174, SEQ ID No.", "1176, SEQ ID No.", "1413, SEQ ID No.", "1414, SEQ ID No.", "1415, SEQ ID No.", "1416, SEQ ID No.", "1417, SEQ ID No.", "1457, SEQ ID No.", "1651, SEQ ID No.", "1856, SEQ ID No.", "1869, SEQ ID No.", "2021, SEQ ID No.", "2080, SEQ ID No.", "2152, SEQ ID No.", "2162, SEQ ID No.", "2173, SEQ ID No.", "2251, SEQ ID No.", "2295, SEQ ID No.", "2306, SEQ ID No.", "2317, SEQ ID No.", "2328, SEQ ID No.", "2340, SEQ ID No.", "2342, SEQ ID No.", "2351, SEQ ID No.", "2500, SEQ ID No.", "3228, SEQ ID No.", "3230, SEQ ID No.", "3311, SEQ ID No.", "3317, SEQ ID No.", "3318, SEQ ID No.", "3319, SEQ ID No.", "3320, SEQ ID No.", "3322, SEQ ID No.", "3323, SEQ ID No.", "3326, SEQ ID No.", "3327, SEQ ID No.", "3328, SEQ ID No.", "3375, SEQ ID No.", "3376, SEQ ID No.", "3377, SEQ ID No.", "3378, SEQ ID No.", "3422, SEQ ID No.", "3489, SEQ ID No.", "3503, SEQ ID No.", "3609, SEQ ID No.", "3623, SEQ ID No.", "3624, SEQ ID No.", "3772, SEQ ID No.", "3783, SEQ ID No.", "3788 and SEQ ID No.", "3794; or b) the polypeptides encoded by the sequences SEQ ID No.", "5835 to SEQ ID No.", "10784 homologous to the sequences as defined in a) above, as indicated in the final column of table II.", "These 82 polypeptides of sequence as defined in paragraph a) above, whose function is associated with an activity of the toxin or antibiotic type, or their homologous polypeptide of table II as defined in paragraph b) above, could be identified, for example, by the presence of a consensus motif associated with these functions or by the presence of sequences juxtaposing them on the genome and involved in this type of activity.", "More generally, the present invention also relates to the nucleotide sequences derived from SEQ ID No.", "1 to SEQ ID No.", "41 or SEQ ID No.", "5826 to SEQ ID No.", "5834, and encoding a polypeptide of P. luminescens, such that they can be isolated from SEQ ID No.", "1 to SEQ ID No.", "41 or SEQ ID No.", "5826 to SEQ ID No.", "5834.In addition, the nucleotide sequences characterized in that they comprise a nucleotide sequence chosen from: a) a nucleotide sequence encoding a polypeptide chosen from the sequences SEQ ID No.", "42 to SEQ ID No.", "3855 or from the polypeptides encoded by the sequences SEQ ID No.", "5835 to SEQ ID No.", "10784, preferably from the 82 polypeptide sequences above selected for their function associated with an activity of the toxin or antibiotic type, or their homologous peptide as defined in table II, in the final column; b) a nucleotide sequence comprising at least 75% identity with a nucleotide sequence as defined in a), preferably at least 80%, 85%, 90%, 95%, 98% or 99% identity; c) a complementary or RNA nucleotide sequence corresponding to a sequence as defined in a) or b); d) a nucleotide sequence of a representative fragment of a sequence as defined in a) or c); and e) a sequence as defined in a) or c), which has been modified are also subjects of the invention.", "The terms “nucleic acid”, “nucleic acid sequence”, “polynucleotide”, “oligonucleotide”, “polynucleotide sequence” and “nucleotide sequence”, terms which will be used indifferently in the present description, are intended to denote a precise chain of nucleotides, which may or may not be modified, making it possible to define a fragment or a region of a nucleic acid, which may or may not comprise unnatural nucleotides, and which may correspond equally to a double-stranded DNA, a single-stranded DNA and products of transcription of said DNAs.", "Thus, the nucleic acid sequences according to the invention also encompass PNAs (Peptide Nucleic Acids).", "It should be understood that the present invention does not concern the nucleotide sequences in their natural chromosomal environment, i.e.", "in their natural state.", "They are sequences which have been isolated and/or purified, i.e.", "they have been taken directly or indirectly, for example by copying, their environment having been at least partially modified.", "The nucleic acids obtained by chemical synthesis are thus also intended to be denoted.", "For the purpose of the present invention, the term “percentage identity” between two nucleic acid or amino acid sequences is intended to denote a percentage of nucleotides or of amino acid residues which are identical between the two sequences to be compared, obtained after best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length.", "The term “best alignment” or “optimal alignment” is intended to denote the alignment for which the percentage identity determined as below is the highest.", "Sequence comparisons between two nucleic acid or amino acid sequences are conventionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by “window of comparison” so as to identify and compare the local regions of sequence similarity.", "The optimal alignment of the sequences for the comparison can be carried out, besides manually, by means of the local homology algorithm of Smith and Waterman (1981, Ad.", "App.", "Math., 2:482), by means of the local homology algorithm of Neddleman and Wunsch (1970, J. Mol.", "Biol., 48:443), by means of the similarity search method of Pearson and Lipman (1988, Proc.", "Natl.", "Acad.", "Sci.", "USA, 85:2444), by means of computer programs using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.).", "In order to obtain the optimal alignment, the BLAST program is preferably used, with the BLOSUM 62 matrix.", "The PAM or PAM250 matrices can also be used.", "The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences when optimally aligned, the nucleic acid or amino acid sequence to be compared possibly comprising additions or deletions with respect to the reference sequence, for optimal alignment between these two sequences.", "The percentage identity is calculated by determining the number of identical positions for which the nucleotide or amino acid residue is identical in the two sequences, dividing this number of identical positions by the total number of positions compared, and multiplying the result obtained by 100 so as to obtain the percentage identity between these two sequences.", "The expression “nucleic acid sequences exhibiting a percentage identity of at least 75%, preferably 80%, 85%, 90%, 95%, 98% or 99%, after optimal alignment, with a reference sequence” is intended to denote the nucleic acid sequences exhibiting, compared to the reference nucleic acid sequence, certain modifications such as in particular a deletion, a truncation, an extension, a chimeric fusion and/or a substitution, in particular of the point type, and the nucleic acid sequence of which exhibits at least 75%, preferably 80%, 85%, 90%, 95%, 98% or 99%, identity, after optimal alignment, with the reference nucleic acid sequence.", "They are preferably sequences for which the complementary sequences are capable of hybridizing specifically with the reference sequences.", "Preferably, the specific or high stringency hybridization conditions will be such that they provide at least 75%, preferably 80%, 85%, 90%, 95%, 98% or 99%, identity, after optimal alignment, between one of the two sequences and the sequence complementary thereto.", "A hybridization under high stringency conditions means that the conditions of temperature and of ionic strength are chosen such that they make it possible to maintain the hybridization between two complementary DNA fragments.", "By way of illustration, high stringency conditions for the hybridization step for the purposes of defining the polynucleotide fragments described above are advantageously as follows.", "The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42° C. for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5×SSC (1×SSC corresponds to a solution of 0.15M NaCl+0.015M sodium citrate), 50% of formamide, 7% of sodium dodecyl sulfate (SDS), 10× Denhardt's, 5% of dextran sulfate and 1% of salmon sperm DNA; (2) hybridization per se for 20 hours at a temperature which depends on the length of the probe (i.e.", ": 42° C. for a probe>100 nucleotides in length) followed by 2 washes of 20 minutes at 20° C. in 2×SSC+2% SDS and 1 wash of 20 minutes at 20° C. in 0.1×SSC+0.1% SDS.", "The final wash is carried out in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. for a probe>100 nucleotides in length.", "The high stringency hybridization conditions described above for a polynucleotide of defined length can be adjusted by those skilled in the art for longer or shorter oligonucleotides, according to the teaching of Sambrook et al.", "(1989, Molecular cloning: a laboratory manual.", "2nd Ed.", "Cold Spring Harbor).", "In addition, the expression “representative fragment of sequences according to the invention” is intended to denote any nucleotide fragment having at least 15 consecutive nucleotides, preferably at least 20, 25, 30, 50, 75, 100, 150, 200, 300 and 450 consecutive nucleotides, of the sequence from which it is derived.", "The term “representative fragment” is intended to mean in particular a nucleic acid sequence encoding a biologically active fragment of a polypeptide, as defined later.", "The term “representative fragment” is also intended to mean the intergenic sequences, and in particular the nucleotide sequences carrying the regulatory signals (promoters, terminators, or even enhancers, etc).", "Among said representative fragments, preference is given to those having nucleotide sequences corresponding to open reading frames, referred to as ORF sequences, included in general between an initiation codon and a stop codon, or between two stop codons, and encoding polypeptides, preferably with at least 100 amino acids, such as, for example, without being limited thereto, the ORF sequences which will subsequently be described.", "The numbering of the ORF nucleotide sequences which will subsequently be used in the present description corresponds to the numbering of the amino acid sequences of the proteins encoded by said ORFs for the sequences SEQ ID No.", "42 to SEQ ID No.", "3855.The numbering of the ORF nucleotide sequences SEQ ID No.", "5835 to SEQ ID No.", "10784 will subsequently be used in the present description for the numbering of the amino acid sequences of the proteins encoded by said ORFs SEQ ID No.", "5835 to SEQ ID No.", "10784.The representative fragments according to the invention can be obtained, for example, by specific amplification such as PCR or after digestion, with suitable restriction enzymes, of nucleotide sequences according to the invention, this method being described in particular in the work by Sambrook et al.", "Said representative fragments can also be obtained by chemical synthesis when they are not too long, according to methods well known to those skilled in the art.", "The sequences containing sequences of the invention, or representative fragments, are also intended to include the sequences which are naturally framed by sequences which exhibit at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the sequences according to the invention.", "The term “modified nucleotide sequence” is intended to mean any nucleotide sequence obtained by mutagenesis according to techniques well known to those skilled in the art, and comprising modifications compared to the normal sequences, preferably at most 10% of modified nucleotides compared to these normal sequences, for example mutations in the regulatory and/or promoter sequences for expression of the polypeptide, in particular leading to a modification of the level of expression or of the activity of said polypeptide.", "The term “modified nucleotide sequence” is also intended to mean any nucleotide sequence encoding a modified polypeptide as defined below.", "The representative fragments according to the invention can also be probes or primers, which can be used in methods for detecting, identifying, assaying or amplifying nucleic acid sequences.", "For the purpose of the invention, a probe or primer is defined as being a single-stranded nucleic acid fragment or a denatured double-stranded fragment comprising, for example, from 12 bases to a few kb, in particular from 15 to a few hundred bases, preferably from 15 to 50 or 100 bases, and having a specificity of hybridization under given conditions so as to form a hybridization complex with a target nucleic acid.", "The probes and primers according to the invention can be labeled directly or indirectly with a radioactive or nonradioactive compound using methods well known to those skilled in the art, in order to obtain a detectable and/or quantifiable signal (patent FR 78 10975 and bDNA of Chiron EP 225 807 and EP 510 085).", "The unlabeled sequences of polynucleotides according to the invention can be used directly as a probe or primer.", "The sequences are generally labeled to obtain sequences which can be used for many applications.", "The labeling of the primers or of the probes according to the invention is carried out with radioactive elements or with nonradioactive molecules.", "Among the radioactive isotopes used, mention may be made of 32P, 33P 35S, 3H or 125I.", "The nonradioactive entities are selected from ligands such as biotin, avidin, streptavidin or digoxigenin, haptens, dyes, and luminescent agents such as radioluminescent, chemiluminescent, bioluminescent, fluorescent or phosphorescent agents.", "The polynucleotides according to the invention can thus be used as a primer and/or a probe in methods using in particular the PCR (polymerase chain reaction) technique (Rolfs et al., 1991, Berlin: Springer-Verlag).", "This technique requires choosing pairs of oligonucleotide primers framing the fragment which must be amplified.", "Reference may, for example, be made to the technique described in U.S. Pat.", "No.", "4,683,202.The amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatography technique such as gel filtration or ion exchange chromatography, and then sequenced.", "The specificity of the amplification can be controlled using the nucleotide sequences of polynucleotides of the invention as a matrix, plasmids containing these sequences or else the derived amplification products.", "The amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments.", "The invention is also directed toward the nucleic acids which can be obtained by amplification using primers according to the invention.", "Other techniques for amplifying the target nucleic acid can advantageously be employed as an alternative to PCR (PCR-like), using a pair of primers of nucleotide sequences according to the invention.", "The term “PCR-like” is intended to denote all the methods using direct or indirect reproductions of nucleic acid sequences, or else in which the labeling systems have been amplified; these techniques are, of course, known.", "In general, they involve amplification of the DNA with a polymerase; when the sample of origin is an RNA, a reverse transcription should be carried out beforehand.", "A very large number of methods currently exist for this amplification, such as, for example, the SDA (strand displacement amplification) technique (Walker et al., 1992, Nucleic Acids Res., 20:1691), the TAS (transcription-based amplification system) technique described by Kwoh et al.", "(1989, Proc.", "Natl.", "Acad.", "Sci.", "USA, 86, 1173), the 3SR (self-sustained sequence replication) technique described by Guatelli et al.", "(1990, Proc.", "Natl.", "Acad.", "Sci.", "USA, 87: 1874), the NASBA (nucleic acid sequence based amplification) technique described by Kievitis et al.", "(1991, J. Virol.", "Methods, 35, 273), the TMA (transcription mediated amplification) technique, the LCR (ligase chain reaction) technique described by Landegren et al.", "(1988, Science, 241, 1077), the RCR (repair chain reaction) technique described by Segev (1992, Kessler C. Springer Verlag, Berlin, N.Y., 197-205), the CPR (cycling probe reaction) technique described by Duck et al.", "(1990, Biotechniques, 9, 142) and the Q-beta-replicase amplification technique described by Miele et al.", "(1983, J. Mol.", "Biol., 171, 281).", "Some of these techniques have since been improved.", "When the target polynucleotide to be detected is an mRNA, an enzyme of the reverse transcriptase type is advantageously used, prior to carrying out an amplification reaction using the primers according to the invention, or to carrying out a method of detection using the probes of the invention, in order to obtain a cDNA from the mRNA contained in the biological sample.", "The cDNA obtained will then serve as a target for the primers or the probes used in the method of amplification or of detection according to the invention.", "The probe hybridization technique can be carried out in various ways (Matthews et al., 1988, Anal.", "Biochem., 169, 1-25).", "The most general method consists in immobilizing the nucleic acid, extracted from the cells of various tissues or from cells in culture, on a support (such as nitrocellulose, nylon or polystyrene) and in incubating, under well-defined conditions, the immobilized target nucleic acid with the probe.", "After hybridization, the excess probe is removed and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, of the fluorescence or of the enzyme activity associated with the probe).", "According to another embodiment of the nucleic acid probes according to the invention, the latter can be used as capture probes.", "In this case, a probe, termed “capture probe”, is immobilized on a support and is used to capture, by specific hybridization, the target nucleic acid obtained from the biological sample to be tested, and the target nucleic acid is then detected using a second probe, termed “detection probe”, labeled with a readily detectable element.", "Among the advantageous nucleic acid fragments, mention should thus in particular be made of antisense oligonucleotides, i.e.", "oligonucleotides whose structure provides, by hybridization with the target sequence, inhibition of the expression of the corresponding product.", "Mention should also be made of sense oligonucleotides which, by interaction with proteins involved in regulating the expression of the corresponding product, will induce either inhibition or activation of this expression.", "Preferably, the probes or primers according to the invention are immobilized on a support in a covalent or noncovalent manner.", "In particular, the support can be a DNA chip or a high or medium density filter, also a subject of the present invention (patents WO 97/29212, WO 98/27317, WO 97/10365 and WO 92/10588).", "The term “DNA chip” or “high density filter” is intended to denote a support on which are attached DNA sequences, it being possible to pinpoint each one of them by its geographical location.", "These chips or filters differ mainly by their size, the material of the support and, optionally, the number of DNA sequences which are attached thereto.", "The probes or primers according to the first invention can be attached to solid supports, in particular the DNA chips, by various methods of production.", "In particular, in situ synthesis can be carried out by photochemical addressing or by inkjet.", "Other techniques consist in carrying out an ex situ synthesis and attaching the probes to the support of the DNA chip by mechanical or electronic addressing or by inkjet.", "These various methods are well known to those skilled in the art.", "A nucleotide sequence (probe or primer) according to the invention therefore makes it possible to detect and/or amplify specific nucleic acid sequences.", "In particular, the detection of these said sequences is facilitated when the probe is attached to a DNA chip or to a high density filter.", "The use of DNA chips or of high density filters in fact makes it possible to determine the gene expression in an organism having a genomic sequence close to P. luminescens, and to type the strain in question.", "The genomic sequence of P. luminescens, supplemented by the identification of the genes of these organisms, as presented in the present invention, serves as a basis for constructing these DNA chips or filters.", "The preparation of these filters or chips consists in synthesizing oligonucleotides, corresponding to the 5′ and 3′ ends of the genes or to more internal fragments, in order to amplify fragments of an appropriate length, for example of between approximately 300 and 800 bases.", "These oligonucleotides are chosen using the genomic sequence and its annotations disclosed in the present invention.", "The temperature for pairing these oligonucleotides at the corresponding places on the DNA should be approximately the same for each oligonucleotide.", "This makes it possible to prepare DNA fragments corresponding to each gene using appropriate PCR conditions in a highly automated environment.", "The amplified fragments are then immobilized on filters or supports made of glass, silicon or synthetic polymers and these media are used for the hybridization.", "The availability of such filters and/or chips and of the corresponding annotated genomic sequence makes it possible to study the expression of large sets, or even all, of the genes of Photorhabdus luminescens , in particular of P. luminescens TT01, by preparing the complementary DNAs and hybridizing them to the DNA or to the oligonucleotides immobilized on the filters or the chips.", "Similarly, the filters and/or the chips make it possible to study the strain or species variability by preparing the DNA of these organisms and hybridizing them to the DNA or to the oligonucleotides immobilized on the filters or the chips.", "The differences between the genomic sequences of the various strains or species can greatly affect the intensity of the hybridization and, consequently, disturb the interpretation of the results.", "It may therefore be necessary to have the precise sequence of the genes of the strain intended to be studied.", "The method for detecting the genes described later in detail, involving determining the sequence of random fragments of a genome and organizing them according to the sequence of the genome of P. luminescens, in particular of P. luminescens TT01, disclosed in the present invention, may be very useful.", "The nucleotide sequences according to the invention can be used in DNA chips to carry out mutation analysis.", "This analysis is based on constituting chips capable of analyzing each base of a nucleotide sequence according to the invention.", "For this purpose, use may in particular be made of the techniques of microsequencing on a DNA chip.", "The mutations are detected by extension of immobilized primers which hybridize to the matrix of the analyzed sequences, just at a position adjacent to that of the mutated nucleotide sought.", "A single-stranded, RNA or DNA matrix of the sequences to be analyzed will advantageously be prepared according to conventional methods, using products amplified according to PCR-type techniques.", "The single-stranded DNA or RNA matrices thus obtained are then deposited onto the DNA chip, under conditions which allow their specific hybridization to the immobilized primers.", "A thermostable polymerase, for example Tth or Taq DNA polymerase, specifically extends the 3′ end of the immobilized primer with a labeled nucleotide analog complementary to the nucleotide at the position of the variable site; for example, thermocycling is carried out in the presence of fluorescent dideoxyribonucleotides.", "The experimental conditions will be adjusted in particular to the chips used, to the immobilized primers, to the polymerases used and to the labeling system chosen.", "One advantage of microsequencing, compared to techniques based on probe hybridization, is that it makes it possible to identify all the variable nucleotides with optimal discrimination under homogeneous reaction conditions; when used on DNA chips, it allows optimal resolution and specificity for the routine and industrial detection of mutations in a multiplex.", "A DNA chip or a filter can be an extremely advantageous tool for determining, detecting and/or identifying a microorganism.", "Thus, the DNA chips according to the invention which also contain at least one nucleotide sequence of a microorganism other than Photorhabdus luminescens, immobilized on the support of said chip, are also preferred.", "The microorganism chosen is preferably done so from the bacteria of the genus Photorhabdus (hereinafter referred to as P. luminescens-related bacteria), or the variants of Photorhabdus luminescens TT01.A DNA chip or a filter according to the invention is a very useful element of certain kits or sets for detecting and/or identifying microorganisms, in particular bacteria belonging to the species Photorhabdus luminescens , which are also the subject of the invention.", "Moreover, the DNA chips or the filters according to the invention, containing probes or primers specific for Photorhabdus luminescens , are very advantageous elements of kits or sets for detecting and/or quantifying the expression of Photorhabdus luminescens genes.", "Specifically, the control of gene expression is a critical point for optimizing the growth and yield of a strain, either by allowing the expression of one or more new genes, or by modifying the expression of genes already present in the cell.", "The present invention provides all of the sequences naturally active in P. luminescens which allow gene expression.", "It thus makes it possible to determine all the sequences expressed in P. luminescens.", "It also provides a tool for pinpointing the genes whose expression follows a given pattern.", "To do this, the DNA of all or some of the genes of P. luminescens can be amplified using primers according to the invention, and then attached to a support such as, for example, glass or nylon or a DNA chip, in order to construct a tool for following the expression profile of these genes.", "This tool, consisting of this support containing the coding sequences, serves as a matrix of hybridization to a mixture of labeled molecules reflecting the messenger RNAs expressed in the cell (in particular the labeled probes according to the invention).", "By repeating this experiment at various moments and combining all of these data using suitable processing, the expression profiles of all these genes are then obtained.", "The knowledge of the sequences which follow a given regulatory scheme can also be exploited to search for, in a directed manner, for example by homology, other sequences following, overall, but in a slightly different way, the same regulatory scheme.", "In addition, it is possible to isolate each control sequence present upstream of the segments acting as probes and to follow the activity thereof using a suitable means such as a reporter gene (luciferase, β-galactosidase, GFP).", "These isolated sequences can then be modified and assembled by metabolic engineering with sequences of interest with a view to the optimal expression thereof.", "The invention also relates to the polypeptides encoded by a nucleotide sequence according to the invention, preferably by a representative fragment of the preceding sequences, corresponding to an ORF sequence.", "In particular, the polypeptides of Photorhabdus luminescens TT01 of SEQ ID No.", "42 to SEQ ID No.", "3855 or encoded by SEQ ID No.", "5835 to SEQ ID No.", "10784 are a subject of the invention.", "The invention also comprises the polypeptides characterized in that they comprise a polypeptide chosen from: a) a polypeptide of sequence SEQ ID No.", "42 to SEQ ID No.", "3855 or encoded by a sequence SEQ ID No.", "5835 to SEQ ID No.", "10784; b) a polypeptide exhibiting at least 80%, preferably 85%, 90%, 95% and 98%, identity with a polypeptide according to the invention; c) a fragment of at least 5 amino acids of a polypeptide as defined in a); d) a biologically active fragment of a polypeptide as defined in a); and e) a polypeptide as defined in a), b), c) or d), which has been modified.", "The nucleotide sequences encoding the polypeptides described above are also a subject of the invention.", "In the present description, the terms “polypeptides”, “polypeptide sequences”, “peptides” and “proteins” are interchangeable.", "The term “polypeptide” comprises any amino acid sequence making it possible to generate an antibody response.", "It should be understood that the invention does not concern the polypeptides in natural form, i.e.", "they are not taken in their natural environment.", "On the other hand, it concerns those which it has been possible to isolate or obtain by purification from natural sources, or else those obtained by genetic recombination or by chemical synthesis, and they can then comprise unnatural amino acids as will be described below.", "The expression “polypeptide exhibiting a certain percentage identity with another”, for which the expression “homologous polypeptide” will also be used, is intended to denote the polypeptides exhibiting, compared to the natural polypeptides, certain modifications, in particular a deletion, addition or substitution of at least one amino acid, a truncation, an extension, a chimeric solution and/or a mutation, or the polypeptides exhibiting post-translational modifications.", "Among the homologous polypeptides, preference is given to those in which the amino acid sequence exhibits at least 80%, preferably 85%, 90%, 95%, 98% or 99%, identity, after optimal alignment, with the amino acid sequences of the polypeptides according to the invention.", "In the case of a substitution, one or more consecutive or nonconsecutive amino acid(s) may be replaced with “equivalent” amino acids.", "The expression “equivalent amino acids” is here aimed at denoting any amino acid capable of being substituted for one of the amino acids of the basic structure without, however, essentially modifying the biological activities of the corresponding peptides as they will be defined subsequently.", "These equivalent amino acids can be determined either based on their structural homology with the amino acids for which they substitute, or based on results of comparative biological activity assays between the various polypeptides liable to be produced.", "By way of example, mention is made of the substitution possibilities which can be made without this resulting in a profound modification of the biological activity of the corresponding modified polypeptide.", "It is thus possible to replace leucine with valine or isoleucine, aspartic acid with glutamic acid, glutamine with asparagine, arginine with lysine, etc, it naturally being possible to envision the reverse substitutions under the same conditions.", "The homologous polypeptides also correspond to the polypeptides encoded by the nucleotide sequences which exhibit a certain percentage identity with the nucleotide sequences of the invention or which are identical, as previously defined, and thus comprise, in the present definition, mutated polypeptides or polypeptides corresponding to inter- or intraspecies variations which can exist in Photorhabdus, and which correspond in particular to truncations, substitutions, deletions and/or additions of at least one amino acid residue.", "It is understood that the percentage identity between two polypeptides is calculated in the same way as between two nucleic acid sequences.", "Thus, the percentage identity between two polypeptides is calculated, after optimal alignment of these two sequences, on a window of maximum homology.", "To define said window of maximum homology, the same algorithms as for the nucleic acid sequences can be used.", "The expression “biologically active fragment of a polypeptide according to the invention” is intended to denote in particular a polypeptide fragment, as defined below, exhibiting at least one of the biological characteristics of the polypeptides according to the invention, in particular in that it is capable of exerting, in general, even a partial activity, such as, for example: an enzymatic (metabolic) activity or an activity which may be involved in the biosynthesis or the biodegradation of organic or inorganic compounds, or preferably a toxic or antibiotic activity, in particular for insects or microorganisms (bacteria or fungi), or else an activity involved in the biosynthesis of these toxins or antibiotics; such proteins with enzymatic activity may in particular be used in methods for screening and/or selecting compounds capable of modifying this activity, in particular of inhibiting it; a structural activity (cell envelope, chaperone molecule, ribosome).", "Proteins corresponding especially to extramembrane proteins may in particular be used as an immunogen for producing mono- or polyclonal antibodies directed specifically against these extramembrane proteins; a transport activity (energy transport, ion transport); or an activity in protein secretion; an activity in the process of replication, amplification, preparation, transcription, translation or maturation, in particular of DNA, of RNA or of proteins.", "The expression “polypeptide fragment according to the invention” is intended to denote a polypeptide comprising at least 5 amino acids, preferably 10, 15, 25, 50, 100 and 150 amino acids.", "The polypeptide fragments can correspond to isolated or purified fragments naturally present in the strains of Photorhabdus, or to fragments which can be obtained by cleavage of said polypeptide with a proteolytic enzyme, such as trypsin or chymotrypsin or collagenase, or with a chemical reagent (cyanogen bromide, CNBr) or by placing said polypeptide in a very acidic environment (for example at pH=2.5).", "Polypeptide fragments can also be prepared by chemical synthesis, from hosts transformed with an expression vector according to the invention which contain a nucleic acid which allows expression of said fragment and is placed under the control of the appropriate regulatory and/or expression elements.", "The term “modified polypeptide” of a polypeptide according to the invention is intended to denote a polypeptide obtained by genetic recombination or by chemical synthesis as described later, which exhibits at least one modification compared to the normal sequence, and preferably at most 10% of modified amino acids compared to the normal sequence.", "These modifications may in particular be made on amino acids necessary for the specificity or the effectiveness of the activity, or responsible for the structural conformation, for the charge or for the hydrophobicity of the polypeptide according to the invention.", "It is thus possible to create polypeptides with equivalent, increased or decreased activity, or with equivalent, stricter or broader specificity.", "Among the modified polypeptides, mention should be made of the polypeptides in which up to five amino acids can be modified, truncated at the N- or C-terminal end, or else deleted, or added.", "As is indicated, the aim of the modifications to a polypeptide is in particular: to allow its use in methods of biosynthesis or of biodegradation of organic or inorganic compounds, or in the biosynthesis of toxins or of antibiotics, to allow its use in methods of replication, of amplification, of repair and of regulation of transcription, translation or maturation in particular of DNA, RNA or proteins; to allow its enhanced secretion, to modify its solubility, or the effectiveness or specificity of its activity, or else to facilitate its purification.", "Chemical synthesis also has the advantage of being able to use unnatural amino acids or nonpeptide bonds.", "Thus, it may be advantageous to use unnatural amino acids, for example in the D form, or amino acid analogs, in particular sulfur-containing forms.", "The invention also relates to the nucleic acid or peptide sequences according to the present invention with the exception of the nucleic acid or peptide sequences described in documents WO 99/54472, WO 99/42589, WO 99/03328, WO 98/08932 and EP 0 823 215.The present invention provides the nucleotide sequence of the Photorhabdus luminescens TT01 genome in the form of 41 contigs or in the form of 9 contigs, and also some polypeptide sequences.", "The nucleic acid or peptide sequences below, characterized by their function, can also be identified by their nucleotide and amino acid sequence with reference to table I. Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01 with activity of the toxin and/or antibiotic type, or involved in the synthesis of these toxins and/or antibiotics.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in amino acid biosynthesis.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the biosynthesis of cofactors, prosthetic groups and transporters.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a cell envelope polypeptide or a polypeptide present at the surface of Photorhabdus luminescens TT01, or one of its fragments.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the cellular machinery.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in central intermediate metabolism.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in energy metabolism.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in fatty acid and phospholipid metabolism.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the metabolism of nucleotides, purines, pyrimidines or nucleosides.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in regulatory functions.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the replication process.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the transcription process.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the translation process.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the process of protein transport and binding.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in adaptation to atypical conditions.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, [lacuna] in sensitivity to medicinal products and analogs.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in functions relating to transposons.", "Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide specific for Photorhabdus luminescens TT01, or one of its fragments.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, [lacuna] activity of the toxin and/or antibiotic type, or involved in the synthesis of these toxins and/or antibiotics.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in amino acid biosynthesis.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the biosynthesis of cofactors, prosthetic groups and transporters.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a cell envelope polypeptide or a surface polypeptide of Photorhabdus luminescens TT01, or one of its fragments.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the cellular machinery.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in central intermediate metabolism.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in energy metabolism.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in fatty acid and phospholipid metabolism.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the metabolism of nucleotides, purines, pyrimidines or nucleosides.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in regulatory functions.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the replication process.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the transcription process.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the translation process.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the process of protein transport and binding.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in adaptation to atypical conditions.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, [lacuna] in sensitivity to medicinal products and analogs.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in functions relating to transposons.", "In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide specific for Photorhabdus luminescens TT01, or one of its fragments.", "A subject of the invention is also the operons involved in the synthesis of antibiotics and/or of toxins.", "Table I provides the list of some polypeptides according to the invention, and also their location in the contigs represented by SEQ ID No.", "1 to SEQ ID No.", "41, and the similarities observed after comparison in the databases.", "Table II provides the list of some polypeptides according to the invention, and also their location in the contigs represented by SEQ ID No.", "5826 to SEQ ID No.", "5834, and the similarities observed after comparison in the databases.", "In table II, contigs 1 to 9 are identified by the sequences SEQ ID No.", "5826 to SEQ ID No.", "5834.Entirely preferably, a subject of the invention is also the polypeptides whose functions annotated in table I, final column, or whose functions annotated in table II, penultimate column, correspond to activities of the toxin and/or antibiotic type, or to polypeptides involved in the synthesis of these toxins and/or antibiotics, which polypeptides are preferably chosen from: a) the polypeptides of sequence SEQ ID No.", "61, SEQ ID No.", "62, SEQ ID No.", "67, SEQ ID No.", "171, SEQ ID No.", "221, SEQ ID No.", "268, SEQ ID No.", "288, SEQ ID No.", "380, SEQ ID No.", "426, SEQ ID No.", "438, SEQ ID No.", "448, SEQ ID No.", "453, SEQ ID No.", "455, SEQ ID No.", "456, SEQ ID No.", "458, SEQ ID No.", "501, SEQ ID No.", "516, SEQ ID No.", "530, SEQ ID No.", "542, SEQ ID No.", "551, SEQ ID No.", "720, SEQ ID No.", "761, SEQ ID No.", "762, SEQ ID No.", "814, SEQ ID No.", "859, SEQ ID No.", "860, SEQ ID No.", "861, SEQ ID No.", "862, SEQ ID No.", "869, SEQ ID No.", "1079, SEQ ID No.", "1168, SEQ ID No.", "1174, SEQ ID No.", "1176, SEQ ID No.", "1413, SEQ ID No.", "1414, SEQ ID No.", "1415, SEQ ID No.", "1416, SEQ ID No.", "1417, SEQ ID No.", "1457, SEQ ID No.", "1651, SEQ ID No.", "1856, SEQ ID No.", "1869, SEQ ID No.", "2021, SEQ ID No.", "2080, SEQ ID No.", "2152, SEQ ID No.", "2162, SEQ ID No.", "2173, SEQ ID No.", "2251, SEQ ID No.", "2295, SEQ ID No.", "2306, SEQ ID No.", "2317, SEQ ID No.", "2328, SEQ ID No.", "2340, SEQ ID No.", "2342, SEQ ID No.", "2351, SEQ ID No.", "2500, SEQ ID No.", "3228, SEQ ID No.", "3230, SEQ ID No.", "3311, SEQ ID No.", "3317, SEQ ID No.", "3318, SEQ ID No.", "3319, SEQ ID No.", "3320, SEQ ID No.", "3322, SEQ ID No.", "3323, SEQ ID No.", "3326, SEQ ID No.", "3327, SEQ ID No.", "3328, SEQ ID No.", "3375, SEQ ID No.", "3376, SEQ ID No.", "3377, SEQ ID No.", "3378, SEQ ID No.", "3422, SEQ ID No.", "3489, SEQ ID No.", "3503, SEQ ID No.", "3609, SEQ ID No.", "3623, SEQ ID No.", "3624, SEQ ID No.", "3772, SEQ ID No.", "3783, SEQ ID No.", "3788 and SEQ ID No.", "3794; or b) the polypeptides encoded by the sequences SEQ ID No.", "5835 to SEQ ID No.", "10784, homologous to the sequences as defined in a), as indicated in the final column of table II.", "The subject of the present invention is also the nucleotide and/or polypeptide sequences according to the invention, characterized in that said sequences are recorded on a recording medium, the form and nature of which facilitate the reading, analysis and/or exploitation of said sequence(s).", "These media may also contain other information extracted from the present invention, in particular the similarities with already known sequences, and/or information concerning the nucleotide and/or polypeptide sequences of a cell of a plant, of an animal or of a microorganism other than P. luminescens, in particular a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, or a variant of P. luminescens, in order to facilitate the comparative analysis and the exploitation of the results obtained.", "Among these said recording media, preference is given in particular to computer-readable media, such as magnetic, optical, electrical or hybrid media, in particular computer disks, CD-ROMs and computer servers.", "Such recording media are also a subject of the invention.", "The recording media according to the invention, with the information provided, are very useful for choosing nucleotide primers or probes for determining genes in Photorhabdus luminescens TT01 or strains related to this organism.", "Similarly, the use of these media for studying the genetic polymorphism of strains related to Photorhabdus luminescens TT01, in particular by determining the regions of colinearity, is very useful insofar as these media provide not only the nucleotide sequence of the Photorhabdus luminescens TT01 genome, but also the genomic organization in said sequence.", "Thus, the uses of recording media according to the invention are also subjects of the invention.", "The analysis of homology between various sequences is in fact advantageously performed using sequence comparison programs, such as the Blast program, or the programs of the GCG package, described above.", "The invention is also directed toward the cloning and/or expression vectors which contain a nucleotide sequence according to the invention.", "The vectors according to the invention preferably comprise elements which allow the expression and/or the secretion of the nucleotide sequences in a given host cell.", "The vector should then comprise a promoter, translation initiation and termination signals, and also regions suitable for regulating transcription.", "It must be possible for it to be maintained stably in the host cell and it may optionally contain particular signals which specify secretion of the translated protein.", "These various elements are chosen and optimized by those skilled in the art as a function of the cellular host used.", "To this effect, the nucleotide sequences according to the invention can be inserted into vectors which replicate autonomously in the host chosen, or may be vectors which integrate in the host chosen.", "Such vectors are prepared by methods commonly used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods, such as lipofection, electroporation, heat shock or chemical methods.", "The vectors according to the invention are, for example, vectors of plasmid or viral origin.", "They are useful for transforming host cells in order to clone or express the nucleotide sequences according to the invention.", "Among these vectors, preference is also given to the cloning and/or expression vectors according to the invention, characterized in that they contain a nucleotide sequence chosen from the sequences SEQ ID No.", "3856 to SEQ ID No.", "5825, and SEQ ID No.", "5835 to SEQ ID No.", "10784, or their fragment derived from the P. luminescens genome, in particular the sequences encoding the polypeptides with toxin or antibiotic activity or involved in these activities, in particular those mentioned above whose functions annotated in table I below correspond to these activities.", "The invention also comprises the host cells transformed with a vector according to the invention.", "The cellular host may be chosen from prokaryotic or eukaryotic systems, for example bacterial cells, but also yeast cells or animal cells, in particular mammalian cells.", "Insect cells or plant cells may also be used.", "The preferred host cells according to the invention are in particular prokaryotic cells, preferably bacteria belonging to the genus Photorhabdus or to the species Photorhabdus luminescens , more particularly Photorhabdus luminescens TT01.The invention also relates to the plants and animals, except humans, which comprise a transformed cell according to the invention.", "The transformed cells according to the invention can be used in methods for preparing recombinant polypeptides according to the invention.", "The methods for preparing a polypeptide according to the invention in recombinant form, characterized in that they use a vector and/or a cell transformed with a vector according to the invention, are themselves included in the present invention.", "Preferably, a cell transformed with a vector according to the invention is cultured under conditions which allow the expression of said polypeptide, and said recombinant polypeptide is recovered.", "As has been mentioned, the cellular host can be chosen from prokaryotic or eukaryotic systems.", "In particular, it is possible to identify nucleotide sequences according to the invention which facilitate secretion in such a prokaryotic or eukaryotic system.", "A vector according to the invention carrying such a sequence can therefore be advantageously used for producing recombinant proteins intended to be secreted.", "As a result, the purification of these recombinant proteins of interest will be facilitated by the fact that they are present in the cell culture supernatant rather than inside the host cells.", "The polypeptides according to the invention may also be prepared by chemical synthesis.", "Such a method of preparation is also a subject of the invention.", "Those skilled in the art are aware of the methods of chemical synthesis, for example the techniques using solid phases (see in particular Steward et al., 1984, Solid phase peptides synthesis, Pierce Chem.", "Company, Rockford, 111, 2nd ed., (1984)) or techniques using partial solid phases, by fragment condensation or by conventional synthesis in solution.", "The polypeptides obtained by chemical synthesis, and possibly comprising corresponding unnatural amino acids, are also included in the invention.", "The hybrid polypeptides according to the invention are very useful for obtaining monoclonal or polyclonal antibodies capable of specifically recognizing the polypeptides according to the invention.", "These specific polyclonal or monoclonal antibodies can be obtained by the standard methods well known to those skilled in the art, after immunizing a mammal using these polypeptides (or their corresponding nucleic acid) or, for example, according to the conventional method of hybridoma culture described by Köhler and Milstein (1975, Nature, 256, 495) for the monoclonal antibodies.", "Such monoclonal or polyclonal antibodies, their fragments, or the chimeric antibodies, which recognize the polypeptides according to the invention, are also subjects of the invention.", "The antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, or Fab or F(ab′)2 fragments.", "They can also be in the form of immunoconjugates or of antibodies which are labeled in order to obtain a detectable and/or quantifiable signal.", "Thus, the antibodies according to the invention can be used in a method for detecting and/or identifying bacteria belonging to the genus Photorhabdus and/or to the species Photorhabdus luminescens , in a biological sample, characterized in that it comprises the following steps: a) bringing the biological sample into contact with an antibody according to the invention; b) demonstrating the antigen-antibody complex possibly formed.", "The antibodies according to the invention can also be used in order to detect expression of a gene of Photorhabdus luminescens TT01.Specifically, the presence of the expression product of a gene recognized by an antibody specific for said expression product can be detected by the presence of an antigen-antibody complex formed after the Photorhabdus luminescens strain TT01 has been brought into contact with an antibody according to the invention.", "The bacterial strain used may have been “prepared”, i.e.", "centrifuged, lyzed and/or placed in an appropriate reagent for constituting the medium suitable for the immunoreaction.", "In particular, preference is given to a method for detecting expression in the gene, corresponding to Western blotting, which may be performed after polyacrylamide gel electrophoresis of a lysate of the bacterial strain, in the presence or absence of reducing conditions (SDS-PAGE).", "After migration and separation of the proteins on the polyacrylamide gel, said proteins are transferred onto a suitable membrane (for example made of nylon) and the presence of the protein or of the polypeptide of interest is detected by bringing said membrane into contact with an antibody according to the invention.", "Thus, the present invention also comprises the kits or sets for carrying out a method as described (for detecting the expression of a gene of Photorhabdus luminescens TT01, or for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens), comprising the following elements: a) a polyclonal or monoclonal antibody according to the invention; b) optionally, the reagents for constituting the medium suitable for the immunoreaction; c) optionally, the reagents for demonstrating the antigen-antibody complexes produced by the immunoreaction.", "The polypeptides and the antibodies according to the invention can advantageously be immobilized on a support, in particular a protein chip.", "Such a protein chip is the subject of the invention and may also contain at least one polypeptide of a microorganism other than Photorhabdus luminescens , or an antibody directed against a compound of a microorganism other than Photorhabdus luminescens .", "The protein chips or high density filters containing proteins according to the invention can be constructed in the same way as the DNA chips according to the invention.", "In practice, it is possible to carry out the synthesis of the polypeptides directly attached to the protein chip, or to carry out an ex situ synthesis followed by a step of attaching the synthesized polypeptide to said chip.", "The latter method is preferable when the intention is to attach proteins of considerable size to the support, these proteins being advantageously prepared by genetic engineering.", "However, if the intention is to attach only peptides to the support of said chip, it may be more advantageous to synthesize said peptides directly in situ.", "The protein chips according to the invention can advantageously be used in kits or sets for detecting and/or identifying bacteria related to the species Photorhabdus luminescens , or to a microorganism, or more generally in kits or sets for detecting and/or identifying microorganisms.", "When the polypeptides according to the invention are attached to DNA chips, the presence of antibodies in the samples tested is sought, the attachment of an antibody according to the invention to the support of said protein chip allowing identification of the protein for which said antibody is specific.", "Preferably, an antibody according to the invention is attached to the support of the protein chip, and the presence of the corresponding antigen, specific for Photorhabdus luminescens , is detected.", "A protein chip described above can be used to detect gene products, in order to establish an expression profile for said genes, in addition to a DNA chip according to the invention.", "The protein chips according to the invention are also extremely useful for proteomic experiments which study interactions between the various proteins of a cell of a plant, of an animal, such as an insect, or of a microorganism other than P. luminescens.", "Thus, the invention also comprises a protein chip according to the invention, characterized in that it also contains at least one polypeptide of a cell of a plant, of an animal or of a microorganism other than P. luminescens, immobilized on the support of said chip, preferably said cell or other microorganism is chosen from a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens.", "In a simplified manner, representative peptides of the various proteins of an organism are attached to a support.", "Said support is then brought into contact with labeled proteins and, after an optional rinsing step, interactions between said labeled proteins and the peptides attached to the protein chip are detected.", "Thus, the protein chips comprising a polypeptide sequence according to the invention or an antibody according to the invention are a subject of the invention, as are the kits or sets containing them.", "The present invention also covers a method for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens , in a biological sample, which uses a nucleotide sequence according to the invention.", "It should be understood that, in the present invention, the term “biological sample” concerns samples taken from a living organism (in particular blood, tissues, organs or others taken from a mammal) or a sample containing biological material, i.e.", "DNA or RNA.", "Such a biological sample also comprises food compositions containing bacteria (for example cheeses, dairy products), but also food compositions containing yeasts (beers, breads) or others.", "The term “biological sample” also concerns bacteria isolated from these samples or food compositions.", "The method of detection and/or identification using the nucleotide sequences according to the invention may be diverse in nature.", "A method comprising the following steps is preferred: a) optionally isolating the DNA from the biological sample to be analyzed, or obtaining a cDNA from the RNA of the biological sample; b) specifically amplifying the DNA of bacteria belonging to the species Photorhabdus luminescens using at least one primer according to the invention; c) demonstrating the amplification products.", "This method is based on the specific amplification of the DNA, in particular via a chain amplification reaction.", "A method comprising the following steps is also preferred: a) bringing a nucleotide probe according to the invention into contact with a biological sample, the nucleic acid contained in the biological sample having, where appropriate, previously been made accessible to hybridization, under conditions which allow hybridization of the probe to the nucleic acid of a bacterium belonging to the species Photorhabdus luminescens ; b) demonstrating the hybrid possibly formed between the nucleotide probe and the DNA of the biological sample.", "Such a method should not be limited to detecting the presence of the DNA contained in the biological sample to be tested, it can also be used to detect the RNA contained in said sample.", "This method encompasses in particular Southern and Northern blotting.", "Another preferred method according to the invention comprises the following steps: a) bringing a nucleotide probe immobilized on a support according to the invention into contact with a biological sample, the nucleic acid of the sample having, where appropriate, previously been made accessible to hybridization, under conditions which allow hybridization of the probe to the nucleic acid of a bacterium belonging to the species Photorhabdus luminescens ; b) bringing the hybrid formed between the nucleotide probe immobilized on a support and the nucleic acid contained in the biological sample, where appropriate after removing the DNA of the biological sample which has not hybridized with the probe, into contact with a labeled nucleotide probe according to the invention; c) demonstrating the new hybrid formed in step b).", "This method is advantageously used with a DNA chip according to the invention, the nucleic acid being sought hybridizing with a probe present at the surface of said chip, and being detected using a labeled probe.", "This method is advantageously carried out by combining a prior step of amplifying the DNA or the complementary DNA optionally obtained by reverse transcription, using primers according to the invention.", "Thus, the present invention also encompasses the kits or sets for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens , characterized in that it comprises the following elements: a) a nucleotide probe according to the invention; b) optionally, the reagents required for carrying out a hybridization reaction; c) optionally, at least one primer according to the invention and also the reagents required for a DNA amplification reaction.", "Similarly, the present invention also encompasses the kits or sets for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens TT01, characterized in that it comprises the following elements: a) a nucleotide probe, termed capture probe, according to the invention; b) an oligonucleotide probe, termed detection probe, according to the invention; c) optionally, at least one primer according to the invention and also the reagents required for a DNA amplification reaction.", "Finally, the kits or sets for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens , characterized in that they comprise the following elements: a) at least one primer or one probe according to the invention; b) optionally, the reagents required to carry out a DNA amplification reaction; c) optionally, a component for verifying the sequence of the amplified fragment, more particularly an oligonucleotide probe according to the invention, are also subjects of the present invention.", "Preferably, said primers and/or probes and/or polypeptides and/or antibodies according to the present invention, used in the methods and/or kits or sets according to the present invention, are chosen from the primers and/or probes and/or polypeptides and/or antibodies specific for the species Photorhabdus luminescens.", "Preferably, these elements are chosen from the nucleotide sequences encoding a secreted protein, from the secreted polypeptides, or from the antibodies directed against secreted polypeptides of Photorhabdus luminescens.", "A subject of the present invention is also the strains of Photorhabdus luminescens TT01 containing one or more mutation(s) in a nucleotide sequence according to the invention, in particular an ORF sequence, or regulatory elements thereof (in particular promoters).", "According to the invention, preference is given to the strains of Photorhabdus luminescens TT01 exhibiting one or more mutation(s) in the nucleotide sequences encoding polypeptides preferably with activity of the toxin or antibiotic type, or involved in their biosynthesis, or else, in another aspect, involved in the cellular machinery, in particular secretion, central intermediate metabolism, energy metabolism, and processes of amino acid synthesis, of transcription and of translation, and of polypeptide synthesis.", "Said mutations may lead to inactivation of the gene or, in particular, when they are located in the regulatory elements of said gene, to overexpression of this gene.", "According to the present invention, the strains of Photorhabdus luminescens TT01 exhibiting one or more mutation(s) may be used to validate the function of a wild-type gene of Photorhabdus luminescens .", "The invention also relates to the use of a nucleotide sequence according to the invention, of a polypeptide according to the invention, of an antibody according to the invention, and/or of a cell according to the invention, for selecting an organic or inorganic compound capable of modulating, regulating, inducing or inhibiting gene expression in a plant or animal cell or in a microorganism other than P. luminescens, the resistance or sensitivity of which to at least one toxin or antibiotic produced by P. luminescens it is, for example, desired to modify.", "The invention also comprises a method for selecting compounds capable of binding to a polypeptide or one of its fragments according to the invention, capable of binding to a nucleotide sequence according to the invention, or capable of recognizing an antibody according to the invention, and/or capable of modulating, regulating, inducing or inhibiting gene expression, and/or of modifying growth or cell replication of eukaryotic or prokaryotic cells, or capable of inducing, inhibiting or increasing, in an animal or plant organism, resistance or sensitivity to at least one toxin or antibiotic produced by P. luminescens, said method comprising the following steps: a) bringing said compound into contact with said polypeptide or said nucleotide sequence and/or with a transformed cell according to the invention; b) determining the ability of said compound to bind to said polypeptide or said nucleotide sequence, or to modulate, regulate, induce or inhibit gene expression, or to modulate growth or cell replication, or to induce, inhibit or increase, in an animal or plant organism, resistance or sensitivity to at least one toxin or antibiotic produced by P. luminescens.", "The transformed cells according to the invention may advantageously be used as a model and may be used in methods for studying, identifying and/or selecting compounds liable to be responsible for resistance or sensitivity to at least one toxin or antibiotic produced by P. luminescens.", "The compounds which may be selected can be organic compounds such as polypeptides or carbohydrates or any other organic or inorganic compounds which are already known, or new organic compounds developed using molecular modeling techniques and obtained by chemical or biochemical synthesis, these techniques being known to those skilled in the art.", "The invention relates to the compounds which can be selected using a method of selection according to the invention.", "The invention also relates to a composition, in particular pesticidal or pharmaceutical composition, comprising a compound chosen from the following compounds: a) a nucleotide sequence according to the invention; b) a polypeptide according to the invention; c) a vector according to the invention; d) an antibody according to the invention; and e) a compound which can be selected using a method of selection according to the invention, optionally in combination with a pharmaceutically acceptable vehicle.", "The invention also relates to a pharmaceutical composition according to the invention, for preventing or treating an infection with a microorganism, such as a bacterium or a fungus, sensitive to at least one toxin or antibiotic produced by P. luminescens.", "The invention also relates to a pesticidal composition, in particular against insects, bacteria and/or fungi, according to the invention, for preventing or treating plants infested with animals, such as insects, or with a microorganism, such as a bacterium or a fungus, sensitive to at least one toxin or antibiotic produced by P. luminescens.", "The invention also comprises the use of a transformed cell according to the invention, for preparing a toxin or an antibiotic produced by P. luminescens.", "The expression “pharmaceutically acceptable vehicle” is intended to denote a compound or a combination of compounds making up a pharmaceutical composition causing no side effects and which makes it possible, for example, to facilitate administration of the active compound, to increase its lifetime and/or its effectiveness in the organism, to increase its solubility in solution or else to improve its conservation.", "These pharmaceutically acceptable vehicles are well known and will be adjusted by those skilled in the art as a function of the nature and of the method of administration of the active compound chosen.", "Preferably, these pharmaceutical compounds will be administered systemically, in particular intravenously, intramuscularly, intradermally or subcutaneously, or orally.", "Their methods of administration, dosages and pharmaceutical forms which are optimal can be determined according to the criteria generally taken into account in establishing a treatment suitable for a patient, such as, for example, the age or the body weight of the patient, the seriousness of his or her general condition, the tolerance to the treatment and the side effects observed.", "Finally, the invention comprises the use of a composition according to the invention, for preparing a medicinal product intended for the prevention or treatment of an infection with a microorganism, such as a bacterium or a fungus, sensitive to at least one toxin or antibiotic produced by P. luminescens.", "Moreover, a subject of the present invention is also a genomic DNA library of a bacterium of the genus Photorhabdus, preferably Photorhabdus luminescens , preferably the strain TT01.The genomic DNA libraries described in the present invention, in particular the BAC library deposited with the CNCM [French National Collection of Cultures and Microorganisms] on May 12, 2000, under No.", "I-2478 and which covers the Photorhabdus luminescens TT01 genome.", "The invention also relates to a method for identifying at least one nucleotide sequence of P. luminescens not present in the genome of another species of bacterium, in particular a pathogenic bacterium, and/or for identifying at least one nucleotide sequence of a genome of a bacterium, in particular a pathogenic bacterium, of a species other than P. luminescens and not present in the P. luminescens genome, characterized in that it comprises the following steps: a) the nucleotide sequences of P. luminescens according to the invention, or contained in a genomic library according to the invention, are aligned with the genomic sequence of the other bacterial species, or one of its fragments; and b) the data obtained with this alignment are processed in order to isolate and identify said sequence(s) only present in one or the other genome.", "Such a method, also called “subtractive genomic method”, can be used here to identify a sequence responsible for the pathogenicity of a bacterium, such as a gram-negative bacterium, for which the P. luminescens bacterium, which is not pathogenic, can serve as a model for comparison.", "The present invention thus relates to the methods for isolating a polynucleotide of interest present in a strain of Photorhabdus and absent from another strain or species, which use at least one DNA library based, for example, on a plasmid pcDNA2.1 containing the Photorhabdus genome.", "The method according to the invention for isolating a polynucleotide of interest can comprise the following steps: a) isolating at least one polynucleotide contained in a clone of the library of DNA of Photorhabdus origin; b) isolating: at least one genomic polynucleotide or cDNA of a bacterium, said bacterium belonging to a strain or species different from the Photorhabdus strain used to construct the DNA library of step a) or, alternatively, at least one polynucleotide contained in a clone of a DNA library prepared from the genome of a bacterium belonging to a strain or species different from the Photorhabdus strain used to construct the DNA library of step a), and hybridizing the polynucleotide of step a) to the polynucleotide of step b); c) selecting the polynucleotides of step a) which have not formed a hybridization complex with the polynucleotides of step b); d) characterizing the selected polynucleotide.", "The polynucleotide of step a) can be prepared by digesting at least one recombinant clone with a suitable restriction enzyme and, optionally, amplifying the polynucleotide insert which results therefrom.", "Thus, the method of the invention allows those skilled in the art to carry out comparative genomic studies between a bacterium of the genus Photorhabdus and between, for example, a pathogenic strain or species.", "In particular, it is possible to study and determine the regions of polymorphism between said strains.", "The present invention also relates to the use of the nucleic acid sequences or of the polypeptides according to the invention: for preparing biopesticides, in particular entomotoxins, antibiotics, antifungal agents or cytotoxins, for secreting proteins, as virulence factors, for control via quorum-sensing, for identifying targets for human diseases for which Photorhabdus luminescens is a model (in particular the plague or whooping cough), and for identifying targets against pathogenic Gram-negative bacteria using the subtractive genomic method (such as, for example, by comparison with E. coli or other pathogenic gram-negative bacteria).", "The present invention also relates to the use of the polypeptides according to the present invention, for screening compounds capable of modulating the activity of these polypeptides, in particular the polypeptides with enzymatic activity.", "Other characteristics and advantages of the invention appear in the following examples: EXAMPLES Example 1 Materials and Methods The strategy for sequencing the genome of Photorhabdus luminescens strain TT01 is based on random (shotgun) sequencing.", "The first step of this study consisted in cloning the genomic DNA of the Photorhabdus luminescens bacterium into various vectors (plasmids and BACs).", "Photorhabdus luminescens genomic DNA libraries used.", "Three genomic DNA libraries were prepared: I)—A genomic DNA library in a high copy number bacterial vector (pcDNA2.1, Invitrogen).", "Average insert size 1.5 kb.", "II)—A genomic DNA library in a low copy number bacterial vector (pSYX34).", "Average insert size 10 kb.", "III)—A genomic DNA library in a BAC vector (pBeloBAC11, California Institute of Technology).", "Average insert size 50 to 100 kb.", "Two batches of DNA of the TT01 strain of the Photorhabdus luminescens bacterium were extracted on Oct. 30, 1998 and Apr.", "14, 1999.I) Establishing a Genomic DNA Library in the Bacterial Vector pcDNA2.1 (Invitrogen) A) Preparation of the Vector: pcDNA2.1 We prepared the plasmid pcDNA2.1 by carrying out two midipreps (QIAGEN KIT) in parallel according to the conditions recommended by the manufacturer.", "The vector pcDNA2.1 was digested with the BstX1 restriction enzyme.", "B) Preparation of the Chromosomal DNA of Photorhabdus luminescens Strain TT01 and Ligation into the Vector pcDNA2.1 1) Dissolving of the DNA A dry pellet of genomic DNA of the strain TT01, prepared on Oct. 30, 1998, was taken up in 200 μl of 10:1 TE, and then dissolved for 30 minutes at 65° C. Its concentration was estimated at 0.15 μg/μl.", "2) Nebulization of the DNA 50 μl of genomic DNA strain TT01 in an amount of H2O sufficient for 2 ml were nebulized for 45 sec at a pressure of 1 bar of nitrogen, and centrifuged for 2 min at 600 rpm in order to recover the entire volume.", "3) Precipitation of the DNA This DNA was then precipitated with sodium acetate (2 ml of DNA+0.2 ml of 3M Na acetate, pH 5.2,+5 ml of absolute ethanol; 2 h at −20° C.), centrifuged for 30 min at 14 000 rpm and at 4° C., and then redissolved in 100 μl of water.", "4) Analysis of the DNA 4 μl were loaded onto a 1% agarose TBE gel.", "DNA fragments of the expected size of 500 bp to 3 kb were visualized.", "5) Repair of the DNA In order to blunt end the DNA fragments, repair was carried out using T4 DNA polymerase.", "The following are mixed in 2 separate tubes: 48 μl of DNA from step 3), 100 μl of H2O, 20 μl of 5× ligation buffer, 2 μl of dNTP mix (10 mM), 5 μl of T4 DNA polymerase (Boehringer).", "Incubation is carried out for 25 min at ambient temperature and the reaction is then stopped by heating for 15 min at 75° C. 6) Precipitation of the DNA This DNA was then precipitated overnight at −20° C. with sodium acetate (1/10 volume of Na acetate and 2.5 volume of absolute ethanol) and centrifuged, and the DNA pellet obtained was then air-dried and redissolved in 30 μl of water.", "7) Ligation of the DNA This DNA was ligated overnight at 16° C. with the Bstx A+Bstx B linkers (Invitrogen) in the presence of 2 μl of ligase.", "8) Preparation of the Inserts After migration of the DNA ligated to the linkers in a 1% agarose TAE gel, at 70 volts, the region of interest (between 1 and 3 Kb) was cut into four fragments.", "9) Purification of the Inserts The agarose fragments containing the regions of interest were purified using Geneclean (BIO101) according to the conditions recommended by the manufacturer.", "An aliquot was loaded onto an agarose minigel in order to validate the quality of the purification.", "10) Ligation of the Inserts into the Vector pcDNA2.1 (Invitrogen) 4 μl of DNA (insert after the Geneclean purification step, step 9), 2 μl of plasmid pcDNA2.1 (after the BstX1 digestion step and then Geneclean purification), 2 μl of ligation buffer (10×), add 2 μl of ligase, 10 μl H2O (total volume: 20 μl).", "The mixture is incubated overnight at 16° C. 11) Transformation of Ultracompetent XL2 Blue Cells (Stratagene) The genomic DNA library obtained in step 10 was integrated into ultracompetent XL2 Blue cells (Stratagene) according to the conditions recommended by the manufacturer.", "Analysis of the inserts of 24 clones by digestion with the PvuII restriction enzyme and by sequencing of the ends was carried out and gave satisfactory results.", "II) Establishing a Genomic DNA Library in the Bacterial Vector pSYX34 The PARTIAL FILL-IN technique, developed in the laboratory, allowed us to construct a library of genomic DNA of the TT01 strain of the bacterium Photorhabdus luminescens , cloned into the plasmid pSYX34 (average insert size 10 Kb).", "Note: in this case, digestion of the vector pSYX34 with the Sal I restriction enzyme frees the following ends: 5′TCGAC-G-5′ The PARTIAL FILL-IN was carried out in the presence of dCTP and dTTP deoxynucleotides.", "5′TCGAC-CTG-5′ A) Preparation of the Vector: pSYX34 1) Production of the Vector Plasmid pSYX34 was prepared by carrying out two midipreps, QIAGEN KIT, in parallel according to the conditions recommended by the manufacturer.", "2) Digestion of the Plasmid pSYX34 with the Sal I Restriction Enzyme The final volume will be 100 μl: 20 μl pSYX34, 10 μl of buffer H, 66 μl of H2O, 4 μl Sal I.", "The mixture is incubated for 2 h at 37° C. An aliquot and also a molecular weight marker were loaded onto an agarose minigel in order to validate the quality of the purification.", "3) Chloroform Extraction Chloroform extraction makes it possible to stop the enzyme digestion and to remove any traces of protein.", "After digestion, 95 μl are recovered, 105 μl of 10 mM TE are added, 200 μl of chloroform are added.", "The mixture is vortexed and centrifuged for 1 min at 1 000 rpm.", "The aqueous phase (upper phase) is recovered.", "4) Precipitation with Sodium Acetate 1/10 volume of sodium acetate (3M, pH: 5.2), i.e.", "20 μl, are added, followed by 2.5 volumes of clean absolute ethanol, i.e.", "500 μl (bottle kept at −20° C.).", "The mixture is left at −20° C. for at least 1 hour (it is possible to leave it at −20° C. overnight).", "The mixture is centrifuged at 4° C. (cold room) for 30 min at 14 000 rpm.", "The supernatant is removed using the vacuum pump.", "400 μl of 70% ethanol are added and the mixture is centrifuged at 4° C. for 5 min at 14 000 rpm.", "The supernatant is removed using the vacuum pump and the pellet is left to dry for approximately one hour on the bench.", "The pellet is resuspended in 20 μl of 10 mM TE (1/10).", "5) Partial Fill-in Final volume of the reaction: 50 μl; 20 μl of pSYX34 digested with Sal I 5 μl of synthesis buffer, 2.5 μl of 1 mM nucleotide (C-T) mix, 20.5 μl of water, 2 μl of Klenow (2 U/μl).", "The mixture is left for 30 min at ambient temperature.", "Verification is carried out on a 1% agarose minigel, followed by freezing at −20° C. Mixture of C-T Nucleotides: Mix: 2 μl of T nucleotides (100 mM), 2 μl of C nucleotides (100 mM), 16 μl of 10 mM TRIS, pH=7.5.The nucleotides are thus diluted to 1/10, for a concentration of 10 mM.", "A second dilution to 1/10 in 10 mM TRIS buffer will be carried out so as to have a concentration of 1 mM.", "In addition, during the reaction, the nucleotides are diluted to 1/20 (2.5 μl in 50 μl), thus obtaining a final concentration of nucleotides of 50 μM.", "6) Purification of the Vector pSYX34: Preparative Gel Prepare a 1% agarose TAE gel, large wells will be provided for the samples.", "Take 30 μl of pGB2 after the partial fill-in (20 μl will therefore remain in the freezer), add 6 μl of loading solution.", "In parallel, load the molecular weight marker in the following proportions: 5 μl H2O, 5 μl of the 1 Kb DNA marker, 2 μl of the loading solution.", "Allow to migrate at 70 volts.", "Cut the gel on the marker side using a scalpel and locate, by taking a photo and using a ruler, the region of interest; in our case, the band corresponding to 4 Kb will be taken.", "Each sample will be cut in half.", "The weight of each sample will indicate its approximate volume.", "There will therefore be four samples to purify by the Geneclean II technique.", "7) Purification of the Vector pSYX34 Take up in 3 volumes of sodium iodide solution (1 ml for a sample of 0.2 g), place at 49° C., shaking every 2 minutes, until the agarose has completely dissolved.", "Add 8 μl of microbeads: GLASSMILK R, leave at ambient temperature for 5-10 min, the DNA attaches to the microbeads.", "Centrifuge for 2 min at 14 000 rpm, remove the supernatant by suction.", "Wash 3 times with the New Wash solution (600 μl/eppendorf, vortex, centrifuge for a few seconds at 10 000 rpm); this solution was prepared and is kept at −20° C. Resuspend the pellet in 10 μl of H2O, vortex, leave for 5 min at 49° C. This allows the DNA to detach from the microbeads.", "Centrifuge for 2 min at 14 000 rpm.", "Recover the supernatants in new eppendorfs (No.", "1 and No.", "2, etc) Add 10 μl of H2O again to the pellets, so as to be sure to recover everything, vortex, leave for 2 min at 49° C. Centrifuge for 2 min at 14 000 rpm.", "Recover the supernatants in the preceding eppendorfs (No.", "1 and No.", "2, etc).", "There are approximately 20 μl of supernatant, centrifuge for 2 min at 14 000 rpm.", "Transfer the supernatants into new eppendorfs, so as to be sure that there are no longer any microbeads.", "Load 1 μl of each preparation (+5 μl of H2O+2 μL of loading solution) onto a 1% agarose minigel, allow to migrate at 80 volts for 2 hours.", "The most concentrated samples will be kept, they will then be frozen at −20° C. B) Preparation of the Chromosomal DNA of Photorhabdus luminescens Strain TT01 1) Dissolving of the DNA A dry pellet of genomic DNA of the strain TT01 was taken up in 200 μl of 10:1 TE and then dissolved for 30 min at 65° C. Its concentration was estimated at 0.15 μg/μl.", "2) Partial Digestion of the Genomic DNA with the Sau3A Restriction Enzyme 15 μl of DNA were digested with 2, 1, 0.5, 0.25, 0.125, 0.0625 or 0.03125 units of Sau3A restriction enzyme for 1 h at 37° C. 3) Chloroform Extraction Chloroform extraction makes it possible to stop the enzyme digestion and to remove any traces of protein.", "After digestion, 100 μl of chromosomal DNA are recovered.", "100 μl of 10 mM TE are added, 200 μl of chloroform are added.", "The mixture is vortexed and centrifuged for 1 min at 1 000 rpm.", "The aqueous phase, the upper phase, comprising the chromosomal DNA is recovered.", "4) Precipitation with Sodium Acetate 1/10 volume of sodium acetate (3M, pH: 5.2), i.e.", "20 μl, are added.", "2.5 volumes of clean absolute ethanol, i.e.", "500 μl, (bottle kept at −20° C.) are then added.", "The mixture is left at −20° C. for at least 1 hour (it is possible to leave it at −20° C. overnight).", "The mixture is centrifuged at 4° C. (cold room) for 30 min at 14 000 rpm.", "The supernatant is removed using the vacuum pump.", "400 μl of 70% ethanol are added, the mixture is centrifuged at 4° C. for 5 min at 14 000 rpm.", "The supernatant is removed using the vacuum pump and the pellet is left to dry for approximately one hour on the bench.", "The pellet is resuspended in 20 μl of H2O.", "5) Verification of the Partial Digestions, after Precipitation with Sodium Acetate.", "A 1% agarose TBE gel is prepared.", "1/10 of the total volume of the precipitations, i.e.", "2 μl of DNA+8 μl of H2O+2 μl of loading solution, is loaded.", "In parallel, a molecular weight marker is loaded: 1 μl+9 μl of H2O+2 μl of loading solution.", "6) Partial Fill-in Final volume of the reaction: approximately 50 μl 36 μl of partially digested DNA (DNA digested with 0.25, 0.125, 0.0625 or 0.03125 units of Sau3A restriction enzyme) 5 μl of synthesis buffer 10 μl of 1 mM nucleotide (A-G) mix=>final concentration: 200 μM 2 μl of Klenow (2 U/μl).", "The mixture is left at ambient temperature for 30 min.", "Mixture of A-G Nucleotides Mix: 2 μl of A nucleotides (100 mM) 2 μl of G nucleotides (100 mM) 16 μl of 10 mM TRIS, pH=7.5.The nucleotides are thus diluted to 1/10, giving a concentration of 10 mM.", "A second dilution to 1/10 in H2O will be carried out, a concentration of 1 mM is therefore obtained.", "7) Chloroform Extraction Chloroform extraction makes it possible to stop the enzyme digestion and to remove any traces of protein.", "After digestion, 53 μl of chromosomal DNA are recovered.", "47 μl of 10 mM TE are added.", "100 μl of chloroform are added.", "The mixture is vortexed and centrifuged for 1 min at 1 000 rpm.", "The aqueous phase, the upper phase, comprising the chromosomal DNA is recovered.", "8) Precipitation with Sodium Acetate 1/10 volume of sodium acetate (3M, pH: 5.2), i.e.", "10 μl, are added.", "2.5 volumes of clean absolute ethanol, i.e.", "250 μl, (bottle kept at −20° C.) are then added.", "The mixture is left at −20° C. for at least 1 hour (it is possible to leave it at −20° C. overnight).", "The mixture is centrifuged at 4° C. (cold room) for 30 min at 14 000 rpm.", "The supernatant is removed using the vacuum pump.", "400 μl of 70% ethanol are added, the mixture is centrifuged at 4° C. for 5 min at 14 000 rpm.", "The supernatant is removed using the vacuum pump and the pellet is left to dry for approximately one hour on the bench.", "The pellet is resuspended in 20 μl of H2O.", "9) Purification of the Chromosomal DNA: Preparative Gel Prepare a 1% agarose TAE gel, large wells will be provided for the samples.", "Take the entire DNA, i.e.", "20 μl, add 4 μl of loading solution.", "In parallel, load the molecular weight marker in the following proportions: 5 μl H2O 5 μl of the 1 Kb DNA marker 2 μl of the loading solution.", "Allow to migrate at 70 volts.", "Cut the gel on the marker side using a scalpel and locate, by taking a photo and using a ruler, the region of interest.", "Since the DNA fragments are long, SPIN X columns from COSTAR will be used to purify the DNA, this will avoid cleaving the chromosomal DNA.", "10) Purification: SPIN X Column After having recovered the various pieces of agarose, in SPIN X columns, add 200 μl of 10 mM TE and then grind using a spatula.", "This may be kept at 4° C. Then, centrifuge for 20 min at 5 000 rpm.", "The agarose will be retained on the filters, whereas the chromosomal DNA will be found at the bottom of the tube.", "11) Chloroform Extraction Chloroform extraction makes it possible to remove any traces of protein.", "800 μl of chromosomal DNA are recovered.", "800 μl of chloroform are added.", "The mixture is vortexed and centrifuged for 1 min at 1 000 rpm.", "The aqueous phase, the upper phase, comprising the chromosomal DNA is recovered.", "12) Precipitation with Sodium Acetate 1/10 volume of sodium acetate (3M, ph: 5.2), i.e.", "80 μl, are added.", "800 μl of isopropanol are then added.", "The mixture is left at −20° C. for at least 1 hour (it is possible to leave it at −20° C. overnight).", "The mixture is centrifuged at 4° C. (cold room) for 30 min at 14 000 rpm.", "The supernatant is removed using the vacuum pump.", "400 μl of 70% ethanol are added and the mixture is centrifuged at 4° C. for 5 min at 14 000 rpm.", "The supernatant is removed using the vacuum pump, and the pellet is allowed to dry for approximately half an hour on the bench and 2 min under vacuum.", "The pellet is resuspended in 20 μl of TE (0.1×, i.e.", "1 mM).", "The suspension is incubated for 10 min at 65° C. The suspension is incubated for approximately 4 hours at 4° C., so that the DNA is well resuspended.", "Verification is carried out on a 1% agarose minigel with a molecular weight marker placed in parallel.", "C) Ligation of the chromosomal DNA of Photorhabdus luminescens Strain TT01 into the Vector pSYX34 Take 6 μl of chromosomal DNA.", "Add 2 μl of pSYX34.Add 2 μl of ligation buffer (LB).", "Add 2 μl of ligase.", "Then add 8 μl of H2O (total volume: 20 μl) In parallel, prepare a ligation control by replacing the chromosomal DNA with 0.1× TE (1 mM).", "Vortex, leave overnight at 16° C. Transformation of Ultracompetent XL10-Gold Kanr Cells (Stratagene) The genomic DNA library obtained in the preceding step was integrated into ultracompetent XL10 Gold Kanr cells (Stratagene) according to the conditions recommended by the manufacturer.", "Analysis of the inserts of 24 clones by digesting with the SalI restriction enzyme and by sequencing of the ends was carried out and gave satisfactory results.", "III) Establishing a Genomic DNA Library in the Bacterial Vector pBeloBAC11 (California Institute of Technology) Construction of the BAC Library Partial digestion of the DNA in pieces of agarose: the pieces of agarose are washed three times for 15 minutes at ambient temperature in a solution of TE (1×) with moderate agitation; the pieces of agarose are equilibrated twice in 300 μl of a Hind III digestion buffer (1×) (Boehringer or Biolabs) for 30 minutes at ambient temperature; the digestion buffer is removed and an ice-cold Hind III digestion buffer (1 ml per piece of agarose) containing 20 U of Hind III (Boehringer or Biolabs) is added; incubation is carried out for two hours in ice; the pieces of agarose are transferred to a water bath at 37° C. and incubation is carried out for 10 minutes to 30 minutes (depending on the DNA contained in the pieces of agarose); and the digestion is stopped by adding 100 μl of a 250 mM EDTA solution (pH 8).", "Size Selection: Separation of the Partially Digested DNA by PFGE Using the CHEF DRIII Apparatus (Bio-Rad): gel of 1% LMP agarose in a tris-acetate-EDTA buffer (1×) at 13° C., reverse the current every 3 to 15 seconds at 6 V/cm for 16 hours, load at least two pieces of agarose and the marker, stain the marker and one lane or a part of the lane to verify the partial digestion, excise the bands (unstained part) of various sizes (i.e.", "from 50 to 100 kb and from 150 to 250 kb), the agarose bands can be stored at 4° C. in a TE solution.", "Preparation of the Vector: isolation of pBeloBACII by the cesium chloride method; digestion with Hind III; dephosphorylation with CIP (optionally with HK phosphatase).", "Ligation and Transformation: the agarose banks containing the DNA are melted at 60° C. for 10 minutes; the molten solution of agarose/DNA is equilibrated for 15 minutes at 45° C.; gelase (Epicentre Technologies) is added in a proportion of 1 U per 100 μl of gel band (do not add digestion buffer, which causes certain problems at the time of ligation); the digestion is carried out for 1 hour at 45° C.; the ligation is carried out in 50 μl of a solution containing pBeloBACII (2 μl), T4 ligase (1 μl at 1:10), a 10× T4 buffer (5 μl ), and the DNA/agarose solution (42 μl), incubation is for 20 hours at 16° C.; the ligation medium is heated for 15 minutes at 65° C.; the ligation medium is dialyzed against a Tris-EDTA buffer, using Millipore membranes of pore size VS 0.025 mM; 1 or 2 μl of the ligation solution are introduced by electroporation into E. coli DH10B cells (Gibco BRL) using electroporation cuvettes with a width of 2 mm, with the following settings: 2.5 kV, 25 μF and 200Ω; after electroporation, the cells are resuspended in 600 μl of SOC or NYZ medium, and then incubation is carried out for 45 minutes at 37° C. with agitation; 10 and 100 μl of each cell suspension are plated out in an LB agar medium containing chloramphenicol (12.5 μg/ml), X-Gal (5-bromo-4-chloro-3-indolyl-α-D-glucuronic acid; 50 μg/ml) and IPTG (isopropyl-α-D-thiogalactopyranoside; 25 μg/ml); and the dishes thus obtained are incubated overnight.", "Extraction of the Plasmid DNA and Sequencing of the Inserts The plasmid DNA was extracted by the alkaline lysis technique in 24-well plates.", "The inserts of the clones were sequenced using the PE Big Dye kit according to the conditions recommended by the manufacturer, using the oligonucleotides specific for each vector.", "The reactions are then introduced into the thermocycler in order to undergo 35 cycles composed of the following three steps: denaturation for 10 seconds at 96° C., hybridization for 10 seconds at 50° C., elongation for 4 minutes at 56° C. A precipitation with 76% ethanol is then carried out for 20 minutes at ambient temperature.", "The plates are then centrifuged for 35 minutes at 2 200 g, then drained on absorbent paper, and then centrifuged turned upside down on absorbent paper for 1 minute at 500 g in order to leave no trace of ethanol.", "The DNA pellets are then: either taken up in a solution of formamide-EDTA-dextran blue, and then denatured for 2 minutes at 96° C. The plates are then immediately placed in ice, an aliquot then being loaded onto an acrylamide gel of a PE-377 automatic sequencer, or taken up in a 0.3 mM EDTA solution, an aliquot then being automatically loaded into a PE-3700 automatic sequencer.", "Assembly of the Sequences Obtained The genomic DNA sequences were assembled in a contig using the PHRED-PHRAP software and visualized using the CONSED software.", "Analysis and Annotation The contigs were first of all analyzed and annotated automatically using the GMPTB software.", "Example 2 The Genes of Interest The various characteristics associated with the way of life of Photorhabdus luminescens and reported in the literature (Forst et al., 1997; Hu et al., 1998, etc.)", "led to the presence of genes involved in the biosynthesis of antibiotics and of toxins being sought in the genome of this bacterium.", "The Operons for Biosynthesis of Antibiotics The search for peptide synthetases was undertaken in silico.", "To do this, we initially used the various motifs conventionally described in the literature (Stachelhaus & Marahiel, 1995, FEMS Microbiology Letters 125:3-14) as a probe in order to locate, by sequence homology, in the Photorhabdus luminescens genome, genes which may be involved in the biosynthesis of antibiotics.", "Adenylation Modules 1 - LKAGGAYYVPID 2 - YSGTTGxPKGV 3 - GELCIGGXGxARGYL 4 - YxTGD 5 - VKIRGxRIELGEIE Thioester Formation Module 6 - DNFYxLGGHSL N-Methylation Module VLE/DxGxGxG Peptide Elongation Module His-HHILxDGW Peptide Racemization Module (Optional) His-HHILxDGW A - AYxTExNDILLTAxG B - EGHGRExIIE C - RTVGWFTSMYPxxLD D - FNYLGQFD Among these various modules, some enabled us to identify, by homology search using the BLASTP—basic local alignment search tool program—(Altschul S F et al., 1990, J. Mol.", "Biol., 215:403-10), genes encoding proteins containing some of these motifs.", "The genes thus identified, probably encoding proteins involved in the biosynthesis of antibiotics, were cataloged and appear in particular in table I.", "Emphasis was placed particularly on the genes homologous to the syr E gene of Pseudomonas syringae or to the nosD gene.", "REFERENCES Eric Guenzi, Giuliano Galli, Ingeborg Grgurina, Dennis C. Gross, and Guido Grandi, 1998.Characterization of the Syringomycin Synthetase Gene Cluster.", "A link between prokaryotic and eukaryotic peptide synthetases.", "J. Biol.", "Chem., 273:32857-32863.Bondi M, Messi P, Sabia C, Baccarani Contri M, Manicardi G., 1999.Antimicrobial properties and morphological characteristics of two Photorhabdus luminescens strains.", "New Microbiol., 22:117-27.The Operons for Biosynthesis of Toxins As for the genes for biosynthesis of antibiotics, we sought, by sequence homology, in the Photorhabdus luminescens genome, genes potentially encoding toxic proteins.", "We identified many genes encoding proteins having these criteria.", "Entomotoxins (Tc loci).", "Homologs of the Tox A gene of Clostridium difficile.", "Hemolysins.", "Genes potentially encoding proteins homologous to the RTX toxins of Vibrio cholerae.", "Genes potentially encoding proteins homologous to the tphA and icmF genes of Legionella pneumophila (Purcell M, Shuman H A, 1998.The Legionella pneumophila icmGCDJBF genes are required for killing of human macrophages.", "Infect Immun., 66:2245-55).", "See annotation of these genes in table I below.", "REFERENCES Ffrench-Constant R, Bowen D., 1999.Photorhabdus toxins: novel biological insecticides.", "Curr Opin Microbiol., 2:284-8.Review.", "Blackburn M, Golubeva E, Bowen D, Ffrench-Constant R H, 1998.A novel insecticidal toxin from Photorhabdus luminescens, toxins complex a (Tca), and its histopathological effects on the midgut of manduca sexta.", "Appl Environ Microbiol., 64:3036-41.Bowen D J, Ensign J C, 1998.Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens .", "Appl Environ Microbiol., 64:3029-35.Bowen D, Rocheleau T A, Blackburn M, Andreev 0, Golubeva E, Bhartia R, Ffrench-Constant R H, 1998.Insecticidal toxins from the bacterium Photorhabdus luminescens.", "Science., 280:2129-32.Guo L, Fatig R O 3rd, Orr G L, Schafer B W, Strickland J A, Sukhapinda K, Woodsworth A T, Petell J K, 1999.Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins.", "Purification and characterization of toxin A and toxin B. J. Biol Chem., 274:9836-42.GENERAL REFERENCES 1.Altschul, S. F., T. L. Madden, A.", "A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman.", "1997.Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.", "[Review] [90 refs].", "Nucleic Acids Research.", "25:3389-402.2.Birnboim, H. C. 1983.A rapid alkaline extraction method for the isolation of plasmid DNA.", "Methods Enzymol.", "100:243-255.3.Braun, L. F. Nato, B. Payrastre, J. C. Mazie, and P. Cossart.", "1999.The 213-amino-acid leucine-rich repeat region of the Photorhabdus luminescens InIB protein is sufficient for entry into mammalian cells, stimulation of P1 3-kinase and membrane ruffling.", "Mol.", "Microbiol.", "34:10-23.4.Buchrieser, C., C. Rusniok, L. Frangeul, E. Couvé, A. Billault, F. Kunst, E. Carniel, and P. Glaser.", "1999.The 102 kb locus of Yersinia pestis: sequence analysis and comparison of selected regions among different Yersinia pestis and Yersinia pseudotuberculosis strains.", "Infect.", "Immun.", "67:4851-4861.5.Ewing, B., and P. Green.", "1998.Base-calling of automated sequencer traces using phred.", "II.", "Error probabilities.", "Genome Res.", "8:186-194.6.Fitch, W. S. 1970.Distinguishing homologous from analogous proteins.", "Syst.", "Zool.", "19:99-113.7.Frangeul, L., K. E. Nelson, C. Buchrieser, A. Danchin, P. Glaser, and K. F. 1999.Cloning and assembly strategies in microbial genome projects.", "Microbiology.", "145:2625-2634.8.Gordon, D., C. Abajian, and P. Green.", "1998.Consed: a graphical tool for sequence finishing.", "Genome Res.", "8:195-202.9.Jacquet, C., J. Bille, and J. Rocourt.", "1992.Typing of Photorhabdus luminescens by restriction polymorphism of the ribonucleic acid gene region.", "Zentralbl Bakteriol.", "276:356-365.10.Li, P., K. C. Kupfer, C. J. Davies, D. Burbee, G. A. Evans, and H. R. Garner.", "1997.PRIMO: A primer design program that applies base quality statistics for automated large-scale DNA sequencing.", "Genomics.", "40:476-485.11.Lukashin, A. V., and M. Borodovsky.", "1998.GeneMark.hmm: new solutions for gene finding.", "Nucleic Acids Res.", "15:1107-1115.Scientific Description of the Photorhabdus luminescens Strain TT01 BAC Library Deposited with the CNCM [French National Collection of Cultures and Microorganisms] on May 12, 2000, Under the Number I-2478 Collection of Escherichia coli DH 10B™ clones (Calvin et al., J. Bacteriol.", "170, 2796, 1988) containing genomic DNA fragments from the Photorhabdus luminescens strain TT01 bacterium, cloned into the vector pBelo BACII (Kim et al., Genomics, 34, 213, 1996) at the Hind III site.", "The average insert size is 60 kb.", "Databanks Local revisions of main public banks were used.", "The protein bank used consists of the nonredundant fusion of these banks, such as the Genpept bank (automatic translation of GenBank and NCBI).", "Use was made of the BLAST software package (public domain, Altschul et al., 1990) for searching for homologies between a sequence and protein or nucleic acid databanks.", "The significance thresholds used depend on the length and on the complexity of the region tested and also on the size of the reference bank.", "They were adjusted and adapted to each analysis.", "The results of the search for homologies between a sequence according to the invention and protein or nucleic acid databanks are given and summarized in table I and table II below.", "Table I List of putative functions (column “homology with nonredundant protein database-description”, and column “annotations” for the sequences selected as being involved in an activity or having an activity of the toxin or antibiotic type) of the proteins of sequences SEQ ID No.", "42 to SEQ ID No.", "3855 encoded by their respective nucleotide sequence of the Photorhabdus luminescens strain TT01 genome (genome represented by the sequences of the 41 contigs (SEQ ID No.", "1 to SEQ ID No.", "41)).", "The nucleic acid sequences of the proteins of sequence SEQ ID No.", "42 to SEQ ID No.", "3855 can be easily identified by their start position (“start” column) and end position (“end” column) on each one of the contig sequences (“contig” column).", "Legend of Table I: All of the putative functions associated with the proteins of sequence SEQ ID No.", "42 to SEQ ID No.", "3855 were obtained through a homology search using in particular BLASTP software (Altschul et al., 1990).", "The significant identities, or the presence of various motifs (cf.", "examples) representative of these functions, were in particular taken into account.", "The description of the most probable function(s) is given in the “homology with” and “Annotation” column.", "Table II List of putative functions (column “function obtained by comparison on databank”) of the proteins encoded by the sequences SEQ ID No.", "5835 to SEQ ID No.", "10784 of the Photorhabdus luminescens strain TT01 genome (genome here represented by the sequences of the 9 contigs (SEQ ID No.", "5826 to SEQ ID No.", "5834)).", "The nucleic acid sequences of sequence sequences SEQ ID No.", "5835 to SEQ ID No.", "10784 can be easily identified by their start position (“CONTIG”, “from” column) and end position (“CONTIG” “to” column) on each one of the contig sequences.", "Legend of Table II: All of the putative functions associated with the proteins encoded by the sequences SEQ ID No.", "5835 to SEQ ID No.", "10784 were obtained through a homology search using in particular BLASTP software (Altschul et al., 1990).", "The significant identities, or the presence of various motifs (cf.", "examples) representative of these functions, were in particular taken into account.", "The description of the most probable function(s) is given in the “homology with” and “Annotation” column.", "In the final column, “HOMOLOGOUS TO SEQUENCE SEQ ID”, the term “#N/A” means that no homology for the sequence concerned had been identified with one of the sequences SEQ ID Nos.", "42 to 3855.When a homologous sequence was found among the sequences SEQ ID Nos.", "42 to 3855 for the sequence concerned, this is mentioned via its SEQ ID number in this column.", "LENGTHY TABLE REFERENCED HERE US20070020625A1-20070125-T00001 Please refer to the end of the specification for access instructions.", "LENGTHY TABLE REFERENCED HERE US20070020625A1-20070125-T00002 Please refer to the end of the specification for access instructions.", "LENGTHY TABLE The patent application contains a lengthy table section.", "A copy of the table is available in electronic form from the USPTO web site () An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3)." ] ]	Patent_10467478
[ [ "Benzo'fisoindole derivatives with affinity to the ep4 receptor", "The present invention relates to a compound of formula (I): corresponding pharmaceutical compositions, preparation processes, and/or methods of using the aforementioned compounds and/or compositions." ], [ "1.A compound of formula (I): wherein: R1 and R3 are the same or different and represent ═O, hydrogen, C1-6alkyl, C1-6-dialkyl, ═CHC1-C5alkyl, ═S, or a 5- or 6-membered aryl; R4 to R9 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or CF3; Q1 is hydrogen, C1-6alkyl, C1-6dialkyl, C1-6alkoxy, NHAc, NR210 where R10 is hydrogen or C1-6alkyl, difluoro, fluoro, ═O, or OH; Q2, Q3, Q4 and Q5 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or a 5- or 6-membered aryl; provided that the compound of formula (I) is not: [4-(1-oxo-1,3-dihydro-2H-benzo [f]isoindol-2-yl)phenyl]-2-propionic acid, and sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; and pharmaceutically acceptable derivatives thereof.", "2.A compound according to claim 1 wherein R1 is ═O.", "3.A compound according to claim 1 wherein R3 is methyl, dimethyl, 2-furanyl, ═O or hydrogen.", "4.A compound according to claim 1 wherein R4 and R9 are each hydrogen, methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy.", "5.A compound according to claim 1 wherein R6 and R7 are each hydrogen, methyl, methoxy, chlorine, bromine, iodine, NO2, or CF3.6.A compound according to claim 1 wherein Q1 is hydrogen, methyl, ethyl, NHAc, NH2 or methoxy.", "7.A compound according to claim 1 wherein Q2, Q3, Q4 and Q5 are each hydrogen, methyl, methoxy, chlorine, bromine, iodine, 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, or phenyl.", "8.A compound of formula (I) wherein said compound is selected from the group consisting of: [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid and [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid and [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo [f]isoindol-2-yl)phenyl]acetic acid; [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid and [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; and [4-(4-butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid.", "9.A process for preparing a compound of formula (I) and pharmaceutically acceptable derivatives thereof according to claim 1 which comprises: (A) where R1 and R3 are both ═O and R4 and R9 are the same or different and represent C1-6alkoxy, by reacting a compound of formula (II): with a 4-aminophenylacetic acid of formula (III): in glacial acetic acid at elevated temperature; (B) where one of R1 and R3 is ═O and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, by reducing a compound of formula (IV): with a suitable reducing agent, followed by separation of isomers and deprotection; (C) where one of R1 and R3 is ═O and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, by reacting a compound of formula (V): with a 4-aminophenylacetic acid of formula (III) as defined above in presence of triethylamine and dimethylformamide, followed by deprotection; (D) where one of R1 and R3 is ═O and the other is hydrogen and one of R4 and R9 is C1-6alkoxy, by reacting a compound of formula (VI): where RD is in the −4 or -9 position and is C1-6 alkoxy, with a 4-aminophenylacetate of formula (VII): in presence of sodium triacetoxyborohydride in a suitable solvent, followed by deprotection; (E) where R1 and R3 are both hydrogen and R4 and R9 are the same or different and represent C1-6 alkoxy, by reacting a compound of formula (VIII): with a 4-aminophenylacetate of formula (VII) as described above in a suitable solvent, at elevated temperature followed by deprotection; (F) where R3 is ═CHC1-5alkyl, by reacting a compound of formula (IV) as defined above with a Grignard reagent C1-C6alkyl-MgBr under conventional conditions, followed by separation of isomers and deprotection; (G) where R3 is C1-6dialkyl, by reacting a compound of formula (IX): with a 4-aminophenylacetate of formula (VII) as defined above in presence of aluminium trichloride, followed by deprotection; or (H) converting compounds of formula (I), prepared according to processes (A) to (G), into other compounds of formula (I) using conventional procedures; and optionally converting compounds of formula (I) prepared by any one of processes (A) to (H) into pharmaceutically acceptable derivatives thereof.", "10.A compound of formula (I) or a pharmaceutically acceptable derivative thereof as defined in claim 1 for use in human or veterinary medicine.", "11.", "(Cancelled) 12.A method of treating a human or animal subject suffering from a condition which is mediated by the action of PGE2 at EP4 receptors which comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof as defined in claim 1.13.A pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable derivative thereof, as defined in claim 1 in admixture with one or more physiologically acceptable carriers or excipients." ], [ "This invention relates to naphthalene derivatives, to processes for their preparation, to pharmaceutical compositions containing them and to their use in medicine.", "The EP4 receptor is a 7-transmembrane receptor and its natural ligand is the prostaglandin PGE2.PGE2 also has affinity for the other EP receptors (types EP1, EP2 and EP3).", "The EP4 receptor is associated with smooth muscle relaxation, inflammation, lymphocyte differentiation, bone metabolism processes, allergic activities, promotion of sleep, renal regulation and gastric or enteric mucus secretion.", "We have now found a novel group of compounds which bind with high affinity to the EP4 receptor.", "Compounds exhibiting EP4 binding activity have been described in, for example, WO00/18744, WO00/03980, WO00/15608, WO0016760, WO00/21532, WO98/55468, EP0855389 and EP0985663.GB2330307 describes the use of EP4 antagonists in the treatment of conditions with accelerated bone resorption.", "Derivatives of indoprofen, such as [4-(1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid, sodium salt shown below, have been described by Rufer et. al.", "In Eur.", "J. Med.", "Chem.—Chimica Therapeutica, 1978, 13, no 2, pg 193-198.Accordingly, the present invention provides a compound of formula (I) wherein R1 and R3 are the same or different and represent ═O, hydrogen, C1-6alkyl, C1-6dialkyl, ═CHC1-C5alkyl, ═S, or a 5- or 6-membered aryl; R4 to R9 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or CF3; Q1 is hydrogen, C1-6alkyl, C1-6dialkyl, C1-6alkoxy, NHAc, NR210 where R10 is hydrogen or C1-6alkyl, difluoro, fluoro, ═O, or OH; Q2, Q3, Q4 and Q5 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or a 5- or 6-membered aryl; with the proviso that the compounds [4-(1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid, sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid are excluded; and pharmaceutically acceptable derivatives thereof.", "By pharmaceutically acceptable derivative is meant any pharmaceutically acceptable salt, solvate or ester, or salt or solvate of such ester of the compounds of formula (I), or any other compound which upon administration to the recipient is capable of providing (directly or indirectly) a compound of formula (I) or an active metabolite or residue thereof.", "It will be appreciated by those skilled in the art that the compounds of formula (I) may be modified to provide pharmaceutically acceptable derivatives thereof at any of the functional groups in the compounds, and that the compounds of formula (I) may be derivatised at more than one position.", "It will be appreciated that, for pharmaceutical use, the salts referred to above will be the pharmaceutically acceptable salts, but other salts may find use, for example in the preparation of compounds of formula (I) and the pharmaceutically acceptable salts thereof.", "Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse, J. Pharm.", "Sci., 1977, 66, 1-19.Suitable pharmaceutically acceptable salts of the compounds of formula (I) include acid addition salts formed with inorganic or organic acids, preferably inorganic acids, e.g.", "hydrochlorides, hydrobromides and sulphates.", "Further representative examples of pharmaceutically acceptable salts include those formed from acetic, maleic, fumaric, benzoic, ascorbic, pamoic, succinic, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, propionic, tartaric, salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, cyclohexylsulfamic, phosphoric and nitric acids.", "Further representative examples of pharmaceutically acceptable salts include alkali metal salts, formed from the addition of alkali metal bases, such as alkali metal hydroxides e.g.", "sodium salts.", "It will be appreciated that the compound of formula (I) may be produced in vivo by metabolism of a suitable prodrug.", "Such prodrugs may be for example physiologically acceptable metabolically labile esters of compounds of the general formula (I).", "These may be formed by esterification of the carboxylic acid group in the parent compound of general formula (I) with, where appropriate, prior protection of any other reactive groups present in the molecule followed by deprotection if required.", "Examples of such metabolically labile esters include C1-4alkyl esters e.g.", "methyl ethyl or t-butyl esters esters, C3-6 alkenyl esters e.g.", "allyl substituted or unsubstituted aminoalkyl esters (e.g.", "aminoethyl, 2-(N,N-diethylamino) ethyl, or 2-(4-morpholino)ethyl esters or acyloxyalkyl esters such as, acyloxymethyl or 1-acyloxyethyl e.g.", "pivaloyloxymethyl, 1-pivaloyloxyethyl, acetoxymethyl, 1-acetoxyethyl,1-(1-methoxy-1-methyl)ethylcarbonyloxyethyl, 1-benzoyloxyethyl, isopropoxycarbonyloxymethyl, 1-isopropoxycarbonyloxyethyl, cyclohexylcarbonyloxymethyl, 1-cyclohexylcarbonyloxyethyl ester, cyclohexyloxycarbonyloxymethyl, 1-cyclohexyloxycarbonyloxyethyl, 1-(4-tetrahydropyranyloxy)carbonyloxyethyl or 1-(4-tetrahydropyranyl)carbonyloxyethyl.", "As used herein, the term halogen atom includes fluorine, and more especially chlorine, bromine or iodine.", "The term C1-6alkyl, C1-6alkoxy or ═CHC1-5alkyl as used herein includes straight chain and branched chain alkyl or alkoxy groups containing 1 to 6 carbon atoms, and in particular includes methyl, ethyl, n-propyl and i-propyl or methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy.", "The term 5-membered aryl as used herein means an aryl selected from the following: The term 6-membered aryl as used herein means an aryl selected from: A preferred subgroup of compounds of formula (I) include the compounds wherein R1 is ═O or hydrogen; R3 is ═O, hydrogen, C1-6alkyl, C1-6dialkyl, or a 5- or 6-membered aryl; R4 and R9 are the same or different and represent hydrogen or C1-6alkoxy; R5 and R8 are hydrogen, or CF3; R6 and R7 are the same or different and represent hydrogen, C1-6alkyl, C1-6alkoxy, halogen, NO2, or CF3; Q1 is hydrogen, C1-6alkyl, C1-6alkoxy, NHAc, or NR210 where R10 is hydrogen or C1-6alkyl; Q2, Q3, Q4 and Q5 are the same or different and represent hydrogen, C1-6alkyl, C1-6alkoxy, halogen, or a 5- or 6-membered aryl; with the proviso that the compounds [4-(1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid, sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid are excluded; and pharmaceutically acceptable derivatives thereof.", "R1 is preferably ═O.", "R3 is aptly selected from methyl, dimethyl, or 2-furanyl.", "R3 is preferably ═O or hydrogen, and is more preferably hydrogen.", "R4 and R9 are each suitably hydrogen, methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy.", "Suitably at least one of R4 and R9 represents C1-6alkoxy when Q1 is methyl.", "Preferably R4 and R9 are both C1-6alkoxy, eg ethoxy.", "R6 and R7 are suitably hydrogen, methyl, methoxy, chlorine, bromine, iodine, NO2, or CF3.Preferably R6 and R7 are both hydrogen.", "Q1 is suitably hydrogen, methyl, ethyl, NHAc, NH2 or methoxy.", "Q1 is preferably hydrogen.", "Q2, Q3, Q4 and Q5 are each suitably hydrogen, methyl, methoxy, chlorine, bromine, iodine, 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, or phenyl.", "Preferably Q2, Q3, Q4 and Q5 are all hydrogen.", "A further preferred group of compounds of formula (I) are those wherein R1 is ═O; R3 is methyl, dimethyl, 2-furanyl, preferably ═O or hydrogen, and is more preferably hydrogen; R4 and R9 are each hydrogen, methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy, preferably R4 and R9 are both C1-6alkoxy, eg ethoxy; R5 and R8 are hydrogen; R6 and R7 are hydrogen, methyl, methoxy, chlorine, bromine, iodine, NO2, or CF3, preferably R6 and R7 are both hydrogen; Q1 is suitably hydrogen, methyl, ethyl, NHAc, NH2 or methoxy, preferably hydrogen; Q2, Q3, Q4 and Q5 are each suitably hydrogen, methyl, methoxy, chlorine, bromine, iodine, 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, or phenyl, preferably Q2, Q3, Q4 and Q5 are all hydrogen; with the proviso that the compounds [4-(1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid, sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid are excluded; and pharmaceutically acceptable derivatives thereof.", "It is to be understood that the present invention encompasses all isomers of the compounds of formula (I) and their pharmaceutically acceptable derivatives, including all geometric, tautomeric and optical forms, and mixtures thereof (e.g.", "racemic mixtures).", "Preferred compounds of the present invention include: [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-propoxy-9-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-propoxy-9-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-9-methoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-9-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-9-propoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dimethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-ethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-methoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-butyric acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-(N-acetylamino)acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-aminoacetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-methoxyacetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-3-methylphenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-3-methoxyphenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-3,5-dimethylphenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-3-iodophenyl]acetic acid; [3-chloro-4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-phenyl]acetic acid; [3-bromo-4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-phenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-2-methylphenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-2-methoxyphenyl]acetic acid; [2-chloro-4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-phenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-2-iodophenyl]acetic acid; [2-bromo-4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-phenyl]acetic acid; [4-(6,7-dichloro-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-6-methyl-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-7-methyl-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(7-bromo-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-7-iodo-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(6-bromo-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-6-iodo-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-3,3-dimethyl-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-3-methyl-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-6-nitro-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-7-nitro-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-6-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-7-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(6-chloro-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(7-chloro-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; and pharmaceutically acceptable derivatives thereof.", "Particularly preferred compounds according to the invention are: [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; and pharmaceutically acceptable derivatives thereof.", "It is to be understood that the present invention covers all combinations of particular and preferred groups as described herein above.", "Since the compounds of the present invention, in particular compounds of formula (I), are intended for use in pharmaceutical compositions, it will be understood that they are each provided in substantially pure form, for example at least 50% pure, more suitably at least 75% pure and preferably at least 95% pure (% are on a wt/wt basis).", "Impure preparations of the compounds of formula (I) may be used for preparing the more pure forms used in the pharmaceutical compositions.", "Although the purity of intermediate compounds of the present invention is less critical, it will be readily understood that the substantially pure form is preferred as for the compounds of formula (I).", "Preferably, whenever possible, the compounds of the present invention are obtained in crystalline form.", "When some of the compounds of this invention are allowed to crystallise or are recrystallised from organic solvents, solvent of crystallisation may be present in the crystalline product.", "This invention includes within its scope such solvates.", "Similarly, some of the compounds of this invention may be crystallised or recrystallised from solvents containing water.", "In such cases water of hydration may be formed.", "This invention includes within its scope stoichiometric hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilisation.", "In addition, different crystallisation conditions may lead to the formation of different polymorphic forms of crystalline products.", "This invention includes within its scope all polymorphic forms of the compounds of formula (I).", "The compounds of the invention bind to the EP4 receptor and are therefore useful in treating EP4 receptor mediated diseases.", "In view of their ability to bind to the EP4 receptor, the compounds of the invention are useful in the treatment of the disorders that follow.", "Thus, the compounds of formula (I) are useful as analgesics.", "For example they are useful in the treatment of chronic articular pain (e.g.", "rheumatoid arthritis, osteoarthritis, rheumatoid spondylitis, gouty arthritis and juvenile arthritis) including the property of disease modification and joint structure preservation; musculoskeletal pain; lower back and neck pain; sprains and strains; neuropathic pain; sympathetically maintained pain; myositis; pain associated with cancer and fibromyalgia; pain associated with migraine; pain associated with influenza or other viral infections, such as the common cold; rheumatic fever; pain associated with functional bowel disorders such as non-ulcer dyspepsia, non-cardiac chest pain and irritable bowel syndrome; pain associated with myocardial ischemia; post operative pain; headache; toothache; and dysmenorrhea.", "The compounds of the invention are particularly useful in the treatment of neuropathic pain.", "Neuropathic pain syndromes can develop following neuronal injury and the resulting pain may persist for months or years, even after the original injury has healed.", "Neuronal injury may occur in the peripheral nerves, dorsal roots, spinal cord or certain regions in the brain.", "Neuropathic pain syndromes are traditionally classified according to the disease or event that precipitated them.", "Neuropathic pain syndromes include: diabetic neuropathy; sciatica; non-specific lower back pain; multiple sclerosis pain; fibromyalgia; HIV-related neuropathy; post-herpetic neuralgia; trigeminal neuralgia; and pain resulting from physical trauma, amputation, cancer, toxins or chronic inflammatory conditions.", "These conditions are difficult to treat and although several drugs are known to have limited efficacy, complete pain control is rarely achieved.", "The symptoms of neuropathic pain are incredibly heterogeneous and are often described as spontaneous shooting and lancinating pain, or ongoing, burning pain.", "In addition, there is pain associated with normally non-painful sensations such as “pins and needles” (paraesthesias and dysesthesias), increased sensitivity to touch (hyperesthesia), painful sensation following innocuous stimulation (dynamic, static or thermal allodynia), increased sensitivity to noxious stimuli (thermal, cold, mechanical hyperalgesia), continuing pain sensation after removal of the stimulation (hyperpathia) or an absence of or deficit in selective sensory pathways (hypoalgesia).", "The compounds of formula (I) are also useful in the treatment of inflammation, for example in the treatment of skin conditions (e.g.", "sunburn, burns, eczema, dermatitis, psoriasis); ophthalmic diseases such as glaucoma, retinitis, retinopathies, uveitis and of acute injury to the eye tissue (e.g.", "conjunctivitis); lung disorders (e.g.", "asthma, bronchitis, emphysema, allergic rhinitis, respiratory distress syndrome pigeon fanciers disease, farmer's lung, COPD); gastrointestinal tract disorders (e.g.", "aphthous ulcer, Crohn's disease, atopic gastritis, gastritis varialoforme, ulcerative colitis, coeliac disease, regional ileitis, irritable bowel syndrome, inflammatory bowel disease, gastrointestinal reflux disease); organ transplantation; other conditions with an inflammatory component such as vascular disease, migraine, periarteritis nodosa, thyroiditis, aplastic anaemia, Hodgkin's disease, scierodoma, myaesthenia gravis, multiple sclerosis, sorcoidosis, nephrotic syndrome, Bechet's syndrome, polymyositis, gingivitis, myocardial ischemia, pyrexia, systemic lupus erythematosus, polymyositis, tendinitis, bursitis, and Sjogren's syndrome.", "The compounds of formula (I) are also useful in the treatment of immunological diseases such as autoimmune diseases, immunological deficiency diseases or organ transplantation.", "The compounds of formula (I) are also effective in increasing the latency of HIV infection.", "The compounds of formula (I) are also useful in the treatment of diseases of abnormal platelet function (e.g.", "occlusive vascular diseases).", "The compounds of formula (I) are also useful for the preparation of a drug with diuretic action.", "The compounds of formula (I) are also useful in the treatment of impotence or erectile dysfunction.", "The compounds of formula (I) are also useful in the treatment of bone disease characterised by abnormal bone metabolism or resorption such as osteoporosis (especially postmenopausal osteoporosis), hyper-calcemia, hyperparathyroidism, Paget's bone diseases, osteolysis, hypercalcemia of malignancy with or without bone metastases, rheumatoid arthritis, periodontitis, osteoarthritis, ostealgia, osteopenia, cancer cacchexia, calculosis, lithiasis (especially urolithiasis), solid carcinoma, gout and ankylosing spondylitis, tendinitis and bursitis.", "In a further aspect compounds of formula (I) may be useful in inhibiting bone resorption and/or promoting bone generation.", "The compounds of formula (I) are also useful for attenuating the hemodynamic side effects of NSAIDs and COX-2 inhibitors.", "The compounds of formula (I) are also useful in the treatment of cardiovascular diseases such as hypertension or myocardiac ischemia; functional or organic venous insufficiency; varicose therapy; haemorrhoids; and shock states associated with a marked drop in arterial pressure (e.g.", "septic shock).", "The compounds of formula (I) are also useful in the treatment of neurodegenerative diseases and neurodegeneration such as dementia, particularly degenerative dementia (including senile dementia, Alzheimer's disease, Pick's disease, Huntingdon's chorea, Parkinson's disease and Creutzfeldt-Jakob disease, ALS, motor neuron disease); vascular dementia (including multi-infarct dementia); as well as dementia associated with intracranial space occupying lesions; trauma; infections and related conditions (including HIV infection); metabolism; toxins; anoxia and vitamin deficiency; and mild cognitive impairment associated with ageing, particularly Age Associated Memory Impairment.", "The compounds of formula (I) are also useful in the treatment of neuroprotection and in the treatment of neurodegeneration following stroke, cardiac arrest, pulmonary bypass, traumatic brain injury, spinal cord injury or the like.", "The compounds of formula (I) are also useful in the treatment of tinnitus.", "The compounds of formula (I) are also useful in preventing or reducing dependence on, or preventing or reducing tolerance or reverse tolerance to, a dependence—inducing agent.", "Examples of dependence inducing agents include opioids (e.g.", "morphine), CNS depressants (e.g.", "ethanol), psychostimulants (e.g.", "cocaine) and nicotine.", "The compounds of formula (I) are also useful in the treatment of complications of Type 1 diabetes (e.g.", "diabetic microangiopathy, diabetic retinopathy, diabetic nephropathy, macular degeneration, glaucoma), nephrotic syndrome, aplastic anaemia, uveitis, Kawasaki disease and sarcoidosis.", "The compounds of formula (I) are also useful in the treatment of kidney dysfunction (nephritis, particularly mesangial proliferative glomerulonephritis, nephritic syndrome), liver dysfunction (hepatitis, cirrhosis), gastrointestinal dysfunction (diarrhoea) and colon cancer.", "It is to be understood that reference to treatment includes both treatment of established symptoms and prophylactic treatment, unless explicitly stated otherwise.", "According to a further aspect of the invention, we provide a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use in human or veterinary medicine.", "According to another aspect of the invention, we provide a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use in the treatment of a condition which is mediated by the action of PGE2 at EP4 receptors.", "According to a further aspect of the invention, we provide a method of treating a human or animal subject suffering from a condition which is mediated by the action of PGE2 at EP4 receptors which comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof.", "According to a further aspect of the invention we provide a method of treating a human or animal subject suffering from a pain, inflammatory, immunological, bone, neurodegenerative or renal disorder, which method comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof.", "According to another aspect of the invention, we provide the use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof for the manufacture of a therapeutic agent for the treatment of a condition which is mediated by the action of PGE2 at EP4 receptors.", "According to another aspect of the invention we provide the use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof for the manufacture of a therapeutic agent for the treatment or prevention of a condition such as a pain, inflammatory, immunological, bone, neurodegenerative or renal disorder.", "The compounds of formula (I) and their pharmaceutically acceptable derivatives are conveniently administered in the form of pharmaceutical compositions.", "Such compositions may conveniently be presented for use in conventional manner in admixture with one or more physiologically acceptable carriers or excipients.", "Thus, in another aspect of the invention, we provide a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof adapted for use in human or veterinary medicine.", "While it is possible for the compounds of formula (I) or a pharmaceutically acceptable derivative thereof to be administered as the raw chemical, it is preferable to present it as a pharmaceutical formulation.", "The formulations of the present invention comprise the compounds of formula (I) or a pharmaceutically acceptable derivative thereof together with one or more acceptable carriers or diluents thereof and optionally other therapeutic ingredients.", "The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.", "The formulations include those suitable for oral, parenteral (including subcutaneous e.g.", "by injection or by depot tablet, intradermal, intrathecal, intramuscular e.g.", "by depot and intravenous), rectal and topical (including dermal, buccal and sublingual) administration although the most suitable route may depend upon for example the condition and disorder of the recipient.", "The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.", "All methods include the step of bringing into association the compound of formula (I) or a pharmaceutically acceptable acid addition salt thereof (“active ingredient”) with the carrier which constitutes one or more accessory ingredients.", "In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.", "Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets (e.g.", "chewable tablets in particular for paediatric administration) each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.", "The active ingredient may also be presented as a bolus, electuary or paste.", "A tablet may be made by compression or moulding, optionally with one or more accessory ingredients.", "Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent.", "Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.", "The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.", "Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.", "The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of a sterile liquid carrier, for example, water-for-injection, immediately prior to use.", "Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.", "Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter, hard fat or polyethylene glycol.", "Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose and acacia.", "The compounds of the invention may also be formulated as depot preparations.", "Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.", "Thus, for example, the compounds of the invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.", "In addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.", "The EP4 receptor compounds for use in the instant invention may be used in combination with other therapeutic agents, for example COX-2 inhibitors, such as celecoxib, rofecoxib, valdecoxib or parecoxib; 5-lipoxygenase inhibitors; NSAID's, such as diclofenac, indomethacin, nabumetone or ibuprofen; leukotriene receptor antagonists; DMARD's such as methotrexate; adenosine A1 receptor agonists; sodium channel blockers, such as lamotrigine; NMDA receptor modulators, such as glycine receptor antagonists; gabapentin and related compounds; tricyclic antidepressants such as amitriptyline; neurone stabilising antiepileptic drugs; mono-aminergic uptake inhibitors such as venlafaxine; opioid analgesics; local anaesthetics; 5HT1 agonists, such as triptans, for example sumatriptan, naratriptan, zolmitriptan, eletriptan, frovatriptan, almotriptan or rizatriptan; EP1 receptor ligands; EP2 receptor ligands; EP3 receptor ligands; EP1 antagonists; EP2 antagonists and EP3 antagonists.", "When the compounds are used in combination with other therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route.", "The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof together with a further therapeutic agent or agents.", "The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention.", "The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.", "When a compound of formula (I) or a pharmaceutically acceptable derivative thereof is used in combination with a second therapeutic agent active against the same disease, the dose of each compound may differ from that when the compound is used alone.", "Appropriate doses will be readily appreciated by those skilled in the art.", "A proposed daily dosage of compounds of formula (I) or their pharmaceutically acceptable salts for the treatment of man is from 0.01 to 10 mg/kg body weight per day and more particularly 0.1 to 3 mg/kg body weight per day, calculated as the free base, which may be administered as a single or divided dose, for example one to four times per day The dose range for adult human beings is generally from 8 to 1000 mg/day, such as from 20 to 800 mg/day, preferably 35 to 200 mg/day, calculated as the free base.", "The precise amount of the compounds of formula (I) administered to a host, particularly a human patient, will be the responsibility of the attendant physician.", "However, the dose employed will depend on a number of factors including the age and sex of the patient, the precise condition being treated and its severity, and the route of administration.", "The present invention provides a process for preparing compounds of formula (I) and pharmaceutically acceptable derivatives thereof.", "Compounds of formula (I) and pharmaceutically acceptable derivatives thereof may be prepared by any method known in the art for the preparation of compounds of analogous structure.", "Suitable methods for the preparation of compounds of formula (I) and pharmaceutically acceptable derivatives thereof are described below and form a further aspect of the invention.", "In the formulae that follow, R1 to R10 and Q1-Q5 are as defined in formula (I) above unless otherwise stated.", "According to a first process (A), compounds of formula (I), where R1 and R3 are both ═O and R4 and R9 are the same or different and represent C1-6alkoxy, may be prepared by reacting a compound of formula (II) with a 4-aminophenylacetic acid of formula (III) in glacial acetic acid at elevated temperature.", "According to another process (B) compounds of formula (I), where one of R1 and R3 is ═O and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, may be prepared by reducing a compound of formula (IV) with a suitable reducing agent, followed by separation of isomers and deprotection (eg.", "with aqueous base at elevated temperature).", "Suitable reducing agents include zinc in acetic acid at elevated temperature and sodium borohydride in methanol followed by trifluoroacetic acid (TFA) and triethylsilane.", "According to another process (C) compounds of formula (I), where one of R1 and R3 is ═O and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, may be prepared by reacting a compound of formula (V) with a 4-aminophenylacetic acid of formula (III) as defined above in the presence of triethylamine and dimethylformamide, followed by deprotection (eg.", "using acetic acid at elevated temperature).", "According to a further process (D) compounds of formula (I), where one of R1 and R3 is ═O and the other is hydrogen and one of R4 and R9 is C1-6alkoxy, may be prepared by reacting a compound of formula (VI) where RD is in the −4 or −9 position and is C1-6 alkoxy, with a 4-aminophenylacetate of formula (VII) in the presence of sodium triacetoxyborohydride in a suitable solvent, such as dichloromethane, followed by deprotection (eg.", "with aqueous base at elevated temperature).", "According to a further process (E) compounds of formula (I), where R1 and R3 are both hydrogen and R4 and R9 are the same or different and represent C1-6 alkoxy, may be prepared by reacting a compound of formula (VIII) with a 4-aminophenylacetate of formula (VII) as described above in a suitable solvent, such as dimethylformamide, at elevated temperature followed by deprotection (eg.", "using aqueous base such as lithium hydroxide in aqueous tetrahydrofuran).", "According to a further process (F) compounds of formula (I), where R3 is ═CHC1-5alkyl, may be prepared by reacting a compound of formula (IV) as defined above with a Grignard reagent C1-C6alkyl-MgBr under conventional conditions, followed by separation of isomers and deprotection (eg.", "with aqueous base at elevated temperature).", "According to a further process (G) compounds of formula (I), where R3 is C1-6dialkyl, may be prepared by reacting a compound of formula (IX) with a 4-aminophenylacetate of formula (VII) as defined above in the presence of aluminium trichloride, followed by deprotection (eg.", "with aqueous base at elevated temperature).", "According to a further process (H) compounds of formula (I) prepared according to processes (A) to (G) may be converted into other compounds of formula (I) using conventional procedures.", "For example, compounds of formula (I) where R3 is C1-6alkyl, may be prepared by reducing a compound of formula (I) where R3 is ═CHC1-5alkyl, protected at the carboxyl group, with a suitable reducing agent, such as hydrogen in the presence of a palladium on carbon catalyst, followed by deprotection (eg.", "with aqueous base at elevated temperature).", "Also, compounds of formula (I) where R3 is ═O may be converted into compounds of formula (I) where R3 is ═S by conventional methods, for example using Lawesson's reagent.", "Compounds of formulae (II) to (IX) may be prepared by any method known in the art for the preparation of compounds of analogous structure.", "Compounds of formula (II) may, for example be prepared according to Scheme 1 that follows.", "Compounds of formula (IV) may be prepared by reacting compounds of formula (II) with a 4-aminophenylacetate of formula (VII) as defined above in the presence of acetic acid.", "Compounds of formula M may, for example be prepared according to Scheme 2 that follows.", "Compounds of formula (VI) may, for example, be prepared according to Scheme 3 that follows.", "Compounds of formula (VIII) may be prepared, for example, by reacting a compound of formula (X) with phosphorous tribromide in an ether solvent, for example a mixture of diethyl ether and tetrahydrofuran.", "Compounds of formula (IX) may be prepared by reacting a compound of formula (XI) with a Grignard reagent R3-MgBr under conventional conditions.", "Compounds of formulae (III) and (VII) may be prepared according to methods known in the art for the preparation of analogous compounds, for example, compounds where Q1 is methyl may be prepared by the method of Takahashi,l et al.", "Heterocycles (1996), 43(II), 2343-2346; compounds where Q1 is ethyl may be prepared by the method of Kirschenheuter, Gary P et al.", "in EP465802; compounds where Q1 is NHAc may be prepared by the method described in US3479339; compounds where Q1 is NH2 may be prepared by the method of Herbert, Richard B et al.", "J. Chem.", "Soc., Perkin Trans.1 (1992), (1), 109-13; compounds where Q1 is MeO may be prepared from the corresponding nitro compounds prepared by the method of Tomioka, Hideo et al.", "J.", "Am.", "Chem.", "Soc.", "(1990), 112(21), 7692-702; compounds where Q2 is Me or Q3 is Cl may be prepared by the method described in U.S. Pat.", "No.", "3,860,639 (Schultz, Everett M.); compounds where Q2 is MeO may be prepared by the method of Nannini, G et al, Arzneim.-Forsch.", "(1973), 23(8), 1090-100 by hydrolysis of the ethyl ester; compounds where Q2 is Cl may be prepared by the method of Atkinson, Joseph G et al.", "Tet Left.", "(1979), (31), 2857-60 by reduction of the corresponding nitro compound; compounds where Q2 is I may be prepared by the method of Sindelar, Karel et al.", "Collect.", "Czech.", "Chem.", "Commun.", "(1978), 43(2), 471-97 by reduction of the corresponding nitro compound; compounds where Q2 is Br may be prepared by the method of Sindelar, Karel et al.", "Collect.", "Czech.", "Chem.", "Commun.", "(1978), 43(2), 471-97; compounds where Q3 is Me may be prepared by the method of Borck, Joachim et al.", "in ZA 6804711; compounds where Q3 is MeO may be prepared by the method of Gallacher, Gerard et al.", "Biorg.", "Amines (1995), 11(1), 49-62 by reduction of the corresponding nitro compound; compounds where Q3 is I may be prepared by the method of Boehm, Marcus F et al.", "J. Chem.", "Soc., Chem.", "Commun.", "(1991), (1), 52-3; compounds where Q3 is Br may be prepared by the method of Figala, Georg et al.", "in DE 2746067; compounds where Q3 and Q4 are Me may be prepared by the method of Yost, Yul et al.", "Org.", "Prep.", "Proced.", "Int.", "(1985), 17(4-5), 23949 from the corresponding benzoic acid using the Arndt-Eistert reaction.", "Certain intermediates described above are novel compounds, and it is to be understood that all novel intermediates herein form further aspects of the present invention.", "Compounds of formula (II), (V), (VI), (VIII) and (IX) are key intermediates and represent a particular aspect of the invention.", "Certain intermediates, such as compounds of formula (IV), may be prodrugs of compounds of formula (I).", "Conveniently, compounds of the invention are isolated following work-up in the form of the free base.", "Pharmaceutically acceptable acid addition salts of the compounds of the invention may be prepared using conventional means.", "Solvates (e.g.", "hydrates) of a compound of the invention may be formed during the work-up procedure of one of the aforementioned process steps.", "The following Examples which should not be construed as constituting a limitation thereto are provided to illustrate the invention.", "1H NMR spectra were obtained at 400 MHz on a Bruker DPX400 spectrophotometer.", "J values are given in Hz.", "Mass spectra were obtained on a Micromass series II MS (electrospray positive or negative).", "Where HPLC retention times are given as a characterisation of intermediates or Examples this refers to an HP 1050 or a HP1100 running a 5.5 minute Gradient: Eluents: A —0.1% VN Formic Acid+10 mmol Ammonium Acetate B —95% MeCN+0.05% VN Formic Acid Flow rate: 3 ml/min Column: 3.3 cm×4.6 mm internal diameter, 3 μm ABZ+PLUS Injection Volume: 5 μl Temperature: Room temperature.", "Gradient: Time % A % B 0.00 100 0.00 0.70 100 0.00 4.40 0.00 100 5.30 0.00 100 5.50 100 0.00 Intermediate 1 Ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate Sodium (60 g, 2.6 mol) was dissolved in ethanol (1.2L) and the mixture was cooled to 40° C. Diethylphthalate (960 ml, 4.83 mol) was added and the mixture heated under nitrogen until the temperature reached 115° C. Diethyl succinate (211.3 g, 1.21 mol) was added dropwise over 45 min.", "The reaction was heated at 115° C. for a further 45 min, cooled to room temperature and poured onto water (1.2L).", "Ethyl acetate (1L) was added and stirred, the layers were separated and the organics were extracted with sodium hydroxide solution (2N, 1L).", "The combined aqueous was acidified to pH 3 and the mixture extracted with ethyl acetate (2×1L).", "The combined organics were washed with a saturated solution of sodium hydrogen carbonate (2×1.5L), then brine, dried (MgSO4), filtered and the solvent evaporated under vacuum.", "The residue was purified using a 2.5 kg Biotage column eluting with 5% ethyl acetate/hexane to give ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate as a white solid, (60 g, 16%).", "δH CDCl3 10.44,(2H, s), 8.34,(2H, m), 7.68,(2H, m), 4.37,(4H, q), 1.37,(6H, t).", "Intermediate 2 Ethyl 1,4-diethoxy-2,3-naphthalenedicarboxylate Ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate (30 g, 98.6 mmol) and potassium carbonate (150 g, 1.09 mmol) were stirred in acetone (600 ml) under nitrogen.", "Iodoethane (150 g, 0.96 mol) was added and the mixture was stirred at reflux overnight.", "The reaction was cooled, diluted with ethyl acetate and filtered.", "The filtrate was evaporated to leave a brown oil, which was dissolved in toluene and washed with potassium hydroxide solution (5%, 150 ml) and brine.", "Drying over magnesium sulphate and evaporation of the solvent gave a yellow solid.", "Purification using an 800 g Biotage column gave ethyl 1,4-diethoxy-2,3-naphthalenedicarboxylate as a white solid (32 g, 90%).", "δH CDCl3 8.16,(2H, m), 7.60,(2H, m), 4.40,(4H, q), 4.18,(4H, q), 1.50,(6H, t), 1.40,(6H, t).", "Intermediate 3 1,4-Diethoxy-2,3-naphthalenedicarboxylic Acid Ethyl 1,4-diethoxy-2,3-naphthalenedicarboxylate (32 g, 89 mmol) was added to a solution of sodium hydroxide (20 g) in ethanol (200 ml) and water (40 ml) and stirred for 1.5h at 60° C. The reaction was cooled and the thick white suspension was filtered.", "The solid was dissolved in a mixture of ethyl acetate (200 ml) and water (800 ml).", "The layers were separated and the aqueous was acidified with hydrochloric acid (2M, 120 ml).", "The aqueous was extracted with ethyl acetate (2×) and the combined organics were dried (MgSO4).", "Evaporation of the solvent under vacuum gave 1,4-diethoxy-2,3-naphthalenedicarboxylic acid as a white solid (25 g, 92%).", "δH [2H6]—DMSO 13.26,(2H, s), 8.15,(2H, m), 7.72,(2H, m), 4.13,(4H, q), 1.42,(6H, t).", "Intermediate 4 1,4-Diethoxy-2,3-naphthalenedicarboxylic Anhydride 1,4-Diethoxy-2,3-naphthalenedicarboxylic acid (25 g, 82 mmol) was added to a solution of thionyl chloride (23.3 g) in chloroform (150 ml) and stirred at reflux for 1 h. The resulting solution was cooled and evaporated to dryness.", "Further chloroform was added and evaporation repeated to give 1,4-diethoxy-2,3-naphthalenedicarboxylic anhydride as a yellow solid (23.3 g, 99%).", "δH [2H6]—DMSO 8.42,(2H, m), 7.93,(2H, m), 4.53,(4H, q), 1.46,(6H, t).", "Intermediate 5 Ethyl[4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[I]isoindol-2-yl)phenyl]acetate 1,4-Diethoxy-2,3-naphthalenedicarboxylic anhydride (23.3 g, 81.5 mmol) and ethyl (4-aminophenyl)acetate (14.8 g, 82 mmol) were refluxed under nitrogen in acetic acid (160 ml) overnight.", "The mixture was cooled to room temperature and poured into water (1L).", "The white solid was filtered, washed with water and dissolved in dichloromethane (800 ml).", "The solution was washed with water, brine and dried (MgSO4) and the solvent evaporated under vacuum to give ethyl [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate as an off-white solid, 33 g, 96%.", "δH [2H6]—DMSO 8.40,(2H, m), 7.87,(2H, m), 7.42,(4H, s), 4.47,(4H, q), 4.12,(2H, q), 3.76,(2H, s), 1.45,(6H, t), 1.21,(3H, t).", "Intermediate 6 3-Hydroxy 4-methoxynaphtho[2,3-c]furan-[(3H)-one N,N,N′-Trimethylethylene diamine (0.82 ml, 6.3 mmol) was dissolved in tetrahydrofuran (16 ml) and cooled to −20° C. n-Butyl lithium (1.6M in hexanes, 3.9 ml, 6.24 mmol) was added and the reaction stirred at −20° C. for 15 min.", "1-Methoxy-2-naphthaldehyde (0.969, 5.9 mmol) was added followed by n-butyl lithium (1.6M in hexanes, 11.25 ml, 18 mmol) and the reaction stirred at 25° C. for 3h.", "Solid carbon dioxide was added and the reaction left until the excess carbon dioxide had sublimed.", "Stirring continued for 15 min before addition of hydrochloric acid (2N, 50 ml).", "The mixture was extracted with dichloromethane (50 ml, ×3).", "The combined extracts were dried (MgSO4) and the solvent evaporated under vacuum.", "The oily residue was preabsorbed onto silica and purified by SPE (silica, 10 g) eluting with an ethyl acetate/cyclohexane gradient to give 3-hydroxy-4-methoxynaphtho[2,3-c]furan-1 (3M-one (50 mg, 3.7%).", "δH [2H6]—DMSO 8.28,(2H, m), 8.18,(2H, m), 7.70,(2H, m), 7.16,(1H, s), 4.27,(3H, s).", "Intermediate 7 Ethyl 1,4-dimethoxy-2-methylnaphthalene-3-carboxylate n-Butyl lithium (1.6M in hexanes, 4.1 ml, 6.56 mmol) was added dropwise to 2-bromo-1,4-dimethoxy-3-methylnaphthalene (1.537 g, 5.47 mmol) in tetrahydrofuran (30 ml) at −50° C. The reaction was stirred for 30 min at −50° C. before dropwise addition of ethyl chloroformate (1 ml, 10.46 mmol).", "The reaction was allowed to warm to 0° C. over 18h quenched by addition of hydrochloric acid (2N) and extracted with ethyl acetate.", "The extract was dried (Na2SO4) and the solvent evaporated under vacuum.", "The residue was purified by SPE (silica, 10 g) eluting with 10% ethyl acetate in cyclohexane to give ethyl 1,4-dimethoxy-2-methylnaphthalene-3-carboxylate (1.339, 89%).", "δH CDCl3 8.08,(2H, d), 7.53,(2H, m), 4.48,(2H, q), 3.99,(3H, s), 3.88,(3H, s), 2.38,(3H, s), 1.44,(3H, t).", "Intermediate 8 2,3-Bis(bromomethyl)-1,4-diethoxy-naphthalene [1,4-Diethoxy-3-(hydroxymethyl)-2-naphthyl]methanol (92 mg, 0.33 mmol) was dissolved in a mixture of diethyl ether (1.5 ml) and tetrahydrofuran (2 ml) under nitrogen at 0° C. and phosphorus tribromide (0.035 ml, 0.35 mmol) was added dropwise.", "The reaction was allowed to warm to ambient temperature and stirred for 4 h before quenching with ice.", "The reaction mixture was extracted with ethyl acetate (3×5 ml) and the combined organic extracts dried over magnesium sulphate.", "The solvent was removed in vacuo, and the residue purified by SPE cartridge (silica), gradient elution cyclohexane to 75% cyclohexane/dichloromethane, to give 2,3-bis(bromomethyl)-1,4-diethoxy-naphthalene as a white solid (92 mg, 70%).", "δH CDCl3 8.07 (2H, dd, J=6.5, 3.2), 7.54 (2H, dd, J=6.5, 3.2), 5.02 (4H, s), 4.21 (4H, q, J=7.0), 1.60 (6H, t, J=7.0); LC retention time 4.16 min.", "EXAMPLE 1 (Process C) [4-(4,9-Dimethoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid To a solution of ethyl 1,4-dimethoxy-2-methylnaphthalene-3-carboxylate (73 mg, 0.266 mmol) in carbon tetrachloride (10 ml) was added N-bromosuccinimide (47 mg, 0.266 mmol) and dibenzylperoxide (5 mg).", "The mixture was heated at reflux under nitrogen for 45 min, illuminating with a 200W lamp.", "The reaction was cooled to room temperature, 4-aminophenyl acetic acid (40 mg, 0.266 mmol), triethylamine (74 μl, 0.53 mmol) and DMF (5 ml) added and the reaction stirred for 48h.", "Acetic acid (glacial, 1 ml) was added to the reaction and the mixture refluxed for 3h under nitrogen.", "Sodium metabisulphite solution (1 ml) was added and the reaction evaporated to dryness under vacuum.", "The product was partially purified by SPE (NH2, 10 g) eluting with methanol then 5% acetic acid in methanol.", "The fractions containing product were passed through a silica plug washing with 5% acetic acid in methanol.", "After evaporation of the solvents under vacuum, the residue was columned on a silica gel flash column eluting with a gradient (1:1 ethyl acetate/cyclohexane to 5% methanol, 1% acetic acid in 1:1 ethyl acetate/cyclohexane) to give [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid (15 mg, 15%).", "δH CDCl3 8.40,(1H, d), 8.17,(1H, d), 7.90.", "(2H, d), 7.65,(1H, t), 7.59,(1H, t), 7.38,(2H, d), 5.02,(2H, s), 4.27,(3H, s), 4.08,(3H, s), 3.69,(2H, s).", "MS 378, [MH+].", "LC retention time 3.28 min.", "EXAMPLE 2 Step 1 (Process E) Ethyl[4-(4,9-diethoxy-1,3-dihydro-2H-benzo[t]isoindol-2-yl)phenyl]acetate A solution of 2,3-bis(bromomethyl)-1,4-diethoxy-naphthalene (41 mg, 0.1 mmol) and 4-aminophenylacetic acid ethyl ester (20 mg, 0.11 mmol) in N,N-dimethylformamide (1 ml) was heated at 60° C. under nitrogen for 14 h. The solvent was removed in vacuo and the residue taken up in ethyl acetate (10 ml) and washed with 8% aqueous sodium bicarbonate solution (5 ml).", "The organic layer was dried over magnesium sulphate and the solvent removed in vacuo.", "Purification by flash column chromatography on silica gel eluting with 90% cyclohexane/ethyl acetate gave the product as a white solid (15 mg, 36%).", "δH CDCl3 8.12 (2H, dd, J=6.5, 3.2), 7.50 (2H, dd, J=6.5, 3.2), 7.23 (2H, d, J=8.5), 6.73 (2H, d, J=8.5), 4.80 (4H, s), 4.17 (6H, m), 3.55 (2H, s), 1.56 (6H, m), 1.24 (3H, m); LC retention time 4.25 min, ms 420, [MH+].", "EXAMPLE 2 Step 2 [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid A solution of ethyl[4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (10 mg, 0.024 mmol) in tetrahydrofuran (4 ml) and a solution of lithium hydroxide (6 mg, 0.24 mmol) in water (1 ml) were stirred vigorously for 14h.", "The reaction mixture was diluted with water (10 ml) and extracted with ethyl acetate (2×5 ml) and the organic extracts discarded.", "The aqueous phase was acidified with 2N hydrochloric acid to pH=5 and extracted with ethyl acetate (3×5 ml).", "The combined organic extracts were dried over magnesium sulphate and the solvent removed in vacuo to give the product as a red solid (4 mg, 43%).", "δH CDCl3 8.13 (2H, dd, J=6.5, 3.2), 7.49, (2H, dd, J=6.5, 3.2), 7.24 (2H, d, 8.3), 6.75 (2H, d, J=8.3), 4.81 (4H, s), 4.19 (4H, q, J=7.0), 3.69 (2H, s), 1.53 (6H, t, J=7.0); LC retention time 3.92 min, ms 392, [MH+].", "General Methodology Method A A mixture of ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate (1.0 g, 3.29 mmol), potassium carbonate (5 g, 36.2 mmol) and the alkyl halide (3.25 mmol) in acetone (20 ml) was heated at reflux under nitrogen for 8h.", "The cooled reaction was filtered and the residue washed with acetone and ethyl acetate.", "The combined filtrate and washings were evaporated to dryness under vacuum.", "The residue was partitioned between hydrochloric acid (2N) and dichloromethane.", "The dichloromethane extract was evaporated to dryness under vacuum.", "The product was isolated by chromatography on a silica gel flash column eluting with an ethyl acetate/cyclohexane gradient (0-10% ethyl acetate).", "Method B A mixture of monoalkylated material (170 mg, 0.49 mmol), potassium carbonate (850 mg, 6.15 mmol) and the alkyl halide (1.0 mmol) in acetone (5 ml) was heated at reflux under nitrogen for 4h.", "The cooled reaction was filtered and the residue washed with acetone and ethyl acetate.", "The combined filtrate and washings were evaporated to dryness under vacuum and the residue purified by SPE (silica) eluting with an ethyl acetate/cyclohexane gradient.", "Method C A mixture of ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate (2 g, 6.57 mmol), potassium carbonate (10 g, 72.4 mmol) and the alkyl halide (64.1 mmol) in acetone (40 ml) was heated at reflux under nitrogen for 4h.", "The cooled reaction was filtered and the residue washed with acetone and ethyl acetate.", "The combined filtrate and washings were evaporated to dryness under vacuum and the residue purified by SPE (silica) eluting with an ethyl acetate/cyclohexane gradient.", "Method D A mixture of the diester (13.87 mmol) and sodium hydroxide solution (2N, 25 ml) in ethanol (25 ml) was heated at reflux for 4h.", "The resulting solution was acidified to pH1 with hydrochloric acid (2N) and extracted with ethyl acetate (×2).", "The extracts were dried (MgSO4) an d the solvent evaporated under vacuum.", "Method E (Process A) A mixture of diacid (0.57 mmol) and 4-aminophenylacetic acid (1.32 mmol) in glacial acetic acid (5 ml) was heated under reflux for 24h.", "The cooled reaction was diluted with water, the precipitate formed filtered off and washed with water.", "Method F A mixture of diacid (0.56 mmol) and ethyl 4-aminophenylacetate (1.11 mmol) in glacial acetic acid (5 ml) was heated at reflux for 24h.", "The cooled reaction was diluted with water, the precipitate formed filtered off and washed with water.", "Method G (Process B—Step 1) The phthalimide ester (0.32 mmol) was dissolved in glacial acetic acid (4 ml) and zinc powder (100 mesh, 300 mg, 4.59 mmol) added.", "The reaction was heated at reflux under nitrogen for 72h.", "The hot reaction was filtered and the residue washed with hot acetic acid.", "The combined filtrate and washings were evaporated to dryness under vacuum.", "Methylamine (33% in ethanol, 2 ml) was added to the resulting solid and the suspension was stirred at room temperature for 20 min.", "The methylamine solution was evaporated under vacuum and the residue purified by SPE (silica) eluting with an ethyl acetate/cyclohexane gradient to give a mixture of isomeric y-lactams.", "Method H (Processes B and D—Step 2) The γ-lactams (0.06 mmol) were mixed with potassium carbonate (1.09 mmol) in ethanol (2 ml) and water (1 ml) and heated at reflux for 4h.", "The cooled solution was acidified to pH1 with hydrochloric acid (2M), the precipitate filtered off and washed with water.", "Dried at 40° C. under vacuum.", "The following compounds were prepared using the above general methodologies: Intermediate 9 Ethyl [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 49% yield from 1,4-di-isopropoxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.41,(2H, m), 7.83,(2H, m), 7.42,(4H, s), 5.02,(2H, m), 4.12,(2H, q), 3.76,(2H, s), 1.36,(12H, d), 1.22,(3H, t).", "Intermediate 10 Ethyl [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 68% yield from 1,4-dipropoxynaphthalene-2.3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.35,(2H, m), 7.82,(2H, m), 7.37,(4H, s), 4.34,(4H, t), 4.07,(2H, q), 3.71,(2H, s), 1.83,(4H, m), 1.17,(3H, t), 1.01,(6H, t).", "Intermediate 11 Ethyl [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 39% yield from 1,4-dibutoxynaphthalene-2,3-dicarboxylic acid using method F. δH [H6]—DMSO 8.39,(2H, m), 7.87,(2H, m), 7.42,(4H, s), 4.43,(4H, t), 4.12,(2H, q), 3.76,(2H, s), 1.85,(4H, m), 1.53,(4H, m), 1.22,(3H, t), 0.97,(6H, t).", "Intermediate 12 Ethyl [444,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared from 1,4-dihexyloxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.38,(2H, m), 7.86,(2H, m), 7.42,(4H, s), 4.41,(4H, t), 4.12,(2H, q), 3.76,(2H, s), 1.85,(4H, m), 1.49,(4H, m), 1.32,(8H, m), 1.21,(3H, t), 0.87,(6H, t).", "Intermediate 13 Ethyl [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared from 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.36,(2H, m), 7.79,(2H, m), 7.37,(4H, s), 4.98,(1H, m), 4.42,(2H, q), 4.07,(2H, q), 3.71,(2H, s), 1.40,(3H, t), 1.31,(6H, d), 1.17,(3H, t).", "Intermediate 14 Ethyl [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared from 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.35,(2H, m), 7.80,(2H, m), 7.37,(4H, s), 4.97,(1H, m), 4.34,(2H, t), 4.07,(2H, q), 3.71,(2H, s), 1.83,(2H, m), 1.31,(6H, d), 1.71,(3H, t), 1.01,(3H, t).", "Intermediate 15 Ethyl [4-(4-hexyloxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4-hexyloxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared from 1-hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.37,(1H, m), 8.32,(1H, m), 7.80,(2H, m), 7.37,(4H, s), 4.97,(1H, m), 4.37,(2H, t), 4.07,(2H, q), 3.71,(2H, s), 1.81,(2H, m), 1.45,(2H, m), 1.35-1.26,(10H, m), 1.17,(3H, t), 0.82,(3H, t).", "Intermediate 16 1,4-Di-isopropoxynaphthalene-2,3-dicarboxylic Acid 1,4-Di-isopropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1,4-d]-propoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.16,(2H, m), 7.67,(2H, m), 4.43,(2H, m), 1.26,(12H, d).", "Intermediate 17 1,4-Dipropoxynaphthalene-2,3-dicarboxylic Acid 1,4-Dipropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1,4-dipropoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.14,(2H, m), 7.71,(2H, m), 4.01,(4H, t), 1.83,(4H, m), 1.05,(6H, t).", "Intermediate 18 1,4-Dibutoxynaphthalene-2,3-dicarboxylic Acid 1,4-Dibutoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1,4-dibutoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.12,(2H, m), 7.12,(2H, m), 4.05,(4H, t), 1.80,(4H, m), 1.52,(4H, m), 0.97,(6H, t).", "Intermediate 19 1,4-Dihexyloxynaphthalene-2,3-dicarboxylic Acid 1,4-Dihexyloxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1,4-dihexyloxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.39,(2H, m), 7.92,(2H, m), 4.46,(4H, t), 1.85,(4H, m), 1.49,(4H, m), 1.32,(8H, m), 0.88,(6H, t).", "Intermediate 20 1-Ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylic Acid 1-Ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.18,(1H, m), 8.12,(1H, m), 7.69,(2H, m), 4.40,(1H, m), 4.11,(2H, q), 1.41,(3H, t), 1.27,(6H, d).", "Intermediate 21 4-Isopropoxy-1-propoxynaphthalene-2,3-dicarboxylic Acid 4-Isopropoxy-1-propoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.18,(1H, m), 8.11,(1H, m), 7.70,(2H, m), 4.41,(1H, m), 4.02,(2H, t), 1.83,(2H, m), 1.27,(6H, d), 1.06,(3H, t).", "Intermediate 22 1-Butoxy-4-isopropoxynaphthalene-2,3-dicarboxylic Acid 1-Butoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1-butoxy-4-isopropoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.18,(1H, m), 8.09,(1H, m), 7.69,(2H, m), 4.40,(1H, m), 4.05,(2H, t), 180,(2H, m), 1.52,(2H, m), 1.26,(6H, d), 0, 97,(3H, t).", "Intermediate 23 1-Hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylic Acid 1-Hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1-hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.18,(1H, m), 8.09,(1H, m), 7.70,(2H, m), 4.40,(1H, m), 4.04,(2H, t), 1.81,(2H, m), 1.49,(2H, m), 1.34,(4H, m), 1.27,(6H, d), 0.90,(3H, t).", "Intermediate 24 Ethyl 1,4-di-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1,4-di-isopropoxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 2-iodopropane using method C. δH [2H6]—DMSO 8.18,(2H, m), 7.73,(2H, m), 4.37,(2H, m), 4.28,(4H, q), 1.30,(6H, t), 1.25,(12H, d).", "Intermediate 25 Ethyl 1,4-dipropoxynaphthalene-2,3-dicarboxylate Ethyl 1,4-dipropoxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 1-iodopropane using method C. δH [2H6]—DMSO 8.16,(2H, m), 7.77,(2H, m), 4.29,(4H, q), 4.00,(4H, t), 1.83,(4H, m), 1.30,(4H, t), 1.04,(4H, t).", "Intermediate 26 Ethyl 1,4-dibutoxynaphthalene-2,3-dicarboxylate Ethyl 1,4-dibutoxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 1-iodobutane using method C. δH [2H6]—DMSO 8.15,(2H, m), 7.76,(2H, m), 4.29,(4H, q), 4.03,(4H, t), 1.79,(4H, m), 1.50,(4H, m), 1.30,(6H, t), 0.96,(6H, t).", "Intermediate 27 Ethyl 1,4-dihexyloxynaphthalene-2,3-dicarboxylate Ethyl 1,4-dihexyloxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 2-iodohexane using method C. δH [2H6]—DMSO 8.14,(2H, m), 7.76,(2H, m), 4.29,(4H, q), 4.03,(4H, t), 1.81,(4H, m), 1.47,(4H, m), 1.38-1.21,(14H, m), 0.88,(6H, t).", "Intermediate 28 Ethyl 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylate was prepared in 91% yield from ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate and iodoethane using method B. δH CDCl3 8.22,(1H, m), 8.13,(1H, m), 7.59,(2H, m), 4.40,(5H, m), 4.18,(2H, q), 1.49,(3H, t), 1.43-1.38,(6H, m), 1.34,(6H, d).", "Intermediate 29 Ethyl 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylate Ethyl 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylate was prepared in 91% yield from ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate and 1-iodopropane using method B. δH CDCl3 8.22,(1H, m), 8.14,(1H, m), 7.60,(2H, m), 4.40,(5H, m), 4.07,(2H, t), 1.92,(2H, m), 1.40,(6H, m), 1.34,(6H, d), 1.10,(3H, t).", "Intermediate 30 Ethyl 1-butoxy-4-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1-butoxy-4-isopropoxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate and 1-iodobutane using method B. δH [2H6]—DMSO 8.20,(1H, m), 8.12,(1H, m), 7.75,(2H, m), 4.38-4.25,(5H, m), 4.04,(2H, t), 1.80,(2H, m), 1.50,(2H, m), 1.30,(6H, t), 1.25,(6H, d), 0.96,(3H, t).", "Intermediate 31 Ethyl 1-hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1-hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylate was prepared in 80 yield from ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate and 1-iodohexane using method B. δH CDCl3 8.21,(1H, m), 8.13,(1H, m), 7.59,(2H, m), 4.40,(5H, m), 4.10,(2H, t), 1.90,(2H, m), 1.53,(2H, m), 1.45-1.31,(16H, m), 0.92,(3H, t).", "Intermediate 32 Ethyl 1-ethoxy-4-hydroxynaphthalene-2,3-dicarboxylate Ethyl 1-ethoxy-4-hydroxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and iodoethane using method A. δH [2H6]—DMSO 8.36,(1H, d), 8.06,(1H, d), 7.83,(1H, m), 7.72,(1H, m), 4.35,(4H, m), 4.03,(2H, q), 1.39,(3H, t), 1.32,(6H, m).", "Intermediate 33 Ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate was prepared in 36% yield from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 2-iodopropane using method A. δH CDCl3 12.3,(1H, s), 8.45,(1H, d), 8.10,(1H, d), 7.65,(1H, m), 7.55,(1H, m), 4.42,(1H, m), 1.41,(6H, t), 1.32,(6H, d).", "EXAMPLE 4 Step 1 (Process B) Ethyl [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 16% yield from ethyl [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.34,(1H, d), 8.16,(1H, d), 7.93,(2H, d), 7.68,(1H, t), 7.60,(1H, t), 7.34,(2H, d), 5.10,(2H, s), 5.01,(1H, m), 4.68,(1H, m), 4.10,(2H, q), 3.69,(2H, s), 1.40,(3H, d), 1.35,(3H, d), 1.19,(3H, t).", "EXAMPLE 4 Step 2 [4-(4,9-Di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in 81% yield from ethyl [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.34,(1H, d), 8.17,(1H, d), 7.91,(2H, d), 7.69,(1H, m), 7.61,(1H, m), 7.34,(2H, d), 5.10,(2H, s), 5.02,(1H, m), 4.68,(1H, m), 3.59,(2H, s), 1.38,(6H, d), 1.34,(6H, d).", "MS 434, [MH+].", "LC retention time 3.91 min.", "EXAMPLE 5 Step 1 (Process B) Ethyl [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 13% yield from ethyl [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.32,(1H, d), 8.19,(1H, d), 7.93,(2H, d), 7.72,(1H, t), 7.65,(1H, t), 7.36,(2H, d), 5.19,(2H, s), 4.30,(2H, t), 4.22,(2H, t), 4.10,(2H, q), 3.69,(2H, s), 1.87,(4H, m), 1.20,(3H, t), 1.13-1.05,(6H, m).", "EXAMPLE 5 Step 2 [4-(4,9-Dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in 89% yield from ethyl [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.32,(1H, d), 8.19,(1H, d), 7.91,(2H, d), 7.71,(1H, t), 7.65,(1H, t), 7.65,(2H, d), 5.19,(2H, s), 4.30,(2H, t), 4.22,(2H, t), 3.59,(2H, s), 1.89,(4H, m), 1.09,(6H, m).", "MS 434, [MH+].", "LC retention time 3.97 min.", "EXAMPLE 6 Step 1 (Process B) Ethyl [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 26% yield from ethyl [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.22,(1H, d), 8.09,(1H, d), 7.84,(2H, d), 7.63,(1H, t), 7.57,(1H, t), 7.27,(2H, d), 5.10,(2H, s), 4.26,(2H, t), 4.17,(2H, t), 4.02,(2H, q), 3.60,(2H, s), 1.77,(4H, quintet), 1.49,(4H, m), 1.12,(3H, t), 0.91,(6H, q).", "EXAMPLE 6 Step 2 [4-(4,9-Dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in 98% yield from ethyl [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.30,(1H, d), 8.18,(1H, d), 7.91,(2H, d), 7.71,(1H, t), 7.65,(1H, t), 7.35,(2H, d), 5.19,(2H, s), 4.34,(2H, t), 4.26,(2H, t), 3.59,(2H, s), 1.85,(4H, m), 1.57,(4H, m), 0.99,(6H, m).", "MS 462, [MH+].", "LC retention time 4.25 min.", "EXAMPLE 7 Step 1 (Process B) Ethyl [44,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 28% yield from ethyl [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.19,(1H, d), 8.07,(1H, d), 7.82,(2H, d), 7.60,(1H, t), 7.54,(1H, t), 7.25,(2H, d), 5.08,(2H, s), 4.23,(2H, t), 4.15,(2H, t), 3.99,(2H, q), 3.58,(2H, s), 1.75,(4H, quintet), 1.50-1.36,(4H, m), 1.29-1.21,(8H, m), 1.09,(3H, t), 0.79,(6H, q).", "EXAMPLE 7 Step 2 [4-(4,9-Dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in 97% yield from ethyl [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.30,(1H, d), 8.17,(1H, d), 7.91,(2H, d), 7.71,(1H, t), 7.64,(1H, t), 7.34,(2H, d), 5.18,(2H, s), 4.33,(2H, t), 4.25,(2H, t), 3.59,(2H, s), 1.86,(4H, m), 1.54,(4H, m), 1.35,(8H, m), 0.90,(6H, m).", "MS 518, [MH+].", "LC retention time 4.76 min.", "EXAMPLE 8 Step 1 (Process B) Ethyl [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-thoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate A mixture of ethyl [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) was prepared in 20% yield from ethyl [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.25,(1H, m), 8.09,(1H, m), 7.85,(2H, d), 7.64-7.58,(2H, m), 7.26,(2H, d), 5.09,(1.2H, s), 5.02,(0.8H, s), 4.90,(0.6H, m), 4.60,(0.4H, m), 4.32,(0.8H, q), 4.22,(1.2H, q), 4.01,(2H, q), 3.60,(2H, s), 1.38,(3H, m), 1.29,(2.4H, d), 1.24,(3.6H, d), 1.11,(3H, t).", "EXAMPLE 8 Step 2 [4-(4-Ethoxy-9-isopropoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid and [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid A mixture of [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid and [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid (60:40) was prepared in 98% yield from ethyl [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) using method H. δH [2H6]—DMSO 8.34,(1H, m), 8.18,(1H, m), 7.92,(2H, d), 7.70,(1H, t), 7.68,(1H, t), 7.34,(2H, d), 5.18,(1.2H, s), 5.11,(0.8H, s), 5.01,(0.6H, m), 4.69,(0.4H, m), 4.40,(0.8H, q), 4.31,(1.2H, q), 3.59,(2H, s), 1.46,(3H, m), 1.37,(2.4H, d), 1.33,(3.6H, d).", "MS 420, [MH+].", "LC retention time 3.73 min.", "EXAMPLE 9 Step 1 (Process B) Ethyl [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate A mixture of ethyl [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) was prepared in 21% yield from ethyl [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.25,(1H, m), 8.10,(1H, m), 7.85,(2H, m), 7.62,(1H, m), 7.53,(1H, m), 7.27,(2H, d), 5.10,(1.2H, s), 5.02,(0.8H, s), 4.91,(0.6H, m), 4.58,(0.4H, m), 4.23,(0.8H, t), 4.13,(1.2H, t), 4.01,(2H, q), 3.60,(2H, s), 1.80,(2H, m), 1.29,(2.4H, d), 1.24,(3.6H, d), 1.11,(3H, t), 1.01,(3H, m).", "EXAMPLE 9 Step 2 [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid and [4-(4-isopropoxy-9-propoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid A mixture of [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid and [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid (60:40) was prepared in quantitative yield from ethyl [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) using method H. δH [2H6]—DMSO 8.34,(1H, m), 8.18,(1H, m), 7.92,(2H, m), 7.70.", "(1H, t), 7.63,(1H, m), 7.34,(2H, d), 5.18,(1.2H, s), 5.11,(0.8H, s), 5.01,(0.6H, m), 4.68,(0.4H, m), 4.32,(0.8H, q), 4.22,(1.2H, q), 3.59,(2H, s), 1.89,(2H, m), 1.37,(2.4H, d), 1.33,(3.6H, d), 1.10,(3H, m).", "MS 434, [MH+].", "LC retention time 3.89 min.", "EXAMPLE 10 Step 1 (Process B) Ethyl [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate A mixture of ethyl [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) was prepared in 16% yield from ethyl [4-(4-hexyloxy-9-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.33,(1H, m), 8.17,(1H, m), 7.94,(2H, m), 7.70,(1H, m), 7.64,(1H, m), 7.35,(2H, d), 5.18,(1.2H, s), 5.11,(0.8H, s), 5.00,(0.6H, m), 4.69,(0.4H, m), 4.35,(0.8H, t), 4.25,(1.2H, t), 4.10,(2H, q), 3.69,(2H, s), 1.87,(2H, m), 1.60-1.45,(2H, m), 1.37,(2.4H, d), 1.33,(3.6H, d), 1.20,(3H, t), 0.91,(3H, m).", "EXAMPLE 10 Step 2 [4-(4-Hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid and [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid A mixture of [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid and [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid (60:40) was prepared in 78% yield from ethyl [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) using method H. δH [2H6]—DMSO 8.35,(0.6H, d), 8.30,(0.4H, d), 8.17,(2H, m), 7.91,(2H, m), 7.70,(1H, t), 7.63,(1H, m), 7.34,(2H, d), 5.18,(1.2H, s), 5.10,(0.8H, s), 5.00,(0.6H, m), 4.68,(0.4H, m), 4.35,(0.8H, q), 4.25,(1.2H, q), 3.59,(2H, s), 1.87,(2H, m), 1.54,(2H, m), 1.35,(10H, m), 0.91,(3H, m).", "MS 476, [MH+].", "LC retention time 4.32 min.", "EXAMPLE 11 Step 1 (Process D) Ethyl [44-methoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate 3-Hydroxy-4-methoxynaphtho[2,3-c]furan-1 (3H-one (50 mg, 0.22 mmol) and ethyl 4-aminophenylacetate (47 mg, 0.26 mmol) in dichloromethane (5 ml) were stirred at room temperature under nitrogen for 1 hour.", "Sodium triacetoxyborohydride (138 mg, 0.66 mmol) was added and stirring continued for 24h.", "The reaction was adjusted to pH14 with sodium hydroxide solution (2N) and extracted with dichloromethane (3×5 ml).", "The combined extracts were evaporated under vacuum and the residue triturated with cyclohexane/ether to give ethyl [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate as a white solid (45 mg, 54%).", "δH [2H6]—DMSO 8.24,(1H, d), 8.16,(1H, d), 8.13,(1H, s), 7.99,(2H, d), 7.64,(2H, m), 7.37,(2H, d), 5.44,(2H, s), 4.26,(3H, s), 4.10,(2H, q), 3.69,(2H, s), 1.20,(3H, t).", "EXAMPLE 11 Step 2 [4-(4-Methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4-Methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in quantitative yield from ethyl [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.19,(1H, d), 8.09,(2H, m), 7.92,(2H, d), 7.59,(2H, m), 7.31,(2H, d), 5.39,(2H, s), 4.21,(3H, s), 3.56,(2H, s).", "MS 348, [MH+].", "LC retention time 3.50 min.", "EXAMPLE 12 [44,9-Diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-diethoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.35,(2H, m), 7.81,(2H, m), 7.36,(4H, s), 4.43,(4H, q), 3.62,(2H, s), 1.40,(6H, t).", "MS 420, [MH+].", "LC retention time 3.58 min.", "EXAMPLE 13 [4-(4,9-Di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-di-isopropoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.41,(2H, m), 7.83,(2H, m), 7.41,(4H, s), 5.02,(2H, m), 3.66,(2H, s), 1.36,(12H, d).", "MS 448, [MH+].", "LC retention time 3.74 min.", "EXAMPLE 14 [4-(4,9-Dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-dipropoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.39,(2H, m), 7.87,(2H, m), 7.41,(4H, s), 4.39,(4H, t), 3.67,(2H, s), 1.87,(4H, m), 1.05,(6H, t).).", "MS 448, [MH+].", "LC retention time 3.89 min.", "EXAMPLE 15 [4-(4,9-Dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-dibutoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.39,(2H, m), 7.88,(2H, m), 7.41,(4H, s), 4.43,(4H, t), 3.67,(2H, s), 1.84,(4H, m), 1.52,(4H, m), 0.96,(6H, t).", "MS 476, [MH+].", "LC retention time 4.15 min.", "EXAMPLE 16 [4-(4,9-Dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-dihexyloxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.38,(2H, m), 7.86,(2H, m), 7.41,(4H, s), 4.42,(4H, t), 3.67,(2H, s), 1.85,(4H, m), 1.49,(4H, m), 1.32,(8H, m), 0.87,(6H, t).).", "MS 532, [MH+].", "LC retention time 4.62 min.", "EXAMPLE 17 [4-(4-Ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4-Ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.41,(2H, m), 7.84,(2H, m), 7.41,(4H, s), 5.03,(1H, m), 4.48,(2H, q), 3.67,(2H, s), 1.45,(3H, t), 1.36,(6H, d).", "MS 434, [MH+].", "LC retention time 3.66 min.", "EXAMPLE 18 [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.41,(2H, m), 7.84,(2H, m), 7.41,(4H, s), 5.02,(1H, m), 4.39,(2H, t), 3.66,(2H, s), 1.88,(2H, m), 1.36,(6H, d), 1.05,(3H, t).", "MS 448, [MH+].", "LC retention time 3.81 min.", "EXAMPLE 19 [4-(4-Butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4-Butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1-butoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.42,(1H, m), 8.37,(1H, m), 7.85,(2H, m), 7.41,(4H, s), 5.02,(1H, m), 4.43,(2H, t), 3.66,(2H, s), 1.85,(2H, m), 1.53,(2H, m), 1.36,(6H, d), 0.97,(3H, t).", "MS 461, [MH+].", "LC retention time 3.94 min.", "Biological Data The ability of the compounds of the invention to bind to EP4 receptors has been demonstrated in the Human EP4 Scintillation Proximity Assay.", "Quantification of radioligand binding by scintillation proximity assay (SPA) is a long-established principle.", "Briefly, the affinity of novel compounds for a receptor is assessed by the specific competition between known quantities of radiolabelled ligand and novel compound for that receptor.", "Increasing concentrations of novel compound reduce the amount of radiolabel that binds to the receptor.", "This gives rise to a diminishing scintillation signal from SPA beads coated with membranes that bear the receptor.", "The signal may be detected with a suitable scintillation counter and the data generated may be analysed with suitable curve-fitting software.", "The human EP4 SPA assay (hereafter referred to as ‘the assay’) utilises membranes prepared from Chinese Hamster Ovary (CHO cells) infected with Semliki Forest Virus (SFV).", "Genetically engineered SFV-1 viral particles containing the genetic sequence of the human EP4 receptor were used to infect CHO cells resulting in expression of the receptor protein in cellular membranes.", "Cells washed free of media are homogenised in a pH-buffered medium containing peptidase inhibitors.", "A suitable buffer is of the following composition: 50 mM HEPES, 1 mM EDTA, 25 μg/ml bacitracin, 100 μM leupeptin, 1 mM PMSF, 2 μM Pepstatin A, pH adjusted to 7.4 with KOH.", "Following removal of cell debris by a low-speed centrifugation, a pellet of membranes is prepared by a high-speed (48000 g) centrifugation of the resulting supernatant.", "Membrane suspensions such as that described may be stored at −80° C. until used.", "For assay, membranes expressing human EP4 receptors are diluted in a pH-buffered medium and mixed with SPA beads coated with a suitable substance to facilitate the adhesion of membranes to the beads.", "The concentrations of membrane protein and SPA beads chosen should result in SPA binding signal of at least 300 corrected counts per minute (CCPM) when tritiated radioligand at a concentration close to its Kd (affinity value) is combined with the mixture.", "Non-specific binding (nsb) may be determined by competition between the radiolabelled ligand and a saturating concentration of unlabelled ligand.", "In order to quantify the affinity of novel EP4 receptor ligands, compounds are diluted in a stepwise manner across the wells of a 96-well plate.", "Radioligand, novel compound, and unlabelled ligand are then added to a 96-well plate suitable for the measurement of SPA binding signals prior to the addition of bead/membrane mixture to initiate the binding reaction.", "Equilibrium may be achieved by incubation at room temperature for 120 minutes prior to scintillation counting.", "The data so generated may be analysed by means of a computerised curve-fitting routine in order to quantify the concentration of compound that displaces 50% of the specific radioligand binding (IC50).", "The affinity (pKi) of the novel compound may be calculated from the IC50 by application of the Cheng-Prusoff correction.", "Suitable reagents and protocols are: reaction buffer containing 50 mM HEPES, 10 mM MgCl2, pH adjusted to 7.4 with KOH; SPA beads coated with wheatgerm agglutinin; 1.25 nM [3H]-prostaglandin E2 as radioligand; 10 μM prostaglandin E2 as unlabelled ligand; a three-fold dilution series of novel compound starting at 10 μM and ending at 0.3 nM is adequate.", "The following examples have a pKi of 6.0 or greater at EP4 receptors as determined using the above-mentioned procedure: 1, 4, 5, 8, 9, 12, 13, 14, 17." ] ]	Patent_10467487
[ [ "Protein-coupled receptor", "The present application provides a gene encoding a G protein-coupled receptor termed nGPCR-14; constructs and recombinant host cells incorporating the genes; the nGPCR-14 polypeptides encoded by the gene; antibodies to the nGPCR-x polypeptides; and methods of making and using all of the foregoing." ], [ "1.An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to a sequence of SEQ ID NO: 192, and fragments thereof; said nucleic acid molecule encoding at least a portion of nGPCR-14, wherein the isolated nucleic acid comprises at least one or more nucleotides from one or more of the following regions of SEQ ID NO: 191: nucleotides 1 to 193 of SEQ ID NO: 191; nucleotides 612 to 644 of SEQ ID NO:191; nucleotides 697 to 706 of SEQ ID NO:191; nucleotides 1011 to 1049 of SEQ ID NO: 191; nucleotides 1051 to 1057 of SEQ ID NO:191; nucleotides 1090 to 1096 of SEQ ID NO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191.2.The isolated nucleic acid molecule of claim 1 comprising a sequence that encodes a polypeptide comprising a sequences of SEQ ID NO: 192, and fragments thereof.", "3.The isolated nucleic acid molecule of claim 1 comprising a sequence homologous to a sequence of SEQ ID NO: 191, and fragments thereof.", "4.The isolated nucleic acid molecule of claim 1 comprising a sequence of SEQ ID NO:191, and fragments thereof.", "5.The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule is DNA.", "6.The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule is RNA.", "7.An expression vector comprising a nucleic acid molecule of any one of claims 1 to 5.8.The expression vector of claim 7 wherein said nucleic acid molecule comprises a sequence of SEQ ID NO:191.9.The expression vector of claim 7 wherein said vector is a plasmid.", "10.The expression vector of claim 7 wherein said vector is a viral particle.", "11.The expression vector of claim 10 wherein said vector is selected from the group consisting of adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses.", "12.The expression vector of claim 7 wherein said nucleic acid molecule is operably connected to a promoter selected from the group consisting of simian virus 40, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Barr virus, rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein.", "13.A host cell transformed with an expression vector of claim 7.14.The transformed host cell of claim 13 wherein said cell is a bacterial cell.", "15.The transformed host cell of claim 14 wherein said bacterial cell is E. coli.", "16.The transformed host cell of claim 13 wherein said cell is yeast.", "17.The transformed host cell of claim 16 wherein said yeast is S. cerevisiae.", "18.The transformed host cell of claim 13 wherein said cell is an insect cell.", "19.The transformed host cell of claim 18 wherein said insect cell is S. frugiperda.", "20.The transformed host cell of claim 13 wherein said cell is a mammalian cell.", "21.The transformed host cell of claim 20 wherein mammalian cell is selected from the group consisting of chinese hamster ovary cells, HeLa cells, African green monkey kidney cells, human 293 cells, and murine 3T3 fibroblasts.", "22.An isolated nucleic acid molecule comprising a nucleotide sequence complementary to at least a portion of a sequence of SEQ ID NO:191, said portion comprising at least 10 nucleotides, wherein said isolated nucleic acid comprises at least one or more nucleotides from one or more of the following regions of SEQ ID NO: 191: nucleotides 1 to 193 of SEQ ID NO: 191; nucleotides 612 to 644 of SEQ ID NO:191; nucleotides 697 to 706 of SEQ ID NO: 191; nucleotides 1011 to 1049 of SEQ ID NO: 191; nucleotides 1051 to 1057 of SEQ ID NO:191; nucleotides 1090 to 1096 of SEQ ID NO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191.23.The nucleic acid molecule of claim 22 wherein said molecule is an antisense oligonucleotide directed to a region of a sequence of SEQ ID NO: 191.24.The nucleic acid molecule of claim 23 wherein said oligonucleotide is directed to a regulatory region of a sequence of SEQ ID NO: 191.25.A composition comprising a nucleic acid molecule of any one of claims 1 to 5 or 22 and an acceptable carrier or diluent.", "26.A composition comprising a recombinant expression vector of claim 7 and an acceptable carrier or diluent.", "27.A method of producing a polypeptide that comprises a sequence of SEQ ID NO: 192, and homologs and fragments thereof, said method comprising the steps of: a) introducing a recombinant expression vector of claim 7 into a compatible host cell; b) growing said host cell under conditions for expression of said polypeptide; and c) recovering said polypeptide, wherein the polypeptide comprising a sequence of SEQ ID NO:192 comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO:192; amino acid residues 68 to 77 of SEQ ID NO:192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192.28.The method of claim 27 wherein said host cell is lysed and said polypeptide is recovered from the lysate of said host cell.", "29.The method of claim 27 wherein said polypeptide is recovered by purifying the culture medium without lysing said host cell.", "30.An isolated polypeptide encoded by a nucleic acid molecule of claim 1, wherein the polynucleotide comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO:192; amino acid residues 68 to 77 of SEQ ID NO:192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192.31.The polypeptide of claim 30 wherein said polypeptide comprises a fragment of SEQ ID NO:192.32.The polypeptide of claim 30 wherein said polypeptide comprises an amino acid sequence homologous to a sequence of SEQ ID NO: 192.33.The polypeptide of claim 30 wherein said sequence homologous to a sequence of SEQ ID NO:192 comprises at least one conservative amino acid substitution compared to the sequence of SEQ ID NO: 192.34.The polypeptide of claim 30 wherein said polypeptide comprises a fragment of a polypeptide with a sequence of SEQ ID NO:192.35.A composition comprising a polypeptide of claim 30 and an acceptable carrier or diluent.", "36.An isolated antibody which binds to an epitope on a polypeptide of claim 30.37.The antibody of claim 36 wherein said antibody is a monoclonal antibody.", "38.A composition comprising an antibody of claim 36 and an acceptable carrier or diluent.", "39.A method of inducing an immune response in a mammal against a polypeptide of claim 30 comprising administering to said mammal an amount of said polypeptide sufficient to induce said immune response.", "40.A method for identifying a compound which binds nGPCR-14 comprising the steps of: a) contacting nGPCR-14 with a compound; and b) determining whether said compound binds nGPCR-14.41.The method of claim 40 wherein the nGPCR-14 comprises an amino acid sequence of SEQ ID NO: 192.42.The method of claim 40 wherein binding of said compound to nGPCR-14 is determined by a protein binding assay.", "43.The method of claim 40 wherein said protein binding assay is selected from the group consisting of a gel-shift assay, Western blot, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, and ELISA.", "44.A compound identified by the method of claim 40.45.A method for identifying a compound which binds a nucleic acid molecule encoding nGPCR-14 comprising the steps of: a) contacting said nucleic acid molecule encoding nGPCR-14 with a compound; and b) determining whether said compound binds said nucleic acid molecule.", "46.The method of claim 45 wherein binding is determined by a gel-shift assay.", "47.A compound identified by the method of claim 45.48.A method for identifying a compound which modulates the activity of nGPCR-14 comprising the steps of: a) contacting nGPCR-14 with a compound; and b) determining whether nGPCR-14 activity has been modulated.", "49.The method of claim 48 wherein the nGPCR-14 comprises an amino acid sequence of SEQ ID NO: 192.50.The method of claim 48 wherein said activity is neuropeptide binding.", "51.The method of claim 48 wherein said activity is neuropeptide signaling.", "52.A compound identified by the method of claim 48.53.A method of identifying an animal homolog of nGPCR-14 comprising the steps: a) comparing the nucleic acid sequences of the animal with a sequence of SEQ ID NO: 191, and portions thereof, said portions being at least 10 nucleotides; and b) identifying nucleic acid sequences of the animal that are homologous to said sequence of SEQ ID NO: 191, and portions thereof.", "54.The method of claim 53 wherein comparing the nucleic acid sequences of the animal with a sequence selected of SEQ ID NO: 191, and portions thereof, said portions being at least 10 nucleotides, is performed by DNA hybridization.", "55.The method of claim 53 wherein comparing the nucleic acid sequences of the animal with a sequence selected of SEQ ID NO: 191, and portions thereof, said portions being at least 10 nucleotides, is performed by computer homology search.", "56.A method of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor, comprising the steps of: (a) assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering an amino acid sequence, expression, or biological activity of at least one nGPCR that is expressed in the brain, wherein the nGPCR comprises an amino acid sequence of SEQ ID NO:192, and allelic variants thereof, and wherein the nucleic acid corresponds to a gene encoding the nGPCR; and (b) diagnosing the disorder or predisposition from the presence or absence of said mutation, wherein the presence of a mutation altering the amino acid sequence, expression, or biological activity of the nGPCR in the nucleic acid correlates with an increased risk of developing the disorder.", "57.A method according to claim 56, wherein the nGPCR is nGPCR-14 comprising an amino acid sequence set forth in SEQ ID NO:192 or an allelic variant thereof.", "58.A method according to claim 56, wherein the assaying step comprises at least one procedure selected from the group consisting of: a) comparing nucleotide sequences from the human subject and reference sequences and determining a difference of at least a nucleotide of at least one codon between is the nucleotide sequences from the human subject that encodes an nGPCR-14 allele and an nGPCR-14 reference sequence; (b) performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; (c) performing a polynucleotide migration assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; and (d) performing a restriction endonuclease digestion to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences.", "59.A method according to claim 58 wherein the assaying step comprises: performing a polymerase chain reaction assay to amplify nucleic acid comprising nGPCR-14 coding sequence, and determining nucleotide sequence of the amplified nucleic acid.", "60.A method of screening for an nGPCR-14 mental disorder genotype in a human patient, comprising the steps of: (a) providing a biological sample comprising nucleic acid from said patient, said nucleic acid including sequences corresponding to allelles of nGPCR-14; and (b) detecting the presence of one or more mutations in the nGPCR-14 allelle; wherein the presence of a mutation in an nGPCR-40 allelle or nGPCR-54 allele is indicative of a mental disorder genotype.", "61.The method according to claim 59 wherein said biological sample is a cell sample.", "62.The method according to claim 59 wherein said detecting the presence of a mutation comprises sequencing at least a portion of said nucleic acid, said portion comprising at least one codon of said nGPCR-14 alleles.", "63.The method according to claim 59 wherein said nucleic acid is DNA.", "64.The method according to claim 59 wherein said nucleic acid is RNA.", "65.A kit for screening a human subject to diagnose a mental disorder or a genetic predisposition therefor, comprising, in association: (a) an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-14 gene, the oligonucleotide comprising 6-50 nucleotides in a sequence that is identical or complementary to a sequence of a wild type human nGPCR-14 gene sequence or nGPCR-14 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution; and (b) a media packaged with the oligonucleotide, said media containing information for identifying polymorphisms that correlate with schizophrenia or a genetic predisposition therefor, the polymorphisms being identifiable using the oligonucleotide as a probe.", "66.A method of identifying a nGPCR allelic variant that correlates with a mental disorder, comprising steps of: (a) providing a biological sample comprising nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny; (b) detecting in the nucleic acid the presence of one or more mutations in an nGPCR that is expressed in the brain, wherein the nGPCR comprises an amino acid sequence of SEQ ID NO: 192, and allelic variants thereof, and wherein the nucleic acid includes sequence corresponding to the gene or genes encoding nGPCR; wherein the one or more mutations detected indicates an allelic variant that correlates with a mental disorder.", "67.A method according to claim 66 wherein the at least one nGPCR is nGPCR-14, or an allelic variant thereof.", "68.A purified and isolated polynucleotide comprising a nucleotide sequence encoding a nGPCR-14 allelic variant identified according to claim 67.69.A host cell transformed or transfected with a polynucleotide according to claim 68 or with a vector comprising the polynucleotide.", "70.A purified polynucleotide comprising a nucleotide sequence encoding nGPCR-14 of a human with a mental disorder; wherein said polynucleotide hybridizes to the complement of SEQ ID NO:191 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS; and wherein the polynucleotide that encodes the nGPCR-14 amino acid sequence of the human differs from SEQ ID NO:192 by at least one residue and comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO: 192; amino acid residues 68 to 77 of SEQ ID NO: 192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192.71.A vector comprising a polynucleotide according to claim 70.72.A host cell that has been transformed or transected with a polynucleotide according to claim 70 and that expresses the nGPCR-14 protein encoded by the polynucleotide.", "73.A host cell according to claim 72 that has been co-transfected with a polynucleotide encoding the nGPCR-14 amino acid sequence set forth in SEQ ID NO:192 and that expresses the nGPCR-14 protein having the amino acid sequence set forth in SEQ ID NO:192.74.A method for identifying a modulator of biological activity of nGPCR-14 comprising the steps of: a) contacting a cell according to claim 72 in the presence and in the absence of a putative modulator compound; b) measuring nGPCR-14 biological activity in the cell; wherein decreased or increased nGPCR-14 biological activity in the presence versus absence of the putative modulator is indicative of a modulator of biological activity.", "75.A method to identify compounds useful for the treatment of a mental disorder, said method comprising steps of: (a) contacting a composition comprising nGPCR-14 with a compound suspected of binding nGPCR-14; (b) detecting binding between nGPCR-14 and the compound suspected of binding nGPCR-14; wherein compounds identified as binding nGPCR-14 are candidate compounds useful for the treatment of a mental disorder.", "76.A method for identifying a compound useful as a modulator of binding between nGPCR-14 and a binding partner of nGPCR-14 comprising the steps of: (a) contacting the binding partner and a composition comprising nGPCR-14 in the presence and in the absence of a putative modulator compound; (b) detecting binding between the binding partner and nGPCR-14; wherein decreased or increased binding between the binding partner and nGPCR-14 in the presence of the putative modulator, as compared to binding in the absence of the putative modulator is indicative a modulator compound useful for the treatment of schizophrenia.", "77.A method according to claim 75 or 76 wherein the composition comprises a cell expressing nGPCR-14 on its surface.", "78.A method according to claim 77 wherein the composition comprises a cell transformed or transfected with a polynucleotide that encodes nGPCR-14.79.A method of purifying a G protein from a sample containing said G protein comprising the steps of: a) contacting said sample with a polypeptide of claim 1 for a time sufficient to allow said G protein to form a complex with said polypeptide; b) isolating said complex from remaining components of said sample; c) maintaining said complex under conditions which result in dissociation of said G protein from said polypeptide; and d) isolating said G protein from said polypeptide.", "80.The method of claim 79 wherein said sample comprises an amino acid sequence of SEQ ID NO: 192.81.The method of claim 79 wherein said polypeptide comprises an amino acid sequence homologous to a sequence of SEQ ID NO:192." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The G protein-coupled receptors (GPCRs) form a vast superfamily of cell surface receptors which are characterized by an amino-terminal extracellular domain, a carboxyl terminal intracellular domain, and a serpentine structure that passes through the cell membrane seven times.", "Hence, such receptors are sometimes also referred to as seven transmembrane (7TM) receptors.", "These seven transmembrane domains define three extracellular loops and three intracellular loops, in addition to the amino- and carboxy-terminal domains.", "The extracellular portions of the receptor have a role in recognizing and binding one or more extracellular binding partners (e.g., ligands), whereas the intracellular portions have a role in recognizing and communicating with downstream molecules in the signal transduction cascade.", "The G protein-coupled receptors bind a variety of ligands including calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and even photons, and are important in the normal (and sometimes the aberrant) function of many cell types.", "[See generally Strosberg, Eur.", "J. Biochem.", "196:1-10 (1991) and Bohm et al., Biochem J.", "322:1-18 (1997).]", "When a specific ligand binds to its corresponding receptor, the ligand typically stimulates the receptor to activate a specific heterotrimeric guanine-nucleotide-binding regulatory protein (G-protein) that is coupled to the intracellular portion of the receptor.", "The G protein in turn transmits a signal to an effector molecule within the cell, by either stimulating or inhibiting the activity of that effector molecule.", "These effector molecules include adenylate cyclase, phospholipases and ion channels.", "Adenylate cyclase and phospholipases are enzymes that are involved in the production of the second messenger molecules cAMP, inositol triphosphate and diacyglycerol.", "It is through this sequence of events that an extracellular ligand stimuli exerts intracellular changes through a G protein-coupled receptor.", "Each such receptor has its own characteristic primary structure, expression pattern, ligand-binding profile, and intracellular effector system.", "Because of the vital role of G protein-coupled receptors in the communication between cells and their environment, such receptors are attractive targets for therapeutic intervention, for example by activating or antagonizing such receptors.", "For receptors having a known ligand, the identification of agonists or antagonists may be sought specifically to enhance or inhibit the action of the ligand.", "Some G protein-coupled receptors have roles in disease pathogenesis (e.g., certain chemokine receptors that act as HIV co-receptors may have a role in AIDS pathogenesis), and are attractive targets for therapeutic intervention even in the absence of knowledge of the natural ligand of the receptor.", "Other receptors are attractive targets for therapeutic intervention by virtue of their expression pattern in tissues or cell types that are themselves attractive targets for therapeutic intervention.", "Examples of this latter category of receptors include receptors expressed in immune cells, which can be targeted to either inhibit autoimmune responses or to enhance immune responses to fight pathogens or cancer; and receptors expressed in the brain or other neural organs and tissues, which are likely targets in the treatment of schizophrenia, depression, bipolar disease, or other neurological disorders.", "This latter category of receptor is also useful as a marker for identifying and/or purifying (e.g., via fluorescence-activated cell sorting) cellular subtypes that express the receptor.", "Unfortunately, only a limited number of G protein receptors from the central nervous system (CNS) are known.", "Thus, a need exists for G protein-coupled receptors that have been identified and show promise as targets for therapeutic intervention in a variety of animals, including humans." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof.", "The nucleic acid molecule encodes at least a portion of nGPCR-x.", "In some embodiments, the nucleic acid molecule comprises a sequence that encodes a polypeptide comprising even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof.", "In some embodiments, the nucleic acid molecule comprises a sequence homologous to odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a fragment thereof.", "In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof.", "According to some embodiments, the present invention provides vectors which comprise the nucleic acid molecule of the invention.", "In some embodiments, the vector is an expression vector.", "According to some embodiments, the present invention provides host cells which comprise the vectors of the invention.", "In some embodiments, the host cells comprise expression vectors.", "The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence complementary to at least a portion of a sequence from an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, said portion comprising at least 10 nucleotides.", "The present invention provides a method of producing a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.", "The method comprising the steps of introducing a recombinant expression vector that includes a nucleotide sequence that encodes the polypeptide into a compatible host cell, growing the host cell under conditions for expression of the polypeptide and recovering the polypeptide.", "The present invention provides an isolated antibody which binds to an epitope on a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.", "The present invention provides an method of inducing an immune response in a mammal against a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.", "The method comprises administering to a mammal an amount of the polypeptide sufficient to induce said immune response.", "The present invention provides a method for identifying a compound which binds nGPCR-x.", "The method comprises the steps of: contacting nGPCR-x with a compound and determining whether the compound binds nGPCR-x.", "The present invention provides a method for identifying a compound which binds a nucleic acid molecule encoding nGPCR-x.", "The method comprises the steps of contacting said nucleic acid molecule encoding nGPCR-x with a compound and determining whether said compound binds said nucleic acid molecule.", "The present invention provides a method for identifying a compound which modulates the activity of nGPCR-x.", "The method comprises the steps of contacting nGPCR-x with a compound and determining whether nGPCR-x activity has been modulated.", "The present invention provides a method of identifying an animal homolog of nGPCR-x.", "The method comprises the steps screening a nucleic acid database of the animal with an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof and determining whether a portion of said library or database is homologous to said odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or portion thereof.", "The present invention provides a method of identifying an animal homolog of nGPCR-x.", "The methods comprises the steps screening a nucleic acid library of the animal with a nucleic acid molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof; and determining whether a portion of said library or database is homologous to said odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof.", "Another aspect of the present invention relates to methods of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor.", "The methods comprise the steps of assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering an amino acid sequence, expression, or biological activity of at least one nGPCR that is expressed in the brain.", "The nGPCR comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof.", "A diagnosis of the disorder or predisposition is made from the presence or absence of the mutation.", "The presence of a mutation altering the amino acid sequence, expression, or biological activity of the nGPCR in the nucleic acid correlates with an increased risk of developing the disorder.", "The present invention further relates to methods of screening for a nGPCR-40 or nGPCR-54 hereditary schizophrenia genotype in a human patient.", "The methods comprise the steps of providing a biological sample comprising nucleic acid from the patient, in which the nucleic acid includes sequences corresponding to alleles of nGPCR-40 or nGPCR-54.The presence of one or more mutations in the nGPCR-40 allele or the nGPCR-54 allele is detected indicative of a hereditary schizophrenia genotype.", "The present invention provides kits for screening a human subject to diagnose schizophrenia or a genetic predisposition therefor.", "The kits include an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-40 gene or a human nGPCR-54 gene.", "The oligonucleotide comprises 6-50 nucleotides in a sequence that is identical or complementary to a sequence of a wild type human nGPCR-40 or nGPCR-54 gene sequence or nGPCR-40 or nGPCR-54 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution.", "The kit also includes a media packaged with the oligonucleotide.", "The media contains information for identifying polymorphisms that correlate with schizophrenia or a genetic predisposition therefor, the polymorphisms being identifiable using the oligonucleotide as a probe.", "The present invention further relates to methods of identifying nGPCR allelic variants that correlates with mental disorders.", "The methods comprise the steps of providing biological samples that comprise nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny, and detecting in the nucleic acid the presence of one or more mutations in an nGPCR that is expressed in the brain.", "The nGPCR comprises an amino acid sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof.", "The nucleic acid includes sequences corresponding to the gene or genes encoding nGPCR.", "The one or more mutations detected indicate an allelic variant that correlates with a mental disorder.", "The present invention further relates to purified polynucleotides comprising nucleotide sequences encoding alleles of nGPCR-40 or nGPCR-54 from a human with schizophrenia.", "The polynucleotide hybridizes to the complement of SEQ ID NO:83 or of SEQ ID NO:85 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.", "The polynucleotide that encodes nGPCR-40 or nGPCR-54 amino acid sequence of the human differs from SEQ ID NO:84 or SEQ ID NO:86 by at least one residue.", "The present invention also provides methods for identifying a modulator of biological activity of nGPCR-40 or nGPCR-54 comprising the steps of contacting a cell that expresses nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and measuring nGPCR-40 or nGPCR-54 biological activity in the cell.", "The decreased or increased nGPCR-40 or nGPCR-54 biological activity in the presence versus absence of the putative modulator is indicative of a modulator of biological activity.", "The present invention further provides methods to identify compounds useful for the treatment of schizophrenia.", "The methods comprise the steps of contacting a composition comprising nGPCR-40 with a compound suspected of binding nGPCR-40 or contacting a composition comprising nGPCR-54 with a compound suspected of binding nGPCR-54.The binding between nGPCR-40 and the compound suspected of binding nGPCR-40 or between nGPCR-54 and the compound suspected of binding nGPCR-54 is detected.", "Compounds identified as binding nGPCR-40 or nGPCR-54 are candidate compounds useful for the treatment of schizophrenia.", "The present invention further provides methods for identifying a compound useful as a modulator of binding between nGPCR-40 and a binding partner of nGPCR-40 or between nGPCR-54 and a binding partner of nGPCR-54.The methods comprise the steps of contacting the binding partner and a composition comprising nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and detecting binding between the binding partner and nGPCR-40 or nGPCR-54.Decreased or increased binding between the binding partner and nGPCR-40 or nGPCR-54 in the presence of the putative modulator, as compared to binding in the absence of the putative modulator is indicative a modulator compound useful for the treatment of schizophrenia.", "Another aspect of the present invention relates to methods of purifying a G protein from a sample containing a G protein.", "The methods comprise the steps of contacting the sample with an nGPCR for a time sufficient to allow the G protein to form a complex with the nGPCR; isolating the complex from remaining components of the sample; maintaining the complex under conditions which result in dissociation of the G protein from the nGPCR; and isolating said G protein from the nGPCR.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention relates generally to the fields of genetics and cellular and molecular biology.", "More particularly, the invention relates to novel G protein coupled receptors, to polynucleotides that encode such novel receptors, to reagents such as antibodies, probes, primers and kits comprising such antibodies, probes, primers related to the same, and to methods which use the novel G protein coupled receptors, polynucleotides or reagents.", "BACKGROUND OF THE INVENTION The G protein-coupled receptors (GPCRs) form a vast superfamily of cell surface receptors which are characterized by an amino-terminal extracellular domain, a carboxyl terminal intracellular domain, and a serpentine structure that passes through the cell membrane seven times.", "Hence, such receptors are sometimes also referred to as seven transmembrane (7TM) receptors.", "These seven transmembrane domains define three extracellular loops and three intracellular loops, in addition to the amino- and carboxy-terminal domains.", "The extracellular portions of the receptor have a role in recognizing and binding one or more extracellular binding partners (e.g., ligands), whereas the intracellular portions have a role in recognizing and communicating with downstream molecules in the signal transduction cascade.", "The G protein-coupled receptors bind a variety of ligands including calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and even photons, and are important in the normal (and sometimes the aberrant) function of many cell types.", "[See generally Strosberg, Eur.", "J. Biochem.", "196:1-10 (1991) and Bohm et al., Biochem J.", "322:1-18 (1997).]", "When a specific ligand binds to its corresponding receptor, the ligand typically stimulates the receptor to activate a specific heterotrimeric guanine-nucleotide-binding regulatory protein (G-protein) that is coupled to the intracellular portion of the receptor.", "The G protein in turn transmits a signal to an effector molecule within the cell, by either stimulating or inhibiting the activity of that effector molecule.", "These effector molecules include adenylate cyclase, phospholipases and ion channels.", "Adenylate cyclase and phospholipases are enzymes that are involved in the production of the second messenger molecules cAMP, inositol triphosphate and diacyglycerol.", "It is through this sequence of events that an extracellular ligand stimuli exerts intracellular changes through a G protein-coupled receptor.", "Each such receptor has its own characteristic primary structure, expression pattern, ligand-binding profile, and intracellular effector system.", "Because of the vital role of G protein-coupled receptors in the communication between cells and their environment, such receptors are attractive targets for therapeutic intervention, for example by activating or antagonizing such receptors.", "For receptors having a known ligand, the identification of agonists or antagonists may be sought specifically to enhance or inhibit the action of the ligand.", "Some G protein-coupled receptors have roles in disease pathogenesis (e.g., certain chemokine receptors that act as HIV co-receptors may have a role in AIDS pathogenesis), and are attractive targets for therapeutic intervention even in the absence of knowledge of the natural ligand of the receptor.", "Other receptors are attractive targets for therapeutic intervention by virtue of their expression pattern in tissues or cell types that are themselves attractive targets for therapeutic intervention.", "Examples of this latter category of receptors include receptors expressed in immune cells, which can be targeted to either inhibit autoimmune responses or to enhance immune responses to fight pathogens or cancer; and receptors expressed in the brain or other neural organs and tissues, which are likely targets in the treatment of schizophrenia, depression, bipolar disease, or other neurological disorders.", "This latter category of receptor is also useful as a marker for identifying and/or purifying (e.g., via fluorescence-activated cell sorting) cellular subtypes that express the receptor.", "Unfortunately, only a limited number of G protein receptors from the central nervous system (CNS) are known.", "Thus, a need exists for G protein-coupled receptors that have been identified and show promise as targets for therapeutic intervention in a variety of animals, including humans.", "SUMMARY OF THE INVENTION The present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof.", "The nucleic acid molecule encodes at least a portion of nGPCR-x.", "In some embodiments, the nucleic acid molecule comprises a sequence that encodes a polypeptide comprising even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof.", "In some embodiments, the nucleic acid molecule comprises a sequence homologous to odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a fragment thereof.", "In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof.", "According to some embodiments, the present invention provides vectors which comprise the nucleic acid molecule of the invention.", "In some embodiments, the vector is an expression vector.", "According to some embodiments, the present invention provides host cells which comprise the vectors of the invention.", "In some embodiments, the host cells comprise expression vectors.", "The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence complementary to at least a portion of a sequence from an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, said portion comprising at least 10 nucleotides.", "The present invention provides a method of producing a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.", "The method comprising the steps of introducing a recombinant expression vector that includes a nucleotide sequence that encodes the polypeptide into a compatible host cell, growing the host cell under conditions for expression of the polypeptide and recovering the polypeptide.", "The present invention provides an isolated antibody which binds to an epitope on a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.", "The present invention provides an method of inducing an immune response in a mammal against a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.", "The method comprises administering to a mammal an amount of the polypeptide sufficient to induce said immune response.", "The present invention provides a method for identifying a compound which binds nGPCR-x.", "The method comprises the steps of: contacting nGPCR-x with a compound and determining whether the compound binds nGPCR-x.", "The present invention provides a method for identifying a compound which binds a nucleic acid molecule encoding nGPCR-x.", "The method comprises the steps of contacting said nucleic acid molecule encoding nGPCR-x with a compound and determining whether said compound binds said nucleic acid molecule.", "The present invention provides a method for identifying a compound which modulates the activity of nGPCR-x.", "The method comprises the steps of contacting nGPCR-x with a compound and determining whether nGPCR-x activity has been modulated.", "The present invention provides a method of identifying an animal homolog of nGPCR-x.", "The method comprises the steps screening a nucleic acid database of the animal with an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof and determining whether a portion of said library or database is homologous to said odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or portion thereof.", "The present invention provides a method of identifying an animal homolog of nGPCR-x.", "The methods comprises the steps screening a nucleic acid library of the animal with a nucleic acid molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof; and determining whether a portion of said library or database is homologous to said odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof.", "Another aspect of the present invention relates to methods of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor.", "The methods comprise the steps of assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering an amino acid sequence, expression, or biological activity of at least one nGPCR that is expressed in the brain.", "The nGPCR comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof.", "A diagnosis of the disorder or predisposition is made from the presence or absence of the mutation.", "The presence of a mutation altering the amino acid sequence, expression, or biological activity of the nGPCR in the nucleic acid correlates with an increased risk of developing the disorder.", "The present invention further relates to methods of screening for a nGPCR-40 or nGPCR-54 hereditary schizophrenia genotype in a human patient.", "The methods comprise the steps of providing a biological sample comprising nucleic acid from the patient, in which the nucleic acid includes sequences corresponding to alleles of nGPCR-40 or nGPCR-54.The presence of one or more mutations in the nGPCR-40 allele or the nGPCR-54 allele is detected indicative of a hereditary schizophrenia genotype.", "The present invention provides kits for screening a human subject to diagnose schizophrenia or a genetic predisposition therefor.", "The kits include an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-40 gene or a human nGPCR-54 gene.", "The oligonucleotide comprises 6-50 nucleotides in a sequence that is identical or complementary to a sequence of a wild type human nGPCR-40 or nGPCR-54 gene sequence or nGPCR-40 or nGPCR-54 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution.", "The kit also includes a media packaged with the oligonucleotide.", "The media contains information for identifying polymorphisms that correlate with schizophrenia or a genetic predisposition therefor, the polymorphisms being identifiable using the oligonucleotide as a probe.", "The present invention further relates to methods of identifying nGPCR allelic variants that correlates with mental disorders.", "The methods comprise the steps of providing biological samples that comprise nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny, and detecting in the nucleic acid the presence of one or more mutations in an nGPCR that is expressed in the brain.", "The nGPCR comprises an amino acid sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof.", "The nucleic acid includes sequences corresponding to the gene or genes encoding nGPCR.", "The one or more mutations detected indicate an allelic variant that correlates with a mental disorder.", "The present invention further relates to purified polynucleotides comprising nucleotide sequences encoding alleles of nGPCR-40 or nGPCR-54 from a human with schizophrenia.", "The polynucleotide hybridizes to the complement of SEQ ID NO:83 or of SEQ ID NO:85 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.", "The polynucleotide that encodes nGPCR-40 or nGPCR-54 amino acid sequence of the human differs from SEQ ID NO:84 or SEQ ID NO:86 by at least one residue.", "The present invention also provides methods for identifying a modulator of biological activity of nGPCR-40 or nGPCR-54 comprising the steps of contacting a cell that expresses nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and measuring nGPCR-40 or nGPCR-54 biological activity in the cell.", "The decreased or increased nGPCR-40 or nGPCR-54 biological activity in the presence versus absence of the putative modulator is indicative of a modulator of biological activity.", "The present invention further provides methods to identify compounds useful for the treatment of schizophrenia.", "The methods comprise the steps of contacting a composition comprising nGPCR-40 with a compound suspected of binding nGPCR-40 or contacting a composition comprising nGPCR-54 with a compound suspected of binding nGPCR-54.The binding between nGPCR-40 and the compound suspected of binding nGPCR-40 or between nGPCR-54 and the compound suspected of binding nGPCR-54 is detected.", "Compounds identified as binding nGPCR-40 or nGPCR-54 are candidate compounds useful for the treatment of schizophrenia.", "The present invention further provides methods for identifying a compound useful as a modulator of binding between nGPCR-40 and a binding partner of nGPCR-40 or between nGPCR-54 and a binding partner of nGPCR-54.The methods comprise the steps of contacting the binding partner and a composition comprising nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and detecting binding between the binding partner and nGPCR-40 or nGPCR-54.Decreased or increased binding between the binding partner and nGPCR-40 or nGPCR-54 in the presence of the putative modulator, as compared to binding in the absence of the putative modulator is indicative a modulator compound useful for the treatment of schizophrenia.", "Another aspect of the present invention relates to methods of purifying a G protein from a sample containing a G protein.", "The methods comprise the steps of contacting the sample with an nGPCR for a time sufficient to allow the G protein to form a complex with the nGPCR; isolating the complex from remaining components of the sample; maintaining the complex under conditions which result in dissociation of the G protein from the nGPCR; and isolating said G protein from the nGPCR.", "DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions Various definitions are made throughout this document.", "Most words have the meaning that would be attributed to those words by one skilled in the art.", "Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as are typically understood by those skilled in the art.", "“Synthesized” as used herein and understood in the art, refers to polynucleotides produced by purely chemical, as opposed to enzymatic, methods.", "“Wholly” synthesized DNA sequences are therefore produced entirely by chemical means, and “partially” synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means.", "By the term “region” is meant a physically contiguous portion of the primary structure of a biomolecule.", "In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.", "The term “domain” is herein defined as referring to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule.", "Domains may be co-extensive with regions or portions thereof; domains may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region.", "Examples of GPCR protein domains include, but are not limited to, the extracellular (i.e., N-terminal), transmembrane and cytoplasmic (i.e., C-terminal) domains, which are co-extensive with like-named regions of GPCRS; each of the seven transmembrane segments of a GPCR; and each of the loop segments (both extracellular and intracellular loops) connecting adjacent transmembrane segments.", "As used herein, the term “activity” refers to a variety of measurable indicia suggesting or revealing binding, either direct or indirect; affecting a response, i.e.", "having a measurable affect in response to some exposure or stimulus, including, for example, the affinity of a compound for directly binding a polypeptide or polynucleotide of the invention, or, for example, measurement of amounts of upstream or downstream proteins or other similar functions after some stimulus or event.", "Unless indicated otherwise, as used herein, the abbreviation in lower case (gpcr) refers to a gene, cDNA, RNA or nucleic acid sequence, while the upper case version (GPCR) refers to a protein, polypeptide, peptide, oligopeptide, or amino acid sequence.", "The term “nGPCR-x” refers to any of the nGPCRs taught herein, while specific reference to a nGPCR (for example nGPCR-5) refers only to that specific nGPCR.", "As used herein, the term “antibody” is meant to refer to complete, intact antibodies, and Fab, Fab′, F(ab)2, and other fragments thereof.", "Complete, intact antibodies include monoclonal antibodies such as murine monoclonal antibodies, chimeric antibodies and humanized antibodies.", "As used herein, the term “binding” means the physical or chemical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof.", "Binding includes ionic, nononic, Hydrogen bonds, Van der Waals, hydrophobic interactions, etc.", "The physical interaction, the binding, can be either direct or indirect, indirect being through or due to the effects of another protein or compound.", "Direct binding refers to interactions that do not take place through or due to the effect of another protein or compound but instead are without other substantial chemical intermediates.", "Binding may be detected in many different manners.", "As a non-limiting example, the physical binding interaction between a nGPCR-x of the invention and a compound can be detected using a labeled compound.", "Alternatively, functional evidence of binding can be detected using, for example, a cell transfected with and expressing a nGPCR-x of the invention.", "Binding of the transfected cell to a ligand of the nGPCR that was transfected into the cell provides functional evidence of binding.", "Other methods of detecting binding are well-known to those of skill in the art.", "As used herein, the term “compound” means any identifiable chemical or molecule, including, but not limited to, small molecule, peptide, protein, sugar, nucleotide, or nucleic acid, and such compound can be natural or synthetic.", "As used herein, the term “complementary” refers to Watson-Crick basepairing between nucleotide units of a nucleic acid molecule.", "As used herein, the term “contacting” means bringing together, either directly or indirectly, a compound into physical proximity to a polypeptide or polynucleotide of the invention.", "The polypeptide or polynucleotide can be in any number of buffers, salts, solutions etc.", "Contacting includes, for example, placing the compound into a beaker, microtiter plate, cell culture flask, or a microarray, such as a gene chip, or the like, which contains the nucleic acid molecule, or polypeptide encoding the nGPCR or fragment thereof.", "As used herein, the phrase “homologous nucleotide sequence,” or “homologous amino acid sequence,” or variations thereof, refers to sequences characterized by a homology, at the nucleotide level or amino acid level, of at least the specified percentage.", "Homologous nucleotide sequences include those sequences coding for isoforms of proteins.", "Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA.", "Alternatively, isoforms can be encoded by different genes.", "Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a species other than humans, including, but not limited to, mammals.", "Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.", "A homologous nucleotide sequence does not, however, include the nucleotide sequence encoding other known GPCRs.", "Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions and which polypeptides have the same binding and/or activity.", "A homologous amino acid sequence does not, however, include the amino acid sequence encoding other known GPCRs.", "Percent homology can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using the default settings, which uses the algorithm of Smith and Waterman (Adv.", "Appl.", "Math., 1981, 2, 482 489, which is incorporated herein by reference in its entirety).", "As used herein, the term “isolated” nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that has been removed from its native environment.", "Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.", "As used herein, the terms “modulates” or “modifies” means an increase or decrease in the amount, quality, or effect of a particular activity or protein.", "As used herein, the term “oligonucleotide” refers to a series of linked nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR).", "This short sequence is based on (or designed from) a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.", "Oligonucleotides comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides.", "They are chemically synthesized and may be used as probes.", "As used herein, the term “probe” refers to nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use.", "They are used in the detection of identical, similar, or complementary nucleic acid sequences.", "Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers.", "They may be single- or double-stranded and carefully designed to have specificity in PCR, hybridization membrane based, or ELISA-like technologies.", "The term “preventing” refers to decreasing the probability that an organism contracts or develops an abnormal condition.", "The term “treating” refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.", "The term “therapeutic effect” refers to the inhibition or activation factors causing or contributing to the abnormal condition.", "A therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition.", "In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following: (a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells.", "Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.", "The term “abnormal condition” refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism.", "An abnormal condition can relate to cell proliferation, cell differentiation, cell signaling, or cell survival.", "An abnormal condition may also include obesity, diabetic complications such as retinal degeneration, and irregularities in glucose uptake and metabolism, and fatty acid uptake and metabolism.", "Abnormal cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation.", "Abnormal differentiation conditions include, but are not limited to, neurodegenerative disorders, slow wound healing rates, and slow tissue grafting healing rates.", "Abnormal cell signaling conditions include, but are not limited to, psychiatric disorders involving excess neurotransmitter activity.", "Abnormal cell survival conditions may also relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated.", "A number of protein kinases are associated with the apoptosis pathways.", "Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death.", "The term “administering” relates to a method of incorporating a compound into cells or tissues of an organism.", "The abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism.", "Cells existing outside the organism can be maintained or grown in cell culture dishes.", "For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.", "For cells outside of the organism, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques and carrier techniques.", "The abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism.", "The effect of administering a compound on organism function can then be monitored.", "The organism is preferably a mouse, rat, rabbit, guinea pig or goat, more preferably a monkey or ape, and most preferably a human.", "By “amplification” it is meant increased numbers of DNA or RNA in a cell compared with normal cells.", "“Amplification” as it refers to RNA can be the detectable presence of RNA in cells, since in some normal cells there is no basal expression of RNA.", "In other normal cells, a basal level of expression exists, therefore in these cases amplification is the detection of at least 1 to 2-fold, and preferably more, compared to the basal level.", "As used herein, the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a probe, primer, or oligonucleotide will hybridize to its target sequence, but to no other sequences.", "Stringent conditions are sequence-dependent and will be different in different circumstances.", "Longer sequences hybridize specifically at higher temperatures.", "Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.", "The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium.", "Since the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium.", "Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes, primers or oligonucleotides (e.g.", "10 to 50 nucleotides) and at least about 60° C. for longer probes, primers or oligonucleotides.", "Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.", "The amino acid sequences are presented in the amino to carboxy direction, from left to right.", "The amino and carboxy groups are not presented in the sequence.", "The nucleotide sequences are presented by single strand only, in the 5′ to 3′ direction, from left to right.", "Nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission or (for amino acids) by three letters code.", "Polynucleotides The present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single- and double-stranded, including splice variants thereof) that encode unknown G protein-coupled receptors heretofore termed novel GPCRs, or nGPCRs.", "These genes are described herein and designated herein collectively as nGPCR-x (where x is 1, 3, 4, 5, 9, 11, 12, 14, 15, 18, 16, 17, 20, 21, 22, 24, 27, 28, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 53, 54, 55, 56, 57, 58, 59, or 60).", "That is, these genes are described herein and designated herein as nGPCR-1 (also referred to as beGPCR-1), nGPCR-3 (also referred to as beGPCR-3), nGPCR-4 (also referred to as beGPCR-4), nGPCR-5 (also referred to as beGPCR-5 and TL-GPCR-5), nGPCR-9 (also referred to as beGPCR-9), nGPCR-11 (also referred to as beGPCR-11), nGPCR-12 (also referred to as beGPCR-12), nGPCR-14 (also referred to as beGPCR-14), nGPCR-15 (also referred to as beGPCR-15), nGPCR-18 (also referred to as beGPCR-18), nGPCR-16 (also referred to as beGPCR-16), nGPCR-17 (also referred to as beGPCR-17), nGPCR-20 (also referred to as beGPCR-20), nGPCR-21 (also referred to as beGPCR-21), nGPCR-22 (also referred to as beGPCR-22), nGPCR-24 (also referred to as beGPCR-24), nGPCR-27 (also referred to as beGPCR-27), nGPCR-28 (also referred to as beGPCR-28), nGPCR-31 (also referred to as beGPCR-31), nGPCR-32 (also referred to as beGPCR-32), nGPCR-33 (also referred to as beGPCR-33), nGPCR-34 (also referred to as beGPCR-34), nGPCR-35 (also referred to as beGPCR-35), nGPCR-36 (also referred to as beGPCR-36), nGPCR-37 (also referred to as beGPCR-37), nGPCR-38 (also referred to as beGPCR-38), nGPCR-40 (also referred to as beGPCR-40), nGPCR-1 (also referred to as beGPCR-41), nGPCR-53, nGPCR-54, nGPCR-55, nGPCR-56, nGPCR-57, nGPCR-58, nGPCR-59, and nGPCR-60.Table 1 below identifies the novel gene sequence nGPCR-x designation, the SEQ ID NO: of the gene sequence, the SEQ ID NO: of the polypeptide hereby, and the U.S.", "Provisional Application in which the gene sequence has been TABLE 1 Nucleotide Amino acid Sequence Sequence (SEQ ID (SEQ ID Originally nGPCR NO:) NO:) filed in: 1 1 2 A 1 73 74 E 3 3 4 A 3 185 186 P 4 5 6 A 5 7 8 A 5 75 76 F 9 9 10 A 9 77 78 G 11 11 12 A 11 79 80 H 12 13 14 A 14 15 16 A 14 191 192 herein 15 17 18 A 18 19 20 A 16 21 22 B 16 81 82 I 17 23 24 B 20 25 26 B 21 27 28 B 22 29 30 B 24 31 32 B 27 33 34 B 28 35 36 B 31 37 38 B 32 39 40 B 33 41 42 C 34 43 44 C 35 45 46 C 36 47 48 C 37 49 50 C 38 51 52 C 40 53 54 C 40 83 84 J 41 55 56 C 53 57 58 D 54 59 60 D 54 85 86 K 55 61 62 D 56 63 64 D 56 87 88 L 56 89 90 M 57 65 66 D 58 67 68 D 58 91 92 N 58 93 94 O 59 69 70 D 60 71 72 D Legend A = Ser.", "No.", "60/165,838 B = Ser.", "No.", "60/166,701 C = Ser.", "No.", "60/166,678 D = Ser.", "No.", "60/173,396 E = Ser.", "No.", "60/184,129 F = Ser.", "No.", "60/188,114 G = Ser.", "No.", "60/185,421 H = Ser.", "No.", "60/186,811 I = Ser.", "No.", "60/186,530 J = Ser.", "No.", "60/207,094 K = Ser.", "No.", "60/203,111 L = Ser.", "No.", "60/190,310 M = Ser.", "No.", "60/201,190 N = Ser.", "No.", "60/185554 O = Ser.", "No.", "60/190,800 P = Ser.", "No.", "60/198,568 When a specific nGPCR is identified (for example nGPCR-5), it is understood that only that specific nGPCR is being referred to.", "As described in Example 4 below, the genes encoding nGPCR-1 (nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ ID NO:77, amino acid sequence SEQ ID NO:10, SEQ ID NO:78), nGPCR-11 (nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequence SEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO: 21, SEQ ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82), nGPCR-40 (nucleic acids sequence SEQ ID NO:53, SEQ ID NO:83, amino acid sequence SEQ ID NO:54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequence SEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO: 86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ ID NO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90), nGPCR-58 (nucleic acid sequence SEQ ID NO:67, SEQ ID NO:91, SEQ ID NO:93, amino acid sequence SEQ ID NO:68, SEQ ID NO: 92, SEQ ID NO:94) and nGPCR-3 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO:185, amino acid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissue indicating that these n-GPCR-x proteins are neuroreceptors.", "The invention provides purified and isolated polynucleotides (e.g., cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, whether single or double-stranded) that comprise a nucleotide sequence encoding the amino acid sequence of the polypeptides of the invention.", "Such polynucleotides are useful for recombinantly expressing the receptor and also for detecting expression of the receptor in cells (e.g., using Northern hybridization and in situ hybridization assays).", "Such polynucleotides also are useful in the design of antisense and other molecules for the suppression of the expression of nGPCR-x in a cultured cell, a tissue, or an animal; for therapeutic purposes; or to provide a model for diseases or conditions characterized by aberrant nGPCR-x expression.", "Specifically excluded from the definition of polynucleotides of the invention are entire isolated, non-recombinant native chromosomes of host cells.", "A preferred polynucleotide has the sequence of the sequence set forth in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191, which correspond to naturally occurring nGPCR-x sequences.", "It will be appreciated that numerous other polynucleotide sequences exist that also encode nGPCR-x having the sequence set forth in even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192, due to the well-known degeneracy of the universal genetic code.", "The invention also provides a purified and isolated polynucleotide comprising a nucleotide sequence that encodes a mammalian polypeptide, wherein the polynucleotide hybridizes to a polynucleotide having the sequence set forth in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185, and SEQ ID NO:191, or the non-coding strand complementary thereto, under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate; and (b) washing 2 times for 30 minutes each at 60° C. in a wash solution comprising 0.1% SSC, 1% SDS.", "Polynucleotides that encode a human allelic variant are highly preferred.", "The present invention relates to molecules which comprise the gene sequences that encode the nGPCRs; constructs and recombinant host cells incorporating the gene sequences; the novel GPCR polypeptides encoded by the gene sequences; antibodies to the polypeptides and homologs; kits employing the polynucleotides and polypeptides, and methods of making and using all of the foregoing.", "In addition, the present invention relates to homologs of the gene sequences and of the polypeptides and methods of making and using the same.", "Genomic DNA of the invention comprises the protein-coding region for a polypeptide of the invention and is also intended to include allelic variants thereof.", "It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein intron (i.e., non-coding regions) of the transcripts are removed, or “spliced out.” RNA transcripts that can be spliced by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode a nGPCR-x polypeptide, are referred to in the art as splice variants which are embraced by the invention.", "Splice variants comprehended by the invention therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts Allelic variants are modified forms of a wild-type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation.", "Allelic variants, like wild type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants that arise from in vitro manipulation).", "The invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding nGPCR-x (conventionally followed by second strand synthesis of a complementary strand to provide a double-stranded DNA).", "Preferred DNA sequences encoding human nGPCR-x polypeptides are set out in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191.A preferred DNA of the invention comprises a double stranded molecule along with the complementary molecule (the “non-coding strand” or “complement”) having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA.", "Also preferred are other polynucleotides encoding the nGPCR-x polypeptide of even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192 which differ in sequence from the polynucleotides of odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 192, by virtue of the well-known degeneracy of the universal nuclear genetic code.", "In a preferred embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which encodes a fragment of polypeptide comprising a sequence of SEQ ID NO: 192.The fragment of the polypeptide comprising a sequence of SEQ ID NO: 192 comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO: 192; amino acid residues 68 to 77 of SEQ ID NO: 192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192.In a preferred embodiment, the isolated nucleic acid comprises a nucleotide sequence of SEQ ID NO: 191, and fragments thereof, that encode a polypeptide having a sequence of SEQ ID NO: 192, or fragments thereof.", "The fragment of the nucleotide sequence of SEQ ID NO: 191 comprises at least one or more nucleotides from one or more of the following regions of SEQ ID NO: 191: nucleotides 1 to 193 of SEQ ID NO: 191; nucleotides 612 to 644 of SEQ ID NO:191; nucleotides 697 to 706 of SEQ ID NO:191; nucleotides 1011 to 1049 of SEQ ID NO: 191; nucleotides 1051 to 1057 of SEQ ID NO:191; nucleotides 1090 to 1096 of SEQ ID NO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191.The invention further embraces other species, preferably mammalian, homologs of the human nGPCR-x DNA.", "Species homologs, sometimes referred to as “orthologs,” in general, share at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology with human DNA of the invention.", "Generally, percent sequence “homology” with respect to polynucleotides of the invention may be calculated as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the nGPCR-x sequence set forth in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.", "Polynucleotides of the invention permit identification and isolation of polynucleotides encoding related nGPCR-x polypeptides, such as human allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR).", "Examples of related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to nGPCR-x and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of nGPCR-x.", "Non-human species genes encoding proteins homologous to nGPCR-x can also be identified by Southern and/or PCR analysis and are useful in animal models for nGPCR-x disorders.", "Knowledge of the sequence of a human nGPCR-x DNA also makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding nGPCR-x expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like.", "Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express nGPCR-x.", "Polynucleotides of the invention may also provide a basis for diagnostic methods useful for identifying a genetic alteration(s) in a nGPCR-x locus that underlies a disease state or states, which information is useful both for diagnosis and for selection of therapeutic strategies.", "According to the present invention, the nGPCR-x nucleotide sequences disclosed herein may be used to identify homologs of the nGPCR-x, in other animals, including but not limited to humans and other mammals, and invertebrates.", "Any of the nucleotide sequences disclosed herein, or any portion thereof, can be used, for example, as probes to screen databases or nucleic acid libraries, such as, for example, genomic or cDNA libraries, to identify homologs, using screening procedures well known to those skilled in the art.", "Accordingly, homologs having at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 100% homology with nGPCR-x sequences can be identified.", "The disclosure herein of full-length polynucleotides encoding nGPCR-x polypeptides makes readily available to the worker of ordinary skill in the art every possible fragment of the full-length polynucleotide.", "One preferred embodiment of the present invention provides an isolated nucleic acid molecule comprising a sequence homologous to odd numbered sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof.", "Another preferred embodiment provides an isolated nucleic acid molecule comprising a sequence selected from the group of odd numbered sequences consisting of SEQ ID NO: 1 to SEQ ID NO:93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof.", "As used in the present invention, fragments of nGPCR-x-encoding polynucleotides comprise at least 10, and preferably at least 12, 14, 16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotide encoding nGPCR-x.", "Preferably, fragment polynucleotides of the invention comprise sequences unique to the nGPCR-x-encoding polynucleotide sequence, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., “specifically”) to polynucleotides encoding nGPCR-x (or fragments thereof).", "Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full-length sequence derived from introns, regulatory regions, and/or other non-translated sequences.", "Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases.", "Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize.", "Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling.", "Fragment polynucleotides are particularly useful as probes for detection of full-length or fragments of nGPCR-x polynucleotides.", "One or more polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding nGPCR-x, or used to detect variations in a polynucleotide sequence encoding nGPCR-x.", "The invention also embraces DNAs encoding nGPCR-x polypeptides that hybridize under moderately stringent or high stringency conditions to the non-coding strand, or complement, of the polynucleotides set forth in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:192.Exemplary highly stringent hybridization conditions are as follows: hybridization at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.", "It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel et al.", "(Eds.", "), Protocols in Molecular Biology, John Wiley & Sons (1994), pp.", "6.0.3 to 6.4.10.Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe.", "The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.", "), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp.", "9.47 to 9.51.With the knowledge of the nucleotide sequence information disclosed in the present invention, one skilled in the art can identify and obtain nucleotide sequences which encode nGPCR-x from different sources (i.e., different tissues or different organisms) through a variety of means well known to the skilled artisan and as disclosed by, for example, Sambrook et al., “Molecular cloning: a laboratory manual”, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (19.89), which is incorporated herein by reference in its entirety.", "For example, DNA that encodes nGPCR-x may be obtained by screening of mRNA, cDNA, or genomic DNA with oligonucleotide probes generated from the nGPCR-x gene sequence information provided herein.", "Probes may be labeled with a detectable group, such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with procedures known to the skilled artisan and used in conventional hybridization assays, as described by, for example, Sambrook et al.", "A nucleic acid molecule comprising any of the nGPCR-x nucleotide sequences described above can alternatively be synthesized by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers produced from the nucleotide sequences provided herein.", "See U.S. Pat.", "No.", "4,683,195 to Mulliset al.", "and U.S. Pat.", "No.", "4,683,202 to Mullis.", "The PCR reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample.", "The method can be used to amplify either single- or double-stranded DNA.", "The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template dependent, polymerase mediated replication of a desired nucleic acid molecule.", "A wide variety of alternative cloning and in vitro amplification methodologies are well known to those skilled in the art Examples of these techniques are found in, for example, Berger et al., Guide to Molecular Cloning Techniques, Methods in Enzymology 152, Academic Press, Inc., San Diego, Calif. (Berger), which is incorporated herein by reference in its entirety.", "Automated sequencing methods can be used to obtain or verify the nucleotide sequence of nGPCR-x.", "The nGPCR-x nucleotide sequences of the present invention are believed to be 100% accurate.", "However, as is known in the art, nucleotide sequence obtained by automated methods may contain some errors.", "Nucleotide sequences determined by automation are typically at least about 90%, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of a given nucleic acid molecule.", "The actual sequence may be more precisely determined using manual sequencing methods, which are well known in the art.", "An error in a sequence which results in an insertion or deletion of one or more nucleotides may result in a frame shift in translation such that the predicted amino acid sequence will differ from that which would be predicted from the actual nucleotide sequence of the nucleic acid molecule, starting at the point of the mutation.", "The nucleic acid molecules of the present invention, and fragments derived therefrom, are useful for screening for restriction fragment length polymorphism (RFLP) associated with certain disorders, as well as for genetic mapping.", "The polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art.", "Vectors Another aspect of the present invention is directed to vectors, or recombinant expression vectors, comprising any of the nucleic acid molecules described above.", "Vectors are used herein either to amplify DNA or RNA encoding nGPCR-x and/or to express DNA which encodes nGPCR-x.", "Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination).", "Preferred viral particles include, but are not limited to, adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses.", "Preferred expression vectors include, but are not limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech).", "Other expression vectors include, but are not limited to, pSPORT™ vectors, pGEM™ vectors (Promega), pPROEX vectors™ (LTI, Bethesda, Md.", "), Bluescript™ vectors (Stratagene), pQE™ vectors (Qiagen), pSE420™ (Invitrogen), and pYES2™ (Invitrogen).", "Expression constructs preferably comprise GPCR-x-encoding polynucleotides operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator.", "Expression control DNA sequences include promoters, enhancers, operators, and regulatory element binding sites generally, and ore typically selected based on the expression systems in which the expression construct is to be utilized.", "Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression.", "Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct.", "Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell.", "Preferred constructs of the invention also include sequences necessary for replication in a host cell.", "Expression constructs are preferably utilized for production of an encoded protein, but may also be utilized simply to amplify a nGPCR-x-encoding polynucleotide sequence.", "In preferred embodiments, the vector is an expression vector wherein the polynucleotide of the invention is operatively linked to a polynucleotide comprising an expression control sequence.", "Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating polynucleotides of the invention are also provided.", "Preferred expression vectors are replicable DNA constructs in which a DNA sequence encoding nGPCR-x is operably linked or connected to suitable control sequences capable of effecting the expression of the nGPCR-x in a suitable host DNA regions are operably linked or connected when they are functionally related to each other.", "For example, a promoter is operably linked or connected to a coding sequence if it controls the transcription of the sequence.", "Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.", "The need for control sequences in the expression vector will vary depending upon the host selected and the transformation method chosen.", "Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding and sequences which control the termination of transcription and translation.", "Preferred vectors preferably contain a promoter that is recognized by the host organism.", "The promoter sequences of the present invention may be prokaryotic, eukaryotic or viral.", "Examples of suitable prokaryotic sequences include the PR and PL promoters of bacteriophage lambda (The bacteriophage Lambda, Hershey, A. D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1973), which is incorporated herein by reference in its entirety; Lambda II, Hendrix, R. W., Ed., Cold Spring Habor Press, Cold Spring Harbor, N.Y. (1980), which is incorporated herein by reference in its entirety); the trp, recA, heat shock, and lacZ promoters of E. coli and the SV40 early promoter (Benoist et al.", "Nature, 1981, 290, 304-310, which is incorporated herein by reference in its entirety).", "Additional promoters include, but are not limited to, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Barr virus, Rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein.", "Additional regulatory sequences can also be included in preferred vectors.", "Preferred examples of suitable regulatory sequences are represented by the Shine-Dalgarno of the replicase gene of the phage MS-2 and of the gene cII of bacteriophage lambda.", "The Shine-Dalgarno sequence may be directly followed by DNA encoding nGPCR-x and result in the expression of the mature nGPCR-x protein.", "Moreover, suitable expression vectors can include an appropriate marker that allows the screening of the transformed host cells.", "The transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et al., supra.", "An origin of replication can also be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism.", "If the vector is integrated into the host cell chromosome, the latter may be sufficient.", "Alternatively, rather than using vectors which contain viral origins of replication, one skilled in the art can transform mammalian cells by the method of co-transformation with a selectable marker and nGPCR-x DNA.", "An example of a suitable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S. Pat.", "No.", "4,399,216).", "Nucleotide sequences encoding GPCR-x may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesiderable joining, and ligation with appropriate ligases.", "Techniques for such manipulation are disclosed by Sambrook et al., supra and are well known in the art Methods for construction of mammalian expression vectors are disclosed in, for example, Okayama et al, Mol.", "Cell.", "Biol., 1983, 3, 280, Cosman et al., Mol.", "Immutol., 1986, 23, 935, Cosman et al., Nature, 1984, 312, 768, EP-A-0367566, and WO 91/18982, each of which is incorporated herein by reference in its entirety.", "Host Cells According to another aspect of the invention, host cells are provided, including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention (or vector of the invention) in a manner that permits expression of the encoded nGPCR-x polypeptide.", "Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector.", "Methods for introducing DNA into the host cell that are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts.", "Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, vertebrate, and mammalian cells systems.", "The invention provides host cells that are transformed or transfected (stably or transiently) with polynucleotides of the invention or vectors of the invention.", "As stated above, such host cells are useful for amplifying the polynucleotides and also for expressing the nGPCR-x polypeptide or fragment thereof encoded by the polynucleotide.", "In still another related embodiment, the invention provides a method for producing a nGPCR-x polypeptide (or fragment thereof) comprising the steps of growing a host cell of the invention in a nutrient medium and isolating the polypeptide or variant thereof from the cell or the medium.", "Because nGPCR-x is a seven transmembrane receptor, it will be appreciated that, for some applications, such as certain activity assays, the preferable isolation may involve isolation of cell membranes containing the polypeptide embedded therein, whereas for other applications a more complete isolation may be preferable.", "According to some aspects of the present invention, transformed host cells having an expression vector comprising any of the nucleic acid molecules described above are provided.", "Expression of the nucleotide sequence occurs when the expression vector is introduced into an appropriate host cell.", "Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes.", "If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences.", "Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, and Staphylococcus.", "If an eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequence.", "Preferably, eukaryotic cells are cells of higher eukaryotes.", "Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells.", "Preferred host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts.", "Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, eds.", "(1973), which is incorporated herein by reference in its entirety).", "In addition, a yeast host may be employed as a host cell.", "Preferred yeast cells include, but are not limited to, the genera Saccharomyces, Pichia, and Kluveromyces.", "Preferred yeast hosts are S. cerevisiae and P. pastoris.", "Preferred yeast vectors can contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.", "Shuttle vectors for replication in both yeast and E. coli are also included herein.", "Alternatively, insect cells may be used as host cells.", "In a preferred embodiment, the polypeptides of the invention are expressed using a baculovirus expression system (sea Luckow et al., Bio/Technology, 1988, 6, 47, Baculovirus Expression Vectors: A Laboratory Manual, O'Rielly et al (Eds.", "), W.H.", "Freeman and Company, New York, 1992, and U.S. Pat.", "No.", "4,879,236, each of which is incorporated herein by reference in its entirety).", "In addition, the MAXBAC™ complete baculovirus expression system (Invitrogen) can, for example, be used for production in insect cells.", "Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with nGPCR-x.", "Host cells of the invention are also useful in methods for the large-scale production of nGPCR-x polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells, or from the medium in which the cells are grown, by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like.", "Still other methods of purification include those methods wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent.", "The purified protein can be cleaved to yield the desired protein, or can be left as an intact fusion protein.", "Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.", "Knowledge of nGPCR-x DNA sequences allows for modification of cells to permit, or increase, expression of endogenous nGPCR-x.", "Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring nGPCR-x promoter with all or part of a heterologous promoter so that the cells express nGPCR-x at higher levels.", "The heterologous promoter is inserted in such a manner that it is operatively linked to endogenous nGPCR-x encoding sequences.", "(See, for example, PCT International Publication No.", "WO 94/12650, PCT International Publication No.", "WO 92/20808, and PCT International Publication No.", "WO 91/09955.)", "It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.", "If linked to the nGPCR-x coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the nGPCR-x coding sequences in the cells.", "Knockouts The DNA sequence information provided by the present invention also makes possible the development (e.g., by homologous recombination or “knock-out” strategies; see Capecchi, Science 244:1288-1292 (1989), which is incorporated herein by reference) of animals that fail to express functional nGPCR-x or that express a variant of nGPCR-x.", "Such animals (especially small laboratory animals such as rats, rabbits, and mice) are useful as models for studying the in vivo activities of nGPCR-x and modulators of nGPCR-x.", "Antisense Also made available by the invention are anti-sense polynucleotides that recognize and hybridize to polynucleotides encoding nGPCR-x.", "Full-length and fragment anti-sense polynucleotides are provided.", "Fragment antisense molecules of the invention include (i) those that specifically recognize and hybridize to nGPCR-x RNA (as determined by sequence comparison of DNA encoding nGPCR-x to DNA encoding other known molecules).", "Identification of sequences unique to nGPCR-x encoding polynucleotides can be deduced through use of any publicly available sequence database, and/or through use of commercially available sequence comparison programs.", "After identification of the desired sequences, isolation through restriction digestion or amplification using any of the various polymerase chain reaction techniques well known in the art can be performed.", "Antisense polynucleotides are particularly relevant to regulating expression of nGPCR-x by those cells expressing nGPCR-x mRNA.", "Antisense nucleic acids (preferably 10 to 30 base-pair oligonucleotides) capable of specifically binding to nGPCR-x expression control sequences or nGPCR-x RNA are introduced into cells (e.g., by a viral vector or colloidal dispersion system such as a liposome).", "The antisense nucleic acid binds to the nGPCR-x target nucleotide sequence in the cell and prevents transcription and/or translation of the target sequence.", "Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention.", "The antisense oligonucleotides may be further modified by adding poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5′ end.", "Suppression of nGPCR-x expression at either the transcriptional or translational level is useful to generate cellular or animal models for diseases/conditions characterized by aberrant nGPCR-x expression.", "Antisense oligonucleotides, or fragments of odd numbered nucleotide sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191, or sequences complementary or homologous thereto, derived from the nucleotide sequences of the present invention encoding nGPCR-x are useful as diagnostic tools for probing gene expression in various tissues.", "For example, tissue can be probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiography techniques to investigate native expression of this enzyme or pathological conditions relating thereto.", "Antisense oligonucleotides are preferably directed to regulatory regions of odd numbered nucleotide sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191, or mRNA corresponding thereto, including, but not limited to, the initiation codon, TATA box, enhancer sequences, and the like.", "Transcription Factors The nGPCR-x sequences taught in the present invention facilitate the design of novel transcription factors for modulating nGPCR-x expression in native cells and animals, and cells transformed or transfected with nGPCR-x polynucleotides.", "For example, the Cys2-His2 zinc finger proteins, which bind DNA via their zinc finger domains, have been shown to be amenable to structural changes that lead to the recognition of different target sequences.", "These artificial zinc finger proteins recognize specific target sites with high affinity and low dissociation constants, and are able to act as gene switches to modulate gene expression.", "Knowledge of the particular nGPCR-x target sequence of the present invention facilitates the engineering of zinc finger proteins specific for the target sequence using known methods such as a combination of structure-based modeling and screening of phage display libraries (Segal et al., Proc.", "Natl.", "Acad.", "Sci.", "(USA) 96:2758-2763 (1999); Liu et al, Proc.", "Natl.", "Acad.", "Sci.", "(USA) 94:5525-5530 (1997); Greisman et al., Science 275:657-661 (1997); Choo et al., J. Mol.", "Biol.", "273:525-532 (1997)).", "Each zinc finger domain usually recognizes three or more base pairs.", "Since a recognition sequence of 18 base pairs is generally sufficient in length to render it unique in any known genome, a zinc finger protein consisting of 6 tandem repeats of zinc fingers would be expected to ensure specificity for a particular sequence (Segal et al.)", "The artificial zinc finger repeats, designed based on nGPCR-x sequences, are fused to activation or repression domains to promote or suppress nGPCR-x expression (Liu et al.)", "Alternatively, the zinc finger domains can be fused to the TATA box-binding factor (TBP) with varying lengths of linker region between the zinc finger peptide and the TBP to create either transcriptional activators or repressors (Kim et al., Proc.", "Natl.", "Acad.", "Sci.", "(USA) 94:3616-3620 (1997).", "Such proteins and polynucleotides that encode them, have utility for modulating nGPCR-x expression in vivo in both native cells, animals and humans; and/or cells transfected with nGPCR-x-encoding sequences.", "The novel transcription factor can be delivered to the target cells by transfecting constructs that express the transcription factor (gene therapy), or by introducing the protein.", "Engineered zinc finger proteins can also be designed to bind RNA sequences for use in therapeutics as alternatives to antisense or catalytic RNA methods (McColl et al., Proc.", "Natl.", "Acad.", "Sci.", "(USA) 96:9521-9526 (1997); Wu et al., Proc.", "Natl.", "Acad.", "Sci.", "(USA) 92:344-348 (1995)).", "The present invention contemplates methods of designing such transcription factors based on the gene sequence of the invention, as well as customized zinc finger proteins, that are useful to modulate nGPCR-x expression in cells (native or transformed) whose genetic complement includes these sequences.", "Polypeptides The invention also provides purified and isolated mammalian nGPCR-x polypeptides encoded by a polynucleotide of the invention.", "Presently preferred is a human nGPCR-x polypeptide comprising the amino acid sequence set out in even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192, or fragments thereof comprising an epitope specific to the polypeptide.", "By “epitope specific to” is meant a portion of the nGPCR receptor that is recognizable by an antibody that is specific for the nGPCR, as defined in detail below.", "Although the sequences provided are particular human sequences, the invention is intended to include within its scope other human allelic variants; non-human mammalian forms of nGPCR-x, and other vertebrate forms of nGPCR-x.", "It will be appreciated that extracellular epitopes are particularly useful for generating and screening for antibodies and other binding compounds that bind to receptors such as nGPCR-x.", "Thus, in another preferred embodiment, the invention provides a purified and isolated polypeptide comprising at least one extracellular domain (e.g., the N-terminal extracellular domain or one of the three extracellular loops) of nGPCR-x.", "Purified and isolated polypeptides comprising the N-terminal extracellular domain of nGPCR-x are highly preferred.", "Also preferred is a purified and isolated polypeptide comprising a nGPCR-x fragment selected from the group consisting of the N-terminal extracellular domain of nGPCR-x, transmembrane domains of nGPCR-x, an extracellular loop connecting transmembrane domains of nGPCR-x, an intracellular loop connecting transmembrane domains of nGPCR-x, the C-terminal cytoplasmic region of nGPCR-x, and fusions thereof.", "Such fragments may be continuous portions of the native receptor.", "However, it will also be appreciated that knowledge of the nGPCR-x gene and protein sequences as provided herein permits recombining of various domains that are not contiguous in the native protein.", "Using a FORTRAN computer program called “tmtrest all” [Parodi et al., Comput.", "Appl.", "Biosci.", "5:527-535 (1994)], nGPCR-x was shown to contain transmembrane-spanning domains.", "The invention also embraces polypeptides that have at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% or at least 50% identity and/or homology to the preferred polypeptide of the invention.", "Percent amino acid sequence “identity” with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the nGPCR-x sequence after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.", "Percent sequence “homology” with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the nGPCR-x sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and also considering any conservative substitutions as part of the sequence identity.", "In one aspect, percent homology is calculated as the percentage of amino acid residues in the smaller of two sequences which align with identical amino acid residue in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to maximize alignment [Dayhoff, in Atlas of Protein Sequence and Structure, Vol.", "5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), incorporated herein by reference].", "Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention.", "Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention.", "Glycosylated and non-glycosylated forms of nGPCR-x polypeptides are embraced by the invention.", "The invention also embraces variant (or analog) nGPCR-x polypeptides.", "In one example, insertion variants are provided wherein one or more amino acid residues supplement a nGPCR-x amino acid sequence.", "Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the nGPCR-x amino acid sequence.", "Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels.", "Insertion variants include nGPCR-x polypeptides wherein one or more amino acid residues are added to a nGPCR-x acid sequence or to a biologically active fragment thereof.", "Variant products of the invention also include mature nGPCR-x products, ie., nGPCR-x products wherein leader or signal sequences are removed, with additional amino terminal residues.", "The additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from specific proteins.", "nGPCR-x products with an additional methionine residue at position −1 (Met−1-nGPCR-x) are contemplated, as are variants with additional methionine and lysine residues at positions −2 and −1 (Met−2-Lys−1-nGPCR-x).", "Variants of nGPCR-x with additional Met, Met-Lys, Lys residues (or, one or more basic residues in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.", "The invention also embraces nGPCR-x variants having additional amino acid residues that result from use of specific expression systems.", "For example, use of commercially available vectors that express a desired polypeptide as part of a glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position −1 after cleavage of the GST component from the desired polypeptide.", "Variants that result from expression in other vector systems are also contemplated.", "Insertional variants also include fusion proteins wherein the amino terminus and/or the carboxy terminus of nGPCR-x is/are fused to another polypeptide.", "In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a nGPCR-x polypeptide are removed.", "Deletions can be effected at one or both termini of the nGPCR-x polypeptide, or with removal of one or more non-terminal amino acid residues of nGPCR-x.", "Deletion variants, therefore, include all fragments of a nGPCR-x polypeptide.", "The invention also embraces polypeptide fragments of the even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, wherein the fragments maintain biological (e.g., ligand binding and/or intracellular signaling) immunological properties of a nGPCR-x polypeptide.", "In one preferred embodiment of the invention, an isolated nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to even numbered sequences selected from the group consisting of: SEQ ID NO:2 to SEQ ID NO:94, SEQ ID NO: 186 and SEQ ID NO:192, and fragments thereof, wherein the nucleic acid molecule encoding at least a portion of nGPCR-x.", "In a more preferred embodiment, the isolated nucleic acid molecule comprises a sequence that encodes a polypeptide comprising even numbered sequences selected from the group consisting of SEQ ID NO:2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, and fragments thereof.", "As used in the present invention, polypeptide fragments comprise at least 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of the even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192.Preferred polypeptide fragments display antigenic properties unique to, or specific for, human nGPCR-x and its allelic and species homologs.", "Fragments of the invention having the desired biological and immunological properties can be prepared by any of the methods well known and routinely practiced in the art.", "In still another aspect, the invention provides substitution variants of nGPCR-x polypeptides.", "Substitution variants include those polypeptides wherein one or more amino acid residues of a nGPCR-x polypeptide are removed and replaced with alternative residues.", "In one aspect, the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative.", "Conservative substitutions for this purpose may be defined as set out in Tables 2, 3, or 4 below.", "Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention.", "Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure.", "A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.", "Exemplary conservative substitutions are set out in Table 2 (from WO 97/09433, page 10, published Mar.", "13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996), immediately below.", "TABLE 2 Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINO ACID Aliphatic Non-polar GAP ILV Polar - uncharged CSTM NQ Polar - charged DE KR Aromatic HFWY Other NQDE Alternatively, conservative amino acids can be grouped as described in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp.71-77] as set out in Table 3, below.", "Table 3 Conservative Substitutions II TABLE 3 Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINO ACID Non-polar (hydrophobic) A. Aliphatic: ALIVP B. Aromatic: FW C. Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: STY B. Amides: NQ C. Sulfhydryl: C D. Borderline: G Positively Charged (Basic): KRH Negatively Charged (Acidic): DE As still another alternative, exemplary conservative substitutions are set out in Table 4, below.", "TABLE 4 Conservative Substitutions III Original Residue Exemplary Substitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala It should be understood that the definition of polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues.", "By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties.", "Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues, or organs.", "Similarly, the invention further embraces nGPCR-x polypeptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.", "Variants that display ligand binding properties of native nGPCR-x and are expressed at higher levels, as well as variants that provide for constitutively active receptors, are particularly useful in assays of the invention; the variants are also useful in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant nGPCR-x activity.", "In a related embodiment, the present invention provides compositions comprising purified polypeptides of the invention.", "Preferred compositions comprise, in addition to the polypeptide of the invention, a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluent that serves as a pharmaceutical vehicle, excipient, or medium.", "Any diluent known in the art may be used.", "Exemplary diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil, and cocoa butter.", "Variants that display ligand binding properties of native nGPCR-x and are expressed at higher levels, as well as variants that provide for constitutively active receptors, are particularly useful in assays of the invention; the variants are also useful in assays of the invention and in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant nGPCR-x activity.", "The G protein-coupled receptor functions through a specific heterotrimeric guanine-nucleotide-binding regulatory protein (G-protein) coupled to the intracellular portion of the G protein-coupled receptor molecule.", "Accordingly, the G protein-coupled receptor has a specific affinity to G protein.", "G proteins specifically bind to guanine nucleotides.", "Isolation of G proteins provides a means to isolate guanine nucleotides.", "G Proteins may be isolated using commercially available anti-G protein antibodies or isolated G protein-coupled receptors.", "Similarly, G proteins may be detected in a sample isolated using commercially available detectable anti-G protein antibodies or isolated G protein-coupled receptors.", "According to the present invention, the isolated n-GPCR-x proteins of the present invention are useful to isolate and purify G proteins from samples such as cell lysates.", "Example 15 below sets forth an example of isolation of G proteins using isolated nGPCR-x proteins.", "Such methodolgy may be used in place of the use of commercially available ant-G protein antibodies which are used to isolate G proteins.", "Moreover, G proteins may be detected using nGPCR-x proteins in place of commercially available detectable anti-G protein antibodies.", "Since nGPCR-x proteins specifically bind to G proteins, they can be employed in any specific use where G protein specific affinity is required, such as those uses where commercially available anti-G protein antibodies are employed.", "Antibodies Also comprehended by the present invention are antibodies (e.g.", "monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for nGPCR-x or fragments thereof.", "Preferred antibodies of the invention are human antibodies that are produced and identified according to methods described in WO93/11236, published Jun.", "20, 1993, which is incorporated herein by reference in its entirety.", "Antibody fragments, including Fab, Fab′, F(ab′)2, and Fv, are also provided by the invention.", "The term “specific for,” when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind nGPCR-x polypeptides exclusively (i.e., are able to distinguish nGPCR-x polypeptides from other known GPCR polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between nGPCR-x and such polypeptides).", "It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule.", "Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art.", "For a comprehensive discussion of such assays, see Harlow et al.", "(Eds.", "), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.Antibodies that recognize and bind fragments of the nGPCR-x polypeptides of the invention are also contemplated, provided that the antibodies are specific for nGPCR-x polypeptides.", "Antibodies of the invention can be produced using any method well known and routinely practiced in the art.", "The invention provides an antibody that is specific for the nGPCR-x of the invention.", "Antibody specificity is described in greater detail below.", "However, it should be emphasized that antibodies that can be generated from polypeptides that have previously been described in the literature and that are capable of fortuitously cross-reacting with nGPCR-x (e.g., due to the fortuitous existence of a similar epitope in both polypeptides) are considered “cross-reactive” antibodies.", "Such cross-reactive antibodies are not antibodies that are “specific” for nGPCR-x.", "The determination of whether an antibody is specific for nGPCR-x or is cross-reactive with another known receptor is made using any of several assays, such as Western blotting assays, that are well known in the art.", "For identifying cells that express nGPCR-x and also for modulating nGPCR-x-ligand binding activity, antibodies that specifically bind to an extracellular epitope of the nGPCR-x are preferred.", "In one preferred variation, the invention provides monoclonal antibodies.", "Hybridomas that produce such antibodies also are intended as aspects of the invention.", "In yet another variation, the invention provides a humanized antibody.", "Humanized antibodies are useful for in vivo therapeutic indications.", "In another variation, the invention provides a cell-free composition comprising polyclonal antibodies, wherein at least one of the antibodies is an antibody of the invention specific for nGPCR-x.", "Antisera isolated from an animal is an exemplary composition, as is a composition comprising an antibody fraction of an antisera that has been resuspended in water or in another diluent, excipient, or carrier.", "In still another related embodiment, the invention provides an anti-idiotypic antibody specific for an antibody that is specific for nGPCR-x.", "It is well known that antibodies contain relatively small antigen binding domains that can be isolated chemically or by recombinant techniques.", "Such domains are useful nGPCR-x binding molecules themselves, and also may be reintroduced into human antibodies, or fused to toxins or other polypeptides.", "Thus, in still another embodiment, the invention provides a polypeptide comprising a fragment of a nGPCR-x-specific antibody, wherein the fragment and the polypeptide bind to the nGPCR-x.", "By way of non-limiting example, the invention provides polypeptides that are single chain antibodies and CDR-grafted antibodies.", "Non-human antibodies may be humanized by any of the methods known in the art.", "In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence.", "Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.", "Antibodies of the invention are useful for, e.g., therapeutic purposes (by modulating activity of nGPCR-x), diagnostic purposes to detect or quantitate nGPCR-x, and purification of nGPCR-x.", "Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended.", "In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific.", "Compositions Mutations in the nGPCR-x gene that result in loss of normal function of the nGPCR-x gene product underlie nGPCR-x-related human disease states.", "The invention comprehends gene therapy to restore nGPCR-x activity to treat those disease states.", "Delivery of a functional nGPCR-x gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or er vivo by use of physical DNA transfer methods (erg, liposomes or chemical treatments).", "See, for example, Anderson, Nature, supplement to vol.", "392, no.", "6679, pp.25-20 (1998).", "For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verna, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).", "Alternatively, it is contemplated that in other human disease states, preventing the expression of, or inhibiting the activity of, nGPCR-x will be useful in treating disease states.", "It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of nGPCR-x.", "Another aspect of the present invention is directed to compositions, including pharmaceutical compositions, comprising any of the nucleic acid molecules or recombinant expression vectors described above and an acceptable carrier or diluent.", "Preferably, the carrier or diluent is pharmaceutically acceptable.", "Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field, which is incorporated herein by reference in its entirety.", "Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.", "Liposomes and nonaqueous vehicles such as fixed oils may also be used.", "The formulations are sterilized by commonly used techniques.", "Also within the scope of the invention are compositions comprising polypeptides, polynucleotides, or antibodies of the invention that have been formulated with, e.g., a pharmaceutically acceptable carrier.", "The invention also provides methods of using antibodies of the invention.", "For example, the invention provides a method for modulating ligand binding of a nGPCR-x comprising the step of contacting the nGPCR-x with an antibody specific for the nGPCR-x, under conditions wherein the antibody binds the receptor.", "GPCRs that may be expressed in the brain, such as nGPCR-x, provide an indication that aberrant nGPCR-x signaling activity may correlate with one or more neurological or psychological disorders.", "The invention also provides a method for treating a neurological or psychiatric disorder comprising the step of administering to a mammal in need of such treatment an amount of an antibody-like polypeptide of the invention that is sufficient to modulate ligand binding to a nGPCR-x in neurons of the mammal.", "nGPCR-x may also be expressed in other tissues, including but not limited to, peripheral blood lymphocytes, pancreas, ovary, uterus, testis, salivary gland, thyroid gland, kidney, adrenal gland, liver, bone marrow, prostate, fetal liver, colon, muscle, and fetal brain, and may be found in many other tissues.", "Within the brain, nGPCR-x mRNA transcripts may be found in many tissues, including, but not limited to, frontal lobe, hypothalamus, pons, cerebellum, caudate nucleus, and medulla.", "Tissues and brain regions where specific nGPCRs of the present invention are expressed are identified in the Examples below.", "Kits The present invention is also directed to kits, including pharmaceutical kits.", "The kits can comprise any of the nucleic acid molecules described above, any of the polypeptides described above, or any antibody which binds to a polypeptide of the invention as described above, as well as a negative control.", "The kit preferably comprises additional components, such as, for example, instructions, solid support, reagents helpful for quantification, and the like.", "In another aspect, the invention features methods for detection of a polypeptide in a sample as a diagnostic tool for diseases or disorders, wherein the method comprises the steps of: (a) contacting the sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of a polypeptide having the sequence of even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, said probe comprising the nucleic acid sequence encoding the polypeptide, fragments thereof, and the complements of the sequences and fragments; and (b) detecting the presence or amount of the probe:target region hybrid as an indication of the disease.", "In preferred embodiments of the invention, the disease is selected from the group consisting of thyroid disorders (e.g.", "thyreotoxicosis, myxoedema); renal failure; inflammatory conditions (e.g., Crohn's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including migraine; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer's; movement disorders, including ataxias, supranuclear palsy, etc.", "); infections, such as viral infections caused by HIV-1 or HIV-2; metabolic and cardiovascular diseases and disorders (e.g., type 2 diabetes, obesity, anorexia, hypotension, hypertension, thrombosis, myocardial infarction, cardiomyopathies, atherosclerosis, etc.", "); proliferative diseases and cancers (e.g., different cancers such as breast, colon, lung, etc., and hyperproliferative disorders such as psoriasis, prostate hyperplasia, etc.", "); hormonal disorders (e.g., male/female hormonal replacement, polycystic ovarian syndrome, alopecia, etc); and sexual dysfunction, among others.", "As described above and in Example 4 below, the genes encoding nGPCR-1 (nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ ID NO:77, amino acid sequence SEQ ID NO:10, SEQ ID NO:78), nGPCR-11 (nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequence SEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO: 21, SEQ ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82), nGPCR-40 (nucleic acid sequence SEQ ID NO:53, SEQ ID NO:83, amino acid sequence SEQ ID NO:54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequence SEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO: 86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ ID NO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90), nGPCR-58 (nucleic acid sequence SEQ ID NO:67, SEQ ID NO:91, SEQ ID NO:93, amino acid sequence SEQ ID NO:68, SEQ ID NO: 92, SEQ ID NO:94) and nGPCR-3 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO:185, amino acid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissue indicating that these n-GPCR-x proteins are neuroreceptors.", "Kits may be designed to detect either expression of polynucleotides encoding these proteins or the proteins themselves in order to identify tissue as being neurological.", "For example, oligonucleotide hybridization kits can be provided which include a container having an oligonucleotide probe specific for the n-GPCR-x-specific DNA and optionally, containers with positive and negative controls and/or instructions.", "Similarly, PCR kits can be provided which include a container having primers specific for the n-GPCR-x-specific sequences, DNA and optionally, containers with size markers, positive and negative controls and/or instructions.", "Hybridization conditions should be such that hybridization occurs only with the genes in the presence of other nucleic acid molecules.", "Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize.", "Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides.", "Such conditions are defined supra.", "The diseases for which detection of genes in a sample could be diagnostic include diseases in which nucleic acid (DNA and/or RNA) is amplified in comparison to normal cells.", "By “amplification” is meant increased numbers of DNA or RNA in a cell compared with normal cells.", "The diseases that could be diagnosed by detection of nucleic acid in a sample preferably include central nervous system and metabolic diseases.", "The test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.", "The samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed.", "Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.", "Alternatively, immunoassay kits can be provided which have containers container having antibodies specific for the n-GPCR-x-protein and optionally, containers with positive and negative controls and/or instructions.", "Kits may also be provided useful in the identification of GPCR binding partners such as natural ligands or modulators (agonists or antagonists).", "Substances useful for treatment of disorders or diseases preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question.", "Substances that modulate the activity of the polypeptides preferably include, but are not limited to, antisense oligonucleotides, agonists and antagonists, and inhibitors of protein kinases.", "Methods of Inducing Immune Response Another aspect of the present invention is directed to methods of inducing an immune response in a mammal against a polypeptide of the invention by administering to the mammal an amount of the polypeptide sufficient to induce an immune response.", "The amount will be dependent on the animal species, size of the animal, and the like but can be determined by those skilled in the art.", "Methods of Identifying Ligands The invention also provides assays to identify compounds that bind nGPCR-x.", "One such assay comprises the steps of: (a) contacting a composition comprising a nGPCR-x with a compound suspected of binding nGPCR-x; and (b) measuring binding between the compound and nGPCR-x.", "In one variation, the composition comprises a cell expressing nGPCR-x on its surface.", "In another variation, isolated nGPCR-x or cell membranes comprising nGPCR-x are employed.", "The binding may be measured directly, e.g., by using a labeled compound, or may be measured indirectly by several techniques, including measuring intracellular signaling of nGPCR-x induced by the compound (or measuring changes in the level of nGPCR-x signaling).", "Specific binding molecules, including natural ligands and synthetic compounds, can be identified or developed using isolated or recombinant nGPCR-x products, nGPCR-x variants, or preferably, cells expressing such products.", "Binding partners are useful for purifying nGPCR-x products and detection or quantification of nGPCR-x products in fluid and tissue samples using known immunological procedures.", "Binding molecules are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of nGPCR-x, especially those activities involved in signal transduction.", "The DNA and amino acid sequence information provided by the present invention also makes possible identification of binding partner compounds with which a nGPCR-x polypeptide or polynucleotide will interact.", "Methods to identify binding partner compounds include solution assays, in vitro assays wherein nGPCR-x polypeptides are immobilized, and cell-based assays.", "Identification of binding partner compounds of nGPCR-x polypeptides provides candidates for therapeutic or prophylactic intervention in pathologies associated with nGPCR-x normal and aberrant biological activity.", "The invention includes several assay systems for identifying nGPCR-x binding partners.", "In solution assays, methods of the invention comprise the steps of (a) contacting a nGPCR-x polypeptide with one or more candidate binding partner compounds and (b) identifying the compounds that bind to the nGPCR-x polypeptide.", "Identification of the compounds that bind the nGPCR-x polypeptide can be achieved by isolating the nGPCR-x polypeptide/binding partner complex, and separating the binding partner compound from the nGPCR-x polypeptide.", "An additional step of characterizing the physical, biological, and/or biochemical properties of the binding partner compound is also comprehended in another embodiment of the invention.", "In one aspect, the nGPCR-x polypeptide/binding partner complex is isolated using an antibody immunospecific for either the nGPCR-x polypeptide or the candidate binding partner compound.", "In still other embodiments, either the nGPCR-x polypeptide or the candidate binding partner compound comprises a label or tag that facilitates its isolation, and methods of the invention to identify binding partner compounds include a step of isolating the nGPCR-x polypeptide/binding partner complex through interaction with the label or tag.", "An exemplary tag of this type is a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation.", "Other labels and tags, such as the FLAG® tag (Eastman Kodak, Rochester, N.Y.), well known and routinely used in the art, are embraced by the invention.", "In one variation of an in vitro assay, the invention provides a method comprising the steps of (a) contacting an immobilized nGPCR-x polypeptide with a candidate binding partner compound and (b) detecting binding of the candidate compound to the nGPCR-x polypeptide.", "In an alternative embodiment, the candidate binding partner compound is immobilized and binding of nGPCR-x is detected.", "Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead, or a chromatographic resin, as well as non-covalent, high affinity interactions such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety.", "Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using of a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non-immobilized compound, (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached, as well as other techniques well known and routinely practiced in the art.", "The invention also provides cell-based assays to identify binding partner compounds of a nGPCR-x polypeptide.", "In one embodiment, the invention provides a method comprising the steps of contacting a nGPCR-x polypeptide expressed on the surface of a cell with a candidate binding partner compound and detecting binding of the candidate binding partner compound to the nGPCR-x polypeptide.", "In a preferred embodiment, the detection comprises detecting a calcium flux or other physiological event in the cell caused by the binding of the molecule.", "Another aspect of the present invention is directed to methods of identifying compounds that bind to either nGPCR-x or nucleic acid molecules encoding nGPCR-x, comprising contacting nGPCR-x, or a nucleic acid molecule encoding the same, with a compound, and determining whether the compound binds nGPCR-x or a nucleic acid molecule encoding the same.", "Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel-shift assays, Western blots, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, ELISA, and the like, which are described in, for example, Current Protocols in Molecular Biology, 1999, John Wiley & Sons, NY, which is incorporated herein by reference in its entirety.", "The compounds to be screened include (which may include compounds which are suspected to bind nGPCR-x, or a nucleic acid molecule encoding the same), but are not limited to, extracellular, intracellular, biologic or chemical origin.", "The methods of the invention also embrace ligands, especially neuropeptides, that are attached to a label, such as a radiolabel (e.g., 125I, 35S, 32P, 33P, 3H), a fluorescence label, a chemiluminescent label, an enzymic label and an immunogenic label.", "Modulators falling within the scope of the invention include, but are not limited to, non-peptide molecules such as non-peptide mimetics, non-peptide allosteric effectors, and peptides.", "The nGPCR-x polypeptide or polynucleotide employed in such a test may either be free in solution, attached to a solid support, borne on a cell surface or located intracellularly or associated with a portion of a cell.", "One skilled in the art can, for example, measure the formation of complexes between nGPCR-x and the compound being tested.", "Alternatively, one skilled in the art can examine the diminution in complex formation between nGPCR-x and its substrate caused by the compound being tested.", "In another embodiment of the invention, high throughput screening for compounds having suitable binding affinity to nGPCR-x is employed.", "Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate.", "The peptide test compounds are contacted with nGPCR-x and washed.", "Bound nGPCR-x is then detected by methods well known in the art.", "Purified polypeptides of the invention can also be coated directly onto plates for use in the aforementioned drug screening techniques.", "In addition, non-neutralizing antibodies can be used to capture the protein and immobilize it on the solid support.", "Generally, an expressed nGPCR-x can be used for HTS binding assays in conjunction with its defined ligand, in this case the corresponding neuropeptide that activates it.", "The identified peptide is labeled with a suitable radioisotope, including, but not limited to, 125I, 3H, 35S or 32P, by methods that are well known to those skilled in the art.", "Alternatively, the peptides may be labeled by well-known methods with a suitable fluorescent derivative (Baindur et al., Drug Dev.", "Res., 1994, 33, 373-398; Rogers, Drug Discovery Today, 1997, 2, 156-160).", "Radioactive ligand specifically bound to the receptor in membrane preparations made from the cell line expressing the recombinant protein can be detected in HTS assays in one of several standard ways, including filtration of the receptor-ligand complex to separate bound ligand from unbound ligand (Williams, Med.", "Res.", "Rev., 1991, 11, 147-184; Sweetnam et al., J.", "Natural Products, 1993, 56, 441455).", "Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary (Nakayama, Cur.", "Opinion Drug Disc.", "Dev., 1998,1,85-91 Bosse et al., J. Biomolecular Screening, 1998, 3, 285-292.).", "Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers, Drug Discovery Today, 1997, 2, 156-160; Hill, Cur.", "Opinion Drug Disc.", "Dev, 1998, 1, 92-97).", "Other assays may be used to identify specific ligands of a nGPCR-x receptor, including assays that identify ligands of the target protein through measuring direct binding of test ligands to the target protein, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods.", "Alternatively, such binding interactions are evaluated indirectly using the yeast two-hybrid system described in Fields et al., Nature, 340:245-246 (1989), and Fields et al., Trends in Genetics, 10:286-292 (1994), both of which are incorporated herein by reference.", "The two-hybrid system is a genetic assay for detecting interactions between two proteins or polypeptides.", "It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction.", "Variations on this methodology have been developed to clone genes that encode DNA binding proteins, to identify peptides that bind to a protein, and to screen for drugs.", "The two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast.", "The assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein.", "The DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene.", "The second hybrid protein, which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS.", "However, when both hybrid proteins are present, the noncovalent interaction of the first and second proteins tethers the activation domain to the UAS, activating transcription of the reporter gene.", "For example, when the first protein is a GPCR gene product, or fragment thereof, that is known to interact with another protein or nucleic acid, this assay can be used to detect agents that interfere with the binding interaction.", "Expression of the reporter gene is monitored as different test agents are added to the system.", "The presence of an inhibitory agent results in lack of a reporter signal.", "The function of nGPCR-x gene products is unclear and no ligands have yet been found which bind the gene product.", "The yeast two-hybrid assay can also be used to identify proteins that bind to the gene product.", "In an assay to identify proteins that bind to a nGPCR-x receptor, or fragment thereof, a fusion polynucleotide encoding both a nGPCR-x receptor (or fragment) and a UAS binding domain (i.e., a first protein) may be used.", "In addition, a large number of hybrid genes each encoding a different second protein fused to an activation domain are produced and screened in the assay.", "Typically, the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein-coding region being fused to the activation domain.", "This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function of the second binding protein.", "The system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein.", "Other assays may be used to search for agents that bind to the target protein.", "One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Pat.", "No.", "5,585,277, incorporated herein by reference.", "This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states.", "When a test ligand binds to the folded form of a target protein (i.e., when the test ligand is a ligand of the target protein), the target protein molecule bound by the ligand remains in its folded state.", "Thus, the folded target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand.", "Binding of the ligand to the target protein can be determined by any method that distinguishes between the folded and unfolded states of the target protein.", "The function of the target protein need not be known in order for this assay to be performed.", "Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules.", "Another method for identifying ligands of a target protein is described in Wieboldt et al., Anal.", "Chem., 69:1683-1691 (1997), incorporated herein by reference.", "This technique screens combinatorial libraries of 20-30 agents at a time in solution phase for binding to the target protein.", "Agents that bind to the target protein are separated from other library components by simple membrane washing.", "The specifically selected molecules that are retained on the filter are subsequently liberated from the target protein and analyzed by HPLC and pneumatically assisted electrospray (ion spray) ionization mass spectroscopy.", "This procedure selects library components with the greatest affinity for the target protein, and is particularly useful for small molecule libraries.", "Other embodiments of the invention comprise using competitive screening assays in which neutralizing antibodies capable of binding a polypeptide of the invention specifically compete with a test compound for binding to the polypeptide.", "In this manner, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with nGPCR-x.", "Radiolabeled competitive binding studies are described in A. H. Lin et al.", "Antimicrobial Agents and Chemotherapy, 1997, vol.", "41, no.", "10.pp.", "2127-2131, the disclosure of which is incorporated herein by reference in its entirety.", "As described above and in Example 4 below, the genes encoding nGPCR-1 (nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ ID NO:77, amino acid sequence SEQ ID NO:10, SEQ ID NO:78), nGPCR-11 (nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequence SEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO: 21, SEQ ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82), nGPCR-40 (nucleic acid sequence SEQ ID NO:53, SEQ ID NO:83, amino acid sequence SEQ ID NO:54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequence SEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO: 86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ ID NO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90), nGPCR-58 (nucleic acid sequence SEQ ID NO:67, SEQ ID NO:91, SEQ ID NO:93, amino acid sequence SEQ ID NO:68, SEQ ID NO: 92, SEQ ID NO:94), and nGPCR-3 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO:185, amino acid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissue indicating that these nGPCR-x proteins are neuroreceptors.", "Accordingly, natural binding partners of these molecules include neurotransmitters.", "Identification of Modulating Agents The invention also provides methods for identifying a modulator of binding between a nGPCR-x and a nGPCR-x binding partner, comprising the steps of: (a) contacting a nGPCR-x binding partner and a composition comprising a nGPCR-x in the presence and in the absence of a putative modulator compound; (b) detecting binding between the binding partner and the nGPCR-x; and (c) identifying a putative modulator compound or a modulator compound in view of decreased or increased binding between the binding partner and the nGPCR-x in the presence of the putative modulator, as compared to binding in the absence of the putative modulator.", "nGPCR-x binding partners that stimulate nGPCR-x activity are useful as agonists in disease states or conditions characterized by insufficient nGPCR-x signaling (e.g., as a result of insufficient activity of a nGPCR-x ligand).", "nGPCR-x binding partners that block ligand-mediated nGPCR-x signaling are useful as nGPCR-x antagonists to treat disease states or conditions characterized by excessive nGPCR-x signaling.", "In addition nGPCR-x modulators in general, as well as nGPCR-x polynucleotides and polypeptides, are useful in diagnostic assays for such diseases or conditions.", "In another aspect, the invention provides methods for treating a disease or abnormal condition by administering to a patient in need of such treatment a substance that modulates the activity or expression of a polypeptide having the sequence of even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192.Agents that modulate (i.e., increase, decrease, or block) nGPCR-x activity or expression may be identified by incubating a putative modulator with a cell containing a nGPCR-x polypeptide or polynucleotide and determining the effect of the putative modulator on nGPCR-x activity or expression.", "The selectivity of a compound that modulates the activity of nGPCR-x can be evaluated by comparing its effects on nGPCR-x to its effect on other GPCR compounds.", "Selective modulators may include, for example, antibodies and other proteins, peptides, or organic molecules that specifically bind to a nGPCR-x polypeptide or a nGPCR-x-encoding nucleic acid.", "Modulators of nGPCR-x activity will be therapeutically useful in treatment of diseases and physiological conditions in which normal or aberrant nGPCR-x activity is involved.", "nGPCR-x polynucleotides, polypeptides, and modulators may be used in the treatment of such diseases and conditions as infections, such as viral infections caused by HIV-1 or HIV-2; pain; cancers; Parkinson's disease; hypotension; hypertension; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome, among others.", "nGPCR-x polynucleotides and polypeptides, as well as nGPCR-x modulators, may also be used in diagnostic assays for such diseases or conditions.", "Methods of the invention to identify modulators include variations on any of the methods described above to identify binding partner compounds, the variations including techniques wherein a binding partner compound has been identified and the binding assay is carried out in the presence and absence of a candidate modulator.", "A modulator is identified in those instances where binding between the nGPCR-x polypeptide and the binding partner compound changes in the presence of the candidate modulator compared to binding in the absence of the candidate modulator compound.", "A modulator that increases binding between the nGPCR-x polypeptide and the binding partner compound is described as an enhancer or activator, and a modulator that decreases binding between the nGPCR-x polypeptide and the binding partner compound is described as an inhibitor.", "The invention also comprehends high-throughput screening (HTS) assays to identify compounds that interact with or inhibit biological activity (i.e., affect enzymatic activity, binding activity, etc.)", "of a nGPCR-x polypeptide.", "HTS assays permit screening of large numbers of compounds in an efficient manner.", "Cell-based HTS systems are contemplated to investigate nGPCR-x receptor-ligand interaction.", "HTS assays are designed to identify “hits” or “lead compounds” having the desired property, from which modifications can be designed to improve the desired property.", "Chemical modification of the “hit” or “lead compound” is often based on an identifiable structure/activity relationship between the “hit” and the nGPCR-x polypeptide.", "Another aspect of the present invention is directed to methods of identifying compounds which modulate (i.e., increase or decrease) activity of nGPCR-x comprising contacting nGPCR-x with a compound, and determining whether the compound modifies activity of nGPCR-x.", "The activity in the presence of the test compared is measured to the activity in the absence of the test compound.", "Where the activity of the sample containing the test compound is higher than the activity in the sample lacking the test compound, the compound will have increased activity.", "Similarly, where the activity of the sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound will have inhibited activity.", "The present invention is particularly useful for screening compounds by using nGPCR-x in any of a variety of drug screening techniques.", "The compounds to be screened include (which may include compounds which are suspected to modulate nGPCR-x activity), but are not limited to, extracellular, intracellular, biologic or chemical origin.", "The nGPCR-x polypeptide employed in such a test may be in any form, preferably, free in solution, attached to a solid support, borne on a cell surface or located intracellularly.", "One skilled in the art can, for example, measure the formation of complexes between nGPCR-x and the compound being tested.", "Alternatively, one skilled in the art can examine the diminution in complex formation between nGPCR-x and its substrate caused by the compound being tested.", "The activity of nGPCR-x polypeptides of the invention can be determined by, for example, examining the ability to bind or be activated by chemically synthesized peptide ligands.", "Alternatively, the activity of nGPCR-x polypeptides can be assayed by examining their ability to bind calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and photons.", "Alternatively, the activity of the nGPCR-x polypeptides can be determined by examining the activity of effector molecules including, but not limited to, adenylate cyclase, phospholipases and ion channels.", "Thus, modulators of nGPCR-x polypeptide activity may alter a GPCR receptor function, such as a binding property of a receptor or an activity such as G protein-mediated signal transduction or membrane localization.", "In various embodiments of the method, the assay may take the form of an ion flux assay, a yeast growth assay, a non-hydrolyzable GTP assay such as a [35S]-GTP S assay, a cAMP assay, an inositol triphosphate assay, a diacylglycerol assay, an Aequorin assay, a Luciferase assay, a FLIPR assay for intracellular Ca2+ concentration, a mitogenesis assay, a MAP Kinase activity assay, an arachidonic acid release assay (e.g., using [3H]-arachidonic acid), and an assay for extracellular acidification rates, as well as other binding or function-based assays of nGPCR-x activity that are generally known in the art.", "In several of these embodiments, the invention comprehends the inclusion of any of the G proteins known in the art, such as G16, G15, or chimeric Gqd5, Gqs5, Gqo5, Gq25, and the like.", "nGPCR-x activity can be determined by methodologies that are used to assay for FaRP activity, which is well known to those skilled in the art.", "Biological activities of nGPCR-x receptors according to the invention include, but are not limited to, the binding of a natural or an unnatural ligand, as well as any one of the functional activities of GPCRs known in the art.", "Non-limiting examples of GPCR activities include transmembrane signaling of various forms, which may involve G protein association and/or the exertion of an influence over G protein binding of various guanidylate nucleotides; another exemplary activity of GPCRs is the binding of accessory proteins or polypeptides that differ from known G proteins.", "The modulators of the invention exhibit a variety of chemical structures, which can be generally grouped into non-peptide mimetics of natural GPCR receptor ligands, peptide and non-peptide allosteric effectors of GPCR receptors, and peptides that may function as activators or inhibitors (competitive, uncompetitive and non-competitive) (e.g., antibody products) of GPCR receptors.", "The invention does not restrict the sources for suitable modulators, which may be obtained from natural sources such as plant, animal or mineral extracts, or non-natural sources such as small molecule libraries, including the products of combinatorial chemical approaches to library construction, and peptide libraries.", "Examples of peptide modulators of GPCR receptors exhibit the following primary structures: GLGPRPLRFamide, GNSFLRFamide, GGPQGPLRFamide, GPSGPLRFamide, PDVDHVFLRFamide, and pyro-EDVDHVFLRFamide.", "Other assays can be used to examine enzymatic activity including, but not limited to, photometric, radiometric, HPLC, electrochemical, and the like, which are described in, for example, Enzyme Assays: A Practical Approach, eds.", "R. Eisenthal and M. J. Danson, 1992, Oxford University Press, which is incorporated herein by reference in its entirety.", "The use of cDNAs encoding GPCRs in drug discovery programs is well-known; assays capable of testing thousands of unknown compounds per day in high-throughput screens (HTSs) are thoroughly documented.", "The literature is replete with examples of the use of radiolabelled ligands in HTS binding assays for drug discovery (see Williams, Medicinal Research Reviews, 1991, 11, 147-184.; Sweetnam, et al., J.", "Natural Products, 1993, 56, 441-455 for review).", "Recombinant receptors are preferred for binding assay HTS because they allow for better specificity (higher relative purity), provide the ability to generate large amounts of receptor material, and can be used in a broad variety of formats (see Hodgson, Bio/Technology, 1992, 10, 973-980; each of which is incorporated herein by reference in its entirety).", "A variety of heterologous systems is available for functional expression of recombinant receptors that are well known to those skilled in the art.", "Such systems include bacteria (Strosberg, et al., Trends in Pharmacological Sciences, 1992, 13, 95-98), yeast (Pausch, Trends in Biotechnology, 1997, 15, 487-494), several kinds of insect cells (Vanden Broeck, Int.", "Rev.", "Cytology, 1996, 164, 189-268), amphibian cells (Jayawickreme et al., Current Opinion in Biotechnology, 1997, 8, 629634) and several mammalian cell lines (CHO, BEK293, COS, etc.", "; see Gerhardt, et al., Eur.", "J. Pharmacology, 1997, 334, 1-23).", "These examples do not preclude the use of other possible cell expression systems, including cell lines obtained from nematodes (PCT application WO 98/37177).", "In preferred embodiments of the invention, methods of screening for compounds that modulate nGPCR-x activity comprise contacting test compounds with nGPCR-x and assaying for the presence of a complex between the compound and nGPCR-x.", "In such assays, the ligand is typically labeled.", "After suitable incubation, free ligand is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular compound to bind to nGPCR-x.", "It is well known that activation of heterologous receptors expressed in recombinant systems results in a variety of biological responses, which are mediated by G proteins expressed in the host cells.", "Occupation of a GPCR by an agonist results in exchange of bound GDP for GIT at a binding site on the Ga subunit; one can use a radioactive, non-hydrolyzable derivative of GTP, GTPγ[35S], to measure binding of an agonist to the receptor (Sim et al., Neuroreport, 1996, 7, 729-733).", "One can also use this binding to measure the ability of antagonists to bind to the receptor by decreasing binding of GTPγ[35S] in the presence of a known agonist.", "One could therefore construct a HTS based on GTPγ[35S] binding, though this is not the preferred method.", "The G proteins required for functional expression of heterologous GPCRs can be native constituents of the host cell or can be introduced through well-known recombinant technology.", "The G proteins can be intact or chimeric.", "Often, a nearly universally competent G protein (e.g., Gα16) is used to couple any given receptor to a detectable response pathway.", "G protein activation results in the stimulation or inhibition of other native proteins, events that can be linked to a measurable response.", "Examples of such biological responses include, but are not limited to, the following: the ability to survive in the absence of a limiting nutrient in specifically engineered yeast cells (Pausch, Trends in Biotechnology, 1997, 15, 487-494); changes in intracellular Ca2+ concentration as measured by fluorescent dyes (Murphy, et al, Cur.", "Opinion Drug Disc.", "Dev., 1998, 1, 192-199).", "Fluorescence changes can also be used to monitor ligand-induced changes in membrane potential or intracellular pH; an automated system suitable for HTS has been described for these purposes (Schroeder, et al., J. Biomolecular Screening, 1996, 1, 75-80).", "Melanophores prepared from Xenopus laevis show a ligand-dependent change in pigment organization in response to heterologous GPCR activation; this response is adaptable to HTS formats (Jayawickreme et al, Cur.", "Opinion Biotechnology, 1997, 8, 629634).", "Assays are also available for the measurement of common second messengers, including cAMP, phosphoinositides and arachidonic acid, but these are not generally preferred for HTS.", "Preferred methods of HTS employing these receptors include permanently transfected CHO cells, in which agonists and antagonists can be identified by the ability to specifically alter the binding of GTPγ[35S] in membranes prepared from these cells.", "In another embodiment of the invention, permanently transfected CHO cells could be used for the preparation of membranes which contain significant amounts of the recombinant receptor proteins; these membrane preparations would then be used in receptor binding assays, employing the radiolabelled ligand specific for the particular receptor.", "Alternatively, a functional assay, such as fluorescent monitoring of ligand-induced changes in internal Ca2+ concentration or membrane potential in permanently transfected CHO cells containing each of these receptors individually or in combination would be preferred for HTS.", "Equally preferred would be an alternative type of mammalian cell, such as HEK293 or COS cells, in similar formats.", "More preferred would be permanently transfected insect cell lines, such as Drosophila S2 cells.", "Even more preferred would be recombinant yeast cells expressing the Drosophila melanogaster receptors in HTS formats well known to those skilled in the art (e.g., Pausch, Trends in Biotechnology, 1997, 15, 487-494).", "The invention contemplates a multitude of assays to screen and identify inhibitors of ligand binding to nGPCR-x receptors.", "In one example, the nGPCR-x receptor is immobilized and interaction with a binding partner is assessed in the presence and absence of a candidate modulator such as an inhibitor compound.", "In another example, interaction between the nGPCR-x receptor and its binding partner is assessed in a solution assay, both in the presence and absence of a candidate inhibitor compound.", "In either assay, an inhibitor is identified as a compound that decreases binding between the nGPCR-x receptor and its binding partner.", "Another contemplated assay involves a variation of the dihybrid assay wherein an inhibitor of protein/protein interactions is identified by detection of a positive signal in a transformed or transfected host cell, as described in PCT publication number WO 95/20652, published Aug. 3, 1995.Candidate modulators contemplated by the invention include compounds selected from libraries of either potential activators or potential inhibitors.", "There are a number of different libraries used for the identification of small molecule modulators, including: (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.", "Chemical libraries consist of random chemical structures, some of which are analogs of known compounds or analogs of compounds that have been identified as “hits” or “leads” in other drug discovery screens, some of which are derived from natural products, and some of which arise from non-directed synthetic organic chemistry.", "Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms.", "Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof.", "For a review, see Science 282:63-68 (1998).", "Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture.", "These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods.", "Of particular interest are non-peptide combinatorial libraries.", "Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.", "For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr.", "Opin.", "Biotechnol.", "8:701-707 (1997).", "Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to modulate activity.", "Still other candidate inhibitors contemplated by the invention can be designed and include soluble forms of binding partners, as well as such binding partners as chimeric, or fusion, proteins.", "A “binding partner” as used herein broadly encompasses non-peptide modulators, as well as such peptide modulators as neuropeptides other than natural ligands, antibodies, antibody fragments, and modified compounds comprising antibody domains that are immunospecific for the expression product of the identified nGPCR-x gene.", "The polypeptides of the invention are employed as a research tool for identification, characterization and purification of interacting, regulatory proteins.", "Appropriate labels are incorporated into the polypeptides of the invention by various methods known in the art and the polypeptides are used to capture interacting molecules.", "For example, molecules are incubated with the labeled polypeptides, washed to remove unbound polypeptides, and the polypeptide complex is quantified.", "Data obtained using different concentrations of polypeptide are used to calculate values for the number, affinity, and association of polypeptide with the protein complex.", "Labeled polypeptides are also useful as reagents for the purification of molecules with which the polypeptide interacts including, but not limited to, inhibitors.", "In one embodiment of affinity purification, a polypeptide is covalently coupled to a chromatography column.", "Cells and their membranes are extracted, and various cellular subcomponents are passed over the column.", "Molecules bind to the column by virtue of their affinity to the polypeptide.", "The polypeptide-complex is recovered from the column, dissociated and the recovered molecule is subjected to protein sequencing.", "This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotides for cloning the corresponding gene from an appropriate cDNA library.", "Alternatively, compounds may be identified which exhibit similar properties to the ligand for the nGPCR-x of the invention, but which are smaller and exhibit a longer half time than the endogenous ligand in a human or animal body.", "When an organic compound is designed, a molecule according to the invention is used as a “lead” compound.", "The design of mimetics to known pharmaceutically active compounds is a well-known approach in the development of pharmaceuticals based on such “lead” compounds.", "Mimetic design, synthesis and testing are generally used to avoid randomly screening a large number of molecules for a target property.", "Furthermore, structural data deriving from the analysis of the deduced amino acid sequences encoded by the DNAs of the present invention are useful to design new drugs, more specific and therefore with a higher pharmacological potency.", "Comparison of the protein sequence of the present invention with the sequences present in all the available databases showed a significant homology with the transmembrane portion of G protein coupled receptors.", "Accordingly, computer modeling can be used to develop a putative tertiary structure of the proteins of the invention based on the available information of the transmembrane domain of other proteins.", "Thus, novel ligands based on the predicted structure of nGPCR-x can be designed.", "In a particular embodiment, the novel molecules identified by the screening methods according to the invention are low molecular weight organic molecules, in which case a composition or pharmaceutical composition can be prepared thereof for oral intake, such as in tablets.", "The compositions, or pharmaceutical compositions, comprising the nucleic acid molecules, vectors, polypeptides, antibodies and compounds identified by the screening methods described herein, can be prepared for any route of administration including, but not limited to, oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal.", "The nature of the carrier or other ingredients will depend on the specific route of administration and particular embodiment of the invention to be administered.", "Examples of techniques and protocols that are useful in this context are, inter alia, found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A (ed.", "), 1980, which is incorporated herein by reference in its entirety.", "The dosage of these low molecular weight compounds will depend on the disease state or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.", "For treating human or animals, between approximately 0.5 mg/kg of body weight to 500 mg/kg of body weight of the compound can be administered.", "Therapy is typically administered at lower dosages and is continued until the desired therapeutic outcome is observed.", "The present compounds and methods, including nucleic acid molecules, polypeptides, antibodies, compounds identified by the screening methods described herein, have a variety of pharmaceutical applications and may be used, for example, to treat or prevent unregulated cellular growth, such as cancer cell and tumor growth.", "In a particular embodiment, the present molecules are used in gene therapy.", "For a review of gene therapy procedures, see e.g.", "Anderson, Science, 1992, 256, 808-813, which is incorporated herein by reference in its entirety.", "The present invention also encompasses a method of agonizing (stimulating) or antagonizing a nGPCR-x natural binding partner associated activity in a mammal comprising administering to said mammal an agonist or antagonist to one of the above disclosed polypeptides in an amount sufficient to effect said agonism or antagonism.", "One embodiment of the present invention, then, is a method of treating diseases in a mammal with an agonist or antagonist of the protein of the present invention comprises administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize nGPCR-x-associated functions.", "In an effort to discover novel treatments for diseases, biomedical researchers and chemists have designed, synthesized, and tested molecules that inhibit the function of protein polypeptides.", "Some small organic molecules form a class of compounds that modulate the function of protein polypeptides.", "Examples of molecules that have been reported to inhibit the function of protein kinases include, but are not limited to, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642, published Nov. 26, 1992 by Maguire et al.", "), vinylene-azaindole derivatives (PCT WO 94/14808, published Jul.", "7, 1994 by Ballinari et al.", "), 1-cyclopropyl-4-pyridyluinolones (U.S. Pat.", "No.", "5,330,992), styryl compounds (U.S. Pat.", "No.", "5,217,999), styryl-substituted pyridyl compounds (U.S. Pat.", "No.", "5,302,606), certain quinazoline derivatives (EP Application No.", "0 566 266 A1), seleoindoles and selenides (PCT WO 94/03427, published Feb. 17, 1994 by Denny et al.", "), tricyclic polyhydroxylic compounds (PCT WO 92/21660, published Dec. 10, 1992 by Dow), and benzylphosphonic acid compounds (PCT WO 91/15495, published Oct. 17, 1991 by Dow et al), all of which are incorporated by reference herein, including any drawings.", "Exemplary diseases and conditions amenable to treatment based on the present invention include, but are not limited to, thyroid disorders (e.g.", "thyreotoxicosis, myxoedema); renal failure; inflammatory conditions (e g., Chron's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including migraine; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer's; movement disorders, including ataxias, supranuclear palsy, etc); infections, such as viral infections caused by HIV-1 or HIV-2; metabolic and cardiovascular diseases and disorders (e.g., type 2 diabetes, obesity, anorexia, hypotension, hypertension, thrombosis, myocardial infarction, cardiomyopathies, atherosclerosis, etc.", "); proliferative diseases and cancers (e.g., different cancers such as breast, colon, lung, etc., and hyperproliferative disorders such as psoriasis, prostate hyperplasia, etc); hormonal disorders (e.g., male/female hormonal replacement, polycystic ovarian syndrome, alopecia, etc.", "); sexual dysfunction, among others.", "Compounds that can traverse cell membranes and are resistant to acid hydrolysis are potentially advantageous as therapeutics as they can become highly bioavailable after being administered orally to patients.", "However, many of these protein inhibitors only weakly inhibit function.", "In addition, many inhibit a variety of protein kinases and will therefore cause multiple side effects as therapeutics for diseases.", "Some indolinone compounds, however, form classes of acid resistant and membrane permeable organic molecules.", "WO 96/22976 (published Aug. 1, 1996 by Ballinari et at) describes hydrosoluble indolinone compounds that harbor tetralin, naphthalene, quinoline, and indole substituents fused to the oxindole ring.", "These bicyclic substituents are in turn substituted with polar groups including hydroxylated alkyl, phosphate, and ether substituents.", "U.S. patent application Ser.", "No.", "08/702,232, filed Aug. 23, 1996, entitled “Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease” by Tang et al.", "(Lyon & Lyon Docket No.", "221/187) and 08/485,323, filed Jun.", "7, 1995, entitled “Benzylidene-Z-Indoline Compounds for the Treatment of Disease” by Tang et al.", "(Lyon & Lyon Docket No.", "223/298) and International Patent Publication WO 96/22976, published Aug. 1, 1996 by Ballinari et al., all of which are incorporated herein by reference in their entirety, including any drawings, describe indolinone chemical libraries of indolinone compounds harboring other bicyclic moieties as well as monocyclic moieties fused to the oxindole ring.", "Application Ser.", "Nos.", "08/02,232, filed Aug. 23, 1996, entitled “Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease” by Tang et al.", "(Lyon & Lyon Docket No.", "221/187), 08/485,323, filed Jun.", "7, 1995, entitled “Benzylidene-Z-Indoline Compounds for the Treatment of Disease” by Tang et al.", "(Lyon & Lyon Docket No.", "223/298), and WO 96/22976, published Aug. 1, 1996 by Ballinari et al.", "teach methods of indolinone synthesis, methods of testing the biological activity of indolinone compounds in cells, and inhibition patterns of indolinone derivatives, both of which are incorporated by reference herein, including any drawings.", "Other examples of substances capable of modulating kinase activity include, but are not limited to, tyrphostins, quinazolines, quinoxolines, and quinolines.", "The quinazolines, tyrphostins, quinolines, and quinoxolines referred to above include well-known compounds such as those described in the literature.", "For example, representative publications describing quinazolines include Barker et al., EPO Publication No.", "0 520 722 A1; Jones et al., U.S. Pat.", "No.", "4,447,608; Kabbe et al., U.S. Pat.", "No.", "4,757,072; Kaul and Vougioukas, U.S. Pat.", "No.", "5,316,553; Kreighbaum and Corner, U.S. Pat.", "No.", "4,343,940; Pegg and Wardleworth, EPO Publication No.", "0 562 734 A1; Barker et al., Proc.", "of Am.", "Assoc.", "for Cancer Research 32:327 (1991); Bertino, J. R., Cancer Research 3:293-304 (1979); Bertino, J. R., Cancer Research 9(2 part 1):293-304 (1979); Curtinet al., Br.", "J.", "Cancer 53:361-368 (1986); Fernandes et al., Cancer Research 43:1117-1123 (1983); Ferris et al.", "J. Org.", "Chem.", "44(2):173-178; Fry et al., Science 265:1093-1095 (1994); Jackman et al., Cancer Research 51:5579-5586 (1981); Jones et al.", "J. Med.", "Chem.", "29(6):1114-1118; Lee and Skibo, Biochemistry 26(23):7355-7362 (1987); Lemus et al., J. Org.", "Chem.", "54:3511-3518 (1989); Ley and Seng, Synthesis 1975:415-522 (1975); Maxwell et al., Magnetic Resonance in Medicine 17:189-196 (1991); Mini et al., Cancer Research 45:325-330 (1985); Phillips and Castle, J. Heterocyclic Chem.", "17(19):1489-1596 (1980); Reece et al., Cancer Research 47(11):2996-2999 (1977); Sculier et al., Cancer Immunol.", "and Immunother.", "23:A65 (1986); Sikora et al., Cancer Letters 23:289-295 (1984); and Sikora et al., Analytical Biochem.", "172:344-355 (1988), all of which are incorporated herein by reference in their entirety, including any drawings.", "Quinoxaline is described in Kaul and Vougioukas, U.S. Pat.", "No.", "5,316,553, incorporated herein by reference in its entirety, including any drawings.", "Quinolines are described in Dolle et al., J. Med.", "Chem.", "37:2627-2629 (1994); MaGuire, J. Med.", "Chem.", "37:2129-2131 (1994); Burke et al., J. Med.", "Chem.", "36:425-432 (1993); and Burke et al.", "BioOrganic Med.", "Chem.", "Letters 2:1771-1774 (1992), all of which are incorporated by reference in their entirety, including any drawings.", "Tyrphostins are described in Allen et al., Clin.", "Exp.", "Immunol.", "91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529 (1993); Baker et al., J.", "Cell Sci.", "102:543-555 (1992); Bilder et al., Amer.", "Physiol.", "Soc.", "pp.", "6363-6143:C721-C730 (1991); Brunton et al., Proceedings of Amer.", "Assoc.", "Cancer Rsch.", "33:558 (1992); Bryckaert et al., Experimental Cell Research 199:255-261 (1992); Dong et al., J. Leukocyte Biology 53:53-60 (1993); Dong et al., J. Immunol.", "151(5):2717-2724 (1993); Gazit et al., J. Med.", "Chem.", "32:2344-2352 (1989); Gazit et al., J. Med.", "Chem.", "36:3556-3564 (1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994); King et al., Biochem.", "J.", "275:413418 (1991); Kuo et al., Cancer Letters 74:197-202 (1993); Levitzkd, A., The FASEB J.", "6:3275-3282 (1992); Lyall et al., J. Biol.", "Chem.", "264:14503-14509 (1989); Peterson et al., The Prostate 22:335-345 (1993); Pillemer et al., Int.", "J.", "Cancer 50:80-85 (1992); Posner et al., Molecular Pharmacology 45:673-683 (1993); Rendu et al., Biol.", "Pharmacology 44(5):881-888 (1992); Sauro and Thomas, Life Sciences 53:371-376 (1993); Sauro and Thomas, J. Pharm.", "and Experimental Therapeutics 267(3):119-1125 (1993); Wolbring et al., J. Biol.", "Chem.", "269(36):22470-22472 (1994); and Yoneda et al., Cancer Research 51:44304435 (1991); all of which are incorporated herein by reference in their entirety, including any drawings.", "Other compounds that could be used as modulators include oxindolinones such as those described in U.S. patent application Ser.", "No.", "08/702,232 filed Aug. 23, 1996, incorporated herein by reference in its entirety, including any drawings.", "Methods of determining the dosages of compounds to be administered to a patient and modes of administering compounds to an organism are disclosed in U.S. application Ser.", "No.", "08/702,282, filed Aug. 23, 1996 and International patent publication number WO 96/22976, published Aug. 1, 1996, both of which are incorporated herein by reference in their entirety, including any drawings, figures or tables.", "Those skilled in the art will appreciate that such descriptions are applicable to the present invention and can be easily adapted to it.", "The proper dosage depends on various factors such as the type of disease being treated, the particular composition being used and the size and physiological condition of the patient.", "Therapeutically effective doses for the compounds described herein can be estimated initially from cell culture and animal models.", "For example, a dose can be formulated in animal models to achieve a circulating concentration range that initially takes into account the IC50 as determined in cell culture assays.", "The animal model data can be used to more accurately determine useful doses in humans.", "Plasma half-life and biodistribution of the drug and metabolites in the plasma, tumors and major organs can also be determined to facilitate the selection of drugs most appropriate to inhibit a disorder.", "Such measurements can be carried out.", "For example, HPLC analysis can be performed on the plasma of animals treated with the drug and the location of radiolabeled compounds can be determined using detection methods such as X-ray, CAT scan and MRI.", "Compounds that show potent inhibitory activity in the screening assays, but have poor pharmacokinetic characteristics, can be optimized by altering the chemical structure and retesting.", "In this regard, compounds displaying good pharmacokinetic characteristics can be used as a model.", "Toxicity studies can also be carried out by measuring the blood cell composition.", "For example, toxicity studies can be carried out in a suitable animal model as follows: 1) the compound is administered to mice (an untreated control mouse should also be used); 2) blood samples are periodically obtained via the tail vein from one mouse in each treatment group; and 3) the samples are analyzed for red and white blood cell counts, blood cell composition and the percent of lymphocytes versus polymorphonuclear cells.", "A comparison of results for each dosing regime with the controls indicates if toxicity is present.", "At the termination of each toxicity study, further studies can be carried out by sacrificing the animals (preferably, in accordance with the American Veterinary Medical Association guidelines Report of the American Veterinary Medical Assoc.", "Panel on Euthanasia, Journal of American Veterinary Medical Assoc., 202:229-249, 1993).", "Representative animals from each treatment group can then be examined by gross necropsy for immediate evidence of metastasis, unusual illness or toxicity.", "Gross abnormalities in tissue are noted and tissues are examined histologically.", "Compounds causing a reduction in body weight or blood components are less preferred, as are compounds having an adverse effect on major organs.", "In general, the greater the adverse effect the less preferred the compound.", "For the treatment of cancers the expected daily dose of a hydrophobic pharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250 mg/day, and most preferably 1 to 50 mg/day.", "Drugs can be delivered less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness.", "Plasma levels should reflect the potency of the drug.", "Generally, the more potent the compound the lower the plasma levels necessary to achieve efficacy.", "nGPCR-x mRNA transcripts may found in many tissues, including, but not limited to, brain, peripheral blood lymphocytes, pancreas, ovary, uterus, testis, salivary gland, kidney, adrenal gland, liver, bone marrow, prostate, fetal liver, colon, muscle, and fetal brain, and may be found in many other tissues.", "Within the brain, nGPCR-x mRNA transcripts may be found in many tissues, including, but not limited to, frontal lobe, hypothalamus, pons, cerebellum, caudate nucleus, and medulla.", "Tissues and brain regions where specific nGPCR mRNA transcripts are expressed are identified in the Examples, below.", "Odd numbered nucleotide sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 will, as detailed above, enable screening the endogenous neurotransmitters/hormones/ligands which activate, agonize, or antagonize nGPCR-x and for compounds with potential utility in treating disorders including, but not limited to, thyroid disorders (e.g.", "thyreotoxicosis, myxoedema); renal failure; inflammatory conditions (e.g., Chron's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including migraine; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer's; movement disorders, including ataxias, supranuclear palsy, etc.", "); infections, such as viral infections caused by HIV-1 or HIV-2; metabolic and cardiovascular diseases and disorders (e.g., type 2 diabetes, obesity, anorexia, hypotension, hypertension, thrombosis, myocardial infarction, cardiomyopathies, atherosclerosis, etc.", "); proliferative diseases and cancers (e.g., different cancers such as breast, colon, lung, etc, and hyperproliferative disorders such as psoriasis, prostate hyperplasia, etc.", "); hormonal disorders (e.g., male/female hormonal replacement, polycystic ovarian syndrome, alopecia, etc.", "); sexual dysfunction, among others.", "For example, nGPCR-x may be useful in the treatment of respiratory ailments such as asthma, where T cells are implicated by the disease.", "Contraction of airway smooth muscle is stimulated by thrombin.", "Cicala et al (1999) Br J Pharmacol 126:478484.Additionally, in bronchiolitis obliterans, it has been noted that activation of thrombin receptors may be deleterious.", "Hauck et al.", "(1999) Am J Physiol 277:L22-L29.Furthermore, mast cells have also been shown to have thrombin receptors.", "Cirino et al (1996) J Exp Med 183:821-827.nGPCR-x may also be useful in remodeling of airway structure s in chronic pulmonary inflammation via stimulation of fibroblast procollagen synthesis.", "See, e.g., Chambers et al.", "(1998) Biochem J 333:121-127; Trejo et al.", "(1996) J Biol Chem 271:21536-21541.In another example, increased release of sCD40L and expression of CD40L by T cells after activation of thrombin receptors suggests that nGPCR-x may be useful in the treatment of unstable angina due to the role of T cells and inflammation.", "See Aukrust et al.", "(1999) Circulation 100:614-620.A further example is the treatment of inflammatory diseases, such as psoriasis, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, and thyroiditis.", "Due to the tissue expression profile of nGPCR-x, inhibition of thrombin receptors may be beneficial for these diseases.", "See, e.g., Morris et al.", "(1996) Ann Rheum Dis 55:841-843.In addition to T cells, NK cells and monocytes are also critical cell types which contribute to the pathogenesis of these diseases.", "See, e.g., Naldini & Carney (1996) Cell Immunol 172:3542; Hoffman & Cooper (1995) Blood Cells Mol Dis 21:156-167; Colotta et al.", "(1994) Am J Pathol 144:975-985.Expression of nGPCR-x in bone marrow and spleen may suggest that it may play a role in the proliferation of hematopoietic progenitor cells.", "See DiCuccio et al.", "(1996) Exp Hematol 24:914-918.As another example, nGPCR-x may be useful in the treatment of acute and/or traumatic brain injury.", "Astrocytes have been demonstrated to express thrombin receptors.", "Activation of thrombin receptors may be involved in astrogliosis following brain injury.", "Therefore, inhibition of receptor activity may be beneficial for limiting neuroinflammation.", "Scar formation mediated by astrocytes may also be limited by inhibiting thrombin receptors.", "See, e.g., Pindon et al.", "(1998) Eur J Biochem 255:766-774; Ubl & Reiser.", "(1997) Glia 21:361-369; Grabham & Cunningham (1995) J Neurochem 64:583-591.nGPCR-x receptor activation may mediate neuronal and astrocyte apoptosis and prevention of neurite outgrowth.", "Inhibition would be beneficial in both chronic and acute brain injury.", "See, e.g., Donovan et al.", "(1997) J Neurosci 17:5316-5326; Turgeon et al (1998) J Neurosci 18:6882-6891; Smith-Swintosky et al.", "(1997) J Neurochem 69:1890-1896; Gill et al.", "(1998) Brain Res 797:321-327; Suidan et al.", "(1996) Semin Thromb Hemost 22:125-133.The attached Sequence Listing contains the sequences of the polynucleotides and polypeptides of the invention and is incorporated herein by reference in its entirety.", "As described above and in Example 4 below, the genes encoding nGPCR-1 (nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ ID NO:77, amino acid sequence SEQ ID NO: 10, SEQ ID NO:78), nGPCR-11 (nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequence SEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO: 21, SEQ.", "ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82), nGPCR-40 (nucleic acid sequence SEQ ID NO:53, SEQ ID NO:83, amino acid sequence SEQ ID NO: 54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequence SEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO: 86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ ID NO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90), nGPCR-58 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO: 185, amino acid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissue indicating that these nGPCR-x proteins are neuroreceptors.", "The identification of modulators such as agonists and antagonists is therefore useful for the identification of compounds useful to treat neurological diseases and disorders.", "Such neurological diseases and disorders, including but are not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia as well as depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.", "Methods of Screening Human Subjects Thus in yet another embodiment, the invention provides genetic screening procedures that entail analyzing a person's genome—in particular their alleles for GPCRs of the invention—to determine whether the individual possesses a genetic characteristic found in other individuals that are considered to be afflicted with, or at risk for, developing a mental disorder or disease of the brain that is suspected of having a hereditary component.", "For example, in one embodiment, the invention provides a method for determining a potential for developing a disorder affecting the brain in a human subject comprising the steps of analyzing the coding sequence of one or more GPCR genes from the human subject; and determining development potential for the disorder in said human subject from the analyzing step.", "More particularly, the invention provides a method of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor, comprising the steps of: (a) assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering the amino acid sequence, expression, or biological activity of at least one seven transmembrane receptor that is expressed in the brain, wherein the seven transmembrane receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 74, 186, 78, 80, 82, 84, 86, 90, and 94, or an allelic variant thereof, and wherein the nucleic acid corresponds to the gene encoding the seven transmembrane receptor; and (b) diagnosing the disorder or predisposition from the presence or absence of said mutation, wherein the presence of a mutation altering the amino acid sequence, expression, or biological activity of allele in the nucleic acid correlates with an increased risk of developing the disorder.", "In preferred variations, the seven transmembrane receptor is nGPCR-40 or nGPCR-54 comprising amino acid sequences set forth in SEQ ID NO: 84 for nGPCR-40 and SEQ ID NO: 86 for nGPCR-54, or an allelic variant thereof, and the disease is schizophrenia.", "By “human subject” is meant any human being, human embryo, or human fetus.", "It will be apparent that methods of the present invention will be of particular interest to individuals that have themselves been diagnosed with a disorder affecting the brain or have relatives that have been diagnosed with a disorder affecting the brain.", "By “screening for an increased risk” is meant determination of whether a genetic variation exists in the human subject that correlates with a greater likelihood of developing a disorder affecting the brain than exists for the human population as a whole, or for a relevant racial or ethnic human sub-population to which the individual belongs.", "Both positive and negative determinations (i.e., determinations that a genetic predisposition marker is present or is absent) are intended to fall within the scope of screening methods of the invention.", "In preferred embodiments, the presence of a mutation altering the sequence or expression of at least one nGPCR-40 or nGPCR-54 seven transmembrane receptor allele in the nucleic acid is correlated with an increased risk of developing schizophrenia, whereas the absence of such a mutation is reported as a negative determination.", "The “assaying” step of the invention may involve any techniques available for analyzing nucleic acid to determine its characteristics, including but not limited to well-known techniques such as single-strand conformation polymorphism analysis (SSCP) [Orita et al., Proc Natl.", "Acad.", "Sci.", "USA, 86: 2766-2770 (1989)]; heteroduplex analysis [White et al., Genomics, 12: 301-306 (1992)]; denaturing gradient gel electrophoresis analysis [Fischer et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 80: 1579-1583 (1983); and Riesner et al., Electrophoresis, 10: 377-389 (1989)]; DNA sequencing; RNase cleavage [Myers et al., Science, 230: 1242-1246 (1985)]; chemical cleavage of mismatch techniques [Rowley et al., Genomics, 30: 574-582 (1995); and Roberts et al., Nucl.", "Acids Res., 25: 3377-3378 (1997)]; restriction fragment length polymorphism analysis; single nucleotide primer extension analysis [Shumaker et al., Hum.", "Mutat., 7: 346-354 (1996); and Pastinen et al., Genome Res., 7: 606-614 (1997)]; 5′ nuclease assays [Pease et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 91:5022-5026 (1994)]; DNA Microchip analysis [Raisay, G., Nature Biotechnology, 16: 4048 (1999); and Chee et al., U.S. Pat.", "No.", "5,837,832]; and ligase chain reaction [Whiteley et al., U.S. Pat.", "No.", "5,521,065].", "[See generally, Schafer and Hawkins, Nature Biotechnology, 16: 33-39 (1998).]", "All of the foregoing documents are hereby incorporated by reference in their entirety.", "Thus, in one preferred embodiment involving screening nGPCR-40 or nGPCR-54 sequences, for example, the assaying step comprises at least one procedure selected from the group consisting of: (a) determining a nucleotide sequence of at least one codon of at least one nGPCR-40 or nGPCR-54 allele of the human subject; (b) performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; (c) performing a polynucleotide migration assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; and (d) performing a restriction endonuclease digestion to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences.", "In a highly preferred embodiment, the assaying involves sequencing of nucleic acid to determine nucleotide sequence thereof, using any available sequencing technique.", "[See, e.g., Sanger et al., Proc.", "Natl.", "Acad.", "Sci.", "(USA), 74: 5463-5467 (1977) (dideoxy chain termination method); Mirzabekov, TIBTECH, 12: 27-32 (1994) (sequencing by hybridization); Drmanac et al., Nature Biotechnology, 16: 54-58 (1998); U.S. Pat.", "No.", "5,202,231; and Science, 260: 1649-1652 (1993) (sequencing by hybridization); Kieleczawa et al., Science, 258: 1787-1791 (1992) (sequencing by primer walking); (Douglas et al., Biotechniques, 14: 824828 (1993) (Direct sequencing of PCR products); and Akane et al., Biotechniques 16: 238-241 (1994); Maxam and Gilbert, Meth.", "Enzymol., 65: 499-560 (1977) (chemical termination sequencing), all incorporated herein by reference.]", "The analysis may entail sequencing of the entire nGPCR gene genomic DNA sequence, or portions thereof, or sequencing of the entire seven transmembrane receptor coding sequence or portions thereof.", "In some circumstances, the analysis may involve a determination of whether an individual possesses a particular allelic variant, in which case sequencing of only a small portion of nucleic acid—enough to determine the sequence of a particular codon characterizing the allelic variant—is sufficient This approach is appropriate, for example, when assaying to determine whether one family member inherited the same allelic variant that has been previously characterized for another family member, or, more generally, whether a person's genome contains an allelic variant that has been previously characterized and correlated with a mental disorder having a heritable component.", "In another highly preferred embodiment, the assaying step comprises performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences.", "In a preferred embodiment, the hybridization involves a determination of whether nucleic acid derived from the human subject will hybridize with one or more oligonucleotides, wherein the oligonucleotides have nucleotide sequences that correspond identically to a portion of the GPCR gene sequence taught herein, such as the nGPCR-40 or nGPCR-54 coding sequence set forth in SEQ ID NOS: 83 for nGPCR-40 or 85 for nGPCR-54, or that correspond identically except for one mismatch.", "The hybridization conditions are selected to differentiate between perfect sequence complementarity and imperfect matches differing by one or more bases.", "Such hybridization experiments thereby can provide single nucleotide polymorphism sequence information about the nucleic acid from the human subject, by virtue of knowing the sequences of the oligonucleotides used in the experiments.", "Several of the techniques outlined above involve an analysis wherein one performs a polynucleotide migration assay, e.g., on a polyacrylamide electrophoresis gel (or in a capillary electrophoresis system), under denaturing or non-denaturing conditions.", "Nucleic acid derived from the human subject is subjected to gel electrophoresis, usually adjacent to (or co-loaded with) one or more reference nucleic acids, such as reference GPCR-encoding sequences having a coding sequence identical to all or a portion of SEQ ID NOS: 83 or 85 (or identical except for one known polymorphism).", "The nucleic acid from the human subject and the reference sequence(s) are subjected to similar chemical or enzymatic treatments and then electrophoresed under conditions whereby the polynucleotides will show a differential migration pattern, unless they contain identical sequences.", "[See generally Ausubel et al.", "(eds.", "), Current Protocols in Molecular Biology, New York: John Wiley & Sons, Inc. (1987-1999); and Sambrook et al., (eds.", "), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989), both incorporated herein by reference in their entirety.]", "In the context of assaying, the term “nucleic acid of a human subject” is intended to include nucleic acid obtained directly from the human subject (e.g., DNA or RNA obtained from a biological sample such as a blood, tissue, or other cell or fluid sample); and also nucleic acid derived from nucleic acid obtained directly from the human subject.", "By way of non-limiting examples, well known procedures exist for creating cDNA that is complementary to RNA derived from a biological sample from a human subject, and for amplifying (e.g., via polymerase chain reaction (PCR)) DNA or RNA derived from a biological sample obtained from a human subject.", "Any such derived polynucleotide which retains relevant nucleotide sequence information of the human subject's own DNA/RNA is intended to fall within the definition of “nucleic acid of a human subject” for the purposes of the present invention.", "In the context of assaying, the term “mutation” includes addition, deletion, and/or substitution of one or more nucleotides in the GPCR gene sequence (e.g., as compared to the seven transmembrane receptor-encoding sequences set forth of SEQ ID NOS: 74, 186, 78, 80, 82, 84, 86, 90, and 94) and other polymorphisms that occur in introns (where introns exist) and that are identifiable via sequencing, restriction fragment length polymorphism, or other techniques.", "The various activity examples provided herein permit determination of whether a mutation modulates activity of the relevant receptor in the presence or absence of various test substances.", "In a related embodiment, the invention provides methods of screening a person's genotype with respect to GPCR's of the invention, and correlating such genotypes with diagnoses for disease or with predisposition for disease (for genetic counseling).", "For example, the invention provides a method of screening for an nGPCR-40 or nGPCR-54 hereditary schizophrenia genotype in a human patient, comprising the steps of: (a) providing a biological sample comprising nucleic acid from the patient, the nucleic acid including sequences corresponding to said patients nGPCR-40 or nGPCR-54 alleles; (b) analyzing the nucleic acid for the presence of a mutation or mutations; (c) determining an nGPCR-40 or nGPCR-54 genotype from the analyzing step; and (d) correlating the presence of a mutation in an nGPCR-40 or nGPCR-54 allele with a hereditary schizophrenia genotype.", "In a preferred embodiment, the biological sample is a cell sample containing human cells that contain genomic DNA of the human subject.", "The analyzing can be performed analogously to the assaying described in preceding paragraphs.", "For example, the analyzing comprises sequencing a portion of the nucleic acid (e.g., DNA or RNA), the portion comprising at least one codon of the nGPCR-40 or nGPCR-54 alleles.", "Although more time consuming and expensive than methods involving nucleic acid analysis, the invention also may be practiced by assaying protein of a human subject to determine the presence or absence of an amino acid sequence variation in GPCR protein from the human subject.", "Such protein analyses may be performed, e.g., by fragmenting GPCR protein via chemical or enzymatic methods and sequencing the resultant peptides; or by Western analyses using an antibody having specificity for a particular allelic variant of the GPCR.", "The invention also provides materials that are useful for performing methods of the invention.", "For example, the present invention provides oligonucleotides useful as probes in the many analyzing techniques described above.", "In general, such oligonucleotide probes comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides that have a sequence that is identical, or exactly complementary, to a portion of a human GPCR gene sequence taught herein (or allelic variant thereof), or that is identical or exactly complementary except for one nucleotide substitution.", "In a preferred embodiment, the oligonucleotides have a sequence that corresponds in the foregoing manner to a human GPCR coding sequence taught herein, and in particular, the coding sequences set forth in SEQ ID NO: 83 and 85.In one variation, an oligonucleotide probe of the invention is purified and isolated.", "In another variation, the oligonucleotide probe is labeled, e.g., with a radioisotope, chromophore, or fluorophore.", "In yet another variation, the probe is covalently attached to a solid support.", "[See generally Ausubel et al.", "And Sambrook et al., supra.]", "In a related embodiment, the invention provides kits comprising reagents that are useful for practicing methods of the invention.", "For example, the invention provides a kit for screening a human subject to diagnose schizophrenia or a genetic predisposition therefor, comprising, in association: (a) an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-40 or nGPCR-54 seven transmembrane receptor gene, the oligonucleotide comprising 6-50 nucleotides that have a sequence that is identical or exactly complementary to a portion of a human nGPCR-40 or nGPCR-54 gene sequence or nGPCR-40 or nGPCR-54 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution; and (b) a media packaged with the oligonucleotide containing information identifying polymorphisms identifiable with the probe that correlate with schizophrenia or a genetic predisposition therefor.", "Exemplary information-containing media include printed paper package inserts or packaging labels; and magnetic and optical storage media that are readable by computers or machines used by practitioners who perform genetic screening and counseling services.", "The practitioner uses the information provided in the media to correlate the results of the analysis with the oligonucleotide with a diagnosis.", "In a preferred variation, the oligonucleotide is labeled.", "In still another embodiment, the invention provides methods of identifying those allelic variants of GPCRs of the invention that correlate with mental disorders.", "For example, the invention provides a method of identifying a seven transmembrane allelic variant that correlates with a mental disorder, comprising steps of: (a) providing a biological sample comprising nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny; (b) analyzing the nucleic acid for the presence of a mutation or mutations in at least one seven transmembrane receptor that is expressed in the brain, wherein the at least one seven transmembrane receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 74, 186, 78, 80, 82, 84, 86, 90, and 94 or an allelic variant thereof, and wherein the nucleic acid includes sequence corresponding to the gene or genes encoding the at least one seven transmembrane receptor, (c) determining a genotype for the patient for the at least one seven transmembrane receptor from said analyzing step; and (d) identifying an allelic variant that correlates with the mental disorder from the determining step.", "To expedite this process, it may be desirable to perform linkage studies in the patients (and possibly their families) to correlate chromosomal markers with disease states.", "The chromosomal localization data provided herein facilitates identifying an involved GPCR with a chromosomal marker.", "The foregoing method can be performed to correlate GPCR's of the invention to a number of disorders having hereditary components that are causative or that predispose persons to the disorder.", "For example, in one preferred variation, the disorder is schizophrenia, and the at least one seven transmembrane receptor comprises nGPCR-40 having an amino acid sequence set forth in SEQ ID NO: 84 or an allelic variant thereof.", "Also contemplated as part of the invention are polynucleotides that comprise the allelic variant sequences identified by such methods, and polypeptides encoded by the allelic variant sequences, and oligonucleotide and oligopeptide fragments thereof that embody the mutations that have been identified.", "Such materials are useful in in vitro cell-free and cell-based assays for identifying lead compounds and therapeutics for treatment of the disorders.", "For example, the variants are used in activity assays, binding assays, and assays to screen for activity modulators described herein.", "In one preferred embodiment, the invention provides a purified and isolated polynucleotide comprising a nucleotide sequence encoding a nGPCR-40 or nGPCR-54 receptor allelic variant identified according to the methods described above; and an oligonucleotide that comprises the sequences that differentiate the allelic variant from the nGPCR-40 or nGPCR-54 sequences set forth in SEQ ID NOS: 83 and 88.The invention also provides a vector comprising the polynucleotide (preferably an expression vector); and a host cell transformed or transfected with the polynucleotide or vector.", "The invention also provides an isolated cell line that is expressing the allelic variant GPCR polypeptide; purified cell membranes from such cells; purified polypeptide; and synthetic peptides that embody the allelic variation amino acid sequence.", "In one particular embodiment, the invention provides a purified polynucleotide comprising a nucleotide sequence encoding a nGPCR-40 seven transmembrane receptor protein of a human that is affected with schizophrenia; wherein said polynucleotide hybridizes to the complement of SEQ ID NO: 83 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS; and wherein the polynucleotide encodes a nGPCR-40 amino acid sequence that differs from SEQ ID NO: 84 by at least one residue.", "An exemplary assay for using the allelic variants is a method for identifying a modulator of nGPCR-x biological activity, comprising the steps of: (a) contacting a cell expressing the allelic variant in the presence and in the absence of a putative modulator compound; (b) measuring nGPCR-x biological activity in the cell; and (c) identifying a putative modulator compound in view of decreased or increased nGPCR-x biological activity in the presence versus absence of the putative modulator.", "Additional features of the invention will be apparent from the following Examples.", "Examples 1, 2, 4, 11, 12, and 13 are actual, while the remaining Examples are prophetic.", "Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the invention.", "Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention.", "Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.", "EXAMPLES Example 1 Identification of nGPCR-X A.", "Database Search The Celera database was searched using known GPCR receptors as query sequences to find patterns suggestive of novel G protein-coupled receptors.", "Positive hits were further analyzed with the GCG program BLAST to determine which ones were the most likely candidates to encode G protein-coupled receptors, using the standard (default) alignment produced by BLAST as a guide.", "Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., J. Molec.", "Biol., 1990, 215, 403-410, which is incorporated herein by reference in its entirety).", "Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).", "This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.", "T is referred to as the neighborhood word score threshold (Altschul et al., supra).", "These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them.", "The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.", "Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached.", "The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment.", "The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (13) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.", "The BLAST algorithm (Karlin et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 1993, 90, 5873-5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences.", "One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.", "For example, a nucleic acid is considered similar to a GPCR gene or cDNA if the smallest sum probability in comparison of the test nucleic acid to a GPCR nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.Homology searches were performed with the program BLAST version 2.08.A collection of 340 query amino acid sequences derived from GPCR's was used to search the genomic DNA sequence using TBLASTN and alignments with an E-value lower than 0.01 were collected from each BLAST search.", "The amino acid sequences have been edited to remove regions in the sequence that produce non-significant alignments with proteins that are not related to GPCR's.", "Multiple query sequences may have a significant alignment to the same genomic region, although each alignment may not cover exactly the same DNA region.", "A procedure is used to determine the region of maximum common overlap between the alignments from several query sequences.", "This region is called the consensus DNA region.", "The procedure for determining this consensus involves the automatic parsing of the BLAST output files using the program MSPcrunch to produce a tabular report.", "From this tabular report the start and end of each alignment in the genomic DNA is extracted.", "This information was used by a PERL script to derive the maximum common overlap.", "These regions were reported in the form of a unique sequence identifier, a start and the end position in the sequence.", "The sequences defined by these regions were extracted from the original genomic sequence file using the program fetchdb.", "The consensus regions were assembled into a non-redundant set by using the program phrap.", "After assembly with phrap a set of contigs and singletons was defined as candidate DNA regions coding for nGPCR-x.", "These sequences were then submitted for further sequence analysis.", "Further sequence analysis involved the removal of sequences previously isolated and removal of sequences related to olfactory GPCRs.", "The transmembrane regions for the sequences that remained were determined using a FORTRAN computer program called “tmtrest.all” [Parodi et al., Comput.", "Appl.", "Biosci.", "5:527-535(1994)].", "Only sequences that contained transmembrane regions in a pattern found in GPCRs were retained.", "cDNAs were sequenced directly using an ABI377 fluorescence-based sequencer (Perkin-Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI PRISM™ Ready Dye-Deoxy Terminator kit with Taq FS™ polymerase.", "Each ABI cycle sequencing reaction contained about 0.5 μg of plasmid DNA.", "Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 minute, followed by 50 cycles using the following parameters: 98° C. for 30 seconds, annealing at 50° C. for 30 seconds, and extension at 60° C. for 4 minutes.", "Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.", "Extension products were purified using Centriflex™ gel filtration cartridges (Advanced Genetic Technologies Corp., Gaithersburg, Md.).", "Each reaction product was loaded by pipette onto the column, which is then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 minutes at room temperature.", "Column-purified samples were dried under vacuum for about 40 minutes and then dissolved in 5 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).", "The samples were then heated to 90° C. for three minutes and loaded into the gel sample wells for sequence analysis using the ABI377 sequencer.", "Sequence analysis was performed by importing ABI377 files into the Sequencer program (Gene Codes, Ann Arbor, Mich.).", "Generally, sequence reads of 700 bp were obtained.", "Potential sequencing errors were minimized by obtaining sequence information from both DNA strands and by re-sequencing difficult areas using primers annealing at different locations until all sequencing ambiguities were removed.", "The following Table 5 contains the sequences of the polynucleotides and polypeptides of the invention.", "Start and stop codons within the polynucleotide sequence are identified by boldface type.", "The transmembrane domains within the polypeptide sequence are identified by underlining.", "TABLE 5 The following DNA sequence beGPCR-seq1 <SEQ ID NO.", "1> was identified in H. sapiens: GTCTGGGGGTGGGGGATGCTGGGACAGGGGTCAATTGCCTGAAGCAAGTGCTCTCATCCCCCTAGCTCCTGC TGATCTAGTTGGGGCTCCAGAGTGGGGAGGAGAAAGGCACTTTGAAACTTCTCTGCCCTTACCGTCTTAGCC ATCAAACTCTGAGCTGGAGATAGTGACGATGTGACAGGAACTTTCCCTGGGCCTCTCTGGGCCACAATTCCT GGCCGAGAGAAAGAGGAGGAATGAGGTGAGCACCTTCTTCACTCCTAGGGCCATGTGGTAGAGCTGCAGTCG CACCTCCTTCTGCCAATAGGCATAGATGAGTGGGTTGAGCAGGGAGTTGCCCACGCCGAGCAGCCACAGGTA CCGTTCCAGCACTAGGTAGAGGTGACACTCCTGGCAGGCCACCTGCACAATGCCAGTGATAAGGAAGGGGGT CCAGGATAGAGCAAAGCTCCCAATGAGAACAGACACAGTACGGAGAGCTTTGAAGTCGCTGGGAGTCCGTGG GGATCGATAACCTCCAGCCATGGCTCCTGCATGTTCCATCTTTCGAATCTGCTGGCTGTGCATGGAGGCAAT TTGAGCATGTCGCAGTAGAAGAAGACAAAGAGGAGCATGGCTGGGAAGAAGCCAACGCAGGAGAGGGTCAGC ACGAAGTGAGGGTGAAATACAGCAAAGAAGCTGCACTGCCCTTTGTAGGCAGTCTGCTGGAACATGGGGATT CCGAGTGGGAGGAAGCCAATGAGGTAAGACACTAACCACAGCCCGGCAATGCAGGCCCCGGCCACGAACCCA CTCATGATCTTCAAGTAGCGGAAGGGCTGCTTGATGGCAAGGTACCTGTCAAAGGTGATCAGCATGACCGTG AGGACAGAGGCAGCTGCGGAGGAAGTGACAAATGCCATCCGCAGGCTGCACAGGGTCTTCTGTGTGGGCCGA GAAGGGCTGGAGAGCTGGTCTGTGAGTAGGCCAGAGATGGCCACACCAATCAAGGTGTCAGCCACAGCCAGA TTCAAGGTGAAGCAGAGACTGACACCATCATTCTTGTGGATCAACAGCAGCACAGCCACAGCCACTAGTGTG TTAGTAGCAATGATGAGGGAGGCCAGGACAGCAAGGATCACTCCAAATGAGAAAGATGATTCCATGTCTCGA AGTGGCAGGACTTCACTTACCAGGGCATG The following amino acid sequence <SEQ ID NO.", "2> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "1: MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAVADTLIGVAISGLLTDQLSSPSRPT QKTLCSLRMAFVTSSAAASVLTVMLITFDRYLAIKQPFRYLKIMSGFVAGACIAGLWLVSYLIGFLPLGIPMF QQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHAGAMAGGYRSPRTPS DFKALRTVSVLIGSFALSWTPFLITGIVQVACQECHLYLVLERYLWLLGVGNSLLNPLIYAYWQKEVRLQLYH MALGVKKVLTSFLLFLSARNCGPERPRESSCHIVTISSSEFDG The following DNA sequence beGPCR-seq3 <SEQ ID NO.", "3> was identified in H. sapiens: CAGCGCGAGCGCCTTCATGGTGACGGTGTCCATGCGCTGGCAGTGTCTGCGTGCCACCCGGTGCACCTGGAG CGAGGTGAGGCAGAGCACCGCCAGCGGCAGCACGAAGCCCACGGCATGGAGCGTGGCGGTGAAGGCTGCGAA GCGCGGACGCTCAGGCTCGGGCGGCAGGCGCAGCGAACAGGACGCGAAGGCGCTGCTGTAGCCAAGCCACGA GCAGCCAAGTGCAGCGCCTGAGAAGGCCAGCGACTGTCCCCAGGCACAGCCCAGCAGCAGGCCGGCATAGCG CGGTCGCAGGCGTCCGGCGTAGCGCAGTGGGAAGCCCACTGCCAGCCACTGGTCTGCGCTCAGCGCCGCCAC GCTCAGCGCCGCGTTGGACGCCAGGAAGGTGTCCAGGAAGCCAATGACTTGGCATGCGCCGGGCGCCGACGG TGTCCGCCCGCGCATCACACCGAGCAGCGTGAAGGGCATGTCCAGCGCCGCCAGCAGCAGGTGGCCCAGAGA CAGATTCACCAGGAGGACGCCTGAGGCTCGAGTGCGGAGCTCAGCGCTGTAGGCGCAACAAAGCAGCACCAG TGCGTTGGATAGCAGCGCCACGGCCAGTACCATCACCAGGAGACCCGCCAGCAGCGCCTCGCCGGGGCCCAT GGCGCTAGC The following amino acid sequence <SEQ ID NO.", "4> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "3: SAMGPGEALLAGLLVMVLAVALLSNALVLLCCAYSAELRTRASGVLLVNLSLGHLLLAALDMPFTLLGVMRGR TPSAPGACQVIGFLDTFLASNAALSVAALSADQWLAVGFPLRYAGRLRPRYAGLLLGCAWGQSLAFSGAALGC SWLGYSSAFASCSLRLPPEPERPRFAAFTATLHAVGFVLPLAVLCLTSLQVHRVARRHCQRMDTVTMKALA The following DNA sequence beGPCR-seq4 <SEQ ID NO.", "5> was identified in H. sapiens: TGTGCAGGTGTGATCTCCATTCCTTTGTACATCCCTCACACGCTGTTCGATGGGATTTTGGAAAGGAAATCT GTGTATTTTGGCTCACTACTGACTATCTGTTATGTACAGCATCTGTATATAACATTGTCCTCATCAGCTATG ATCGATACCTGTCAGTCTCAAATGCTGTAAGTCGAACACATTAATTTATCCCCCTTAGAAGATTATGTAAAT GTATA The following amino acid sequence <SEQ ID NO.", "6> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "5: CAGVISIPLYIPHTLFEWDFGKEICVFWLTTDYLLCTASVYNIVLISYDRYLSVSNAVSRTHFIPLR RLCKCI The following DNA sequence beGPCR-seq5 <SEQ ID NO.", "7> was identified in H. sapiens: GACGTCGAAGCAGGTGATGATGCCCAGGGCGTGCACCGGGTAGGTGAGATCGGTGCGCGCCAGCGGGGACAGG GCGGTCAGGAGCAGCAGCCAGGTCCCTGCACACGCGGCCACCGCGTAACGACGGCGGCGCCAGCGCTTGGAGC TGAGCGGGTACAGGATCCCCAGGAAGCGCTCCACGCTGATACAGGTCATGGTGAGGATGCTGGAATACATGTT TGCGTAAAAGGCCACGGTCACCACGTTGCAAAGCAGCACCCCGAATACCCAGTGGTGGCGGTTGCAATGGTAG TAGATTTGGAAAGGCAACACGCTGGCCAGCATCAGGTCCGTGACGCTCAGGTTGATCATGAAGATGACCGACG GGGATCTGGGCCCCATGCGCCGGCACAGCACCCACAGAGAGAAGAGGTTGCCCGGGATGCTGACCGCCGCCAC CAGCGAGTACACCACGGGCAGGGCCACCGCGATCGCCGGGTTCCGCAGCATCTGCAGCGTCGCGTTGTC The following amino acid sequence <SEQ ID NO.", "8> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "7: DNATLQMLRNPAIAVALPVVYSLVAAVSIPGNLFSLWVLCRRMGPRSPSVIFMINLSVTDLMLASVLPFQIYY HCNRHHWVFGVLCNLVVTVAFYANMYSSILTMTCISVERFLGILYPLSSKRWRRRRYAVAACAGTWLLLLTAL SPLARTDLTYPVHALGIITCFDV The following DNA sequence beGPCR-seq9 <SEQ ID NO.", "9> was identified in H. sapiens: CCCATGTTCCTGCTCCTGGGCAGCCTCACGTTGTCGGATCTGCTGGCAGGCGCCGCCTACGCCGCCAACAT CCTACTGTCGGGGCCGCTCACGCTGAAACTGTCCCCCGCGCTCTGGTTCGCACGGGAGGGAGGCGTCTTCG TGGCACTCACTGCGTCCGTGCTGAGCCTCCTGGGCATCGCGCTGGAGCGCAGCCTCACCATGGCGCGCAGG GGGCCCGCGCCCGTCTCCAGTCGGGGGCGCACGCTGGCGATGGCAGCCGCGGCCTGG The following amino acid sequence <SEQ ID NO.", "10> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "9: PMFLLLGSLTLSDLLAGAAYAANILLSGPLTLKLSPALWFAREGGVFVALTASVLSLLGIALERSLTMARRGP APVSSRGRTLAMAAAAW The following DNA sequence beGPCR-seq11 <SEQ ID NO.", "11> was identified in H. sapiens: CTGCTCATTGTGGCCTTTGTGCTGGGCGCACTAGGCAATGGGGTCGCCCTGTGTGGTTTCTGCTTCCACAT GAAGACCTGGAAGCCCAGCACTGTTTACCTTTTCAATTTGGCCGTGGCTGATTTCCTCCTTATGATCTGCC TGCCTTTTCGGACAGACTATTACCTCAGACGTAGACACTGGGCTTTTGGGGACATTCCCTGCCGAGTGGGG CTCTTCACGTTGGCCATGAACAGGGCCGGGAGCATCGTGTTCCTTACGGTGGTGGCTGCGGACAGGTATTT CAAAGTGGTCCACCCCCACCACGCGGTGAACACTATCTCCACCCGGGTGGCGGCTGGCATCGTCTGCACCC TGTGGGCCCTGGTCATCCTGGGAACAGTGTATCTTTTGCTGGAGAACCATCTCTGCGTGCAAGAGACGGCC GTCTCCTGTGAGAGCTTCATCATGGAGTCGGCCAATGGCTGGCATGACATCATGTTCCAGCTGGAGTTCTT TATGCCCCTCGGCATCATCTTATTTTGCTCCTTCAAGATTGTTTGGAGCCTGAGGCGGAGGCAGCAGCTGG CCAGACAGGCTCGGATGAAGAAGGCGACCCGGTTCATCATGGTGGTGGCAATTGTGTTCATCACATGCTAC CTGCCCAGCGTGTCTGCTAGACTCTATTTCCTCTGGACGGTGCCCTCGAGTGCCTGCGATCCCTCTGTCCA TGGGGCCCTGCACATAACCCTCAGCTTCACCTACATGAACAGCATGCTGGATCCCCTGGTGTATTATTTTT CAAGCCCCTCCTTTCCCAAATTCTACAACAAGCTCAAAATCTGCAGTCTGAAACCCAAGCAGCCAGGACAC TCAAAAACACAAAGGCCGGAAGAGATGCCAATTTCG The following amino acid sequence <SEQ ID NO.", "12> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "11: LLIVAFVLGALGNGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFRTDYYLRRRHWAFGDIPCRVGLF TLAMNRAGSIVFLTVVAADRYFKVVHPHHAVNTISTRVAAGIVCTLWALVILGTVYLLLENHLCVQETAVSCE SFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQARMKKATRFIMVVAIVFITCYLPSVSA RLYFLWTVPSSACDPSVHGALHITLSFTYMNSMLDPLVYYFSSPSFPKFYNKLKICSLKPKQPGHSKTQRPEE MPIS The following DNA sequence beGPCR-seq12 <SEQ ID NO.", "13> was identified in H. sapiens: TGGAGCTGTGCCACCACCTATCTGGTGAACCTGATGGTGGCCGACCTGCTTTATGTGCTATTGCCCTTCCT CATCATCACCTACTCACTAGATGACAGGTGGCCCTTCGGGGAGCTGCTCTGCAAGCTGGTGCACTTCCTGT TCTATATCAACCTTTACGGCAGCATCCTGCTGCTGACCTGCATCTCTGTGCACCAGTTCCTAGGTGTGTGC CACCCACTGTGTTCGCTGCCCTACCGGACCCGCAGGCATGCCTGGCTGGGCACCAGCACCACCTGGGCCCT GGTGGTCCTCCAGCTGCTGCCCACACTGGCCTTCTCCCACACGGACTACATCAATGGCCAGATGATCTGGT ATGACATGACCAGCCAAGAGAATTTTGATCGGCTTTTTGCCTACGGCATAGTTCTGACATTGTCTGGCTTT CTTTCCCTCCTTGGTCATTTTGGTGTGCTATTCACTGATGGTCAGGAGCCTGATCAAGCCAGAGGAGAACC TCATGAGGACAGG The following amino acid sequence <SEQ ID NO.", "14> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "13: WSCATTYLVNLMVADLLYVLLPFLIITYSLDDRWPFGELLCKLVHFLFYINLYGSILLLTCISVHQFLGVCHP LCSLPYRTRRHAWLGTSTTWALVVLQLLPTLAFSHTDYINGQMIWYDMTSQENFDRLFAYGIVLTLSGFLSLL GHFGVLFTDGQEPDQARGEPHEDR The following DNA sequence beGPCR-seq14 <SEQ ID NO.", "15> was identified in H. sapiens: CCACCACGCGCAGCACGCCGACAGGGCCTCTCCCTCCCATTCTCCCGCAGGCCCGGACGACCACGCTGCCT CCAGCCGGTCGGCAAACTAGGGCAGCTCGCAGCCCACGAACAGCAGCCCCAGCAGCTGGCTCATCTTCAGG CTCTGCACCTTGGCGCGGGGCATCGCGCTGGGCGCACGGGCTCCACCTGGGCTCGCCGACCAGGCCGCTGC ACCCGCTGGGGCCTTCAGCCGGTGCCGCCACCAGACGGAGAGTAGGTGGCCACAAGCGACACCCATGATCT TAACAGGCGCGACGAAGCCCGCGACGGCCTCATAGAACGCGTACACCTGCACGTGCCAGCGCTGCAGGAGC GCGAAGATCCAGTGGCAGCGACGCATCCCCGGCCAGGCTCGGGCGGAGAGTGGCGCGCCTGGCTGCAGAGA CGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGTACTAGCGCACCACAAACCCCGACCCCCGCGCCA GCAGCAGTGCCAGCAGCCAGCCCAGGGCGGCGAGGGCACGCGCGGGCAGCGGCCGGCCGTGCGGAAGACGC ACCGCGCGCCGGCGCTCGAGGGCGATGAGCACCACGAGGTGGGCCGAGGCGCCCCGCCCGGATGCCTGCAG CAGCTGCAGGAAGCGGCACGCCAGGTCCCCCGTGGCCGCGCGGGGCTCGCCCAGCAGTTCCCAGGCCAGCT GTGACAGCGCCGTGCCCCCGCACGCGTACAGGTCCGCCAGGGCCAGCTGCACCAGCAGGAAGTCCATCTTG CGACGCTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACAGGCGGCACAGCACTGTGGTGTTGCCTGCCAC CGCCACCACCAGGATGACCCCCAGGAACACCAGGCGGACGCG The following amino acid sequence <SEQ ID NO.", "16> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "15: RVRLVFLGVILVVAVAGNTTVLCRLXXXXXXXXXXKRRKMDFLLVQLALADLYACGGTALSQLAWELLGEPRA ATGDLACRFLQLLQASGRGASAHLVVLIALERRRAVRLPHGRPLPARALAALGWLLALLLARGSGFVVRYXXX XXXXXXXXTSLQPGAPLSARAWPGMRRCHWIFALLQRWHVQVYAFYEAVAGFVAPVKIMGVACGHLLSVWWRH RLKAPAGAAAWSASPGGARAPSAMPRAKVQSLKMSQLLGLLFVGCELPFADRLEAAWSSGPAGEWEGEALSAC CAWW The following DNA sequence beGPCR-seq15 <SEQ ID NO.", "17> was identified in H. sapiens: TCTAAGTTTTTCTCTGAACTTTGAGCCTGTGAAAAAAGAAGGGATGCTGCCTCAGGCCACCCCAGCCTAGA TACTCACTCTGAGTGCCATGAGGTAGTAGAGGACACTGATGACAGTCATGGGGAGGAGGTAGAATAGGAAG GAGGTGACCTGGATGATGAAATTGTAGATCCACATGGGCTTGATGACCGTACAGGTGGCCGAACCTGGGAC CAGGGACCCATTGGGGAAGTAGTGGAACTTGATGCCATGGATGCTGGTGTTGGGCAGGGAGAAGAGCACGG AGAAGCCCCAGACGATGCCGAGGATCCTGAGGGCCCGGCGCCGGGTGCTCTGCAGTTTGGCGCGGAACGGG TGTAGGATGGCCACGTAGCGCTCCACGCTGACGGTGGTGATGCTGAGGATGGAGGCGAAGCACACGGTCTC AAAGAGGGCCGTCTTGAAGTAGCAGCCCACGGGCCCGAACAAGAAAGGGTAGTTGCGCCACATCTCATAGA CCTCCAGGGGCATTCCAAGGAGCAGGACCAGGAGGTCAGAGACCGCCAGGCTGAAGAGGTAGTAGTTGGTG GGCGTCTTCATAGCCTGGTGCTGCAGAATCACCAGGCACACCAGGACATTGCCAATGACCCCCACCACAAA AATTGGCACATACACCACAGACACGGGGAGGAAGAAGTGGCTGCGCCGAGGTCCGCAGAGGAAGGCCAGAT ACTCCTCGGTGCTGTTCAGGTGTTTCTGGAATGGATCTTCTAGTTTCTGCTGGTAGATCCAGGAAGCATTC TGAAGTTTTTCCATCCCTGA The following amino acid sequence <SEQ ID NO.", "18> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "17: SGMEKLQNASWIYQQKLEDPFQKHLNSTEEYLAFLCGPRRSHFFLPVSVVYVPIFVVGVIGNVLVCLVILQHQ AMKTPNTYYLFSLAVSDLLVLLLGMPLEVYEMWRNYPFLFGPVGCYFKTALFETVCFASILSITTVSVERYVA ILHPFRAKLQSTRRRALRILGIVWGFSVLFSLPNTSIHGIKFHYFPNGSLVPGSATCTVIKPMWIYNFIIQVT SFLFYLLPMTVISVLYYLMALRVSIAGVAG The following DNA sequence beGPCR-seq18 <SEQ ID NO.", "19> was identified in H. sapiens: ATCAAGATGATTTTTGCTATCGTGCAAATTATTGGATTTTCCAACTCCATCTGTAATCCCATTGTCTATGC ATTTATGAATGAAAACTTCAAAAAAAATGTTTTGTCTGCAGTTTGTTATTGCATAGTAAATAAAACCTTCT CTCCAGCACAAAGGCATGGAAATTCAGGAATTACAATGATGCGGAAGAAAGCAAAGTTTTCCCTCAGAGAG AATCCAGTG The following amino acid sequence <SEQ ID NO.", "20> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "19: IKMIFAIVQIIGFSNSICNPIVYAFMNENFKKNVLSAVCYCIVNKTFSPAQRHGNSGITMMRKKAKFSLRENP The following DNA sequence beGPCR-seq16 <SEQ ID NO.", "21> was identified in H. sapiens: GCCACAGCATGCAGTTTTCTGTAGAATTCCACTTTGTCTTTGCACTTGAAGAAGATGAGGTATCTGGTGAC CAGGATCACCACATAGAATAGGAACCGTGAGGTACATGTGGATGTGCAGCATGGCACTCACAAATTTGCAG AAGGGCAGCCCAAACATCCAAGTCTTCTTGATGAGGTAGGTCAAGCGAAATGGCACTGTCAGCAGAAAAAC GCTGTGGACCACCACCAAGTTAATGACCGCCATGGTGGTCACTGACCGGGTGTTCATTTTCACCAGGAGGA AAAGAATGGAAATGACACCCACCAGCCCGCCAATAAGCACTATGAAGTAGAGGCTGATTAAGTGGGGTGTC ACTATAGGATCGCAAGAGGAATTCCTGGAGGTATTGTGGCCAGGCATACTTGGGAAGTCACCTGGAGGAGA AAAAGCACCAGAGTAACTGAC The following amino acid sequence <SEQ ID NO.", "22> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "21: VSYSGAFSPPGDFPSMPGHNTSRNSSCDPIVTPHLISLYFIVLIGGLVGVISILFLLVKMNTRSVTTMAVINL VVVHSVFLLTVPFRLTYLIKKTWMFGLPFCKFVSAMLHIHMYLTVPILCGDPGHQIPHLLQVQRQSGILQKTA CCG The following DNA sequence beGPCR-seq17 <SEQ ID NO.", "23> was identified in H. sapiens: ACTGACCAAGGTCAGGGCATCGACTGAGGCTAGAAGGCCACAGGAAATGCCAGTCAAGGTGTTGGCGCCTG CAATCGCACCTACCACAAACTTGACCGGGGGCAGGGGGGCAGGCCCGCCAGCGAACACGGTCAGCAGCACC AGTCCATTGCAGAGCACGGAGAGCAACACGATGGCCCACACGGCCAGGCGGATGCCCCAGCTTTCAAAGAG GTACTCACA The following amino acid sequence <SEQ ID NO.", "24> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "23: CEYLFESWGIRLAVWAIVLLSVLCNGLVLLTVFAGGPAPLPPVKFVVGAIAGANTLTGISCGLLASVDALTLV S The following DNA sequence beGPCR-seq20 <SEQ ID NO.", "25> was identified in H. sapiens: AACCCCATCATCTACACGCTCACCAACCGCGACCTGCGCCACGCGCTCCTGCGCCTGGTCTGCTGCGGACG CCACTCCTGCGGCAGAGACCCGAGTGGCTCCCAGCAGTCGGCGAGCGCGGCTGAGGCTTCCGGGGGCCTGC GCCGCTGCCTGCCCCCGGGCCTTGATGGGAGCTTCAGCGGCTCGGAGCGCTCATCGCCCCAGCGCGACGGG CTGGACACCAGCGGCTCCACAGGCAGCCCCGGT The following amino acid sequence <SEQ ID NO.", "26> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "25: NPIIYTLTNRDLRHALLRLVCCGRHSCGRDPSGSQQSASAAEASGGLRRCLPPGLDGSFSGSERSSPQRDGLD TSGSTGSPG The following DNA sequence beGPCR-seq21 <SEQ ID NO.", "27> was identified in H. sapiens: CGTGAAGAACAGCGCCACCATGACCAGCATGTGCACCACGCGCGCTCTGCGCCGCGATGCTCGCGGGTCCG CAGCCTCCTNNNNNNNNNNNNNNNNNNNNNNNNNNTGGCAGAGCTTGCGCGCGATGCGGGCGTACATGACC ACGATGAGCGCCAGCGGCGCCAGGTAGATGTGCGAGAAGAGCACAGTGGTGTAGACCCTGCGCATGCCCTT CTCGGGCCAGGCCTCCCAGCAGGAGTAGAGAGGGTAGGAGCGGTTGCGGGCGTCCACCATGAAGTGGTGCT CCTCACGGGTGACGGTCAGCGTGACGGCCGAGGGACACATGATGAGCAGCGCCAGGGCCCAGATGACGGCG ATGGTGACGAGCGCCTTCCGCAGGGTCAGCTTCTCGCGGAAAGGGTGCACGATGCAGCGGAACCT The following amino acid sequence <SEQ ID NO.", "28> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "27: FRCIVHPFREKLTLRKALVTIAVIWALALLIMCPSAVTLTVTREEHHFMVDARNRSYPLYSCWEAWPEKGM RRVYTTVLFSHIYLAPLALIVVMYARIARKLCXXXXXXXXXXEAADPRASRRRARVVHMLVMVALFFT The following DNA sequence beGPCR-seq22 <SEQ ID NO.", "29> was identified in H. sapiens: GCAGGGGGCGTGAGTCCTCAGGCACTTCTTGAGGTCCTTGTTGAGCAGGAAGCAGACAATTGGGTTGACGG CAGCCTGGGCGAAGCTCATCCAAACAGCATGGCCAGGTAGCGGTGGGGCACAGCACAGGCTTTCACAAACA CTCGCCAGTAGCAGGCCACGATGTAGGGTGACCAGAGGAGCAGAAAGAGCAGTGTGATCGCGTAGAACATG CGGCCCAGCTGCTTTTCACCCTTGACCTCGTCCATGCCCAGTAGCCGCCGGCTGGCTGCATGCCCATTCTG CCGGATACCCAGCAGGGTTGGTGGCATGGGCCC The following amino acid sequence <SEQ ID NO.", "30> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "29: GPMPPTLLGIRQNGHASRRLLGMDEVKGEKQLGRMFYAITLLFLLLWSPYIVACYWRVFVKACAVPHRYLAT AVWMSFAQAAVNPIVCFLLNKDLKKCLRTHAPC The following DNA sequence beGPCR-seq24 <SEQ ID NO.", "31> was identified in H. sapiens: TATTCTGTAATGAAGAATGTCATTCACACTGCCATTGGCACATCCAGTGGCCTCACCTAGCATTGTGAAAG CCCTTCGGTTGGTGTATTGCCACTTCATTTTAAAAGGATGCACAAGTCCCTGGTGCCTTTCCACAGCAATG CAGGTCATAGTGAGGATTTCTGTCACAACAGCGGTAGACTGGACAAATGGCACCATCTTGCAAATGAAAGC ACCTGCAGTAAGGAAATAGGATAAATCATACATCAAAACAAAAAGAATAAAGGTTTCATCTGTGTCTTTGT AATTATCACTATCAGTCCATTCTGAGCCTCTGCCAAAAAGTTTGATAATTGTAATTACTCTGTAGACACA The following amino acid sequence <SEQ ID NO.", "32> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "31: VYRVITIIKLFGRGSEWTDSDNYKDTDETFILFVLMYDLSYFLTAGAFICKMVPFVQSTAVVTEILTMTCIAV ERHQGLVHPFKMKWQYTNRRAFTMLGEATGCANGSVNDILHYRI The following DNA sequence beGPCR-seq27 <SEQ ID NO.", "33> was identified in H. sapiens: GAGCAACATGATCTTTTTGAAGTACTTGACGGTGTCGTTCTTGACGGTCACGAAGCACAGAGTGTTGATCA TGCTGTTGCTCATGGCGATGCACTCGACGATGTAGAAGGCAGTGAGGTAGTGCTTCTCCTTCACAAACACG GTGGGGAAGAAGTCGCGCACGATGGTGAAGCCGTAGAAGGGCGCCCAGCATAGCACGTAGGCGGTGAGGAT GCACATGAGCACCAGGACCGTCTTCCTGCGGCAGCGCAGCCTCTTGCGGATCTGCTCTGTCTGGAATCCAG GGACCGCCTTGAACCAGAGCTCCCGGGAGATCCTGGCATAGCACAGGGTCATGGTGACCACGGGGCCCACG AATTCTATGCCAAAGATAAAGAGGAAGTAGGACTTGTAGTAGAGCTGCTGGTCCACAGGCCAGATCTGGCC GCAGAAGATCTTTTCCTGGCTCTTGACAATGACGAGGACCGTCTCGGTGGTGAAGTAGGCGGAAGGGATGG CGATCAGGATGGACACCGTCCACACCAAGGCAATCAGGCCAGTGGCTGTTTGGCACTTCATTCGTGGTCTC AGCGGATGGACAATAGCCAGATACCTAGGGCAAGAACACAAGTGGAGGCAGCC The following amino acid sequence <SEQ ID NO.", "34> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "33: GCLHLCSCPRYLAIVHPLRPRMKCQTATGLIALVWTVSILIAIPSAYFTTETVLVIVKSQEKIFCGQIWPVDQ QLYYKSYFLFIFGIEFVGPVVTMTLCYARISRELWFKAVPGFQTEQIRKRLRCRRKTVLVLMCILTAYVLCWA PFYGFTIVRDFFPTVFVKEKHYLTAFYIVECIAMSNSMINTLCFVTVKNDTVKYFKKIMLL The following DNA sequence beGPCR-seq28 <SEQ ID NO.", "35> was identified in H. sapiens: CAGCCACACTGCAGTGATGAAATCAAATGTCCAACACCAACCATAGTCACCATTACTAACTAAGAAGCCAC AAAACTTCCCTTCCAGGGTGTTCAGCAGCAGGGACAGGGCCCAGGGCAGGGCACACATGACAGTTGACAGG TTTCTTGGGCAGCAGCAGCAGTACCAGATAGGCCGCAGGACAGACAGGCAGCACTCAGTACTGATGGCACT CAGCATGCTCAGGCCTACAAGGTAGGCAAAGGTCATCACGCTGGTGAAGAAGCTAGGGAAATTGATGGAGA TGGAACAGAAGAAGTTACTGAGGTACACCAGGCAATTTATAATCTGGAAGCAGAGGAAGAGGAAGTCGGCC CCGGCCAGGCTGAGGACGTAGACAGAGAAGGCGTTCCTGCGCATGCGGAAGCCCAGGAGCCAGAGCACAAA CCCGTTTCCTACCAGCCCGACCAGGGCAATGAAAAGGATCAGGAAGACCGGGATCAG The following amino acid sequence <SEQ ID NO.", "36> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "35: LIPVFLILFIALVGLVGNGFVLWLLGFRMRRNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSISINFPS FFTSVMTFAYLVGLSMLSAISTECCLSVLRPIWYCCCCPRNLSTVMCALPWALSLLLNTLEGKFCGFLVSNGD YGWCWTFDFITAVWL The following DNA sequence beGPCR-seq31 <SEQ ID NO.", "37> was identified in H. sapiens: GAGAGTCTGATTCTGACTTACATCACATATGTAGGCCTGGGCATTTCTATTTGCAGCCTGATCCTTTGCTTGT CCGTTGAGGTCCTAGTCTGGAGCCAAGTGACAAAGACAGAGATCACCTATTTACGCCATGTGTGCATTGTTAA CATTGCAGCCACTTTGCTGATGGCAGATGTGTGGTTCATTGTGGCTTCCTTTCTTAGTGGCCCAATAACACAC CACAAGGGATGTGTGGCAGCCACATTTTTTGGTCATTTCTTTTACCTTTCTGTATTTTTCTGGATGCTTGCCA AGGCACTCCTTATCCTCTATGGAATCATGATTGTTTTC The following amino acid sequence <SEQ ID NO.", "38> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "37: ESLILTYITYVGLGISICSLILCLSVEVLVWSQVTKTEITYLRHVCIVNIAATLLMADVWFIVASFLSGPITH HKGCVAATFFGHFFYLSVFFWMLAKALLILYGIMIVF The following DNA sequence beGPCR-seq32 <SEQ ID NO.", "39> was identified in H. sapiens: TTGTGTGGCAGTAGAGAGATGTCAGGCTTCAGAGTCAACAAGAACTGGATTTCAAACTGGATTTGAGGACCCC CACCTTTGGTAAGTGACTTATTATCTGCGAGCCTCTGTTTCTCTCTTCTTTAAATGAGGACAGTAAATCCCAT ACGGCAGGGTGGTGGGGAGAATCAGAGATGATACAGCTGGTGATCACATCTGGTTTGTGTTCCCAGGGGCACC AGACTAGGGTTTCTGAGCATGGATCCAACCGTCCCAGTCTTCGGTACAAAACTGACACCAATCAACGGACGTG AGGAGACTCCTTGCTACAATCAGACCCTGAGCTTCACGGTGCTGACGTGCATCATTTCCCTTGTCGGACTGAC AGGAAACGCGGTAGTGCTCTGGCTCCTGGGCTACCGCATGCGCAGGAACGCTGTCTCCATCTACATCCTCAAC CTGGCCGCAGCAGACTTCCTCTTCCTCAGCTTCCAGATTATACGTTCGCCATTACGCCTCATCAATATCAGCC ATCTCATCCGCAAAATCCTCGTTTCTGTGATGACCTTTCCCTACTTTACAGGCCTGAGTATGCTGAGCGCCAT CAGCACCGAGCGCTGCCTGTCTGTTCTGTGGCCCATCTGGTACC The following amino acid sequence <SEQ ID NO.", "40> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "39: LCGSREMSGFRVNKNWISNWIGPPPLVSDLLSASLCFSLLMRTVNPIRQGGGENQRYSWSHLVCVPRGTRLGF LSMDPTVPVFGTKLTPINGREETPCYNQTLSFTVLTCIISLVGLTGNAVVLWLLGYRMRRNAVSIYILNLAAA DFLFLSFQIIRSPLRLINISHLIRKILVSVMTFPYFTGLSMLSAISTERCLSVLWPIWY The following DNA sequence beGPCR-seq33 <SEQ ID NO.", "41> was identified in H. sapiens: ACAGAAAGCAAGGCCACCAGGACCTTAGGCATAGTCATGGGAGTGTTTGTGTTGTGCTGGCTGCCCTTCTTTG TCTTGACGATCACAGATCCTTTCATTAATTTTACAACCCTTGAAGATCTGTACAATGTCTTCCTCTGGCTAGG CTATTTCAACTCTGCTTTCAATCCCATTTTATATGGCATGCTTTATCCTTGGTTTCGCAAGGCATTGAGGATG ATTGTCACAGGCATGATCTTCCACCCTGACTCTTCCACCCTAAGCCTGTTTTCTGCCCATGCTTAGGCTGTGT TCATCATTCAATAGGACTCTTTTCTGG The following amino acid sequence <SEQ ID NO.", "42> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "41: TESKATRTLGIVMGVFVLCWLPFFVLTITDPFINFTTLEDLYNVFLWLGYFNSAFNPILYGMLYPWFRKALRM IVTGMIFHPDSSTLSLFSAHAAVFIIQDSF The following DNA sequence beGPCR-seq34 <SEQ ID NO.", "43> was identified in H. sapiens: TAGGAATCTCAGAGAAGAAAGTAAGGAACCAGAAAACCATAAAAGAATGTAAATGGAAAAGAATCAGCAAATC TTATTCACTTATCACTAAATCTAAAATATGTCAAAATACATGAAGACAACAAATGCTTTAGAACAACTGTTGA ATGTATTGTCCTACAACTTGGCATATGATCATGCTTGCCTCTCTATGTCCAAGTGTTTATTTTTGCAGTTGAC CTTAATTTCAAGTTAGTTTTGAGGTCTCTACAGTAATGTTTTTAATCTGTCTCTACTTCTTCAGAAAATAAAT TAGTTGTTGACGAATCAGTCCTTAAGACCTTGCCGCTTACAATAAGTTTTATTGCCTTCCCAAACCATTGGTA AAAGAAAGCATAAATCAAGGGGTTCATAGCTGAATTATAATAAACACACCAAACTAAAATCTCATAAACATAA GGAGGAGTTATAAAATTCATATAAGCATCAATCACTGCATCAACGAGGTATGGTAGCCAAGAGACAAGAAATG CTGC The following amino acid sequence <SEQ ID NO.", "44> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "43: LHQRGMVAKRQEMLAAFLVSWLPYLVDAVIDAYMNFITPPYVYEILVWCVYYNSAMNPLIYAFFYQWFGKAIK LIVSGKVLRTDSSTTNLFSEEVETDKHYCRDLKTNLKLRSTAKINTWTRGKHDHMPSCRTIHSTVVLKHLLSS CI The following DNA sequence beGPCR-seq35 <SEQ ID NO.", "45> was identified in H. sapiens: CTGGAAAGAGGTCCTCGATCTATCCTCTACGCCGTCCTTGGTTTTGGGGCTGTGCTGGCAGCGTTTGGAAACT TACTGGTCATGATTGCTATCCTTCACTTCTAACAACTGCACACACCTACAAACTTTCTGATTGCGTCGCTGGC CTGTGCTGACTTCTTGGTGGGAGTCACTGTGATGCCCTTCAGCACAGTGAGGTCTGTGGAGAGCTGTTGGTAC TTTGGGGACAGTTACTGTAAATTCCATACATGTTTTGACACATCTTTCTGTTTTGCTTCTTTATTTCATTTAT GCTGTATCTCTGTTGATAGATACATTGCTGTTACTGATCCTCTGACCTATCCAACCAAGTTTACTGTGTCAGT TTCAGGGATATGCATTGTTCTTTCCTGGTTCTTTTCTGTCACATACAGCTTTTCGATCTTTTACACGGGAGCC AACGAAGAAGGAATTGAGGAATTAGTAGTTGCTCTAACCTGTGTAGGAGGCTGCCAGGCTCCACTGAATCAAA ACTGGGTCCTACTTTGTTTTCTTCTATTCTTTATACCCAATGTCGCCATGGTGTTTATATACAGTAAGATATT TTTGGTGGCCAAGCATCAGGCTAGGAAGATAGAAAGTACAGCCAGCCAAGCTCAGTCCTTCTCAGAGAGTTAC AAGGAAAGAGTAGCAAAAAGAGAGAGAAAGGCTGCCAAAACCTTGGGAATTGCTATGGCAGCATTTCTT The following amino acid sequence <SEQ ID NO.", "46> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "45: LERGPRSILYAVLGFGAVLAAFGNLLVMIAILHFQLHTPTNFLIASLACADFLVGVTVMPFSTVRSVESCWYF GDSYCKFHTCFDTSFCFASLFHLCCISVDRYIAVTDPLTYPTKFTVSVSGICIVLSWFFSVTYSFSIFYTGAN EEGIEELVVALTCVGGCQAPLNQNWVLLCFLLFFIPNVAMVFIYSKIFLVAKHQARKIESTASQAQSFSESYK ERVAKRERKAAKTLGIAMAAFL The following DNA sequence beGPCR-seq36 <SEQ ID NO.", "47> was identified in H. sapiens: AACCAGGTGGCCTTACTCCTAAGACCCCTGGCCTTGTCTATGGCCTTTATCAACAGCTGTCTCAATCCAGTTC TCTATGTCTTCATTGGGCATGACTTCTGGGAGCACTTGCTCCACTCCCTGCTAGCTGCCTTAGAACGGGCACT TAGCGAGGAGCCAGATAGTGCCTGAATCCCAGCTCCCAGGCAGATGAGTCCTTTATAACATGACCCAATTTCC TACTCCATTTTCCCACCACTCAATCCTCTTCCCAAACAGCTCTACCATAATCCAACATCCAACAGAATTTAAG AGAATAAACCACAACTTTTAAGTGAGCTCTATGTGCTAGGTCATGTTTTAGAATACAACCTTAAGTGCCTGGA AGATGGAGGCAAGAAACAAACAAGGTCTCATTCTTTAGAGGAAGACAGTTCACCAAGACTCAAACAGAAAAAA AGATAGTTATCTTGTGACAAAACAAGTCATAAAATTGGGTCAGGACCTGCAGCAATGACTTTATGCTAGAATC CAGAGCACTAGCAGGAAACTGCTTAAATTTTACTTAATCAAAGTCAAGTTTGGACATACATGTCAGGTAAAAC CTAGCAGAGATGAGCTACCTTGATTTTAAAACTTCAAGGGATAGCTCAATGTCATCAAGATCCTTTTGATGAC TTG The following amino acid sequence <SEQ ID NO.", "48> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "47: NQVALLLRPLALSMAFINSCLNPVLYVFIGHDFWEHLLHSLLAALERALSEEPDSAIPAPRQMSPLHDPISYS IFPPLNPLPKQLYHNPTSNRIENKPQLLSELYVLGHVLEYNLKCLEDGGKKQTRSHSLEEDSSPRLKQKKRLS CDKTSHKIGSGPAAMTLCNPEHQETAILLNQSQVWTYMSGKTQRATLILKLQGIAQCHQDPFDDL The following DNA sequence beGPCR-seq37 <SEQ ID NO.", "49> was identified in H. sapiens: GCTTGTTCACGGCCACCATCCTCAAGCTGTTGCGCACGGAGGAGGCGCACGGCCGGGAGCAGCGGAGGCGCGC GGTGGGCCTGGCCGCGGTGGTCTTGCTGGCCTTTGTCACCTGCTTCGCCCCCAACAACTTCGTGCTCCTGGCG CACATCGTGAGCCGCCTGTTCTACGGCAAGAGCTACTACCACGTGTACAAGCTCACGCTGTGTCTCAGCTGCC TCAACAACTGTCTGGACCCGTTTGTTTATTACTTTGCGTCCCGGGAATTCCAGCTGCGCCTGCGGGAATATTT GGGCTGCCGCCGGGTGCCCAGAGACACCCTGGACACGCGCCGCGAGAGCCTCTTCTCCGCCAGGACCACGTCC GTGCGCTCCGAGGCCGGTGCGCACCCTGAAGGGATGGAGGGAGCCACCAGGCCCGGCCTCCAGAGGCAGGAGA GTGTGTTCTGAGTCCCGGGGGCGCAGC The following amino acid sequence <SEQ ID NO.", "50> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "49: LFTATILKLLRTEEAHGREQRRRAVGLAAVVLLAFVTCFAPNNFVLLAHIVSRLFYGKSYYHVYKLTLCLSCL NNCLDPFVYYFASREFQLRLREYLGCRRVPRDTLDTRRESLFSARTTSVRSEAGAHPEGMEGATRPGLQRQES VFVPGAQAAPPGLR The following DNA sequence beGPCR-seq38 <SEQ ID NO.", "51> was identified in H. sapiens: TTACTTATTCTGCCCTTTATCCAACTTTTAATTCCCTTTGCTATTCTCCTGCCTCATTTTCTGGCCTCATTTT CCCTATTATCCTGCCTCACATTGATCAAGGGATGAGGCTGGCAGGATCCGGAACCCACAGGGCCCCGTGGGCC ATGAGAGGCTCCTGGACTTGAACCTCAGGACACTCCCACTCTGGCTGCCGGCAGGGATGGAAGCTGGATGAGC AGGCAGGAGCTGGCAGTGGGGGTGGAGAGCCATAGGCTATTGGGGTGGACAGGCTTGGGTGCCTCATGGGAGC TCCCCATGGGAGCTGTGGCCCCTTGGGGCCTCTTATTTCTCACCCCAGGCTTTCCCGGGAGAGGTTCAAGTCA GAAGATGCCCCAAAGATCCACGTGGCCCTGGGTGGCAGCCTGTTCCTCCTGAATCTGGCCTTCTTGGTCAATG TGGGGAGTGGCTCAAAGGGGTCTGATGCTGCCTGCTGGGCCCGGGGGGCTGTCTTCCACTACTTCCTGCTCTG TGCCTTCACCTGGATGGGCCTTGAAGCCTTCCACCTCTACCTGCTCGCTGTCAGGGTCTTCAACACCTACTTC GGGCACTACTTCCTGAAGC The following amino acid sequence <SEQ ID NO.", "52> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "51: ETYSALYPTFNSLCYSPASFSGLIFPIILPHIDQGMRLAGSGTHRAPWAMRGSWTTSGHSHSGCRQGWKLDEQ AGAGSGGGEPAIGVDRLGCLMGAPHGSCGPLGPLISHPRLSRERFKSEDAPKIHVALGGSLFLLNLAFLVNVG SGSKGSDAACWARGAVFHYFLLCAFTWMGLEAFHLYLLAVRVFNTYFGHYFL The following DNA sequence beGPCR-seq40 <SEQ ID NO.", "53> was identified in H. sapiens: AATTGGTCGGAGAGTGCAGCTGCTTGAAATGGAGGATTGAAATCATCACCAGGAGGTTTCCAAACACAGCCAG CACAGCCCCAAAGCCAAACACTATGTACAGAATCACCCGGGATCCCGGCGAGAAGGGGATTTTCACACAGGAC CCATTCACGTTCGCGTAGCACAGCTGCACAGCCACCAGCAGGGATGAATTGCTGCTCATAACGCTGGTATTTA CATATGGAGAAATTTTGTCCTTGTTGATTATCACAAAAAATACAGGATTGTTCCTGATTTTCATTGCTCCTGC GGAAAAAAACACATATTCACCAGGATGCCAGAGGAAATGATCA The following amino acid sequence <SEQ ID NO.", "54> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "53: DHFLWHPGEYVFFSAGAMKIRNNPVFFVIINKDKISPYVNTSVMSSNSSLLVAVQLCYANVNGSCVKIPFSPG SRVILYIVFGFGAVLAVFGNLLVMISILHFKQLHSPTN The following DNA sequence beGPCR-seq41 <SEQ ID NO.", "55> was identified in H. sapiens: CACATCTTAACAAGACTGAAAAACATTGATTTGTTTTTAATTTGAAGAGCAATTTATTTGCTATTCATTCATA GTCTTACTTGATTTTTAAAAACTCATTTCGCTTGGTAATTTTAAAGGTATCCTGAACTTCGTCTATCCAACTG CTTATATATGTTCAGAAAACAAATTCATGGTTGCTGAACTGTTCTTTAAAACCTGACCAGTTACAATAACTTT TATTGCTTTCCTAAACCATGGGTAAAATAAAGCATAAATCAAAGGATTCATGGCTGAGTTATAATAAGCACAC CAACAGCATCATAAATACAGGCAGGGGTTATAAAGCCCATAAAGGCATCAATTAATGAATCAATGCTATATGG TAACCATGAAATCATAAATGCTACCACTGTGACCCCCAGGGTTTTAGCTGCTTTTCTCTCTCTCCTGGCCACT CTGGCTTTGTAACTCTCTGAGGATGATTCTGTCTTGCTACCAGTATTTTCTATCTTTTTCGCCTGTCGTCTAG CCACAAGAAATATGTTACCATACAGAATTATCATAATAAAGGTAGGTATAAAGAAGGATAGAAAATCTGTCAA CA The following amino acid sequence <SEQ ID NO.", "56> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "55: LTDFLSFFIPTFIMIILYGNIFLVARRQAKKIENTGSKTESSSESYKARVARRERKAAKTLGVTVVAFMISWL PYSIDSLIDAFMGFITPACIYEICCWCAYYNSAMNPLIYALFYPWFRKAIKVIVTGQVLKNSSATMNLFSEHI AVGTKFRIPLKLPSEMSFKSSKTMNEQINCSSNKQINVFQSCDV The following DNA sequence nGPCR-seq53 <SEQ ID NO.", "57> was identified in H. sapiens: TTTGTGGCAAGGAGACCCTGATCCCGGTCTTCCTGATCCTTTTCATTGCCCTGGTCGGGCTGGTAGGAAACGG GTTTGTGCTCTGGCTCCTGGGCTTCCGCATGCGCAGGAACGCCTTCTCTGTCTACGTCCTCAGCCTGGCCGGG GCCGACTTCCTCTTCCTCTGCTTCCAGATTATAAATTGCCTGGTGTACCTCAGTAACTTCTTCTGTTCCATCT CCATCAATTTCCCTAGCTTCTTCACCACTGTGATGACCTGTGCCTACCTTGCAGGCCTGAGCATGCTGAGCAC CGTCAGCACCGAGCGCTGCCTGTCCGTCCTGTGGCCCATCTGGTATCGCTGCCGCCGCCCCAGACACCTGTCA GCGGTCGTGTGTGTCCTGCTCTGGGCCCTGTCCCTACTGCTGAGCATCTTGGAAGGGAAGTTCTGTGGCTTCT TATTTAGTGATGGTGACTCTGGTTGGTGTCAGACATTTGATTTCATCACTGCAGCGTGGCTGATTTTTTTATT CATGGTTCTCTGTGGGTCCAGTCTGGCCCTGCTGGTCAGGATCCTCTGTGGCTCCAGGGGTCTGCCACTGACC AGGCTGTACCTGACCATCCTGCTCACAGTGCTGGTGTCCCTCCTCTGCGGCCTGCCCTTTGGCATTCAGTGGT TCCTAATATTATGGATCTGGAAGGATTCTGATGTCTTATTTTGTCATATTCATCCAGTTTCAGTTGTCCTGTC ATCTCTTAACAGCAGTGCCAACCCCATCATTTACTTCTTCGTGGGCTCTTTTAGGAAGCAGTGGCGGSTGCAG CACCCGATCCTCAAGCTGGCTCTCCAGAGGGCTCTGCAGGACATTGCTGAGGTGGATCACAGTGAAGGATGCT TCCGTCAGGGCACCCGGAGATTCAAAGAAGCATTCTGGTGTAGGGATGGACCCCTCTACTTCCATCATATATA TGTGGCTTTGAGAGGCAACTTTGCCCC The following amino acid sequence <SEQ ID NO.", "58> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "57: CGKETLIPVFLILFIALVGLVGNGFVLWLLGFRMRRNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSIS INFPSFFTTVMTCAYLAGLSMLSTVSTERCLSVLWPIWYRCRRPRHLSAVVCVLLWALSLLLSILEGKFCGFL FSDGDSGWCQTFDFITAAWLIFLFMVLCGSSLALLVRILCGSRGLPLTRLYLTILLTVLVSLLCGLPFGIQWF LILWIWKDSDVLFCHIHPVSVVLSSLNSSANPIIYFFVGSFRKQWRXQHPILKLALQRALQDIAEVDHSEGCF RQGTRRFKEAFWCRDGPLYFHHIYVALRGNFA The following DNA sequence nGPCR-seq54 <SEQ ID NO.", "59> was identified in H. sapiens: CTTTGCATCTCACTGTTGAGCAGACAGCCTGCTGAAAGTTGTCGCTGACCACCACATATAGTAACAGGTTACC AAAGGTGTTCAGAGCAGCATAATGGTCTAGAAACGATGTAAGCTTCATGGATCTGATTCTCAATGGAACAACT GATTGAAAGCAGGCTGAGATTCGATCCTGAATGACCCTCAAGATATGGAAGGGTAAAAAACATACGTAAAATG CAAGGAGTAGCAGAATGGTTAGCCTTCGTGCTTTCTGCTTAAGGCAGCTGTCAGTTTGCAGTCCATGGGTCAA AGTGTGGATAATCGTGGTATAGCAAAGTGTCACTATCACCAAGGGGAGGCAGAAAGTACTTGCAGTCAAAATC AGGTTGTACCACTTAATAGTATTGAGTTCATCCGAACTGGTGAGGTCGAGACAGGCTGATCTGTTGGTCCTGT TGGTTGATGTGATCAAGAAGGTCATCGGAATGACAGCTACCAGTGAAATGATCCACACCACAGCACAGGCTAC AACTGCACATCGAGTTTTGTGAATGGAAAAGCAGCTCATTGGGTGAATGATCACACAGTAGCGGAAG The following amino acid sequence <SEQ ID NO.", "60> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "59: FRYCVIIHPMSCFSIHKTRCAVVACAVVWIISLVAVIPMTFLITSTNRTNRSACLDLTSSDELNTIKWYNLIL TASTFCLPLVIVTLCYTTIIHTLTHGLQTDSCLKQKARRLTILLLLAFYVCFLPFHILRVIQDRISACFQSVV PLRIRSMKLTSFLDHYAALNTFGNLLLYVVVSDNFQQAVCSTVRCK The following DNA sequence nGPCR-seq55 <SEQ ID NO.", "61> was identified in H. sapiens, where the underlined ATG identifies a probable start codon: GGGAGGGCTCGTAGACACACTAACCCTACCCTTTCTGTTTCTTCCTCATCTTTCCTTTCCATCTGTTTCTCAT GGTCTCCTGTCTGTCTCTCTCTCTCTCCCCTCTTTCTCTCTCCTCGCTCTTTCTCATCCCCTCCATTTCTGTG TCAATCTCAATCCATTTATATCGGTGGCCACTTTTCTATCTCTTTGTTCTATCTCTCTCTCTCTCTCTTTCCC ACTTTGTCTCTGCACGCCTGTTGTGTTTTTCTGCCTGTCTCTCTCTTGCCCTCATCTCTCTGTCTCTCTCTTG CCCTCATCTCTCTGTCTCTCTGTGTCTGTGTCTCCCCCGCTCATTCCCATTTGCAGGTGCAATGTAGCAGGAC AACTCATGGAGCCCCCCCGGGCCCATCGAGTACCGGACTGGCTGACCCCCTAGGGTTGGCAGTAGCCCCTGAC CCTCAGTATGGCCAACACTACCGGAGAGCCTGAGGAGGTGAGCGGCGCTCTGTCCCCACCGTCCGCATCAGCT TATGTGAAGCTGGTACTGCTGGGACTGATTATGTGCGTGAGCCTGGCGGGTAACGCCATCTTGTCCCTGCTGG TGCTCAAGGAGCGGGCCCTGCACAAGGCTCCTTACTACTTCCTGCTGGACCTGTGCCTGGCCGATGGCATACG CTCTGCCGTCTGCTTCCCCTTTGTGCTGGCTTCTGTGCGCCACGGCTCTTCATGGACCTTCAGTGCACTCAGC TGCAAGATTGTGGCCTTTATGGCCGTGCTCTTTTGCTTCCATGCGGCCTTCATGCTGTTCTGCATCAGCGTCA CCCGCTACATGGCCATCGCCCACCACCGCTTCTACGCCAAGCGCATGACACTCTGGACATGCGCGGCTG The following amino acid sequence <SEQ ID NO.", "62> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "61: MANTTGEPEEVSGALSPPSASAYVKLVLLGLIMCVSLAGNAILSLLVLKERALHKAPYYFLLDLCLADGIRSA VCFPFVLASVRHGSSWTFSALSCKIVAFMAVLFCFHAAFMLFCISVTRYMAIAHHRFYAKRMTLWTCAAE The following DNA sequence nGPCR-seq56 <SEQ ID NO.", "63> was identified in H. sapiens: AAAAATTGCTGTACTGAACTATTGAATGGAACTTGGAAATAAAGTCCCTTCCAAAATAACTATTCTTCAACAG AGAGTAATAGGTAAATGTTTTAGAAGTGAGAGGACTCAAATTGCCAATGATTTACTCTTTTATTTTTCCTCCT AGGTTTCTGGGATAAGTATGTGCAAATAAAAAATAAACATGAGAAGGAACTGTAACCTGATTATGGATTTGGG AAAAAGATAAATCAACACACAAAGGGAAAAGTAAACTGATTGACAGCCCTCAGGAATGATGCCCTTTTGCCAC AATATAATTAATATTTCCTGTGTGAAAAACAACTGGTCAAATGATGTCCGTGCTTCCCTGTACAGTTTAATGG TGCTCATAATTCTGACCACACTCGTTGGCAATCTGATAGTTATTGTTTCTATATCACACTTCAAACAACTTCA TACCCCAACAAATTGGCTCATTCATTCCATGGCCACTGTGGACTTTCTTCTGGGGTGTCTGGTCATGCCTTAC AGTATGGTGAGATCTGCTGAGCACTGTTGGTATTTTGGAGAAGTCTTCTGTAAAATTCACACAAGCACCGACA TTATGCTGAGCTCAGCCTCCATTTTCCATTTGTCTTTCATCTCCATTGACCGCTACTATGCTGTGTGTGATCC ACTGAGATATAAAGCCAAGATGAATATCTTGGTTATTTGTGTGATGATCTTCATTAGTTGGAGTGTCCCTGCT GTTTTTGCATTTGGAATGATCTTTCTGGAGCTAAACTTCAAAGGCGCTGAAGAGATATATTACAAACATGTTC ACTGCAGAGGAGGTTGCTCTGTCTTCTTTAGCAAAATATCTGGGGTACTGACCTTTATGACTTCTTTTTATAT ACCTGGATCTATTATGTTATGTGTCTATTACAGAATATATCTTATCGCTAAAGAACAGGCAAGATTAATTAGT GATGCCAATCAGA The following amino acid sequence <SEQ ID NO.", "64> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "63: REKTDQPSGMMPFCRNIINISCVKNNWSNDVRASLYSLMVLIILTTLVGNLIVIVSISHFKQLHTPTNWLIHS MATVDFLLGCLVMPYSMVRSAEHCWYFGEVFCKIHTSTDIMLSSASIFHLSFISIDRYYAVCDPLRYKAKMNI LVICVMIFISWSVPAVFAFGMIFLELNFKGAEEIYYKHVHCRGGCSVFFSKISGVLTFMTSFYIPGSIMLCVY YRIYLIAKEQARLISDANQ The following DNA sequence nGPCR-seq57 <SEQ ID NO.", "65> was identified in H. sapiens: AACAGTCCCGGGTGGAACCTGGGCATGTATATTTTGATTGTTTTATGCATACTCCTAGTGAAGAACCAATGTC TTGCTCAGATAGAAGCAAGATACTCAGACTTAGTTTCTCTGTAGCTCCTGCTTTTTATTATTCCTGGTTGGAT TGCACCACTACTCAGTTTCTATTTTATAATACTGATTATAAAACATGGGAGGGAAATAACTTTGTATTGGTTT TTATGGATAATTTATTATGTGTCCTAGACTCTGGCCTTGTCAAAAGAAGGACGTAAGAAGGCACGATGTATTA TACTTGGGAATGATAGAAGAGACTGACCTGGTATTTCCACCCGGAAGAGGGAAAGGATTTTAACTACAAATAC AGGAATCCAGCAGATGGCATCAGAGAACACTATAAAAAAGAAACGATTTGCAACAGCCACCTCTCTTCCAAAA CAATTCCTTACTTCTGTGGTCTGCAAGGCGGTTTTTTGAATGGAACAGAACATAGTAATATAGGAAAACACAA TGATGAGAAAAGCCAGCAAGTTCACACCTGTTGGGGAAAAGCACACTTTTAACATCTCAGGCGTAAAAGTCAA CAGTAAAATTACTGTGGTACAGGTTGAGTATCCCTTACCCAAAATGTTTGAAACCAGAAATGTTTTGGATTTC GGATTTCGGAATATTTACACATTCATAATGATATATCTTGGAAATGGTTCCCAAGTCTAAACACAAAATTTAT TTATGTTTCATATACACCTTATACACATAGTCTGAAAGTAATTTTGTACAATATTTTAAATAATTTTGGGCAT GAAACAAAGTTTGCATACATTGAACCATCAGACAGCAAAAGCTTCAGGTGTGGAATTTTCCACTTGTGGCATC ATGTTGATGCTCAAAAAGTTCCATATTTTAGAGCATTTCAAATTTTGGATTTTCAAATTACAAATGCTTAACC TGTACTTAGATGTTAAATACAGTGCCTCTTCCACGGGCACTTTCAGGAAGCATTCTTTTATATAAGCCC The following amino acid sequence <SEQ ID NO.", "66> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "65: YIKECFLKVPVEEALYLTSKYRLSICNLKIQNLKCSKIWNFLSINMMPQVENSTPEAFAVWFNVCKLCFMPKI INIVQNYFQTMCIRCININKFCVTWEPFPRYIIMNVIFRNPKSKTFLVSNILGKGYSTCTTVILLLTFTPEML KVCFSPTGVNLLAFLIIVFSYITMFCSIQKTALQTTEVRNCFGREVAVANRFFFIVFSDAICWIPVFVVKILS LFRVEIPGQSLLSFPSIIHRAFLRPSFDKARVDTIIHKNQYKVISLPCFIISIIKKLSSGAIQPGIIKSRSYR ETKSEYLASIARHWFFTRSMHKTIKIYMPRFHPGL The following DNA sequence nGPCR-seq58 <SEQ ID NO.", "67> was identified in H. sapiens: ACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACTCCTACC CCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCAATGGGTTGATGGC GTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCTCAGCCTGGCCCTC TCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGACACTGGCCGCTGG GGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCTTCCTGCTGGCCGC CCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCCAGTCCGCCTGCCC CTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTCTTCCCCGAGGCTG CCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGCTGAGGATGCTGGA GGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGCCACAGCCTGTCGC ACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACCATTCTGTCAGCCT ATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGGACGTCTACTCTGG CTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCTCAGCCCCTTCCTC TGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCGGCAGCTCTCTGCG AGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTCCAACTCTGCCAGA GCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCACACTCCAGCCACGA TCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACAGCCCAGCCACAGC TGAACCTCATGGCCCAGCCACAGTCAGATTCTGTGGCCCAGCCACAGGCAGACACTAACGTCCAGACCCCTGC ACCTGCTGCC The following amino acid sequence <SEQ ID NO.", "68> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "67: TTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLL LSLALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHW YPGHRPVRLPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFL LLLVCHVLTQATACRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFLWDVYSGYLLW EALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLP EPMAEAQSQMDPVAQPQVNPTLQPRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADT NVQTPAPAA The following DNA sequence nGPCR-seq59 <SEQ ID NO.", "69> was identified in H. sapiens: TACAGGCCTGAGCATGCTGGGCTCCATCAGCACCAAGCACTGCCTGTCCATCCTGTGGCCCATCTAGTACCGC TGCCACCACCCCACACACCTGTCAGCAGTCGTGTGTCCTGCTCTGGGCCCTGTCCCTGCTGCAGAGCATCCTG GAATGGATGTTCTGTGGCTTCCTGTCTAGTGGTGCTGATTCTGTTTGGTGTGAAACATCAGATTTCATCACAG TCACATGGCTGATTTTTTTATGTGTGGTTCTCTGCGGGTCCAGCCCGGTTCTGCTGGTCAGGATCCTTTGTGG ATCCCGGAAGATGCCCTTGACCAGGCTGTACATGACCATCCTGCTCAGAGTGCTGGTCTTCCTCCTCTGTGAC CTGCCCTTTGGCATTCAGTGATTCCTATTTTTCTGGATCCACGTGGATTTGTCACGTTCGTCTAGTTTCCATT TTCCTGTCCACTCTTAACAGCAGTGCCAACCCCATTATTTACTTCTTCATGGGCTCCTTTAGGCAGCTTCAAA ACAGGAAGACTCTCTAGCTGGTTCTCCAGAGGGCTCTGCAGGACACGCCTGAGGTGGAAGAAGGCAGATGGCG GCTTTCTGAGGAAACCCTGGAGCTGTCATGAAGCAGATTGGGGCCATGAGGAAGAGCCTCTGCCCTGTCAGTC AG The following amino acid sequence <SEQ ID NO.", "70> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "69: YRPEHAGLHQHQALPVHPVAHLVPLPPPHTPVSSRVSCSGPCPCCRASWNGCSVASCLVVLILFGVKHQISSQ SHGFFYVWFSAGPARFCWSGSFVDPGRCPPGCTPSCSECWSSSSVTCPLAFSDSYFSGSTWICHVRLVSIFLS TLNSSANPIIYFFMGSFRQLQNRKTLLVLQRALQDTPEVEEGRWRLSEETLELSSRLGPGRASALSV The following DNA sequence nGPCR-seq60 <SEQ ID NO.", "71> was identified in H. sapiens: ATGCCGAAGGCAGGCCGCAGAAGAGAAGAGGAGGACGGTGAGGAGGATGAGCCCAGGGAAGCCCCGGGGTGGG GGCCGCTGGGGGCCTCGCTCCACCCGCAGCAGCAGCATAAGGCTGGCCCCACACATGGTGCAACACAGCAGAG CCAGCAGCACCGCTGCCACCAGCCACAGCGTCCGGCACAAGTGGCGGCTGGGCTCCCCGAAGAACTGGGTGCA GGCGCCGCTGAGCAGCAGGTGCAGCAGCAGGCAGAGGGCCCAGGTGAGGGCGCACACACAGGTGGTCAGGTGG CGTGGGCGGCGGCACGAGTACCAGGCTGGGAAGAGGGCGGCCAGGCACTGCTCCACGCTGACGGCCGCCAGGA GACTCAGGCCCACGATGTAGCAGAAGAAGCGCAGCGTTGCCAGGCTGGTCTGCACGAAGCCCGGGAAGTCCAG CCGGCCTTGCAGCAAGTCGGGGACGATGGCCACCATGTGGCAGCCAAGGAAGATGAGATCCGCGCAGGCCACG TCCAGGAGGTAGATGGCGAAAGGGTTTCTGTAGACATTGGAGCTGAGC The following amino acid sequence <SEQ ID NO.", "72> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "71: LSSNVYRNPFAIYLLDVACADLIFLGCHMVAIVPDLLQGRLDFPGFVQTSLATLRFFCYIVGLSLLA AVSVEQCLAALFPAWYSCRRPRHLTTCVCALTWALCLLLHLTTCVCALTWALCLLLHLLLSGACTLL LSGACTQFFGEPSRHLCRTLWLVAAVLLALLCCTMCGASLMLLLRVERGPQRPPPRGFPGLILLTVL LFSSAACLRH The following DNA sequence nGPCR-1 <SEQ ID NO.", "73> was identified in H. sapiens: ATGGAATCATCTTTCTCATTTGGAGTGATCCTTGCTGTCCTGGCCTCCCTCATCATTGCTACTAACACACTAG TGGCTGTGGCTGTGCTGCTGTTGATCCACAAGAATGATGGTGTCAGTCTCTGCTTCACCTTGAATCTGGCTGT GGCTGACACCTTGATTGGTGTGGCCATCTCTGGCCTACTCACAGACCAGCTCTCCAGCCCTTCTCGGCCCACA CAGAAGACCCTGTGCAGCCTGCGGATGGCATTTGTCACTTCCTCCGCAGCTGCCTCTGTCCTCACGGTCATGC TGATCACCTTTGACAGGTACCTTGCCATCAAGCAGCCCTTCCGCTACTTGAAGATCATGAGTGGGTTCGTGGC CGGGGCCTGCATTGCCGGGCTGTGGTTAGTGTCTTACCTCATTGGCTTCCTCCCACTCGGAATCCCCATGTTC CAGCAGACTGCCTACAAAGGGCAGTGCAGCTTCTTTGCTGTATTTCACCCTCACTTCGTGCTGACCCTCTCCT GCGTTGGCTTCTTCCCAGCCATGCTCCTCTTTGTCTTCTTCTACTGCGACATGCTCAAGATTGCCTCCATGCA CAGCCAGCAGATTCGAAAGATGGAACATGCAGGAGCCATGGCTGGAGGTTATCGATCCCCACGGACTCCCAGC GACTTCAAAGCTCTCCGTACTGTGTCTGTTCTCATTGGGAGCTTTGCTCTATCCTGGACCCCCTTCCTTATCA CTGGCATTGTGCAGGTGGCCTGCCAGGAGTGTCACCTCTACCTAGTGCTGGAACGGTACCTGTGGCTGCTCGG CGTGGGCAACTCCCTGCTCAACCCACTCATCTATGCCTATTGGCAGAAGGAGGTGCGACTGCAGCTCTACCAC ATGGCCCTAGGAGTGAAGAAGGTGCTCACCTCATTCCTCCTCTTTCTCTCGGCCAGGAATTGTGGCCCAGAGA GGCCCAGGGAAAGTTCCTGTCACATCGTCACTATCTCCAGCTCAGAGTTTGATGGCTAA The following amino acid sequence <SEQ ID NO.", "74> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "73: MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAVADTLIGVAISGLLTDQLSSPSRPT QKTLCSLRMAFVTSSAAASVLTVMLITFDRYLAIKQPFRYLKIMSGFVAGACIAGLWLVSYLIGFLPLGIPMF QQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHAGAMAGGYRSPRTPS DFKALRTVSVLIGSFALSWTPFLITGIVQVACQECHLYLVLERYLWLLGVGNSLLNPLIYAYWQKEVRLQLY HMALGVKKVLTSFLLFLSARNCGPERPRESSCHIVTISSSEFDG The following DNA sequence TL-GPCR-seq5 <SEQ ID NO.", "75> was identified in H. sapiens.", "AACTGGAAGGGCAGCCGTCTGCCGCCCACGAACACCTTCTCAAGCACTTTGAGTGACCACGGCTTGCAAGCTG GTGGCTGGCCCCCCGAGTCCCGGGCTCTGAGGCACGGCCGTCGACTTAAGCGTTGCATCCTGTTACCTGGAGA CCCTCTGAGCTCTCACCTGCTACTTCTGCCGCTGCTTCTGCACAGAGCCCGGGCGAGGACCCCTCCAGGATGC AGGTCCCGAACAGCACCGGCCCGGACAACGCGACGCTGCAGATGCTGCGGAACCCGGCGATCGCGGTGGCCCT GCCCGTGGTGTACTCGCTGGTGGCGGCGGTCAGCATCCCGGGCAACCTCTTCTCTCTGTGGGTGCTGTGCCGG CGCATGGGGCCCAGATCCCCGTCGGTCATCTTCATGATCAACCTGAGCGTCACGGACCTGATGCTGGCCAGCG TGTTGCCTTTCCAAATCTACTACCATTGCAACCGCCACCACTGGGTATTCGGGGTGCTGCTTTGCAACGTGGT GACCGTGGCCTTTTACGCAAACATGTATTCCAGCATCCTCACCATGACCTGTATCAGCGTGGAGCGCTTCCTG GGGGTCCTGTACCCGCTCAGCTCCAAGCGCTGGCGCCGCCGTCGTTACGCGGTGGCCGCGTGTGCAGGGACCT GGCTGCTGCTCCTGACCGCCCTGTCCCCGCTGGCGCGCACCGATCTCACCTACCCGGTGCACGCCCTGGGCAT CATCACCTGCTTCGACGTCCTCAAGTGGACGATGCTCCCCAGCGTGGCCATGTGGGCCGTGTTCCTCTTCACC ATCTTCATCCTGCTGTTCCTCATCCCGTTCGTGATCACCGTGGCTTGTTACACGGCCACCATCCTCAAGCTGT TGCGCACGGAGGAGGCGCACGGCCGGGAGCAGCGGAGGCGCGCGGTGGGCCTGGCCGCGGTGGTCTTGCTGGC CTTTGTCACCTGCTTCGCCCCCAACAACTTCGTGCTCCTGGCGCACATCGTGAGCCGCCTGTTCTACGGCAAG AGCTACTACCACGTGTACAAGCTCACGCTGTGTCTCAGCTGCCTCAACAACTGTCTGGACCCGTTTGTTTATT ACTTTGCGTCCCGGGAATTCCAGCTGCGCCTGCGGGAATATTTGGGCTGCCGCCGGGTGCCCAGAGACACCCT GGACACGCGCCGCGAGAGCCTCTTCTCCGCCAGGACCACGTCCGTGCGCTCCGAGGCCGGTGCGCACCCTGAA GGGATGGAGGGAGCCACCAGGCCCGGCCTCCAGAGGCAGGAGAGTGTGTTCTGAGTCCCGGGGGCGCAGCTTG GAGAGCCGGGGGCGCAGCTTGGAGGATCCAGGGGCGCATGGAGAGGCCACGGTGCCAGAGGTTCAGGGAGAAC AGCTGCGTTGCTCCCAGGCACTGCAGAGGCCCGGTGGGGAAGGGTCTCCAGGCTTTATTCCTCCCAGGCACTG CAGAGGCACCGGTGAGGAAGGGTCTCCAGGCTTCACTCAGGGTAGAGAAACAAGCAAAGCCCAGCAGCGCACA GGGTGCTTGTTATCCTGCAGAGGGTGCCTCTGCCTCTCTGTGTCAGGGGACAGCTTGTGTCACCACGCCCGGC TAATTTTTGTATTTTTTTTAGTAGAGCTGGGCTGTCACCCCCGAGCTCCTTAGACACTCCTCACACCTGTCCA TACCCGAGGATGGATATTCAACCAGCCCCACCGCCTACCCGACTCGGTTTCTGGATATCCTCTGTGGGCGAAC TGCGAGCCCCATTCCCAGCTCTTCTCCCTGCTGACATCGTCCCTTAGCACACCTGTCCATACCCGAGGATGGA TATTCAACCAGCCCCACCGCCTACCCGACTCGGTTTCTGGATATCCTCTGTGGGCGAACTGCGAGCCCCATTC CCAGCTCTTCTCCCTGCTGACATCGTCCCTTAGTTGTGGTTCTGGCCTTCTCCATTCTCCTCCAGGGGTTCTG GTCTCCGTAGCCCGGTGCACGCCGAAATTTCTGTTTATTTCACTCAGGGGCACTGTGGTTGCTGTGGTTGGAA TTCTTCTTTCAGAGGAGCGCCTGGGGCTCCTGCAAGTCAGCTACTCTCCGTGCCCACTTCCCCTCACACACAC ACCCCCCTCGTGCCGAATTC The following amino acid sequence <SEQ ID NO.", "76> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "75.MQVPNSTGPDNATLQMLRNPAIAVALPVVYSLVAAVSIPGNLFSLWVLCRRMGPRSPSVIFMINLSVTDLMLA SVLPFQIYYHCNRHHWVFGVLLCNVVTVAFYANMYSSILTMTCISVERFLGVLYPLSSKRWRRRRYAVAACAG TWLLLLTALSPLARTDLTYPVHALGIITCFDVLKWTMLPSVAMWAVFLFTIFILLFLIPFVITVACYTATILK LLRTEEAHGREQRRRAVGLAAVVLLAFVTCFAPNNFVLLAHIVSRLFYGKSYYHVYKLTLCLSCLNNCLDPFV YYFASREFQLRLREYLGCRRVPRDTLDTRRESLFSARTTSVRSEAGAHPEGMEGATRPGLQRQESVF The following DNA sequence nGPCR-9 <SEQ ID NO.", "77> was identified in H. sapiens: ATGGAGTCGGGGCTGCTGCGGCCGGCGCCGGTGAGCGAGGTCATCGTCCTGCATTACAACTACACCGGCAAGC TCCGCGGTGCGCGCTACCAGCCGGGTGCCGGCCTGCGCGCCGACGCCGTGGTGTGCCTGGCGGTGTGCGCCTT CATCGTGCTAGAGAATCTAGCCGTGTTGTTGGTGCTCGGACGCCACCCGCGCTTCCACGCTCCCATGTTCCTG CTCCTGGGCAGCCTCACGTTGTCGGATCTGCTGGCAGGCGCCGCCTACGCCGCCAACATCCTACTGTCGGGGC CGCTCACGCTGAAACTGTCCCCCGCGCTCTGGTTCGCACGGGAGGGAGGCGTCTTCGTGGCACTCACTGCGTC CGTGCTGAGCCTCCTGGCCATCGCGCTGGAGCGCAGCCTCACCATGGCGCGCAGGGGGCCCGCGCCCGTCTCC AGTCGGGGGCGCACGCTGGCGATGGCAGCCGCGGCCTGGGGCGTGTCGCTGCTCCTCGGGCTCCTGCCAGCGC TGGGCTGGAATTGCCTGGGTCGCCTGGACGCTTGCTCCACTGTCTTGCCGCTCTACGCCAAGGCCTACGTGCT CTTCTGCGTGCTCGCCTTCGTGGGCATCCTGGCCGCTATCTGTGCACTCTACGCGCGCATCTACTGCCAGGTA CGCGCCAACGCGCGGCGCCTGCCGGCACGGCCCGGGACTGCGGGGACCACCTCGACCCGGGCGCGTCGCAAGC CGCGCTCGCTGGCCTTGCTGCGCACGCTCAGCGTGGTGCTCCTGGCCTTTGTGGCATGTTGGGGCCCCCTCTT CCTGCTGCTGTTGCTCGACGTGGCGTGCCCGGCGCGCACCTGTCCTGTACTCCTGCAGGCCGATCCCTTCCTG GGACTGGCCATGGCCAACTCACTTCTGAACCCCATCATCTACACGCTCACCAACCGCGACCTGCGCCACGCGC TCCTGCGCCTGGTCTGCTGCGGACGCCACTCCTGCGGCAGAGACCCGAGTGGCTCCCAGCAGTCGGCGAGCGC GGCTGAGGCTTCCGGGGGCCTGCGCCGCTGCCTGCCCCCGGGCCTTGATGGGAGCTTCAGCGGCTCGGAGCGC TCATCGCCCCAGCGCGACGGGCTGGACACCAGCGGCTCCACAGGCAGCCCCGGTGCACCCACAGCCGCCCGGA CTCTGGTATCAGAACCGGCTGCAGACTGA The following amino acid sequence <SEQ ID NO.", "78> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "77: MESGLLRPAPVSEVIVLHYNYTGKLRGARYQPGAGLRADAVVCLAVCAFIVLENLAVLLVLGRHPRFHAPMFL LLGSLTLSDLLAGAAYAANILLSGPLTLKLSPALWFAREGGVFVALTASVLSLLAIALERSLTMARRGPAPVS SRGRTLAMAAAAWGVSLLLGLLPALGWNCLGRLDACSTVLPLYAKAYVLFCVLAFVGILAAICALYARIYCQV RANARRLPARPGTAGTTSTRARRKPRSLALLRTLSVVLLAFVACWGPLFLLLLLDVACPARTCPVLLQADPFL GLAMANSLLNPIIYTLTNRDLRHALLRLVCCGRHSCGRDPSGSQQSASAAEASGGLRRCLPPGLDGSFSGSER SSPQRDGLDTSGSTGSPGAPTAARTLVSEPAAD The following DNA sequence nGPcR-11 <SEQ ID NO.", "79> was identified in H. sapiens: ATGTACAACGGGTCGTGCTGCCGCATCGAGGGGGACACCATCTCCCAGGTGATGCCGCCGCTGCTCATTGTGG CCTTTGTGCTGGGCGCACTAGGCAATGGGGTCGCCCTGTGTGGTTTCTGCTTCCACATGAAGACCTGGAAGCC CAGCACTGTTTACCTTTTCAATTTGGCCGTGGCTGATTTCCTCCTTATGATCTGCCTGCCTTTTCGGACAGAC TATTACCTCAGACGTAGACACTGGGCTTTTGGGGACATTCCCTGCCGAGTGGGGCTCTTCACGTTGGCCATGA ACAGGGCCGGGAGCATCGTGTTCCTTACGGTGGTGGCTGCGGACAGGTATTTCAAAGTGGTCCACCCCCACCA CGCGGTGAACACTATCTCCACCCGGGTGGCGGCTGGCATCGTCTGCACCCTGTGGGCCCTGGTCATCCTGGGA ACAGTGTATCTTTTGCTGGAGAACCATCTCTGCGTGCAAGAGACGGCCGTCTCCTGTGAGAGCTTCATCATGG AGTCGGCCAATGGCTGGCATGACATCATGTTCCAGCTGGAGTTCTTTATGCCCCTCGGCATCATCTTATTTTG CTCCTTCAAGATTGTTTGGAGCCTGAGGCGGAGGCAGCAGCTGGCCAGACAGGCTCGGATGAAGAAGGCGACC CGGTTCATCATGGTGGTGGCAATTGTGTTCATCACATGCTACCTGCCCAGCGTGTCTGCTAGACTCTATTTCC TCTGGACGGTGCCCTCGAGTGCCTGCGATCCCTCTGTCCATGGGGCCCTGCACATAACCCTCAGCTTCACCTA CATGAACAGCATGCTGGATCCCCTGGTGTATTATTTTTCAAGCCCCTCCTTTCCCAAATTCTACAACAAGCTC AAAATCTGCAGTCTGAAACCCAAGCAGCCAGGACACTCAAAAACACAAAGGCCGGAAGAGATGCCAATTTCGA ACCTCGGTCGCAGGAGTTGCATCAGTGTGGCAAATAGTTTCCAAAGCCAGTCTGATGGGCAATGGGATCCCCA CATTGTTGAGTGGCACTGA The following amino acid sequence <SEQ ID NO.", "80> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "79: MYNGSCCRIEGDTISQVMPPLLIVAFVLGALGNGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFRTD YYLRRRHWAFGDIPCRVGLFTLAMNRAGSIVFLTVVAADRYFKVVHPHHAVNTISTRVAAGIVCTLWALVILG TVYLLLENHLCVQETAVSCESFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQARMKKAT RFIMVVAIVFITCYLPSVSARLYFLWTVPSSACDPSVHGALHITLSFTYMNSMLDPLVYYFSSPSFPKFYNKL KICSLKPKQPGHSKTQRPEEMPISNLGRRSCISVANSFQSQSDGQWDPHIVEWH The following DNA sequence nGPCR-16 <SEQ ID NO.", "81> was identified in H. sapiens: ATGACAGGTGACTTCCCAAGTATGCCTGGCCACAATACCTCCAGGAATTCCTCTTGCGATCCTATAGACACCC CACTTAATCAGCCTCTACTTCATAGTGCTTATTGGCGGGCTGGTGGGTGTCATTTCCATTCTTTTCCTCCTGG TGAAAATGAACACCCGGTCAGTGACCACCATGGCGGTCATTAACTTGGTGGTGGTCCACAGCGTTTTTCTGCT GACAGTGCCATTTCGCTTGACCTACCTCATCAAGAAGACTTGGATGTTTGGGCTGCCCTTCTGCAAATTTGTG AGTGCCATGCTGCACATCCACATGTACCTCACGTTCCTATTCTATGTGGTGATCCTGGTCACCAGATACCTCA TCTTCTTCAAGTGCAAAGACAAAGTGGAATTCTACAGAAAACTGCATGCTGTGGCTGCCAGTGCTGGCATGTG GACGCTGGTGATTGTCATTGTGGTACCCCTGGTTGTCTCCCGGTATGGAATCCATGAGGAATACAATGAGGAG CACTGTTTTAAATTTCACAAAGAGCTTGCTTACACATATGTGAAAATCATCAACTATATGATAGTCATTTTTG TCATAGCCGTTGCTGTGATTCTGTTGGTCTTCCAGGTCTTCATCATTATGTTGATGGTGCAGAAGCTACGCCA CTCTTTACTATCCCACCAGGAGTTCTGGGCTCAGCTGAAAAACCTATTTTTTATAGGGGTCATCCTTGTTTGT TTCCTTCCCTACCAGTTCTTTAGGATCTATTACTTGAATGTTGTGACGCATTCCAATGCCTGTAACAGCAAGG TTGCATTTTATAACGAAATCTTCTTGAGTGTAACAGCAATTAGCTGCTATGATTTGCTTCTCTTTGTCTTTGG GGGAAGCCATTGGTTTAAGCAAAAGATAATTGGCTTATGGAATTGTGTTTTGTGCCGTTAGCCACAAACTACA GTATTCATATTTGCTTCCTTTATATTGGGAATAAAAATGGGTATAGGGGAGGTAAGAATGGTATTTCATTACT TGATCAAAACCATGCCTTGATGTACCCAAAACAAAAGGACTATAAAATGCAAGAGCCCTCATTGTAGTCCTTA TGGGATCCCTCCCATCTCTGAGTGATGGCCGTACAAAGACCAGTGTTGTTGAATCCACCTGGAGTTGCAATAT TACATTATTTTCCAGTACAGAATGTCTGTGTGGCCCATGAAAGCAACATAGGTTTTAAGAGTTTTAGAGTTTC ATTAGCTCATTCTAAGTTCCTCTGTTTGAAGCATGGTCTCTTAGGTTTTGGACTGAACTCAGACCTTTAGTTC TTTTCATCCCACTTCACCTTAGGTAAGTAAATTCTGGCCACCACCCAGCTCCAAAGACACAAACTCTCCTTCG CTAACCAGGTTAGATGTCCCATTCATCTCATGCCCTGATAAAAACTGATAAGGGGAGAGAATAGTTAAAAATT TTTCTAGGGTATCATAACTCTGGTAGGAAGTCATCTGTCTAGAAATCAAGAGAAAAAGAACGTGTGGCCTCCT GTTATAACAAGGGTTTCTAGATTTGTCCTGTGAAAGGTCGTTTAAGGACTTGGGGATCAACTTCCTCAATTAT CACCAATTGCACTGTTGCTCCAAAAATCATTTAAAAGCTTACTGGACATATCTACATAATGGTGAAACTGTAA TTTAGAGACTATCCCTGACTAATGTGCTGGTAGGCATTAAAATGAGTTCCCAAGGGAAGTGATTAAAATTTTT TTCTCTTCTGTTTTTTGAGAGAATTTCTAGATGTCCTGGGCCACAGTTAATTAAGATTTTTAGGGGGGACAGA AAGTTATACTGAAATCTTTAGAGCTCCCTTCCGCCGTTAAAATTATATATATATATATTTAAATTATACCTTA AGTTCTGGGGTACATGTGCAGAATGTGCAGGTTTGTTACATAGGTATACACGTGCCATGGTGGTTTGCGGCAC CTGTCAACCCATCTACATTAGGTATTTCTCCTAATGCTCTCCCTCCCCTAGCCCCCCACCCCTGGACAGGCCC CATTGTGTGATGTTCCCCTCCCTGTGTCCATGTGTTTTCATTGTTCAACTCCCACTTCTAAGTGAGAACATGC GGTGTTTGGTTTTCTGTTCCTGTGTTAGTTTGCTGAGAATGATGGTTTCCAGGTTAAAATTATATATTTTTAA ATAAATGAAAACTGTGTTTTTAAAAGAGGACTTTTGAGAAGTATATAGAAAAACCATTAATTTAGACTCTGTG AGATTAGGTTGCATGAAGAAGGTTTTCTGAATATTTGAAGAGTGGATAAATAAATGTCCCCCAAAGCAATAAA ATCATAATCCTTTAAAATATAGGAAAAATAACTAATGGGAACTAGGCTTAATACTCGGGATGAAATAATCTGT ACAACAAACTCCCATGACACATGTTTACCTATGTAACAAACCTGCACATGTACCCCTGAACTTAAAATAAAAT TTAAAGTATAATAATAAAATAATATGGATTTTCTTT The following amino acid sequence <SEQ ID NO.", "82> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "81: MTGDFPSMPGHNTSRNSSCDPIVTPHLISLYFIVLIGGLVGVISILFLLVKMNTRSVTTMAVINLVVVHSVFL LTVPFRLTYLIKKTWMFGLPFCKFVSAMLHIHMYLTFLFYVVILVTRYLIFFKCKDKVEFYRKLHAVAASAGM WTLVIVIVVPLVVSRYGIHEEYNEEHCFKFHKELAYTYVKIINYMIVIFVIAVAVILLVFQVFIIMLMVQKLR HSLLSHQEFWAQLKNLFFIGVILVCFLPYQFFRIYYLNVVTHSNACNSKVAFYNEIFLSVTAISCYDLLLFVF GGSHWFKQKIIGLWNCVLCR The following DNA sequence nGPcR-40 <SEQ ID NO.", "83> was identified in H. sapiens: GCAGGAGCACTGAAAATCAGGAACAATCCTGTATTTTTTGTGATAATCAACAAGGACAAAACTTCTCCATATG TAAATAACAGCGTTATGAGCAGCAATTCATCCCTGCTGGTGGCTGTGCAGCTGTGCTACGCGAACGTGAATGG GTCCTGTGTGAAAATCCCCTTCTCGCCGGGATCCCGGGTGATTCTGTACATAGTGTTTGGCTTTGGGGCTGTG CTGGCTGTGTTTGGAAACCTCCTGGTGATGATTTCAATCCTCCATTTCAAGCAGCTGCACTCTCCGACCAATT TTCTCGTTGCCTCTCTGGCCTGCGCTGATTTCTTGGTGGGTGTGACTGTGATGCCCTTCAGCATGGTCAGGAC GGTGGAGAGCTGCTGGTATTTTGGGAGGAGTTTTTGTACTTTCCACACCTGCTGTGATGTGGCATTTTGTTAC TCTTCTCTCTTTCACTTGTGCTTCATCTCCATCGACAGGTACATTGCGGTTACTGACCCCCTGGTCTATCCTA CCAAGTTCACCGTATCTGTGTCAGGAATTTGCATCAGCGTGTCCTGGATCCTGCCCCTCATGTACAGCGGTGC TGTGTTCTACACAGGTGTCTATGACGATGGGCTGGAGGAATTATCTGATGCCCTAAACTGTATAGGAGGTTGT CAGACCGTTGTAAATCAAAACTGGGTGTTGACAGATTTTCTATCCTTCTTTATACCTACCTTTATTATGATAA TTCTGTATGGTAACATATTTCTTGTGGCTAGACGACAGGCGAAAAAGATAGAAAATACTGGTAGCAAGACAGA ATCATCCTCAGAGAGTTACAAAGCCAGAGTGGCCAGGAGAGAGAGAAAAGCAGCTAAAACCCTGGGGGTCACA GTGGTAGCATTTATGATTTCATGGTTACCATATAGCATTGATTCATTAATTGATGCCTTTATGGGCTTTATAA CCCCTGCCTGTATTTATGAGATTTGCTGTTGGTGTGCTTATTATAACTCAGCCATGAATCCTTTGATTTATGC TTTATTTTACCCATGGTTTAGGAAAGCAATAAAAGTTATTGTAACTGGTCAGGTTTTAAAGAACAGTTCAGCA ACCATGAATTTGTTTTCTGAACATATATAA The following amino acid sequence <SEQ ID NO.", "84> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "83: MSSNSSLLVAVQLCYANVNGSCVKIPFSPGSRVILYIVFGFGAVLAVFGNLLVMISILHFKQLHSPTNFLVAS LACADFLVGVTVMPFSMVRTVESCWYFGRSFCTFHTCCDVAFCYSSLFHLCFISIDRYIAVTDPLVYPTKFTV SVSGICISVSWILPLMYSGAVFYTGVYDDGLEELSDALNCIGGCQTVVNQNWVLTDFLSFFIPTFIMIILYGN IFLVARRQAKKIENTGSKTESSSESYKARVARRERKAAKTLGVTVVAFMISWLPYSIDSLIDAFMGFITPACI YEICCWCAYYNSAMNPLIYALFYPWFRKAIKVIVTGQVLKNSSATMNLFSEHI The following DNA sequence nGPCR-54 <SEQ ID NO.", "85> was identified in H. sapiens: ACCATGAATGAGCCACTAGACTATTTAGCAAATGCTTCTGATTTCCCCGATTATGCAGCTGCTTTTGGAAATT GCACTGATGAAAACATCCCACTCAAGATGCACTACCTCCCTGTTATTTATGGCATTATCTTCCTCGTGGGATT TCCAGGCAATGCAGTAGTGATATCCACTTACATTTTCAAAATGAGACCTTGGAAGAGCAGCACCATCATTATG CTGAACCTGGCCTGCACAGATCTGCTGTATCTGACCAGCCTCCCCTTCCTGATTCACTACTATGCCAGTGGCG AAAACTGGATCTTTGGAGATTTCATGTGTAAGTTTATCCGCTTCAGCTTCCATTTCAACCTGTATAGCAGCAT CCTCTTCCTCACCTGTTTCAGCATCTTCCGCTACTGTGTGATCATTCACCCAATGAGCTGCTTTTCCATTCAC AAAACTCGATGTGCAGTTGTAGCCTGTGCTGTGGTGTGGATCATTTCACTGGTAGCTGTCATTCCGATGACCT TCTTGATCACATCAACCAACAGGACCAACAGATCAGCCTGTCTCGACCTCACCAGTTCGGATGAACTCAATAC TATTAAGTGGTACAACCTGATTTTGACTGCAAGTACTTTCTGCCTCCCCTTGGTGATAGTGACACTTTGCTAT ACCACGATTATCCACACTTTGACCCATGGACTGCAAACTGACAGCTGCCTTAAGCAGAAAGCACGAAGGCTAA CCATTCTGCTACTCCTTGCATTTTACGTATGTTTTTTACCCTTCCATATCTTGAGGGTCATTCAGGATCGAAT CTCAGCCTGCTTTCAATCAGTTGTTCCATTGAGAATCAGATCCATGAAGCTTACATCGTTTCTAGACCATTAT GCTGCTCTGAACACCTTTGGTAACCTGTTACTATATGTGGTGGTCAGCGACAACTTTCAGCAGGCTGTCTGCT CAACAGTGAGATGCAAAGTAAGCGGGAACCTTGAGCAAGCAAAGAAAATTAGTTACTCAAACAACCCTTGA The following amino acid sequence <SEQ ID NO.", "86> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "85: MNEPLDYLANASDFPDYAAAFGNCTDENIPLKMHYLPVIYGIIFLVGFPGNAVVISTYIFKMRPWKSSTIIML NLACTDLLYLTSLPFLIHYYASGENWIFGDFMCKFIRFSFHFNLYSSILFLTCFSIFRYCVIIHPMSCFSIHK TRCAVVACAVVWIISLVAVIPMTFLITSTNRTNRSACLDLTSSDELNTIKWYNLILTASTFCLPLVIVTLCYT TIIHTLTHGLQTDSCLKQKARRLTILLLLAFYVCFLPFHILRVIQDRISACFQSVVPLRIRSMKLTSFLDHYA ALNTFGNLLLYVVVSDNFQQAVCSTVRCKVSGNLEQAKKISYSN The following DNA sequence nGPCR-56 <SEQ ID NO.", "87> was identified in H. sapiens: AAAAATTGCTGTACTGAACTATTGAATGGAACTTGGAAATAAAGTCCCTTCCAAAATAACTATTCTTCAACAG AGAGTAATAGGTAAATGTTTTAGAAGTGAGAGGACTCAAATTGCCAATGATTTACTCTTTTATTTTTCCTCCT AGGTTTCTGGGATAAGTATGTGCAAATAAAAAATAAACATGAGAAGGAACTGTAACCTGATTATGGATTTGGG AAAAAGATAAATCAACACACAAAGGGAAAAGTAAACTGATTGACAGCCCTCAGGAATGATGCCCTTTTGCCAC AATATAATTAATATTTCCTGTGTGAAAAACAACTGGTCAAATGATGTCCGTGCTTCCCTGTACAGTTTAATGG TGCTCATAATTCTGACCACACTCGTTGGCAATCTGATAGTTATTGTTTCTATATCACACTTCAAACAACTTCA TACCCCAACAAATTGGCTCATTCATTCCATGGCCACTGTGGACTTTCTTCTGGGGTGTCTGGTCATGCCTTAC AGTATGGTGAGATCTGCTGAGCACTGTTGGTATTTTGGAGAAGTCTTCTGTAAAATTCACACAAGCACCGACA TTATGCTGAGCTCAGCCTCCATTTTCCATTTGTCTTTCATCTCCATTGACCGCTACTATGCTGTGTGTGATCC ACTGAGATATAAAGCCAAGATGAATATCTTGGTTATTTGTGTGATGATCTTCATTAGTTGGAGTGTCCCTGCT GTTTTTGCATTTGGAATGATCTTTCTGGAGCTAAACTTCAAAGGCGCTGAAGAGATATATTACAAACATGTTC ACTGCAGAGGAGGTTGCTCTGTCTTCTTTAGCAAAATATCTGGGGTACTGACCTTTATGACTTCTTTTTATAT ACCTGGATCTATTATGTTATGTGTCTATTACAGAATATATCTTATCGCTAAAGAACAGGCAAGATTAATTAGT GATGCCAATCAGAAGCTCCAAATTGGATTGGAAATGAAAAATGGAATTTCACAAAGCAAAGAAAGGAAAGCTG TGAAGACATTGGGGATTGTGATGGGAGTTTTCCTAATATGCTGGTGCCCTTTCTTTATCTGTACAGTCATGGA CCCTTTTCTTCACTACATTATTCCACCTACTTTGAATGATGTA The following amino acid sequence <SEQ ID NO.", "88> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "87: MMPFCHNIINISCVKNNWSNDVRASLYSLMVLIILTTLVGNLIVIVSISHFKQLHTPTNWLIHSMATVDFLLG CLVMPYSMVRSAEHCWYFGEVFCKIHTSTDIMLSSASIFHLSFISIDRYYAVCDPLRYKAKMNILVICVMIFI SWSVPAVFAFGMIFLELNFKGAEEIYYKHVHCRGGCSVFFSKISGVLTFMTSFYIPGSIMLCVYYRIYLIAKE QARLISDANQKLQIGLEMKNGISQSKERKAVKTLGIVMGVFLICWCPFFICTVMDPFLHYIIPPTLNDARGSR ANSA The following DNA sequence nGPCR-56 <SEQ ID NO.", "89> was identified in H. sapiens: GGAATGATGCCCTTTTGCCACAATATAATTAATATTTCCTGTGTGAAAAACAACTGGTCAAATGATGTCCGTG CTTCCCTGTACAGTTTAATGGTGCTCATAATTCTGACCACACTCGTTGGCAATCTGATAGTTATTGTTTCTAT ATCACACTTCAAACAACTTCATACCCCAACAAATTGGCTCATTCATTCCATGGCCACTGTGGACTTTCTTCTG GGGTGTCTGGTCATGCCTTACAGTATGGTGAGATCTGCTGAGCACTGTTGGTATTTTGGAGAAGTCTTCTGTA AAATTCACACAAGCACCGACATTATGCTGAGCTCAGCCTCCATTTTCCATTTGTCTTTCATCTCCATTGACCG CTACTATGCTGTGTGTGATCCACTGAGATATAAAGCCAAGATGAATATCTTGGTTATTTGTGTGATGATCTTC ATTAGTTGGAGTGTCCCTGCTGTTTTTGCATTTGGAATGATCTTTCTGGAGCTAAACTTCAAAGGCGCTGAAG AGATATATTACAAACATGTTCACTGCAGAGGAGGTTGCTCTGTCTTCTTTAGCAAAATATCTGGGGTACTGAC CTTTATGACTTCTTTTTATATACCTGGATCTATTATGTTATGTGTCTATTACAGAATATATCTTATCGCTAAA GAACAGGCAAGATTAATTAGTGATGCCAATCAGAAGCTCCAAATTGGATTGGAAATGAAAAATGGAATTTCAC AAAGCAAAGAAAGGAAAGCTGTGAAGACATTGGGGATTGTGATGGGAGTTTTCCTAATATGCTGGTGCCCTTT CTTTATCTGTACAGTCATGGACCCTTTTCTTCACTACATTATTCCACCTACTTTGAATGATGTATTGATTTGG TTTGGCTACTTGAACTCTACATTTAATCCAATGGTTTATGCATTTTTCTATCCTTGGTTTAGAAAAGCACTGA AGATGATGCTGTTTGGTAAAATTTTCCAAAAAGATTCATCCAGGTGTAAATTATTTTTGGAATTGAGTTCATA G The following amino acid sequence <SEQ ID NO.", "90> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "89: MMPFCHNIINISCVKNNWSNDVRASLYSLMVLIILTTLVGNLIVIVSISHFKQLHTPTNWLIHSMATVDFLLG CLVMPYSMVRSAEHCWYFGEVFCKIHTSTDIMLSSASIFHLSFISIDRYYAVCDPLRYKAKMNILVICVMIFI SWSVPAVFAFGMIFLELNFKGAEEIYYKHVHCRGGCSVFFSKISGVLTFMTSFYIPGSIMLCVYYRIYLIAKE QARLISDANQKLQIGLEMKNGISQSKERKAVKTLGIVMGVFLICWCPFFICTVMDPFLHYIIPPTLNDVLIWF GYLNSTFNPMVYAFFYPWFRKALKMMLFGKIFQKDSSRCKLFLELSS The following DNA sequence nGPCR-58 <SEQ ID NO.", "91> was identified in H. sapiens: CTGTAAAGTAGATTGTATGAGGACTCCATGAGGTCATCCACTTCAAGTCCTTGGCATAGGATAATTACTCAAA AGGTGATGACAATGGCGCAGGGAGGGATGGTGACTTGCCTGGAGATGCACAGCACCGTCTCTCCCATACTCGG TCATTCACACCATCATTGATTCACCAGGCACCACTCCGTGTCCAGCAGGACTCTGGGGACCCCAAATGGACAC TACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACTCCTACCCC CAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCAATGGGTTGATGGCGT GGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCTCAGCCTGGCCCTCTC TGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGACACTGGCCGCTGGGG ACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCTTCCTGCTGGCCGCCC TCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCCAGTCCGCCTGCCCCT CTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTCTTCCCCGAGGCTGCC GTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGCTGAGGATGCTGGAGG TCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGCCACAGCCTGTCGCAC CTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACCATTCTGTCAGCCTAT GTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGGACGTCTACTCTGGCT ACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCTCAGCCCCTTCCTCTG CCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCGGCAGCTCTCTGCGAG GAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTCCAACTCTGCCAGAGC CGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCACACTCCAGCCACGATC GGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACAGCCCAGCCACAGCTG AACCTCATGGCCCAGCCACAGTCAGATTCTGTGGCCCAGCCACAGGCAGACACTAACGTCCAGACCCCTGCAC CTGCTGCCAGTTCTGTGCCCAGTCCCTGTGATGAAGCTTCCCCAACCCCATCCTCGCATCCTACCCCAGGGGC CCTTGAGGACCCAGCCACACCTCCTGCCTCTGAAGGAGAAAGCCCCAGCAGCACCCCGCCAGAGGCGGCCCCG GGCGCAGGCCCCACGTGAGGGTCCAGGAACACGCAGGCCCACCAGAGCAGTGAAAGAGCCCAGGGCAGACAGA GGAACCAGCCAGTCAGA The following amino acid seqence <SEQ ID NO.", "92> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "91: LAWRCTAPSLPYSVIHTIIDSPGTTPCPAGLWGPQMDTTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVA LLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSLALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLW GVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVRLPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLD FWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATACRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQ LLYLAFIMDVYSGYLLWEALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEP QTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQPRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSV AQPQADTNVQTPAPAASSVPSPCDEASPTPSSHPTPGALEDPATPPASEGESPSSTPPEAAPGAGPT The following DNA sequence nGPCR-58 <SEQ ID NO.", "93> was identified in H. sapiens: ATGGACACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACT CCTACCCCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCA ATGGGTTGATGGCGTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCT CAGCCTGGCCCTCTCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGA CACTGGCCGCTGGGGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCT TCCTGCTGGCCGCCCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCC AGTCCGCCTGCCCCTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTC TTCCCCGAGGCTGCCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGC TGAGGATGCTGGAGGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGC CACAGCCTGTCGCACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACC ATTCTGTCAGCCTATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGG ACGTCTACTCTGGCTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCT CAGCCCCTTCCTCTGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCG GCAGCTCTCTGCGAGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTC CAACTCTGCCAGAGCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCAC ACTCCAGCCACGATCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACA GCCCAGCCACAGCTGAACCTCATGGCCCAGCCACAGTCAGACTCTGTGGCCCAGCCACAGGCAGACACTAACG TCCAGACCCCTGCACCTGCTGCCAGTTCTGTGCCCAGTCCCTGTGATGAAGCTTCCCCAACCCCATCCTCGCA TCCTACCCCAGGGGCCCTTGAGGACCCAGCCACACCTCCTGCCTCTGAAGGAGAAAGCCCCAGCAGCACCCCG CCAGAGGCGGCCCCGGGCGCAGGCCCCACGTGA The following amino acid sequence <SEQ ID NO.", "94> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "93: MDTTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSL ALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVR LPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATA CRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFLWDVYSGYLLWEALVYSDYLILLNSCLSP FLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQ PRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADTNVQTPAPAA The following DNA sequence nGPCR-3 <SEQ ID NO.", "185> was identified in H. sapiens: AGGCTCGCGCCCGAAGCAGAGCCATGAGAACCCCAGGGTGCCTGGCGAGCCGCTAGCGCCATGGGCCCCGGCG AGGCGCTGCTGGCGGGTCTCCTGGTGATGGTACTGGCCGTGGCGCTGCTATCCAACGCACTGGTGCTGCTTTG TTGCGCCTACAGCGCTGAGCTCCGCACTCGAGCCTCAGGCGTCCTCCTGGTGAATCTGTCTCTGGGCCACCTG CTGCTGGCGGCGCTGGACATGCCCTTCACGCTGCTCGGTGTGATGCGCGGGCGGACACCGTCGGCGCCCGGCG CATGCCAAGTCATTGGCTTCCTGGACACCTTCCTGGCGTCCAACGCGGCGCTGAGCGTGGCGGCGCTGAGCGC AGACCAGTGGCTGGCAGTGGGCTTCCCACTGCGCTACGCCGGACGCCTGCGACCGCGCTATGCCGGCCTGCTG CTGGGCTGTGCCTGGGGACAGTCGCTGGCCTTCTCAGGCGCTGCACTTGGCTGCTCGTGGCTTGGCTACAGCA GCGCCTTCGCGTCCTGTTCGCTGCGCCTGCCGCCCGAGCCTGAGCGTCCGCGCTTCGCAGCCTTCACCGCCAC GCTCCATGCCGTGGGCTTCGTGCTGCCGCTGGCGGTGCTCTGCCTCACCTCGCTCCAGGTGCACCGGGTGGCA CGCAGACACTGCCAGCGCATGGACACCGTCACCATGAAGGCGCTCGCGCTGCTCGCCGACCTGCACCCCAGTG TGCGGCAGCGCTGCCTCATCCAGCAGAAGCGGCGCCGCCACCGCGCCACCAGGAAGATTGGCATTGCTATTGC GACCTTCCTCATCTGCTTTGCCCCGTATGTCATGACCAGGCTGGCGGAGCTCGTGCCCTTCGTCACCGTGAAC GCCCAGTGGGGCATCCTCAGCAAGTGCCTGACCTACAGCAAGGCGGTGGCCGACCCGTTCACGTACTCTCTGC TCCGCCGGCCGTTCCGCCAAGTCCTGGCCGGCATGGTGCACCGGCTGCTGAAGAGAACCCCGCGCCCAGCATC CACCCATGACAGCTCTCTGGATGTGGCCGGCATGGTGCACCAGCTGCTGAAGAGAACCCCGCGCCCAGCGTCC ACCCACAACGGCTCTGTGGACACAGAGAATGATTCCTGCCTGCAGCAGACACACTGAGGGCCTGGCAGGGCTC ATCGCCCCCACCTTCTAAGA The following amino acid sequence <SEQ ID NO.", "186> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "185: MGPGEALLAGLLVMVLAVALLSNALVLLCCAYSAELRTRASGVLLVNLSLGHLLLAALDMPFTLLGVMRGRTP SAPGACQVIGFLDTFLASNAALSVAALSADQWLAVGFPLRYAGRLRPRYAGLLLGCAWGQSLAFSGAALGCSW LGYSSAFASCSLRLPPEPERPRFAAFTATLHAVGFVLPLAVLCLTSLQVHRVARRHCQRMDTVTMKALALLAD LHPSVRQRCLIQQKRRRHRATRKIGIAIATFLICFAPYVMTRLAELVPFVTVNAQWGILSKCLTYSKAVADPF TYSLLRRPFRQVLAGMVHRLLKRTPRPASTHDSSLDVAGMVHQLLKRTPRPASTHNGSVDTENDSCLQQTH The following DNA sequence nGPCR-58 <SEQ ID NO.", "93> was identified in H. sapiens: ATGGACACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACT CCTACCCCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCA ATGGGTTGATGGCGTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCT CAGCCTGGCCCTCTCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGA CACTGGCCGCTGGGGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCT TCCTGCTGGCCGCCCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCC AGTCCGCCTGCCCCTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTC TTCCCCGAGGCTGCCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGC TGAGGATGCTGGAGGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGC CACAGCCTGTCGCACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACC ATTCTGTCAGCCTATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGG ACGTCTACTCTGGCTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCT CAGCCCCTTCCTCTGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCG GCAGCTCTCTGCGAGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTC CAACTCTGCCAGAGCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCAC ACTCCAGCCACGATCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACA GCCCAGCCACAGCTGAACCTCATGGCCCAGCCACAGTCAGACTCTGTGGCCCAGCCACAGGCAGACACTAACG TCCAGACCCCTGCACCTGCTGCCAGTTCTGTGCCCAGTCCCTGTGATGAAGCTTCCCCAACCCCATCCTCGCA TCCTACCCCAGGGGCCCTTGAGGACCCAGCCACACCTCCTGCCTCTGAAGGAGAAAGCCCCAGCAGCACCCCG CCAGAGGCGGCCCCGGGCGCAGGCCCCACGTGA The following amino acid sequence <SEQ ID NO.", "94> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "93: MDTTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSL ALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVR LPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATA CRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFLWDVYSGYLLWEALVYSDYLILLNSCLSP FLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQ PRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADTNVQTPAPAA The following DNA sequence nGPCR-14 <SEQ ID NO.", "191> was identified in H. sapiens: ACTAACTTTGGGAACTCGTATAGACCCAGCGTCGCTCCCCGCGCCGCCTCGCCTCCACTTTGGTTTCCCGCGT CCTGCCCGCCCTCTTCGGTGCCTCCTCTTCCTCCGGGACAAGGATGGAGGATCTCTTTAGCCCCTCAATTCTG CCGCCGGCGCCCAACATTTCCGTGCCCATCTTGCTGGGCTGGGGTCTCAACCTGACCTTGGGGCAAGGACCCC CTGCCTCTGGGCCGCCCAGCCCGCGTGCGGGGGCACGGCGCTGTCACAGCTGGCCTGGGAACTGCTGGGCGAG CCCCGCGCGGCCACGGGGGACCTGGCGTGCCGCTTCCTGCAGCTGCTGCAGGCATCCGGGCGGGGCGCCTCGG CCCACCTAGTGGTGCTCATCGCCCTCGAGCGCCGGCGCGCGGTGCGTCTTCCGCACGGCCGGCCGCTGCCCGC GCGTGCCCTCGCCGCCCTGGGCTGGCTGCTGGCACTGCTGCTGGCGCTGCCCCCGGCCTTCGTGGTGCGCGGG GACTCCCCCTCGCCGCTGCCGCCGCCGCCGCCGCCAACGTCCCTGCAGCCAGGCGCGCCCCCGGCCGCCCGCG CCTGGCCGGGGGAGCGTCGCTGCCACGGGATCTTCGCGCCCCTGCCGCGCTGGCACCTGCAGGTCTACGCGTT CTACGAGGCCGTCGCGGGCTTCGTCGCGCCTGTTACGGTCCTGGGCGTCGCTTGCGGCCACCTACTCTCCGTC TGGTGGCGGCACCGGCCGCAGGCCCCCGCGGCTGCAGCGCCCTGGTCGGCGAGCCCAGGTCGAGCCCCTGCGC CCAGCGCGCTGCCCCGCGCCAAGGTGCAGAGCCTGAAGATGAGCCTGCTGCTGGCGCTGCTGTTCGTGGGCTG CGAGCTGCCCTACTTTGCCGCCCGGCTGGCGGCCGCGTGGTCGTCCGGGCCCGCGGGAGACTGGGAGGGAGAG GGCCTGTCGGCGGCGCTGCGCGTGgTGGCGATGGCCAACAGCGCTCTCAATCCCTTCGTCTACCTCTTCTTCC AGGCGGGCGACTGCTGGCTCCGGCGACAGCTGCGGAAGCGGCTGGGCTCTCTGTGCTGCGCGCCGCAGGGAGG CGCGGAGGACGAGGAGGGGCCCCGGGGCCACCAGGCGCTCTACCGCCAACGCTGGCCCCACCCTCATTATCAC CATGCTCGGCGGGAACCCGCTGGACGAGGGCGGCTTGCGCCCACCCCCTCCGCGCCCCAGACCCCTGCCTTGC TCCTGCGAAAGTGCCTTCTAGGTGCTTGGTGGTCAGAGACGGGTCATCTGTCGCTAAGGCGCAACCTCCAGGG AACTCGAGGCCTGCCAGGGTCTGTCCAGATCACAAGGGGCAGGAGAGTCTGTGAGAGAGTGACACTGAAGTTG TCCCCTTCCTCCACTCTCCTATTCCCTTCTCATGTTTACATTTCCCTATGCTCTTCCAGTTTCTCTTCTTCCC TACAGTTCCTCTCATATCTCCCCATTTGGAGACAGTGAGCCACTGGAAAGTTGTAAAAACAAAAACAGTTATT TTTGCAGTTTTCTTTCACGCATTTATAGTGCTCTGGATAATGCCATTTATTTTTGCTGATTACCCAACTTTCA GTATTTGCTGTGTTATCATCTGTATTTACTTATTTTGA The following amino acid sequence <SEQ ID NO.", "192> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.", "191: MEDLFSPSILPPAPNISVPILLGWGLNLTLGQGAPASGPPSRRVRLVFLGVILVVAVAGNTTVLCRLCGGGG PWAGPKRRKMDFLLVQLALADLYACGGTALSQLAWELLGEPRAATGDLACRFLQLLQASGRGASAHLVVLIA LERRRAVRLPHGRPLPARALAALGWLLALLLALPPAFVVRGDSPSPLPPPPPPTSLQPGAPPAARAWPGERR CHGIFAPLPRWHLQVYAFYEAVAGFVAPVTVLGVACGHLLSVWWRHRPQAPAAAAPWSASPGRAPAPSALPR AKVQSLKMSLLLALLFVGCELPYFAARLAAAWSSGPAGDWEGEGLSAALRVVAMANSALNPFVYLFFQAGDC WLRRQLRKRLGSLCCAPQGGAEDEEGPRGHQALYRQRWPHPHYHHARREPAGRGRLAPTPSAPQTPALLLRK CLLGAWWSETGHLSLRRNLQGTRGLPGSVQITRGRRVCERVTLKLSPSSTLLFPSHVYISLCSSSFSSSLQF LSYLPIWRQ Example 2 Cloning of nGPCR-x To isolate a cDNA clone encoding full length nGPCR-x, a DNA fragment corresponding to a nucleotide sequence set forth in odd numbered nucleotide sequences ranging from SEQ ID NO: 1-93, or a portion thereof, can be used as a probe for hybridization screening of a phage cDNA library.", "The DNA fragment is amplified by the polymerase chain reaction (PCR) method.", "The PCR reaction mixture of 50 μl contains polymerase mixture (0.2 mM dNTPs, 1×PCR Buffer and 0.75 μl Expand High Fidelity Polymerase (Roche Biochemicals)), 1 μg of plasmid, and 50 pmoles of forward primer and 50 pmoles of reverse primer.", "The primers are preferably 10 to 25 nucleotides in length and are determined by procedures well known to those skilled in the art.", "Amplification is performed in an Applied Biosystems PE2400 thermocycler, using the following program: 95° C. for 15 seconds, 52° C. for 30 seconds and 72° C. for 90 seconds; repeated for 25 cycles.", "The amplified product is separated from the plasmid by agarose gel electrophoresis, and purified by Qiaquick™ gel extraction kit (Qiagen).", "A lambda phage library containing cDNAs cloned into lambda ZAPII phagovector is plated with E. coli XL-1 blue host, on 15 cm LB-agar plates at a density of 50,000 pfu per plate, and grown overnight at 37° C.; (plated as described by Sambrook et al., supra).", "Phage plaques are transferred to nylon membranes (Amersham Hybond NJ), denatured for 2 minutes in denaturation solution (0.5 M NaOH, 1.5 M NaCl), renatured for 5 minutes in renaturation solution (1 M Tris pH 7.5, 1.5 M NaCl), and washed briefly in 2×SSC (20×SSC: 3 M NaCl, 0.3 M Na-citrate).", "Filter membranes are dried and incubated at 80° C. for 120 minutes to cross-link the phage DNA to the membranes.", "The membranes are hybridized with a DNA probe prepared as described above.", "A DNA fragment (25 ng) is labeled with α-32P-dCTP (NEN) using Rediprime™ random priming (Amersham Pharmacia Biotech), according to manufacturers instructions.", "Labeled DNA is separated from unincorporated nucleotides by S200 spin columns (Amersham Pharmacia Biotech), denatured at 95° C. for 5 minutes and kept on ice.", "The DNA-containing membranes (above) are pre-hybridized in 50 ml ExpressHyb™ (Clontech) solution at 68° C. for 90 minutes.", "Subsequently, the labeled DNA probe is added to the hybridization solution, and the probe is left to hybridize to the membranes at 68° C. for 70 minutes.", "The membranes are washed five times in 2×SSC, 0.1% SDS at 42° C. for 5 minutes each, and finally washed 30 minutes in 0.1×SSC, 0.2% SDS.", "Filters are exposed to Kodak XAR™ film (Eastman Kodak Company, Rochester, N.Y., USA) with an intensifying screen at −80° C. for 16 hours.", "One positive colony is isolated from the plates, and replated with about 1000 pfu on a 15 cm LB plate.", "Plating, plaque lift to filters and hybridization are performed as described above.", "About four positive phage plaques are isolated form this secondary screening.", "cDNA containing plasmids (pBluescript SK−) are rescued from the isolated phages by in vivo excision by culturing XL-1 blue cells co-infected with the isolated phages and with the Excision helper phage, as described by manufacturer (Stratagene).", "XL-blue cells containing the plasmids are plated on LB plates and grown at 37° C. for 16 hours.", "Colonies (18) from each plate are replated on LB plates and grown.", "One colony from each plate is stricken onto a nylon filter in an ordered array, and the filter is placed on a LB plate to raise the colonies.", "The filter is then hybridized with a labeled probe as described above.", "About three positive colonies are selected and grown up in LB medium.", "Plasmid DNA is isolated from the three clones by Qiagen Midi Kit™ (Qiagen) according to the manufacturer's instructions.", "The size of the insert is determined by digesting the plasmid with the restriction enzymes NotI and SalI, which establishes an insert size.", "The sequence of the entire insert is determined by automated sequencing on both strands of the plasmids.", "nGPCR-1: PCR and Subcloning cDNAs were sequenced directly using an AB1377 fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI PRISM Ready Dye-Deoxy Terminator kit with Taq FS polymerase.", "Each ABI cycle sequencing reaction contained about 0.5 μg of plasmid DNA.", "Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 min, followed by 50 cycles: 99° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.", "Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.", "Extension products were purified using AGTC® gel filtration block (Edge BiosSystems, Gaithersburg, Md.).", "Each reaction product was loaded by pipette onto the column, which was then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B table top centrifuge) at 1500×g for 4 min at room temperature.", "Column-purified samples were dried under vacuum for about 40 min and then dissolved in 5 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).", "The samples were then heated to 90° C. for three min and loaded into the gel sample wells for sequence analysis by the ABI377 sequencer.", "Sequence analysis was performed by importing ABI373A files into the Sequencher program (Gene Codes, Ann Arbor, Mich.).", "The PCR reaction was performed in 50 μL samples containing 41.9 μL H2O, 5 μL 10× Buffer containing 15 mM MgCl2 (Boehringer Mannheim Expand High Fidelity PCR System), 0.5 μL 10 mM dNTP mix, 1.5 μL human genomic DNA (Clontech #6550-1, 0.1 μg/μL), 0.3 μL primer VR1A (1 μg/μL), 0.3 μL primer VR1B (1 μg/μL), and 0.5 μL High Fidelity Taq polymerase (Boehringer Mannheim, 3.5U/μl).", "The primer sequences for and, respectively were: 5′TCAAAGCTTATGGAATCATCTTTCTCATTTGGAGTGATCCTTGCTGTC, (VR1A) (SEQ ID NO: 95) corresponding to the 5′ end of the coding region and containing a HindIII restriction site, and: 5′ TTCACTCGAGTTAGCCATCAAACTCTGAGCTGGAGATAGTGACGATGTG (VR1B) (SEQ ID NO: 96) corresponding to the 3′ end of the coding region and containing an XhoI restriction site (Genosys).", "The PCR reaction was carried out using a GeneAmp PCR9700 thermocycler (Perkin Elmer Applied Biosystems) and started with 1 cycle of 94° C. for 2 min followed by 5 cycles at 94° C. for 30 sec, 60° C. for 2 min, 72° C. for 1.5 min, followed by 20 cycles at 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 1.5 min.", "The PCR reaction was loaded onto a 0.75% agarose gel.", "The DNA band was excised from the gel and the DNA eluted from the agarose using a QIAquick gel extraction kit (Qiagen).", "The eluted DNA was ethanol-precipitated and resuspended in 4 μL H2O for ligation.", "The ligation reaction consisted of 4 μL of fresh ethanol-precipitated PCR product and 1 μL of pCRII-TOPO vector (Invitrogen).", "The reaction was gently mixed and allowed to incubate for 5 min.", "at room temperature followed by the addition of 1 μL of 6×TOPO cloning stop solution and mixing for 10 sec.", "at room temperature.", "The sample was then placed on ice and 2 μL was transformed in 50 μL of One Shot cells (Invitrogen) and plated onto ampicillin plates.", "Four white colonies were chosen and the presence of an insert was verified by PCR in the following manner.", "Each colony was resuspended in 2 ml LB broth for 2 hrs.", "A 500 μL aliquot was spun down in the microfuge, the supernatant discarded, and the pellet resuspended in 25 μL of H2O.", "A 16 μL aliquot was removed and boiled for 5 min and the sample was placed on ice.", "The sample was microfuged briefly to pellet any bacterial debris and PCR was carried out with 15 μL sample using primers VR1A and VR1B, described above.", "Colonies from positive clones identified by PCR were used to inoculate a 4 ml culture of LB medium containing 100 μg/ml ampicillin.", "Plasmid DNA was purified using the Wizard Plus Minipreps DNA purification system (Promega).", "Since the primers used to amplify the fragment of nGPCR-1 from genomic DNA were engineered to have HindIII and XhoI sites, the cDNA obtained from the minipreps was digested with these restriction enzymes.", "One clone was verified by gel electrophoresis to give a DNA band of the correct size.", "cDNA from this clone was then sequenced, yielding the sequence of SEQ ID NO: 73.nGPCR-3: PCR and Subcloning First-strand cDNA synthesis was performed following the directions for 3′-RACE ready cDNA from the SMART™ RACE cDNA Amplification Kit (Clontech).", "First 3 μl of H2O, 1 μl human whole brain poly A+ RNA (1 μg/μl) (Clontech, 6516-1) and 1 μl 3′-CDS primer were mixed together, incubated at 70° C. for 2 minutes, then placed on ice for 2 minutes.", "Added to the tube was 2 μl 5× First-Strand buffer, 1 μl 20 mM DTT, 1 μl dNTP mix (10 mM) and 1 μl Superscript II RT (200 units/μl) (GIBCO/BRL).", "The tube was incubated at 42° C. for 1.5 hours then the reaction was diluted with 250 μl of Tricine-EDTA buffer.", "PCR was performed in a 50 μl reaction using components that come with the Advantage®-GC cDNA PCR Kit.", "The PCR reaction contained 22.4 μl H2O, 10 μl 5×GC cDNA PCR Reaction buffer, 10 μl 5M GC Melt, 1 μl 50×dNTP mix (10 mM each), 5 μl human brain cDNA, 0.3 μl of LW1649 (SEQ ID NO: 187) (1 μg/μl), 0.3 μl of LW1650 (SEQ ID NO: 188) (1 μg/μl), 1 μl 50× Advantage-GC cDNA polymerase mix.", "The PCR reaction was performed in a Perkin-Elmer 9600 GeneAmp PCR System starting with 1 cycle of 94° C. for 2 min then 8 cycles at 94° C. for 15 sec, 72° C. for 2 min (decreasing 1° C. with each cycle), 72° C. for 3 min, followed by 30 cycles of 94° C. for 15 sec, 68° C. for 3 min.", "The PCR reaction was loaded onto a 1.2% agarose gel.", "The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed in a microcentrifuge.", "The eluted DNA was EtOH precipitated and resuspended in 4H2O for ligation.", "The PCR primer sequence for LW1649 was: GCATAAGCTTGCCATGGGCCCCGGCGAGG (SEQ ID NO: 187) and for LW1650 was: GCATTCTAGACCTCAGTGTGTCTGCTGC (SEQ ID NO: 188).", "The underlined portion of the primers matches the 5′ and 3′ areas, respectively, of the coding region.", "The ligation reaction used solutions from the TOPO TA Cloning Kit (Invitrogen) which consisted of 4 μl PCR product DNA, 1 μl Salt Solution and 1 μl pCRII-TOPO vector that was incubated for 5 minutes at room temperature and then placed on ice.", "Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.", "The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.", "A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.", "Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.", "The DNA subcloned into pCRII-TOPO was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.", "Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.", "The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).", "The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 95.nGPCR-9: PCR and Subcloning The PCR reaction was performed in 50 μl containing 34.5 μl H2O, 5 μl Buffer II (PE Applied Biosystems AmpliTaq Gold system), 6 μl 25 mM MgCl2, 2 μl 10 mM dNTP mix, 1.5 μl human genomic DNA (Clontech #6550-1, 0.1 μg/μl), 0.3 μl primer VR9A (1 μg/μl), 0.3 μl primer VR9B (1 μg/μl), and 0.4 μl AmpliTaq Gold™ DNA Polymerase.", "The primer sequences for VR9A and VR9B were as follows: VR9A 5′TTCAAAGCTTATGGAGTCGGGGCTGCTG 3′ (SEQ ID NO: 101), corresponding to the 5′ end of the coding region and containing a HindIII restriction site, and the reverse primer was: VR9B 5′ TTCACTCGAGTCAGTCTGCAGCCGGTTCTG 3′, (SEQ ID NO: 102), corresponding to the 3′ end of the coding region and containing an XhoI restriction site (Genosys).", "The PCR reaction was carried out using a GeneAmp PCR 9700 thermocycler (Perkin Elmer Applied Biosystems) and started with 1 cycle of 95° C. for 10 min, then 10 cycles at 95° C. for 30 sec, 72° C. for 2 min decreasing 1° C. each cycle, 72° C. for 1 min.", "followed by 30 cycles at 95° C. for 30 sec, 60° C. for 30 sec, 72° C. for 1 min.", "The PCR reaction was loaded on a 0.75% gel.", "The DNA band was excised from the gel and the DNA was eluted from the agarose using a QIAquick gel extraction kit (Qiagen).", "The eluted DNA was ethanol-precipitated and resuspended in 4 μl H2O for ligation.", "The ligation reaction consisted of 4 μl of fresh ethanol-precipitated PCR product and 1 μl of pCRII-TOPO vector (Invitrogen).", "The reaction was gently mixed and allowed to incubate for 5 min at room temperature followed by the addition of 1 μl of 6×TOPO cloning stop solution and mixing for 10 sec at room temperature.", "The sample was then placed on ice and 2 μl was transformed in 50 μl of One Shot cells (Invitrogen) and plated onto ampicillin plates.", "Five white colonies were chosen and were used to inoculate a 4 ml culture of LB medium containing 100 μg/ml ampicillin.", "Plasmid DNA was purified using the Wizard Plus Minipreps DNA purification system (Promega).", "Since the primers used to PCR SEQ-9 from genomic DNA were engineered to have HindIII and XhoI sites, the cDNA obtained from the minipreps was digested with these restriction enzymes.", "One clone was verified by gel electrophoresis to give a DNA band of the correct size.", "cDNA from this clone was then submitted for sequencing.", "One mutation was found (bp 621 T→G) and repaired as described as below.", "The mutation in the identified clone was repaired using the QuikChange Site-Directed Mutagenesis Kit (Stratagene).", "The PCR reaction contained 39.3 μl H2O, 5 μl 10× reaction buffer, 50 ng mini-prep cDNA, 1.25 μl primer VR9E (100 ng/μl), 1.25 μl primer VR9F (100 ng/μl), 1 μl 20 mM dNTP mix, 1 μl Pfu DNA polymerase.", "The cycle conditions were 95° C. for 30 sec, then 12 cycles at 95° C. for 30 sec, 55° C. for 1 min, 68° C. for 10 min.", "One μl of DpnI was added and the tube incubated at 37° C. for 1 hr.", "One μl of the DpnI-treated DNA was transformed into 50 μl Epicurian coli XL1-Blue supercompetent cells and the entire insert was re-sequenced.", "The primer sequences used were: VR9E: 5′ GCATCCTGGCCGCTATCTGTGCACTCTACG 3′ (SEQ ID NO: 103) and VR9F: 5′ CGTAGAGTGCACAGATAGCGGCCAGGATGC 3′ (SEQ ID NO: 104) where the base underlined was the base being corrected.", "The clone described above was sequenced directly using an ABI377 fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI BigDye™ Terminator Cycle Sequencing Ready Reaction kit with Taq FS™ polymerase.", "Each ABI cycle sequencing reaction contained 0.5 μg of plasmid DNA.", "Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 min, followed by 50 cycles: 96° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.", "Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.", "Extension products were purified using AGTC (R) gel filtration block (Edge BiosSystems, Gaithersburg, Md.).", "Each reaction product was loaded by pipette onto the column, which was then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 min at room temperature.", "Column-purified samples were dried under vacuum for about 40 min and then dissolved in 3 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).", "The samples were then heated to 90° C. for 3.5 min and loaded into the gel sample wells for sequence analysis by the ABI377 sequencer.", "Sequence analysis was performed by importing ABI377 files into the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 77.nGPCR-11: PCR and Subcloning PCR was performed in a 50 g reaction containing 32 μl H2O, 5 A 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris-tricine pH 8.4), 5 μl 15 mM MgSO4, 2 μl 10 mM dNTP, 5 μl human genomic DNA (0.3 μg/μL) (Clontech), 0.3 μl of LW1564 (1 μg/μl), 0.3 μl of LW1565 (1 μg/μl), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim).", "The PCR reaction was performed in a GeneAmp 9600 PCR thermocycler (PE Applied Biosystems) starting with 1 cycle of 94° C. for 2 min followed by 17 cycles at 94° C. for 30 sec, 72° C. for 2 min decreasing 1° C. each cycle, 68° C. for 2 min, then 25 cycles of 94° C. for 30 sec, 55° C. for 30 sec, 68° C. for 2 min.", "The PCR reaction was loaded onto a 1.2% agarose gel.", "The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed in a microcentrifuge.", "The eluted DNA was EtOH precipitated and resuspended in 4 μl H2O for ligation.", "The forward PCR primer sequence was: LW1564: GCATAAGCTTCCATGTACAACGGGTCGTGCTGC (SEQ ID NO: 107), and the reverse PCR primer was: LW1565: GCATTCTAGATCAGTGCCACTCAACAATGTGGG (SEQ ID NO: 108).", "The ligation reaction used solutions from the TOPO TA Cloning Kit (vitrogen) which consisted of 4 μl PCR product DNA and 1 μl pCkII-TOPO vector that was incubated for 5 minutes at room temperature.", "To the ligation reaction one microliter of 6×TOPO Cloning Stop Solution was added then the reaction was placed on ice.", "Two microliters of the ligation reaction was transformed in One Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.", "The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 Tl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.", "A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.", "Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.", "The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.", "Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/11) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.", "The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).", "The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 79.nGPCR-16: PCR and Subcloning PCR was performed in a 50 μl reaction containing 32 μl H2O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris-tricine pH 8.4), 5 μl 15 mM MgSO4, 2 μl 10 mM dNTP, 5 μl 2445704H1 DNA (0.17 Tg/Tl), 0.3 μl of LW1587 (1 μg/μl), 0.3 μl of LW1588 (1 μg/μl), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim).", "The PCR reaction was performed on a Robocycler thermocycler (Stratagene) starting with 1 cycle of 94° C. for 2 min followed by 15 cycles of 94° C. for 30 sec, 55° C. for 1.3 min, 68° C. for 2 min.", "The PCR reaction was loaded onto a 1.2% agarose gel.", "The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed in a microcentrifuge.", "The eluted DNA was EtOH precipitated and resuspended in 12 μl H2O for ligation.", "The PCR primer sequence for the forward primer was: LW1587: GATCAAGCTTATGACAGGTGACTTCCCAAGTATGC (SEQ ID NO: 111), and the sequence for the reverse primer was: LW1588: GATCCTCGAGGCTAACGGCACAAAACACAATTCC (SEQ ID NO: 112).", "The ligation reaction used solutions from the TOPO TA Cloning Kit (Invitrogen) which consisted of 4 μl PCR product DNA and 1 μl pCRII-TOPO vector that was incubated for 5 minutes at room temperature.", "To the ligation reaction one microliter of 6×TOPO Cloning Stop Solution was added then the reaction was placed on ice.", "Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.", "The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.", "A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.", "Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.", "The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.", "Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.", "The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).", "The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 81.nGPCR-40: PCR and Subcloning PCR was performed in a 50 μl reaction containing utilizing Herculase DNA Polymerase blend (Stratagene), using the buffer recommendations provided by the manufacturer, 200 ng each of primers PSK 18 and 19 (SEQ ID NOS: 115 and 116), 150 ng of human genomic DNA (Clontech), and 2% DMSO.", "The PCR reaction was performed on a Robocycler thermocycler (Stratagene) starting with 1 cycle of 94° C. for 2 min followed by 35 cycles of 94° C. for 30 sec, 65° C. for.", "30 sec, 72° C. for 2 min.", "The PCR reaction was purified using the QiaQuick PCR Purification Kit (Qiagen), and then eluted in TE.", "The PCR primer sequences were: PSK 18 GATC GAATTCGCAGGAGCAATG AAAATCAGGAAC (SEQ ID NO: 115), and: PSK19: GATCGAATTCTTATATATGTTCAGAAAACAAATTCATGG (SEQ ID NO: 116)).", "The underlined portion of the primer matches the 5′ and 3′ areas, respectively, of a portion of the 5′ untranslated region and coding region.", "Initiation and termination codons are shown above in bold.", "The PCR product was ligated into the pCR-BluntII-TOPO vector (Invitrogen) using the Zero Blunt Topo PCR TA cloning kit as follow: 3 μl PCR product DNA, 1 μl pCRII-TOPO vector, and 1 μl TOPOII salt solution (1.2M NaCl, 0.06M MgCl2).", "The mixture was incubated for 5 minutes at room temperature.", "To the ligation reaction one microliter of 6×TOPO Cloning Stop Solution was added, and then the reaction was placed on ice.", "Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.", "The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates supplemented with Xgal and IPTG.", "Single colonies were screened by PCR for the presence of the insert, and a plasmid DNA from colony 58 was purified using a Qiagen Endo-Free plasmid purification kit.", "nGPCR-40 was sequenced directly using an ABI377 fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI BigDye™ Terminator Cycle Sequencing Ready Reaction kit with Taq FS™ polymerase.", "Each ABI cycle sequencing reaction contained about 0.5 μg of plasmid DNA.", "Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 min, followed by 50 cycles: 96° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.", "Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.", "Extension products were purified using AGTC® gel filtration block (Edge BiosSystems, Gaithersburg, Md.).", "Each reaction product was loaded by pipette onto the column, which was then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 min at room temperature.", "Column-purified samples were dried under vacuum for about 40 min and then dissolved in 3 μl of DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).", "The samples were then heated to 90° C. for 3.5 min and loaded into the gel sample wells for sequence analysis by the ABI377 sequencer.", "Sequence analysis was performed by importing ABI377 files into the Sequencher program (Gene Codes, Aim Arbor, M), which yielded a sequence identical to SEQ ID NO:83 with the exception that the nucleotide at position 10 was identified as an “A” which incorrectly indicated the presence of an initiation codon at that position.", "Subsequent analysis of genomic DNA samples indicated that this position was incorrectly assigned and that the correct nucleotide at that position was a “C”.", "The sequence reported at SEQ ID NO.", "83 correctly identifies the nucleotide at position 10 and indicates that the first initiation codon occurs at position 88-90.nGPCR-54: PCR and Subcloning Two microliters of a human genomic library (˜108 PFU/ml) (Clontech) was added to 6 ml of an overnight culture of K802 cells (Clontech), then distributed as 250 μl aliquots into each of 24 tubes.", "The tubes were incubated at 37° C. for 15 min.", "Seven milliliters of 0.8% agarose was added to each tube, mixed, then poured onto LB agar+10 mM MgSO4 plates and incubated overnight at 37° C. To each plate 5 ml of SM (0.1M NaCl, 8.1 mM MgSO4-7H2O, 50 mM Tris-Cl (pH 7.5), 0.0001% gelatin) phage buffer was added and the top agarose was removed with a microscope slide and placed in a 50 ml centrifuge tube.", "A drop of chloroform was added and the tube was place in a 37° C. shaker for 15 min, then centrifuged for 20 min at 4000 RPM (Sorvall RT6000 table top centrifuge) and the supernatant stored at 4° C. as a stock solution.", "Two μl of phage from each tube was heated to 99° C. for 4 min then cooled to 10° C. Added to the phage was a PCR mix containing 8.8 μl H2O, 4 μl 5× Rapid-load Buffer (Origene), 2 μl 10×PCR buffer II (Perkin-Elmer), 2 μl 25 mM MgCl2, 0.8 μl 10 mM dNTP, 0.12 μl LW1634 (1 μg/μl) (SEQ ID NO: 119), 0.12 μl LW1635 (1 μg/μl) (SEQ ID NO: 120), 0.2 μl AmpliTaq Gold polymerase (Perlin Elmer).", "The PCR reaction involved 1 cycle at 95° C. for 10 min followed by 35 cycles at 95° C. for 45 sec, 53.5° C. for 2 min, 72° C. for 45 sec.", "The reaction was loaded onto a 2% agarose gel.", "From the tube that gave a PCR product of the correct size, 10 μl was used to set up five 1:10 dilutions that were plated onto LB agar+10 mM MgSO4 plates and incubated overnight.", "A BA85 nitrocellulose filter (Schleicher & Schuell) was placed on top of each plate for 1 hour.", "The filter was removed, placed phage side up in a petri dish, and covered with 4 ml of SM for 15 min to elute the phage.", "One milliliter of SM was removed from each plate and used to set up a PCR reaction as above.", "The plate of the lowest dilution to give a PCR product was subdivided, filter-lifted and the PCR reaction was repeated.", "The series of dilutions and subdividing of the plate was continued until a single plaque was isolated that gave a positive PCR band.", "Once a single plaque was isolated, 10 μl phage supernatant was added to 100 μl SM and 200 μl of K802 cells per plate with a total of 8 plates set up.", "The plates were incubated overnight at 37° C. The top agarose was removed by adding 8 ml of SM then scrapping off the agarose with a microscope slide and collected in a centrifuge tube.", "To the tube, 3 drops of chloroform was added, vortexed, incubated at 37° C. for 15 min then centrifuged for 20 min at 4000 RPM (Sorvall RT6000 table top centrifuge) to recover the phage, which was used to isolate genomic phage DNA using the Qiagen Lambda Midi Kit.", "The sequence for primer LW1634 was: CTGAAAGTTGTCGCTGACC (SEQ ID NO: 119), and for primer LW1635 was: CGATTATCCACACTTTGACCC (SEQ ID NO: 120).", "The PCR reaction for the coding region was performed in a 50 μl reaction containing 33 μl H2O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris tricine pH 8.4), 5 μl 15 mM MgSO4, 2 μl 10 mM DNTP, 4 μl genomic phage DNA (0.25 μg/μl), 0.3 μl LW1698 (1 μg/μl) (SEQ ID NO: 121), 0.3 μl LW1699 (1 μg/μl) (SEQ ID NO: 122), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim).", "The PCR reaction was started with 1 cycle of 94° C. for 2 min followed by 30 cycles at 94° C. for 30 sec, 55° C. for 30 sec., 68° C. for 2 min.", "The PCR reaction was loaded onto a 2% agarose gel.", "The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed.", "The eluted DNA was EtOH precipitated and resuspended in 8 μl H2O.", "The PCR primer sequence for primer LW1698 was: GCATACCATGAATGAGCCACTAGAC (SEQ ID NO: 121), and for primer LW1699 was: GCATCTCGAGTCAAGGGTTGTTTGAGTAAC (SEQ ID NO: 122).", "The underlined portion of the primer matches the 5′ and 3′ areas, respectively, of the coding region of nGPCR-54.The ligation reaction used solutions from the TOPO TA Cloning Kit (Invitrogen) which consisted of 4 μl PCR product DNA, 1 μl of salt solution and 1 μl pCRII-TOPO vector that was incubated for 5 minutes at room temperature then the reaction was placed on ice.", "Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.", "The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.", "A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.", "Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.", "nGPCR-54 genomic phage DNA was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.", "The cycle-sequencing reaction contained 14 μl of H2O, 16 μl of BigDye Terminator mix, 7 μl genomic phage DNA (0.1 μg/μl), and 3 μl primer (25 ng/μl).", "The reaction was performed in a Perkin-Elmer 9600 thermocycler at 95° C. for 5 min, followed by 99 cycles of 95° C. for 30 sec, 55° C. for 20 sec, and 60° C. for 4 min.", "The product was purified using a Centriflex™ gel filtration cartridges, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent.", "The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer.", "The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.", "Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.", "The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).", "The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 85.nGPCR-56: PCR and Subcloning The PCR reaction for the coding region of nGPCR-56 used components that come with PLATINUM® Pfx DNA Polymerase (GibcoBRL) containing 35.5 μl H2O, 5 μl 10×Pfx Amplification buffer, 1.5 μl 50 mM MgSO4, 2 μl 10 mM &M, 5 μl human genomic DNA (0.3 μg/μl) (Clontech), 0.3 μl of LW1603 (1 μg/μl) (SEQ ID NO: 152), 0.3 μl of LW1604 (1 μg/μl) (SEQ ID NO: 153), 0.4 μl PLATINUM® Pfa DNA Polymerase (2.5 U/Tl).", "The PCR reaction was performed in a Robocycler Gradient 96 (Stratagene) starting with 1 cycle of 94° C. for 5 min followed by 30 cycles at 94° C. for 40 sec, 55° C. for 2 min, 68° C. for 3 min.", "Following the final cycle, 0.5 μl of AmpliTaq DNA Polymerase (5 U/μl) was added and the tube was incubated at 72° C. for 5 min.", "The sequence of LW1603 is: GATCAAGCTTGGAATGATGCCCTITTGCCAC (SEQ ID NO: 152), and for LW1604 is: GATCCTCGAGCATCATTCAAAGTAGGTGG.", "(SEQ ID NO: 153).", "The underlined portion of the primer matches the 5′ and 3′ areas, respectively, of a portion of the coding region of nGPCR-56.The PCR reaction for the coding region was performed in a 50 μl reaction containing 32 μl H2O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris tricine pH 8.4), 5 μl 15 mM MgSO4, 2 μl 10 mM DNTP, 5 μl human genomic DNA (0.3 μg/μl) (Clontech), 0.3 μl LW1603 (1 μg/μl) (SEQ ID NO: 152), 0.3 μl LW1696 (1 μg/μl) (SEQ ID NO: 154), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim).", "The PCR reaction was started with 1 cycle of 94° C. for 2 min followed by 25 cycles at 94° C. for 40 sec, 55° C. for 60 sec., 68° C. for 2 min.", "The PCR reaction was loaded onto a 2% agarose gel.", "The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed.", "The eluted DNA was EtOH precipitated and resuspended in 12 μl H2O for ligation.", "The PCR primer sequence for LW1603 is: GATCAAGCTTGGAATGATGCCCTTTTGCCAC (SEQ ID NO: 152), and LW1696: GATCCTCGAGCTATGAACTCAATTCCAAAAATAATTTACACC (SEQ ID NO: 154).", "The underlined portion of the primer matches the 5′ and 3′ areas, respectively, of a portion of the coding region.", "The ligation reaction used solutions from the TOPO TA Cloning Kit (Invitrogen) which consisted of 4 μl PCR product DNA, 1 μl of salt solution and 1 μl pCRH-TOPO vector that was incubated for 5 minutes at room temperature then the reaction was placed on ice.", "Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.", "The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.", "A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.", "Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.", "The mutation in nGPCR-56 was repaired using the QuikChange Site-Directed Mutagenesis Kit (Stratagene).", "The PCR reaction contained 40 μl H2O, 5 μl 10× Reaction buffer, 1 μl mini-prep DNA, 1 μl LW1700 (125 ng/μl) (SEQ ID NO: 155), 1 μl LW1701 (125 ng/μl) (SEQ ID NO: 156), 1 μl 10 mM dNTP, 1 μl Pfu DNA polymerase.", "The cycle conditions were 95° C. for 30 sec then 14 cycles at 95° C. for 30 sec, 55° C. for 1 min, 68° C. for 12 min.", "The tube was placed on ice for 2 min, then 1 μl of DpnI was added and the tube incubated at 37° C. for one hour.", "One microliter of the DpnI-treated DNA was transformed into Epicurian coli XL1-Blue supercompetent cells and the entire insert was re sequenced.", "The primer sequences are: GCTACTTGAACTCTACATTTAATCCAATGGTTTATGCATITTTCTATCC (LW1700) (SEQ ID NO: 155), and: GGATAGAAAAATGCATAAACCATTGGATTAAATGTAGAGTTCAAGTAGC (LW1701) (SEQ ID NO: 156).", "The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.", "Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.", "The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).", "The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 89.nGPCR-58: PCR and Subcloning Isolation of a clone for nGPCR-58 from genomic DNA was performed by PCR in a 50 μl reaction containing Herculase DNA Polymerse blend (Stratagene), with buffer recommendations as supplied by the manufacturer, 200 ng each primers PSK14 (SEQ ID NO: 157) and PSK15 (SEQ ID NO: 158), 150 ng of human genomic DNA (Clontech) and 6% DMSO.", "The PCR reaction was performed on a Robocycler thermocycler (Stratagene) starting with 1 cycle of 94° C. for 2 min followed by 35 cycles of 94° C. for 30 sec, 65° C. for 30 sec, 72° C. for 2 min.", "The PCR reaction was purified by the QiaQuick PCR Purification Kit (Qiagen) and eluted in Th.", "The PCR primer sequences were: PSK14: 5′GATCGAATTCATGGACACTACCATGGAAGCTGACC (SEQ ID NO: 157), and: PSK15: 5′GATCCTCGAGTCACGTGGGGCCTGCGCCCGG (SEQ ID NO: 158).", "The underlined portion of the primers match the 5′ and 3′ areas, respectively, of a portion of the 5′ untranslated region and coding region.", "Translation initiation and termination codons are shown above in bold.", "The blunt ended PCR product was prepared for cloning by the addition of a single base “A” residue by AmpliTaq Gold (Perkin Elmer) in a reaction with 1×PCR Buffer II, 1 mM MgCl2, 200 uM each dATP, dGTP, dCTP, and dTTP.", "The reaction was incubated at 94° C. for 10 minutes followed by 72° C. for 10 minutes.", "The products were cloned into the pCRII-TOPO vector (Invitrogen) using the TOPO TA cloning kit as follows: 3 μl PCR product DNA, 1 μl pCRII-TOPO vector, and 1 μl TOPOII salt solution (1.2M NaCl, 0.06M MgCl2) was incubated for 5 minutes at room temperature.", "To the ligation reaction one microliter of 6×TOPO Cloning Stop Solution was added then the reaction was placed on ice.", "Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.", "The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates supplemented with X-gal and IPTG.", "Single colonies were screened by PCR for the presence of the insert, and a plasmid DNA from colony 58-6 was purified using a Qiagen Endo-Free plasmid purification kit and deposited as nGPCR-58.nGPCR-58 was sequenced directly using an ABI377 fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI BigDye™ Terminator Cycle Sequencing Ready Reaction kit with Taq FS™ polymerase.", "Each ABI cycle sequencing reaction contained about 0.5 μg of plasmid DNA.", "Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 min, followed by 50 cycles: 96° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.", "Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.", "Extension products were purified using AGTC (R) gel filtration block (Edge RiosSystems, Gaithersburg, Md.).", "Each reaction product was loaded by pipette onto the column, which was then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 min at room temperature.", "Column-purified samples were dried under vacuum for about 40 min and then dissolved in 3 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).", "The samples were then heated to 90° C. for 3.5 min and loaded into the gel sample wells for sequence analysis by the ABI377 sequencer.", "Sequence analysis was performed by importing ABI377 files into the Sequencer program (Gene Codes, Ann Arbor, Mich.), yielding the sequence of SEQ ID NO: 93.Example 3 Hybridization Analysis to Demonstrate nGPCR-X Expression in Brain The expression of nGPCR-x in mammals, such as the rat, may be investigated by in situ hybridization histochemistry.", "To investigate expression in the brain, for example, coronal and sagittal rat brain cryosections (20 pun thick) are prepared using a Reichert-Jung cryostat.", "Individual sections are thaw-mounted onto silanized, nuclease-free slides (CEL Associates, Inc., Houston, Tex.", "), and stored at −80° C. Sections are processed starting with post-fixation in cold 4% paraformaldehyde, rinsed in cold phosphate-buffered saline (PBS), acetylated using acetic anhydride in triethanolamine buffer, and dehydrated through a series of alcohol washes in 70%, 95%, and 100% alcohol at room temperature.", "Subsequently, sections are delipidated in chloroform, followed by rehydration through successive exposure to 100% and 95% alcohol at room temperature.", "Microscope slides containing processed cryosections are allowed to air dry prior to hybridization.", "Other tissues may be assayed in a similar fashion.", "A nGPCR-x-specific probe is generated using PCR.", "Following PCR amplification, the fragment is digested with restriction enzymes and cloned into pBluescript II cleaved with the same enzymes.", "For production of a probe specific for the sense strand of nGPCR-x, the nGPCR-x clone in pBluescript II is linearized with a suitable restriction enzyme, which provides a substrate for labeled run-off transcripts (i.e., cRNA riboprobes) using the vector-borne T7 promoter and commercially available T7 RNA polymerase.", "A probe specific for the antisense strand of nGPCR-x is also readily prepared using the nGPCR-x clone in pBluescript II by cleaving the recombinant plasmid with a suitable restriction enzyme to generate a linearized substrate for the production of labeled run-off cRNA transcripts using the T3 promoter and cognate polymerase.", "The riboprobes are labeled with [35S]-UTP to yield a specific activity of about 0.40×106 cpm/pmol for antisense riboprobes and about 0.65×106 cpm/pmol for sense-strand riboprobes.", "Each riboprobe is subsequently denatured and added (2 pmol/ml) to hybridization buffer which contained 50% formamide, 10% dextran, 0.3 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, 1× Denhardt's Solution, and 10 mM dithiothreitol.", "Microscope slides containing sequential brain cryosections are independently exposed to 45 μl of hybridization solution per slide and silanized cover slips are placed over the sections being exposed to hybridization solution.", "Sections are incubated overnight (15-18 hours) at 52° C. to allow hybridization to occur.", "Equivalent series of cryosections are exposed to sense or antisense nGPCR-40-specific cRNA riboprobes.", "Following the hybridization period, coverslips are washed off the slides in 1×SSC, followed by RNase A treatment involving the exposure of slides to 20 μg/ml RNase A in a buffer containing 10 mM Tris-HCl (pH 7.4), 0.5 M EDTA, and 0.5 M NaCl for 45 minutes at 37° C. The cryosections are then subjected to three high-stringency washes in 0.1×SSC at 52° C. for 20 minutes each.", "Following the series of washes, cryosections are dehydrated by consecutive exposure to 70%, 95%, and 100% ammonium acetate in alcohol, followed by air drying and exposure to Kodak BioMax™ MR-1 film.", "After 13 days of exposure, the film is developed.", "Based on these results, slides containing tissue that hybridized, as shown by film autoradiograms, are coated with Kodak NTB-2 nuclear track emulsion and the slides are stored in the dark for 32 days.", "The slides are then developed and counterstained with hematoxylin.", "Emulsion-coated sections are analyzed microscopically to determine the specificity of labeling.", "The signal is determined to be specific if autoradiographic grains (generated by antisense probe hybridization) are clearly associated with cresyl violate-stained cell bodies.", "Autoradiographic grains found between cell bodies indicates non-specific binding of the probe.", "Expression of nGPCR-x in the brain provides an indication that modulators of nGPCR-x activity have utility for treating neurological disorders, including but not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.", "Some other diseases for which modulators of nGPCR-x may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.", "Use of nGPCR-x modulators, including nGPCR-x ligands and anti-nGPCR-x antibodies, to treat individuals having such disease states is intended as an aspect of the invention.", "Example 4 Tissue Expression Profiling Tissue specific expression of the cDNAs encoding nGPCR-1, nGPCR-3, nGPCR-9, nGPCR-11, nGPCR-16, nGPCR-40, nGPCR-54, nGPCR-56, and nGPCR-58 was detected using a PCR-based system.", "Tissue specific expression of cDNAs encoding nGPCR-x may be accomplished using similar methods.", "Primers were synthesized by Genosys Corp., The Woodlands, Tex.", "PCR reactions were assembled using the components of the Expand Hi-Fi PCR System™ (Roche Molecular Biochemicals, Indianapolis, Ind.).", "nGPCR-1 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.", "Human tissues in the array may include: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.", "Human brain regions in the array may include: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.", "Expression of the nGPCR-1 in the various tissues was detected by using PCR primers designed based on the available sequence of the receptor that will prime the synthesis of a 212 bp fragment in the presence of the appropriate cDNA.", "The forward primer was: GCTCAACCCACTCATCTATGCC (SEQ ID NO: 97), and the reverse primer was: AAACTTCTCTGCCCTTACCGTC (SEQ ID NO: 98) The PCR reaction mixture was added to each well of the PCR plate.", "The plate was placed in a GeneAmp PCR9700 PCR thermocycler (Perkin Elmer Applied Biosystems).", "The plate was then exposed to the following cycling parameters: Pre-soak 94° C. for 3 min; denaturation at 94° C. for 30 seconds; annealing at primer Tm for 45 seconds; extension 72° C. for 2 minutes; for 35 cycles.", "PCR products were then separated and analyzed by electrophoresis on a 1.5-% agarose gel.", "The 4-log dilution range of cDNA deposited on the plate ensured that the amplification reaction is within the linear range and, hence, facilitated the semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.", "Expression of nGPCR-1 was found to be highest in the testis, adrenal gland and heart.", "Significant levels of expression were also found in the brain, kidney, spleen ovary, prostate, muscle, PBL, stomach and bone marrow.", "Within the brain, expression levels were highest in the cerebellum, amygdala, thalamus and spinal cord, with significant levels of expression in the frontal lobe, hippocampus, substantia nigra, hypothalamus and pons.", "Expression of nGPCR-1 in the brain provided an indication that modulators of nGPCR-1 activity have utility for treating neurological disorders, including but not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.", "Some other diseases for which modulators of nGPCR-1 may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.", "Use of nGPCR-1 modulators, including nGPCR-1 ligands and ant-nGPCR-1 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.", "nGPCR-3 Tissue specific expression of the cDNA encoding nGPCR-3 was detected using a PCR-based method.", "Multiple Choice™ first strand cDNAs (OriGene Technologies, Rockville, Md.)", "from 6 human tissues were serially diluted over a 3-log range and arrayed into a multi-well PCR plate.", "This array was used to generate a comprehensive expression profile of the putative GPCR in human tissues.", "Human tissues arrayed included: brain, heart, kidney, peripheral blood leukocytes, lung and testis.", "PCR primers were designed based on the available sequence of the putative GPCR.", "The sequence of the forward primer used was: 5′TGCTGCTTTGTTGCGCCTAC3′ (SEQ ID NO: 189), corresponding to base pairs 77 through 96 of the predicted coding sequence of nGPCR-3.The sequence of the reverse primer used was: 5′TTGGACGCCAGGAAGGTG3′ (SEQ ID NO: 190), corresponding to base pairs 258 through 285 of the predicted coding sequence of nGPCR-3.This primer set primes the synthesis of a 298 base pair fragment in the presence of the appropriate cDNA.", "For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).", "This panel represents serial dilutions over a 3 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.", "Primers were synthesized by Genosys Corp., The Woodlands, Tex.", "PCR reactions were assembled using the components of the Expand Hi-Fi PCR System (Roche Molecular Biochemicals, Indianapolis, Ind.).", "Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.", "The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).", "The following cycling program was executed: Pre-soak at (94° C. for 3 min.)", "followed by 35 cycles of [(94° C. for 45 sec.", "), (53° C. for 2 min.", "), and (72° C. for 45 sec.)].", "PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.", "The results indicated that nGPCR-3 was expressed in the brain, heart, kidney, peripheral blood lymphocytes, lung, and testis.", "In the brain, nGPCR-3 was expressed in frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla, as well as in the spinal cord.", "nGPCR-9 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.", "Human tissues arrayed include: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.", "The forward primer used was to detect expression of nGPCR-9 was: 5′ AACCCCATCATCTACACGC 3′(SEQ ID NO: 105), and, the reverse primer was: 5′ TGCCTGTGGAGCCGCTGG 3′(SEQ ID NO: 106).", "This primer set will prime the synthesis of a 238 base pair fragment in the presence of the appropriate cDNA.", "For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).", "This panel represents serial dilutions over a 2-log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.", "Twenty-five microliters of the PCR reaction mixture was added to each well of the PCR plate.", "The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).", "The following cycling program was executed: Pre-soak at (94° C. for 3 min.)", "followed by 35 cycles of [(94° C. for 45 sec.)", "(52° C. for 2 min.)", "(72° C. for 45 sec.)].", "PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel and stained with ethidium bromide.", "nGPCR-9 was expressed in the brain, peripheral blood leukocytes, heart, kidney, adrenal gland, spleen, pancreas, liver, lung, skin, bone marrow, testis, placenta, salivary gland, uterus, small intestine, muscle, stomach, and fetal liver.", "Within the brain, nGPCR-9 was expressed in all areas examined including the frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.", "Expression of nGPCR-9 in the brain provided an indication that modulators of nGPCR-9 activity have utility for treating disorders, including but not limited to, schizophrenia, affective disorders, movement disorders, metabolic disorders, inflammatory disorders, cancers, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.", "Use of nGPCR-9 modulators, including nGPCR-9 ligands and anti-nGPCR-9 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.", "nGPCR-41 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.", "Human tissues in the array included, inter alia: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.", "Human brain regions in the array included, inter alia: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.", "Expression of nGPCR-11 in the various tissues was detected by using PCR primers designed based on the available sequence of the receptor that will prime the synthesis of a 206 bp fragment in the presence of the appropriate cDNA.", "The forward primer used to detect expression of nGPCR-11 was: 5′-GAAGCCCAGCACTGTTTACC-3′ (SEQ ID NO: 109), and the reverse primer was: 5′-TGAAATACCTGTCCGCAGCC-3 (SEQ ID NO: 10).", "Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.", "The plate was placed in a GeneAmp 9700 PCR thermocycler (PE Applied Biosystems).", "The following cycling program was executed: Pre-soak 94° C. for 3 min; denaturation at 94° C. for 30 seconds; annealing at primer Tm for 45 seconds; extension at 72° C. for 2 minutes; for 35 cycles.", "PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.", "The 4-log dilution range of cDNA deposited on the plate ensured that the amplification reaction was within the linear range and, facilitated semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.", "nGPCR-11 was expressed in the thyroid gland, brain, heart, kidney, adrenal gland, spleen, liver, ovary, muscle, testis, salivary gland, colon, prostate, small intestine, skin stomach, bone marrow, fetal brain and placenta.", "Within the brain, nGPCR-11 was expressed in the temporal lobe, amygdala, substantia nigra, pons, spinal cord, frontal lobe, and cerebellum.", "Expression of the nGPCR-11 in the brain provided an indication that modulators of nGPCR-11 activity have utility for treating disorders, including but not limited to, schizophrenia, affective disorders, metabolic disorders, inflammatory disorders, cancers, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.", "Some other diseases for which modulators of nGPCR-11 may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the lice.", "Use of nGPCR-11 modulators, including nGPCR1-11 ligands and anti-nGPCR-11 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.", "Expression of nGPCR-11 in the thyroid gland, indicates that agonists or antagonists could be of use in the treatment of thyroid dysfunction such as thyreotoxicosis and myxoedema.", "They could also be of use in the stimulation of thyroid hormone release leading to overall increase in metabolic rate and weight reduction.", "The expression of nGPCR-11 in liver and muscle indicate a use for agonists or antagonists in regulation of glucose metabolism applicable in diabetes type II.", "nGCPR-16 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.", "Human tissues in the array included, inter alia: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.", "Human brain regions in the array included, inter alia: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.", "Expression of nGPCR-16 in the various tissues was detected by using PCR primers designed based on the available sequence of the receptor that will prime the synthesis of a 205 bp fragment in the presence of the appropriate cDNA.", "The forward primer used to detect expression of nGPCR-16 was: 5′ CAGCCCAAACATCCAAGTC 3′.", "(SEQ ID NO: 113).", "The reverse primer used to detect expression of nGPCR-16 was: 5′ ACCCCACTTAATCAGCCTC 3′ (SEQ ID NO: 114).", "For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).", "This panel represents serial dilutions over a 2 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.", "Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.", "The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).", "The following cycling program was executed: Pre-soak at (94° for 3 min.)", "followed by 35 cycles of [(94° C. for 45 sec.)", "(53° C. for 2 min.)", "(72° C. for 45 sec.)].", "PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel, and stained with ethidium bromide.", "The 4-log dilution range of cDNA deposited on the plate ensured that the amplification reaction was within the linear range and, facilitated semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.", "nGPCR-16 was expressed in the ovary, lung, prostate, bone marrow, salivary gland, heart, adrenal gland, spleen, liver, small intestine, skin, muscle, peripheral blood leukocytes, testis, placenta, fetal liver, brain, thyroid gland, kidney, pancreas, colon, uterus, and stomach.", "Within the brain, nGPCR-16 was expressed in all areas examined including the frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.", "Expression of nGPCR-16 in the brain provides an indication that modulators of nGPCR-16 activity have utility for treating neurological disorders, including but not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.", "Some other diseases for which modulators of nGPCR-16 may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.", "Use of nGPCR-16 modulators, including nGPCR-16 ligands and anti-nGPCR-16 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.", "nGPCR-40 The RapidScan™ Gene Expression Panel (OriGene Technologies, Rockville, Md.)", "was used to generate a comprehensive expression profile of the putative GPCR in human tissues.", "Human tissues arrayed include: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.", "The forward primer used was: 5′ACAGCCCCAAAGCCAAACAC3′, (SEQ ID NO: 117), and the reverse primer was: 5′CCGCAGGAGCAATGAAAATCAG3′, (SEQ ID NO: 118).", "This primer set primed the synthesis of a 220 base pair fragment in the presence of the appropriate cDNA.", "For detection of expression within brain regions, the same primer set was used with the Human Brain RapidScan™ Panel (OriGene Technologies, Rockville, Md.).", "This panel represents serial dilutions over a 2 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.", "Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.", "The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).", "The following cycling program was executed: Pre-soak at (94° C. for 3 min.)", "followed by 35 cycles of [(94° for 45 sec.)", "(54° C. for 2 min.)", "(72° for 45 sec.)].", "PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.", "The dilution range of cDNA deposited on the plates ensured that the amplification reaction was within the linear range and, hence, facilitated semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.", "nGPCR-40 was expressed in the brain, peripheral blood lymphocytes, pancreas, ovary, uterus, testis, salivary gland, kidney, adrenal gland, liver, bone marrow, prostate, fetal liver, colon, muscle, and fetal brain, may be found in many other tissues, including, but not limited to, lung, small intestine, fetal brain cord, and bone.", "Within the brain, nGPCR-40 was expressed in the frontal lobe, hypothalamus, pons, cerebellum, caudate nucleus, and medulla.", "Expression of nGPCR-40 in the brain provides an indication that modulators of nGPCR-40 activity have utility for treating neurological disorders, including but not limited to, movement disorders, affective disorders, metabolic disorders, inflammatory disorders and cancers.", "Use of nGPCR-40 modulators, including nGPCR-40 ligands and anti-nGPCR-40 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.", "nGPCR-54 Multiple Choice™ first strand cDNAs (OriGene Technologies, Rockville, Md.)", "from 12 human tissues were serially diluted over a 3-log range and arrayed into a multi-well PCR plate.", "Human tissues arrayed include: brain, heart, kidney, peripheral blood leukocytes, liver, lung, muscle, ovary, prostate, small intestine, spleen and testis.", "PCR primers were designed based on the sequence of nGPCR-54 provided herein.", "The forward primer used was: 5′CTGTCTCTCTGTCCTCTTCC3′, (SEQ ID NO: 123).", "The reverse primer used was: 5′GCACCGATCTTCATTGAATITC3′, (SEQ ID NO: 124).", "This primer set primes the synthesis of a 145 base pair fragment in the presence of the appropriate cDNA.", "For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).", "This panel represents serial dilutions over a 3 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.", "Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.", "The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).", "The following cycling program was executed: Pre-soak at (94° C. for 3 min.)", "followed by 35 cycles of [(94° C. for 45 sec.)", "(52.5° C. for 2 min.)", "(72° C. for 45 sec.)].", "PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.", "nGPCR-54 was expressed in the brain, kidney, lung, muscle, testis, heart, liver, ovary, prostate, small intestine, spleen, and peripheral blood leukocytes.", "Within the brain, nGPCR-54 was expressed in the cerebellum, hippocampus, substantia nigra, thalamus, hypothalamus, pons, frontal lobe, temporal lobe, caudate nucleus, medulla, spinal cord, and amygdala.", "Expression of the nGPCR-54 in the brain provides an indication that modulators of nGPCR-54 activity have utility for treating neurological disorders, including but not limited to, movement disorders, affective disorders, metabolic disorders, inflammatory disorders and cancers.", "Use of nGPCR-54 modulators, including nGPCR-54 ligands and anti-nGPCR-54 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.", "nGPCR-56 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.", "Human tissues arrayed include: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.", "The forward primer used was: 5′ ACTRCAAACAACTTCATACCCC 3′ (SEQ ID NO: 125), and the reverse primer used was: 5′ACACACAGCATAGTAGCG 3′ (SEQ ID NO: 126).", "This primer set will prime the synthesis of a 231 base pair fragment in the presence of the appropriate cDNA.", "For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).", "This panel represents serial dilutions over a 2 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.", "Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.", "The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).", "The following cycling program was executed: Pre-soak at (94° C. for 3 min.)", "followed by 35 cycles of [(94° C. for 45 sec.)", "(53° C. for 2 min.)", "(72° C. for 45 sec.)].", "PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.", "nGPCR-56 was expressed in peripheral blood lymphocytes, testis, salivary gland, kidney, spleen, skin, stomach, placenta, ovary, bone marrow, fetal liver, small intestine, and fetal brain.", "Expression of nGPCR-56 in the brain provides an indication that modulators of nGPCR-56 activity have utility for treating neurological disorders, including but not limited to, movement disorders, affective disorders, metabolic disorders, inflammatory disorders and cancers.", "Use of nGPCR-56 modulators, including nGPCR-56 ligands and anti-nGPCR-56 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.", "nGPCR-58 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.", "Human tissues in the array included: brain, heart, kidney, spleen, liver, lung, small intestine, muscle, testis, ovary, prostate, and PBL.", "Human brain regions in the array included: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.", "Expression of the nGPCR-58 in the various tissues was detected by using PCR primers designed based on the available sequence of the receptor that will prime the synthesis of a 282 bp fragment in the presence of the appropriate cDNA.", "The forward primer was: CAGAGCTTGATGATGAGGAC (SEQ ID NO: 127), and the reverse primer was: CCCATAGGAAGTAGTAGAAG (SEQ ID NO: 128).", "The PCR reaction mixture was added to each well of the PCR plate.", "The plate was placed in a GeneAmp PCR9700 PCR thermocycler (Perkin Elmer Applied Biosystems).", "The plate was then exposed to the following cycling parameters: Pre-soak 94° for 3 min; denaturation at 94° for 30 seconds; annealing at primer Tm for 45 seconds; extension at 72° for 2 minutes; for 35 cycles.", "PCR productions were then separated and analyzed by electrophoresis on a 1.5-% agarose gel.", "The 4-log dilution range of cDNA deposited on the plate ensured that the amplification reaction was within the linear range and, hence, facilitated semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.", "nGPCR-58 was expressed in all tissues included on the array, including brain, muscle, prostate, kidney, peripheral blood lymphocytes, liver, lung, small intestine, spleen, testis, heart, and ovary.", "Within the brain, nGPCR-58 was expressed in many regions including, but not limited to cerebellum, substantia nigra, thalamus, pons, spinal cord, frontal lobe, temporal lobe, hippocampus, caudate nucleus, amygdala, hypothalamus, and medulla.", "Expression of the nGPCR-58 in the brain provided an indication that modulators of nGPCR-58 activity have utility for treating disorders, including but not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, senile dementia, depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, metabolic disorders, inflammatory disorders, cancers and the like.", "Use of nGPCR-58 modulators, including nGPCR-58 ligands and anti-nGPCR-58 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.", "Example 5 Northern Blot Analysis Northern blots are performed to examine the expression of nGPCR-x mRNA.", "The sense orientation oligonucleotide and the antisense-orientation oligonucleotide, described above, are used as primers to amplify a portion of the GPCR-x cDNA sequence of an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.Multiple human tissue northern blots from Clontech (Human II # 7767-1) are hybridized with the probe.", "Pre-hybridization is carried out at 42 C for 4 hours in 5×SSC, 1× Denhardt's reagent, 0.1% SDS, 50% formamide, 250 mg/ml salmon sperm DNA.", "Hybridization is performed overnight at 42° C. in the same mixture with the addition of about 1.5×106 cpm/ml of labeled probe.", "The probe is labeled with α-32P-DCTP by Rediprime™ DNA labeling system (Amersham Pharmacia), purified on Nick Column (Amersham Pharmacia) and added to the hybridization solution.", "The filters are washed several times at 42 C in 0.2×SSC, 0.1% SDS.", "Filters are exposed to Kodak XAR film (Eastman Kodak Company, Rochester, N.Y., USA) with intensifying screen at −80° C. Example 6 Recombinant Expression of nGPCR-x in Eukaryotic Host Cells A.", "Expression of nGPCR-x in Mammalian Cells To produce nGPCR-x protein, a nGPCR-x-encoding polynucleotide is expressed in a suitable host cell using a suitable expression vector and standard genetic engineering techniques.", "For example, the nGPCR-x-encoding sequence described in Example 1 is subcloned into the commercial expression vector pzeoSV2 (Invitrogen, San Diego, Calif.) and transfected into Chinese Hamster Ovary (CHO) cells using the transfection reagent FuGENE6™ (Boehringer-Mannheim) and the transfection protocol provided in the product insert.", "Other eukaryotic cell lines, including human embryonic kidney (HEK 293) and COS cells, are suitable as well.", "Cells stably expressing nGPCR-x are selected by growth in the presence of 100 μg/ml zeocin (Stratagene, LaJolla, Calif.).", "Optionally, nGPCR-x may be purified from the cells using standard chromatographic techniques.", "To facilitate purification, antisera is raised against one or more synthetic peptide sequences that correspond to portions of the nGPCR-x amino acid sequence, and the antisera is used to affinity purify nGPCR-x.", "The nGPCR-x also may be expressed in-frame with a tag sequence (e.g.", "polyhistidine, hemagluttinin, FLAG) to facilitate purification.", "Moreover, it will be appreciated that many of the uses for nGPCR-x polypeptides, such as assays described below, do not require purification of nGPCR-x from the host cell.", "B.", "Expression of nGPCR-x in 293 Cells For expression of nGPCR-x in mammalian cells 293 (transformed human, primary embryonic kidney cells), a plasmid bearing the relevant nGPCR-x coding sequence is prepared, using vector pSecTag2A (Invitrogen).", "Vector pSecTag2A contains the murine IgK chain leader sequence for secretion, the c-myc epitope for detection of the recombinant protein with the anti-myc antibody, a C-terminal polyhistidine for purification with nickel chelate chromatography, and a Zeocin resistant gene for selection of stable transfectants.", "The forward primer for amplification of this GPCR cDNA is determined by routine procedures and preferably contains a 5′ extension of nucleotides to introduce the HindIII cloning site and nucleotides matching the GPCR sequence.", "The reverse primer is also determined by routine procedures and preferably contains a 5′ extension of nucleotides to introduce an XhoI restriction site for cloning and nucleotides corresponding to the reverse complement of the nGPCR-x sequence.", "The PCR conditions are 55° C. as the annealing temperature.", "The PCR product is gel purified and cloned into the HindIII-XhoI sites of the vector.", "The DNA is purified using Qiagen chromatography columns and transfected into 293 cells using DOTAP™ transfection media (Boehringer Mannheim, Indianapolis, Ind.).", "Transiently transfected cells are tested for expression after 24 hours of transfection, using western blots probed with anti-His and anti-nGPCR-x peptide antibodies.", "Permanently transfected cells are selected with Zeocin and propagated.", "Production of the recombinant protein is detected from both cells and media by western blots probed with anti-His, anti-Myc or anti-GPCR peptide antibodies.", "C. Expression of nGPCR-x in COS Cells For expression of the nGPCR-x in COS7 cells, a polynucleotide molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 can be cloned into vector p3-CI.", "This vector is a pUC18-derived plasmid that contains the HCMV (human cytomegalovirus) promoter-intron located upstream from the bGH (bovine growth hormone) polyadenylation sequence and a multiple cloning site.", "In addition, the plasmid contains the dhrf (dihydrofolate reductase) gene which provides selection in the presence of the drug methotrexane (WIX) for selection of stable transformants.", "The forward primer is determined by routine procedures and preferably contains a 5′ extension which introduces an XbaI restriction site for cloning, followed by nucleotides which correspond to an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.The reverse primer is also determined by routine procedures and preferably contains 5′-extension of nucleotides which introduces a SalI cloning site followed by nucleotides which correspond to the reverse complement of an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.The PCR consists of an initial denaturation step of 5 min at 95° C. 30 cycles of 30 sec denaturation at 95° C., 30 sec annealing at 58° C. and 30 sec extension at 72° C., followed by 5 min extension at 72° C. The PCR product is gel purified and ligated into the XbaI and SalI sites of vector p3-CI.", "This construct is transformed into E. coli cells for amplification and DNA purification.", "The DNA is purified with Qiagen chromatography columns and transfected into COS 7 cells using Lipofectamine™ reagent from BRL, following the manufacturer's protocols.", "Forty-eight and 72 hours after transfection, the media and the cells are tested for recombinant protein expression.", "nGPCR-x expressed from a COS cell culture can be purified by concentrating the cell-growth media to about 10 mg of protein/ml, and purifying the protein by, for example, chromatography.", "Purified nGPCR-x is concentrated to 0.5 mg/ml in an Amicon concentrator fitted with a YM-10 membrane and stored at −80° C. D. Expression of nGPCR-x in Insect Cells For expression of nGPCR-x in a baculovirus system, a polynucleotide molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 can be amplified by PCR.", "The forward primer is determined by routine procedures and preferably contains a 5′ extension which adds the NdeI cloning site, followed by nucleotides which correspond to an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.The reverse primer is also determined by routine procedures and preferably contains a 5′ extension which introduces the KpnI cloning site, followed by nucleotides which correspond to the reverse complement of an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.The PCR product is gel purified, digested with NdeI and KpnI, and cloned into the corresponding sites of vector pACHTL-A (Pharmingen, San Diego, Calif.).", "The pAcHTL expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV), and a 6×His tag upstream from the multiple cloning site.", "A protein kinase site for phosphorylation and a thrombin site for excision of the recombinant protein precede the multiple cloning site is also present.", "Of course, many other baculovirus vectors could be used in place of pAcHTL-A, such as pAc373, pVL941 and pAcIM1.Other suitable vectors for the expression of GPCR polypeptides can be used, provided that the vector construct includes appropriately located signals for transcription, translation, and trafficking, such as an in-frame AUG and a signal peptide, as required.", "Such vectors are described in Luckow et al., Virology 170:31-39, among others.", "The virus is grown and isolated using standard baculovirus expression methods, such as those described in Summers et al.", "(A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No.", "1555 (1987)).", "In a preferred embodiment, pAcHLT-A containing nGPCR-x gene is introduced into baculovirus using the “BaculoGold™” transfection kit (Pharmingen, San Diego, Calif.) using methods established by the manufacturer.", "Individual virus isolates are analyzed for protein production by radiolabeling infected cells with 35S-methionine at 24 hours post infection.", "Infected cells are harvested at 48 hours post infection, and the labeled proteins are visualized by SDS-PAGE.", "Viruses exhibiting high expression levels can be isolated and used for scaled up expression.", "For expression of a nGPCR-x polypeptide in a Sf9 cells, a polynucleotide molecule having the sequence of an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 can be amplified by PCR using the primers and methods described above for baculovirus expression.", "The nGPCR-x cDNA is cloned into vector pAcHLT-A (Pharmingen) for expression in Sf9 insect.", "The insert is cloned into the NdeI and KpnI sites, after elimination of an internal NdeI site (using the same primers described above for expression in baculovirus).", "DNA is purified with Qiagen chromatography columns and expressed in Sf9 cells.", "Preliminary Western blot experiments from non-purified plaques are tested for the presence of the recombinant protein of the expected size which reacted with the GPCR-specific antibody.", "These results are confirmed after further purification and expression optimization in HiG5 cells.", "Example 7 Interaction Trap/Two-Hybrid System In order to assay for nGPCR-x-interacting proteins, the interaction trap/two-hybrid library screening method can be used.", "This assay was first described in Fields et al., Nature, 1989, 340, 245, which is incorporated herein by reference in its entirety.", "A protocol is published in Current Protocols in Molecular Biology 1999, John Wiley & Sons, NY, and Ausubel, F. M et al.", "1992, Short protocols in molecular biology, Fourth edition, Greene and Wiley-interscience, NY, each of which is incorporated herein by reference in its entirety.", "Kits are available from Clontech, Palo Alto, Calif. (Matchmaker Two-Hybrid System 3).", "A fusion of the nucleotide sequences encoding all or partial nGPCR-x and the yeast transcription factor GAL4 DNA-binding domain (DNA-BD) is constructed in an appropriate plasmid (ie., pGBKT7) using standard subcloning techniques.", "Similarly, a GALA active domain (AD) fusion library is constructed in a second plasmid (i.e., pGADT7) from cDNA of potential GPCR-binding proteins (for protocols on forming cDNA libraries, see Sambrook et al.", "1989, Molecular cloning: a laboratory manual, second edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety.", "The DNA-BD/nGPCR-x fusion construct is verified by sequencing, and tested for autonomous reporter gene activation and cell toxicity, both of which would prevent a successful two-hybrid analysis.", "Similar controls are performed with the AD/library fusion construct to ensure expression in host cells and lack of transcriptional activity.", "Yeast cells are transformed (ca.", "105 transformants/mg DNA) with both the nGPCR-x and library fusion plasmids according to standard procedures (Ausubel et al., 1992, Short protocols in molecular biology, fourth edition, Greene and Wiley-interscience, NY, which is incorporated herein by reference in its entirety).", "In vivo binding of DNA-BD/nGPCR-x with AD/library proteins results in transcription of specific yeast plasmid reporter genes (i.e., lacZ, HIS3, ADE2, LEU2).", "Yeast cells are plated on nutrient-deficient media to screen for expression of reporter genes.", "Colonies are dually assayed for β-galactosidase activity upon growth in Xgal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) supplemented media (filter assay for β-galactosidase activity is described in Breeden et al., Cold Spring Harb.", "Symp.", "Quant.", "Biol., 1985, 50, 643, which is incorporated herein by reference in its entirety).", "Positive AD-library plasmids are rescued from transformants and reintroduced into the original yeast strain as well as other strains containing unrelated DNA-BD fusion proteins to confirm specific nGPCR-x/library protein interactions.", "Insert DNA is sequenced to verify the presence of an open reading frame fused to GAL4 AD and to determine the identity of the nGPCR-x-binding protein.", "Example 8 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis A gel electrophoresis mobility shift assay can rapidly detect specific protein-DNA interactions.", "Protocols are widely available in such manuals as Sambrook et al.", "1989, Molecular cloning: a laboratory manual, second edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. and Ausubel, F. M. et al., 1992, Short Protocols in Molecular Biology, fourth edition, Greene and Wiley-interscience, NY, each of which is incorporated herein by reference in its entirety.", "Probe DNA (<300 bp) is obtained from synthetic oligonucleotides, restriction endonuclease fragments, or PCR fragments and end-labeled with 32P.", "An aliquot of purified nGPCR-x (ca.", "15 μg) or crude nGPCR-x extract (ca.", "15 ng) is incubated at constant temperature (in the range 22-37 C) for at least 30 minutes in 10-15 μl of buffer (i.e.", "TAE or TBE, pH 8.0-8.5) containing radiolabeled probe DNA, nonspecific carrier DNA (ca 1 μg), BSA (300 μg/ml), and 10% (v/v) glycerol.", "The reaction mixture is then loaded onto a polyacrylamide gel and run at 30-35 mA until good separation of free probe DNA from protein-DNA complexes occurs.", "The gel is then dried and bands corresponding to free DNA and protein-DNA complexes are detected by autoradiography.", "Example 9 Antibodies to nGPCR-X Standard techniques are employed to generate polyclonal or monoclonal antibodies to the nGPCR-x receptor, and to generate useful antigen-binding fragments thereof or variants thereof, including “humanized” variants.", "Such protocols can be found, for example, in Sambrook et al.", "(1989) and Harlow et al.", "(Eds.", "), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988).", "In one embodiment, recombinant nGPCR-x polypeptides (or cells or cell membranes containing such polypeptides) are used as antigen to generate the antibodies.", "In another embodiment, one or more peptides having amino acid sequences corresponding to an immunogenic portion of nGPCR-x (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids) are used as antigen.", "Peptides corresponding to extracellular portions of nGPCR-x, especially hydrophilic extracellular portions, are preferred.", "The antigen may be mixed with an adjuvant or linked to a hapten to increase antibody production.", "A. Polyclonal or Monoclonal Antibodies As one exemplary protocol, recombinant nGPCR-x or a synthetic fragment thereof is used to immunize a mouse for generation of monoclonal antibodies (or larger mammal, such as a rabbit, for polyclonal antibodies).", "To increase antigenicity, peptides are conjugated to Keyhole Lympet Hemocyanin (Pierce), according to the manufacturer's recommendations.", "For an initial injection, the antigen is emulsified with Freund's Complete Adjuvant and injected subcutaneously.", "At intervals of two to three weeks, additional aliquots of nGPCR-x antigen are emulsified with Freund's Incomplete Adjuvant and injected subcutaneously.", "Prior to the final booster injection, a serum sample is taken from the immunized mice and assayed by western blot to confirm the presence of antibodies that immunoreact with nGPCR-x.", "Serum from the immunized animals may be used as polyclonal antisera or used to isolate polyclonal antibodies that recognize nGPCR-x.", "Alternatively, the mice are sacrificed and their spleen removed for generation of monoclonal antibodies.", "To generate monoclonal antibodies, the spleens are placed in 10 ml serum-free RPMI 1640, and single cell suspensions are formed by grinding the spleens in serum-free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada).", "The cell suspensions are filtered and washed by centrifugation and resuspended in serum-free RPMI.", "Thymocytes taken from three naive Balb/c mice are prepared in a similar manner and used as a Feeder Layer.", "NS-1 myeloma cells, kept in log phase in RPMI with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three days prior to fusion, are centrifuged and washed as well.", "To produce hybridoma fusions, spleen cells from the immunized mice are combined with NS-1 cells and centrifuged, and the supernatant is aspirated.", "The cell pellet is dislodged by tapping the tube, and 2 ml of 37° C. PEG 1500 (50% in 75 mM HEPES, pH 8.0) (Boehringer-Mannheim) is stirred into the pellet, followed by the addition of serum-free RPMI.", "Thereafter, the cells are centrifuged, resuspended in RPMI containing 15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer-Mannheim) and 1.5×106 thymocytes/ml, and plated into 10 Corning flat-bottom 96-well tissue culture plates (Corning, Corning N.Y.).", "On days 2, 4, and 6 after the fusion, 100 μl of medium is removed from the wells of the fusion plates and replaced with fresh medium.", "On day 8, the fusions are screened by ELISA, testing for the presence of mouse IgG that binds to nGPCR-x.", "Selected fusion wells are further cloned by dilution until monoclonal cultures producing anti-nGPCR-x antibodies are obtained.", "B. Humanization of Anti-nGPCR-x Monoclonal Antibodies The expression pattern of nGPCR-x as reported herein and the proven track record of GPCRs as targets for therapeutic intervention suggest therapeutic indications for nGPCR-x inhibitors (antagonists).", "nGPCR-x-neutralizing antibodies comprise one class of therapeutics useful as nGPCR-x antagonists.", "Following are protocols to improve the utility of anti-nGPCR-x monoclonal antibodies as therapeutics in humans by “humanizing” the monoclonal antibodies to improve their serum half-life and render them less immunogenic in human hosts (i.e., to prevent human antibody response to non-human anti-nGPCR-x antibodies).", "The principles of humanization have been described in the literature and are facilitated by the modular arrangement of antibody proteins.", "To minimize the possibility of binding complement, a humanized antibody of the IgG4 isotype is preferred.", "For example, a level of humanization is achieved by generating chimeric antibodies comprising the variable domains of non-human antibody proteins of interest with the constant domains of human antibody molecules.", "(See, e.g., Morrison et al., Adv.", "Immunol., 44:65-92 (1989)).", "The variable domains of nGPCR-x-neutralizing anti-nGPCR-x antibodies are cloned from the genomic DNA of a B-cell hybridoma or from cDNA generated from mRNA isolated from the hybridoma of interest.", "The V region gene fragments are linked to exons encoding human antibody constant domains, and the resultant construct is expressed in suitable mammalian host cells (e.g., myeloma or CHO cells).", "To achieve an even greater level of humanization, only those portions of the variable region gene fragments that encode antigen-binding complementarity determining regions (“CDR”) of the non-human monoclonal antibody genes are cloned into human antibody sequences.", "(See, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-36 (1988); and Tempest et al., Bio/Technology 9: 266-71 (1991)).", "If necessary, the P-sheet framework of the human antibody surrounding the CDR3 regions also is modified to more closely mirror the three dimensional structure of the antigen-binding domain of the original monoclonal antibody.", "(See Kettleborough et al., Protein Engin., 4:773-783 (1991); and Foote et al., J. Mol.", "Biol., 224:487-499 (1992)).", "In an alternative approach, the surface of a non-human monoclonal antibody of interest is humanized by altering selected surface residues of the non-human antibody, e.g., by site-directed mutagenesis, while retaining all of the interior and contacting residues of the non-human antibody.", "See Padlan, Molecular Immunol., 28(4/5):489-98 (1991).", "The foregoing approaches are employed using nGPCR-x-neutralizing anti-nGPCR-x monoclonal antibodies and the hybridomas that produce them to generate humanized nGPCR-x-neutralizing antibodies useful as therapeutics to treat or palliate conditions wherein nGPCR-x expression or ligand-mediated nGPCR-x signaling is detrimental.", "C. Human nGPCR-x-Neutralizing Antibodies from Phage Display Human nGPCR-x-neutralizing antibodies are generated by phage display techniques such as those described in Aujame et al., Human Antibodies 8(4):155-168 (1997); Hoogenboom, TIBTECH 15:62-70 (1997); and Rader et al., Curr.", "Opin.", "Biotechnol.", "8:503-508 (1997), all of which are incorporated by reference.", "For example, antibody variable regions in the form of Fab fragments or linked single chain Fv fragments are fused to the amino terminus of filamentous phage minor coat protein pIII.", "Expression of the fusion protein and incorporation thereof into the mature phage coat results in phage particles that present an antibody on their surface and contain the genetic material encoding the antibody.", "A phage library comprising such constructs is expressed in bacteria, and the library is screened for nGPCR-x-specific phage-antibodies using labeled or immobilized nGPCR-x as antigen-probe.", "D. Human nGPCR-x-Neutralizing Antibodies from Transgenic Mice Human nGPCR-x-neutralizing antibodies are generated in transgenic mice essentially as described in Bruggemann et al., Immunol.", "Today 17(8):391-97 (1996) and Bruggemann et al., Curr.", "Opin.", "Biotechnol.", "8:455-58 (1997).", "Transgenic mice carrying human V-gene segments in germline configuration and that express these transgenes in their lymphoid tissue are immunized with a nGPCR-x composition using conventional immunization protocols.", "Hybridomas are generated using B cells from the immunized mice using conventional protocols and screened to identify hybridomas secreting anti-nGPCR-x human antibodies (e.g., as described above).", "Example 10 Assays to Identify Modulators of nGPCR-x Activity Set forth below are several nonlimiting assays for identifying modulators (agonists and antagonists) of nGPCR-x activity.", "Among the modulators that can be identified by these assays are natural ligand compounds of the receptor, synthetic analogs and derivatives of natural ligands; antibodies, antibody fragments, and/or antibody-like compounds derived from natural antibodies or from antibody-like combinatorial libraries; and/or synthetic compounds identified by high-throughput screening of libraries; and the like.", "All modulators that bind nGPCR-x are useful for identifying nGPCR-x in tissue samples (e.g., for diagnostic purposes, pathological purposes, and the like).", "Agonist and antagonist modulators are useful for up-regulating and down-regulating nGPCR-x activity, respectively, to treat disease states characterized by abnormal levels of nGPCR-x activity.", "The assays may be performed using single putative modulators, and/or may be performed using a known agonist in combination with candidate antagonists (or visa versa).", "A. cAMP Assays In one type of assay, levels of cyclic adenosine monophosphate (cAMP) are measured in nGPCR-x-transfected cells that have been exposed to candidate modulator compounds.", "Protocols for cAMP assays have been described in the literature.", "(See, e.g., Sutherland et al., Circulation 37: 279 (1968); Frandsen et al., Life Sciences 18: 529-541 (1976); Dooley et al., Journal of Pharmacology and Experimental Therapeutics 283 (2): 735-41 (1997); and George et al., Journal of Biomolecular Screening 2 (4): 23540 (1997)).", "An exemplary protocol for such an assay, using an Adenylyl Cyclase Activation FlashPlate® Assay from NEN™ Life Science Products, is set forth below.", "Briefly, the nGPCR-x coding sequence (e.g., a cDNA or intronless genomic DNA) is subcloned into a commercial expression vector, such as pzeoSV2 (Invitrogen), and transiently transfected into Chinese Hamster Ovary (CHO) cells using known methods, such as the transfection protocol provided by Boehringer-Mannheim when supplying the FuGENE 6 transfection reagent.", "Transfected CHO cells are seeded into 96-well microplates from the FlashPlate® assay kit, which are coated with solid scintillant to which antisera to cAMP has been bound.", "For a control, some wells are seeded with wild type (untransfected) CHO cells.", "Other wells in the plate receive various amounts of acAMP standard solution for use in creating a standard curve.", "One or more test compounds (i.e., candidate modulators) are added to the cells in each well, with water and/or compound-free medium/diluent serving as a control or controls.", "After treatment, cAMP is allowed to accumulate in the cells for exactly 15 minutes at room temperature.", "The assay is terminated by the addition of lysis buffer containing [125I]-labeled cAMP, and the plate is counted using a Packard Topcount™ 96-well microplate scintillation counter.", "Unlabeled cAMP from the lysed cells (or from standards) and fixed amounts of [125I]-cAMP compete for antibody bound to the plate.", "A standard curve is constructed, and cAMP values for the unknowns are obtained by interpolation.", "Changes in intracellular cAMP levels of cells in response to exposure to a test compound are indicative of nGPCR-x modulating activity.", "Modulators that act as agonists of receptors which couple to the Gs subtype of G proteins will stimulate production of cAMP, leading to a measurable 3-10 fold increase in cAMP levels.", "Agonists of receptors which couple to the Gi/o subtype of G proteins will inhibit forskolin-stimulated cAMP production, leading to a measurable decrease in cAMP levels of 50-100%.", "Modulators that act as inverse agonists will reverse these effects at receptors that are either constitutively active or activated by known agonists.", "B. Aequorin Assays In another assay, cells (e.g., CHO cells) are transiently co-transfected with both a nGPCR-x expression construct and a construct that encodes the photoprotein apoaquorin.", "In the presence of the cofactor coelenterazine, apoaquorin will emit a measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium.", "(See generally, Cobbold, et al.", "“Aequorin measurements of cytoplasmic free calcium,” In: McCormack J. G. and Cobbold P. H., eds., Cellular Calcium: A Practical Approach.", "Oxford:IRL Press (1991); Stables et al., Analytical Biochemistry 252: 115-26 (1997); and Haugland, Handbook of Fluorescent Probes and Research Chemicals.", "Sixth edition.", "Eugene Oreg.", ": Molecular Probes (1996).)", "In one exemplary assay, nGPCR-x is subcloned into the commercial expression vector pzeoSV2 (Invitrogen) and transiently co-transfected along with a construct that encodes the photoprotein apoaquorin (Molecular Probes, Eugene, Oreg.)", "into CHO cells using the transfection reagent FuGENE 6 (Boehringer-Mannheim) and the transfection protocol provided in the product insert.", "The cells are cultured for 24 hours at 37° C. in MEM (Gibco/BRL, Gaithersburg, Md.)", "supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin, at which time the medium is changed to serum-free MEM containing 5 μM coelenterazine (Molecular Probes, Eugene, Oreg.).", "Culturing is then continued for two additional hours at 37° C. Subsequently, cells are detached from the plate using VERSEN (Gibco/BRL), washed, and resuspended at 200,000 cells/ml in serum-free MEM.", "Dilutions of candidate nGPCR-x modulator compounds are prepared in serum-free MEM and dispensed into wells of an opaque 96-well assay plate at 50 μl/well.", "Plates are then loaded onto an MLX microtiter plate luminometer (Dynex Technologies, Inc., Chantilly, Va.).", "The instrument is programmed to dispense 50 μl cell suspensions into each well, one well at a time, and immediately read luminescence for 15 seconds.", "Dos response curves for the candidate modulators are constructed using the area under the curve for each light signal peak.", "Data are analyzed with SlideWrite, using the equation for a one-site ligand, and EC50 values are obtained.", "Changes in luminescence caused by the compounds are considered indicative of modulatory activity.", "Modulators that act as agonists at receptors which couple to the Gq subtype of G proteins give an increase in luminescence of up to 100 fold.", "Modulators that act as inverse agonists will reverse this effect at receptors that are either constitutively active or activated by known agonists.", "C. Luciferase Reporter Gene Assay The photoprotein luciferase provides another useful tool for assaying for modulators of nGPCR-x activity.", "Cells (e.g., CHO cells or COS 7 cells) are transiently co-transfected with both a nGPCR-x expression construct (e.g., nGPCR-x in pzeoSV2) and a reporter construct which includes a gene for the luciferase protein downstream from a transcription factor binding site, such as the cAMP-response element (CRE), AP-1, or NF-kappa B. Agonist binding to receptors coupled to the G. subtype of G proteins leads to increases in cAMP, thereby activating the CRE transcription factor and resulting in expression of the luciferase gene.", "Agonist binding to receptors coupled to the Gq subtype of G protein leads to production of diacylglycerol that activates protein kinase C, which activates the AP-1 or NF-kappa B transcription factors, in turn resulting in expression of the luciferase gene.", "Expression levels of luciferase reflect the activation status of the signaling events.", "(See generally, George et al, Journal of Biomolecular Screening 2(4): 235-240 (1997); and Stratowa et al, Current Opinion in Biotechnology 6: 574-581 (1995)).", "Luciferase activity may be quantitatively measured using, e.g., luciferase assay reagents that are commercially available from Promega (Madison, Wis.).", "In one exemplary assay, CHO cells are plated in 24-well culture dishes at a density of 100,000 cells/well one day prior to transfection and cultured at 37° C. in MEM (Gibco/BRL) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin.", "Cells are transiently co-transfected with both a nGPCR-x expression construct and a reporter construct containing the luciferase gene.", "The reporter plasmids CRE-luciferase, AP-1-luciferase and NF-kappaB-luciferase may be purchased from Stratagene (LaJolla, Calif.).", "Transfections are performed using the FuGENE 6 transfection reagent (Boehringer-Mannheim) according to the supplier's instructions.", "Cells transfected with the reporter construct alone are used as a control.", "Twenty-four hours after transfection, cells are washed once with PBS pre-warmed to 37° C. Serum-free MEM is then added to the cells either alone (control) or with one or more candidate modulators and the cells are incubated at 37° C. for five hours.", "Thereafter, cells are washed once with ice-cold PBS and lysed by the addition of 100 μl of lysis buffer per well from the luciferase assay kit supplied by Promega.", "After incubation for 15 minutes at room temperature, 15 μl of the lysate is mixed with 50 μl of substrate solution (Promega) in an opaque-white, 96-well plate, and the luminescence is read immediately on a Wallace model 1450 MicroBeta scintillation and luminescence counter (Wallace Instruments, Gaithersburg, Md.).", "Differences in luminescence in the presence versus the absence of a candidate modulator compound are indicative of modulatory activity.", "Receptors that are either constitutively active or activated by agonists typically give a 3 to 20-fold stimulation of luminescence compared to cells transfected with the reporter gene alone.", "Modulators that act as inverse agonists will reverse this effect.", "D. Intracellular Calcium Measurement Using FLIPR Changes in intracellular calcium levels are another recognized indicator of G protein-coupled receptor activity, and such assays can be employed to screen for modulators of nGPCR-x activity.", "For example, CHO cells stably transfected with a nGPCR-x expression vector are plated at a density of 4×104 cells/well in Packard black-walled, 96-well plates specially designed to discriminate fluorescence signals emanating from the various wells on the plate.", "The cells are incubated for 60 minutes at 37° C. in modified Dulbecco's PBS (D-PBS) containing 36 mg/L pyruvate and 1 g/L glucose with the addition of 1% fetal bovine serum and one of four calcium indicator dyes (Fluo-3™ AM, Fluo-4™ AM, Calcium Green™-1 AM, or Oregon Green™ 488 BAPTA-1 AM), each at a concentration of 4 μM.", "Plates are washed once with modified D-PBS without 1% fetal bovine serum and incubated for 10 minutes at 37° C. to remove residual dye from the cellular membrane.", "In addition, a series of washes with modified D-PBS without 1% fetal bovine serum is performed immediately prior to activation of the calcium response.", "A calcium response is initiated by the addition of one or more candidate receptor agonist compounds, calcium ionophore A23187 (10 μM; positive control), or ATP (4 μM; positive control).", "Fluorescence is measured by Molecular Device's FLIPR with an argon laser (excitation at 488 nm).", "(See, e.g., Kuntzweiler et al., Drug Development Research, 44(1): 14-20 (1998)).", "The F-stop for the detector camera was set at 2.5 and the length of exposure was 0.4 milliseconds.", "Basal fluorescence of cells was measured for 20 seconds prior to addition of candidate agonist, ATP, or A23187, and the basal fluorescence level was subtracted from the response signal.", "The calcium signal is measured for approximately 200 seconds, taking readings every two seconds.", "Calcium ionophore A23187 and ATP increase the calcium signal 200% above baseline levels.", "In general, activated GPCRs increase the calcium signal approximately 10-15% above baseline signal.", "E. Mitogenesis Assay In a mitogenesis assay, the ability of candidate modulators to induce or inhibit nGPCR-x-mediated cell division is determined.", "(See, e.g., Lajiness et al., Journal of Pharmacology and Experimental Therapeutics 267(3): 1573-1581 (1993)).", "For example, CHO cells stably expressing nGPCR-x are seeded into 96-well plates at a density of 5000 cells/well and grown at 37° C. in MEM with 10% fetal calf serum for 48 hours, at which time the cells are rinsed twice with serum-free MEM.", "After rinsing, 80 μl of fresh MEM, or MEM containing a known mitogen, is added along with 20 μl MEM containing varying concentrations of one or more candidate modulators or test compounds diluted in serum-free medium.", "As controls, some wells on each plate receive serum-free medium alone, and some receive medium containing 10% fetal bovine serum.", "Untransfected cells or cells transfected with vector alone also may serve as controls.", "After culture for 16-18 hours, 1 μCi of [3H]-thymidine (2 Ci/mmol) is added to the wells and cells are incubated for an additional 2 hours at 37° C. The cells are trypsinized and collected on filter mats with a cell harvester (Tomtec); the filters are then counted in a Betaplate counter.", "The incorporation of [3H]-thymidine in serum-free test wells is compared to the results achieved in cells stimulated with serum (positive control).", "Use of multiple concentrations of test compounds permits creation and analysis of dose-response curves using the non-linear, least squares fit equation: A=B×[C/(D+C)]+G where A is the percent of serum stimulation; B is the maximal effect minus baseline; C is the EC50; D is the concentration of the compound; and G is the maximal effect.", "Parameters B, C and G are determined by Simplex optimization.", "Agonists that bind to the receptor are expected to increase [3H]-thymidine incorporation into cells, showing up to 80% of the response to serum.", "Antagonists that bind to the receptor will inhibit the stimulation seen with a known agonist by up to 100%.", "F. [35S]GTPγS Binding Assay Because G protein-coupled receptors signal through intracellular G proteins whose activity involves GTP binding and hydrolysis to yield bound GDP, measurement of binding of the non-hydrolyzable GTP analog [35S]GTPγS in the presence and absence of candidate modulators provides another assay for modulator activity.", "(See, e.g., Kowal et al., Neuropharmacology 37:179-187 (1998).)", "In one exemplary assay, cells stably transfected with a nGPCR-x expression vector are grown in 10 cm tissue culture dishes to subconfluence, rinsed once with 5 ml of ice-cold Ca2+/Mg2+-free phosphate-buffered saline, and scraped into 5 ml of the same buffer.", "Cells are pelleted by centrifugation (500×g, 5 minutes), resuspended in TEE buffer (25 mM Tris, pH 7.5, 5 in M EDTA, 5 mM EGTA), and frozen in liquid nitrogen.", "After thawing, the cells are homogenized using a Dounce homogenizer (one ml TEE per plate of cells), and centrifuged at 1,000×g for 5 minutes to remove nuclei and unbroken cells.", "The homogenate supernatant is centrifuged at 20,000×g for 20 minutes to isolate the membrane fraction, and the membrane pellet is washed once with TEE and resuspended in binding buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA).", "The resuspended membranes can be frozen in liquid nitrogen and stored at −70° C. until use.", "Aliquots of cell membranes prepared as described above and stored at −70° C. are thawed, homogenized, and diluted into buffer containing 20 mM HEPES, 10 mM MgCl, 1 mM EDTA, 120 mM NaCl, 10 μM GDP, and 0.2 mM ascorbate, at a concentration of 10-50 μg/ml.", "In a final volume of 90 μl, homogenates are incubated with varying concentrations of candidate modulator compounds or 100 μM GTP for 30 minutes at 30° C. and then placed on ice.", "To each sample, 10 μl guanosine 5′-O-(3[35S]thio) triphosphate (NEN, 1200 Ci/mmol; [35S]-GTPγS), was added to a final concentration of 100-200 pM.", "Samples are incubated at 30° C. for an additional 30 minutes, 1 ml of 10 mM HEPES, pH 7.4, 10 mM MgCl2, at 4° C. is added and the reaction is stopped by filtration.", "Samples are filtered over Whatman GF/B filters and the filters are washed with 20 ml ice-cold 10 mM HEPES, pH 7.4, 10 mM MgCl2.Filters are counted by liquid scintillation spectroscopy.", "Nonspecific binding of [35S]-GTPγS is measured in the presence of 100 μM GTP and subtracted from the total.", "Compounds are selected that modulate the amount of [35]-GTPγS binding in the cells, compared to untransfected control cells.", "Activation of receptors by agonists gives up to a five-fold increase in [35S]GTPγS binding.", "This response is blocked by antagonists.", "G. MAP Kinase Activity Assay Evaluation of MAP kinase activity in cells expressing a GPCR provides another assay to identify modulators of GPCR activity.", "(See, e.g.", "Lajiness et al., Journal of Pharmacology and Experimental Therapeutics 267(3):1573-1581 (1993) and Boulton et al., Cell 65:663-675 (1991).)", "In one embodiment, CHO cells stably transfected with nGPCR-x are seeded into 6-well plates at a density of 70,000 cells/well 48 hours prior to the assay.", "During this 48-hour period, the cells are cultured at 37° C. in MEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin.", "The cells are serum-starved for 1-2 hours prior to the addition of stimulants.", "For the assay, the cells are treated with medium alone or medium containing either a candidate agonist or 200 nM Phorbol ester-myristoyl acetate (i.e., PMA, a positive control), and the cells are incubated at 37° C. for varying times.", "To stop the reaction, the plates are placed on ice, the medium is aspirated, and the cells are rinsed with 1 ml of ice-cold PBS containing 1 mM EDTA.", "Thereafter, 200 μl of cell lysis buffer (12.5 mM MOPS, pH 7.3, 12.5 mM glycerophosphate, 7.5 mM MgCl2, 0.5 MM EGTA, 0.5 mM sodium vanadate, 1 mM benzamidine, 1 mM dithiothreitol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 2 μg/ml pepstatin A, and 1 μM okadaic acid) is added to the cells.", "The cells are scraped from the plates and homogenized by 10 passages through a 23 3/4 G needle, and the cytosol fraction is prepared by centrifugation at 20,000×g for 15 minutes.", "Aliquots (5-10 μl containing 1-5 μg protein) of cytosol are mixed with 1 mM MAPK Substrate Peptide (APRTPGGRR (SEQ ID NO: 129), Upstate Biotechnology, Inc., N.Y.) and 50 μM [γ-32P]ATP (NEN, 3000 Ci/mmol), diluted to a final specific activity of ˜2000 cpm/pmol, in a total volume of 25 μl.", "The samples are incubated for 5 minutes at 30° C., and reactions are stopped by spotting 20 μl on 2 cm2 squares of Whatman P81 phosphocellulose paper.", "The filter squares are washed in 4 changes of 1% H3PO4, and the squares are subjected to liquid scintillation spectroscopy to quantitate bound label.", "Equivalent cytosolic extracts are incubated without MAPK substrate peptide, and the bound label from these samples are subtracted from the matched samples with the substrate peptide.", "The cytosolic extract from each well is used as a separate point.", "Protein concentrations are determined by a dye binding protein assay (Bio-Rad Laboratories).", "Agonist activation of the receptor is expected to result in up to a five-fold increase in MAPK enzyme activity.", "This increase is blocked by antagonists.", "H. [3H]Anachidonic Acid Release The activation of GPCRs also has been observed to potentiate arachidonic acid release in cells, providing yet another useful assay for modulators of GPCR activity.", "(See, e.g., Kanterman et al., Molecular Pharmacology 39:364-369 (1991).)", "For example, CHO cells that are stably transfected with a nGPCR-x expression vector are plated in 24-well plates at a density of 15,000 cells/well and grown in MEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin for 48 hours at 37° C. before use.", "Cells of each well are labeled by incubation with [3H]-arachidonic acid (Amersham Corp., 210 Ci/mmol) at 0.5 μCi/ml in 1 ml MEM supplemented with 10 mM HEPES, pH 7.5, and 0.5% fatty-acid-free bovine serum albumin for 2 hours at 37° C. The cells are then washed twice with 1 ml of the same buffer.", "Candidate modulator compounds are added in 1 ml of the same buffer, either alone or with 10 μM ATP and the cells are incubated at 37° C. for 30 minutes.", "Buffer alone and mock-transfected cells are used as controls.", "Samples (0.5 ml) from each well are counted by liquid scintillation spectroscopy.", "Agonists which activate the receptor will lead to potentiation of the ATP-stimulated release of [3H]-arachidonic acid.", "This potentiation is blocked by antagonists.", "I. Extracellular Acidification Rate In yet another assay, the effects of candidate modulators of nGFCR-x activity are assayed by monitoring extracellular changes in pH induced by the test compounds.", "(See, e.g., Dunlop et al, Journal of Pharmacological and Toxicological Methods 40(1):47-55 (1998).)", "In one embodiment, CHO cells transfected with a nGPCR-x expression vector are seeded into 12 mm capsule cups (Molecular Devices Corp.) at 4×105 cells/cup in MEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 U/ml penicillin, and 10 μg/ml streptomycin.", "The cells are incubated in this medium at 37° C. in 5% CO2 for 24 hours.", "Extracellular acidification rates are measured using a Cytosensor microphysiometer (Molecular Devices Corp.).", "The capsule cups are loaded into the sensor chambers of the microphysiometer and the chambers are perfused with running buffer (bicarbonate-free MEM supplemented with 4 mM L-glutamine, 10 units/ml penicillin, 10 μg/ml streptomycin, 26 mM NaCl) at a flow rate of 100 μl/minute.", "Candidate agonists or other agents are diluted into the running buffer and perfused through a second fluid path.", "During each 60-second pump cycle, the pump is run for 38 seconds and is off for the remaining 22 seconds.", "The pH of the running buffer in the sensor chamber is recorded during the cycle from 43-58 seconds, and the pump is re-started at 60 seconds to start the next cycle.", "The rate of acidification of the running buffer during the recording time is calculated by the Cytosoft program.", "Changes in the rate of acidification are calculated by subtracting the baseline value (the average of 4 rate measurements immediately before addition of a modulator candidate) from the highest rate measurement obtained after addition of a modulator candidate.", "The selected instrument detects 61 mV/pH unit.", "Modulators that act as agonists of the receptor result in an increase in the rate of extracellular acidification compared to the rate in the absence of agonist.", "This response is blocked by modulators which act as antagonists of the receptor.", "Example 11 In Situ Hybridization DNA Probe Preparation for nGPCR-11, -16, 40, -54, and -56 DNA probes for in situ hybridization were prepared as follows.", "Two sets of primer pairs were prepared.", "The first set has the sequence for T7 polymerase promoter on the 5′ primer to make the sense RNA, and the second set has the T7 polymerase promoter sequence on the 3′ primer to make the antisense RNA.", "PCR was performed in a 50 μl reaction containing 36.5 μl H2O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatine, 0.6 M Tris-tricine pH 8.4), 5 μl 25 mM MgCl2, 2 μl 10 mM dNTP, 0.4 μl Incyte clone 1722192 DNA, 0.5 μl AmpliTaq (PE Applied Biosystems), and 0.3 μl oligo1 (1 mg/ml) and 0.3 μl oligo2 (1 mg/ml) [to make the sense RNA], or 0.3 μl oligo3 (1 mg/ml) and 0.3 μl oligo4 (1 mg/ml) [to make the antisense RNA].", "The PCR reaction involved one cycle at 94° C. for 2 min followed by 35 cycles at 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 30 sec.", "The two PCR reactions were loaded onto a 1.2% agarose gel.", "The DNA band was excised from the gel, placed in a GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed.", "The eluted DNA was EtOH precipitated and resuspended in transcription buffer.", "The primer sequences for each nGPCR tested are listed below.", "For nGPCR-11, the sense primers were: GCGTAATACGACTCACTATAGGGAGACCGCGTGTCTGCTAGACTCTATTTCC 3′(LW1658) (SEQ ID NO: 159), and: 5′ TGCCACACTGATGCAACTCC 3′ (LW661) (SEQ ID NO: 160).", "The antisense primers were: GCGTAATACGACTCACTATAGGGAGACCTGCCACACTGATGCAACTCC (LW1659) SEQ ID NO: 161) and: 5′GCGTGTCTGCTAGACTCTATITCC 3′ (LW1660) (SEQ ID NO: 162).", "The primer pairs yielded a product of 275 bp.", "For nGPCR-16, the sense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCGCACGCCACTCTTTACTATCCC (LW1645) (SEQ ID NO: 163), and: 5′ GCACAAAACACAATFCCATAAGCC 3′ (LW1648) (SEQ ID NO: 164).", "The antisense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCGCACAAAACACAATTCCATAAGCC 3′ (LW1646) (SEQ ID NO: 165), and: 5′ GCTACGCCACTCTTrACTATCCC 3′ (LW1647) (SEQ ID NO: 166).", "The primer pairs yielded a product of 283 bp.", "For nGPCR-40, the sense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCTTATGAGCAGCAATTCATCCC 3′(LW1704) (SEQ ID NO: 167), and: 5′CACACCCACCAAGAAATCAG 3′(LW1707) (SEQ ID NO: 168).", "The antisense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCCACACCCACCAAGAAATCAG 3′(LW1705) (SEQ ID NO: 169), and: 5′ TTATGAGCAGCAATTCATCCC 3′ (LW1706) (SEQ ID NO: 170).", "The primer pairs yielded a product of 25 lbp.", "For nGPCR-54, the sense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCCGATTATCCACACTTTGACCC 3′ (LW1803) (SEQ ID NO: 171), and: 5′CTGAAAGTrGTCGCTGACC 3′ (LW1634) (SEQ ID NO: 172).", "The anti-sense primers were: GCGTAATACGACTCACTATAGGGAGACCCTGCTGAAAGTTGTCGCTGACC 3′ (LW1804) (SEQ ID NO: 173), and: 5′CGATTATCCACACTTTGACGC 3′ (LW1635) (SEQ ID NO: 174).", "The primer pairs yielded a product of 286 bp.", "For nGPCR-56, the sense primers were: GCGTAATACGACTCACTATAGGGAGACCCTGTAAAATTCACACAAGCACC 3′ (LW1763) (SEQ ID NO: 175), and: 5′AGAAGACAGAGCAACCTCC 3′ (L[W1766) (SEQ ID NO: 176).", "The anti-sense primers were: GCGTAATACGACTCACTATAGGGAGACCAGAAGACAGAGCAACCTCC (LW1764) (SEQ ID NO: 177) and: CTGTAAAATTCACACAAGCACC (LW1765) (SEQ ID NO: 178).", "The primer pairs yielded a product of 272 bp.", "DNA Probe Preparation For nGPCR-1 Probes for nGPCR-1 were prepared as above with the following modifications.", "Using a sense primer: GCATGGATCCTCTTTTGCTGTATTTCACCCTC) (LW1595) (SEQ ID NO: 179) and an antisense primer: 5′GCATGAATTCACAATGCCAGTGATAAGGAAG 3′ (LW1596) (SEQ ID NO: 180), a 271 bp fragment was generated by PCR.", "The fragment was digested with BamHI and EcoRI and ligated into a BluescriptII vector that had been cut with BamHI and EcoRI.", "The orientation of the insert was such that T7 polymerase generates the anti-sense strand and T3 polymerase generates the sense strand.", "Histochemistry Coronal and sagittal oriented rat brain sections were cryosectioned (20 μm thick) using a Reichert-Jung cryostat.", "The individual sections were thaw-mounted onto silanated, nuclease-free slides (CEL Associates, Inc., Houston, Tex.", "), and stored at −80° C. The sections were processed starting with post-fixation in cold 4% paraformaldehyde, rinsed in cold PBS, acetylated using acetic anhydride in triethanolamine buffer and dehydrated through 70%, 95%, and 100% alcohols at room temperature (RT).", "This was followed with delipidation in chloroform then rehydration in 100% and 95% alcohol at room temperature.", "Sections were air-dried prior to hybridization.", "Two PCR fragments (˜250 bp) were generated, one that contained T7 polymerase on the 5′ end (sense) and the other with T7 polymerase on the 3′ end (antisense).", "The PCR fragments were labeled with 35S-UTP to yield a specific activity of 0.655×106 cpm/pmol for antisense and 0.675×106 cpm/pmol for sense probe.", "Both riboprobes were denatured and added to hybridization buffer containing 50% formamide, 10% dextran, 0.3M NaCl, 10 mM Tris, 1 mM EDTA, 1× Denhardts, and 10 mM DTT.", "Sequential brain cryosections were hybridized with 45 μl/slide of the sense and antisense riboprobe hybridization mixture, then covered with silanized glass coverslips.", "The sections were hybridized overnight (15-18 hrs) at 42° C. in an incubator.", "Coverslips were washed off the slides in 1×SSC, followed by RNase A treatment, and high temperature stringency washes (3×, 20 mins at 41° C.) in 0.1×SSC.", "Slides were dehydrated with 70%, 95% NH4OAc, and 100% NH4OAc alcohols, air-dried and exposed to Kodak BioMax MR-1 film.", "After 9 days of exposure, the film was developed.", "This was followed with coating selected tissue slides with Kodak NTB-2 nuclear track emulsion and storing the slides in the dark for 23 days.", "The slides were then developed and counterstained with hematoxylin.", "Emulsion-coated sections were analyzed microscopically to determine the specificity of labeling.", "Presence of autoradiographic grains (generated by antisense probe hybridization) over cell bodies (versus between cell bodies) was used as an index of specific hybridization.", "Results In Situ Hybridization Results Indicated Localization in the Following Brain Areas: nGPCR-1 was localized to the dentate gyrus of hippocampus, piriform cortex, and red nucleus.", "nGPCR-11 was localized to the piriform cortex, hippocampus, red nucleus, subthalamic nuclei, dorsal raphe, interpeduncular nucleus, and habenula.", "nGPCR-16 was localized to the cortex, piriform cortex, hippocampus, thalamus, subthalamic nuclei, hypothalamus, bed nucleus stria terminals and posterior striatum.", "nGPCR-40 was localized to the cortex, piriform cortex, hippocampus, substantia nigra compacta, hypothalamus, laterial septus, bed nucleus stria terminalis, thalamus, ventral tegmental area, interpeduncular nucleus, dorsal raphe, medical geniculate, islands of Calleja, subthamalmic nuclei, choroid plexus.", "nGPCR-54 was localized to the piriform cortex and hippocampus, including the dentate gyms, CA1 and CA3.nGPCR-56 was localized to the piriform cortex, cortex, interpeduncular nuceus, red nucleus, hippocampus, habenula, substantia nigra pars compacta, mamillary body stria terminalis, hypothalamus, subthamalmic nuclei, corsal raphe, and ventral tegmental area.", "Example 12 Chromosomal Localization Methods Chromosomal location of the genes encoding nGPCRs was determined using the Stanford G3 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, Ala.).", "This panel contains 83 radiation hybrid clones of the entire human genome created by the Stanford Human Genome Center.", "PCR reactions were assembled containing 25 ng of DNA from each clone and the components of the Expand Hi-Fi PCR System™ (Roche Molecular Biochemicals, Indianapolis, Ind.)", "in a final reaction volume of 15 μl.", "PCR primers were synthesized by Genosys Corp., The Woodlands, Tex.", "PCR reactions were incubated in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).", "The following cycling program was executed: Pre-soak at (940 for 3 min.)", "(94° for 30 sec.)", "(52° C. for 60 sec.)", "(72′ for 2 min.)]", "for 35 cycles.", "PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel, and stained with ethidium bromide.", "Lanes were scored for the presence or absence of the expected PCR product and the results submitted to the Stanford Human Genome Center via e-mail for analysis (http://wwwshgc.stanford.edu./RH/rhserverformnew.html).", "nGPCR-40 PCR primers were designed based on the available sequence of the Celera sequence HUM_IDS\|Contig\|11000258115466.The forward primer used was: 5′ACAGCCCCAAAGCCAAACAC3′ (SEQ ID NO: 181).", "The reverse primer was: 5′CCGCAGGAGCAATG-AAAATCAG3′ (SEQ ID NO: 182).", "This prier set will prime the synthesis of a 220 base pair fragment in the presence of the appropriate genomic DNA.", "G3 Radiation Hybrid Panel Analysis places nGPCR-40 on chromosome 6, most nearly linked to Stanford marker SHGC-1836 with a LOD score of 11.84.This marker lies at position 6q21.In a genome scanning data set, Cao et al.", "(Genomics 1997 Jul.", "1: 43(1): 1-8) found excess allele sharing for markers on 6q13-q26.Greatest allele sharing was at interval 6q21-q22.3 with a maximum multipoint MLS value of 3.06 close to marker D6S278.Replication data from a second data set found maximum multipoint MLS at the interval D6S424-D6S275.These results provide suggestive evidence for a susceptibility locus for schizophrenia in chromosome 6q from two independent data sets.", "nGPCR-54 PCR primers were designed based on the available sequence of the Celera sequence GA—11824020.The forward primer used was: 5′CTGTCTCTCTGTCCTCTTCC3′, (SEQ ID NO: 183).", "The reverse primer used was: 5′GCACCGATCTTCATTGAATTTC3′, (SEQ ID NO: 184).", "This primer set will prime the synthesis of a 145 base pair fragment in the presence of the appropriate genomic DNA.", "G3 Radiation Hybrid Panel Analysis places nGPCR-54 on chromosome 13, most nearly linked to Stanford marker SHGC-68276 with a LOD score of 6.31.This marker lies at position 13q32.Numerous investigations have found significant suggestion of linkage of schizophrenia to this region of chromosome 13q32.See, for example, Brzustowicz et al., Am J Hum Genet 1999 October; 65(4): 1096-1103; Blouinet al., Nat Genet 1998 September; 20(1): 70-3; Shaw et al., Am J Med Genet.", "1998 Sep. 7; 81(5): 364-76; Lin et al., Hum Genet 1997 March, 99(3): 417-20; Pulver et al., Cold Spring Harb Symp Quant Biol 1996; 61:797-814.Genes localized to chromosomal regions in linkage with schizophrenia are candidate genes for disease susceptibility.", "Genes in these regions with the potential to play a biochemical/functional role in the disease process (like G protein coupled receptors) have a high probability of being a disease-modifying locus.", "nGPCR-40 and -54, because of their chromosomal location, are attractive targets therefore for screening ligands useful in modulating cellular processes involved in schizophrenia.", "Example 13 Clone Deposit Information In accordance with the Budapest Treaty, clones of the present invention have been deposited at the Agricultural Research Culture Collection (NRRL) International Depository Authority, 1815 N. University Street, Peoria, Ill. 61604, U.S.A. Accession numbers and deposit dates are provided below in Table 6.TABLE 6 DEPOSIT INFORMATION Accession Number Nudapest Treaty Clone NRRL Deposit Date nGPCR-1 (SEQ ID NO:73) B-30243 Jan. 18, 2000 nGPCR-5 (SEQ ID NO: 75) B-30244 Jan. 18, 2000 nGPCR-16 (SEQ ID NO: 81) B-30245 Jan. 18, 2000 nGPCR-11 (SEQ ID NO: 79) B-30258 Feb. 2, 2000 nGPCR-17 (SEQ ID NO: 23) B-30259 Feb. 3, 2000 nGPCR-9 (SEQ ID NO: 77) B-30262 Feb. 22, 2000 nGPCR-58 (SEQ ID NO: 91) B-30274 Mar.", "23, 2000 nGPCR-56 (SEQ ID NO: 89) B-30288 May 5, 2000 nGPCR-3 (SEQ ID NO: 185) B-30290 May 5, 2000 nGPCR-54 (SEQ ID NO: 85) B-30291 May 5, 2000 nGPCR-40 (SEQ ID NO: 83) B-30299N Jun.", "2, 2000 The clone deposited with NRLL Accession Number N30299N comprises a sequence identical to SEQ ID NO: 83 but with the substitution of an “A” at neucleotide position 10.Example 14 Using nGPCR-x Proteins to Isolate Neurotransmitters The isolated nGPCR-x proteins can be used to isolate novel or known neurotransmitters (Saito et al., Nature 400: 265-269, 1999).", "The cDNAs that encode the isolated nGPCR-x can be cloned into mammalian expression vectors and used to stably or transiently transfect mammalian cells including CHO, Cos or HEK293 cells.", "Receptor expression can be determined by Northern blot analysis of transfected cells and identification of an appropriately sized mRNA band (predicted size from the cDNA).", "Brain regions shown by mRNA analysis to express each of the nGPCR-x proteins could be processed for peptide extraction using any of several protocols ((Reinsheidk R. K. et al., Science 270: 243-247, 1996; Sakurai, T., et al., Cell 92; 573-585, 1998; Hinuma, S., et al., Nature 398: 272-276, 1998).", "Chromotographic fractions of brain extracts could be tested for ability to activate nGPCR-x proteins by measuring second messenger production such as changes in cAMP production in the presence or absence of forskolin, changes in inositol 3-phosphate levels, changes in intracellular calcium levels or by indirect measures of receptor activation including receptor stimulated mitogenesis, receptor mediated changes in extracellular acidification or receptor mediated changes in reporter gene activation in response to cAMP or calcium (these methods should all be referenced in other sections of the patent).", "Receptor activation could also be monitored by co-transfecting cells with a chimeric GIq/i3 to force receptor coupling to a calcium stimulating pathway (Conklin et al., Nature 363; 274-276, 1993).", "Neurotransmitter mediated activation of receptors could also be monitored by measuring changes in [35S]-GTPKS binding in membrane fractions prepared from transfected mammalian cells.", "This assay could also be performed using baculoviruses containing nGPCR-x proteins infected into SF9 insect cells.", "The neurotransmitter which activates nGPCR-x proteins can be purified to homogeneity through successive rounds of purification using nGPCR-x proteins activation as a measurement of neurotransmitter activity.", "The composition of the neurotransmitter can be determined by mass spectrometry and Edman degradation if peptidergic.", "Neurotramitters isolated in this manner will be bioactive materials which will alter neurotransmission in the central nervous system and will produce behavioral and biochemical changes.", "Example 15 Using nGPCR-x Proteins to Isolate and Purify G Proteins cDNAs encoding nGPCR-x proteins are epitope-tagged at the amino terminus end of the cDNA with the cleavable influenza-hemagglutinin signal sequence followed by the FLAG epitope (IBI, New Haven, Conn.).", "Additionally, these sequences are tagged at the carboxyl terminus with DNA encoding six histidine residues.", "(Amino and Carboxyl Terminal Modifications to Facilitate the Production and Purification of a G Protein-Coupled Receptor, B. K. Kobilka, Analytical Biochemistry, Vol.", "231, No.", "1, October 1995, pp.", "269-271).", "The resulting sequences are cloned into a baculovirus expression vector such as pVL1392 (Invitrogen).", "The baculovirus expression vectors are used to infect SF-9 insect cells as described (Guan, X. M., Kobilka, T. S., and Kobilka, B. K. (1992) J. Biol.", "Chem.", "267, 21995-21998).", "Infected SF-9 cells could be grown in 1000-ml cultures in SF900 II medium (Life Technologies, Inc.) containing 5% fetal calf serum (Gemini, Calabasas, Calif.) and 0.1 mg/ml gentamicin (Life Technologies, Inc.) for 48 hours at which time the cells could be harvested.", "Cell membrane preparations could be separated from soluble proteins following cell lysis.", "nGPCR-x protein purification is carried out as described for purification of the θ2 receptor (Kobilka, Anal.", "Biochem., 231 (1): 269-271, 1995) including solubilization of the membranes in 0.8-1.0% n-dodecyl-D-maltoside (DM) (CalBiochem, La Jolla, Calif.) in buffer containing protease inhibitors followed by Ni-column chromatography using chelating Sepharose™ (Pharmacia, Uppsala, Sweden).", "The eluate from the Ni-column is further purified on an M1 anti-FLAG antibody column (IBI).", "Receptor containing fractions are monitored by using receptor specific antibodies following western blot analysis or by SDS-PAGE analysis to look for an appropriate sized protein band (appropriate size would be the predicted molecular weight of the protein).", "This method of purifying G protein is particularly useful to isolate G proteins that bind to the nGPCR-x proteins in the absence of an activating ligand.", "Some of the preferred embodiments of the invention described above are outlined below and include, but are not limited to, the following embodiments.", "As those skilled in the art will appreciate, numerous changes and modifications may be made to the preferred embodiments of the invention without departing from the spirit of the invention.", "It is intended that all such variations fall within the scope of the invention.", "The entire disclosure of each publication cited herein is hereby incorporated by reference." ] ]	Patent_10467492
[ [ "Pharmaceutical formulation", "The present invention provides a pharmaceutical formulation which comprises a core comprising a first active ingredient, a coating comprising a second active ingredient which is incompatible with the first active ingredient and a barrier between the core and the coating which prevents physical contact between the core and the coating, characterised in that the barrier is formed on the core by film-coating and the coating is formed on the barrier by press-coating." ], [ "1.A pharmaceutical formulation which comprises a core comprising a first active ingredient, a coating comprising a second active ingredient which is incompatible with the first active ingredient and a barrier between the core and the coating which prevents physical contact between the core and the coating, characterised in that the barrier is formed on the core by film-coating and the coating is formed on the barrier by press-coating.", "2.A pharmaceutical formulation as claimed in claim 1 wherein the core further comprises one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents or diluents.", "3.A pharmaceutical formulation as claimed in claim 1 wherein the coating further comprises one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents or diluents.", "4.A pharmaceutical formulation as claimed in claim 1 wherein the core comprises ranitidine or a physiologically acceptable salt thereof and the coating comprises acetylsalicylic acid or a physiologically acceptable salt thereof.", "5.A pharmaceutical formulation as claimed in claim 4 wherein the ranitidine is present in the core in an amount of from 10 to 200 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 10 to 200 mg of ranitidine.", "6.A pharmaceutical formulation as claimed in claim 4 wherein the ranitidine is present in the form of ranitidine hydrochloride.", "7.A pharmaceutical formulation as claimed in claim 4 wherein acetylsalicylic acid is present in the coating in an amount of from 50 to 1000 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the core in an amount which is equivalent to from 50 to 1000 mg of acetylsalicylic acid.", "8.A pharmaceutical formulation as claimed in claim 4 wherein the acetylsalicylic acid or the physiologically acceptable salt of acetylsalicylic acid is microencapsulated with ethyl cellulose.", "9.A pharmaceutical formulation as claimed in claim 1 wherein the barrier also reduces the transmission of moisture between the core and the coating.", "10.A pharmaceutical formulation as claimed in claim 1 wherein the barrier is present in an amount of from 1 to 20% by weight based on the total weight of the core." ], [ "The present invention relates to improvements in the formulation of pharmaceutical compositions.", "In particular, it relates to improvements in the formulation of pharmaceutical compositions wherein two or more active ingredients are present in the composition and wherein at least two of the active ingredients are incompatible with one another.", "In the context of the present application, the term “incompatible”, when applied to active ingredients, means that the active ingredients cannot normally be formulated into a pharmaceutical composition wherein the active ingredients are in physical contact.", "The incompatibility is usually, but not necessarily, the result of a chemical reaction between the active ingredients which results in a reduction of the therapeutic activity of at least one of the active ingredients.", "It will be appreciated by a person skilled in the art that many pharmaceutical compositions which comprise incompatible active ingredients may be formulated as described herein.", "However, the present invention is particularly directed to compositions which comprise acetylsalicylic acid or a physiologically acceptable salt of acetylsalicylic acid and ranitidine or a physiologically acceptable salt of ranitidine, such as ranitidine hydrochloride.", "Systemic non-steroidal anti-inflammatory drugs such as acetylsalicylic acid are known to give undesirable side effects.", "In particular they are known to be ulcerogenic and can therefore give rise to gastric ulceration when administered orally over a period of time to a patient.", "Ranitidine is the approved name for N-[2-[[[5-(dimethylamino)methyl]-2-furanyl]methyl]thio]ethyl-N′-methyl-2-nitro-1,1-ethanediamine which is described and claimed in British Patent No 1,565,966.It is known to be a potent histamine H2-antagonist which may be used in the treatment of conditions where there is an advantage in lowering gastric acidity, particularly in gastric and peptic ulceration.", "It is also known from British Patent No 2,105,193 that mucosal lesions of the gastrointestinal tract caused by systemic non-steroidal anti-inflammatory drugs can be significantly reduced by co-administering ranitidine.", "British Patent No 2,105,193 discloses pharmaceutical compositions comprising a systemic non-steroidal anti-inflammatory drug and ranitidine or a physiologically acceptable salt thereof.", "However, experiments show that there is a clear chemical incompatibility between ranitidine hydrochloride (the most preferred physiologically acceptable salt of ranitidine) and acetylsalicylic acid.", "These two active ingredients react chemically with each other to produce degradation products.", "The reaction becomes manifest in a matter of days and implies a serious stability problem for pharmaceutical compositions containing both active ingredients.", "The present invention addresses the problems outlined above and provides a pharmaceutical formulation which comprises a core comprising a first active ingredient, a coating comprising a second active ingredient which is incompatible with the first active ingredient and a barrier between the core and the coating which prevents physical contact between the core and the coating, characterised in that the barrier is formed on the core by film-coating and the coating is formed on the barrier by press-coating.", "It will be appreciated by those skilled in the art that further barriers and coatings may be formed on the composition if so desired.", "This may be necessary if, for example, more than two active ingredients are present and each active ingredient is incompatible with all or some of the other active ingredients.", "In one embodiment of the present invention the core comprises ranitidine or a physiologically acceptable salt thereof and the coating comprises acetylsalicylic acid or a physiologically acceptable salt thereof.", "In a preferred embodiment, ranitidine or a physiologically acceptable salt thereof is present in the core in an amount sufficient to reduce gastrointestinal distress caused by acetylsalicylic acid.", "In a more preferred embodiment, ranitidine is present in the core in an amount of from 10 to 200 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 10 to 200 mg of ranitidine.", "In a more preferred embodiment, ranitidine is present in the core in an amount of from 50 to 100 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 50 to 100 mg of ranitidine.", "In a more preferred embodiment, ranitidine is present in the core in an amount of from 70 to 80 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 70 to 80 mg of ranitidine.", "It is preferable that the ranitidine be present in the form of a physiologically acceptable salt.", "Such salts include salts of inorganic or organic acids such as the hydrochloride, hydrobromide, sulphate, acetate, maleate, succinate and fumarate salts.", "In a particularly preferred embodiment, ranitidine is present in the form of ranitidine hydrochloride.", "As well as the first active ingredient, the core may also comprise one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents, diluents or taste-masking agents.", "In a preferred embodiment, the one or more lubricants are present in the core in an amount of from 0.1 to 5% by weight based on the total weight of the core.", "In a more preferred embodiment, the one or more lubricants are present in the core in an amount of from 0.1 to 3% by weight based on the total weight of the core.", "In a still more preferred embodiment, the one or more lubricants are present in the core in an amount of from 0.5 to 1.0% by weight based on the total weight of the core.", "Examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, stearic acid, palmitic acid and sodium stearyl fumarate.", "A preferred lubricant is magnesium stearate.", "In a preferred embodiment, the one or more diluents are present in the core in an amount of from 10 to 90% by weight based on the total weight of the core.", "In a more preferred embodiment, the one or more diluents are present in the core in an amount of from 30 to 70% by weight based on the total weight of the core.", "In a still more preferred embodiment, the one or more diluents are present in the core in an amount of from 40 to 60% by weight based on the total weight of the core.", "Examples of suitable diluents include microcrystalline cellulose, powdered cellulose, lactose, mannitol, sucrose and calcium phosphate.", "A preferred diluent is microcrystalline cellulose.", "In a preferred embodiment, the one or more disintegrants are present in the core in an amount of from 0.1 to 10% by weight based on the total weight of the core.", "In a more preferred embodiment, the one or more disintegrants are present in the core in an amount of from 0.1 to 5% by weight based on the total weight of the core.", "In a still more preferred embodiment, the one or more disintegrants are present in the core in an amount of from 1 to 3% by weight based on the total weight of the core.", "An example of a suitable disintegrant is sodium croscaramelose.", "In a preferred embodiment, acetylsalicylic acid is present in the coating in an amount of from 50 to 1000 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the core in an amount which is equivalent to from 50 to 1000 mg of acetylsalicylic acid.", "In a more preferred embodiment, acetylsalicylic acid is present in the coating in an amount of from 300 to 700 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the coating in an amount which is equivalent to from 300 to 700 mg of acetylsalicylic acid.", "In a more preferred embodiment, acetylsalicylic acid is present in the coating in an amount of from 450 to 550 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the coating in an amount which is equivalent to from 450 to 550 mg of acetylsalicylic acid.", "In an embodiment the acetylsalicylic acid or the physiologically acceptable salt of acetylsalicylic acid may be microencapsulated with ethyl cellulose.", "An example of a commercially available microencapsulated acetylsalicylic acid product is Rhodine NCR-P™.", "As well as the second active ingredient, the coating may also comprise one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents, diluents or taste-masking agents.", "In a preferred embodiment, the one or more diluents are present in the coating in an amount of from 5 to 90% by weight based on the total weight of the coating.", "In a more preferred embodiment, the one or more diluents are present in the coating in an amount of from 5 to 50% by weight based on the total weight of the coating.", "In a still more preferred embodiment, the one or more diluents are present In the coating in an amount of from 10 to 20% by weight based on the total weight of the coating.", "Examples of suitable diluents include copovidone, microcrystalline cellulose, powdered cellulose, maize starch, lactose, Cellactose™80 (a compound formed from 25% powdered cellulose and 75% lactose monohydrate) and Ludipress™ (a compound formed from 93% lactose monohydrate, 3.5% povidone and 3.5% crospovidone).", "A preferred diluent is Cellactose™80.In a preferred embodiment, the one or more disintegrants are present in the coating in an amount of from 0.1 to 10% by weight based on the total weight of the coating.", "In a more preferred embodiment, the one or more disintegrants are present in the coating in an amount of from 0.1 to 5% by weight based on the total weight of the coating.", "In a still more preferred embodiment, the one or more disintegrants are present in the coating in an amount of from 1 to 3% by weight based on the total weight of the coating.", "Examples of suitable disintegrants include maize starch, crospovidone, sodium starch glycolate, bentonite, aluminium magnesium silicate and sodium croscaramelose.", "A preferred disintegrant is sodium croscaramelose.", "In a preferred embodiment, the one or more lubricants are present in the coating in an amount of from 0.1 to 10% by weight based on the total weight of the coating.", "In a more preferred embodiment, the one or more lubricants are present in the coating in an amount of from 0.1 to 5% by weight based on the total weight of the coating.", "In a still more preferred embodiment, the one or more lubricants are present in the coating in an amount of from 0.1 to 1.0% by weight based on the total weight of the coating.", "Examples of suitable lubricants include hydrogenated vegetable oil, stearic acid, palmitic acid and sodium stearyl fumarate.", "A preferred lubricant is sodium stearyl fumarate.", "In a preferred embodiment, the one or more anti-adhesion agents are present in the coating in an amount of from 0.1 to 10% by weight based on the total weight of the coating.", "Examples of suitable anti-adhesion agents are talc and hydrogenated vegetable oil.", "In a preferred embodiment, the one or more flow agents are present in the coating in an amount of from 0.01 to 5% by weight based on the total weight of the coating.", "In a more preferred embodiment, the one or more flow agents are present in the coating in an amount of from 0.01 to 1% by weight based on the total weight of the coating.", "In a still more preferred embodiment, the one or more flow agents are present in the coating in an amount of from 0.01 to 0.5% by weight based on the total weight of the coating.", "An example of a suitable flow agent is colloidal anhydrous silica.", "The barrier is present in order to prevent physical contact between the core and the coating and therefore prevent reaction between the first and second active ingredients which are present in the core and the coating respectively.", "Preferably the barrier also reduces the transmission of moisture between the core and the coating.", "More preferably the barrier is substantially impermeable to the transmission of moisture between the core and the coating.", "In a preferred embodiment the barrier is present in an amount of from 1 to 20% by weight based on the total weight of the core.", "In a more preferred embodiment the barrier is present in an amount of from 3 to 15% by weight based on the total weight of the core.", "In a still more preferred embodiment the barrier is present in an amount of from 5 to 10% by weight based on the total weight of the core.", "In general terms any material that reduces water transmission and is compatible with the active ingredients contained in the core and the coating can be used to physically separate them.", "Thus, a barrier material which comprises one or more of stearic acid, polyvinyl alcohol, ethyl cellulose, microcrystalline cellulose, hydroxypropyl cellulose and methacrylic acid copolymer type C, can be used.", "An example of a suitable barrier material is OPADRY™ Aqueous Moisture Barrier (AMB) produced by the Colorcon Company (Reference OY-B-28920).", "OPADRY™ AMB is made up of polyvinyl alcohol, titanium dioxide, purified talc, lecithin and xanthan gum.", "The formula is supplied as a powder which is reconstituted using purified cold water before it is sprayed on the core.", "The actual solids content of the coating suspensions can be varied according to the atomising capabilities of the spraying equipment.", "Preferably, the solids content is approximately 20% w/w.", "Another example of a suitable barrier material is SEPIFILM™ Low Permeability (LP) produced by Seppic.", "SEPIFILM™ LP is made up of hydroxypropylmethyl cellulose, microcrystalline cellulose, stearic acid and pigments and/or lakes (if required).", "Another example of a suitable barrier material is LUSTRECLEAR™ produced by FMC Corporation.", "LUSTRECLEAR™ is made up of microcrystalline cellulose, carrageenan, polyethylene glycol, hydroxyethyl cellulose, maltodextrin and pigments and/or lakes (if required).", "Another example of a suitable barrier material is EUDRAGIT™ L 30D-55 produced by Degussa.", "If this barrier material is chosen, it should not be applied in an amount greater than approximately 1 mg/cm2 of barrier material on the tablet surface.", "If more is used there is a danger of forming an enteric coating around the core which could reduce the bioavailability of the active ingredient in the core.", "EUDRAGIT™ L 30D-55 is made up of methacrylic acid—ethyl acrylate copolymer as a 30% dispersion in water.", "The mean relative molecular mass of the copolymer is approximately 250 000 and the ratio of carboxylic groups to ester groups is approximately 1:1.The material may also comprise surface-active agents such as sodium dodecyl sulphate and polysorbate 80.It contains not less than 46.0% m/m and not more than 50.6% m/m of methacrylic acid units, calculated with reference to the residue on evaporation.", "Methods of manufacturing by press coating, also known as dry coating or compression coating are well known in the pharmaceutical industry.", "See for example; Pharmaceutics, the Science of Dosage Form Design, edited by M E Aulton, Published by Churchill Livingstone (part of the Longman Group) 1988, ISBN 0-443-03643-8.In particular pages 675 to 676 explain the method of press coating as follows.", "Press coating involves the compaction of granular material around an already preformed core using compressing equipment similar to that used for the core itself, e.g.", "Manesty Drycota.", "It is used mainly to separate chemically incompatible materials, one or more being placed in the core and the other(s) in the coating layer.", "However, there is still an interface of contact left between the two layers.", "In cases where even this is important then the process of press coating can be taken one step further.", "It is possible to apply two press coatings to a tablet core using suitable equipment, e.g.", "Manesty Bicota.", "This equipment produces press coated tablets with perfect separation between the active core and the coating as these two can be separated by an inert coating.", "The technique is also described in Reminton: The Science and Practice of Pharmacy 19th Edition 1995, published by Mack Publishing Company, ISBN 0-912734-04-3.See in particular pages 1616 and 1631 which explain the method of manufacture by press coating as follows.", "Press coated tablets, also referred to as dry-coated, are prepared by feeding previously compressed tablets into a special tableting machine and compressing another granulation layer around the preformed tablets.", "They have all the advantages of compressed tablets, i.e.", "slotting, monogramming, speed of disintegration, etc, while retaining the attributes of sugar-coated tablets in masking the taste of the taste drug substance in the core tablets.", "An example of a press-coated tablet press is the Manesty Drycota.", "Press coated tablets can also be used to separate incompatible drug substances; in addition, they can provide a means to give an enteric coating to the core tablets.", "The following journal articles also provide some discussion of the technique of press-coating: Press-coated tablets for the sequential pulsed administration of two different drugs, International Journal of Pharmaceutics, Volume 99, Issues 2-3, 1993, pages 173-179, Maggi L, et al.", "Press-coated tablets for time-programmed released of drug, Biomaterials, Volume 14, Issue 13,1993 1017-1023, Conte U, et al.", "The technique of film-coating is also well known in the pharmaceutical industry and is described for example in Pharmaceutics, the Science of Dosage Form Design, edited by M E Aulton, Published by Churchill Livingstone (part of the Longman Group) 1988, ISBN 0-443-03643-8.In particular pages 672 to 675 explain the method of film coating as follows.", "Film coating involves the deposition, usually by a spray method, of a thin film of polymer surrounding the tablet core.", "It is possible to utilize conventional panning equipment but more usually specialized equipment is employed to take advantage of fast coating times and a high degree of automation is possible.", "The coating solution contains a polymer in a suitable liquid medium together with other ingredients such as pigments and plasticizers.", "This solution is sprayed on to a rotated mixed tablet bed.", "The drying conditions permit the removal of the solvent so as to leave a thin deposition of coating material around each tablet core.", "Typically the coating solution formulation comprises: polymer, solvent, plasticizer and colorants.", "The technique is also described in Reminton: The Science and Practice of Pharmacy 19th Edition 1995, published by Mack Publishing Company, ISBN 0-912734-04-3.See in particular pages 1652 to 1659 which explain the method of film coating as follows.", "Film coating involves the deposition of a thin, but uniform, film onto the surface of the substrate.", "Unlike sugar coating, the flexibility afforded in film coating allows additional substrates other than just compressed tablets, to be considered (eg, powders, granules, nonpareils, capsules).", "Coatings are essentially applied continuously to a moving bed of material by means of a spray technique, although manual application procedures have also been used.", "The following are examples of formulations which are representative of the present invention.", "They are intended to illustrate the invention but are not intended to be limiting on the scope of the invention which is defined by the claims.", "EXAMPLE 1 A double tablet having the following specifications for the core, barrier and coating was manufactured.", "The first active ingredient is ranitidine hydrochloride and the second active ingredient is acetylsalicylic acid: Core Weight per % w/w (based on Core Ingredient tablet (mg) total core weight) Function Granulated 84.000 56.00 Active Ingredient Ranitidine Hydrochloride Microcrystalline 64.875 43.25 Diluent Cellulose Magnesium 1.125 0.75 Lubricant Stearate Barrier Barrier Weight per % w/w (based on Ingredient tablet (mg) total core weight) Function Opadry AMB ™ 12.000 8.00 Moisture Barrier Coating % w/w (based on Coating Weight per total coating Ingredient tablet (mg) weight) Function Acetylsalicylic 500.000 82.40 Active Ingredient acid Cellactose ™ 80 91.020 15.00 Diluent Sodium 12.140 2.00 Disintegrant Croscaramelose Sodium Stearyl 3.030 0.50 Lubricant Fumarate Colloidal 0.610 0.10 Flow Promoter Anhydrous Silica The manufacturing method was as follows, the steps being numbered sequentially: A) Manufacture of the Ranitidine Tablet Cores: Mixture of the Ranitidine Tablet Core Ingredients 1) The selected amount of granulated ranitidine hydrochloride, microcrystalline cellulose and magnesium stearate were weighed out according to the batch size to be manufactured.", "2) The granulated ranitidine hydrochloride and the microcrystalline cellulose were added to a drum blender or equivalent mixing equipment.", "3) The mixture was blended for 10 minutes at a speed of rotation of 15 rpm to provide a homogenous mixture.", "Note that the mixing parameters may be varied according to the equipment used and batch size intended for manufacture.", "4) To the homogenous mixture of ranitidine hydrochloride and microcrystalline cellulose was added the magnesium stearate.", "Mixing was continued in the same drum blender or equivalent mixing equipment for 5 minutes at a speed of rotation of 15 rpm.", "Again, the mixing parameters may be varied according to the equipment used and batch size intended for manufacture.", "Compression of the Powder Mixture to Produce the Ranitidine Tablet Core 5) A tableting machine was used to compress the previous powder mixture into tablet cores in compliance with the following specifications: Weight: 150 mg Uniformity of Mass: In compliance with European Pharmacopeia Hardness: greater than 8 Kp Friability: less than 1% w/w Disintegration time: less than 15 minutes The punches used were standard concave, 8 mm diameter.", "B) Film-Coating the Ranitidine Tablet Cores: 6) The coating suspension of Opadry AMB was prepared as follows.", "The required amount of Opadry AMB and purified water to prepare a 20% w/w dispersion of Opadry AMB in purified water were measured out.", "Note that the recommended solids level is 20% w/w, but the actual solids level in the suspension can be changed to allow for the atomising capabilities of the spraying equipment.", "Opadry AMB was reconstituted as follows.", "The total quantity of cold water was poured into a suitable container.", "A propeller or similar type of stirrer was placed in the water, and rotated so that a vortex was produced, but without drawing air into the liquid.", "The Opadry AMB powder was added to the vortex as quickly as possible so that undispersed powder did not float on the surface of the liquid.", "During the addition step, the suspension viscosity rose, thus it was necessary to increase the stirrer speed in order to maintain the vortex.", "After all the Opadry AMB powder had been added, the stirrer speed was reduced until the vortex was nearly eliminated and stirring continued for a further 45 minutes, after which time the coating suspension was ready for use.", "It was preferable to provide gentle agitation to the coating suspension while it was being sprayed.", "7) The selected amount of ranitidine tablet cores were placed into a suitable film coating equipment e.g.", "ACCELA-COTA or similar equipment.", "12 mg of Opadry AMB film was coated on the ranitidine tablet cores using the following film coating parameters: Inlet air temperature: ≧90° C. Product temperature before spraying: ≧70° C. Drum speed: 10 rpm Spray equipment: Manesty spray gun Atomising air pressure: 3.5 Bar The spraying parameters may be varied according to the equipment used and batch size intended for manufacture.", "C) Press Coating the Ranitidine Film Coated Cores: Mixture of the Acetyl Salicylic Acid (ASA) Tablet Coat Ingredients 8) The selected amounts of ASA, Cellactose™ 80, sodium croscaramelose, sodium stearyl fumarate and colloidal anhydrous silica were weighed out.", "9) The ASA, Cellactose™ 80, sodium croscaramelose, sodium stearyl fumarate and colloidal anhydrous silica were added to a drum blender or equivalent mixing equipment.", "10) The mixture was blended for 10 minutes at 15 rpm to obtain a homogenous mixture.", "Note that these parameters are Intended as a guide only.", "The proper parameters will depend upon the equipment and the batch size intended for manufacture.", "Press Coating the ASA Powder Mixture onto the Film Coated Ranitidine Cores 11) A tableting machine which is capable of producing double compressed tablets e.g.", "Manesty Drycota or similar equipment was used to provide a tablet in compliance with the following specifications: Weight: 768.8 mg Uniformity of Mass: compliant with European Pharmacopeia Hardness: greater than 10 Kp Friability: less than 1% w/w The punches used were standard concave, 12 mm diameter The press coating process can be summarised in the following steps.", "Approximately half of the ASA powder mixture was filled into a tablet die.", "Then the filmed-coated ranitidine tablet was fed into the tablet die.", "Afterwards, the rest of the ASA mixture was filled into the die.", "The punches compressed the ASA coat onto the filmed-coated ranitidine tablet, producing a double compressed tablet where the tablet core is the filmed-coated ranitidine tablet and the external coat is the ASA.", "This cycle was repeated by the tableting machine as more film coated ranitidine tablets and ASA mixture (dry coating material) were fed into the dies from the hoppers.", "This formulation was subjected to a stability study under the following conditions: 25° C./60% relative humidity and 40° C./75% relative humidity for 1, 2, 3 and 6 months.", "It was found that the ranitidine remains stable for 6 months at 25° C./60% relative humidity and at 40° C./75% relative humidity.", "The acetylsalicylic acid is stable for 6 months at 25° C./60% relative humidity and is stable for 2 months at 40° C./75% relative humidity.", "EXAMPLE 2 A double tablet having the following specifications for the core, barrier and coating was manufactured: Core Weight per % w/w (based on Core Ingredient tablet (mg) total core weight) Function Granulated 84.000 56.00 Active Ingredient Ranitidine Hydrochloride Microcrystalline 64.875 43.25 Diluent Cellulose Magnesium 1.125 0.75 Lubricant Stearate Barrier Barrier Weight per % w/w (based on Ingredient tablet (mg) total core weight) Function Opadry AMB ™ 12.000 8.00 Moisture Barrier Coating % w/w (based on Coating Weight per total coating Ingredient tablet (mg) weight) Function Acetylsalicylic 507.6-526.3(1) 82.7-85.7 Active Ingredient Acid micro- encapsulated with Ethyl Cellulose (Rhodine NCR- P ™) Cellactose ™ 80 73.7-92.4(2) 12.0-15.0 Diluent Sodium 12.3 2.0 Disintegrant Croscaramelose Sodium Stearyl 1.2 0.2 Lubricant Fumarate Colloidal 0.6 0.1 Flow Promoter Anhydrous Silica (1) The purity of acetylsalicylic acid micro-encapsulated with ethyl cellulose varies between 95% and 98.5% w/w.", "Therefore the quantity of acetylsalicylic acid micro-encapsulated with ethyl cellulose that has to be included in each tablet can vary between 507.6 and 526.3 mg in order to provide a dose of 500 mg of acetylsalicylic acid.", "(2) The amount of Cellactose™ 80 added can vary between 73.7 and 92.4 mg so as to provide a total of 600 mg for the combined weight of Cellactose™ 80 and acetylsalicylic acid micro-encapsulated with ethyl cellulose.", "The manufacturing method was the same as for Example 1 above except that the weight of acetylsalicylic acid micro-encapsulated with ethyl cellulose has to be adjusted to provide 500 mg of acetylsalicylic acid and the difference in the final tablet weight is compensated with the amount of Cellactose™ 80.This formulation was subjected to a stability study under the following conditions: 25° C./60% relative humidity and 40° C./75% relative humidity for 1, 2, 3 and 6 months.", "It was found that the ranitidine remains stable for 6 months at 25° C./60% relative humidity and at 40° C./75% relative humidity.", "The acetylsalicylic acid is stable for 6 months at 25° C./60% relative humidity and is stable for 2 months at 40° C./75% relative humidity." ] ]	Patent_10467504
[ [ "Method of education and scholastic management for cyber education system utilizing internet", "This invention relates to the method of cyber education through internet capable to check the true attendance of a cyber class by a fingerprint and calculate the accumulative learning time of each registered lectures by the host computer for the scholastic management of the cyber education system.", "In addition, utilizing this invention, it provides a pre-school system by using a game and animation created for pre-school curriculums capable to evaluate the education development of each children with a method for calculating the accumulative learning time of each subject by individual ID, and a software system for studying subjects at a stage of character formation for kids and evaluating the mental development of human being by means of playing game." ], [ "1.The method for checking the attendance of the cyber class through internet, comprising the step of, (a) registering the fingerprint of registrant for completing a course of study and being issued ID number.", "(b) confirming the attendance of the cyber class by inputting the registrant's fingerprint through internet; (c) confirming the time to depart from the class by inputting the fingerprint when the registrant finishes attending a lecture; (d) verifying the true attendance of class by the fingerprint data inputted from the registrant by comparing the fingerprint data with the fingerprint data memorized in the host computer in accordance with ID data of each registrant.", "2.The method of claim 1, further comprising the step of alarming with a sound signal every set of time to the student for inputting the fingerprint, and verifying the true attendance of the cyber class by checking the fingerprint for a certain interval of the time.", "3.The method of claim 1 or claim 2, further comprising the step of giving credits for the completion of the course by calculating the accumulated time of each student's log in time checked by means of fingerprint.", "4.The method of claim 1 and 2, further comprising the step of creating database for each subject and lessons, and divide total running time of the program to small quantity, and when student logs-in to database with fingerprint and only views a few parts of the lesson then host computer records partial time spent and accumulates and giving credits when partial lessons are accumulating to complete lesson time which host computer recognizes as attendance.", "5.The method of claim 1, wherein checking the attendance of the class through the verification of the iris of the eye, the voice signal or a passwords of the students instead of fingerprint.", "6.The method of the scholastic management for granting academic degree at cyber school comprising of; (a) calculating by the host computer the starting time and ending time of each lesson by student's fingerprint data inputted and accumulate the total logging time in the memory device of the host computer; (b) granting credits when the host computer recognizes individual's accumulative time spent of each lesson is qualified according to the school's guide line for completion of the course; (c) giving a qualification to the student to submit a thesis through on-line or off-line when student finishes academic requirements according to the records of credits by the host computer; (d) granting a degree to the student when student passes all examinations concerning a thesis and complete academic cyber courses.", "7.The method of claim 6, further comprising the step of giving an examination by the host computer and receiving the answer through internet to earn enough credits to graduate.", "8.The method of claim 6, wherein further comprising the step of examining the thesis by attending the student on the thesis examination committee of the institute and giving the degree after the said examination.", "9.The method of the scholastic management for operating the universalized university such as a cyber university or a cyber graduate school for worldwide, comprising the step of; (a) constructing the database of the lectures in different languages comprising at least one or more different languages of the each lecture of the each professor; (b) registering the fingerprint of the student for completing a course of study and being issued ID code; (c) logging into the system with registered ID and selecting the lecture by the language and the professor of the database; (d) attending the cyber class by confirming the starting time and the ending time of each lecture by inputting the student's fingerprint to check the true attendance of the cyber class; (e) granting credits when the host computer recognizes the student's accumulative time spent of each lesson is qualified according to the school's guide line for completion of the course; (f) giving a qualification to the student to submit a thesis through on-line or off-line when student finishes academic requirements according to the records of credits calculated by the host computer; (g) granting a degree to the student when student passes all examinations concerning a thesis and complete academic cyber courses.", "10.The educational system for operating the cyber pre-school system utilizing internet comprising the step of; (a) a database comprising animation and/or game of each subject suitable for constructing the pre-school curriculums for the kids for studying by playing the game and/or animation; (b) a host computer for, (i) registering the cyber pre-school for completing a course of study by the parents and being issued ID code of the kids, (ii) calculating the time of log-in and log-out of each subject by the ID code for recording the accumulated playing time of each subject, (iii) qualifying the kids for completion of the program when individual's accumulative time spent of the each subject according to school's guide line, and (iv) recognizing the kids as completion of the academic programs when the kids complete all the courses, according to the records of the host computer.", "11.The educational system of claim 10, further comprising a means for registering kid's fingerprint to indicate starting time and ending time for accessing DB to check an accurate attendance.", "12.The educational system of claim 10, further comprising a database of the educational curriculums such as a language course, a music course or mathematics course for issuing a certificate for completion of the said educational curriculums by recognizing the kids as completion of the course when kid's accumulative time spent of the course is qualified by the host computer.", "13.The educational system of claim 10, further comprising a database of internet on-line games for providing internet on-line game service to kids for interchanging among other kids.", "14.The educational system of claim 10, further comprising a database for providing internet on-line game service and a means for a visual chatting service for kids to interchange among other kids.", "15.The educational system of claim 10, further registering the kids of off-line pre-school to cyber pre-school for logging into the database through internet and recognizing the completion of courses with data calculated by the host computer.", "16.The business method for operating the cyber pre-school utilizing internet comprising the step of; (a) creating a DB (database) for studying the each subject divided into session according to the pre-school curriculums; (b) issuing ID to log in to the DB by paying the tuition; (c) calculating the accumulated time of studying by the kids according to the time of log-in and log-out of each subject with the host computer; (d) advising the accumulated studying time of each subject through internet to the parents to select the subject for their kids to study more according to the said accumulated learning time data of each subject; (e) qualifying the kids for completion of the pre-school when individual's accumulative time spent of the each subject is qualified according to the guide line of the school by the host computer.", "17.The business method of claim 16, further comprising a step for evaluating the development of study by calculating the signal from the kids for selecting answer among the questionnaires comprising animations or games with explanation voice.", "18.The business method of claim 17, wherein the step for calculating further comprising a step for calculating the accumulated time of student's study by the subject and the number of the complete study of the subject.", "19.The business method of claim 17, wherein the database is comprising educational game created to learn the subject of the pre-school curriculums for studying through playing the games.", "20.The business method of claim 17, wherein the database is comprising educational animation and/or animated game created to study according to the curriculums for studying through playing the game and/or viewing animation.", "21.The business method of claim 20, further comprising a cyber class created to study the subject according to the curriculums for studying through the explanation of the cyber teacher and the class activities of the characters in the cyber class.", "22.The method for evaluating the mental development of a human being through internet comprising the step of, (a) making a DB (database) of games created to evaluate the mental development such as a character, a disposition and/or a morality of a human being in a category of each checking subject; (b) registering ID to log in the database with/without the fingerprint of the player; (c) accessing the database with said ID with/without the fingerprint of the player and playing the games through internet; (d) calculating the each action of the cyber characters played by the player and giving different points according to the favorable action and the non-favorable action based on the mental development evaluating standard in a category of each subject by the host computer of the DB system; (e) advising the result of the test in a category of each checking subject, calculated by the host computer for evaluating the mental developments of the player.", "23.The method of claim 22, wherein the database comprising games to check the mental development and/or knowledge of pre-school kids and evaluating the mental development and/or knowledge of the kids according to the action of the cyber characters played by kids.", "24.The method of claim 22 or claim 23, wherein the DB contents comprising animated questionnaires with/without a sound explanation to be selected by the player for evaluating the mental development of the player.", "25.The software system for evaluating the mental development of human being with the PC comprising, (a) an animated game created to evaluate the player's intention according to the action of the game character in the categories of each situation through playing the game by the player; (b) a calculating means for calculating the each action of the game characters played by the player by giving different points according to the favorable action and the non-favorable action based on the mental development evaluation standard in a category of each subject to check; (c) a displaying means for displaying the result of the calculation by the point in a category of each subject to evaluate the mental development of the player.", "26.The software system of claim 25, wherein the calculating means calculating the accumulated points in category of each subject and the number of the completion of the game to display according to the player.", "27.The software system for studying the educational subjects with PC comprising, an animated game created to learn the subject through playing the game by the kids; a means for calculating the playing time of the each subject of the software by the each subject; and a means for displaying the accumulated playing time according to the subject to check the studying intensive of the kids.", "28.The software system of claim 27 further comprising a cyber class created to study the subject to learn through the activities of the kids and the guidance of the teacher in the cyber class.", "29.The software system of claim 27 or claim 28, further comprising a animated questionnaires for the kids to select the answer for calculating points to display on the monitor of the PC to evaluate the kid's understanding." ], [ "<SOH> FIELD OF INVENTION <EOH>The present invention relates to the method of cyber education and scholastic management system related to the cyber education through Internet capable to check the true attendance of the cyber class by a fingerprint and calculate the accumulative learning time of each registered lectures by the host computer for determining automatically the completion of program for cyber education's scholastic management.", "In addition, utilizing the system that calculates accumulative learning time of each registered lectures by the host computer, a children's educational software program can be created for the pre-school children at the stage of character formation where they start learning language, morality and human nature by imitation on the model of parents and home environment, the system can accumulate how much they absorb knowledge on each subject and how many subjects are covered according to the accumulated learning time data of the each subject by the child's individual ID, to provide the data for evaluating each child's progress in data by the category of each subject, so that the parents may know what kind of the subjects to be covered more for their kids." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWING <EOH>The figures in the drawings are briefly described as follows: FIG.", "1 is a block diagram of the registering fingerprint data into education management system.", "FIG.", "2 is a block diagram of the system that calculates logging time on educational institution's database detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF INVENTION The present invention relates to the method of cyber education and scholastic management system related to the cyber education through Internet capable to check the true attendance of the cyber class by a fingerprint and calculate the accumulative learning time of each registered lectures by the host computer for determining automatically the completion of program for cyber education's scholastic management.", "In addition, utilizing the system that calculates accumulative learning time of each registered lectures by the host computer, a children's educational software program can be created for the pre-school children at the stage of character formation where they start learning language, morality and human nature by imitation on the model of parents and home environment, the system can accumulate how much they absorb knowledge on each subject and how many subjects are covered according to the accumulated learning time data of the each subject by the child's individual ID, to provide the data for evaluating each child's progress in data by the category of each subject, so that the parents may know what kind of the subjects to be covered more for their kids.", "DESCRIPTION OF THE RELATED ART The conventional way of Internet education is when a student registers for a program, the student receives an ID, attends class with that given ID and takes a form of tests to get grades or get completion of courses.", "However, this method can not distinguish the true attendance of the cyber class from the wrong access by the substitutes.", "Also, when student does not want to be presented at a complete lecture at the same time, attending a part of lecture, the conventional system have a problem with not enough data base information to accumulate actual time spent.", "Further, taking advantage of world wide internet, cyber lecture course can be used on a world wide for many different people using different language, and it needs more developed technology rather than administering by human force in order to administer, control and provide the multi language lecture on the same subject by the same professor.", "In addition, conventional curriculums for pre-school children courses on Internet have a problem with not providing enough information to evaluate the learning time of each subject according to the curriculums and how much children understand the program by the individual child's ID.", "Especially, when a child is registered on a off-line pre-school program, parents do not know how many hours the child spent on what subject, and what area needs to be covered more for the children's human mental development.", "Just by completing the program, parents assume that their child obtain more knowledge from the school.", "Children are imitative by nature and begin to build up their character and temper by recognizing objects, and learning from daily activity of their parents and home environment from the age of 2-3 years old.", "It is difficult for the parents to be a standard model for every aspect and subject for providing enough learning experiences for their children, therefore, it needs to develop a software for human brains that provide a progressive learning effect and memorizing the knowledge just like a software for the computer.", "Learning experience on the model of parents from childhood helps to develop personality and morality of that person for lifetime so it is crucial that children's learning experience should be from very early stage of their lives.", "The conventional ordinary off-line course or on-line course does not provide an accurate information such as what kind of learning experience children absorb and what needs to cover more in order to form a good character and a good disposition.", "SUMMERY OF THE INVENTION A primary object of the present invention is to resolve prior mentioned problems by when a student registers for Internet course, the student inputs his/her own fingerprint data or iris of the eyes data in school's host computer.", "When the student begins the class and ends the class, the student is required to put personal data such as fingerprints or an iris of the eyes, then host computer from school compares the data information from the memory mean of host computer to the student's data when he/she inputted to recognize if true person took the lesson.", "When a student is taking a course, the host computer calculates data information containing how many hours on each subject the student spent and progress to determine grades and manage educational matters.", "Further, regarding the pre-school curriculums that are made by an educational institution, the pre-school curriculums should construct games and animations data to help student to learn by himself by playing the PC, create a ID to log into the system and calculate number of logging time and hours spent on each subject to provide accurate data for parents to understand progress.", "Therefore, parents can determine shortage on certain subject and what more needs to be done according to the progressive report data of the host computer.", "Also, by given ID for students, the host computer accumulates hours spent on each subject and determines qualification for completion of the program to give out graduation certificates to the educational institution to manage its business.", "BRIEF DESCRIPTION OF THE DRAWING The figures in the drawings are briefly described as follows: FIG.", "1 is a block diagram of the registering fingerprint data into education management system.", "FIG.", "2 is a block diagram of the system that calculates logging time on educational institution's database DETAILED DESCRIPTION OF THE INVENTION To overcome the limitations in the prior art describe above, base on understanding and predicting current situation and certain limitation that will occur in the future.", "In managing cyber institution, the present invention provides a method to verify an accurate attendance and registration by comparing fingerprints or iris of the eyes, and provides a method of scholastic management that cyber institution calculates automatically the true attendance of the cyber class by student's individual ID by a host computer to prevent waste of human source and operating cost by the more accurate and economical managing system.", "In addition, utilizing the technology that calculates an accumulative learning time of each registered lectures by the host computer, the present invention is providing a pre-school education system through internet.", "The pre-school management can construct a data base program on variety of subjects for pre-school children, and under the guidance of parents, children can log into an access to the program and complete courses.", "Furthermore, it provides a progressive data by children's ID, and parents can determine what programs are covered and how many hours spend to measure the children's learning stage.", "It also provides a technology to construct a software to evaluate the mental development of the children's memory from early stage of learning to form a good character and temper.", "It is understood that many modifications and variations can be made therein, without departing from the spirit of the invention and from the scope of the appended claims.", "In the following description of the exemplary embodiment, reference is made to the accompanying drawings, which is shown by way of illustrations of specific embodiment in the invention.", "It is made without departing from the scope of the present invention.", "FIG.", "1 illustrates a block diagram of the registering fingerprint data into education management system.", "According to the illustration of FIG.", "1, the invention comprises a cyber educational institution (110); an institution's host computer (115); a data base (111) that stores each subject's lessons as in a form of digital data and controlled by host computer, when students (114-1 to 114-n) log into the data base through internet (113); an access control mean (112) to control fraud by checking ID and fingerprint data For checking attendance, when students (114-l-114-n) registers for the program, student inputs fingerprint to the host computer (115) and receives an ID to log into the data base (111).", "The host computer then stores registered ID and fingerprint as in a form of digital data to memory.", "When a student tries to log into institution's database (111) through the Internet (113) and put the given ID as same as conventional way.", "When student wants to log in and try to view a lecture for a subject, the student needs to input fingerprint data through personal computer.", "Then, the host computer compares the inputted data to the data from the memory.", "If both of data matches, then, the computer recognizes ID and allows student to access the lecture and stores the data such as the log-in time, the learning time by the subject of the lectures.", "When the student wishes to end the lecture, with ending alert signal, the student needs to input fingerprint data through personal computer.", "Then, host computer (115) compares the inputted data to the data from the memory.", "If both data matches, then, computer calculates accumulative logging time and store to the memory with ID.", "In case, if both fingerprint data does not match, the host computer indicates as “ERROR” to the student and asks to re-enter fingerprint If the lecture session is ended or log-off without verifying fingerprint, then host the computer considers as fraud and stores the learning time as for the session.", "Also, if the cyber educational institution tries to check constantly for more accurate attendance, set the program for certain set time such as in every 30 min for host computer (115) to transmit a signal to input fingerprint through student's PC monitor and compare the data in the same way.", "Further more, if the institution creates a program for 2 hour lecture session and divides lecture into 30 min session or 1 hour session, the student can stop or continue each session for convenient and host computer stores and accumulates session time for each ID automatically and when all the sessions are complete then the host computer can give the grades.", "According to the program mentioned above, when a student is complete with all the courses, then host computer accumulates all the credits and notify the student to do finals step or any kind of graduation process such as an examination, or a presentation of a thesis for granting a degree to the student.", "If the student finishes the final step through on-line or off-line according to the institution's guideline, then the student is qualified for the completion of courses and granted for a degree.", "These steps can be used for public school system, private schools or any kind of educational institutions such as a college, an university and a graduate school.", "Also, instead of the fingerprint recognition system, when an iris recognition system or a voice recognition system become popular and economical, the system can be replaced and gets same effects.", "Internet is the worldwide source without any borders among countries, when the educational institution want to manage worldwide, construct data base for each subject in different languages according to the professor, when students register and log in with choice of language, subject and professor then institution can check attendance with the prior mentioned systems to manage the institution worldwide.", "FIG.", "2 illustrates the block diagram for a pre school educational system utilizing the technology of the present invention.", "According to FIG.", "2, the present invention comprises a school (216) for creating and managing a cyber pre-school system; an educational program data base (214) created suitable for constructing the pre-school curriculums by subjects; a host computer (213-1, 213-2) for controlling access to the database by the non-authorized user, and calculating accumulative the learning time and number of log in times and controlling the system; an on-line game server (215) for storing the data of games for students; user's personal computers(211-1, 211-2, - - - 211-n) to log in the data base (214) and the game server (215) through Internet (212).", "The database (214) includes a pre-school or a kindergarten's educational curriculums on each subject as a form of games or animations.", "Each subject is divided to cover all the details on educational program.", "In addition to the basic programs, a dance program, a music program, a language program, a mathematics program and an honor program can be created and added for the additional education in addition to the pre-school curriculums.", "Game server (215) provides children to play game, stores all different kinds of pre-school game programs and provides multi media chatting services.", "When student registers to the school (216) then student receives an ID code issue by school to log in to the system including program database (214) and games server (215).", "Student pays tuition to the school and the tuition is main source of the cyber school's income.", "When the student registers a fingerprint for checking attendance the scholastic management of the cyber pre-school can be used for same effects as described prior.", "Student's parents can link to school's educational program database (214) through internet (212) using their personal computers (211-2, 211-2, - - - 211,n) to view and select each subject and help student to log in.", "Educational program database (214) includes each subject's lessons through games, animations, and videos to help understand and teach children who are just starting to learn.", "For example, “Christmas” can be put as search word, then children can learn about Christmas, and or “food” can be main search word to explain relationship between human body and food and all related topics.", "Parents can select certain program if they feel more lessons should be provided certain area for the kids.", "If parents select daily curriculums or lessons for children, children can join programs by simply pushing few buttons to play games, watch cartoons and videos and can repeat the programs for greater effects by the progressive learning effect.", "After finishing with internet lessons, parents can discuss daily subject which children learned and add more explanation to help understand better and this process is reaching the goal of educational program and help children learn step by step.", "The host computer (213-1, 213-2) then make data and stores information containing each subject logging time, time spent with user's ID to calculate accumulative time spent and indicate learning progress.", "Each completed subject and time spent is stored as data then those stored data can be used for providing and checking completion of the entire program to give out graduation certificate and base for children's progress in learning.", "Those stored data can provide and use for checking individual's progress, if parents think there are more area which needs to be covered, it is simple to recognize that information, and from stored data, the school as a service provider can make individual's progress report data for parents for convenience.", "Addition to the basic curriculums, an honor program, a dance lessons, a music lessons, a language lessons or a mathematics can be added to the data base (215) for the extra curriculums and more professional lessons.", "Calculating and checking progress is same as a basic system.", "For extra curriculums, the school can provide special certificate for completion of those courses in addition to basic graduation certificate.", "Off-line or conventional pre-school or kindergarten can join cyber program and instead of parents providing and selecting each subject or course for individual child, off-line school's teacher or guardian can do same part.", "All data and progress report can provide base for off-line schools to help issue certificates.", "Also, when students are log into games server (215) through internet (212), the students can be able to chat online with system setting, also when proper equipment is installed for video chatting, a child can join with other friends to talk online with monitor showing actual friends image.", "Children can develop friendship and teamwork to solve problems for the games and children would want to stay with the program for social gathering.", "This advantage can be helpful for increasing number of membership of the cyber pre-school service provider and increasing profit for the school.", "The present invention provides a progressive structured learning on each curriculum for children by a software through Internet.", "It also provides a scientific accurate accumulative time records from the beginning to the end of each software program.", "Computer organizes all the progressive learning records, so it becomes simple to look at each individual's progress and check area for more supplement learning.", "Children begin to recognize and learn about objects and everything around daily life when they are two or three years old.", "From parent's behavior, children also learn and adopt a way of life and value of life.", "Parents become teachers in early stage in every aspect and children develop their own personality from that stage.", "So it is very crucial to the childhood to help to learn, and to help to memorize all kinds of knowledge from the very beginning stage, and accordingly, the present invention provides teaching methods and steps.", "Just like a brand new computer needs to be installed with all different kinds of software, it is important for human being to learn proper knowledge and moral standard from beginning because it will be effect for rest of their lives.", "Therefore, to provide a good knowledge and a good character for very young children, “learning software” can be created.", "The leaning software can be made when top of professionals design curriculums for each subject which the kids have to know and divide them into small lessons and each small sessions are created in games and animations for the children who do not know letters or text yet.", "On the way to help children to learn and memorize the learning software, since the software is designed with games and animations when children start to play the games, the children will try to understand the subjects and will begin to memorize by playing.", "When subjects are described or explained in forms of animation, the effects become greater.", "Furthermore, the method to check the progress for individuals can be done with a calculator means of the computer when children's curriculums are divided into each subject and more divided into small sessions.", "For example, when 100 is perfect score to get when all the sessions are completed, computer records gives certain grades for each child's ID with the number of sessions play, the play time, and the number of sessions that are completed.", "Each of these gets certain value for grades and calculator mean accumulates all the scores to check the progress.", "Also, when there are animations containing visual and audio effects to test children with the multiple questionnaires by a form of animation or game then computer the combines the answers, more accurate information about progress can be made and let parents to figure out what area needs to cover more.", "In addition, to overcome current problems of the on-line pre-school system, the present invention improves the quality by providing online games that can be played with multiple users with visual chatting to help social life of the kids.", "For the business, since the program is designed with grading system with a proper graduation certificate, make it more interesting with online games to reach out to more customers.", "Eventually number of the customers will be increased with the customers who want to stay with the programs for longer time and these will become profit to the business of the pre-school service provider.", "This invention also can be used for evaluating the character, the disposition and the moral standard of the human being not only kids through internet or CD with personal computer.", "The database or CD can be created with a games designed to check the mental development such as the character, the disposition and morality of the player by giving different points according to the favorable action and the non-favorable action based on the mental development evaluating standard in a category of each subject for checking those points by the action of the cyber characters played in the cyber situation by the player.", "The method of this invention for evaluating the mental development, and the moral standard of a human being with a game is also providing the technology for making diagnoses of a mental diseases/disorder, and for evaluating the character, the disposition and the moral standards of human being for the assignment of a position for the right man in the right post." ] ]	Patent_10467520
[ [ "Fuel cell arrangement and method for operating a fuel cell arrangement", "A fuel cell arrangement (10) and its operation are described.", "This fuel cell arrangement (10) comprises one or more fuel cells (1) to which a combustible gas or a cathode gas is fed and which supply an electric power Pext to an external consuming device (2).", "One or more internal electrical consuming devices (3) can optionally be switched on and off by means of a control device (5) as a function of the operating condition of the fuel cell arrangement (10).", "Excessive heat is removed from the fuel cell arrangement by means of a cooling device (4).", "According to the invention, a portion of the electric power PBZ generated by the fuel cells (1) is converted at the internal electrical consuming devices (3) during the operation of the fuel cell arrangement (10).", "When the electric power Pext supplied to the external electrical consuming device (2) increases or decreases, the electric power Pint converted at the internal electrical consuming devices (3) is reduced or increased inversely in the sense of making the electric power PBZ generated by the fuel cells (1) uniform.", "This is carried out by the control device (5) of the fuel cell arrangement (10)." ], [ "1-40.", "(Cancelled) 41.A method of operating a fuel cell arrangement having at least one fuel cell comprising: feeding a combustible gas and a cathode gas to said fuel cell arrangement; supplying an electric power Pext to an external consuming device; optionally switching on and off at least one internal electrical consuming device; and removing excessive heat from said fuel cell arrangement by a cooling device; wherein an electric power PBZ is generated by said at least one fuel cell, and an electric power Pint is consumed by said at least one internal electrical consuming device, said power Pint being a portion of said power PBZ.", "42.The method of claim 41, wherein an increase or decrease of said power Pext causes a decrease or increase in said power Pint, respectively.", "43.The method of claim 41, wherein said power Pext and said power Pint are inversely related.", "44.The method of claim 42, wherein said power PBZ is uniform.", "45.The method of claim 43, wherein, for a load-following operation, an increase of said power Pext supplied to said external electrical consuming device is compensated by a reduction of said power Pint, and a reduction of said power Pext is compensated by an increase of said power Pint.", "46.The method of claim 45, wherein, for a standby operation, a switching-off of said external electrical consuming device is compensated by an increase in said power Pint.", "47.The method of claim 46, wherein a change of said power Pint takes place essentially immediately with a change of the power Pext.", "48.The method of claim 47, wherein said power PBZ is kept essentially constant.", "49.The method of claim 48, wherein said power Pint maximally amounts to approximately one fourth to approximately one half of a nominal electric power of said at least one fuel cell.", "50.The method of claim 48, wherein said power Pint is removed from said fuel cell arrangement by an increased use of said cooling device.", "51.A fuel cell arrangement having at least one fuel cell comprising: at least one internal electrical consuming device, said at least one internal electrical consuming device being optionally switched on and off; a cooling device for removing excess heat from said fuel cell arrangement; and a control device, said control device controlling said at least one internal electrical consuming device as a function of an operating condition of said fuel cell arrangement; wherein an electric power PBZ is generated by said fuel cell arrangement, an electric power Pint is consumed by said at least one internal electrical consuming device, said power Pint being a portion of said power PBZ, and an electric power Pext is consumed by an external consuming device.", "52.The fuel cell arrangement of claim 51, wherein an increase or decrease of said power Pext causes a decrease or increase in said power Pint, respectively.", "53.The fuel cell arrangement of claim 51, wherein said power Pext and said power Pint are inversely related.", "54.The fuel cell arrangement of claim 52, wherein said power PBZ is uniform.", "55.The fuel cell arrangement of claim 53, wherein, for a load-following operation, said control device controlling said at least one internal consuming device such that an increase of said power Pext is compensated by a reduction of said power Pint, and a reduction of said power Pext is compensated by an increase of said power Pint.", "56.The fuel cell arrangement of claim 55, wherein, for a standby operation, said control device controlling said at least one internal consuming device such that a switching-off of said external electrical consuming device is compensated by an increase in said power Pint.", "57.The fuel cell arrangement of claim 56, wherein said control device controlling said at least one internal consuming device such that a change of said power Pint takes place essentially immediately with a change of the power Pext.", "58.The fuel cell arrangement of claim 57, wherein said control device controlling said at least one internal consuming device such that said power PBZ is kept essentially constant.", "59.The fuel cell arrangement of claim 58, wherein said control device controlling said at least one internal consuming device such that said power Pint maximally amounts to approximately one fourth to approximately one half of a nominal electric power of said fuel cell arrangement.", "60.The fuel cell arrangement of claim 58, wherein said cooling device is constructed such that it removes said power Pint from said fuel cell arrangement.", "61.The fuel cell arrangement of claim 60, said at least one internal electrical consuming device further comprising at least one of a start heating device, a ventilator device, and another aggregate.", "62.The fuel cell arrangement of claim 61, further comprising an electrical inverter for converting a direct voltage supplied by said at least one fuel cell into an alternating voltage, said alternating voltage being supplied to said external consuming device.", "63.The fuel cell arrangement of claim 62, wherein said at least one internal electrical consuming device being connected, via said control device, to said external electrical consuming device in parallel with an output of said inverter." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Fuel cell arrangements are known which comprise one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device.", "One or more internal electrical consuming devices of the fuel cell arrangement can optionally be switched on and off.", "A cooling device is provided for eliminating excessive heat from the fuel cell arrangement.", "One difficulty during the operation of fuel cell arrangements of the above-mentioned type is the fact that, with respect to their performance, they cannot arbitrarily rapidly follow a change of the electric load of the connected external consuming device.", "The reason is based less on the dynamics of the electrochemical events taking place in the fuel cells than in the slowness of supplying the fuel cells with their reaction gases: the combustible gas and the cathode gas.", "The load-following operation therefore requires electric power at the expense of the efficiency in order to be able to immediately react in the event of a load demand.", "In the case of a power supply system failure, i.e., in the event of a sudden failure of the load demand of the connected external consuming device, the fuel cell arrangement, if possible, should not switch off but change to a standby operation, in which the current requirement for the internal consuming devices of the fuel cell arrangement is covered.", "The intrinsic consumption of the fuel cell arrangement is minimized for economical reasons, so that the system generates little electric power.", "In the load-following operation, the amount of the current production of a fuel cell arrangement is defined by the external consuming devices connected to it.", "Such a condition, which is also called an “island operation”, particularly in the case of small system sizes, results in load demands, which fluctuate considerably with respect to their percentages as a result of the connecting or disconnecting of individual consumption points.", "Rapid load changes occur during failures of the external consuming device, for example, in the event of a power supply system failure.", "The fuel cell arrangement will then supply no electric power to the external consuming device.", "In this case, the fuel cell arrangement is to change to a standby operation in which it generates just enough electric power in order to satisfy the intrinsic consumption." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the invention to provide a method of operating a fuel cell arrangement of the initially mentioned type by means of which the fuel cell arrangement can be operated in the sense of a fast response to changing load requirements.", "Furthermore, by means of the invention, a fuel cell arrangement may be created which is capable of rapidly responding to changing load requirements.", "With respect to the method, the object is achieved by means of the method of operating a fuel cell arrangement indicated herein.", "With respect to the device, the object is achieved by means of the fuel cell arrangement indicated herein.", "The invention creates a method of operating a fuel cell arrangement having one or more fuel cells, to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device, and having one or more internal electrical consuming devices which can optionally be switched on and off, as well as having a cooling device by which excessive heat is eliminated from the fuel cell arrangement.", "According to the invention, it is provided that, in the operation of the fuel cell arrangement, a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform.", "It is an advantage of the method according to the invention that, while the generating of the electric power is uniform, the fuel cells can rapidly respond to a change of the load demand by the external electrical consuming device.", "According to a preferred embodiment of the invention, it is provided that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the power converted at the internal electrical consuming devices.", "According to another aspect of the invention, it is provided that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.", "It is preferably provided that the change of the electric power inverted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device.", "Here, it is particularly advantageous for the electric power generated by the fuel cells to be kept essentially constant.", "According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells.", "Preferably, the electric power converted at the internal electrical consuming devices is removed from the fuel cell arrangement by an increased use of the cooling device.", "Furthermore, by means of the invention, a fuel cell arrangement is created which has one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply an electric power to an external consuming device, and which has one or more internal electrical consuming devices which can optionally be switched on and off, and which has a cooling device by means of which the excessive heat is removed from the fuel cell arrangement, and which has a control device for controlling the operation of the internal consuming devices as a function of the operating condition of the fuel cell arrangement.", "According to the invention, it is provided that the control device controls the internal electrical consuming devices such that a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform.", "It is an advantage of the fuel cell arrangement according to the invention that, while the generating of the electric power is essentially uniform, this fuel cell arrangement can rapidly respond to a change of the load demand of the connected electrical consuming device.", "According to an aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.", "According to another aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.", "Preferably, it is provided that the control device controls the internal electrical consuming devices such that the change of the electric power converted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device.", "Furthermore, it is an advantage for the control device to control the internal electrical consuming devices such that the electric power generated by the fuel cells is essentially kept constant.", "According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells.", "Furthermore, it is advantageous for the cooling device to be constructed such that it removes the electric power converted at the internal electrical consuming devices from the fuel cell arrangement.", "Advantageously, the internal electrical consuming devices comprise a start heating device and/or a ventilating device and/or other aggregates of the fuel cell arrangement.", "According to a preferred embodiment of the invention, it is provided that the fuel cell arrangement comprises an electrical inverter for converting the direct voltage supplied by the fuel cells into an alternating voltage which is fed to the external consuming device.", "Finally, according to an advantageous embodiment of the invention, it is provided that the internal electrical consuming devices can be connected by means of the control device optionally parallel to the external electric consuming device with the output of the inverter." ], [ "CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to German Patent Application No.", "101 06 219.2, which was filed Feb. 10, 2001.FIELD OF THE INVENTION The invention relates to a method of operating a fuel cell arrangement and further to a fuel cell arrangement.", "BACKGROUND OF THE INVENTION Fuel cell arrangements are known which comprise one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device.", "One or more internal electrical consuming devices of the fuel cell arrangement can optionally be switched on and off.", "A cooling device is provided for eliminating excessive heat from the fuel cell arrangement.", "One difficulty during the operation of fuel cell arrangements of the above-mentioned type is the fact that, with respect to their performance, they cannot arbitrarily rapidly follow a change of the electric load of the connected external consuming device.", "The reason is based less on the dynamics of the electrochemical events taking place in the fuel cells than in the slowness of supplying the fuel cells with their reaction gases: the combustible gas and the cathode gas.", "The load-following operation therefore requires electric power at the expense of the efficiency in order to be able to immediately react in the event of a load demand.", "In the case of a power supply system failure, i.e., in the event of a sudden failure of the load demand of the connected external consuming device, the fuel cell arrangement, if possible, should not switch off but change to a standby operation, in which the current requirement for the internal consuming devices of the fuel cell arrangement is covered.", "The intrinsic consumption of the fuel cell arrangement is minimized for economical reasons, so that the system generates little electric power.", "In the load-following operation, the amount of the current production of a fuel cell arrangement is defined by the external consuming devices connected to it.", "Such a condition, which is also called an “island operation”, particularly in the case of small system sizes, results in load demands, which fluctuate considerably with respect to their percentages as a result of the connecting or disconnecting of individual consumption points.", "Rapid load changes occur during failures of the external consuming device, for example, in the event of a power supply system failure.", "The fuel cell arrangement will then supply no electric power to the external consuming device.", "In this case, the fuel cell arrangement is to change to a standby operation in which it generates just enough electric power in order to satisfy the intrinsic consumption.", "SUMMARY OF THE INVENTION It is an object of the invention to provide a method of operating a fuel cell arrangement of the initially mentioned type by means of which the fuel cell arrangement can be operated in the sense of a fast response to changing load requirements.", "Furthermore, by means of the invention, a fuel cell arrangement may be created which is capable of rapidly responding to changing load requirements.", "With respect to the method, the object is achieved by means of the method of operating a fuel cell arrangement indicated herein.", "With respect to the device, the object is achieved by means of the fuel cell arrangement indicated herein.", "The invention creates a method of operating a fuel cell arrangement having one or more fuel cells, to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device, and having one or more internal electrical consuming devices which can optionally be switched on and off, as well as having a cooling device by which excessive heat is eliminated from the fuel cell arrangement.", "According to the invention, it is provided that, in the operation of the fuel cell arrangement, a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform.", "It is an advantage of the method according to the invention that, while the generating of the electric power is uniform, the fuel cells can rapidly respond to a change of the load demand by the external electrical consuming device.", "According to a preferred embodiment of the invention, it is provided that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the power converted at the internal electrical consuming devices.", "According to another aspect of the invention, it is provided that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.", "It is preferably provided that the change of the electric power inverted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device.", "Here, it is particularly advantageous for the electric power generated by the fuel cells to be kept essentially constant.", "According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells.", "Preferably, the electric power converted at the internal electrical consuming devices is removed from the fuel cell arrangement by an increased use of the cooling device.", "Furthermore, by means of the invention, a fuel cell arrangement is created which has one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply an electric power to an external consuming device, and which has one or more internal electrical consuming devices which can optionally be switched on and off, and which has a cooling device by means of which the excessive heat is removed from the fuel cell arrangement, and which has a control device for controlling the operation of the internal consuming devices as a function of the operating condition of the fuel cell arrangement.", "According to the invention, it is provided that the control device controls the internal electrical consuming devices such that a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform.", "It is an advantage of the fuel cell arrangement according to the invention that, while the generating of the electric power is essentially uniform, this fuel cell arrangement can rapidly respond to a change of the load demand of the connected electrical consuming device.", "According to an aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.", "According to another aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.", "Preferably, it is provided that the control device controls the internal electrical consuming devices such that the change of the electric power converted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device.", "Furthermore, it is an advantage for the control device to control the internal electrical consuming devices such that the electric power generated by the fuel cells is essentially kept constant.", "According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells.", "Furthermore, it is advantageous for the cooling device to be constructed such that it removes the electric power converted at the internal electrical consuming devices from the fuel cell arrangement.", "Advantageously, the internal electrical consuming devices comprise a start heating device and/or a ventilating device and/or other aggregates of the fuel cell arrangement.", "According to a preferred embodiment of the invention, it is provided that the fuel cell arrangement comprises an electrical inverter for converting the direct voltage supplied by the fuel cells into an alternating voltage which is fed to the external consuming device.", "Finally, according to an advantageous embodiment of the invention, it is provided that the internal electrical consuming devices can be connected by means of the control device optionally parallel to the external electric consuming device with the output of the inverter.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 illustrates a block diagram of a fuel cell arrangement according to an embodiment of the invention.", "DETAILED DESCRIPTION OF THE INVENTION FIG.", "1 illustrates a fuel cell arrangement which, as a whole, has the reference number 10, and comprises one or more fuel cells 1.A combustible gas and a cathode gas are fed (not shown in the figure) to the fuel cells 1, from which electric energy is obtained in an electrochemical manner.", "This electric energy is supplied by the fuel cells 1 to an external consuming device 2 as electric power Pext.", "The external consuming device 2 is illustrated in FIG.", "1 as an electrical line network, which supplies several consuming points that are not separately shown in the figure.", "The fuel cell arrangement 10 comprises one or more internal electrical consuming devices 3 which are used for the operation of the fuel cell arrangement 10 and which can optionally be switched on and off.", "Such internal electrical consuming devices 3 may, for example, comprise a start heating device 7 required for the starting operation and/or a ventilator device 8 required for the circulation of reaction gases, such as the combustible gas and the cathode gas or a cooling gas, and/or other aggregates 9 of the fuel cell arrangement.", "The fuel cell arrangement 10 also has a cooling device 4 by means of which excessive heat generated during the operation of the fuel cell arrangement 10 is removed from the latter.", "A control device 5 is used for controlling the operation of the internal consuming devices 3 as a function of the operating condition of the fuel cell arrangement 10.During the operation of the fuel cell arrangement 10, a portion of the electric power PBZ generated by the fuel cells 1 is converted at the internal electrical consuming devices 3.When the electric power Pext supplied to the external electrical consuming device 2 increases or decreases, the electric power Pint converted at the internal electrical consuming devices 3 is inversely reduced or increased in the sense of making the electric power PBZ generated by the fuel cells uniform.", "The control device 5 controls the internal electrical consuming devices 3 in this sense.", "For a load-following operation, thus an operation in which the fuel cell arrangement adapts the electric power provided by it to the load requirement of the connected external consuming device 2, that is, of the connected electrical power supply system, an increase of the power Pext supplied to the external electrical consuming device 2 is compensated by a reduction of the electric power Pint converted at the internal electrical consuming devices 3.In a corresponding manner, a reduction of the power Pext supplied to the external electrical consuming device 2 is compensated by an increase of the electric power Pint converted at the internal electrical consuming devices 3.For a standby operation, thus an operation in which, for example, in the event of the failure of the connected power supply system, or if its load requirement should be zero, no electric power Pext can be supplied anymore to the outside, a switching-off of the external electrical consuming device 2, for example, the above-mentioned power supply system failure, is compensated by an increase of the electric power Pint converted at the internal electrical consuming device 3.As a result, the fuel cell arrangement does not have to be moved to a minimal operating point but can still generate electric power in the amount of the electric power Pint converted at the internal electrical consuming devices 3.As required, thus when the external electrical consuming device 2, that is the connected power supply system, is restored, this power will immediately be available again.", "The change of the electric power Pint converted at the internal electrical consuming devices 3 essentially takes place immediately with the change of the power Pint supplied to the external electrical consuming device 2.This is carried out by the control device 5.The electric power PBZ generated by the fuel cells 1 is kept essentially constant; that is, PBZ−Pint=Pext.", "In the embodiment described here, the internal electrical consuming devices 3 are dimensioned such that maximally an electric power Pint is converted which amounts approximately to one quarter to approximately half of the nominal electric power of the fuel cells 1.The electric power Pint converted at the internal electrical consuming devices 3 is removed from the fuel cell arrangement 10 by an increased use of the cooling device 4.The electric power supplied by the fuel cells 1 to the external consuming device 2 is converted by an inverter 6 provided in the fuel cell arrangement 10 from the direct voltage supplied by the fuel cells 1 to an alternating voltage that is supplied to the external consuming device 2.The internal electrical consuming devices 3 can be connected by means of the control device 5 optionally parallel to the external electrical consuming device 2 with the output of the inverter 6.The adaptation of the electric power Pint converted at the internal electrical consuming devices 3 to the power Pext supplied to the external electrical consuming device 2 in the sense of making the electric power PBZ uniform which is generated by the fuel cells 1, can take place in steps or continuously, depending on the type and construction of the control device 5 by means of which this adaptation is carried out." ] ]	Patent_10467525
[ [ "Recombinant proteinase k", "The invention concerns recombinant proteinase K. Furthermore a method for producing recombinant proteinase K is disclosed, which is characterized in that a) a host cell is transformed with a recombinant nucleic acid which codes for the zymogenic precursor of proteinase K, b) the host cell is cultured in such a manner that the zymogenic precursor of proteinase K is formed in the form of inclusion bodies in the host cell, c) the inclusion bodies are isolated and natured under conditions which result in the formation of the protease part of the zymogenic precursor in its natural conformation, d) the natured proteinase K is activated and purified." ], [ "1-22.", "(canceled) 23.A method for the naturation of denatured zymogenic proteinase K comprising transferring the denatured zymogenic proteinase K to a folding buffer, the buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH of 7.5 to 10.5 and the method being carried out at a temperature between 0° C. and 37° C. 24.The method of claim 23 wherein the redox shuffling system comprises mixed disulfides or thiosulfonates.", "25.The method of claim 23 wherein the pH range is 8 to 9.26.The method of claim 23 wherein the temperature is between 0° C. and 25° C. 27.The method of claim 23 wherein the buffer further comprises denaturing agents at a concentration of less than 50 mM.", "28.The method of claim 23 wherein the low molecular weight substances are selected from the group consisting of L-arginine at a concentration of 0.5 to 2.0 M, Tris at a concentration of 0.5 M to 2.0 M, triethanolamine at a concentration of 0.5 M to 2.0 M, and α-cyclodextrin at a concentration of 60 mM to 120 mM.", "29.The method of claim 23 wherein the Ca2+ ion concentration is 1 to 20 mM.", "30.The method of claim 23 wherein the denatured zymogenic proteinase K is transferred to the folding buffer while reducing the concentration of denaturing agents that may be present.", "31.A folding buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH value in the range of 7.5 to 10.5.32.The buffer of claim 31 wherein the pH value is 8 to 9 and the redox shuffling system comprises mixed disulfides or thiosulfonates.", "33.A method for activating a natured zymogenic precursor of active proteinase K comprising adding a detergent to an inactive complex comprising a native proteinase K and an inhibitory propeptide of the active proteinase K, thereby releasing the active proteinase K from the inactive complex.", "34.The method of claim 33 wherein the detergent is SDS at a concentration of 0.1 to 2% (w/v).", "35.A method for producing active recombinant proteinase K comprising (a) producing an inactive zymogenic proform of proteinase K in an inclusion body, (b) naturing in vitro the zymogenic proform of proteinase K, and (c) activating the zymogenic proform by autocatalytic cleavage, thereby converting it to the active proteinase K. 36.The method of claim 35 wherein the naturing step comprises transferring the denatured zymogenic proteinase K to a folding buffer, the buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH of 7.5 to 10.5 and the naturing step being carried out at a temperature between 0° C. and 37° C. 37.The method of claim 35 wherein the inclusion body is solubilized by a denaturing agent and a reducing agent.", "38.The method of claim 37 wherein the denaturing agent is guanidinium hydrochloride at a concentration of 6-8 M or urea at a concentration of 8-10 M and the reducing agent is DTT or DTE at a concentration of 50-200 mM.", "39.A method for producing active recombinant proteinase K comprising (a) transforming a host cell with a vector containing a DNA sequence coding for a zymogenic precursor of proteinase K (b) expressing the zymogenic precursor in inclusion bodies, (c) isolating the inclusion bodies and solubilizing the zymogenic precursor, (d) naturing the zymogenic precursor with a folding buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH of 7.5 to 10.5 and the naturing step being carried out at a temperature between 0° C. and 37° C., and (e) activating the zymogenic precursor by autocatalytic cleavage, thereby converting it to the active proteinase K 40.The method of claim 39 wherein the host cell is a prokaryotic cell.", "41.The method of claim 39 wherein the host cell is Escherichia coli.", "42.A codon-optimized recombinant nucleic acid comprising DNA coding for a recombinant zymogenic proteinase K which has been optimized for expression in Escherichia coli.", "43.A vector containing the recombinant nucleic acid of claim 42.44.A host cell transformed with the vector of claim 43." ], [ "The present invention concerns the preparation of recombinant proteinase K from Tritirachium album Limber and corresponding methods for the expression, in vitro naturation and activation of the recombinant proteinase K. Proteinase K (E.C.", "3.4.21.64, also known as endopeptidase K) is an extracellular endopeptidase which is synthesized by the fungus Tritirachium album Limber.", "It is a member of the class of serine proteases with the typical catalytic triad Asp39-His69-Ser224 (Jany, K. D. et al.", "(1986) FEBS Letters Vol.", "199(2), 139-144).", "Since the sequence of the polypeptide chain of 279 amino acids in length (Gunkel, F. A. and Gassen, H. G. (1989) Eur.", "J. Biochem.", "Vol.", "179(1), 185-194) and the three dimensional structure (Betzel, C. et al.", "(1988) Eur.", "J. Biochem.", "Vol.", "178(1), 155-71) has a high degree of homology to bacterial subtilisins, proteinase K is classified as a member of the subtilisin family (Pahler, A. et al.", "(1984) EMBO J. Vol.", "3(6), 1311-1314; Jany, K. D. and Mayer, B.", "(1985), Biol.", "Chem.", "Hoppe-Seyler, Vol.", "366(5), 485-492).", "Proteinase K was named on the basis of its ability to hydrolyse native keratin and thus allows the fungus to grow on keratin as the only source of carbon and nitrogen (Ebeling, W. et al.", "(1974) Eur.", "J. Biochem.", "Vol.", "47(1), 91-97) Roelcke and Uhlenbruch, 1069, Z. Med.", "Mikrobiol.", "Immunol.", "Vol.", "155(2), 156-170).", "Proteinase K has a specific activity of more than 30 U/mg and is thus one of the most active of the known endopeptidases (Betzel, C. et al.", "(1986) FEBS Lett.", "Vol.", "197(1-2), 105-110) and unspecifically hydrolyses native and denatured proteins (Kraus, E. and Femfert, U, (1976) Hoppe Seylers Z. Physiol.", "Chem.", "Vol.", "357(7):937-947).", "Proteinase K from Tritirachium album Limber is translated in its natural host as a preproprotein.", "The sequence of the cDNA of the gene which codes for proteinase K was decoded in 1989 by Gunkel, F. A. and Gassen, H. G. (1989) Eur.", "J. Biochem.", "Vol.", "179(1), 185-194.According to this the gene for prepro-proteinase K is composed of two exons and codes for a signal sequence of 15 amino acids in length, a prosequence of 90 amino acids in length and a mature proteinase K of 279 amino acids in length.", "A 63 bp intron is located in the region of the prosequence.", "The prepeptide is cleaved off during translocation into the endoplasmatic reticulum (ER).", "At present very little is known about the subsequent processing to form mature proteinase K with cleavage of the propeptide.", "Consequently mature proteinase K consists of 279 amino acids.", "The compact structure is stabilized by two disulfide bridges and two bound calcium ions.", "This explains why proteinase K compared to other subtilisins has a considerably higher stability towards extreme pH values, high temperatures, chaotropic substances and detergents (Dolashka, P. et al.", "(1992) Int.", "J. Pept.", "Protein.", "Res.", "Vol.", "40(5), 465-471).", "Proteinase K is characterized by a high thermostability (up to 65° C., Bajorath et al.", "(1988), Eur.", "J. Biochem.", "Vol.", "176, 441-447) and a wide pH range (pH 7.5-12.0, Ebeling, W. et al.", "(1974) Eur.", "J. Biochem.", "Vol.", "47(1), 91-97).", "Its activity is increased in the presence of denaturing substances such as urea or SDS (Hilz, H. et al.", "(1975) J. Biochem.", "Vol.", "56(1), 103-108; Jany, K. D. and Mayer, B.", "(1985) Biol.", "Chem.", "Hoppe-Seyler, Vol.", "366(5), 485-492).", "The above-mentioned properties make proteinase K of particular interest for biotechnological applications in which an unspecific protein degradation is required.", "Special examples are nucleic acid isolation (DNA or RNA) from crude extracts and sample preparation in DNA analysis (Goldenberger, D. et al.", "(1995) PCR Methods Appl.", "Vol.", "4(6), 368-370; U.S. Pat.", "No.", "5,187,083; U.S. Pat.", "No.", "5,346,999).", "Other applications are in the field of protein analysis such as structure elucidation.", "Proteinase K is obtained commercially in large amounts by fermentation of the fungus Tritirachium album Limber (e.g.", "CBS 348.55, Merck strain No.", "2429 or the strain ATCC 22563).", "The production of proteinase K is suppressed by glucose or free amino acids.", "Since protein-containing media also induce the expression of proteases, it is necessary to use proteins such as BSA, milk powder or soybean flour as the only nitrogen source.", "The secretion of the protease starts as soon as the stationary phase of growth is reached (Ebeling, W. et al.", "(1974) Eur.", "J. Biochem.", "Vol.", "47(1), 91-97).", "Since Tritirachium album Limber is consequently unsuitable for fermentation on a large scale and moreover is difficult to genetically manipulate, various attempts have been made to overexpress recombinant proteinase K in other host cells.", "However, no significant activity was detected in these experiments due to lack of expression, formation of inactive inclusion bodies or problems with the naturation (Gunkel, F. A. and Gassen, H. G. (1989) Eur.", "J. Biochem.", "Vol.", "179(1), 185-194; Samal, B.", "B. et al.", "(1996) Adv.", "Exp.", "Med.", "Biol.", "Vol.", "379, 95-104).", "Moreover, Tritirachium album Limber is a slowly growing fungus which only secretes small amounts of proteases into the medium.", "The long fermentation period of one to two weeks is disadvantageous.", "In addition it is known that T. album also produces other proteases apart from proteinase K which can contaminate the preparation (Samal, B.", "B. et al.", "(1991) Enzyme Microb.", "Technol.", "Vol.", "13, 66-70).", "The object of the present invention is to provide a method for the economical production of recombinant proteinase K and of inactive zymogenic precursors of proteinase K that can be autocatalytically activated.", "The object was achieved by providing a method for producing recombinant proteinase K in which the inactive zymogenic proform of proteinase K is produced in an insoluble form in inclusion bodies, and the zymogenic proform of proteinase K is natured and the zymogenic proform processed i.e.", "activated in subsequent steps.", "The methods for the naturation and activation of proteinase K are also a subject matter of the present invention.", "The present invention concerns a method for producing recombinant proteinase K characterized in that the zymogenic proform is folded by in vitro naturation and is converted by autocatalytic cleavage into the active form.", "The present invention concerns in particular a method for producing a recombinant proteinase K in which a zymogenic precursor of proteinase K is converted by oxidative folding from isolated and solubilized inclusion bodies into the native structure i.e.", "it is natured and subsequently the active proteinase K is obtained from the natively folded zymogen by autocatalytic cleavage by adding detergents.", "Hence the present invention concerns a method for obtaining recombinant proteinase K by transforming a host cell with a DNA coding for the zymogenic proform of proteinase K characterized by the following process steps: a) Culturing the said host cell under conditions which result in an expression of the DNA coding for the zymogenic proform of proteinase K such that a zymogenic precursor of proteinase K is formed in the host cell in the form of insoluble inclusion bodies.", "b) Isolating the inclusion bodies, solubilizing the enzyme and naturing of the zymogenic precursor of proteinase K under conditions in which the protease part of the zymogenic precursor of proteinase K is formed.", "c) Activating the proteinase K by removing the propeptide and further purification.", "The DNA coding for the zymogenic proform of proteinase K corresponds to the DNA shown in SEQ ID NO: 2 or a DNA corresponding thereto within the scope of the degeneracy of the genetic code.", "SEQ ID NO: 2 includes the DNA sequence which codes for proteinase K and the propeptide.", "Furthermore the DNA can be codon-optimized for expression in a particular host.", "Method for codon-optimization are known to a person skilled in the art and are described in example 1.Hence the present invention concerns methods in which the host cell is transformed by a DNA which is selected from the above-mentioned group.", "A proteinase K is obtained by the method according to the invention which is homogeneous and hence particularly suitable for analytical and diagnostic applications.", "The zymogenic proform of proteinase K according to the invention can optionally contain additional N-terminal modifications and in particular sequences which facilitate purification of the target protein (affinity tags), sequences which increase the efficiency of translation, sequences which increase the folding efficiency or sequences which result in a secretion of the target protein into the culture medium (natural presequence and other signal peptides).", "Proteinase K in the sense of the invention means the sequence according to Gassen et al.", "(1989) shown in SEQ ID NO: 1 as well as other variants of proteinase K from Tritirachium album Limber like the amino acid sequence disclosed by Ch.", "Betzel et al.", "(Biochemistry 40 (2001), 3080-3088) and in particular proteinase T (Samal, B.", "B. et al.", "(1989) Gene Vol.", "85(2), 329-333; Samal, B.", "B. et al.", "(1996) Adv.", "Exp.", "Med.", "Biol.", "Vol.", "379, 95-104) and proteinase R (Samal, B.", "B. et al.", "(1990) Mol.", "Microbiol.", "Vol.", "4(10), 1789-1792; U.S. Pat.", "No.", "5,278,062) and in addition variants produced by recombinant means (as described for example in WO 96/28556).", "The sequence shown in SEQ ID NO: 1 comprises the signal sequence (1-15, 15 amino acids), the prosequence (16-105; 90 amino acids) and the sequence of the mature proteinase K (106-384; 279 amino acids).", "The amino acid sequence described by Betzel et al.", "(Biochemistry 40 (2001), 3080-3088) has in particular aspartate instead of a serine residue at position 207 of the active protease.", "Pro-proteinase K in the sense of the invention means in particular a proteinase K whose N-terminus is linked to its prosequence.", "In the case of the closely related subtilisin E and other variants it is known that the prosequence has an important influence on the folding and formation of active protease (Ikemura, H. et al.", "(1987) Biol.", "Chem.", "Vol.", "262(16), 7859-7864).", "In particular it is presumed that the prosequence acts as an intramolecular chaperone (Inouye, M. (1991) Enzyme Vol.", "45, 314-321).", "After the folding it is processed to form the mature subtilisin protease by autocatalytically cleaving the propeptide (Ikemura, H. and Inouye, M. (1988) J. Biol.", "Chem.", "Vol.", "263(26), 12959-12963).", "This process occurs in the case of subtilisin E (Samal, B.", "B. et al.", "(1989) Gene vol.", "85(2), 329-333; Volkov, A. and Jordan, F. (1996) J. Mol.", "Biol.", "Vol.", "262, 595-599), subtilisin BPN′ (Eder, J. et al.", "(1993) Biochemistry Vol.", "32, 18-26), papain (Vernet, T. et al.", "(1991) J. Biol.", "Chem.", "Vol.", "266(32), 21451-21457) and thermolysin (Marie-Claire, C. (1998) J. Biol.", "Chem.", "Vol.", "273(10), 5697-5701).", "If added exogenously the propeptide can also act intermolecularly in trans as a chaperone on the folding of denatured mature subtilisin protease (Ohta, Y. et al.", "(1991) Mol.", "Microbiol.", "Vol.", "5(6), 1507-1510; Hu, Z. et al.", "(1996) J. Biol.", "Chem.", "Vol.", "271(7), 3375-3384).", "The propeptide binds to the active centre of subtilisin (Jain, S. C. et al.", "(1998) J. Mol.", "Biol.", "Vol.", "284, 137-144) and acts as a specific inhibitor (Kojima, S. et al.", "(1998) J. Mol.", "Biol.", "Vol.", "277, 1007-1013; Li, Y. et al.", "(1995) J. Biol.", "Chem.", "Vol.", "270, 25127-25132; Ohta, Y.", "(1991) Mol.", "Microbiol.", "Vol.", "5(6), 1507-1510).", "This effect is used in the sense of the invention in order to prevent autoproteolysis of proteolysis-sensitive folding intermediates by already folded, active proteinase K during the naturation.", "Only certain, usually hydrophobic core regions of the prosequence appear to be necessary for the chaperone function since mutations in wide areas have no influence on the activity (Kobayashi, T. and Inouye, M. (1992) J. Mol.", "Biol.", "Vol.", "226, 931-933).", "In addition it is known that propeptides can be exchanged between various subtilisin variants.", "Thus for example subtilisin BPN′ also recognizes the prosequence of subtilisin E (Hu, Z. et al.", "(1996) J. Biol.", "Chem.", "Vol.", "271(7), 3375-3384).", "Inclusion bodies are microscopically visible particles consisting of insoluble and inactive protein aggregates which are often formed in the cytoplasm of the host cell when heterologous proteins are overexpressed and they contain very pure target protein.", "Methods for producing and purifying such inclusion bodies are described for example in Creighton, T. E. (1978) Prog.", "Biophys.", "Mol.", "Biol.", "Vol.", "33(3), 231-297; Marston, F. A.", "(1986) Biochem.", "J. Vol.", "240(1), 1-12; Rudolph, R. (1997).", "Folding proteins in: Creighton, T. E.", "(ed.)", "Protein Function: A practical approach.", "Oxford University Press, 57-99; Fink, A. L. (1998) Fold.", "Des.", "Vol.", "3(1), R9-23; and EP 0 114 506.In order to isolate inclusion bodies the host cells are lysed after fermentation by conventional methods e.g.", "by ultrasound, high pressure dispersion or lysozyme.", "The lysis preferably takes place in an aqueous neutral to slightly acid buffer.", "The insoluble inclusion bodies can be separated and purified by various methods, preferably by centrifugation or filtration with several washing steps (Rudolph, R. (1997).", "Folding Proteins In: Creighton, T. E.", "(ed.)", "Protein Function: A practical Approach.", "Oxford University Press, 57-99).", "The inclusion bodies obtained in this manner are then solubilized in a known manner.", "Denaturing agents are advantageously used for this at a concentration which is suitable for dissolving the inclusion bodies, in particular guanidinium hydro-chloride and other guanidinium salts and/or urea.", "In order to completely monomerize the inclusion body proteins it is also advantageous to add reducing agents such as dithiothreitol (DTT), dithioerythritol (DTE) or 2-mercaptoethanol during the solubilization in order to break possible disulfide bridges by reduction.", "The invention also concerns a proteinase K in which the cysteines are not reduced but are derivatized in particular with GSSG to form mixed disulfides or thiocyanates (EP 0 393 725).", "Hence according to the invention the inclusion bodies are solubilized by denaturing agents and reducing agents.", "6-8 M guanidinium hydrochloride or 8-10 M urea are preferred as denaturing agents and 50-200 mM DTT (dithiothreitol) or DTE (dithioerythritol) are preferred as reducing agents.", "Hence the present invention concerns the prosequence according to SEQ ID NO: 1 of 90 amino acids in length (amino acids 16-105) as well as other variants which facilitate folding.", "It also concerns a propeptide which is added exogenously for the folding of mature proteinase K and has the functions described above.", "A further subject matter of the invention is a recombinant vector which contains one or more copies of the recombinant DNA defined above.", "The basic vector is advantageously a plasmid preferably containing a multi-copy origin of replication, but is also possible to use viral vectors.", "The choice of expression vector depends on the selected host cell.", "Methods are used to construct the expression vector and to transform the host cell with this vector that are familiar to a person skilled in the art and are described for example in Sambrook et al.", "(1989), Molecular Cloning (see below).", "A suitable vector for expression in E. coli is for example the pKKT5 expression vector or pKK177, pKK223, pUC, pET vectors (Novagen) as well as pQE vectors (Qiagen).", "The expression plasmid pKKT5 is formed from pKK177-3 (Kopetzki et al., 1989, Mol.", "Gen. Genet.", "216:149-155) by exchanging the tac promoter for the T5 promoter from pDS (Bujard et al., 1987, Methods Enzymol.", "155:416-433).", "The EcoRI restriction endonuclease cleavage site in the sequence of the T5 promoter was removed by two point mutations.", "In addition the coding DNA in the vector according to the invention is under the control of a preferably strong, regulatable promoter.", "A promoter that can be induced by IPTG is preferred such as the lac, lacUV5, tac or T5 promoter.", "The T5 promoter is especially preferred.", "A host cell in the sense of the invention means any host cell in which proteins can form as inclusion bodies.", "It is usually a microorganism e.g.", "prokaryotes.", "Prokaryotic cells are preferred and in particular Escherichia coli.", "Particular preference is given to the following strains: E. coli K12 strains JM83, JM105, UT5600, RR1Δ15, DH5α, C600, TG1, NM522, M15 or the E. coli B derivatives BL21, HB101, E. coli M15 is particularly preferred.", "The corresponding host cells are transformed according to the invention with a recombinant nucleic acid which encodes a recombinant zymogenic proteinase K according to SEQ ID NO:2 or with a nucleic acid which is derived from the said DNA by codon-optimization or with a DNA which is derived from the said DNA within the scope of the degeneracy of the genetic code.", "The E. coli host cells are preferably transformed with a codon-optimized recombinant nucleic acid coding for a recombinant zymogenic proteinase K which has been optimized for expression in Escherichia coli.", "Hence the present invention also concerns a suitable vector which is for example selected from the above-mentioned vectors and contains a recombinant nucleic acid that is codon-optimized for E. coli and codes for a recombinant proteinase K or a recombinant zymogenic proteinase K. Another subject matter of the invention is a host cell which is for example selected from the above-mentioned host cells which has been transformed by the above-mentioned vector.", "A further subject matter of the present invention is a method for the naturation of denatured zymogenic proteinase K in which the denatured zymogenic proteinase K is transferred to a folding buffer which is characterized in that the folding buffer has the following features: A) pH value of the buffer is in the range of 7.5 to 10.5 B) presence of low-molecular weight substances which aid folding C) presence of a redox shuffling system D) presence of a complexing agent at a substoichiometric concentration relative to the Ca2+ ions and wherein the method is carried out at a temperature between 0° C. and 37° C. A low concentration of denaturing agents is preferably present during the naturation.", "Denaturing agents may for example be present because they are still in the reaction solution due to the prior solubilization of the inclusion bodies.", "The concentration of denaturing agents such as guanidine hydrochloride should be less than 50 mM.", "Naturation in the sense of the invention is understood as a method in which denatured, essentially inactive protein is converted into a conformation in which the protein has the desired activity after autocatalytic cleavage and activation.", "This is achieved by transferring the solubilized inclusion bodies to a folding buffer while reducing the concentration of the denaturing agent.", "The conditions must be selected such that the protein remains in solution in this process.", "This can be expediently carried out by rapid dilution or dialysis against the folding buffer.", "It is preferred that the folding buffer has a pH of 8 to 9.Particularly preferred buffer substances are Tris/HCl buffer and bicine buffer.", "The naturation method according to the invention is preferably carried out at a temperature between 0° C. and 25° C. The low molecular weight folding agents in the folding buffer are preferably selected from the following group of low molecular weight compounds.", "They can be added alone as well as in mixtures, and other substances that aid folding may be present: L-arginine at a concentration of 0.5 to 2.0 M Tris at a concentration of 0.5 M to 2.0 M triethanolamine at a concentration of 0.5 M to 2.0 M α-cyclodextrin at a concentration of 60 mM to 120 mM Low molecular weight substances that aid folding are described for example in U.S. Pat.", "No.", "5,593,865; Rudolph, R. (1997) Folding Proteins.", "In: Creighton, T. E.", "(ed.)", "Protein Function: A practical Approach.", "Oxford University Press, 57-99 or De Bernardez Clark, E. et al.", "(1999) Methods.", "Enzymol.", "Vol.", "309,217-236.The above-mentioned redox shuffling system is preferably a mixed disulfide or thiosulfonate.", "Systems are for example suitable as a redox shuffling system which consist of a thiol component in an oxidized and reduced form.", "This allows the formation of disulfide bridges within the folding polypeptide chain during naturation by controlling the reduction potential, and on the other hand, enables the reshuffling of incorrect disulfide bridges within or between the folding polypeptide chains (Rudolph, R. (1997), see above).", "Preferred thiol components are for example: glutathione in a reduced (GSH) and oxidized form (GSSH) cysteine and cystine cysteamine and cystamine 2-mercaptoethanol and 2-hydroxyethyldisulfide In the naturation method according to the invention the Ca2+ ions are preferably present at a concentration of 1 to 20 mM.", "For example CaCl2 can be added in amounts of 1 to 20 mM.", "The Ca2+ ions can bind to the calcium binding sites of the folding proteinase K. The presence of a complexing agent preferably EDTA, in a substoichiometric concentration relative to Ca2+ prevents the oxidation of the reducing agent by atmospheric oxygen and protects free SH groups.", "The naturation is preferably carried out at a low temperature i.e.", "below 20° C., preferably 10° C. to 20° C. In the method according to the invention the naturation is usually completed after a period of about 24 h to 48 h. The present invention also concerns a folding buffer which is characterized by the following features: A) pH value of the buffer is in the range of 7.5 to 10.5 B) presence of low-molecular weight substances which aid folding C) presence of a redox shuffling system D) presence of a complexing agent at a substoichiometric concentration relative to the Ca2+ ions.", "It is especially preferred when the folding buffer has a pH of 8 to 9 and/or when the redox shuffling system is a mixed disulfide or thiosulfonate.", "Another subject matter of the invention is a method for activating the natured zymogenic precursor of proteinase K. After the folding process according to the invention an inactive complex is formed from native proteinase K and the inhibitory propeptide.", "The active proteinase K can be released from this complex.", "Addition of detergents is preferred, SDS is particularly preferred at a concentration of 0.1 to 2% (w/v).", "The advantages of the method disclosed here for producing recombinant proteinase K are: 1.The ability to utilize the high expression potential and the rapid and simple culture of Escherichia coli or other suitable microorganisms.", "2.The possibility to genetically manipulate the recombinant DNA.", "3.The uncomplicated purification after naturation.", "4.The absence of eukaryotic impurities when a prokaryote is selected as a host cell.", "A method would also be conceivable in which the nucleic acids which code for mature proteinase K and nucleic acids which code for the propeptide or pro-proteinase K are expressed separately in host cells and are then commonly transferred to a folding buffer for the naturation of mature proteinase K. DESCRIPTION OF THE FIGURES FIG.", "1: Schematic representation of the PCR reaction to produce proteinase K fragments having an N-terminal BamHI cleavage site and an alternative enterokinase cleavage site for fusion with an N-terminal affinity tag.", "FIG.", "2: Dependency of the yield of naturation on temperature.", "FIG.", "3: Dependency of the yield of naturation on pH.", "FIG.", "4: Dependency of the yield of naturation on redox potential.", "FIG.", "5: Dependency of the yield of naturation on the arginine concentration.", "FIG.", "6: Dependency of the yield of naturation on the Tris concentration.", "FIG.", "7: Dependency of the yield of naturation on the α-cyclodextrin concentration.", "FIG.", "8: Dependency of the yield of naturation on the triethanolamine concentration.", "FIG.", "9: Dependency of the yield of naturation on the urea concentration.", "FIG.", "10: SDS polyacrylamide gel of the naturation of pro-proteinase K. FIG.", "11: Reverse phase chromatography of natured proteinase K. FIG.", "12: Renatured and processed proteinase K was analysed by analytical ultracentrifugation.", "The centrifugation was carried out at 12000 rpm, 20° C. for 63 h. The data (o) could be fitted to a homogeneous species having an apparent molecular weight of 29-490 Da.", "No systematic deviation was observed between the fitted and measured data (lower graph).", "FIG.", "13: Determination of the Km value of natured proteinase K. FIG.", "14: Degradation pattern of blood serum proteins by natured proteinase K. FIG.", "15: Purification of natured proteinase K by gel filtration.", "EXAMPLE 1 Synthesis of the Gene which Codes for the Mature Form of Proteinase K. The gene for the mature proteinase K from Tritirachium album Limber without a signal sequence and without an intron was generated by means of gene synthesis.", "The sequence of Gunkel, F. A. and Gassen, H. G. (1989) Eur.", "J. Biochem.", "Vol.", "179(1), 185-194 of 837 bp in length (amino acids 106-384 from Swiss Prot P06873) was used as the template.", "A codon usage optimized for Escherichia coli was used as the basis for retranslating the amino acid sequence to optimize the expression (Andersson, S. G. E. and Kurland, C. G. (1990) Microbiol.", "Rev.", "Vol.", "54(2), 198-210, Kane, J. F. Curr.", "Opin.", "Biotechnol., Vol.", "6, pp.", "494-500).", "The amino acid sequence is shown in SEQ ID NO: 1 and the nucleotide sequence is shown in SEQ ID NO: 2.For the gene synthesis the gene was divided into 18 fragments of sense and reverse, complementary counterstrand oligonucleotides in alternating sequence (SEQ ID NO:3-20).", "An at least 15 bp region was attached to the 5′ end and to the 3′ end which in each case overlapped the neighbouring oligonucleotides.", "Recognition sites for restriction endonucleases were attached to the 5′ and 3′ ends of the synthetic gene outside the coding region for subsequent cloning into expression vectors.", "The oligonucleotide shown in SEQ ID NO:3 which contains an EcoRI cleavage site was used as a 5′ primer for cloning the pro-protein X gene without an N-terminal affinity tag.", "SEQ ID NO: 20 shows the 3′ primer containing a HindIII cleavage site.", "The 3′ primer contains an additional stop codon after the natural stop codon to ensure termination of the translation.", "The oligonucleotide with a BamHI cleavage site shown in SEQ ID NO: 23 or the oligonucleotide with a BamHI cleavage site and enterokinase cleavage site shown in SEQ ID NO: 24 was used as a 5′ primer to clone the proprotein X gene with N-terminal affinity tags and an alternative enterokinase cleavage site as described in example 3.The oligonucleotides were linked together by means of a PCR reaction and the resulting gene was amplified.", "For this the gene was firstly divided into three fragments of 6 oligonucleotides each and the three fragments were linked together in a second PCR cycle.", "Fragment 1 is composed of the oligonucleotides shown in SEQ ID NO: 3-8, fragment 2 is composed of the oligonucleotides shown in SEQ ID NO: 9-14 and fragment 3 is composed of the oligonucleotides shown in SEQ ID NO: 15-20.The following PCR parameters were employed PCR reaction 1 (generation of three fragments) 5 min 95° C. hot start 2 min 95° C. 2 min 42° C. 1.5 min 72° C. {close oversize brace} 30 cycles 7 min 72° C. final extension PCR reaction 2 (linkage of the fragments to form the total gene) 5 min 95° C. hot start 1.5 min 95° C. 2 min 48° C. {close oversize brace} 6 cycles (without terminal primers) 2 min 72° C. addition of terminal primers 1.5 min 95° C. 1.5 min 60° C. {close oversize brace} 25 cycles (with terminal primers) 2 min 72° C. 7 min 72° C. final extension EXAMPLE 2 Cloning of the Synthetic Proteinase K Fragment from the Gene Synthesis The PCR mixture was applied to an agarose gel and the ca.", "1130 bp PCR fragment was isolated from the agarose gel (Geneclean II Kit from Bio 101, Inc. CA USA).", "The fragment was cleaved for 1 hour at 37° C. with EcoRI and HindIII restriction endonucleases (Roche Diagnostics GmbH, Germany).", "At the same time the pUC18 plasmid (Roche Diagnostics GmbH, Germany) was cleaved for 1 hour at 37° C. with EcoRI and HindIII restriction endonucleases, the mixture was separated by agarose gel electrophoresis and the 2635 bp vector fragment was isolated.", "Subsequently the PCR fragment and the vector fragment were ligated together using T4 DNA ligase.", "For this 1 μl (20 ng) vector fragment and 3 μl (100 ng) PCR fragment, 1 μl 10× ligase buffer (Maniatis, T., Fritsch, E. F. and Sambrook, T. (1989).", "Molecular Cloning: A laboratory manual.", "2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), 1 μl T4 DNA ligase, 4 μl sterile redistilled H2O were pipetted, carefully mixed and incubated overnight at 16° C. The cloned gene was examined by restriction analysis and by multiple sequencing of both strands.", "The sequence is shown in SEQ ID NO: 2.a) Construction of the pPK-1 Expression Plasmid In order to express proteinase K, the structural gene was cloned into the pKKT5 expression vector in such a manner that the structural gene is inserted in the correct orientation under the control of a suitable promoter, preferably a promoter that can be induced by IPTG such as the lac, lacUV5, tac or T5 promoter, particularly preferably the T5 promoter.", "For this purpose the structural gene for proteinase K was cleaved from the plasmid pUC18 by EcoRI and HindIII, the restriction mixture was separated by agarose gel electrophoresis and the ca.", "1130 bp fragment was isolated from the agarose gel.", "At the same time the expression plasmid pKKT5 was cleaved with EcoRI and HindIII, the restriction mixture was separated by agarose gel electrophoresis and the ca.", "2.5 kbp vector fragment was isolated from the agarose gel.", "The fragments obtained in this manner were ligated together as described above.", "The correct insertion of the gene was checked by sequencing.", "b) Transformation of the Expression Plasmid pPK-1 in Various E. coli Expression Strains The expression vector was transformed in various expression strains that had been previously transformed with the plasmid pREP4 and/or pUBS520.The plasmid pREP4 contains a gene for the lacI repressor that should ensure a complete suppression of the expression before induction.", "The plasmid pUBS520 (Brinkmann, U. et al.", "(1989) Gene Vol.", "85(1), 109-114) also contains the lacI repressor and additionally the dnaY gene which codes for the tRNA which is necessary to translate the rare arginine codons AGA and AGG in E. coli.", "Competent cells of various E. coli strains were prepared according to the method of Hanahan, D. (1983) J. Mol.", "Biol.", "Vol.", "166, 557-580.100 μl cells prepared in this manner was admixed with 20 ng isolated pPK-1 plasmid DNA.", "After 30 min incubation on ice, they were heat-shocked (90 sec at 42° C.) and then incubated for 2 min on ice.", "Subsequently the cells were transferred to 1 ml SOC medium and incubated for 1 hour at 37° C. while shaking for the phenotypic expression.", "Aliquots of this transformation mixture were plated out on LB plates containing ampicillin as a selection marker and incubated for 15 hours at 37° C. Preferred strains are E. coli K12-strains JM83, JM105, UT5600, RR1Δ15, DH5α, C600, TG1, NM522, M15 or the E. coli B derivatives BL21, HB101; E. coli M15 is particularly preferred.", "EXAMPLE 3 Cloning of an N-Terminal Affinity Tag In order to insert an N-terminal affinity tag, a BamHI cleavage site was inserted before the 5′ end of the gene for pro-proteinase K. This was achieved by PCR using the product obtained in example 1 as a template and the oligonucleotides described in SEQ ID NO:20, 23 and 24 as primers.", "The primer described in SEQ ID NO:23 contains a BamHI cleavage site upstream of the 5′ region of pro-proteinase K, the primer described in SEQ ID NO:24 additionally contains an enterokinase cleavage site directly in front of the first codon of the prosequence.", "SEQ ID NO:20 shows the 3′ primer that was also used in example 1 with a HindIII cleavage site.", "The resulting PCR products were isolated as described above, digested with BamHI and HindIII and purified by agarose electrophoresis.", "The affinity tag was inserted by means of a synthetic linker composed of two complementary oligonucleotides in such a manner that an EcoRI cleavage site was formed at the 5′ end and a BamHI cleavage site was formed at the 3′ end without further restriction digestion.", "For a His tag the sense strand had the sequence shown in SEQ ID NO:21 and the antisense strand had the sequence shown in SEQ ID NO.22.The linker coded for a hexa-His tag with an N-terminal RGS motif.", "The BamHI cleavage site between the linker and pro-proteinase K is translated into a Gly-Ser linker.", "In order to anneal the linker, the two oligonucleotides (SEQ ID NO:21 and 22) were heated for 5 min to 95° C. in equimolar amounts (50 pmol/μl each) and subsequently cooled at 2° C. per min to room temperature.", "As a result the annealing of the complementary oligonucleotides should be as complete as possible.", "The linker was ligated with the BamHI/HindIII-digested PCR product.", "(Rapid Ligation Kit from Roche Diagnostics GmbH, Germany) and purified by agarose gel electrophoresis (QIAquick gel extraction Kit from Qiagen, Germany).", "The resulting ligation product was ligated into an expression vector analogously to example 2b via the EcoRI and HindIII overhangs and transformed correspondingly in expression strains.", "This module system enables various affinity tags that are coded by the synthetic linker to be fused to the structural gene for pro-proteinase K. An enterokinase cleavage site can be alternatively inserted between the tag and propeptide by suitable selection of the corresponding 5′ primer if a subsequent removal of the tag is desired.", "Furthermore a certain region of the proteinase K gene such as the mature proteinase K or the propeptide can be amplified by suitable selection of the overlapping regions of the PCR primers (FIG.", "1).", "EXAMPLE 4 Expression of Proteinase K in Escherichia coli Since proteinase K is a very active unspecific protease, it is preferable to express it in an inactive form preferably as inclusion bodies.", "In order to express the gene which codes for proteinase K, 3 ml Lbamp medium was inoculated with plasmid-containing clones and incubated at 37° C. in a shaker.", "LB medium: 10 g tryptone 10 yeast extract 5 g NaCl make up to a final volume of 1 l with distilled H2O, adjust to pH 7.0 with NaOH addition of antibiotics (100 μg/ml ampicillin) directly before inoculation The cells were induced with 1 mM IPTG at an optical density of 0.5 at 550 nm and incubated for 4 h at 37° C. in a shaker.", "Subsequently the optical density of the individual expression clones was determined, an aliquot corresponding to an OD550 of 3/ml was removed and the cells were centrifuged (10 min 6000 rpm, 4° C.).", "The cells were resuspended in 400 μl TE buffer, lysed by ultrasound and the soluble protein fraction was separated from the insoluble protein fraction by centrifugation (10 min, 14,000 rpm, 4° C.).", "TE buffer: 50 mM Tris/HCl 50 mM EDTA pH 8.0 (at RT) Application buffer containing SDS and β-mercaptoethanol was added to all fractions and the proteins were denatured by heating (5 min 95° C.).", "Subsequently 10 μl aliquots were analysed by means of a 12.5% analytical SDS gel (Laemmli, U.K. (1970) Nature Vol.", "227(259), 680-685).", "A very strong expression in the form of insoluble protein aggregates (inclusion bodies) was observed for the clones of mature proteinase K as well as for the clones of pro-proteinase K. Accordingly no proteinase K activity was measured.", "EXAMPLE 5 Isolation of the Inclusion Bodies The inclusion bodies were prepared by known methods (Rudolph, R. (1997) see above).", "For the cell lysis, 10 g wet biomass was in each case resuspended in 50 ml 100 mM Tris/HCl pH 7.0, 1 mM EDTA.", "Afterwards 15 mg lysozyme was added, incubated for 60 min at 4° C. and the cells were subsequently lysed by high pressure (Gaulin cell lysis apparatus).", "The DNA was digested for 30 min at RT by adding 3 mM MgCl2 and 10 μg/ml DNase to the crude extract.", "The insoluble cell components which contain the inclusion bodies were separated by centrifugation (30 min 20,000 g) and washed once with washing buffer 1 and three times with washing buffer 2.washing buffer 1: 100 mM Tris/HCl 20 mM EDTA 2% (v/v) Triton X-100 0.5 M NaCl pH 7.0 (RT) washing buffer 2: 100 mM Tris/HCl 1 mM EDTA pH 7.0 (RT) The pellet of the last washing step constitutes the crude inclusion bodies which already contain highly pure target protein.", "EXAMPLE 6 Solubilization of Inclusion Bodies a) Solubilization While Reducing with Cysteines 1 g crude inclusion bodies was suspended in 10 ml solubilization buffer and incubated for 2 h at RT while stirring gently.", "Solubilization buffer: 100 mM Tris/HCl 6.0 M guanidinium hydrochloride 100 mM DTT pH 8.0 (RT) The solubilisate was titrated to pH 3 with 25% HCl and dialysed twice for 4 h at RT against 500 ml 6 M guanidine hydrochloride pH 3 and then overnight at 4° C. against 1000 ml guanidine hydrochloride pH 7.The protein concentration was determined by the Bradford method (Bradford, 1976) using a calculated extinction coefficient at 280 nm and was between 10 and 20 mg/ml.", "The number of free cysteines was determined according to the Ellman method.", "In accordance with the sequence 5 mol free cysteines per mol proteinase K were found.", "The purity of the solubilized inclusion bodies was determined by 12.5% SDS PAGE and quantification of the bands after Coomassie staining.", "b) Solubilization with Derivatization of the Cysteines to Form Mixed Disulfides Using Glutathione.", "1 g Crude Inclusion Bodies Were Suspended in 10 ml Solubilization Buffer.", "Solubilization buffer: 100 mM Tris/HCl 6.0 M guanidine hydrochloride 1 mM DTT pH 8.0 (RT) After 15 min incubation at RT while stirring gently, during which a catalytic amount of reduced cysteines was formed due to the small amounts of DTT, 100 mM GSSG was added, the pH was adjusted to 8.0 and it was incubated for a further 2 h at RT while stirring gently.", "Further treatment as described under a).", "EXAMPLE 7 Optimization of the Naturation of Pro-Proteinase K Various parameters were varied in order to optimize the yield in the folding and processing of pro-proteinase K from the solubilisates prepared in example 6a).", "For all preparations the stated folding buffer was filtered, degassed, gassed with N2 and incubated at the desired temperature.", "The redox shuffling system was not added until shortly before the start of the folding reaction and the pH was readjusted.", "The folding was initiated by adding the solubilized inclusion bodies while rapidly mixing.", "The volume of the folding mixtures was 1.8 ml in 2 ml glass tubes with a screw cap.", "The yield was analysed by an activity test using the chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA from the Bachem Company (Heidelberg).", "100 mM Tris/HCl, 5 mM CaCl2, pH 8.5 at 25° C. was used as the test buffer.", "The concentration of the peptide in the test was 2 mM from a 200 mM stock solution in DMSO.", "In order to activate the renaturate, 0.1% SDS was added to the sample (see example 8).", "The absorbance at 410 nm was measured over a period of 20 min and the activity was calculated from the slope.", "The following parameters were varied: a) Temperature and Time The folding buffer containing 100 mM Tris, 1.0 mM L-arginine, 10 mM CaCl2 was equilibrated at various temperatures.", "After adding 3 mM GSH and 1 mM GSSG the pH was readjusted at the corresponding temperature.", "The reaction was started by adding 50 μg/ml pro-proteinase K. After 12 h, 36 h and 60 h, aliquots were removed and tested for activity.", "The results are shown in FIG.", "2.b) pH Value A universal buffer containing 50 mM citrate, 50 mM MES, 50 mM bicine, 500 mM arginine, 2 mM CaCl2 and 1 mM EDTA was incubated at 15° C. and 3 mM GSH and 1 mM GSSG were added.", "The pH was readjusted in a range between pH 4.0 and pH 12.0 and the folding reaction was started by adding 50 μg/ml pro-proteinase K inclusion bodies.", "The activity measured after 18 h, 3 d and 5 d is shown in FIG.", "3.c) Redox Potential Various redox potentials were set in a renaturation buffer containing 1.0 M L-arginine, 100 mM bicine, 2 mM CaCl2 and 10 mM CaCl2 by mixing various ratios of oxidized and reducing glutathione.", "The protein concentration in the folding mixture was 50 μg/ml.", "The folding was carried out at 15° C. The concentrations of GSH and GSSG are shown in table 1, the measurements are shown in FIG.", "4.TABLE 1 concentrations of GSH and GSSG at the various redox potentials.", "Redox potential (log(cGSH2/cGSSG) [M] c(GSH) [mM] c(GSSG) [mM] −00 0 2.500 −6.000 0.050 2.475 −5.500 0.088 2.456 −5.000 0.156 2.422 −4.500 0.273 2.363 −4.000 0.476 2.262 −3.500 0.814 2.093 −3.000 1.351 1.825 −2.500 2.130 1.435 −2.000 3.090 0.955 −1.500 3.992 0.504 −1.000 4.580 0.210 −0.500 4.851 0.074 0.000 4.951 0.025 +0.500 4.984 7.856e−3 +1.000 4.995 2.495e−3 +00 5.000 0 d) Solvent Additives that Promote Folding Various substances were examined for their ability to increase the folding yield of proteinase K. For this purpose solutions containing the substances at various concentrations were prepared and admixed with 2 mM CaCl2, 1 mM EDTA and 100 mM bicine.", "The pH was adjusted to pH 8.75 at the folding temperature of 15° C. The protein concentration was 50 μg/ml.", "FIG.", "5 shows the relative yields of active proteinase K in relation to the concentration of the selected buffer additive.", "EXAMPLE 8 Activation of the Natured Pro-Proteinase K After naturation of pro-proteinase K by the method according to the invention it was found to have no activity or only a very slight activity.", "Chromatographic methods and SDS-PAGE showed that mature proteinase K is already present but is still associated in a complex with the propeptide.", "This can be separated in a method which is referred to here as activation and is also a subject matter of the invention.", "In this example SDS is added at a concentration of 2% (v/v) to the folding mixture and subsequently the folding additive and the SDS are removed by dialysis.", "Alternatively SDS could also be added after removing the additives by dialysis.", "In all cases full activity of proteinase K was detected.", "EXAMPLE 9 Characterization of the Folding Product The proteinase K natured and activated by the method according to the invention was further characterized by various methods.", "a) Analysis of Purity and Molecular Weight Determination by SDS Polyacrylamide Gel Electrophoresis Aliquots from various steps in the maturation process and the final product, the folded and activated recombinant proteinase K were applied to a 12.5% SDS polyacrylamide gel.", "The samples each contained 10 mM DTT or 1% (v/v) 2-mercaptoethanol.", "The recombinant proteinase K prepared by the method according to the invention had no significant contamination and runs identically with the authentic proteinase K at an apparent molecular weight of ca.", "30 kDa (see FIG.", "11).", "b) Analysis of Purity Using RP-HPLC The folded and activated proteinase K and the authentic proteinase K from T. album and the pro-proteinase K inclusion bodies were analysed by means of reversed phase HPLC.", "A Vydac C4 column having the dimensions 15 cm×4.6 cm diameter was used.", "The samples were eluted with an acetonitrile gradient of 0% to 80% in 0.1% TFA.", "The folding product exhibits mobility properties that are identical to the authentic proteinase K used as a standard (see FIG.", "12).", "c) Analytical Ultracentrifugation In order to analyse whether the renatured and processed proteinase K is present in a monomeric form without propeptide, the protein was examined by means of analytical ultracentrifugation.", "The molecular weight was determined to be 29490 Da and corresponds to the mass of the monomeric mature proteinase K within the limits of error of this method (see FIG.", "13).", "Hence this showed that the propeptide was quantitatively cleaved by activation of the proteinase K. d) N-Terminal Sequence Analysis In order to examine whether the propeptide was cleaved at the correct cleavage site the natured and activated recombinant proteinase K was subjected to a sequence analysis.", "For this the folding product was desalted by RP-HPLC as described in example 9b) and the first 6 residues were examined by N-terminal sequencing.", "The result (AAQTNA) agrees with the authentic N-terminus of mature proteinase K. e) Activity and Km Value the Km value of the folded and activated proteinase K was compared with that of the authentic proteinase K. The tetrapeptide Suc-Ala-Ala-Pro-Phe-pNA was used as a substrate.", "The test was carried out in 2.0 ml 50 mM Tris, pH 8.5 containing 1 mM CaCl2 at 25° C. The hydrolysis of the peptide was monitored spectroscopically at 410 nm.", "A Km value of 0.16 mM was found for the recombinant proteinase K which corresponded very well with the Km value of authentic proteinase K (see FIG.", "14).", "f) Degradation Pattern of Blood Serum Proteins In an additional test to characterize the activity, the cleavage pattern of blood serum proteins was examined.", "For this a defined amount of blood serum proteins was digested with 1 μg recombinant proteinase K or the same amount of authentic proteinase K. The cleavage pattern was analysed by means of RP-HPLC under identical conditions as described in example 9b).", "FIG.", "15 shows that the recombinant and the authentic proteinase K result in an identical degradation pattern.", "EXAMPLE 10 Purification of the Folding Product The recombinant pro-proteinase K natured by the method according to the invention was purified by gel filtration.", "As described in FIG.", "11 the concentrated naturation solution was separated on a Superdex 75 pg after naturation in a first run without prior activation and in a second run with prior activation using 0.15% (w/v) SDS (30 min, 4° C.).", "100 mM Tris/HCl, 150 mM NaCl pH 8.75 (4° C.) was used as the mobile buffer.", "The application volume was 10 ml at a column volume of 1200 ml and a flow rate of 5 ml/min.", "After completion of the application, 14 ml fractions were collected.", "Aliquots of the fractions were precipitated with trichloroacetic acid, washed and taken up in Laemmli sample buffer containing 10 mM DTT.", "The samples were applied to a 12.5% SDS polyacrylamide gel which was stained after the run with Coomassie blue R250.In the first run without activation a non-processed recombinant pro-proteinase K is seen in a first peak which probably runs in the form of microaggregates in the exclusion volume.", "In a second peak one observes processed recombinant proteinase K which co-elutes with the propeptide which is non-covalently bound and acts as an inhibitor.", "As a result no activity is found without prior activation.", "Only after adding SDS to the fractions did the second peak exhibit significant proteinase K activity (not shown).", "The second run in which the folded recombinant proteinase K was previously activated with SDS only shows one peak which elutes after an identical volume like proteinase K under the same conditions (not shown).", "On the SDS gel one sees clean mature recombinant proteinase K without propeptide in this peak.", "All impurities and the propeptide appear to have already been digested in the applied mixture by the activated recombinant proteinase K. As expected the fractions of the proteinase K peak exhibited activity without further activation with SDS.", "The recombinant proteinase K purified in this manner appears to be almost 100% pure on the SDS gel and shows an identical migration behaviour to the authentic proteinase K (FIG.", "16)." ] ]	Patent_10467532
[ [ "Antibody complexes and methods for immunolabeling", "The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents.", "The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label.", "Typically, the labeling reagent is an anti-Fc Fab or Fab′ fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody.", "The present invention provides for discrete subsets of labeling reagent and immuno-labeled complexes that facilitate the simultaneous detection of multiple targets in a sample wherein the immuno-labeled complexes are distinguished by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody.", "This is particularly useful for fluorophore labels that can be attached to labeling reagents and subsequently immuno-labeled complexes in ratios for the detection of multiple targets." ], [ "1.A method of forming an immuno-labeled complex, wherein said method comprises the steps of: b) contacting a solution of target-binding antibodies with a discreet labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent whereby a discreet immuno-labeled complex subset is formed; c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof; and, c) optionally repeating said steps a), b), and c) to form a panel of immuno-labeled complex subsets wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody.", "2.The method according to claim 1, wherein said target binding antibody is a murine monoclonal antibody, a rabbit polyclonal antibody or a goat polyclonal antibody.", "3.The method according to claim 2, wherein said target-binding antibodies are in a solution comprising serum proteins or ascites proteins.", "4.The method according to claim 1, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.", "5.The method according to claim 4, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.", "6.The method according to claim 5, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.", "7.The method according to claim 5, wherein said fluorescent protein is a phycobiliprotein.", "8.The method according to claim 5, wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative.", "9.The method according to claim 5, wherein said enzyme is selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase.", "10.A method for detecting a target in a sample, wherein said method comprises the steps of: a) contacting said sample with an immuno-labeled complex comprising a target-binding antibody and a labeling reagent; b) incubating said sample of step a) for a time sufficient to permit said immuno-labeled complex to selectively bind to said target; and, c) illuminating said immuno-labeled complex whereby said target is detected.", "11.The method according to claim 11, wherein said sample comprises a population of cells, cellular extract, subcellular component, proteins, peptides, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.", "12.The method according to claim 11, wherein said sample is immobilized on a solid or semi-solid matrix.", "13.The method according to claim 12, wherein said matrix is a gel, a membrane, an array, a glass surface or a microparticle.", "14.The method according to claim 10, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.", "15.The method according to claim 14, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.", "16.The method according to claim 15, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.", "17.The method according to claim 15, wherein said fluorescent protein is a phycobiliprotein.", "18.The method according to claim 15, wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative.", "19.The method according to claim 15, wherein said enzyme selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase.", "20.The method according to claim 19, wherein said method further comprises adding a colorimetric, fluorescent or chemiluminescent enzyme substrate.", "21.The method according to claim 10, wherein said immuno-labeled complex comprises a labeling reagent that is a Fab or Fab′ anti-Fc fragment wherein said fragment is independently covalently bonded to one or more labels that are selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a phycobiliprotein, a borapolyazaindacene, a peroxidase, a phosphatase, a tandem dye and derivatives thereof.", "22.A method for detecting multiple targets in a sample, said method comprising: a) contacting said sample with a solution comprising A) a pooled subset of immuno-labeled complexes wherein each said complex comprises a target-binding antibody and a labeling reagent, wherein each said subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual immuno-labeled complex subset wherein multiple individual subsets are added to said sample; b) incubating said sample for a time sufficient to allow said immuno-labeled complex to selectively bind to said target; and, c) illuminating said immuno-labeled complex whereby said multiple targets are detected.", "23.The method according to claim 22, wherein said sample comprises a population of cells, cellular extract, subcellular component, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.", "24.The method according to claim 23, wherein said sample is immobilized on a solid or semi-solid matrix.", "25.The method according to claim 24, wherein said matrix is a gel, a membrane, an array, a glass surface or a microparticle.", "26.The method according to claim 23, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.", "27.The method according to claim 26, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an electron transfer agent, a hapten, an enzyme and a radioisotope.", "28.The method according to claim 27, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.", "29.The method according to claim 27, wherein said fluorescent protein is a phycobiliprotein.", "30.The method according to claim 27, wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative.", "31.The method according to claim 27, wherein said enzyme is selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase.", "32.The method according to claim 31, wherein said method further comprises adding a calorimetric, fluorescent or chemiluminescent enzyme substrate.", "33.A method for identifying and quantitating multiple targets in a sample, wherein said method comprises the steps of: a) contacting a population of cells or fragments thereof in a sample with A) a solution comprising a pooled subset of immuno-labeled complexes wherein said complex comprises a target-binding antibody and a labeling reagent, wherein each subset is.", "distinguished from another subset i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual subset of immuno-labeled complexes wherein multiple individual subsets are added to said sample; b) incubating said cells or fragments thereof for a time period sufficient to allow said immuno-labeled complex to bind said targets; c) passing said incubated population of cells or fragments thereof through an examination zone; and, d) collecting data from said cells or fragments thereof passed through said examination zone whereby said identification and quantitation of multiple targets is determined.", "34.The method according to claim 33, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.", "35.The method according to claim 34, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten.", "36.The method according to claim 35, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.", "37.The method according to claim 35, wherein said fluorescent protein is a phycobiliprotein.", "38.The method according to claim 35, wherein said tandem dye is selected from the group consisting of cyanine-phycobiliprotein and xanthene-phycobiliprotein.", "39.A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: a) cleaving an intact anti-region antibody with an enzyme to generate Fab fragments; b) isolating said anti-region Fab fragments of step a); c) contacting a matrix comprising intact immunoglobulin proteins or fragments that selectively bind anti-region Fab fragments with a solution comprising said anti-region fragments of step b) wherein said anti-region Fab fragments are immobilized on said matrix; d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; e) washing said matrix of step d) to remove unbound label, and; f) eluting said labeling reagent from said matrix whereby said isolated labeling reagent is manufactured.", "40.The method according to claim 39, wherein said anti-region Fab fragment are selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.", "41.The method according to claim 40, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.", "42.A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: a) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that selectively bind non-antibody proteins with a solution comprising said non-antibody proteins wherein said non-antibody proteins are immobilized on said matrix; b) contacting said matrix of step a) with a solution comprising a fluorophore label that contains a reactive group; c) washing said matrix to remove unbound label, and; d) eluting said labeling reagent from said matrix whereby said isolated labeling reagent is manufactured that comprises a fluorophore label.", "43.The method according to claim 42, wherein said non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.", "44.The method according to claim 43, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.", "45.A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: a) cleaving an intact anti-region antibody with an enzyme resulting in Fab or Fab′fragments; a) contacting said anti-region Fab or Fab′ fragments of step a) with a solution comprising a label that contains a reactive group; and, c) isolating labeled anti-region Fab or Fab′ fragments by size exclusion or affinity chromatography whereby an isolated labeling reagent is manufactured.", "46.The method according to claim 45, wherein said anti-region Fab or Fab′ fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.", "47.The method according to claim 46, wherein said label is a fluorescent protein or a tandem dye.", "48.The method according to claim 47, wherein said fluorescent protein is a phycobiliprotein.", "49.The method according to claim 47, wherein said tandem dye is selected from the group consisting of cyanine-phycobiliprotein and xanthene-phycobiliprotein.", "50.An isolated labeling reagent made by a process comprising: a) cleaving an intact anti-region antibody with an enzyme resulting in Fab or Fab′fragments; b) contacting said Fab or Fab′ fragments of step a) with a solution comprising a label that contains a reactive group; and, c) isolating labeled anti-region Fab or Fab′ fragments by size exclusion or affinity chromatography.", "51.The labeling reagent according to claim 50, wherein said anti-region Fab or Fab′fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.", "52.The labeling reagent according to claim 51, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten.", "53.The labeling reagent according to claim 52, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.", "54.An isolated labeling reagent made by a process comprising: a) cleaving an intact anti-region antibody with an enzyme to generate F(ab′)2 fragments; b) contacting said anti-region F(ab′)2 with a reducing agent to produce anti-region Fab′ fragments containing a thiol group; c) contacting said Fab′ fragments with a solution comprising a label that contains a reactive group; and, d) isolating said Fab′ fragments of step d) that are covalently attached to a label by size exclusion or affinity chromatography.", "55.The labeling reagent according to claim 54, wherein said anti-region Fab′ fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.", "56.The method according to claim 55, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten.", "57.An isolated labeling reagent made by a process comprising: a) cleaving an intact anti-region antibody with an enzyme to generate Fab fragments; b) isolating said anti-region Fab fragments of step a); c) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that specifically bind anti-region Fab fragments with a solution comprising said anti-region Fab fragments of step b) wherein said anti-region Fab fragments are immobilized; d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; e) washing said matrix to remove unbound label, and; f) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label and being isolated from other proteins and fragments thereof.", "58.The labeling reagent according to claim 57, wherein said anti-region Fab fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.", "59.The labeling reagent according to claim 58, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.", "60.An isolated labeling reagent made by a process comprising: a) contacting a matrix comprising intact immunoglobulin proteins or fragments that selectively bind non-antibody proteins with a solution comprising said non-antibody proteins wherein said non-antibody proteins are immobilized; b) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; c) washing said matrix to remove unbound label, and; d) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label.", "61.The labeling reagent according to claim 60, wherein said non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.", "62.The labeling reagent according to claim 61, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.", "63.An immuno-labeled complex generated by a process comprising: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subset is distinguished from another labeling reagent subset by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent; and, c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof whereby an immuno-labeled complex is produced.", "64.The immuno-labeled complex according to claim 63, wherein said process further comprises repeating said steps a), b), and c) to form subsets of immuno-labeled complexes wherein each subset is distinguished from another subset by i) ratio of label to labeling reagent or ii) a physical properties of said label, or iii) a ratio of labeling reagent to said target-binding antibody or iv) by said target-binding antibody.", "65.The immuno-labeled complex according to claim 63, wherein said target-binding antibody is a murine monoclonal antibody, a rabbit polyclonal antibody or a goat polyclonal antibody.", "66.The immuno-labeled complex according to claim 65, wherein said labeling reagent is a Fab or Fab′ fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.", "67.The immuno-labeled complex according to claim 66, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.", "68.The immuno-labeled complex according to claim 67, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.", "69.A labeling solution comprising a buffer and either an individual labeling reagent subset or a pooled subset of labeling reagents, wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) by a physical property of said label.", "70.The solution according to claim 69, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.", "71.The solution according to claim 70, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an enzyme, a hapten and a radioisotope.", "72.The solution according to claim 71, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.", "73.A kit for preparing an immuno-labeling complex, said kit comprising: a) a labeling solution comprising a labeling reagent that is independently covalently bonded to one or more labels; or b) a panel of labeling solutions wherein each labeling solution comprises a subset of a labeling reagent and each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) by a physical property of said label; and, c) a solution comprising a capture reagent.", "74.The kit according to claim 73, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.", "75.The kit according to claim 74, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an enzyme, a hapten and a radioisotope.", "76.The kit according to claim 75, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.", "77.The kit according to claim 76, wherein the kit further comprises an addition component selected from the group consisting of (a) stains for characterization of cellular organelles, cell viability, or cell proliferation state, (b) enzyme substrates and (c) enzyme conjugates." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Immunolabeling is a method for qualitative or quantitative determination of the presence of a target in a sample, wherein antibodies are utilized for their specific binding capacity.", "The antibodies form a complex with the target (antigen), wherein a detectable label is present on the antibody or on a secondary antibody.", "The detectable label is a key feature of immunolabeling, which can be detected directly or indirectly.", "The label provides a measurable signal by which the binding reaction is monitored providing a qualitative and/or quantitative measure of the degree of binding.", "The relative quantity and location of signal generated by the labeled antibodies can serve to indicate the location and/or concentration of the target.", "The label can also be used to select and isolate labeled targets, such as by flow sorting or using magnetic separation media.", "Examples of labels include but are not limited to radioactive nucleotides ( 125 I, 3 H, 14 C, 32 P), chemiluminescent, fluorescent, or phosphorescent compounds (e.g., dioxetanes, xanthene, or carbocyanine dyes, lanthanide chelates), particles (e.g., gold clusters, colloidal gold, microspheres, quantum dots), and enzymes (e.g., peroxidases, glycosidases, phosphatases, kinases).", "Ideally, the label is attached to the antibody in a manner that does not perturb the antibody's binding characteristics but enables the label to be measured by an appropriate detection technology.", "The choice of labels is influenced by factors such as ease and sensitivity of detection, equipment availability, background in the sample (including other labels) and the degree to which such labels are readily attached to the particular antibody.", "Both direct and indirect labeling of antibodies is utilized for immunolabeling.", "Direct labeling utilizes only a primary antibody, i.e.", "the antibody specific for the target, bound to the label.", "In contrast, indirect labeling utilizes a secondary antibody bound to the label, which is specific for the primary antibody, e.g.", "a goat anti-rabbit antibody.", "The principal differences in immunolabeling methods and materials reside in the way that the label is attached to the antibody-antigen complex, the type of label that is used, and the means by which the antibody-antigen complex is detected.", "Limitations for direct labeling primary antibodies include the need for buffers free of primary amines, or carrier proteins such as bovine serum albumin (BSA), and other compounds such as tris-(hydroxymethyl)aminomethane (TRIS), glycine, and ammonium ions.", "These materials are, however, common components in antibody buffers and purification methods, and it may not be possible or feasible to remove them prior to the coupling reaction.", "In particular, many monoclonal antibodies are available only as ascites fluid or in hybridoma culture supernatants, or diluted with carrier proteins, such as albumins.", "Thus, direct labeling of antibodies in ascites fluid or other medias containing interfering compounds is not attainable.", "The indirect immunolabeling method typically involves a multi-step process in which an unlabeled first antibody (typically a primary antibody) is directly added to the sample to form a complex with the antigen in the sample.", "Subsequently, a labeled secondary antibody, specific for the primary antibody, is added to the sample, where it attaches noncovalently to the primary antibody-antigen complex.", "Alternatively, a detectable label is covalently attached to an immunoglobulin-binding protein such as protein A and protein G to detect the antibody-antigen complex that has previously been formed with the target in the sample.", "Using ligands, such as streptavidin, that are meant to amplify the detectable signal also expands this cascade binding.", "Indirect immunolabeling often results in false positives and high background.", "This is due to the fact that secondary antibodies, even when purified by adsorption against related species, nevertheless can exhibit significant residual cross-reactivity when used in the same sample.", "For example, when mouse tissue is probed with a mouse monoclonal antibody, the secondary antibody must necessarily be a labeled anti-mouse antibody.", "This anti-mouse antibody will detect the antibody of interest but will inevitably and additionally detect irrelevant, endogenous mouse immunoglobulins inherent in mouse tissue.", "This causes a significant background problem, especially in diseased tissues, which reduces the usefulness and sensitivity of the assay.", "Thus, the simultaneous detection of more than one primary antibody in a sample without this significant background interference depends on the availability of secondary antibodies that 1) do not cross-react with proteins intrinsic to the sample being examined, 2) recognize only one of the primary antibodies, and 3) do not recognize each other (Brelje, et al., METHODS IN CELL BIOLOGY 38, 97-181, especially 111-118 (1993)).", "To address the background problem in indirect labeling, a number of strategies have been developed to block access of the anti-mouse secondary antibodies to the endogenous mouse immunoglobulins.", "One such strategy for blocking involves complexing the primary antibody with a selected biotinylated secondary antibody to produce a complex of the primary and secondary antibodies, which is then mixed with diluted normal murine serum (Trojanowski et al., U.S. Pat No.", "5,281,521 (1994)).", "This method is limited by the necessity to utilize an appropriate ratio of primary-secondary complex.", "Too low a ratio of primary-secondary complex will cause a decrease in specific staining and increased background levels due to the uncomplexed secondary anti-mouse antibody binding to endogenous mouse antibodies.", "However, the ability of a whole IgG antibody (as was used in the referenced method) to simultaneously bind and cross-link two antigens results in too high a ratio, causing the complex to precipitate or form complexes that are too large to penetrate into the cell or tissue.", "Another strategy for blocking access to endogenous immunoglobulins in the sample involves pre-incubating the sample with a monovalent antibody, such as Fab′ fragments, from an irrelevant species that recognize endogenous immunoglobulins.", "This approach requires large quantities of expensive Fab′ fragments and gives mixed results and adds at least two steps (block and wash) to the overall staining procedure.", "The addition of a cross-linking reagent has resulted in improved reduction of background levels (Tsao, et al., U.S. Pat.", "No.", "5,869,274 (1997)) but this is problematic when used with fluorophore-labeled antibodies.", "The cross-linking causes an increase in the levels of autofluorescence and thus the background (J. Neurosci.", "Meth.", "83, 97 (1998); Mosiman et al., Methods 77, 191 (1997); Commun.", "Clin.", "Cytometry 30,151 (1997); Beisker et al., Cytometry 8, 235 (1987)).", "In addition, pre-incubation with a cross-linking reagent often masks or prevents the antibody from binding to its antigen (J. Histochem.", "Cytochem.", "45, 327 (1997); J. Histochem.", "Cytochem.", "39, 741 (1991); J. Histochem.", "Cytochem.", "43,193 (1995); Appl.", "Immunohistochem.", "Molecul.", "Morphol.", "9,176 (2001)).", "In a variation of this blocking strategy, a multi-step sequential-labeling procedure is used to overcome the problems of cross-reactivity.", "The sample is incubated with a first antibody to form a complex with the first antigen, followed by incubation of the sample with a fluorophore-labeled goat Fab anti-mouse IgG to label the first antibody and block it from subsequently complexing when the second antibody is added.", "In the third step, a second mouse antibody forms a complex with the second antigen.", "Because the second antibody is blocked from cross-reacting with the first antibody, the second mouse antibody is detected with a standard indirect-labeling method using a goat anti-mouse antibody conjugated to a different fluorescent dye (J. Histochem.", "Cytochem.", "34, 703 (1986)).", "This process requires multiple incubation steps and washing steps and it still cannot be used with mouse antibodies to probe mouse tissue.", "Another blocking method is disclosed in the animal research kit (ARK) developed by DAKO.", "In this kit, a primary antibody is complexed with biotin-labeled goat Fab anti-mouse IgG and excess free Fab is blocked with normal mouse serum.", "However, since the Fab used in this process is generated from the intact IgG (rather than a selected region) there is a potential for the formation of anti-paratope or anti-idiotype antibodies that will block the antigen-binding site and prevent immunolabeling.", "The biotinylated antibody also requires subsequent addition of a labeled avidin or streptavidin conjugate for its subsequent visualization.", "The present invention is advantageous over previously described methods and compositions in that it provides the benefits of indirect labeling with the easy and flexibility of direct labeling for determination of a desired target in a biological sample.", "The present invention provides labeled monovalent proteins specific for a target-binding antibody, which are complexed prior to addition with a biological sample.", "Because these monovalent proteins are not bivalent antibodies, precipitation and cross-linking are not a problem.", "Therefore the compositions of the present invention can be used with immunologically similar monoclonal or polyclonal antibodies of either an identical isotype or different isotypes.", "The monovalent labeling reagents are specific for the Fc region of target-binding antibodies, these reagents will not interfere with the binding region of the primary antibody.", "In addition, the monovalent labeling proteins are not negatively affected by the presence of primary amines like BSA, gelatin, hybridoma culture supernatants or ascites fluid, thus primary antibodies present in these media can be effectively labeled with the labeling reagents of the present invention.", "Thus, the present invention provides numerous advantages over the conventional methods of immunolabeling." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents.", "The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label.", "Typically, the labeling reagent is an anti-Fc Fab or Fab′ fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody.", "The methods for labeling a target-binding antibody with a labeling reagent comprise a) contacting a solution of target-binding antibodies with a labeling reagent, b) incubating said target-binding antibodies and said labeling reagent wherein a region of said target binding antibody is selectively bound by labeling reagent, and c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof that are optionally immobilized on a matrix.", "The labeling of the target-binding antibody can be performed irrespective of the solution that the antibody is present in and includes proteins that are normally present in serum or ascites.", "This feature of the labeling process of the target-binding antibody eliminates the need to purify and concentrate the target-binding antibody.", "The time required for the labeling reagent to selectively bind to the target-binding antibody is typically very short, often less than 10 minutes.", "Often the labeling reagent binds the target-binding antibody in the amount of time it takes to add and mix the labeling reagent with the target-binding antibody.", "This formation of an immuno-labeled complex—a target-binding antibody and a labeling reagent—results in the formation of an target detection solution that is used to detect a target in a sample.", "The labeling steps of the target-binding antibody are optionally repeated to form a panel of subsets, these immuno-labeled complex subsets may be used individually or pooled wherein each subset is distinguished from another subset by i) the target-binding antibody, or ii) a ratio of label to labeling reagent, or iii) a ratio of labeling reagent to the target-binding antibody or iv) by a physical property of the label.", "Thus, it is appreciated that a wide range of subsets can be formed wherein the subsets can be used individually to detect a target in a sample or pooled to simultaneously detect multiple targets in a sample.", "The simultaneous detection of multiple targets in a sample is especially useful in methods that utilize flow cytometry or methods that immobilize a population of cells or tissue on a surface.", "The methods for determining a target in a sample using immuno-labeled subsets comprises forming a subset of immuno-labeled complexes, as described above, contacting a sample with said immuno-labeled complexes, incubating the sample for a time sufficient to allow the immuno-labeled complex to selectively bind to a desired target, and illuminating the immuno-labeled complex whereby the target is detected.", "The sample is any material that may contain a target and typically comprises a population of cells, cellular extract, subcellular component, proteins, peptides, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.", "When multiple targets are detected a pooled subset of immuno-labeled complexes are formed and incubated with the sample or individual subsets are add sequentially to a sample.", "For methods using flow cytometry the population of cells is illuminated when they pass through an optical examination zone and the data collected about the label determines the identity and quantity of the targets." ], [ "CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Ser.", "No.", "60/329,068, filed Oct. 12, 2001; U.S. Ser.", "No.", "60/369,418 filed Apr.", "1, 2002, U.S. Ser.", "No.10/118,204 filed Apr.", "5, 2002, and PCT/US02/31416 filed Oct. 2, 2002, which disclosures are herein incorporated by reference.", "FIELD OF THE INVENTION The present invention relates to immuno-labeled complexes and methods for use in the detection and measurement of one or more targets in a biological sample.", "The invention has applications in the fields of molecular biology, cell biology, immunohistochemistry, diagnostics, and therapeutics.", "BACKGROUND OF THE INVENTION Immunolabeling is a method for qualitative or quantitative determination of the presence of a target in a sample, wherein antibodies are utilized for their specific binding capacity.", "The antibodies form a complex with the target (antigen), wherein a detectable label is present on the antibody or on a secondary antibody.", "The detectable label is a key feature of immunolabeling, which can be detected directly or indirectly.", "The label provides a measurable signal by which the binding reaction is monitored providing a qualitative and/or quantitative measure of the degree of binding.", "The relative quantity and location of signal generated by the labeled antibodies can serve to indicate the location and/or concentration of the target.", "The label can also be used to select and isolate labeled targets, such as by flow sorting or using magnetic separation media.", "Examples of labels include but are not limited to radioactive nucleotides (125I, 3H, 14C, 32P), chemiluminescent, fluorescent, or phosphorescent compounds (e.g., dioxetanes, xanthene, or carbocyanine dyes, lanthanide chelates), particles (e.g., gold clusters, colloidal gold, microspheres, quantum dots), and enzymes (e.g., peroxidases, glycosidases, phosphatases, kinases).", "Ideally, the label is attached to the antibody in a manner that does not perturb the antibody's binding characteristics but enables the label to be measured by an appropriate detection technology.", "The choice of labels is influenced by factors such as ease and sensitivity of detection, equipment availability, background in the sample (including other labels) and the degree to which such labels are readily attached to the particular antibody.", "Both direct and indirect labeling of antibodies is utilized for immunolabeling.", "Direct labeling utilizes only a primary antibody, i.e.", "the antibody specific for the target, bound to the label.", "In contrast, indirect labeling utilizes a secondary antibody bound to the label, which is specific for the primary antibody, e.g.", "a goat anti-rabbit antibody.", "The principal differences in immunolabeling methods and materials reside in the way that the label is attached to the antibody-antigen complex, the type of label that is used, and the means by which the antibody-antigen complex is detected.", "Limitations for direct labeling primary antibodies include the need for buffers free of primary amines, or carrier proteins such as bovine serum albumin (BSA), and other compounds such as tris-(hydroxymethyl)aminomethane (TRIS), glycine, and ammonium ions.", "These materials are, however, common components in antibody buffers and purification methods, and it may not be possible or feasible to remove them prior to the coupling reaction.", "In particular, many monoclonal antibodies are available only as ascites fluid or in hybridoma culture supernatants, or diluted with carrier proteins, such as albumins.", "Thus, direct labeling of antibodies in ascites fluid or other medias containing interfering compounds is not attainable.", "The indirect immunolabeling method typically involves a multi-step process in which an unlabeled first antibody (typically a primary antibody) is directly added to the sample to form a complex with the antigen in the sample.", "Subsequently, a labeled secondary antibody, specific for the primary antibody, is added to the sample, where it attaches noncovalently to the primary antibody-antigen complex.", "Alternatively, a detectable label is covalently attached to an immunoglobulin-binding protein such as protein A and protein G to detect the antibody-antigen complex that has previously been formed with the target in the sample.", "Using ligands, such as streptavidin, that are meant to amplify the detectable signal also expands this cascade binding.", "Indirect immunolabeling often results in false positives and high background.", "This is due to the fact that secondary antibodies, even when purified by adsorption against related species, nevertheless can exhibit significant residual cross-reactivity when used in the same sample.", "For example, when mouse tissue is probed with a mouse monoclonal antibody, the secondary antibody must necessarily be a labeled anti-mouse antibody.", "This anti-mouse antibody will detect the antibody of interest but will inevitably and additionally detect irrelevant, endogenous mouse immunoglobulins inherent in mouse tissue.", "This causes a significant background problem, especially in diseased tissues, which reduces the usefulness and sensitivity of the assay.", "Thus, the simultaneous detection of more than one primary antibody in a sample without this significant background interference depends on the availability of secondary antibodies that 1) do not cross-react with proteins intrinsic to the sample being examined, 2) recognize only one of the primary antibodies, and 3) do not recognize each other (Brelje, et al., METHODS IN CELL BIOLOGY 38, 97-181, especially 111-118 (1993)).", "To address the background problem in indirect labeling, a number of strategies have been developed to block access of the anti-mouse secondary antibodies to the endogenous mouse immunoglobulins.", "One such strategy for blocking involves complexing the primary antibody with a selected biotinylated secondary antibody to produce a complex of the primary and secondary antibodies, which is then mixed with diluted normal murine serum (Trojanowski et al., U.S. Pat No.", "5,281,521 (1994)).", "This method is limited by the necessity to utilize an appropriate ratio of primary-secondary complex.", "Too low a ratio of primary-secondary complex will cause a decrease in specific staining and increased background levels due to the uncomplexed secondary anti-mouse antibody binding to endogenous mouse antibodies.", "However, the ability of a whole IgG antibody (as was used in the referenced method) to simultaneously bind and cross-link two antigens results in too high a ratio, causing the complex to precipitate or form complexes that are too large to penetrate into the cell or tissue.", "Another strategy for blocking access to endogenous immunoglobulins in the sample involves pre-incubating the sample with a monovalent antibody, such as Fab′ fragments, from an irrelevant species that recognize endogenous immunoglobulins.", "This approach requires large quantities of expensive Fab′ fragments and gives mixed results and adds at least two steps (block and wash) to the overall staining procedure.", "The addition of a cross-linking reagent has resulted in improved reduction of background levels (Tsao, et al., U.S. Pat.", "No.", "5,869,274 (1997)) but this is problematic when used with fluorophore-labeled antibodies.", "The cross-linking causes an increase in the levels of autofluorescence and thus the background (J. Neurosci.", "Meth.", "83, 97 (1998); Mosiman et al., Methods 77, 191 (1997); Commun.", "Clin.", "Cytometry 30,151 (1997); Beisker et al., Cytometry 8, 235 (1987)).", "In addition, pre-incubation with a cross-linking reagent often masks or prevents the antibody from binding to its antigen (J. Histochem.", "Cytochem.", "45, 327 (1997); J. Histochem.", "Cytochem.", "39, 741 (1991); J. Histochem.", "Cytochem.", "43,193 (1995); Appl.", "Immunohistochem.", "Molecul.", "Morphol.", "9,176 (2001)).", "In a variation of this blocking strategy, a multi-step sequential-labeling procedure is used to overcome the problems of cross-reactivity.", "The sample is incubated with a first antibody to form a complex with the first antigen, followed by incubation of the sample with a fluorophore-labeled goat Fab anti-mouse IgG to label the first antibody and block it from subsequently complexing when the second antibody is added.", "In the third step, a second mouse antibody forms a complex with the second antigen.", "Because the second antibody is blocked from cross-reacting with the first antibody, the second mouse antibody is detected with a standard indirect-labeling method using a goat anti-mouse antibody conjugated to a different fluorescent dye (J. Histochem.", "Cytochem.", "34, 703 (1986)).", "This process requires multiple incubation steps and washing steps and it still cannot be used with mouse antibodies to probe mouse tissue.", "Another blocking method is disclosed in the animal research kit (ARK) developed by DAKO.", "In this kit, a primary antibody is complexed with biotin-labeled goat Fab anti-mouse IgG and excess free Fab is blocked with normal mouse serum.", "However, since the Fab used in this process is generated from the intact IgG (rather than a selected region) there is a potential for the formation of anti-paratope or anti-idiotype antibodies that will block the antigen-binding site and prevent immunolabeling.", "The biotinylated antibody also requires subsequent addition of a labeled avidin or streptavidin conjugate for its subsequent visualization.", "The present invention is advantageous over previously described methods and compositions in that it provides the benefits of indirect labeling with the easy and flexibility of direct labeling for determination of a desired target in a biological sample.", "The present invention provides labeled monovalent proteins specific for a target-binding antibody, which are complexed prior to addition with a biological sample.", "Because these monovalent proteins are not bivalent antibodies, precipitation and cross-linking are not a problem.", "Therefore the compositions of the present invention can be used with immunologically similar monoclonal or polyclonal antibodies of either an identical isotype or different isotypes.", "The monovalent labeling reagents are specific for the Fc region of target-binding antibodies, these reagents will not interfere with the binding region of the primary antibody.", "In addition, the monovalent labeling proteins are not negatively affected by the presence of primary amines like BSA, gelatin, hybridoma culture supernatants or ascites fluid, thus primary antibodies present in these media can be effectively labeled with the labeling reagents of the present invention.", "Thus, the present invention provides numerous advantages over the conventional methods of immunolabeling.", "SUMMARY OF THE INVENTION The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents.", "The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label.", "Typically, the labeling reagent is an anti-Fc Fab or Fab′ fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody.", "The methods for labeling a target-binding antibody with a labeling reagent comprise a) contacting a solution of target-binding antibodies with a labeling reagent, b) incubating said target-binding antibodies and said labeling reagent wherein a region of said target binding antibody is selectively bound by labeling reagent, and c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof that are optionally immobilized on a matrix.", "The labeling of the target-binding antibody can be performed irrespective of the solution that the antibody is present in and includes proteins that are normally present in serum or ascites.", "This feature of the labeling process of the target-binding antibody eliminates the need to purify and concentrate the target-binding antibody.", "The time required for the labeling reagent to selectively bind to the target-binding antibody is typically very short, often less than 10 minutes.", "Often the labeling reagent binds the target-binding antibody in the amount of time it takes to add and mix the labeling reagent with the target-binding antibody.", "This formation of an immuno-labeled complex—a target-binding antibody and a labeling reagent—results in the formation of an target detection solution that is used to detect a target in a sample.", "The labeling steps of the target-binding antibody are optionally repeated to form a panel of subsets, these immuno-labeled complex subsets may be used individually or pooled wherein each subset is distinguished from another subset by i) the target-binding antibody, or ii) a ratio of label to labeling reagent, or iii) a ratio of labeling reagent to the target-binding antibody or iv) by a physical property of the label.", "Thus, it is appreciated that a wide range of subsets can be formed wherein the subsets can be used individually to detect a target in a sample or pooled to simultaneously detect multiple targets in a sample.", "The simultaneous detection of multiple targets in a sample is especially useful in methods that utilize flow cytometry or methods that immobilize a population of cells or tissue on a surface.", "The methods for determining a target in a sample using immuno-labeled subsets comprises forming a subset of immuno-labeled complexes, as described above, contacting a sample with said immuno-labeled complexes, incubating the sample for a time sufficient to allow the immuno-labeled complex to selectively bind to a desired target, and illuminating the immuno-labeled complex whereby the target is detected.", "The sample is any material that may contain a target and typically comprises a population of cells, cellular extract, subcellular component, proteins, peptides, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.", "When multiple targets are detected a pooled subset of immuno-labeled complexes are formed and incubated with the sample or individual subsets are add sequentially to a sample.", "For methods using flow cytometry the population of cells is illuminated when they pass through an optical examination zone and the data collected about the label determines the identity and quantity of the targets.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1: Shows a schematic representation of the formation of the immuno-labeled complex (target-binding antibody and labeling reagent).", "FIG.", "2: Shows species specificity of goat Fab anti-(mouse Fc), as observed using a microplate coated with IgG of various species.", "The various species were blocked with BSA, reacted with biotinylated goat Fab anti-(mouse Fc), washed, and then treated with streptavidin-horseradish peroxidase (HRP), followed by hydrogen peroxide (H2O2) and the Amplex Red peroxidase detection reagent.", "FIG.", "3: Shows a preferred molar ratio of a goat Fab anti-(mouse Fc) labeling reagent.", "Varying amounts of an Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) were added to a constant amount of anti-biotin monoclonal antibody (mAb).", "This mixture was equilibrated for 20 minutes, and then added to biotinylated-BSA in a microplate well.", "After allowing time to bind, the plates were washed and the remaining fluorescence was quantitated.", "The analysis was performed in triplicate (circles).", "Control experiments were performed, as described above, but without adding the primary anti-biotin antibody (solid squares).", "FIG.", "4: Shows a comparison of the fluorescence intensity (Example 6) for labeling reagent prepared in homogeneous solution (Example 4) and labeling reagent prepared on a column (Example 5).", "FIG.", "5: Shows detection of multiple targets on T cells using a labeling reagent attached to a R-phycoerythrin (R-PE) (FIG.", "5A) to detect CD3-positive T cells, a labeling reagent attached to Alexa Fluor 647 dye (FIG.", "5B) to detect CD4-positive T cells and a labeling reagent attached to Alexa Fluor 488 dye (FIG.", "5B) to detect CD8-positive T cells (Example 18).", "The CD-3 detected T cells are shown in the upper left (UL) and upper right (UR) quadrants.", "The relative percentages of total lymphocytes that are CD3-positive cells are 83.3% (UL+UR).", "The relative percentage of CD8-positive Alexa Fluor 488 dye-stained lymphocytes and CD3-positive R-PE dye-stained lymphocytes is 35.1% (UR quadrant).", "The lower left quadrant (LL, 20.4%) shows CD3-negative lymphocytes (i.e.", "non-T cells) comprised of NK cells, B cells and some monocytes.", "In the lower right (LR, 2.7%) region are non-T cells, which are nonspecifically stained.", "FIG.", "5B further shows CD3-positive T-cells subdivided into Alexa Fluor 647 dye CD4-positive and Alexa Fluor 488 dye CD8-positive.", "CD4-positive cells represent 50.9% of total lymphocytes (UL quadrant) and CD8-positive cells represent 24.5% of the total lymphocytes (LR quadrant).", "The 23.1% of cells in the LL quadrant are non-T cells, while the 1.5% of cells in UR quadrant are likely nonspecifically stained lymphocytes.", "FIG.", "6: Shows high-performance size-exclusion chromatographic analysis of Alexa Fluor 488 dye-labeled goat Fab anti-(mouse Fc) labeling reagent binding to a mouse IgG, target-binding antibody.", "The labeling reagent, alone, appears as a peak at 38 minutes; the target-binding antibody, alone, appears as a peak at 33 minutes.", "When labeling reagent and target-binding antibody are mixed together at a molar ratio of -5:1 (labeling reagent:target-binding antibody), the resulting immunolabeling complex appears as a peak at 29 minutes (Example 10).", "FIG.", "7: Shows the production of labeling reagent wherein the label is attached to the labeling reagent when immobilized on a column.", "DETAILED DESCRIPTION OF THE INVENTION I. Definitions Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions or process steps, as such may vary.", "It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.", "Thus, for example, reference to “a protein labeling complex” includes a plurality of complexes and reference to “a target-binding protein” includes a plurality of proteins and the like.", "Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.", "The following terms are defined for purposes of the invention as described herein.", "The term “affinity” as used herein refers to the strength of the binding interaction of two molecules, such as an antibody and an antigen or a positively charged moiety and a negatively charged moiety.", "For bivalent molecules such as antibodies, affinity is typically defined as the binding strength of one binding domain for the antigen, e.g.", "one Fab fragment for the antigen.", "The binding strength of both binding domains together for the antigen is referred to as “avidity”.", "As used herein “High affinity” refers to a ligand that binds to an antibody having an affinity constant (Ka) greater than 104 M−1, typically 105-1011 M−1; as determined by inhibition ELISA or an equivalent affinity determined by comparable techniques such as, for example, Scatchard plots or using Kd/dissociation constant, which is the reciprocal of the Ka, etc.", "The term “antibody” as used herein refers to a protein of the immunoglobulin (Ig) superfamily that binds noncovalently to certain substances (e.g.", "antigens and immunogens) to form an antibody-antigen complex.", "Antibodies can be endogenous, or polyclonal wherein an animal is immunized to elicit a polyclonal antibody response or by recombinant methods resulting in monoclonal antibodies produced from hybridoma cells or other cell lines.", "It is understood that the term “antibody” as used herein includes within its scope any of the various classes or sub-classes of immunoglobulin derived from any of the animals conventionally used.", "The term “antibody fragments” as used herein refers to fragments of antibodies that retain the principal selective binding characteristics of the whole antibody.", "Particular fragments are well-known in the art, for example, Fab, Fab′, and F(ab′)2, which are obtained by digestion with various proteases, pepsin or papain, and which lack the Fc fragment of an intact antibody or the so-called “half-molecule” fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components in the intact antibody.", "Such fragments also include isolated fragments consisting of the light-chain-variable region, “Fv” fragments consisting of the variable regions of the heavy and light chains, and recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker.", "Other examples of binding fragments include (i) the Fd fragment, consisting of the VH and CH1 domains; (ii) the dAb fragment (Ward, et al., Nature 341, 544 (1989)), which consists of a VH domain; (iii) isolated CDR regions; and (iv) single-chain Fv molecules (scFv) described above.", "In addition, arbitrary fragments can be made using recombinant technology that retains antigen-recognition characteristics.", "The term “antigen” as used herein refers to a molecule that induces, or is capable of inducing, the formation of an antibody or to which an antibody binds selectively, including but not limited to a biological material.", "Antigen also refers to “immunogen”.", "The target-binding antibodies selectively bind an antigen, as such the term can be used herein interchangeably with the term “target”.", "The term “anti-region antibody” as used herein refers to an antibody that was produced by immunizing an animal with a select region that is a fragment of a foreign antibody wherein only the fragment is used as the immunogen.", "Anti-region antibodies include monoclonal and polyclonal antibodies.", "The term “anti-region fragment” as used herein refers to a monovalent fragment that was generated from an anti-region antibody of the present invention by enzymatic cleavage.", "The term “biotin” as used herein refers to any biotin derivative, including without limitation, substituted and unsubstituted biotin, and analogs and derivatives thereof, as well as substituted and unsubstituted derivatives of caproylamidobiotin, biocytin, desthiobiotin, desthiobiocytin, iminobiotin, and biotin sulfone.", "The term “biotin-binding protein” as used herein refers to any protein that binds selectively and with high affinity to biotin, including without limitation, substituted or unsubstituted avidin, and analogs and derivatives thereof, as well as substituted and unsubstituted derivatives of streptavidin, ferritin avidin, nitroavidin, nitrostreptavidin, and Neutravidin™ avidin (a de-glycosylated modified avidin having an isoelectric point near neutral).", "The term “buffer” as used herein refers to a system that acts to minimize the change in acidity or basicity of the solution against addition or depletion of chemical substances.", "The term “capture reagent” refers to a non-specific immunoglobulin that is used to remove excess labeling reagent after the formation of the immuno-labeled complex.", "The capture reagent is optionally attached a matrix to facilitate removal of the excess labeling regent.", "A matrix typically includes a microsphere, an agarose bead or any solid surface that the excess labeling reagent can be passed by.", "The term “chromophore” as used herein refers to a label that emits light in the visible spectra that can be observed without the aid of instrumentation.", "The term “complex” as used herein refers to the association of two or more molecules, usually by non-covalent bonding, e.g., the association between an antibody and an antigen or the labeling reagent and the target-binding antibody.", "The term “detectable response” as used herein refers to an occurrence of, or a change in, a signal that is directly or indirectly detectable either by observation or by instrumentation.", "Typically, the detectable response is an occurrence of a signal wherein the fluorophore is inherently fluorescent and does not produce a change in signal upon binding to a metal ion or biological compound.", "Alternatively, the detectable response is an optical response resulting in a change in the wavelength distribution patterns or intensity of absorbance or fluorescence or a change in light scatter, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters.", "Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density.", "The term “detectably distinct” as used herein refers to a signal that is distinguishable or separable by a physical property either by observation or by instrumentation.", "For example, a fluorophore is readily distinguishable either by spectral characteristics or by fluorescence intensity, lifetime, polarization or photo-bleaching rate from another fluorophore in the sample, as well as from additional materials that are optionally present.", "The term “directly detectable” as used herein refers to the presence of a material or the signal generated from the material is immediately detectable by observation, instrumentation, or film without requiring chemical modifications or additional substances.", "The term “examination zone” as used herein refers to an optical zone of a flow cytometer, or a similar instrument, wherein cells are passed through essentially one at a time in a thin stream whereby the bound immuno-labeled complex is illuminated and the intensity and emission spectra of the fluorophore is detected and recorded.", "This includes instruments wherein the examination zone moves and the sample is held in place.", "The term “fluorophore” as used herein refers to a composition that is inherently fluorescent or demonstrates a change in fluorescence upon binding to a biological compound or metal ion, i.e., fluorogenic.", "Fluorophores may contain substitutents that alter the solubility, spectral properties or physical properties of the fluorophore.", "Numerous fluorophores are known to those skilled in the art and include, but are not limited to coumarin, cyanine, benzofuran, a quinoline, a quinazolinone, an indole, a benzazole, a borapolyazaindacene and xanthenes including fluoroscein, rhodamine and rhodol as well as other fluorophores described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9th edition, CD-ROM, September 2002).", "The term “immuno-labeled complex” refers to the complex of target-binding antibody that is non-covalently attached to a labeling reagent.", "The term “immuno-labeled complex subset” as used herein refers to a discrete set of immuno-labeled complexes that are homogenous and can be distinguished from another subset of immuno-labeled complex by the physical properties of the label, or the ratio of the label to labeling reagent, or the ratio of labeling reagent to target-binding antibody, or the target-binding antibody.", "Typically an immuno-labeled complex subset is present in a buffer to provide a “target detection solution”.", "The term “kit” as used herein refers to a packaged set of related components, typically one or more compounds or compositions.", "The term “label” as used herein refers to a chemical moiety or protein that retains it's native properties (e.g.", "spectral properties, conformation and activity) when attached to a labeling reagent and used in the present methods.", "The label can be directly detectable (fluorophore) or indirectly detectable (hapten or enzyme).", "Such labels include, but are not limited to, radiolabels that can be measured with radiation-counting devices; pigments, dyes or other chromogens that can be visually observed or measured with a spectrophotometer; spin labels that can be measured with a spin label analyzer; and fluorescent labels (fluorophores), where the output signal is generated by the excitation of a suitable molecular adduct and that can be visualized by excitation with light that is absorbed by the dye or can be measured with standard fluorometers or imaging systems, for example.", "The label can be a chemiluminescent substance, where the output signal is generated by chemical modification of the signal compound; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal, such as the formation of a colored product from a colorless substrate.", "The term label can also refer to a “tag” or hapten that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used to generate a detectable signal.", "For example, one can use biotin as a tag and then use an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag, and then use a colorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorogenic substrate such as Amplex Red reagent (Molecular Probes, Inc.) to detect the presence of HRP.", "Numerous labels are know by those of skill in the art and include, but are not limited to, particles, fluorophores, haptens, enzymes and their calorimetric, fluorogenic and chemiluminescent substrates and other labels that are described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS (9th edition, CD-ROM, September 2002), supra.", "The term “labeling reagent” as used herein refers to a monovalent antibody fragment or a non-antibody monomeric protein provided that the labeling reagent has affinity for a selected region of the target-binding antibody and is covalently attached to a label.", "The term “labeling reagent subset” as used herein refers to a discrete set of labeling reagents that are homogenous and can be distinguished from another subset of labeling reagent either by the physical properties of the label or the ratio of the label to labeling reagent.", "The term “labeling solution” as used herein refers to a solution that is used to form an immuno-labeled complex wherein the solution comprises labeling reagents and a buffer.", "The term “matrix” as used herein refers to a solid or semi-solid surface that a biological molecule can be attached to, such as a sample of the present invention or a capture reagent.", "Examples include, but are not limited to, agarose, polyacrylamide gel, polymers, microspheres, glass surface, plastic surface, membrane, margnetic surface, and an array.", "The term “monovalent antibody fragment” as used herein refers to an antibody fragment that has only one antigen-binding site.", "Examples of monovalent antibody fragments include, but are not limited to, Fab fragments (no hinge region), Fab′ fragments (monovalent fragments that contain a heavy chain hinge region), and single-chain fragment variable (ScFv) proteins.", "The term “non-antibody monomeric protein” as used herein refers to a protein that binds selectively and non-covalently to a member of the Ig superfamily of proteins, including but not limited to proteins A, G, and L, hybrids thereof (A/G), recombinant versions and cloned versions thereof, fusions of these proteins with detectable protein labels, and lectins but the protein itself is not an antibody or an antibody fragment.", "The terms “protein” and “polypeptide” are used herein in a generic sense to include polymers of amino acid residues of any length.", "The term “peptide” is used herein to refer to polypeptides having less than 100 amino acid residues, typically less than 10 amino acid residues.", "The terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.", "The term “purified” as used herein refers to a preparation of a target-binding antibody that is essentially free from contaminating proteins that normally would be present in association with the antibody, e.g., in a cellular mixture or milieu in which the protein or complex is found endogenously such as serum proteins or hybridoma supernatant.", "The term “sample” as used herein refers to any material that may contain a target, as defined below.", "Typically, the sample comprises a population of cells, cellular extract, subcellular components, tissue culture, a bodily fluid, and tissue.", "The sample may be in an aqueous solution, a viable cell culture or immobilized on a solid or semi solid surface such as a gel, a membrane, a glass surface, a microparticle or on a microarray.", "The term “target” as used herein refers to any entity that a target-binding antibody has affinity for such as an epitope or antigen.", "This target includes not only the discrete epitope that the target-binding antibody has affinity for but also includes any subsequently bound molecules or structures.", "In this way an epitope serves as a marker for the intended target.", "For example, a cell is a target wherein the target-binding antibody binds a cell surface protein such as CD3 on a T cell wherein the target marker is CD3 and the target is the T cell.", "The term “target-binding antibody” as used herein refers to an antibody that has affinity for a discrete epitope or antigen that can be used with the methods of the present invention.", "Typically the discrete epitope is the target but the epitope can be a marker for the target such as CD3 on T cells.", "The term can be used interchangeably with the term “primary antibody” when describing methods that use an antibody that binds directly to the antigen as opposed to a “secondary antibody” that binds to a region of the primary antibody.", "II.", "Compositions and Methods of Use In accordance with the present invention, labeling reagents, methods for labeling target-binging antibodies and methods for using the labeled antibodies to detect a target in a sample are provided.", "The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins that are covalently attached to a label of the present invention.", "The label covalently attached to a labeling reagent is directly detectable such as a fluorophore or functions as an indirect label that requires an additional component such as a calorimetric enzyme substrate or an enzyme conjugate.", "The labeling reagents have affinity for a specific region of the target-binding antibody.", "The target-binding antibodies are defined as any antibody known to one skilled in the art that has an affinity for a target in a sample.", "The target-binding antibodies are labeled with the labeling reagent in a labeling method to form immuno-labeled complexes and then added to a sample to detect a target.", "The labeling reagent and the methods of the present invention provide for detection of one or multiple targets in a sample.", "Multiple targets are detected when either pooled subsets of immuno-labeled complexes or a panel of subsets that are sequentially added to a sample.", "The subset of immuno-labeled complexes begins with labeling reagent subsets wherein a labeling reagent subset is distinguished by the ratio of label to labeling reagent or by the physical characteristics of the label.", "The discrete labeling reagents subsets are added to the target-binding antibodies wherein the affinity of the antibody and ratio of labeling reagent to target-binding antibody determines the subsets of immuno-labeled complexes.", "This results in an infinite number of immuno-labeled complex subsets that are distinguished by i) the target-binding antibody, or ii) a ratio of label to labeling reagent, or iii) a ratio of labeling reagent to the target-binding antibody or iv) by a physical property of the label.", "These subsets can be used individually in a method of the present invention to detect a single or multiple targets in a sample or pooled and used to simultaneously detect multiple targets in a sample.", "These pooled subsets allow for not only detection but also identification and quantitation of the targets.", "A. Labelinq Reagents 1.Monovalent Antibody Fragments and Monomeric Non-Antibody Proteins The labeling reagents of the present invention are monovalent antibody fragments or non-antibody monomeric proteins that have affinity for a region of a target-binding antibody.", "The regions of the target-binding antibody that can be bound by a labeling reagent include the Fc region, the kappa or lambda light chain region or a heavy chain region.", "When the labeling reagent is derived from an antibody the monovalent fragment can be, anti-Fc, an anti-Fc isotype, anti-kappa light chain, anti-lambda light chain, or a single-chain fragment variable protein.", "Labeling reagents that are a non-antibody peptide or protein, are for example but not limited to, soluble Fc receptor, protein G, protein A, protein L, lectins, or a fragment thereof.", "The labeling reagents typically have affinity for the Fc region of the target-binding antibody but any region, except the binding domain, may be used as a binding site for the labeling reagent.", "The Fc region is preferable because it is the farthest from the binding domain of the target-binding antibody and is unlikely to cause steric hinderance, when bound by a labeling reagent, of the binding domain for the target.", "Antibody is a term of the art denoting the soluble substance or molecule secreted or produced by an animal in response to an antigen, and which has the particular property of combining specifically with the antigen that induced its formation.", "Antibodies themselves also serve are antigens or immunogens because they are glycoproteins and therefore are used to generate anti-species antibodies.", "Antibodies, also known as immunoglobulins, are classified into five distinct classes—IgG, IgA, IgM, IgD, and IgE.", "The basic IgG immunoglobulin structure consists of two identical light polypeptide chains and two identical heavy polypeptide chains (linked together by disulfide bonds).", "When IgG is treated with the enzyme papain, a monovalent antigen-binding fragment can be isolated, referred herein to as a Fab fragment.", "When IgG is treated with pepsin (another proteolytic enzyme), a larger fragment is produced, F(ab′)2.This fragment can be split in half by treating with a mild reducing buffer that results in the monovalent Fab′ fragment.", "The Fab′ fragment is slightly larger than the Fab and contains one or more free sulfhydryls from the hinge region (which are not found in the smaller Fab fragment).", "The term “antibody fragment” is used herein to define both the Fab′ and Fab portions of the antibody.", "It is well known in the art to treat antibody molecules with pepsin and papain in order to produce antibody fragments (Gorevic et al., Methods of Enzyol., 116:3 (1985)).", "The monovalent Fab fragments of the present invention are produced from either murine monoclonal antibodies or polyclonal antibodies generated in a variety of animals that have been immunized with a foreign antibody or fragment thereof, U.S. Pat.", "No.", "4,196,265 discloses a method of producing monoclonal antibodies.", "Typically, labeling reagents are derived from a polyclonal antibody that has been produced in a rabbit or goat but any animal known to one skilled in the art to produce polyclonal antibodies can be used to generate anti-species antibodies.", "However, monoclonal antibodies are equal, and in some cases, preferred over polyclonal antibodies provided that the target-binding antibody is compatible with the monoclonal antibodies that are typically produced from murine hybridoma cell lines using methods well known to one skilled in the art.", "Example 1 describes production of polyclonal antibodies raised in animals immunized with the Fc region of a foreign antibody.", "It is a preferred embodiment of the present invention that the labeling reagents be generated against only the Fc region of a foreign antibody.", "Essentially, the animal is immunized with only the Fc region fragment of a foreign antibody, such as murine.", "The polyclonal antibodies are collected from subsequent bleeds, digested with an enzyme, pepsin or papain, to produce monovalent fragments.", "The fragments are then affinity purified on a column comprising whole immunoglobulin protein that the animal was immunized against or just the Fc fragments.", "As described in detail below, the labeling reagents are also covalently labeled with fluorophore labels when bound to the affinity column to eliminate incorporating label into the binding domain of the monovalent fragment.", "One of skill in the art will appreciate that this method can be used to generate monovalent fragments against any region of a target-binding protein and that selected peptide fragments of the target-binding antibody could also be used to generate fragments.", "Alternatively, a non-antibody protein or peptide such as protein G, or other suitable proteins, can be used alone or coupled with albumin wherein albumin is attached with a label of the present invention.", "Preferred albumins of the invention include human and bovine serum albumins or ovalbumin.", "Protein A, G and L are defined to include those proteins know to one skilled in the art or derivatives thereof that comprise at least one binding domain for IgG, i.e.", "proteins that have affinity for IgG.", "These proteins can be modified but do not need to be and are labeled in the same manner as the monovalent Fab fragments of the invention.", "2.Labels The labels of the present invention include any directly or indirectly detectable label known by one skilled in the art that can be covalently attached to the labeling reagent of the present invention.", "Labels include, without limitation, a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.", "Preferred labels include fluorophores, fluorescent proteins, haptens, and enzymes.", "A fluorophore of the present invention is any chemical moiety that exhibits an absorption maximum beyond 280 nm, and when covalently attached to a labeling reagent retains its spectral properties.", "Fluorophores of the present invention include, without limitation; a pyrene (including any of the corresponding derivative compounds disclosed in U.S. Pat.", "No.", "5,132,432), an anthracene, a naphthalene, an acridine, a stilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine (including any corresponding compounds in U.S. Ser.", "Nos.", "09/968,401 and 09/969,853), a carbocyanine (including any corresponding compounds in U.S. Ser.", "Nos.", "09/557,275; 09/969,853 and 09/968,401; U.S. Pat.", "Nos.", "4,981,977; 5,268,486; 5,569,587; 5,569,766; 5,486,616; 5,627,027; 5,808,044; 5,877,310; 6,002,003; 6,004,536; 6,008,373; 6,043,025; 6,127,134; 6,130,094; 6,133,445; and publications WO 02/26891, WO 97/40104, WO 99/51702, WO 01/21624; EP 1 065 250 A1), a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene (including any corresponding compounds disclosed in U.S. Pat.", "Nos.", "4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896), a xanthene (including any corresponding compounds disclosed in U.S. Pat.", "No.", "6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343 and U.S. Ser.", "No.", "09/922,333), an oxazine (including any corresponding compounds disclosed in U.S. Pat.", "No.", "4,714,763) or a benzoxazine, a carbazine (including any corresponding compounds disclosed in U.S. Pat.", "No.", "4,810,636), a phenalenone, a coumarin (including an corresponding compounds disclosed in U.S. Pat.", "Nos.", "5,696,157; 5,459,276; 5,501,980 and 5,830,912), a benzofuran (including an corresponding compounds disclosed in U.S. Pat.", "Nos.", "4,603,209 and 4,849,362) and benzphenalenone (including any corresponding compounds disclosed in U.S. Pat.", "No.", "4,812,409) and derivatives thereof.", "As used herein, oxazines include resorufins (including any corresponding compounds disclosed in Pat.", "No.", "5,242,805), aminooxazinones, diaminooxazines, and their benzo-substituted analogs.", "When the fluorophore is a xanthene, the fluorophore is optionally a fluorescein, a rhodol (including any corresponding compounds disclosed in U.S. Pat.", "Nos.", "5,227,487 and 5,442,045), or a rhodamine (including any corresponding compounds in U.S. Pat.", "Nos.", "5,798,276; 5,846,737; U.S. Ser.", "No.", "09/129,015).", "As used herein, fluorescein includes benzo- or dibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins.", "Similarly, as used herein rhodol includes seminaphthorhodafluors (including any corresponding compounds disclosed in U.S. Pat.", "No.", "4,945,171).", "Alternatively, the fluorophore is a xanthene that is bound via a linkage that is a single covalent bond at the 9-position of the xanthene.", "Preferred xanthenes include derivatives of 3H-xanthen-6-ol-3-one attached at the 9-position, derivatives of 6-amino-3H-xanthen-3-one attached at the 9-position, or derivatives of 6-amino-3H-xanthen-3-imine attached at the 9-position.", "Preferred fluorophores of the invention include xanthene (rhodol, rhodamine, fluorescein and derivatives thereof) coumarin, cyanine, pyrene, oxazine and borapolyazaindacene.", "Most preferred are sulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinated coumarins and sulfonated cyanines.", "The choice of the fluorophore attached to the labeling reagent will determine the absorption and fluorescence emission properties of the labeling reagent and immuno-labeled complex.", "Physical properties of a fluorophore label include spectral characteristics (absorption, emission and stokes shift), fluorescence intensity, lifetime, polarization and photo-bleaching rate all of which can be used to distinguish one fluorophore from another.", "Typically the fluorophore contains one or more aromatic or heteroaromatic rings, that are optionally substituted one or more times by a variety of substituents, including without limitation, halogen, nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring system, benzo, or other substituents typically present on fluorophores known in the art.", "In one aspect of the invention, the fluorophore has an absorption maximum beyond 480 nm.", "In a particularly useful embodiment, the fluorophore absorbs at or near 488 nm to 514 nm (particularly suitable for excitation by the output of the argon-ion laser excitation source) or near 546 nm (particularly suitable for excitation by a mercury arc lamp).", "Many of fluorophores can also function as chromophores and thus the described fluorophores are also preferred chromophores of the present invention.", "In addition to fluorophores, enzymes also find use as labels for the labeling reagents.", "Enzymes are desirable labels because amplification of the detectable signal can be obtained resulting in increased assay sensitivity.", "The enzyme itself does not produce a detectable response but functions to break down a substrate when it is contacted by an appropriate substrate such that the converted substrate produces a fluorescent, colorimetric or luminescent signal.", "Enzymes amplify the detectable signal because one enzyme on a labeling reagent can result in multiple substrates being converted to a detectable signal.", "This is advantageous where there is a low quantity of target present in the sample or a fluorophore does not exist that will give comparable or stronger signal than the enzyme.", "However, fluorophores are most preferred because they do not require additional assay steps and thus reduce the overall time required to complete an assay.", "The enzyme substrate is selected to yield the preferred measurable product, e.g.", "calorimetric, fluorescent or chemiluminescence.", "Such substrates are extensively used in the art, many of which are described in the MOLECULAR PROBES HANDBOOK, supra.", "A preferred calorimetric or fluorogenic substrate and enzyme combination uses oxidoreductases such as horseradish peroxidase and a substrate such as 3,3′-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color (brown and red, respectively).", "Other calorimetric oxidoreductase substrates that yield detectable products include, but are not limited to: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol.", "Fluorogenic substrates include, but are not limited to, homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reduced benzothiazines, including Amplexe Red reagent and its variants (U.S. Pat.", "No.", "4,384,042) and reduced dihydroxanthenes, including dihydrofluoresceins (U.S. Pat.", "No.", "6,162,931) and dihydrorhodamines including dihydrorhodamine 123.Peroxidase substrates that are tyramides (U.S. Pat.", "Nos.", "5,196,306; 5,583,001 and 5,731,158) represent a unique class of peroxidase substrates in that they can be intrinsically detectable before action of the enzyme but are “fixed in place” by the action of a peroxidase in the process described as tyramide signal amplification (TSA).", "These substrates are extensively utilized to label targets in samples that are cells, tissues or arrays for their subsequent detection by microscopy, flow cytometry, optical scanning and fluorometry.", "Another preferred calorimetric (and in some cases fluorogenic) substrate and enzyme combination uses a phosphatase enzyme such as an acid phosphatase, an alkaline phosphatase or a recombinant version of such a phosphatase in combination with a calorimetric substrate such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenyl phosphate, or o-nitrophenyl phosphate or with a fluorogenic substrate such as 4-methylumbelliferyl phosphate, 6,8-difluoro-7-hydroxy4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat.", "No.", "5,830,912) fluorescein diphosphate, 3-O-methylfluorescein phosphate, resorufin phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO phosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat.", "Nos.", "5,316,906 and 5,443,986).", "Glycosidases, in particular beta-galactosidase, beta-glucuronidase and beta-glucosidase, are additional suitable enzymes.", "Appropriate colorimetric substrates include, but are not limited to, 5-bromo4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) and similar indolyl galactosides, glucosides, and glucuronides, o-nitrophenyl beta-D-galactopyranoside (ONPG) and p-nitrophenyl beta-D-galactopyranoside.", "Preferred fluorogenic substrates include resorufin beta-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and their structural variants (U.S. Pat.", "Nos.", "5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236), 4-methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferyl beta-D-galactopyranoside and fluorinated coumarin beta-D-galactopyranosides (U.S. Pat.", "No.", "5,830,912).", "Additional enzymes include, but are not limited to, hydrolases such as cholinesterases and peptidases, oxidases such as glucose oxidase and cytochrome oxidases, and reductases for which suitable substrates are known.", "Enzymes and their appropriate substrates that produce chemiluminescence are preferred for some assays.", "These include, but are not limited to, natural and recombinant forms of luciferases and aequorins.", "Chemiluminescence-producing substrates for phosphatases, glycosidases and oxidases such as those containing stable dioxetanes, luminol, isoluminol and acridinium esters are additionally useful.", "In addition to enzymes, haptens such as biotin are also preferred labels.", "Biotin is useful because it can function in an enzyme system to further amplify the detectable signal, and it can function as a tag to be used in affinity chromatography for isolation purposes.", "For detection purposes, an enzyme conjugate that has affinity for biotin is used, such as avidin-HRP.", "Subsequently a peroxidase substrate is added to produce a detectable signal.", "Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, affector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.", "Fluorescent proteins also find use as labels for the labeling reagents of the present invention.", "Examples of fluorescent proteins include green fluorescent protein (GFP) and the phycobiliproteins and the derivatives thereof.", "The fluorescent proteins, especially phycobiliprotein, are particularly useful for creating tandem dye labeled labeling reagents.", "These tandem dyes comprise a fluorescent protein and a fluorophore for the purposes of obtaining a larger stokes shift wherein the emission spectra is farther shifted from the wavelength of the fluorescent protein's absorption spectra.", "This is particularly advantageous for detecting a low quantity of a target in a sample wherein the emitted fluorescent light is maximally optimized, in other words little to none of the emitted light is reabsorbed by the fluorescent protein.", "For this to work, the fluorescent protein and fluorophore function as an energy transfer pair wherein the fluorescent protein emits at the wavelength that the fluorophore absorbs at and the fluorphore then emits at a wavelength farther from the fluorescent proteins than could have been obtained with only the fluorescent protein.", "A particularly useful combination is the phycobiliproteins disclosed in U.S. Pat.", "Nos.", "4,520,110; 4,859,582; 5,055,556 and the sulforhodamine fluorophores disclosed in Pat.", "No.", "5,798,276, or the sulfonated cyanine fluorophores disclosed in U.S. Ser.", "Nos.", "09/968/401 and 09/969/853; or the sulfonated xanthene derivatives disclosed in Pat.", "No.", "6,130,101 and those combinations disclosed in U.S. Pat.", "4,542,104.Alternatively, the fluorophore functions as the energy donor and the fluorescent protein is the energy acceptor.", "3.Covalent Attachment of Labels to the Labeling Reagents The labeling reagents can be independently attached to one or more labels of the present invention by a number of methods known to one skilled in the art and modification of such methods.", "Methods include, labeling in a solution or on an affinity column.", "For labeling in solution the labeling reagent is optionally modified to contain a reactive group and the label is modified to contain a reactive group or is synthesized to contain a reactive group, as is typically the case with fluorophore labels wherein the reactive group facilitates covalent attachment.", "The modification of the labeling reagent to contain a reactive group includes (1) chemical addition of such a reactive group or (2) alternatively takes advantage of the disulfide bonds of the F(ab′)2 fragment wherein the fragment is reduced to break the bond and expose the thiol group that readily reacts with a reactive group on a label, as disclosed in U.S. Pat.", "No.", "5,360,895.Typically, covalent attachment of the label to the fragment is the result of a chemical reaction between an electrophilic group and a nucleophilic group.", "However, when a label is used that is photoactivated the covalent attachment results when the labeling solution is illuminated.", "A method for covalently attaching a label, particularly an enzyme, a fluorescent protein or a particle, comprises the following steps: a) cleaving an intact anti-region antibody with an enzyme resulting in a F(ab′)2 fragment; b) contacting said F(ab′)2 fragment with a reducing agent to produce Fab′ fragments containing a thiol group; c) contacting said Fab′ fragments with a solution comprising a label that contains a reactive group; and, d) isolating Fab′ fragments of step d) that are covalently attached to a label by size exclusion or affinity chromatography.", "The whole anti-region antibody is cleaved with pepsin to generate a bivalent F(ab)′2 fragment.", "This fragment is typically affinity purified on a column comprising immunoglobulin proteins such as IgG that is immobilized on agarose.", "The fragment is then reduced to break the disulfide bond of the hinge region that connects the two Fab fragments resulting in a Fab′ fragment with an exposed thiol group.", "This is typically accomplished by adding a mild reducing buffer to the affinity purified F(ab′)2 fragments such as a buffer comprising 0.01 M EDTA and 0.01 M cysteine in phosphate buffer saline (PBS).", "The resulting thiol group readily reacts with a reactive group on a label to covalently attach the label to the fragment.", "Thus, a solution containing a label that has been chemically modified to contain a reactive group, using methods well known to one skilled in the art, is added to the solution of reduced Fab′ fragments.", "This method is particularly useful for covalently attaching enzyme and other protein labels due to their size and the lack of exposed amine groups on the Fab fragments.", "One of skill in the art will appreciate that this method requires the use of Fab′ fragments as apposed to Fab fragments due to the disulfide bonds of the Fab′ fragment and that the use of the enzyme papain or the like results in such a fragment.", "An alternative labeling of monovalent antibody fragments and the monomeric non-antibody proteins is also accomplished in a solution.", "The method comprises the steps: a) contacting a Fab fragment or non-antibody monomeric protein with a solution comprising a label that contains a reactive group; and, b) isolating labeled anti-region Fab fragment or non-antibody monomeric protein by size exclusion or affinity chromatography.", "When a Fab fragment is to be labeled the whole antibody is cleaved with an enzyme, such as papain, to generate Fab monovalent fragments and the fragments are typically purified on an affinity column prior to addition of the label.", "The Fab fragment or non-antibody monomeric proteins are optionally chemically modified to contain a reactive group.", "However, for covalently attaching reactive fluorophore labels it has been found that this modification of the fragment of non-antibody protein is not necessary.", "The reactive label, typically a fluorophore or hapten, are added to a solution of Fab fragments or non-antibody proteins and the labeling reagent is separated from excess label by size exclusion or affinity chromatorgraphy.", "The labeling reagents are then stored in an appropriate buffer.", "Labeling in solution can have some drawbacks, especially when labeling of Fab fragments or non-antibody proteins with fluorophores.", "Thus, Fab fragments and non-antibody proteins of the present invention are preferably covalently attached to a fluorophore label when immobilized on an affinity column.", "The fragments and non-antibody proteins are immobilized on an affinity column that comprises a protein that the fragment has affinity for, typically IgG, and after immobilization a reactive fluorophore is added to the column wherein the fragments are labeled and unreacted fluorophores pass through the column.", "The use of this affinity chromatography method avoids the incorporation of label into the binding domain of the Fab fragment or non-antibody protein.", "When Fab fragments are labeled with fluorophores using this method unexpected advantages were obtained wherein the fluorescent signal form fragments labeled on a column are brighter than fragments labeled in solution when the fluorophore and ratio of fluorophore to labeling reagent are held constant.", "Without wishing to be bound by a theory it is possible that the decreased brightness observed from the fragments labeled in solution is due to quenching of fluorphores that are bound in or near the binding domain by the high concentration of amine groups in the binding domain.", "Thus, a preferred embodiment of the invention for covalently attaching fluorphore labels to Fab fragments comprises the following steps: a) cleaving an intact anti region antibody with an enzyme that generates Fab fragments; b) isolating the anti-region Fab fragments of step a); c) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that specifically bind anti-region Fab fragments with a solution comprising said anti-region fragments of step b) wherein said Fab fragments are immobilized; d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; e) washing said matrix to remove unbound label, and; f) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label and being isolated from other proteins and fragments thereof.", "The matrix is typically an agarose column that comprises either the selected region, such as the Fc region, or the entire antibody provided that the antibody or fragment thereof is the same species and isotype that was used to produce the antibodies that the labeling reagent was generated from.", "However any matrix known to one skilled in the art can be used that allows for immobilization of labeling reagent and removal following attachment of the fluorophore label.", "Fab and Fab′ fragments can both be labeled in this manner.", "However a free thiol group is not necessary and therefore Fab fragments are typically labeled using this method.", "Due to the unique properties of the labeling reagent and the attached labels it is a preferred embodiment of the present invention that enzyme or other protein labels are covalently attached to Fab′ fragments in solution utilizing the free thiol group of the Fab′ fragment.", "It is another preferred embodiment that fluorophore labels be covalently attached to the labeling reagent when the reagent is immobilized on a affinity column wherein the labeling reagent is typically an Fab fragment or a non-antibody monomeric protein.", "The attachment of the label to the fragments or the non-antibody proteins results in multiple subsets that are distinguished by the ratio of the label to the labeling reagent and the physical properties of the label.", "A labeling reagent subset as used herein refers to a discrete set of labeling reagents that are homogenous and can be distinguished from another subset of labeling reagent either by the physical properties of the label or the ratio of the label to labeling reagent.", "The physical properties include differences within a group of labels, such as emission spectra of fluorphores, or across groups of labels, such as the difference between an enzyme and a fluorophore.", "For fluorphore labels, the physical properties typically relates to the emission spectra, this includes modification of the same label, e.g.", "a cyanine with different substitutions that shifts the emission wavelength, or different fluorophores, e.g.", "a cyanine and a coumarin on the same labeling reagent.", "The difference in physical properties also includes the use of tandem dyes, which is specifically defined to include an energy transfer pair wherein one is a protein and the other is a fluorophore or both are fluorophores, or the pairing of other labels that are not necessarily energy transfer pairs.", "A few examples of labeling reagent subsets includes, but are not limited to, a first subset comprising a single fluorophore at a known ration attached to a anti-Fc Fab fragment; a second subset comprises the same fluorophore on the Fab fragment at a different known ration from the first subset, a third subset comprises the same fluorophore but that has a shifted wavelength due to a substitution on the fluorophore.", "Thus, the attachment of labels to the labeling reagents results in an extensive selection of subsets that when complexed with a target-binding antibody results in a unique method to detect one or multiple targets in a sample whereby the target is identified and quantitated.", "B. Immuno-Labeled Complex The subsets of labeling reagent are complexed with target-binding antibodies to produce subsets of immuno-labeled complex that for the target detection solution.", "The methods for forming the immuno-labeled complex comprises the following steps: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent; c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof; and, d) optionally repeating said steps a), b), and c) to form individual or pooled subsets of immuno-labeling complexes wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody.", "A particular advantage for the use of labeling reagent of the present invention to label target-binding antibodies is that the process is relatively insensitive to the solution the antibodies are in.", "Due to the physical nature of the labeling reagents, small monovalent fragments, the reagents do not cross-link and fall out of solution in the presence of high concentration of proteins.", "For this reason, target-binding antibodies can be complexed when present in ascites fluid, tissue culture supernatant, serum or other solutions where there is a high concentration of proteins.", "This eliminates the need to purify target-binding proteins prior to labeling.", "When preparing the immuno-labeled complex using purified target-binding antibody, stock solutions of both the labeling reagent and the target-binding antibody are typically near 1 mg/mL in an appropriate buffer, although more or less concentrated solutions are also suitable.", "Generally, the labeling reagent is mixed in a molar ratio of at least one to 50 moles of labeling reagent to one mole of the target-binding antibody to be complexed.", "More commonly a ratio of at least one to as many as 10 moles of labeling reagent per mole of target-binding antibody is combined.", "With an anti-Fc region Fab to a target-binding antibody, a molar ratio of approximately 2 to 10 is typical, more typically 3 to 5 (particularly for complexes in which the labeling reagent has been labeled while immobilized on an affinity matrix).", "The ease of formation of the complex permits rapid optimization of the complex and assessment of the effect of variation in experimental parameters.", "A particularly unique advantage of the invention is that the stoichiometry of the complex is easily adjusted to provide complexes with different ratios of labeling reagent to target-binding antibody, and thus there is control over the ultimate detectability of the target in the sample.", "Complexes that have been labeled with the same dye but at different molar ratios can be separately detected by the differences in their intensities.", "Complex formation appears to occur almost within the mixing time of the solutions (<1 minute) but the reaction typically is allowed to proceed for at least 5 minutes and can be longer before combining the immuno-labeled complex with the sample.", "Although complex formation can be reversed by addition of an unlabeled antibody that contains the same binding region, reversibility is very slow; furthermore, following binding of the immuno-labeled complex to a target in a sample, the sample can be “fixed” using aldehyde-based fixatives by methods that are commonly practiced by those skilled in the art of immunolabeling.", "The labeling process optionally further comprises the addition of a capture component to remove excess labeling reagent.", "For applications in which immunolabeling complexes of multiple primary antibodies from the same species (e.g.", "mouse monoclonal antibodies) or cross-reacting species (e.g.", "mouse and human antibodies) are to be used simultaneously or sequentially, it is necessary to quench or otherwise remove any excess labeling reagent by use of a capture component or by other means to avoid inappropriate labeling of the sample.", "The most effective capturing components to capture excess labeling reagent are those that contain the binding site of the labeling reagent but are themselves not labeled, preferably an antibody or antibody fragment.", "Capture components may be free in solution or immobilized on a matrix, such as agarose, cellulose, or a natural or synthetic polymer, to facilitate separation of the excess capture component from the immuno-labeled complex.", "The capture component is optionally attached to a microsphere or magnetic particle.", "However, separation of excess labeling reagent is not essential for successful utilization of the invention, particularly when using a single target-binding antibody.", "The steps of the labeling process for the target-binding antibodies can be repeated to form discrete immuno-labeled complex subsets that can be used individually or pooled in an assay to detect individual or multiple targets.", "As used herein the term immuno-labeled complex subsets refers to subsets that are distinguished from each other i) a ratio of label to labeling reagent, or ii) a physical property of the label, or iii) a ratio of labeling reagent to the target-binding antibody, or iv) by the target-binding antibody, or a combination thereof.", "For example a panel of subsets may comprise a target-binding antibody that is bound by a labeling reagent comprising a subset of different ratios of the same label on the labeling reagent resulting in a discrete subset of immuno-labeled complexes.", "This subset of immuno-labeled complexes can be used individually wherein a target is identified by the intensity of the detectable label or used in combination with another subset of immunocomplexes that differ in the target-binding antibody to identify multiple targets.", "C. Methods of Use The labeling reagents, target-binding antibodies and resulting immuno-labeled complex that forms the target detection solution can be used in a wide range of immunoassays, essentially in any assay a traditional secondary antibody is used including some assays that secondary antibodies are not used because of their size and ability to cross-link.", "Examples of such assays used to detect a target in a sample include immunoblots, direct detection in a gel, flow cytometry, immunohistochemistry, confocal microscopy, fluorometry, ELISA and other modified immunoassays.", "A method of the present invention for detecting a single target in a sample comprises the following steps: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent; c) contacting said sample with said immuno-labeled complex of step b); d) incubating said sample of step c) for a time sufficient to allow said immuno-labeled complex to selectively bind to said target; and, e) illuminating said immuno-labeled complex whereby said target is detected.", "A sample is incubated with a preformed immuno-labeled complex that comprises a labeling reagent and a target-binding antibody.", "While this method describes the identification of a single target, subsets of labeling reagents bound to the same target-binding antibody can be used to identify and provide additional information about such targets.", "For example, subsets of labeling reagent can be prepared wherein two discrete subsets are generate each with a distinct fluorophore label that is distinguished by their emission spectra, e.g.", "one that emits in the green spectra and one that emits in the red spectra.", "The labeling reagent subsets are then added to a solution of target-binding antibody in a controlled ratio, e.g.", "two parts one labeling reagent (green emission) and one part the other labeling reagent (red emission) per target binding antibody.", "In this way the immuno-labeled complexes can be used to detect a target.", "If another immuno-labeled complex were added to the sample the original target could be distinguished from the subsequently detected target.", "The methods of the present invention also provide for the detection of multiple targets in a sample.", "Multiple targets include the discrete epitope that the target-binding antibody has affinity for as well as molecules or structures that the epitiope is bound to.", "Thus, multiple target identification includes phenotyping of cells based on the concentration of the same cell surface marker on different cells.", "In this way multiple target identification is not limited to the discrete epitope that the target binding antibody binds, although this is clearly a way that multiple targets can be identified, i.e.", "based on the affinity of the target-binding antibody.", "Therefore, a method for detecting multiple targets in a sample comprises the following steps: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target-binding antibody is selectively bound by labeling reagent, wherein steps a) and b) are repeated to form discrete immuno-labeling complex subsets; c) contacting said sample with a solution comprising A) a pooled subset of immuno-labeled complexes, wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual subset wherein step c) with a solution comprising an individual subset is repeated; d) incubating said sample of step c) for a time sufficient to allow said immuno-labeled complex to selectively bind to said target; and, e) illuminating said immuno-labeled complex whereby said target is detected.", "A selected target-binding antibody and a subset of labeling reagent are incubated to form an immuno-labeled complex subset.", "This procedure is repeated to form a panel of immuno-labeled complex subsets that may be pooled and added to a sample.", "Altematively each immuno-labeled complex subset is added stepwise to a sample.", "The immuno-labeled complex subsets are distinguished by four characteristics resulting in an infinite number of immuno-labeled complex subsets.", "First (i) the subsets can be distinguished by the target-binding antibody that is determined by the end user for the information that is desired from a sample.", "This means that each subset is distinguished based on the affinity of the target-binding antibody.", "The target-binding antibody typically distinguishes immuno-labeled complexes when multiple targets are identified, however this is normally combined with another characteristic to gain information form a sample or increase the number of targets that can be detected at one time.", "The second (ii) distinguishing feature used is the ratio of label to labeling reagent, as discussed in detail above.", "A subset based on this feature would have for example a ratio of two fluorophore per each labeling reagent.", "The third (iii) distinguishing feature is the ratio of labeling reagent to target-binding antibody.", "This is accomplished using a controlled concentration of target-binding antibody mixed with a controlled concentration of a labeling reagent subset and the subset would comprise a target-binding antibody that is bound by a discrete number of labeling proteins.", "The fourth (iv) feature is the physical feature of the label.", "Typically this refers to the physical properties of the fluorophore labels wherein a subset of this group is distinguished by the label itself such as a green emitting fluorophore compared to a red emitting fluorophore.", "One of skill in the art will appreciate that while immuno-labeling complex subsets can be distinguished based on one feature the subsets are typically, and most useful, when discretely identified based on a combination of the distinguishing characteristics.", "Another example of detection of multiple targets utilizes the following immuno-labeled subsets, all of which comprise a different target-binding antibody but differ in the label and ratio of label.", "The first subset comprises a fluorophore label that emits red-fluorescent light, a second subset comprises a fluorophore label that emits green fluorescent light, a third subset comprises a ratio of 1:1 red to green fluorophore label; a fourth subset comprises a ratio of 2:1 red to green fluorophore label and a fifth subset comprises a ratio of 1:2 red to green fluorophore label.", "These subsets allow for the simultaneous detection of five targets in a sample.", "This aspect of the present invention is particularly important due to the limited range of fluorophores available wherein the labeling reagents can be utilized to increase the number of targets that can be detected at one time.", "One of skill in the art can appreciate that these subsets could be expanded by altering the ratio of label to labeling reagent instead of just the ratio of labeling reagent to target-binding antibody.", "This same methodology can also be applied to a single fluorophore label wherein the ratios are altered and a target is detected based on the intensity of the signal instead of the color and the ratio of the color to another color.", "Following the formation of the immuno-labeled complex subsets the subsets can be pooled and added to a sample or added stepwise to a sample, either of which is determined by the end user and the particular assay format.", "This method of the present invention provides for maximum flexibility and ease of determining multiple targets in a sample.", "Another method of the present invention provides for the determination of multiple targets in a sample specifically using the flow cytometry assay format.", "Traditionally targets identified using flow cytometry used either directly labeled primary antibody or labeled microspheres that were covalently attached to a primary antibody wherein the microsphere is the label.", "Examples include the fluorescent encapsulated microsphere beads sold by Luminex.", "The labeling reagents and the present invention overcome both the need for directly labeled primary antibody and the need for expensive microspheres.", "Thus, a method of the present invention for determining identity and quantity of targets in a sample by detecting multiple targets comprises the following steps: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent, wherein steps a) and b) are repeated to form a pooled subset of immuno-labeling complexes; c) contacting a population of cells in a sample with a solution comprising a pooled subset of immuno-labeled complexes, wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody; d) incubating said cells for a time period sufficient to allow said immuno-labeled complex to bind said targets; e) passing said incubated population of cells through an examination zone; and, f) collecting data from said cells that were passed through said examination zone wherein said multiple targets are detected whereby the identity and quantity of said targets is determined.", "In one aspect, a target-binding antibody is pre-complexed to the target-binding antibody to form a subset and that subset or a panel of subsets are added to a sample, that are typically distinguished by the target binding antibody.", "This method then avoids the need for a directly labeled primary.", "Secondly, when the panel of subsets is distinguished, for example, by the ratio of label to labeling reagent or the ratio of labeling reagent to target-binding antibody the immuno-labeled complex can function similar to the microsphere beads of Luminex.", "For example, this is accomplished wherein three immuno-labeled complex subsets are distinguished by the target binding antibody and the fluorophore attached to the labeling reagent and within one of the subsets is another set of subsets that are distinguished based on the ratio of label to labeling reagent.", "In this way three different epitopes are detected and one of the epitopes is further distinguished and a phenotype distinction made based on the intensity of the signal generated from the labeled-immuno complex subsets based on the ratio of fluorophore to labeling reagent.", "This determination of targets is facilitated when a population of cells or cellular organelles is passed through the examination zone of a flow cytometer wherein the fluorescent signal and intensity is recorded for each cell resulting in a histogram of the cell population or cellular organelles based on the detected epitopes.", "In another aspect of the invention, additional detection reagents are combined with the sample concurrently with or following the addition of immuno-labeled complex subsets.", "Such additional detection reagents include, but are not limited to reagents that selectively detect cells or subcellular components, ions, or indicate the cell viability, life cycle, or proliferation state.", "For example, the additional detection reagent is a labeled target-binding antibody that is directly or indirectly detectable and another additional detection reagent is a stain for nucleic acids, for F-actin, or for a cellular organelle.", "1.Sample Preparation The sample is defined to include any material that may contain a target to which an antibody has affinity for.", "Typically the sample is biological in origin and comprises tissue, cell or a population of cells, cell extracts, cell homogenates, purified or reconstituted proteins, recombinant proteins, bodily and other biological fluids, viruses or viral particles, prions, subcellular components, or synthesized proteins.", "Possible sources of cellular material used to prepare the sample of the invention include without limitation plants, animals, fungi, bacteria, archae, or cell lines derived from such organisms.", "The sample can be a biological fluid such as whole blood, plasma, serum, nasal secretions, sputum, saliva, urine, sweat, transdermal exudates, cerebrospinal fluid, or the like.", "Alternatively, the sample may be whole organs, tissue or cells from an animal.", "Examples of sources of such samples include muscle, eye, skin, gonads, lymph nodes, heart, brain, lung, liver, kidney, spleen, solid tumors, macrophages, mesothelium, and the like.", "Prior to combination with the immuno-labeled complexes, the sample is prepared in a way that makes the target, which is determined by the end user, in the sample accessible to the immuno-labeled complexes.", "Typically, the samples used in the invention are comprised of tissue, cells, cell extracts, cell homogenates, purified or reconstituted proteins, recombinant proteins, biological fluids, or synthesized proteins.", "Large macromolecules such as immuno-labeled complexes tend to be impermeant to membranes of live biological cells.", "Treatments that permeabilize the plasma membrane, such as electroporation, shock treatments, or high extracellular ATP, can be used to introduce the immuno-labeled complexes into cells.", "Alternatively, the immuno-labeled complexes can be physically inserted into cells, e.g.", "by pressure microinjection, scrape loading, patch-clamp methods, or phagocytosis.", "However, the desired target may require purification or separation prior to addition of the immuno-labeled complexes, which will depend on the way the antigenic determinants are contained in the sample.", "For example, when the sample is to be separated on a SDS-polyacrylamide gel the sample is first equilibrated in an appropriate buffer, such as a SDS-sample buffer containing Tris, glycerol, DTT, SDS, and bromophenol blue.", "When the sample contains purified target materials, the purified target materials may still be mixtures of different materials.", "For example, purified protein or nucleic acid mixtures may contain several different proteins or nucleic acids.", "Alternatively, the purified target materials may be electrophoresed on gels such as agarose or polyacrylamide gels to provide individual species of target materials that may be subsequently blotted onto a polymeric membrane or detected within the gel matrix.", "Preparation of a sample containing purified nucleic acids or proteins generally includes denaturation and neutralization.", "DNA may be denatured by incubation with base (such as sodium hydroxide) or heat.", "RNA is also denatured by heating (for dot blots) or by electrophoresing in the presence of denaturants such as urea, glyoxal, or formaldehyde, rather than through exposure to base (for Northern blots).", "Proteins are denatured by heating in combination with incubation or electrophoresis in the presence of detergents such as sodium dodecyl sulfate.", "The nucleic acids are then neutralized by the addition of an acid (e.g., hydrochloric acid), chilling, or addition of buffer (e.g., Tris, phosphate or citrate buffer), as appropriate.", "Preferably, the preparation of a sample containing purified target materials further comprises immobilization of the target materials on a solid or semi-solid support.", "Purified nucleic acids are generally spotted onto filter membranes such as nitrocellulose filters or nylon membranes in the presence of appropriate salts (such as sodium chloride or ammonium acetate) for DNA spot blots.", "Alternatively, the purified nucleic acids are transferred to nitrocellulose filters by capillary blotting or electroblotting under appropriate buffer conditions (for Northern or Southern blots).", "To permanently bind nucleic acids to the filter membranes, standard cross-linking techniques are used (for example, nitrocellulose filters are baked at 80° C. in vacuum; nylon membranes are subjected to illumination with 360 nm light).", "The filter membranes are then incubated with solutions designed to prevent nonspecific binding of the nucleic acid probe (such as BSA, casein hydrolysate, single-stranded nucleic acids from a species not related to the probe, etc.)", "and hybridized to probes in a similar solution.", "Purified proteins are generally spotted onto nitrocellulose or nylon filter membranes after heat and/or detergent denaturation.", "Alternatively, the purified proteins are transferred to filter membranes by capillary blotting or electroblotting under appropriate buffer conditions (for Western blots).", "Nonspecifically bound probe is washed from the filters with a solution such as saline-citrate or phosphate buffer.", "Filters are again blocked, to prevent nonspecific adherence of immuno-labeled complexes.", "Finally, samples are mixed with immuno-labeled complexes.", "Nonspecifically bound immuno-labeled complexes are typically removed by washing.", "When the sample contains cellular nucleic acids (such as chromosomal or plasmid-borne genes within cells, RNA or DNA viruses or mycoplasma infecting cells, or intracellular RNA) or proteins, preparation of the sample involves lysing or permeabilizing the cell, in addition to the denaturation and neutralization already described.", "Cells are lysed by exposure to agents such as detergent (for example sodium dodecyl sulfate, Tween, sarkosyl, or Triton), lysozyme, base (for example sodium, lithium, or potassium hydroxide), chloroform, or heat.", "Cells are permeabilized by conventional methods, such as by formaldehyde in buffer.", "As with samples containing purified target materials, preparation of the sample containing cellular target materials typically further comprises immobilization of the target materials on a surface such as a solid or semi-solid matrix.", "The targets may be arrayed on the support in a regular pattern or randomly.", "These supports include such materials as slides, polymeric beads including latex, optical fibers, and membranes.", "The beads are preferably fluorescent or nonfluorescent polystyrene, the slides and optical fibers are preferably glass or plastic, and the membrane is preferably poly(vinylidene difluoride) or nitrocellulose.", "Thus, for example, when the sample contains lysed cells, cells in suspension are spotted onto or filtered through nitrocellulose or nylon membranes, or colonies of cells are grown directly on membranes that are in contact with appropriate growth media, and the cellular components, such as proteins and nucleic acids, are permanently bound to filters as described above.", "Permeabilized cells are typically fixed on microscope slides with known techniques used for in situ hybridization and hybridization to chromosome “squashes” and “spreads,” (e.g., with a reagent such as formaldehyde in a buffered solution).", "Alternatively, the samples used may be in a gel or solution.", "In a particular aspect of the invention, the sample comprises of cells in a fluid, such as ascites, hybridoma supernatant, or serum, wherein the presence or absence of the target in such cells is detected by using an automated instrument that sorts cells according to the detectable fluorescence response of the detectable moieties in the immunolabeling complexes bound to such cells, such as by fluorescence activated cell sorting (FACS).", "For methods using flow cytometry a cell population typically comprises individually isolated cells that have been isolated from other proteins and connective tissue by means well known in the art.", "For example, lymphocyte cells are isolated from blood using centrifugation and a density gradient.", "The cells are washed and pelleted and the labeling solution added to the pelleted cells.", "2.Illumination At any time after addition of the immuno-labeled complex to the sample, the sample is illuminated with a wavelength of light selected to give a detectable optical response, and observed with a means for detecting the optical response.", "Equipment that is useful for illuminating the fluorescent compounds of the present invention includes, but is not limited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers and laser diodes.", "These illumination sources are optically integrated into laser scanners, fluorescent microplate readers or standard or microfluorometers.", "The degree and/or location of signal, compared with a standard or expected response, indicates whether and to what degree the sample possesses a given characteristic, i.e.", "desired target.", "The optical response is optionally detected by visual inspection, or by use of any of the following devices: CCD camera, video camera, photographic film, laser-scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or by means for amplifying the signal such as photomultiplier tubes.", "Where the sample is examined using a flow cytometer, examination of the sample optionally includes sorting portions of the sample according to their fluorescence response.", "When an indirectly detectable label is used then the step of illuminating typically includes the addition of a reagent that facilitates a detectable signal such as colorimetric enzyme substrate.", "Radioisotopes are also considered indirectly detectable wherein an additional reagent is not required but instead the radioisotope must be exposed to X-ray film or some other mechanism for recording and measuring the radioisotope signal.", "This can also be true for some chemiluminescent signals that are best observed after expose to film.", "III.", "Kits of the Invention Suitable kits for preparing an immuno-labeled complex and for detection of a target in a sample also form part of the invention.", "Such kits can be prepared from readily available materials and reagents and can come in a variety of embodiments.", "The contents of the kit will depend on the design of the assay protocol or reagent for detection or measurement.", "Generally, the kits will contain instructions, appropriate reagents and labels, and solid supports, as needed.", "Typically, instructions include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like to allow the user to carry out any one of the methods or preparations described above.", "A preferred kit of the present invention comprises: a) a labeling solution comprising a labeling reagent that is independently attached to one or more labels and b) a solution comprising a capture reagent.", "A preferred emodiment of this kit provides a labeling reagent that is anti-Fc Fab fragment, protein G or protein G complexed with albumin.", "In a more particular embodiment of this kit, the capture component is purified mouse IgG or non-immune mouse serum and the albumin is human albumin, bovine serum albumin, or ovalbumin.", "In a more preferred embodiment the albumin is ovalbumin.", "The labeling solution is either a homogenous mixture of labeling reagents or comprises a pooled subset of labeling reagents.", "Alternatively the kit comprises a panel of labeling reagent subsets that can be used to make a subset of immuno-labeled complexes.", "Additionally the kits may comprise one or more additional components that include (a) stains for characterization of cellular organelles, cell viability, or cell proliferation state, (b) enzyme substrates or (c) enzyme conjugates such as avidin-HRP.", "A wide variety of kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user.", "It is understood by one skilled in the art, that any of the labeling reagents contemplated by the present invention can be used to in a labeling solution to be included in a kit.", "The labeling reagents are not intended to be limited to only the described preferred embodiments.", "IV.", "Applications The instant invention has useful applications in basic research, high-throughput screening, immunohistochemistry, fluorescence in situ hybridization (FISH), microarray technology, flow cytometry, diagnostics, and medical therapeutics.", "The invention can be used in a variety of assay formats for diagnostic applications in the disciplines of microbiology, immunology, hematology and blood transfusion, tissue pathology, forensic pathology, and veterinary pathology.", "The invention is particularly useful in the characterization and selection of optimized antibodies from hybridoma supernatants.", "Additionally, the invention can be used to deliver therapeutics to a specific target.", "In general, the current invention provides a versatile and convenient method to enhance any assay that uses an antibody as part of its detection methodology.", "The instant invention can be used to study biological phenomena, such as, for example, cell proliferation, signal transduction in cells, or apoptosis.", "For illustration purposes only and not limitation, one could study thymidine analog 5-bromo-2′-deoxyuridine (BrdU) incorporation.", "BrdU is a marker for both cell proliferation and apoptosis, as it is readily incorporated into newly synthesized DNA that has progressed through the S-phase of the cell cycle and also into DNA break sites by deoxynucleotidyl transferase (TdT).", "Anti-BrdU antibodies are used to detect cells marked by BrdU incorporation.", "By being able to directly label the anti-BrdU antibodies, the current invention provides a convenient method to allow for detection of the incorporated BrdU by conventional immunohistochemistry or fluorescence, depending on detection method required.", "Additionally, the current invention has the advantage of allowing staining for multiple targets in one cocktail, thereby reducing the need for more samples or processing steps per experiment.", "This is particularly important when analyzing precious samples (e.g., pediatric samples, leukocytes isolated from biopsies, rare antigen-specific lymphocytes and mouse tissues that yield a small number of cells).", "Although it is currently possible to simultaneously measure up to 11 distinct fluorescent colors through a convoluted series of novel developments in flow cytometry hardware, software, and dye chemistry, the use of these advances has been severely limited by the lack of commercial availability of spectrally distinct directly labeled primary and secondary antibodies.", "Although labeled secondary antibodies directed at individual isotype-specific targeting antibodies (e.g., anti-lgG, isotype antibodies) exist, it is not possible to use this type of labeled antibody to detect more than one of the same isotype of an antibody (e.g., an IgG1 isotype antibody) in a single sample due to cross-reactivity.", "The current invention overcomes these limitations by providing for a convenient and extremely versatile method of rapidly labeling either small or large quantities of any primary antibody including primary antibodies of the same isotype to be used in, for example, multicolor flow cytometry and on Western blots.", "This advance in multicolor systems has a number of advantages over current two- and three-color flow cytometric measurements.", "For example, no combination of one-color stains can accurately enumerate or be used to isolate CD3+ CD4+ CD8− T cells (excluding, for example CD3+ CD4+ CD8+ T cells and small CD4+ monocytes).", "The use of cell membrane markers to study leukocyte composition in blood and tissue serves as an example of an analytical monoclonal antibody application, particularly in combination with flow cytometry.", "It is also the example most relevant to studies of the immune system, because the cellular composition of blood and lymphoid tissue provides a ‘window’, allowing the analysis and monitoring of the immune system.", "The methods of the invention can also be used in immunofluorescence histochemistry.", "This technique involves the use of antibodies labeled with fluorophores to detect substances within a specimen.", "The pathologist derives a great deal of information of diagnostic value by examining thin sections of tissue in the microscope.", "Tissue pathology is particularly relevant to, for example, the early diagnosis of cancer or premalignant states, and to the assessment of immunologically mediated disorders, including inflammation and transplant rejection.", "The problems associated with immunofluorescence histochemistry, however, stem from the limitations of the methods currently available for use in such application.", "For example, directly labeling an antibody can result in antibody inactivation and requires a relatively large of amount of antibody and time to do the conjugation.", "It is also expensive and impractical to prepare directly labeled antibodies having variable degrees of label substitution.", "Similarly, indirect labeling of an antibody has problems, such as lack of secondary antibody specificity, and reliance upon primary antibody differences, including antibody isotypes and available fluorophores, to do multicolor labeling.", "Secondary antibody labeling is not practical where the primary antibody is from the same species or of the same isotypes.", "Combinations of fluorophores or other detectable labels on the same target-binding antibody, which can be readily prepared in multiple mixtures by the methods on this invention, greatly increase the number of distinguishable signals in multicolor protocols.", "Lack of secondary antibody specificity arises when the specimen containing the targeted moiety and target-binding antibody are from homologous species.", "For example, BrdU-labeled DNA in rodent tissue is detected by immunohistochemical staining.", "The target-binding antibody is conventionally mouse anti-BrdU, and the detecting antibody system uses an anti-mouse immunoglobulin antibody, labeled with fluorescein.", "Because there is homology between mouse immunoglobulin and immunoglobulins from a number of rodent species (for example, rats, mice, hamsters, etc.", "), the detecting antibody not only binds to the target-binding antibody, but also nonspecifically binds to immunoglobulin in the tissue.", "The current invention eliminates this problem by pre-forming the immunolabeling complex and allows for a simple, rapid and convenient method to proceed with labeling with two, three or more fluorescent antibodies in one experiment.", "Very significantly, it can always be used with primary antibodies of either the same or different isotype, and always on tissue of the same or similar species as the primary antibody.", "The instant invention also has application in the field of microarrays.", "Microarray technology is a powerful platform for biological exploration (Schena (Ed.", "), Microarray Biochip Technology, (2000)).", "Many current applications of arrays, also known as “biochips,” can be used in functional genomics as scientists seek characteristic patterns of gene expression in different physiopathological states or tissues.", "A common method used in gene and protein microarray technology involves the use of biotin, digoxigenin (DIG), or dinitrophenyl (DNP) as an epitope or a “tag” such as an oligohistidine, glutathione transferase, hemagglutinin (HA), or c-myc.", "In this case a detectably labeled anti-biotin, anti-DIG, anti-DNP, anti-oligohistidine, anti-glutathione transferase, anti-HA, or anti-c-myc is used as the detection reagent.", "The instant invention allows for the use of multiple fluorophore- or enzyme-labeled antibodies, thereby greatly expanding the detection modalities and also providing for enhanced multiplexing and two-dimensional analysis capabilities.", "Similarly, the invention can be used with protein microarrays and on Western blots.", "Protein microarrays can provide a practical means to characterize patterns of variation in hundreds of thousands of different proteins in clinical or research applications.", "Antibody arrays have been successfully employed that used a set of 115 antibody/antigen pairs for detection and quantitation of multiple proteins in complex mixtures (Haab et al., Genome Biology, 2, 4.1 (2001)).", "However, protein microarrays use very low sample volumes, which historically have significantly limited the use of antibody technology for this application.", "The invention of the application readily overcomes this limitation and provides a means to label antibodies with the fluorescent dyes using a very low sample volume and to automate formation of the staining complex and the staining process.", "The present invention also provides a means for the specific detection, monitoring, and/or treatment of disease and contemplates the use of immunolabeling complexes to detect the presence of particular targets in vitro.", "In such immunoassays, the sample may be utilized in liquid phase, in a gel, or bound to a solid-phase carrier, such as an array of fluorophore-labeled microspheres (e.g., U.S. Pat.", "No.", "5,981,180 and 5,736,330).", "For example, a sample can be attached to a polymer, such as aminodextran, in order to link the sample to an insoluble support such as a polymer-coated bead, plate, or tube.", "For instance, but not as a limitation, using the methods of the present invention in an in vitro assay, antibodies that specifically recognize an antigen of a particular disease are used to determine the presence and amounts of this antigen.", "Likewise, the immunolabeling complexes of the present invention can be used to detect the presence of a particular target in tissue sections prepared from a histological specimen.", "Preferably, the tissue to be assayed will be obtained by surgical procedures, e.g., biopsy.", "The excised tissue will be assayed by procedures generally known in the art, e.g.", "immunohistochemistry, for the presence of a desired target that is recognized by an immunolabeling complex, as described above.", "The tissue may be fixed or frozen to permit histological sectioning.", "The immunolabeling complex may be labeled, for example with a dye or fluorescent label, chemical, heavy metal or radioactive marker to permit the detection and localization of the target-binding antibody in the assayed tissue.", "In situ detection can be accomplished by applying a detectable immunolabeling complex to the tissue sections.", "In situ detection can be used to determine the presence of a particular target and to determine the distribution of the target in the examined tissue.", "General techniques of in situ detection are well known to those of ordinary skill.", "See, for example, Ponder, “Cell Marking Techniques and Their Application,” in MAMMALIAN DEVELOPMENT: A PRACTICAL APPROACH, Monk (ed.", "), 115 (1987).", "For diagnosing and classifying disease types, tissues are probed with an immuno-labeled complex, as defined above, that comprises a target-binding antibody to a target antigen associated with the disease, e.g., by immunohistochemical methods.", "Where the disease antigen is present in body fluids, such immuno-labeled complexes comprising a target-binding antibody to the disease antigen are preferably used in immunoassays to detect a secreted disease antigen target.", "Detection can be by a variety of methods including, for example, but not limited to, flow cytometry and diagnostic imaging.", "When using flow cytometry for the detection method, the use of microspheres, beads, or other particles as solid supports for antigen-antibody reactions in order to detect antigens or antibodies in serum and other body fluids is particularly attractive.", "Flow cytometers have the capacity to detect particle size and light scattering differences and are highly sensitive fluorescence detectors.", "Microfluidic devices provide a means to perform flow-based analyses on very small samples.", "Alternatively, one can use diagnostic imaging.", "The method of diagnostic imaging with radiolabeled antibodies is well known.", "See, for example, Srivastava (ed.", "), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY, Plenum Press (1988); Chase, “Medical Applications of Radioisotopes,” in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al.", "(eds.)", "Mack Publishing Co., 624 (1990); and Brown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al.", "(eds.", "), Chapman & Hall, 227 (1993).", "This technique, also known as immunoscintigraphy, uses a gamma camera to detect the location of gamma-emitting radioisotopes conjugated to antibodies.", "Diagnostic imaging is used, in particular, to diagnose cardiovascular disease and infectious disease.", "Thus, the present invention contemplates the use of immuno-labeled complexes to diagnose cardiovascular disease.", "For example, immuno-labeled complexes comprising anti-myosin antibodies can be used for imaging myocardial necrosis associated with acute myocardial infarction.", "Immuno-labeled complexes comprising antibodies that bind platelets and fibrin can be used for imaging deep-vein thrombosis.", "Moreover, immuno-labeled complexes comprising antibodies that bind to activated platelets can be used for imaging atherosclerotic plaque.", "Immuno-labeled complexes of the present invention also can be used in the diagnosis of infectious diseases.", "For example, immuno-labeled complexes comprising antibodies that bind specific bacterial antigens can be used to localize abscesses.", "In addition, immuno-labeled complexes comprising antibodies that bind granulocytes and inflammatory leukocytes can be used to localize sites of bacterial infection.", "Similarly, the immuno-labeled complexes of the present invention can be used to detect signal transduction in cells, the products of signal transduction, and defects, inhibitors, and activators of signal transduction.", "Numerous studies have evaluated the use of antibodies for scintigraphic detection of cancer.", "Investigations have covered the major types of solid tumors such as melanoma, colorectal carcinoma, ovarian carcinoma, breast carcinoma, sarcoma, and lung carcinoma.", "Thus, the present invention contemplates the detection of cancer using immuno-labeled complexes comprising antibodies that bind tumor markers (targets) to detect cancer.", "Examples of such tumor markers include carcinoembryonic antigen, a-fetoprotein, oncogene products, tumor-associated cell surface antigens, and necrosis-associated intracellular antigens.", "In addition to diagnosis, antibody imaging can be used to monitor therapeutic responses, detect recurrences of a disease, and guide subsequent clinical decisions and surgical procedures.", "In vivo diagnostic imaging using fluorescent complexes that absorb and emit light in the near infrared (such as those of the Alexa Fluor 700 and Alexa Fluor 750 dyes) is also known.", "EXAMPLES The following examples describe specific aspects of the invention to illustrate the invention and to provide a description of the methods for those of skill in the art.", "The examples should not be construed as limiting the invention, as the examples merely provide specific methodology useful in understanding and practicing the invention.", "Example 1 Preparation of Fc Antigen Purified mouse and rabbit IgG was fragmented with the proteolytic enzyme papain (CURRENT PROTOCOLS IN CELL BIOLOGY, 16.4.1-16.4.10 (2000)).", "A 12 mL solution of mouse IgG was prepared at ˜2 mg/mL in phosphate-buffered saline (PBS).", "A solution containing 0.1 mg of papain in digestion buffer (PBS, 0.02 M EDTA, 0.02 M cysteine) was added to the antibody and allowed to react at 37° C. for 16 hours.", "The digestion was terminated by the addition 20 μL of 0.3 M iodoacetamide in PBS.", "The fragments were dialyzed against 2 L of PBS for 16 hours at 4° C. The Fc fragment was purified on a protein G-Sepharose CL-4B column.", "The bound fraction containing the Fc fragment was eluted from the column using 50-100 mM glycine/HCl buffer, pH 2.5-2.8.The eluate was collected in 1 mL fractions.", "The pH of the protein fractions was immediately raised to neutral by addition of 100 μL of either 500 mM phosphate or Tris buffer, pH 7.6, to each 1 mL fraction.", "The solution was then loaded onto a Sephacryl S-200 Superfine size-exclusion column and fractions corresponding to a molecular weight of -50 kDa were collected and analyzed by SDS-PAGE and HPLC.", "Example 2 Production of Anti-Fc Antibodies Polyclonal antibodies specific for the Fc region of an antibody were raised in goats against the purified FC region of an antibody from a different species (Example 1).", "Methods of immunizing animals are well known in the art, and suitable immunization protocols and immunogen concentrations can be readily determined by those skilled in the art (Current Protocols in Immunology 2.4.1-9 (1995); ILAR Journal 37, 93 (1995)).", "Briefly, individual goats were immunized with purified mouse Fc or purified rabbit Fc fragments.", "The initial immunization in 50% Freund's complete adjuvant (1000 μg conjugate (half subcutaneous, half intramuscularly)) was followed by 500 pg conjugate per goat in Freund's incomplete adjuvant two and four weeks later and at monthly intervals thereafter.", "Antibodies were purified from serum using protein A-Sepharose chromatography.", "Antibodies against mouse Fc isotypes can be prepared by starting with isotype-selected mouse Fc antigens.", "Rabbits have a single Fc isotype.", "Characterization of the selectivity and cross-reactivity of isotype-specific antibodies is by standard techniques, including HPLC.", "Example 3 Preparation of Fab Fragments Fragmentation of the goat anti-(mouse Fc) antibody to the monovalent Fab fragment was carried out using the proteolytic enzyme, papain, as described in Example 1.Following dialysis against PBS, the Fab fragment was purified on a protein A-Sepharose CL-4B column.", "The unbound fraction containing the Fab fragment and the papain was collected.", "This solution was then loaded onto a Sephacryl S-200 Superfine size-exclusion column and fractions corresponding to a molecular weight of ˜50 kDa were collected and analyzed by SDS-PAGE.", "The Fab fragments of goat anti-(rabbit Fc) can be prepared similarly.", "Example 4 Preparation of the Labeled Antibody Immunoglobulin-Binding Protein or the Non-Antibody Immunoglobulin-Binding Peptide and Protein Conjugates in Homogeneous Solution Conjugates of antibody immunoglobulin-binding protein or the non-antibody immunoglobulin-binding peptides or proteins with low molecular weight dyes and haptens such as biotin or digoxigenin are typically prepared from succinimidyl esters of the dye or hapten, although reactive dyes and haptens having other protein-reactive functional groups are also suitable.", "The typical method for protein conjugation with succinimidyl esters is as follows.", "Variations in molar ratios of dye-to-protein, protein concentration, time, temperature, buffer composition and other variables that are well known in the art are possible that still yield useful conjugates.", "A protein solution of the Fab fragment of goat anti-(rabbit Fc), goat anti-(mouse Fc), protein A, protein G, or protein L or an immunoglobulin-binding peptide (e.g., a peptide identified by screening a library of peptides) is prepared at ˜10 mg/mL in 0.1 M sodium bicarbonate (pH ˜8.3).", "The labeling reagents are dissolved in a suitable solvent such as DMF at ˜10 mg/mL.", "Predetermined amounts of the labeling reagents are added to the protein solution with stirring.", "A molar ratio of 10 moles of dye to 1 mole of protein is typical, though the optimal amount can be varied with the particular labeling reagent, the protein being labeled and the protein's concentration.", "The optimal ratio was determined empirically.", "When optimizing the fluorescence yield and determining the effect of degree of substitution (DOS) on the conjugate's brightness, it is typical to vary the ratio of reactive dye to protein over a several-fold range.", "The reaction mixture is incubated at room temperature for a period that is typically one hour or on ice for several hours.", "The dye-protein conjugate is typically separated from unreacted reagents by size-exclusion chromatography, such as on BIO-RAD P-30 resin equilibrated with PBS.", "The initial, protein-containing band is collected and the DOS is determined from the absorbance at the absorbance maximum of each fluorophore, using the extinction coefficient of the free fluorophore.", "The DOS of nonchromophoric labels, such as biotin, is determined as described in Haugland (Haugland et al., Meth.", "Mol.", "Biol.", "45, 205 (1995); Haugland, Meth.", "Mol.", "Biol.", "45, 223 (1995); Haugland, Meth.", "Mol.", "Biol.", "45, 235 (1995); Haugland, Current Protocols in Cell Biol.", "16.5.1-16.5.22 (2000)).", "Using the above procedures, conjugates of goat anti-(mouse Fc) and goat anti-(rabbit Fc) were prepared with several different Alexa Fluor dyes, with Oregon Green dyes, with biotin-X succinimidyl ester, with desthiobiotin-X succinimidyl ester, with succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and with succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC).", "Some dye conjugates of protein A and protein G, including those of some Alexa Fluor dyes, are commercially available, such as from Molecular Probes.", "Inc. (Eugene, Oreg.).", "The interspecies specificity and approximate affinity of some other non-antibody immunoglobulin-binding proteins bind to segments of a target antibody, such as that of protein A and protein G are known (Langone, Adv.", "Immunol.", "32,157 (1982); Surolia et al., Trends Biochem.", "Sci.", "7, 74 (1982); Notani et al., J. Histochem.", "Cytochem.", "27, 1438 (1979); Goding, J. Immunol.", "Meth.", "20, 241 (1978); J. Immunol.", "Meth.", "127, 215 (1990); Bjorck et al., J. Immunol.", "133, 969 (1984)).", "In addition, labeling proteins (goat Fab anti-(mouse Fc), goat Fab anti-(mouse lambda light chain), goat Fab anti-(mouse kappa light chain), protein A, protein G, protein L, lectins, single-chain fragment variable antibodies (ScFv) ) conjugated to the detectable labels of R-phycoerythrin (R-PE), allophycocyanin (APC), tandem conjugates of phycobiliproteins with chemical dyes including several Alexa Fluor dyes, horseradish peroxidase (HRP), Coprinus cinereus peroxidase, Arthromyces ramosus peroxidase, glucose oxidase and alkaline phosphatase (AP) were or can be prepared by standard means (Haugland et al., Meth.", "Mol.", "Biol.", "45, 205 (1995); Haugland, Meth.", "Mol.", "Biol.", "45, 223 (1995); Haugland, Meth.", "Mol.", "Biol.", "45, 235 (1995); Haugland, Current Protocols in Cell Biol 16.5.1-16.5.22 (2000)).", "Fusion proteins, such as of protein G or protein A with detectable labels such as luciferin, aequorin, green-fluorescent protein and alkaline phosphatase are also known that are suitable for practice of the invention (Sun et al., J. Immunol.", "Meth.", "152, 43 (1992); Eliasson et al., J. Biol.", "Chem.", "263, 4323 (1988); Eliasson et al., J. Immunol.", "142, 575 (1989)).", "Immunoglobulin heavy and light chains, like most secreted and membrane bound proteins, are synthesized on membrane-bound ribosomes in the rough endoplasmic endoplasmic reticulum where N-linked glycosylation occurs.", "The specificity of lectins for carbohydrates, including N-linked glycoproteins, is also known (EY laboratories, Inc. Lectin Conjugates Catalog, 1998).", "Example 5 Preparation of the Labeled Antibody Immunoglobulin-Binding Protein or the Non-Antibody Immunoglobulin-Binding Peptide and Protein Conjugates While Bound to an Affinity Matrix Unlabeled Fab fragment for goat anti-(mouse Fc) (prepared as in Example 3) was bound to agarose-immobilized mouse IgG for one hour.", "Following a wash step with bicarbonate buffer, pH 8.3, the complex of immobilized IgG and unlabeled Fab was labeled for one hour at room temperature with the succinimidyl ester of the amine-reactive label.", "Unconjugated dye was eluted with bicarbonate buffer, and then the covalently labeled Fab fragment was eluted with 50-100 mM glycine/HCl buffer, pH 2.5-2.8.The eluate was collected in 1 mL fractions.", "The pH of the protein fractions was immediately raised to neutral by addition of 100 μL of either 500 mM phosphate or Tris buffer, pH 7.6, to each 1 mL fraction.", "Variations of the reagent concentrations, labeling times, buffer composition, elution methods and other variables are possible that can yield equivalent results.", "Conjugates of the Fab fragment of goat anti-(rabbit Fc) and of protein G and protein A are prepared similarly.", "Example 6 Comparison of the Alexa Fluor 488 Dye-Labeled Fab Fragments of Goat Anti-(Mouse Fc) Prepared as in Example 4 and as in Example 5 Conjugates of the Fab fragment of goat anti-(mouse Fc) with the Alexa Fluor 488 succinimidyl ester were separately prepared, as described in Examples 4 and 5.The conjugates had estimated degrees of substitution of ˜1.9 (labeled as in Example 4) and ˜3.0 (labeled as in Example 5), respectively, and virtually identical absorption and emission spectral maxima.", "When excited at 488 nm, conjugates prepared using the fragment prepared as described in Example 5 were about 3.2-times more fluorescent than using the fragments that were prepared in Example 4 (FIG.", "8) as detected by flow cytometry when bound to CD3 on Jurkat T cells.", "Similar results were observed with other dyes.", "Example 7 Preparation of a Labeling Protein from Protein G and Albumins Native protein G has a high affinity binding (nanomolar) site for albumins, in particular ovalbumin.", "Equal weights of protein G and Texas Red ovalbumin (Molecular Probes.", "Inc.) were dissolved in PBS, pH 7.5.After one hour, the resulting complex was separated on a Sephacryl S-200 Superfine size-exclusion column and analyzed by SDS-PAGE and HPLC.", "Alternatively, the protein G is combined with a labeled albumin while the protein G is immobilized on any of the several immunoglobulins to which it binds, and the excess labeled albumin is washed away preceding elution of the albumin-labeled protein G complex from the matrix.", "Example 8 Preparation of an Immunolabeling Complex on a Very Small Scale Submicrogram quantities of a target-binding antibody were complexed with submicrograms of a labeling protein in varying molar ratios of between about 1:1 and 1:20 to prepare an immunolabeling complex that was suitable for staining a sample.", "For instance, 0.1 μg of mouse monoclonal anti-tubulin in 1 μL PBS with 0.1% BSA was complexed with 0.5 μg of the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) (prepared as in Example 4) or with 0.1 μg of the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) (prepared as in Example 5) in 5 μL of PBS for 10 minutes at room temperature.", "The immunolabeling complex can be used immediately for staining tubulin in fixed-cell preparations (Example 16) or any excess unbound Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) in the immunolabeling complex can be captured with non-immune mouse IgG (Example 9) for combination with other antibody conjugates, including those of targeting antibodies that have been directly conjugated to other labels.", "Rabbit antibodies were labeled similarly using labeled goat anti-(rabbit Fc).", "Labeling of targeting antibodies with a labeled protein A, protein L, protein G, protein G complexed with a labeled albumin, or other immunoglobulin-binding peptides or proteins proceeds similarly.", "In the case of a mouse (or rat) monoclonal antibody, it is preferred to use a labeled protein that is selective for the specific isotype of the primary antibody (e.g.", "anti-(mouse IgG,) for a mouse IgG1 isotype primary antibody).", "Although some cross-reactivity for other mouse (or rat) isotypes was observed using a goat antibody that was selective for mouse IgG, isotype monoclonal antibodies, routine and optimal use for labeling unmatched mouse isotypes required greater amounts of immunolabeling complexes and was somewhat less reliable.", "Example 9 Capturing Excess Immunoglobulin-Binding Protein by a Capturing Component Immunolabeling complexes were prepared as described in Example 8.To the immunolabeling complex was added to each tube 25 μL of a 14.1 mg/mL stock solution of unlabeled mouse IgG to capture excess immunolabeling complexes.", "As shown in FIG.", "1, not all of the immunoglobulin-binding protein was necessarily complexed with the target-binding antibody to form an immunolabeling complex.", "Consequently, particularly for applications in which labeling complexes of multiple primary antibodies from the same species (e.g.", "mouse monoclonal antibodies) or crossreacting species (e.g.", "mouse and human antibodies, FIG.", "2, Table 1) were to be used simultaneously or sequentially, it is necessary to quench or otherwise remove any excess immunoglobulin-binding protein by use of a capturing component or by other means to avoid inappropriate labeling of the sample.", "The most effective capturing component to capture excess immunoglobulin-binding protein is one that contains the binding site of the targeting agent.", "For instance, whole mouse IgG or mouse serum was shown to be an effective and inexpensive reagent when the immunoglobulin-binding protein was bound to a segment of a mouse monoclonal antibody.", "The mouse IgG was added in excess to the amount of immunoglobulin-binding protein and incubated for a period of approximately 1-5 minutes, or longer.", "It is preferred to prepare the immunolabeling complex and then add the capturing component shortly before the experiment.", "The rapid quenching effect permits this to be done within minutes of performing labeling of the sample by the immunolabeling complex.", "If desired, the excess capturing component can be removed following labeling of the sample by a simple wash step.", "Alternatively, fixation of the stained sample by aldehyde-based fixatives or other reagents or methods subsequent to incubation with the immunolabeling complex can provide permanent immobilization of the immunolabeling complex on its target in the sample.", "As an alternative to adding a soluble capturing component to the immunolabeling complex, the capturing component can be immobilized on an insoluble matrix such as agarose and the immunolabeling complex contacted with that matrix.", "A preferred matrix when labeling mouse antibodies to mouse antigens is mouse IgG immobilized on agarose.", "Excess labeled anti-rabbit antibodies can be captured using rabbit IgG that is free in solution or immobilized.", "Alternatively, the immunolabeling complex can be separated from any capturing component by chromatographic or electrophoretic means.", "Example 10 HPLC Analysis of a Labeling Complex In order to analyze the success and extent of complex formation of the labeling protein with the target-binding antibody, size exclusion HPLC of the samples was performed.", "For instance, a complex of Alexa Fluor 488 dye-labeled goat Fab anti-(mouse Fc) with a monoclonal mouse anti-tubulin in molar ratios of approximately 1:1, 3:1, 5:1 and 10:1.These were separated by analytical HPLC using a BioSep S-3000 column and eluting with 0.1 M NaPi, 0.1 M NaCl, pH 6.8, at a flow rate of 0.25 mLs/min.", "An example of the separation using the 5:1 molar ratio (FIG.", "6) demonstrates that, using this molar ratio, formation of the labeled complex is essentially quantitative.", "Example 11 Cross-Reactivity of Goat Fab Anti-(Mouse Fc) to Other Species of IgG Microplates were equilibrated overnight with IgG from a mouse or non-mouse species, and then further blocked with BSA.", "Variable amounts of the biotinylated Fab fragment of goat anti-(mouse Fc) were added to each well and allowed to bind.", "After washing, streptavidin-HRP and the Amplex Red peroxidase substrate were added.", "HRP activity was detected by the addition of H2O2 using the Amplex Red Peroxidase Assay Kit (Molecular Probes, Inc., Eugene, Oreg.).", "Reactions containing 200 μM Amplex Red reagent, 1 U/mL HRP and 1 mM H2O2 (3% solution) in 50 mM sodium phosphate buffer, pH 7.4, were incubated for 30 minutes at room temperature.", "Fluorescence was measured with a fluorescence microplate reader using excitation at 560±10 nm and fluorescence detection at 590±10 nm.", "Background fluorescence, determined for a no-H2O2 control reaction, was subtracted from each value (Table 1 and FIG.", "2).", "Table 1 shows that the goat anti-(mouse Fc) antibody because of the highly conserved structure of the Fc region of an antibody it can be used to complex other non-mouse antibodies, including rat, and human antibodies.", "The goat anti-mouse IgG antibody reaction with mouse antibody was set at 100% and the crossreacting antibodies were expressed as a percentage compared the mouse on mouse data.", "The data in Table 1 show that the Fab fragment of the goat anti-(mouse Fc) antibody of the current invention does not strongly bind to the goat or sheep Fc domain; however, one skilled in the art could generate antibodies that will react with the goat and sheep Fc domain or the Fc domain of any other species.", "Biotinylated Fab goat anti-(mouse Fc) was used in this example because it provided a convenient method to quantitate the amount of crossreactivity in a conventional method but it could have been accomplished using a fluorophore Fab labeled goat anti-(mouse Fc).", "It was demonstrated by HPLC (as in Example 10) that Alexa Fluor 488 dye-labeled goat anti-(rabbit Fc) bound to rabbit primary antibodies.", "TABLE 1 Cross-reactivity of goat anti-mouse IgG antibody with other non-mouse antibodies.", "Species Crossreactivity % Fluorescence Mouse ++++ 100 Rat +++ 80.7 Human ++ 66.7 Rabbit + 16.9 Goat − 6.5 Sheep − 5.7 Example 12 Determination of the Optimal Molar Ratio of Immunoglobulin-Binding Protein to Target Antibody Using a Microplate Assay To 1.6 μg of mouse monoclonal anti-biotin (MW ˜145,000) in 8.0 μL PBS was added varying amounts of the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) (MW ˜50,000) (prepared as in Example 4) to form an immunolabeling complex.", "After equilibration for 20 min, a 100 μL aliquot was added to a 96-well microplate coated with biotinylated BSA.", "After 30 minutes, the plates were washed and the residual fluorescence was quantitated using a fluorescence microplate reader using excitation at 485±10 nm and detecting emission at 530±12.5 nm.", "As shown in FIG.", "3, a molar ratio of the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) to the anti-biotin between 5 to 20 was sufficient to form appreciably detectable complexes (FIG.", "3; fluorescence quantitated, performed in triplicate (circles); control experiments performed but without adding the primary anti-biotin antibody (solid squares)).", "A molar ratio of about 5 to about 10 was preferred for this pair of immunoglobulin-binding protein and target antibody.", "This ratio can be varied somewhat to increase or decrease the signal or to affect the consumption of valuable reagents.", "The weight ratio of immunoglobulin-binding protein to target-binding antibody is particularly affected by the actual molecular weight of the immunoglobulin-binding protein.", "For instance, equal weights of the dye-labeled goat Fab anti-(mouse Fc) (prepared as in Example 5) and an intact mouse primary antibody, which corresponds to an approximately 3 to 1 molar ratio, usually yields suitable labeling complexes.", "Fluorescence intensity (or enzymatic activity) of the immunolabeling complex is readily adjusted by a corresponding adjustment of the amount of labeled Fab fragment used.", "Similar analyses of the ratio for other labeling proteins (including those of labeled protein A, protein G, protein L, IgG-binding peptides and antibodies to other segments of the primary antibody), and for conjugates of labels other than Alexa Fluor 488 dye (including enzymes in combination with the appropriate enzyme substrates) are done essentially as described in this example.", "Example 13 Dissociation Rate of the Immunolabeling Complex A pre-equilibrated immunolabeling complex was prepared from 50 μg of an Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) and 15 μg of an anti-biotin monoclonal antibody (mAb).", "The immunolabeling complex was rapidly diluted with capturing component sufficient to give a 6.2 molar excess over the anti-biotin mAb.", "At various times, an aliquot was taken and added to a microplate well containing an excess of biotinylated BSA.", "After 30 minutes, the plates were washed and the remaining fluorescence was quantitated.", "Displacement of the labeling protein from the target-binding antibody through exchange was measured by any time-dependent decrease in fluorescence in the microplate well.", "For example the fragments prepared as described in Example 4 had 68 percent fragments bound to the target-binding antibody after 30 minutes compared to 87 percent of bound fragments that were prepared according to Example 5.One hour showed a similar decrease, 56 percent and 68 percent respectively.", "The labeling protein was shown to undergo a stable interaction with the target-binding antibody, with a lifetime for half exchange under these conditions of 3.5 hours.", "Dissociation rates were measured for labeling protein prepared according to Example 4 and for labeling protein prepared according to Example 5, demonstrating the greater stability of immunolabeling complexes made using the labeling proteins prepared according to Example 5.Example 14 Protocol for Staining Cultured Cells with a Single Immunolabeling Complex Culturable cells, such as bovine pulmonary artery endothelial cells (BPAEC), were grown on a 22 x 22 mm glass coverslip.", "The cells were fixed for 10 minutes using 3.7% formaldehyde in DMEM with fetal calf serum (FCS) at 37° C. The fixed cells were washed 3 times with PBS.", "The cells were permeabilized for 10 min with 0.02% Triton X-100 in PBS, washed 3X with PBS and blocked for 30 min with 1% BSA in PBS.", "Variations of the cell type and cell preparation, fixation, and permeabilization methods, including methods for antigen retrieval, are well known to scientists familiar with the art.", "An immunolabeling complex was prepared as described in Example 8.The immunolabeling complex was added directly to the fixed and permeabilized cells in an amount sufficient to give a detectable signal if there is a binding site for the primary antibody present in the sample.", "After an incubation period that was typically 1060 minutes (usually about 15-30 minutes), the cells were washed with fresh medium and the labeling was evaluated by methods suitable for detection of the label.", "Staining by the immunolabeling complex can be additionally preceded, followed by or combined with staining by additional reagents, such as DAPI, which yields blue-fluorescent nuclei.", "Example 15 Protocol for Staining Cultured Cells with Multiple Immunolabeling Complexes Cells were fixed and permeabilized as described in Example 14.Multiple immunolabeling complexes were individually prepared from a variety of labeling proteins, according to the procedure described in Example 8.The multiple immunolabeling complexes were either used individually or sequentially to stain the cells, according to the procedure described in Example 14, or two or more immunolabeling complexes were formed then co-mixed in a single staining solution and used to simultaneously stain the sample.", "The optimal method for cell fixation and permeabilization and the best ratio for combination of the immunolabeling complexes are typically determined by preliminary experimentation using single immunolabeling complexes or multiple immunolabeling complexes used in combination.", "A first immunolabeling complex was prepared from an Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) and mouse monoclonal anti-α-tubulin, a second immunolabeling complex was prepared from an Alexa Fluor 568 dye-labeled Fab fragment of goat anti-(mouse Fc) and mouse monoclonal anti-vimentin (anti-vimentin was an ascites fluid preparation) and a third immunolabeling complex was prepared from an Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse Fc) and mouse monoclonal anti-cdc6 peptide antibody (Molecular Probes).", "Aliquots of the three different immunolabeling complexes were combined and used to stain BPAE cells for 30 minutes, washed with fresh medium and observed by fluorescence microscopy using optical filters appropriate for the three dyes.", "In this example, some cells showed cytoplasmic staining by the anti-vimentin antibody, nuclear staining by the anti-cdc6 peptide antibody and staining of mitotic spindles by the anti-α-tubulin antibody, indicative of a cell in mitosis.", "Staining by the immunolabeling complexes was additionally preceded, followed by or combined with staining by additional reagents, such as Alexa Fluor 350 phalloidin, which yielded blue-fluorescent actin filaments in the above example.", "The immunolabeling complexes that are used in combination do not have to be targeted toward antibodies from the same species.", "For instance, complexes of Alexa Fluor 488 dye-labeled goat anti-(mouse IgG, Fc) with a mouse IgG, monoclonal target-binding antibody and an Alexa Fluor 594 dye-labeled goat anti-(rabbit Fc) with a rabbit primary target-binding antibody can be prepared and used in combined staining protocols.", "Example 16 Protocol for Staining Tissue with a Single Immunolabeling Complex A mouse intestine cryosection (University of Oregon histology core facility), a cross-section of about 16 μm thickness, was mounted on a slide.", "The intestine was perfused and fixed with 4% formaldehyde prior to dissection, embedding, and sectioning.", "The tissue section was rehydrated for 20 minutes in PBS.", "An immunolabeling complex was prepared as described in Example 8.Briefly, 0.1 μg of mouse monoclonal anti-cdc6 peptide (a nuclear antigen) in 1 μL PBS with 0.1 % BSA was complexed with 0.5 μg of the Alexa Fluor 350 dye-labeled Fab fragment of goat anti-(mouse IgG1 Fc) (prepared as in Example 4) in 5 μL of PBS for 10 minutes at room temperature.", "Excess Fab fragment of goat anti-(mouse IgG, Fc) was captured with 25 μL of a 14.1 mg/mL stock of unlabeled mouse IgG.", "The tissue was permeabilized with 0.1% Triton X-100 for 10 min.", "The tissue was washed two times with PBS and was blocked in 1 % BSA for 30 min.", "The immunolabeling complex was added directly to the tissue for 30 minutes and washed three times in PBS.", "The sample was mounted in Molecular Probes' Prolong antifade mounting medium and observed by fluorescence microscopy using optical filters appropriate for the Alexa Fluor 350 dye.", "Results showed that the mouse monoclonal anti-cdc6 peptide immunolabeling complex showed specific nuclear labeling in the mouse intestine tissue section.", "Variations of the tissue type and tissue preparation, fixation and permeabilization methods, mounting methods, including methods for antigen retrieval, are well known to scientists familiar with the art.", "Example 17 Staining of a Tissue Target in Combination with Tyramide Signal Amplification (TSA) Mouse brain cryosections were labeled with a pre-formed complex of horseradish peroxidase (HRP)-labeled goat anti-(mouse IgG, Fc) antibody and a mouse IgG1 monoclonal anti-(glial fibrillary acidic protein (GFAP)) prepared essentially as in Example 8 using a molar ratio of labeling protein to monoclonal antibody of 3.Staining of the mouse tissues was essentially as in Example 16.The staining localization and intensity was compared to that of (a) goat anti-mouse IgG HRP conjugate and mouse anti-GFAP, (b) the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) antibody complex of mouse anti-GFAP, (c) Alexa Fluor 488 goat anti-mouse IgG secondary antibody and mouse anti-GFAP, and (d) a direct conjugate of the Alexa Fluor 488 dye with mouse anti-GFAP.", "The HRP-conjugated probes were incubated with Alexa Fluor 488 tyramide using TSA Kit #2 (Molecular Probes, Inc.) according to standard procedures.", "The tissue staining patterns in each case were similar and consistent with the expected staining pattern of mouse anti-GFAP and staining was essentially free of nonspecific background.", "The relative fluorescence intensities of staining measured by digital imaging were sequentially: 541 relative intensity units for the HRP-goat anti-(mouse IgG, Fc) complex of mouse anti-GFAP and (using the combinations indicated by the letters above): (a) 539, (b) 234, (c) 294, and (d) 255 relative intensity units.", "Example 18 Staining of Live Cells by Multiple Immunolabeling Complexes A first immunolabeling complex was prepared from an Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) and mouse monoclonal anti-(human CD8), a second immunolabeling complex was prepared from an R-phycoerythrin-conjugated Fab fragment of goat anti-(mouse IgG1 Fc) and mouse anti-(human CD3), and a third immunolabeling complex was prepared from an Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) and mouse anti-(human CD4).", "The complexes were prepared as described in Example 8 and were each blocked with 20 μg (1.3 μL of 14.1 μg/mL) of mouse IgG for 10 minutes at room temperature.", "The first immunolabeling complex was added to 100 μL of whole blood and incubated for 15 min.", "The cells were washed with PBS and 280.5 μL of the second immunolabeling complex was added and incubated for 15 min.", "The cells were again washed, and 46.2 μL of the third labeling complex was added and incubated for 15 min.", "After the final incubation, the red blood cells were lysed with cell-lysis buffer.", "The cells were resuspended in 1% formaldehyde/PBS and analyzed on a FACS Vantage flow cytometer using a 488 nm argon-ion laser for excitation of the first and second immunolabeling complexes and a 633 nm red He-Ne laser for excitation of the third immunolabeling complex (FIGS.", "5a, 5b).", "The emission band pass filters used for selective detection of the dyes are 525±10 nm for the Alexa Fluor 488 (CD8), 585±21 nm for R-PE (CD3) and 675±10 nm for the Alexa Fluor 647 dye (CD4).", "FIGS.", "5a and 5b show that the instant invention can be used in a 3-color immunophenotyping experiment using peripheral blood lymphocytes.", "CD3-positive T cells were stained with the R-phycoerythrin-conjugated Fab fragment of goat anti-(mouse Fc) and mouse anti-(human CD3), upper left (UL) quadrant, FIG.", "5a.", "CD4-positive cells, a T cell subset, are identified using Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG1 Fc) and mouse anti-(human CD4), UL quadrant, FIG.", "5b and CD8-positive T cells, a T cell subset, were identified using Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG1 Fc) and mouse monoclonal anti-(human CD8), lower right (LR) quadrant, FIG.", "5b.", "Exposed antigens of live cells, including cultured cells and cells from biological fluids such as blood and cerebrospinal fluid can be simultaneously or sequentially stained by combinations of immunolabeling complexes, including antibodies to the same target labeled with two or more separately detectable immunoglobulin-binding proteins.", "Example 19 The Dye-Labeled Fab Fragment of Goat Anti-(Mouse Fc) can be Utilized for the Combinatorial Labeling of Primary Antibodies, to Generate a Multitude of Colored Targets A first immuno-labeled complex was made by combining 2.5 μg Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) with 0.5 μg mouse anti-human CD3 (Caltag at 200 μg/mL), according to the procedure described in Example 4.A second immunolabeling complex was made by combining 5.0 μg Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) with 0.5 μg mouse anti-human CD3, according to the procedure in Example 4.Each complex was separately incubated at room temperature for 5 minutes, and each complex was then separately combined with an excess of mouse IgG (14.1 mg/mL) for 5 min at room temperature to capture excess unbound dye-labeled Fab fragments.", "The two immunolabeling complexes were then added in different percentage combinations (see Table 2) to 100 μL of washed heparinized blood.", "The cells were incubated with the respective combinations of complexes for 20 min on ice.", "The red blood cells were then lysed with a cell-lysis buffer.", "The cells were resuspended in 1 % formaldehyde/PBS and analyzed on a FacVantage flow cytometer using a 488 nm argon 633 HeNe laser for excitation and a 530 +/-10 nm band pass emission filter (FL1), and a 640 long pass filter (FL4).", "Five samples of different combined percentages (Table 2) were compared by flow cytometry, with signals being collected in FL1 and FL4.To determine the percentage of cells detected with each type of emission, the FL1 and FL4 intensities for each percentage combination were normalized by dividing the FL1 and FL4 channel intensities for such combinations by the intensities of the 100% Alexa Fluor 488 dye-and 100% Alexa Fluor 647 dye-labeled cells, respectively.", "TABLE 2 Theoretical versus recovered dye-labeled Fab fragment of goat anti- (mouse IgG1 Fc) combinatorial experiment.", "Recovered Experimentally Experimentally percentage of mixed Recovered mixed measured cells percentage percentage of percentage labeled with of cells measured cells of cells Alexa Fluor labeled with labeled with labeled with 647 dye- Alexa Fluor Alexa Fluor Alexa Fluor 647 labeled Fab 488 dye-labeled 488 dye-labeled dye-labeled Fab fragment of Fab fragment of Fab fragment fragment of goat goat anti- goat anti-(mouse of goat anti- anti-(mouse (mouse IgG1 Fc) (mouse IgG1 Fc) IgG1 Fc) IgG1 Fc) 100% 100% 0% 0% 75% 81% 25% 14% 50% 63% 50% 38% 25% 35% 75% 73% 0% 0% 100% 100% Example 20 The Immunolabeling Complex can be Used to Detect Antigens on a Western Blot Bovine heart mitochondria were isolated (Hanson et al., Electrophoresis 22, 950 (2001)).", "The isolated mitochondria were resuspended to ˜10 mg/mL in 100 mM Tris-HCl, pH 7.8,1 mM phenylmethylsulfonyl fluoride (a protease inhibitor), 2% SDS and insoluble material was removed by centrifugation for 10 minutes at 10,000×g in a tabletop centrifuge.", "The protein concentration of the lysate was checked by the BCA assay (Pierce, Rockford, Ill.).", "Samples for gel electrophoresis were prepared by mixing lysate, water, and loading buffer to the appropriate concentrations (final concentration of loading buffer in samples: 58 mM Tris/HCl, 10% glycerol, 2% SDS, 0.02 mg/mL bromphenol blue, 50 mM DTT, pH 8.6).", "The samples were then heated to 90° C. for 5 minutes before loading on the gel and separated on a 13% SDS-PAGE gel.", "Two-fold serial dilution of the extracts ranging from 8 μg of extract down to 20 0.03 μg were loaded on the SDS-PAGE gel.", "The proteins were transferred to PVDF membrane for 1.5 hours using a semi-dry transfer system according to manufacturer's directions (The W.E.P.", "Company, Concord, Calif.).", "The PVDF membrane was blocked for 1 hour in 5% milk.", "Immunolabeling complexes were made with mouse monoclonal antibodies that recognize two different mitochondrial proteins.", "Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG1 Fc) (5 μL of a 1 mg/mL stock, prepared as in Example 4) was incubated with 21 pL (0.88 mg/mL) mouse anti-(CV-alpha) and Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) (5 μL of a 1 mg/mL stock, prepared as in Example 4) was incubated with 19 μL (0.88 mg/mL) mouse anti-(CIII-core2) (Molecular Probes, Eugene, Oreg.).", "Following a 30 minute incubation, 25 μL of a 14.1 mg/mL stock of unlabeled mouse IgG was added to each tube.", "The immunolabeling complexes were then mixed together and brought up to 5 mL in 5% milk.", "The blot was incubated with the mixture of immunolabeling complexes for 1 hour at room temperature.", "The blot was washed twice for 5 seconds each with PBST (PBS with 0.1% Tween) and once with PBST for 15 minutes.", "The blot was air dried and imaged on an EG&G Wallac Imager with the appropriate filters.", "The Western blot revealed two distinct bands of the appropriate molecular weight.", "The Western blot also showed that no cross-labeling of the antibodies occurred and the detection limit was 125 ng.", "Example 21 High-Throughput Screening of Hybridomas for Identifying High Affinity and High IgG Producers Microplate wells containing both a fluorescent labeled antigen of one fluorescent color label and fluorescently labeled Fab fragments of goat anti-(mouse Fc) of a different fluorescent color made by the method described in Example 4 and 5.Hybridoma supernatant is harvested and added to the wells.", "If the hybridoma are producing the desired antibody, i.e.", "antibodies that bind to the labeled antigen, polarization of the florescence corresponding to the labeled antigen will allow visualization of those wells containing antigen specific antibody.", "In addition, the amount of IgG that the hybridomas produce, can be simultaneously identified by polarization of the fluorescence corresponding to the labeled Fab fragments.", "This method thus allows for both quantitation of the amount of antibody present in a specific amount of hybridoma supernatant and the affinity of the monoclonal antibodies for the antigen.", "The reagents employed in the preceding examples are commercially available or can be prepared using commercially available instrumentation, methods, or reagents known in the art or whose preparation is described in the examples.", "It is evident from the above description and results that the subject invention is greatly superior to the presently available methods for determining the presence of a target in a biological sample.", "The subject invention overcomes the shortcomings of the currently used methods by allowing small quantities of antibodies to be labeled and in unlimited media while maintaining specificity and sensitivity.", "The examples are not intended to provide an exhaustive description of the many different embodiments of the invention.", "Thus, although the forgoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill in the art will realize readily that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.", "All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference." ] ]	Patent_10467550
[ [ "Gssp4 polynucleotides and polypeptides and uses thereof", "The present invention relates to the field of metabolic research.", "Metabolic disorders, such as obesity, are a public health problem that is serious and widespread.", "GSSP4 polypeptides have been identified that are believed to be beneficial in the treatment of metabolic disorders.", "These compounds should be effective for reducing cholesterol levels, body mass, body fat, and for treating metabolic-related diseases and disorders.", "The metabolic-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to: obesity, hyperlipidemia, hypercholesterolemia, atherosclerosis, diabetes, glucose intolerance, insulin resistance and hypertension." ], [ "1.A method of reducing circulating free fatty acid levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass.", "2.A method of reducing circulating glucose levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass.", "3.A method of reducing circulating triglyceride levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass.", "4.A method of reducing circulating cholesterol levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass.", "5.An isolated polypeptide comprising: a) an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; b) a polypeptide fragment of at least six consecutive amino acids of SEQ ID NO: 3; c) the polypeptide of SEQ ID NO: 3; d) homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; e) a heterologous polypeptide fused to the polypeptide of a), b), c), or d); or f) a polypeptide according to a), b), c), d), or e) that has been differentially modified.", "6.A composition comprising an isolated polypeptide according to claim 3 and a pharmaceutically or physiologically acceptable carrier.", "7.An isolated or purified polynucleotide: a) encoding a polypeptide comprising an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; b) encoding a polypeptide comprising a fragment of at least six consecutive amino acids of SEQ ID NO: 3; c) encoding a polypeptide comprising SEQ ID NO: 3; d) encoding homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; e) encoding a polypeptide comprising a heterologous polypeptide fused to the polypeptide of a), b), c), or d); f) encoding a polypeptide according to a), b), c), d), or e) that has been differentially modified; g) comprising a polynucleotide according to a), b), c), d), e), or f) operably linked to a promoter, other regulatory element, or selectable marker; or h) comprising a vector comprising a polynucleotide according to a), b), c), d), e), f), or g).", "8.A recombinant cell comprising a polynucleotide according to claim 7.9.An antibody that specifically binds to a polypeptide according to claim 5.10.A method of producing a polypeptide comprising culturing a recombinant cell according to claim 8 under conditions that allow for the expression of said polypeptide.", "11.The method according to claim 10, further comprising the isolation, recovery, or purification of said polypeptide.", "12.A method of treating a obesity, a metabolic disease or a metabolic disorder comprising the administration of a therapeutically effective amount of a composition comprising: a) an isolated polypeptide comprising: 1) an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; 2) a polypeptide fragment of at least six consecutive amino acids of SEQ ID NO: 3; 3) the polypeptide of SEQ ID NO: 3; 4) homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; 5) a heterologous polypeptide fused to the polypeptide of a(1), a(2), a(3), or a(4); or 6) a polypeptide according to a(1), a(2), a(3), a(4), or a(5) that has been differentially modified; b) an isolated or purified polynucleotide: 1) encoding a polypeptide comprising an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; 2) encoding a polypeptide comprising a fragment of at least six consecutive amino acids of SEQ ID NO: 3; 3) encoding a polypeptide comprising SEQ ID NO: 3; 4) encoding homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; 5) encoding a polypeptide comprising a heterologous polypeptide fused to the polypeptide of b(1), b(2), b(3), or b(4); 6) encoding a polypeptide according to b(1), b(2), b(3), b(4), or b(5) that has been differentially modified; 7) comprising a polynucleotide according to b(1), b(2), b(3), b(4), b(5), or b(6) operably linked to a promoter, other regulatory element, or selectable marker; or 8) comprising a vector comprising a polynucleotide according to b(1), b(2), b(3), b(4), b(5), b(6), or b(7); or c) a recombinant cell comprising: 1) a polynucleotide encoding a polypeptide comprising an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; 2) a polynucleotide encoding a polypeptide comprising a fragment of at least six consecutive amino acids of SEQ ID NO: 3; 3) a polynucleotide encoding a polypeptide comprising SEQ ID NO: 3; 4) a polynucleotide encoding homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; 5) a polynucleotide encoding a polypeptide comprising a heterologous polypeptide fused to the polypeptide of c(1), c(2), c(3), or c(4); 6) a polynucleotide encoding a polypeptide according to c(1), c(2), c(3), c(4), or c(5) that has been differentially modified; 7) a polynucleotide according to c(1), c(2), c(3), c(4), c(5), or c(6) operably linked to a promoter, other regulatory element, or selectable marker; or 8) comprising a vector comprising a polynucleotide according to c(1), c(2), c(3), c(4), c(5), c(6), or c(7).", "13.A transgenic animal comprising a polynucleotide according to claim 7." ], [ "<SOH> BACKGROUND OF TH INVENTION <EOH>The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.", "Obesity is a public health problem that is serious, widespread, and increasing.", "In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634).", "Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643).", "Even modest weight loss ameliorates these associated conditions.", "While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Barsh et al (2000) Nature 404:644-651).", "In agreement with these studies, is the fact that an increasing number of obesity-related genes are being identified.", "Some of the more extensively studied genes include those encoding leptin (ob) and its receptor (db), pro-opiomelanocortin (Pomc), melanocortin-4-receptor (Mc4r), agouti protein (A y ), carboxypeptidase E (fat), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loop-helix 2 (Nhlh2), prohormone convertase 1 IPCSK1), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651)." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The instant invention is based on the discovery that GSSP4 polypeptides have unexpected effects in vitro and in vivo, including utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels in humans and other mammals.", "These unexpected effects of administration of GSSP4 polypeptides in mammals also include reduction of elevated free fatty acid levels caused by administration of epinephrine, i.v.", "injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, reduction of circulating cholesterol levels, modulation of blood glucose and weight reduction in mammals, particularly those consuming a high fat/high carbohydrate diet.", "These effects are unexpected and surprising given that proteins of similar structure or homology (such as colipase and mamba intestinal toxin 1) have not been shown to have utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels.", "However, the GSSP4 polypeptides of the invention are effective and can be provided at levels that are feasible for treatments in humans.", "Thus, the invention is drawn to GSSP4 polypeptides, polynucleotides encoding said polypeptides, vectors comprising said GSSP4 polynucleotides, and cells recombinant for said GSSP4 polynucleotides, as well as to pharmaceutical and physiologically acceptable compositions comprising said GSSP4 polypeptide and methods of administering said GSSP4 polypeptides or polynucleotides in a pharmaceutical and physiologically acceptable compositions in order to reduce body weight, cholesterol levels or glucose levels, or to treat metabolic-related diseases and disorders.", "Assays for identifying agonists and antagonists of metabolic-related activity are also part of the invention.", "In a first aspect, the invention features a purified, isolated, or recombinant GSSP4 polypeptides.", "In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, those having significant activity wherein the said activity is selected from the group consisiting of cholesterol reduction, cholesterol regulation, lipid partitioning, lipid metabolism, glucose control, and insulin-like activity.", "In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, the full length polypeptide of SEQ ID NO:3 or a fragment of consecutive amino acids of the full length polypeptide sequence of SEQ ID NO:3.In other preferred embodiments, said polypeptides comprise an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of the polypeptide sequences identified in SEQ ID NO:3.In a further preferred embodiment, the GSSP4 polypeptide is able to lower circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.", "Further preferred GSSP4 polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.", "Further preferred GSSP4 polypeptides are those that cause C2C12 cells differentiated in the presence of said polypeptides to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells.", "Further preferred GSSP4 polypeptides are those that are at least 30% more efficient than untreated cells at increasing leptin uptake in a liver cell line (preferably BPRCL mouse liver cells (ATCC CRL-2217)).", "Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma free fatty acids, particularly following a high fat meal.", "Further preferred GSSP4 polypeptides are those that significantly reduce or eliminate ketone body production, particularly following a high fat meal.", "Further preferred GSSP4 polypeptides are those that increase glucose uptake in skeletal muscle cells.", "Further preferred GSSP4 polypeptides are those that increase glucose uptake in adipose cells.", "Further preferred GSSP4 polypeptides are those that increase glucose uptake in neuronal cells.", "Further preferred GSSP4 polypeptides are those that increase glucose uptake in red blood cells.", "Further preferred GSSP4 polypeptides are those that increase glucose uptake in the brain.", "Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.", "Further preferred GSSP4 polypeptides are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal.", "Further preferred GSSP4 polypeptides are those that improve insulin sensitivity.", "Further preferred GSSP4 polypeptides are those that modulate food intake or food selection.", "Further preferred GSSP4 polypeptides are those that modulate satiety.", "Further preferred GSSP4 polypeptides are those that modulate fatty acid metabolism.", "Further preferred GSSP4 polypeptides are those that modulate cholesterol metabolism, particularly in steroidogenic tissues.", "Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of reproductive hormones (eg.", "estradiol, progesterone, testosterone).", "Further preferred GSSP4 polypeptides are those that modulate cortisol levels.", "Further preferred GSSP4 polypeptides are those that modulate aldosterone levels.", "Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of sodium and potassium.", "Further preferred GSSP4 polypeptides are those that modulate blood pressure preferably to normalize blood pressure within a normal range.", "Further preferred GSSP4 polypeptides are those that form multimers (e.g., heteromultimers or homomultimers) in vitro and/or in vivo.", "Preferred multimers are homodimers or homotrimers.", "Other preferred multimers are homomultimers comprising at least 4, 6, 8, 9, 10 or 12 GSSP4 polypeptides.", "Other preferred mulimers are hetero multimers comprising GSSP4 polypeptides of the invention.", "Further preferred embodiments include heterologous polypeptides comprising a GSSP4 polypeptide of the invention.", "In a second aspect, the invention features purified, isolated, or recombinant polynucleotides encoding said GSSP4 polypeptides described in the first aspect, or the complement thereof.", "A further preferred embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, cDNA, or fragments thereof.", "In one aspect the sequence is derived from a human, mouse or other mammal.", "In a preferred aspect, the genomic sequence includes isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 2000, 5000, 6000 or 7500 nucleotides of any one of the polynucleotide sequences described in SEQ ID NO:1, 2, or the complements thereof, wherein said contiguous span comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding nucleotide sequence in SEQ ID NO: 1, 2, or 3.In further embodiments the polynucleotides are DNA, RNA, DNA/RNA hybrids, single-stranded, and double-stranded.", "Further preferred are GSSP4 polynucleotides and polypeptides that have cholesterol regulating activies.", "Further preferred are GSSP4 polynucleotides and polypeptides that have body weight regulating activies.", "Further preferred are GSSP4 polynucleotides and polypeptides that have body fat regulating activies.", "Further preferred are GSSP4 polynucleotides and polypeptides that have glucose regulating activies.", "Further preferred are GSSP4 polynucleotides and polypeptides that have lipid regulating activies.", "In a third aspect, the invention features a recombinant vector comprising, consisting essentially of, or consisting of, said polynucleotide described in the second aspect.", "In a fourth aspect, the invention features a recombinant cell comprising, consisting essentially of, or consisting of, said recombinant vector described in the third aspect.", "A further embodiment includes a host cell recombinant for a polynucleotide of the invention.", "In a fifth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said GSSP4 polypeptides described in the first aspect and, a pharmaceutical or physiologically acceptable diluent.", "In a sixth aspect, the invention features a method of controlling cholesterol levels comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.", "In further preferred embodiments, the invention features a method of lowering body weight comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.", "In further preferred embodiments, the invention features a method of lowering body fat comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.", "In further preferred embodiments, the invention features a method of lowering controlling blood glucose comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.", "In a seventh aspect, the invention features a method of preventing or treating a metabolic-related disease or disorder comprising, providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.", "Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In a further preferred embodiment, a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect suggest that a compound may have utility in alleviating insulin resistance in individuals, particularly those that are obese or overweight.", "In a further preferred embodiment, the present invention may be used in complementary therapy in individuals to improve their cholesterol, weight or glucose level, comprising a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in combination with known agents.", "The present invention further provides a method of improving the cholesterol levels, body weight or glucose control in individuals comprising the administration of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect alone, without known agents.", "In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with known agents for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the known agent).", "Accordingly, the present invention further provides a product containing a composition a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect and a known agent as a combined preparation for simultaneous, separate or sequential use for the improvement of cholesterol levels, body weight or glucose control in individuals, particularly those who are obese or overweight.", "The ratio of the present composition to known agent is such that the quantity of each active ingredient employed will be such as to provide a therapeutically effective level, but will not be larger than the quantity recommended as safe for administration.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetics (Type II) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetes mellitus (NIDDM, Type II) in combination with alternate therapies.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used as a method in prophylaxis of long-term detrimental effects caused by prolonged high dosage of insulin in humans having IDDM or NIDDM.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypersecretion of insulin and disorders or conditions resulting therefrom.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing obesity and consequences or complications thereof.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypercholesterolemia and consequences or complications thereof.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing NIDDM or IDDM and consequences or complications thereof.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing impaired glucose tolerance (IGT).", "Further preferred embodiment thus provides therapeutics and methods for normalizing insulin resistance.", "Further preferred embodiment thus provides therapeutics and methods for reducing, slowing or preventing the progression to NIDDM.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing the appearance of insulin-resistance syndrome.", "In further preferred embodiments, other conditions, particularly obesity, associated with insulin resistance are treated or prevented according to the methods of the invention.", "Thus, by preventing or treating obesity, the methods of the invention will allow an individual to have a more comfortable life and avoid the onset of various diseases triggered by obesity.", "In further preferred embodiments, the target of the methods according to the present invention includes individuals with normal glucose tolerance (NGT) who are obese or who have fasting hyperinsulinemia, or who have both.", "In an eighth aspect, the invention features a method of controlling blood free fatty acid (FFA) levels and lipid metabolism comprising, providing, or administering to individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.", "In a further preferred embodiment, the identification of said individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores to be treated with said pharmaceutical or physiologically acceptable composition comprises a person who is involved in physical activity which increases metabolic demand.", "Furthermore, increasing mobilization and utilization of fat stores and decreasing total fat stores would provide a means to decrease body weight, preventing weight gain, decrease body fat in overweight and obese individuals.", "Reduction in weight and obesity will thus decrease the risk of chronic disease associated with obesity such as but not limited to the onset of various lipid metabolism disorders, hypertension, Type II diabetes, atherosclerosis, cardiovascular disease and stroke.", "In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a GSSP4 polypeptide, wherein said biological response is selected from the group consisting of: (a) lowering circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids; (b) lowering circulating (either blood, serum or plasma) levels (concentration) of glucose; (c) lowering circulating (either blood, serum or plasma) levels (concentration) of triglycerides; (d) stimulating muscle lipid or free fatty acid oxidation; (e) increasing leptin uptake in the liver or liver cells; (f) reducing the postprandial increase in plasma free fatty acids, particularly following a high fat meal; and, (g) reducing or eliminating ketone body production, particularly following a high fat meal; (h) increasing tissue sensitivity to insulin, particularly muscle, adipose, liver or brain, (i) reducing cholesterol levels, particularly in those with elevated cholesterol (ie.", "greater than 200 mg/dl); (j) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose within physiological range, preferably maintaining glucose between 60-190 mg/dl; (k) modulating circulating (either blood, serum or plasma) levels (concentration) of FFA within physiological range preferably maintaining FFA between 190-420 mg/dl; (l) modulating ketone body production as the result of a high fat meal, wherein said modulating is preferably reducing or eliminating; (m) reducing body weight particularly in individuals with a BMI of greater than 27.In a ninth aspect, the invention features a method of making the GSSP4 polypeptide described in the first aspect, wherein said method is selected from the group consisting of: proteolytic cleavage, recombinant methodology and artificial synthesis.", "In a tenth aspect, the present invention provides a method of making a recombinant GSSP4 polypeptides, the method comprising providing a transgenic, non-human mammal whose milk contains said recombinant GSSP4 polypeptides, and purifying said recombinant GSSP4 polypeptides from the milk of said non-human mammal.", "In one embodiment, said non-human mammal is a cow, goat, sheep, rabbit, or mouse.", "In another embodiment, the method comprises purifying a recombinant GSSP4 polypeptides from said milk, and further comprises cleaving said protein in vitro to obtain a desired GSSP4 polypeptides.", "In an eleventh aspect, the invention features a purified or isolated antibody capable of specifically binding to a protein comprising the sequence of one of the polypeptides of the present invention.", "In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 6 consecutive amino acids, at least 8 consecutive amino acids, or at least 10 consecutive amino acids of the sequence of one of the polypeptides of the present invention.", "In a twelfth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for treatment of metabolic-related diseases and disorders or reducing or increasing body mass.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In a thirteenth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for the preparation of a medicament for the treatment of metabolic-related diseases and disorders or for reducing body mass.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In a fourteenth aspect, the invention provides polypeptides of the first aspect of the invention or a composition of the fifth aspect for use in a method of treatment of the human or animal body.", "In a fifteenth aspect, the invention provides polynucleotides described in the second aspect or an acceptable composition thereof, for use in a method of treatment of the human or animal body.", "In a sixteenth aspect, the invention features methods of reducing body weight for cosmetic purposes comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or polypeptides described in the first aspect.", "Preferably, for said reducing body weight said individual has a BMI of at least 20, 25, 30, 35, or 40.In a seventeenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect or a polypeptide described in the first aspect for reducing body mass in said individuals with a BMI of at least 30, 35, 40, or 45 or for treatment or prevention of metabolic-related diseases or disorders.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GSSP4 single nucleotide polymorphisms (SNPs) or measuring GSSP4 polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "In an eighteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing body weight for cosmetic reasons.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing glucose levels.", "In a nineteenth aspect, the invention features methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or a polypeptide described in the first aspect." ], [ "FIELD OF THE INVENTION The present invention relates to the field of metabolic research, in particular the discovery of compounds effective for reducing cholesterol levels, body fat, and body mass, and useful for treating metabolic-related diseases an disorders.", "The metabolic-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to, obesity, hyperlipidemia, hypercholesterolemia, atherosclerosis, diabetes, glucose intolerance, insulin resistance and hypertension.", "BACKGROUND OF TH INVENTION The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.", "Obesity is a public health problem that is serious, widespread, and increasing.", "In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634).", "Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643).", "Even modest weight loss ameliorates these associated conditions.", "While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Barsh et al (2000) Nature 404:644-651).", "In agreement with these studies, is the fact that an increasing number of obesity-related genes are being identified.", "Some of the more extensively studied genes include those encoding leptin (ob) and its receptor (db), pro-opiomelanocortin (Pomc), melanocortin-4-receptor (Mc4r), agouti protein (Ay), carboxypeptidase E (fat), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loop-helix 2 (Nhlh2), prohormone convertase 1 IPCSK1), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651).", "SUMMARY OF THE INVENTION The instant invention is based on the discovery that GSSP4 polypeptides have unexpected effects in vitro and in vivo, including utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels in humans and other mammals.", "These unexpected effects of administration of GSSP4 polypeptides in mammals also include reduction of elevated free fatty acid levels caused by administration of epinephrine, i.v.", "injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, reduction of circulating cholesterol levels, modulation of blood glucose and weight reduction in mammals, particularly those consuming a high fat/high carbohydrate diet.", "These effects are unexpected and surprising given that proteins of similar structure or homology (such as colipase and mamba intestinal toxin 1) have not been shown to have utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels.", "However, the GSSP4 polypeptides of the invention are effective and can be provided at levels that are feasible for treatments in humans.", "Thus, the invention is drawn to GSSP4 polypeptides, polynucleotides encoding said polypeptides, vectors comprising said GSSP4 polynucleotides, and cells recombinant for said GSSP4 polynucleotides, as well as to pharmaceutical and physiologically acceptable compositions comprising said GSSP4 polypeptide and methods of administering said GSSP4 polypeptides or polynucleotides in a pharmaceutical and physiologically acceptable compositions in order to reduce body weight, cholesterol levels or glucose levels, or to treat metabolic-related diseases and disorders.", "Assays for identifying agonists and antagonists of metabolic-related activity are also part of the invention.", "In a first aspect, the invention features a purified, isolated, or recombinant GSSP4 polypeptides.", "In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, those having significant activity wherein the said activity is selected from the group consisiting of cholesterol reduction, cholesterol regulation, lipid partitioning, lipid metabolism, glucose control, and insulin-like activity.", "In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, the full length polypeptide of SEQ ID NO:3 or a fragment of consecutive amino acids of the full length polypeptide sequence of SEQ ID NO:3.In other preferred embodiments, said polypeptides comprise an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of the polypeptide sequences identified in SEQ ID NO:3.In a further preferred embodiment, the GSSP4 polypeptide is able to lower circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.", "Further preferred GSSP4 polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.", "Further preferred GSSP4 polypeptides are those that cause C2C12 cells differentiated in the presence of said polypeptides to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells.", "Further preferred GSSP4 polypeptides are those that are at least 30% more efficient than untreated cells at increasing leptin uptake in a liver cell line (preferably BPRCL mouse liver cells (ATCC CRL-2217)).", "Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma free fatty acids, particularly following a high fat meal.", "Further preferred GSSP4 polypeptides are those that significantly reduce or eliminate ketone body production, particularly following a high fat meal.", "Further preferred GSSP4 polypeptides are those that increase glucose uptake in skeletal muscle cells.", "Further preferred GSSP4 polypeptides are those that increase glucose uptake in adipose cells.", "Further preferred GSSP4 polypeptides are those that increase glucose uptake in neuronal cells.", "Further preferred GSSP4 polypeptides are those that increase glucose uptake in red blood cells.", "Further preferred GSSP4 polypeptides are those that increase glucose uptake in the brain.", "Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.", "Further preferred GSSP4 polypeptides are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal.", "Further preferred GSSP4 polypeptides are those that improve insulin sensitivity.", "Further preferred GSSP4 polypeptides are those that modulate food intake or food selection.", "Further preferred GSSP4 polypeptides are those that modulate satiety.", "Further preferred GSSP4 polypeptides are those that modulate fatty acid metabolism.", "Further preferred GSSP4 polypeptides are those that modulate cholesterol metabolism, particularly in steroidogenic tissues.", "Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of reproductive hormones (eg.", "estradiol, progesterone, testosterone).", "Further preferred GSSP4 polypeptides are those that modulate cortisol levels.", "Further preferred GSSP4 polypeptides are those that modulate aldosterone levels.", "Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of sodium and potassium.", "Further preferred GSSP4 polypeptides are those that modulate blood pressure preferably to normalize blood pressure within a normal range.", "Further preferred GSSP4 polypeptides are those that form multimers (e.g., heteromultimers or homomultimers) in vitro and/or in vivo.", "Preferred multimers are homodimers or homotrimers.", "Other preferred multimers are homomultimers comprising at least 4, 6, 8, 9, 10 or 12 GSSP4 polypeptides.", "Other preferred mulimers are hetero multimers comprising GSSP4 polypeptides of the invention.", "Further preferred embodiments include heterologous polypeptides comprising a GSSP4 polypeptide of the invention.", "In a second aspect, the invention features purified, isolated, or recombinant polynucleotides encoding said GSSP4 polypeptides described in the first aspect, or the complement thereof.", "A further preferred embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, cDNA, or fragments thereof.", "In one aspect the sequence is derived from a human, mouse or other mammal.", "In a preferred aspect, the genomic sequence includes isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 2000, 5000, 6000 or 7500 nucleotides of any one of the polynucleotide sequences described in SEQ ID NO:1, 2, or the complements thereof, wherein said contiguous span comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding nucleotide sequence in SEQ ID NO: 1, 2, or 3.In further embodiments the polynucleotides are DNA, RNA, DNA/RNA hybrids, single-stranded, and double-stranded.", "Further preferred are GSSP4 polynucleotides and polypeptides that have cholesterol regulating activies.", "Further preferred are GSSP4 polynucleotides and polypeptides that have body weight regulating activies.", "Further preferred are GSSP4 polynucleotides and polypeptides that have body fat regulating activies.", "Further preferred are GSSP4 polynucleotides and polypeptides that have glucose regulating activies.", "Further preferred are GSSP4 polynucleotides and polypeptides that have lipid regulating activies.", "In a third aspect, the invention features a recombinant vector comprising, consisting essentially of, or consisting of, said polynucleotide described in the second aspect.", "In a fourth aspect, the invention features a recombinant cell comprising, consisting essentially of, or consisting of, said recombinant vector described in the third aspect.", "A further embodiment includes a host cell recombinant for a polynucleotide of the invention.", "In a fifth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said GSSP4 polypeptides described in the first aspect and, a pharmaceutical or physiologically acceptable diluent.", "In a sixth aspect, the invention features a method of controlling cholesterol levels comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.", "In further preferred embodiments, the invention features a method of lowering body weight comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.", "In further preferred embodiments, the invention features a method of lowering body fat comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.", "In further preferred embodiments, the invention features a method of lowering controlling blood glucose comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.", "In a seventh aspect, the invention features a method of preventing or treating a metabolic-related disease or disorder comprising, providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.", "Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In a further preferred embodiment, a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect suggest that a compound may have utility in alleviating insulin resistance in individuals, particularly those that are obese or overweight.", "In a further preferred embodiment, the present invention may be used in complementary therapy in individuals to improve their cholesterol, weight or glucose level, comprising a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in combination with known agents.", "The present invention further provides a method of improving the cholesterol levels, body weight or glucose control in individuals comprising the administration of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect alone, without known agents.", "In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with known agents for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the known agent).", "Accordingly, the present invention further provides a product containing a composition a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect and a known agent as a combined preparation for simultaneous, separate or sequential use for the improvement of cholesterol levels, body weight or glucose control in individuals, particularly those who are obese or overweight.", "The ratio of the present composition to known agent is such that the quantity of each active ingredient employed will be such as to provide a therapeutically effective level, but will not be larger than the quantity recommended as safe for administration.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetics (Type II) in combination with insulin therapy.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetes mellitus (NIDDM, Type II) in combination with alternate therapies.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used as a method in prophylaxis of long-term detrimental effects caused by prolonged high dosage of insulin in humans having IDDM or NIDDM.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypersecretion of insulin and disorders or conditions resulting therefrom.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing obesity and consequences or complications thereof.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypercholesterolemia and consequences or complications thereof.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing NIDDM or IDDM and consequences or complications thereof.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing impaired glucose tolerance (IGT).", "Further preferred embodiment thus provides therapeutics and methods for normalizing insulin resistance.", "Further preferred embodiment thus provides therapeutics and methods for reducing, slowing or preventing the progression to NIDDM.", "In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing the appearance of insulin-resistance syndrome.", "In further preferred embodiments, other conditions, particularly obesity, associated with insulin resistance are treated or prevented according to the methods of the invention.", "Thus, by preventing or treating obesity, the methods of the invention will allow an individual to have a more comfortable life and avoid the onset of various diseases triggered by obesity.", "In further preferred embodiments, the target of the methods according to the present invention includes individuals with normal glucose tolerance (NGT) who are obese or who have fasting hyperinsulinemia, or who have both.", "In an eighth aspect, the invention features a method of controlling blood free fatty acid (FFA) levels and lipid metabolism comprising, providing, or administering to individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.", "In a further preferred embodiment, the identification of said individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores to be treated with said pharmaceutical or physiologically acceptable composition comprises a person who is involved in physical activity which increases metabolic demand.", "Furthermore, increasing mobilization and utilization of fat stores and decreasing total fat stores would provide a means to decrease body weight, preventing weight gain, decrease body fat in overweight and obese individuals.", "Reduction in weight and obesity will thus decrease the risk of chronic disease associated with obesity such as but not limited to the onset of various lipid metabolism disorders, hypertension, Type II diabetes, atherosclerosis, cardiovascular disease and stroke.", "In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a GSSP4 polypeptide, wherein said biological response is selected from the group consisting of: (a) lowering circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids; (b) lowering circulating (either blood, serum or plasma) levels (concentration) of glucose; (c) lowering circulating (either blood, serum or plasma) levels (concentration) of triglycerides; (d) stimulating muscle lipid or free fatty acid oxidation; (e) increasing leptin uptake in the liver or liver cells; (f) reducing the postprandial increase in plasma free fatty acids, particularly following a high fat meal; and, (g) reducing or eliminating ketone body production, particularly following a high fat meal; (h) increasing tissue sensitivity to insulin, particularly muscle, adipose, liver or brain, (i) reducing cholesterol levels, particularly in those with elevated cholesterol (ie.", "greater than 200 mg/dl); (j) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose within physiological range, preferably maintaining glucose between 60-190 mg/dl; (k) modulating circulating (either blood, serum or plasma) levels (concentration) of FFA within physiological range preferably maintaining FFA between 190-420 mg/dl; (l) modulating ketone body production as the result of a high fat meal, wherein said modulating is preferably reducing or eliminating; (m) reducing body weight particularly in individuals with a BMI of greater than 27.In a ninth aspect, the invention features a method of making the GSSP4 polypeptide described in the first aspect, wherein said method is selected from the group consisting of: proteolytic cleavage, recombinant methodology and artificial synthesis.", "In a tenth aspect, the present invention provides a method of making a recombinant GSSP4 polypeptides, the method comprising providing a transgenic, non-human mammal whose milk contains said recombinant GSSP4 polypeptides, and purifying said recombinant GSSP4 polypeptides from the milk of said non-human mammal.", "In one embodiment, said non-human mammal is a cow, goat, sheep, rabbit, or mouse.", "In another embodiment, the method comprises purifying a recombinant GSSP4 polypeptides from said milk, and further comprises cleaving said protein in vitro to obtain a desired GSSP4 polypeptides.", "In an eleventh aspect, the invention features a purified or isolated antibody capable of specifically binding to a protein comprising the sequence of one of the polypeptides of the present invention.", "In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 6 consecutive amino acids, at least 8 consecutive amino acids, or at least 10 consecutive amino acids of the sequence of one of the polypeptides of the present invention.", "In a twelfth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for treatment of metabolic-related diseases and disorders or reducing or increasing body mass.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In a thirteenth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for the preparation of a medicament for the treatment of metabolic-related diseases and disorders or for reducing body mass.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In a fourteenth aspect, the invention provides polypeptides of the first aspect of the invention or a composition of the fifth aspect for use in a method of treatment of the human or animal body.", "In a fifteenth aspect, the invention provides polynucleotides described in the second aspect or an acceptable composition thereof, for use in a method of treatment of the human or animal body.", "In a sixteenth aspect, the invention features methods of reducing body weight for cosmetic purposes comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or polypeptides described in the first aspect.", "Preferably, for said reducing body weight said individual has a BMI of at least 20, 25, 30, 35, or 40.In a seventeenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect or a polypeptide described in the first aspect for reducing body mass in said individuals with a BMI of at least 30, 35, 40, or 45 or for treatment or prevention of metabolic-related diseases or disorders.", "Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "In preferred embodiments, said individual is a mammal, preferably a human.", "In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GSSP4 single nucleotide polymorphisms (SNPs) or measuring GSSP4 polypeptide or mRNA levels in clinical samples from said individuals.", "Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.", "In an eighteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing body weight for cosmetic reasons.", "In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing glucose levels.", "In a nineteenth aspect, the invention features methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or a polypeptide described in the first aspect.", "BRIEF DESCRIPTION OF THE SEQENCES SEQ ID NO:1 shows GSSP4 genomic sequence indicating regulatory regions, exons, polymorphic alleles and positions, and reverse and forward microsequencing primers for haplotyping.", "SEQ ID NO:2 shows GSSP4 polynucleotide sequence of cDNA SEQ ID NO:3 shows GSSP4 polypeptide sequence SEQ ID NO:4-18 shows GSSP4 47-mer nucleotide sequence comprising polymorphic allele at position 24.The corresponding alleles and primers indicated in SEQ ID NO:1 are as follows: VLP_1206_C_A, m=C or A SEQ ID NO:4 VLP_148_A_G, r=A or G SEQ ID NO:5 VLP_1851_T_C, y=T or C SEQ ID NO:6 VLP_2551_G_A, r=G or A SEQ ID NO:7 VLP_3124_C_T, y=C or T SEQ ID NO:8 VLP_3563_G_A, r=G or A SEQ ID NO:9 VLP_3792_G_A, r=G or A SEQ ID NO:10 VLP_4417_A_C, m=A or G SEQ ID NO:11 VLP_5757_T_C, y=T or C SEQ ID NO:12 VLP_6322_A_G, r=G or A SEQ ID NO:13 VLP_816_G_A, r=G or A SEQ ID NO:14 VLP_924_G_A, r=G or A SEQ ID NO:15 VLP_99-1_174_T_C, y=T or C SEQ ID NO:16 VLP_99-1_325_C_G, s=C or G SEQ ID NO:17 VLP_99-2_389_T_C, y=T or G SEQ ID NO:18 DETAILED DESCRIPTION OF THE INVENTION Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein.", "As used interchangeably herein, the terms “oligonucleotides”, and “polynucleotides” and nucleic acid include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.", "The terms encompass “modified nucleotides” which comprise at least one modification, including by way of example and not limitation: (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar.", "For examples of analogous linking groups, purines, pyrimidines, and sugars see for example PCT publication No.", "WO 95/04064.The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.", "The terms polynucleotide construct, recombinant polynucleotide and recombinant polypeptide are used herein consistently with their use in the art.", "The terms “upstream” and “downstream” are also used herein consistently with their use in the art.", "The terms “base paired” and “Watson & Crick base paired” are used interchangeably herein and consistently with their use in the art.", "Similarly, the terms “complementary”, “complement thereof”, “complement”, “complementary polynucleotide”, “complementary nucleic acid” and “complementary nucleotide sequence” are used interchangeably herein and consistently with their use in the art.", "The term “purified” is used herein to describe a polynucleotide or polynucleotide vector of the invention that has been separated from other compounds including, but not limited to, other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide).", "Purified can also refer to the separation of covalently closed polynucleotides from linear polynucleotides, or vice versa, for example.", "A polynucleotide is substantially pure when at least about 50%, 60%, 75%, or 90% of a sample contains a single polynucleotide sequence.", "In some cases this involves a determination between conformations (linear versus covalently closed).", "A substantially pure polynucleotide typically comprises about 50, 60, 70, 80, 90, 95, 99% weight/weight of a nucleic acid sample.", "Polynucleotide purity or homogeneity may be indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel.", "For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.", "Similarly, the term “purified” is used herein to describe a polypeptide of the invention that has been separated from other compounds including, but not limited to, nucleic acids, lipids, carbohydrates and other proteins.", "In some preferred embodiments, a polypeptide is substantially pure when at least about 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the polypeptide molecules of a sample have a single amino acid sequence.", "In some preferred embodiments, a substantially pure polypeptide typically comprises about 50%, 60%, 70%, 80%, 90% 95%, 96%, 97%, 98%, 99% or 99.5% weight/weight of a protein sample.", "Polypeptide purity or homogeneity is indicated by a number of methods well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel.", "For certain purposes higher resolution can be provided by using HPLC or other methods well known in the art.", "Further, as used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition.", "Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.", "Alternatively, purification may be expressed as “at least” a percent purity relative to heterologous polynucleotides (DNA, RNA or both) or polypeptides.", "As a preferred embodiment, the polynucleotides or polypeptides of the present invention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 98%, 99%, 99.5% or 100% pure relative to heterologous polynucleotides or polypeptides.", "As a further preferred embodiment the polynucleotides or polypeptides have an “at least” purity ranging from any number, to the thousandth position, between 90% and 100% (e.g., at least 99.995% pure) relative to heterologous polynucleotides or polypeptides.", "Additionally, purity of the polynucleotides or polypeptides may be expressed as a percentage (as descried above) relative to all materials and compounds other than the carrier solution.", "Each number, to the thousandth position, may be claimed as individual species of purity.", "The term “isolated” requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).", "For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.", "Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.", "Specifically excluded from the definition of “isolated” are: naturally occurring chromosomes (e.g., chromosome spreads), artificial chromosome libraries, genomic libraries, and cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies.", "Also specifically excluded are the above libraries wherein a 5′ EST makes up less than 5% (or alternatively 1%, 2%, 3%, 4%, 10%, 25%, 50%, 75%, or 90%, 95%, or 99%) of the number of nucleic acid inserts in the vector molecules.", "Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including said whole cell preparations which are mechanically sheared or enzymatically digested).", "Further specifically excluded are the above whole cell preparations as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis (including blot transfers of the same) wherein the polynucleotide of the invention have not been further separated from the heterologous polynucleotides in the electrophoresis medium (e.g., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).", "The term “primer” denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.", "A primer serves as an initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase, or reverse transcriptase.", "The term “probe” denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., PNA as defined hereinbelow) which can be used to identify a specific polynucleotide sequence present in a sample, said nucleic acid segment comprising a nucleotide sequence complementary to the specific polynucleotide sequence to be identified.", "The term “polypeptide” refers to a polymer of amino acids without regard to the length of the polymer.", "Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.", "This term also does not specify or exclude post-expression modifications of polypeptides.", "For example, polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.", "Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.", "), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.", "The phrases “control of blood glucose”, “glucose regulation” “regulation of blood glucose”, “modulating blood glucose”, and “glucose control” refer to maintaining or regulating blood, serum, and plasma levels of glucose between 70-190 mg/dl.", "Without being limited by theory, the compounds/polypeptides of the invention feature a method of chronic control of blood glucose within a narrow and more physiological range compared to the control of blood glucose attained with current therapies which treat disorders of glucose metabolism or insulin action, including but not limited to hyperglycemia, insulin resistance, insulin dependent diabetes mellitus and noninsulin dependent diabetes mellitus.", "Without being limited by theory, the compounds/polypeptides of the invention are capable of modulating the control of blood glucose (as defined above), directing or partitioning glucose between the liver and the peripheral tissues, and are thus believed to treat “diseases involving the control of blood glucose between the liver and peripheral tissues”.", "The term “peripheral tissues” is meant to include the blood, brain, muscle and adipose tissue.", "In preferred embodiments, the compounds/polypeptides of the invention direct or partition glucose towards the liver, muscle and brain.", "In alternative preferred embodiments, glucose is directed or partitioned towards the adipose tissue.", "In other preferred embodiments, glucose is directed or partitioned towards the liver.", "In other preferred embodiments, glucose is directed or partitioned towards the brain.", "In other preferred embodiments, glucose is directed towards the blood.", "In yet other preferred embodiments, the compounds/polypeptides of the invention increase or decrease the oxidation of glucose, preferably by the muscle.", "Without being limited by theory, the compounds/polypeptides of the invention are capable of modulating the partitioning of dietary or endogenous lipids between the liver and peripheral tissues, and are thus believed to treat “diseases involving the partitioning of lipids between the liver and peripheral tissues.” The term “peripheral tissues” is meant to include the blood, muscle and adipose tissue.", "In preferred embodiments, the compounds/polypeptides of the invention partition the lipids toward the muscle.", "In alternative preferred embodiments, the lipids are partitioned toward the blood.", "In alternative preferred embodiments, the lipids are partitioned toward the adipose tissue.", "In other preferred embodiments, the lipids are partitioned toward the liver.", "In yet other preferred embodiments, the compounds/polypeptides of the invention increase or decrease the oxidation of dietary or endogenous lipids, preferably free fatty acids (FFA) by the muscle.", "Dietary and endogenous lipids include, but are not limited to triglycerides (TG) and FFA.", "“Preferred diseases” believed to involve the the control of cholesterol, body fat, lipid metabolism, blood glucose, partitioning of blood glucose, and partitioning of lipids include obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, reproductive cancers, hypercortisolism, aldosteronism, hyperandrogenism, hyperkalemia, hypernatremia, hyperlipoproteinemia, hyperinsulinemia, hyperglycemia, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (eg.", "ketosis), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be prevented or treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "The term “heterologous”, when used herein, is intended to designate any polypeptide or polynucleotide other than a GSSP4 polypeptide or a polynucleotide encoding a GSSP4 polypeptide of the present invention.", "The terms “comprising”, “consisting of” and “consisting essentially of” are defined according to their standard meaning.", "A defined meaning set forth in the M.P.E.P.", "controls over a defined meaning in the art and a defined meaning set forth in controlling Federal Circuit case law controls over a meaning set forth in the M.P.E.P.", "With this in mind, the terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.", "The term “host cell recombinant for” a particular polynucleotide of the present invention, means a host cell that has been altered by the hands of man to contain said polynucleotide in a way not naturally found in said cell.", "For example, said host cell may be transiently or stably transfected or transduced with said polynucleotide of the present invention.", "The term “obesity” as used herein is defined by the National Heart, Lung and Blood Institute Expert Panel (J Amer Diet Assoc 98:1178-1191, 1998) based on Body Mass Index (BMI).", "BMI is calculated as body weight in kilograms divided by the square of height in meters, ie kg/m2.A BMI of less than 18.5 kg/m2 is considered “Underweight”; a BMI of 18.5-24.9 kg/m2 is considered “Normal” (healthy); a BMI of 25.0-29.9 kg/m2 is considered “Overweight”; a BMI of 30.0-34.9 kg/m2 is considered “Class I Obesity”; a BMI of 35.0-39.9 kg/m2 is considered “Class II Obesity”; a BMI of greater than 39.9 kg/m2 is considered “Class III Obesity”.", "Waist circumference can also be used to indicate a risk of metabolic complications where in men a circumference of greater than or equal to 94 cm indicates an increased risk, and greater than or equal to 102 cm indicates a substantially increased risk Similarly for women, greater than or equal to 88 cm indicates an increased risk, and greater than or equal to 88 cm indicates a substantially increased risk.", "The waist circumference is measured in cm at midpoint between lower border of ribs and upper border of the pelvis.", "Other measures of obesity include, but are not limited to, skinfold thickness which is a measurement in cm of skinfold thickness using calipers and bioimpedance which is based on the principle that lean mass and water will conduct electrical current and measurement of resistance to a weak current (impedance) applied across extremities provides an estimate of body fat using an empirically derived equation.", "The term “Insulin Dependent Diabetes Mellitus”, “IDDM”, “Type I Diabetes” and “Type I diabetics” are synomous, inclusive, or interchangable.", "The term “diabetes-related complications” refer to pathologic or physiologic states experienced by Type I diabetics (as defined previously) or Type II diabetics (as defined previously) or both.", "The term “Noninsulin Dependent Diabetes Mellitus”, “NIDDM”, “Type II Diabetes” and “Type II diabetics” are synomous, inclusive, or interchangable.", "The term “agent acting on the control of blood glucose, directing glucose between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the control of blood glucose as previously described.", "Preferably, the agent increases or decreases the oxidation of glucose, preferably by the muscle.", "Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an metabolic-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "The terms “response to an agent acting on the control of blood glucose, directing glucose between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.", "The terms “side effects to an agent acting on the control of blood glucose, directing glucose between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.", "“Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.", "The term “agent acting on the partitioning of glucose between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the partitioning of glucose between the liver and the peripheral tissues as previously described.", "Preferably, the agent increases or decreases blood glucose levels.", "Preferably, the agent increases or decreases oxidation of glucose, preferably by the muscle.", "Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an metabolic-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "The terms “response to an agent acting on the partitioning of glucose between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.", "The terms “side effects to an agent acting on the partitioning of glucose between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.", "“Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.", "The term “agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the partitioning of dietary lipids between the liver and the peripheral tissues as previously described.", "Preferably, the agent increases or decreases the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle.", "Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an metabolic-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "The terms “response to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.", "The terms “side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.", "“Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.", "The term “agent acting on the partitioning of endogenous lipids between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the partitioning of endogenous lipids between the liver and the peripheral tissues as previously described.", "Preferably, the agent increases or decreases the oxidation of endogenous lipids, preferably free fatty acids (FFA) by the muscle.", "Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an metabolic-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "The terms “response to an agent acting on the partitioning of endogenous lipids between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.", "The terms “side effects to an agent acting on the partitioning of endogenous lipids between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.", "“Side effects to an agent acting on the partitioning of endogenous lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.", "The term “GSSP4-related diseases and disorders” as used herein refers to any disease or disorder comprising an aberrant functioning of a GSSP4, or which could be treated or prevented by modulating GSSP4 levels or activity.", "“Aberrant functioning of a GSSP4” includes, but is not limited to, aberrant levels of expression of a GSSP4 polypeptide (either increased or decreased, but preferably decreased), aberrant activity of a GSSP4 polypeptide (either increased or decreased), and aberrant interactions with ligands or binding partners (either increased or decreased).", "By “aberrant” is meant a change from the type, or level of activity seen in normal cells, tissues, or patients, or seen previously in the cell, tissue, or patient prior to the onset of the illness.", "In preferred embodiments, these GSSP4-related diseases and disorders include obesity and the metabolic-related diseases and disorders described previously.", "The term “cosmetic treatments” is meant to include treatments with compounds or polypeptides of the invention that increase or decrease the body mass of an individual where the individual is not clinically obese or clinically underweight as defined previously.", "Thus, these individuals have a body mass index (BMI) below the cut-off for clinical obesity (e.g.", "below 30 kg/m2) and above the cut-off for clinical thinness (e.g.", "above 18.5 kg/m2).", "In addition, these individuals are preferably healthy (e.g.", "do not have an metabolic-related disease or disorder of the invention).", "“Cosmetic treatments” are also meant to encompass, in some circumstances, more localized increases in adipose tissue, for example, gains or losses specifically around the waist or hips, or around the hips and thighs, for example.", "These localized gains or losses of adipose tissue can be identified by increases or decreases in waist or hip size, for example.", "The term “prevent” or “preventing” as used herein refers to administering a compound prior to the onset of clinical symptoms of a disease or condition so as to prevent a physical manifestation of aberrations associated with obesity or insulin resistance to some extent.", "The term “prevent” or “preventing” does not mean the result is necessarily absolute, but rather effective for providing some degree of prevention or amelioration of the progression of the metabolic or GSSP4-related disorder (i.e., provide protective effects), amelioration of the symptoms of the disorder, and amelioration of the reoccurrence of the metabolic or GSSP4-related disorder.", "The term “treat” or “treating” as used herein refers to administering a compound after the onset of clinical symptoms.", "The term “treat” or “treating” means to ameliorate, alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition.", "The term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g.", "physician, nurse, nurse practitioner, etc in the case of humans; veterinarian in the case of animals, including non-human mammals) that an individual or animal requires or will benefit from treatment.", "This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that include the knowledge that the individual or animal is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention.", "The term “perceives a need for treatment” refers to a sub-clinical determination that an individual desires to reduce weight for cosmetic reasons as discussed under “cosmetic treatment” above.", "The term “perceives a need for treatment” in other embodiments can refer to the decision that an owner of an animal makes for cosmetic treatment of the animal.", "The term “patient” or “individual” as used herein refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.", "The term may specify male or female or both, or exclude male or female.", "The term “non-human animal” refers to any non-human vertebrate, including birds and more usually mammals, preferably primates, animals such as swine, goats, sheep, donkeys, horses, cats, dogs, rabbits or rodents, more preferably rats or mice.", "Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term “non-human”.", "The inventors believe GSSP4 polypeptides are able to significantly reduce the postprandial response of glucose in an individual, particularly following a high fat/high carbohydrate meal, while not effecting the levels of insulin.", "Further, GSSP4 polypeptides of the invention are believed to modulate weight gain, particularly in individuals are fed a high fat/high carbohydrate diet.", "The instant invention encompasses the use of GSSP4 polypeptides in the modulation of glucose as an important new tool to control energy homeostasis and glucose regulation.", "The instant invention encompasses the use of GSSP4 polypeptides in the partitioning of glucose as an important new tool to control energy homeostasis and glucose regulation.", "The instant invention encompasses the use of GSSP4 polypeptides in the partitioning of lipids as an important new tool to control energy homeostasis and lipid regulation.", "The instant invention encompasses the use of GSSP4 polypeptides in the partitioning of dietary lipids as an important new tool to control energy homeostasis and lipid regulation.", "The instant invention encompasses the use of GSSP4 polypeptides in the partitioning of endogenous lipids as an important new tool to control energy homeostasis and lipid regulation.", "PREFERRED EMBODIMENTS OF THE INVENTION I. GSSP4 Polypeptides of the Invention GSSP4 polypeptides that have measurable activity in vitro and in vivo have been identified.", "These activities include, but are not limited to, reduction of the postprandial response of plasma free fatty acids, glucose, and triglycerides, particularly in mice fed a high fat/carbohydrate meal, increase in muscle free fatty acid oxidation in vitro and ex vivo, and sustained weight loss in mice on a high fat/carbohydrate diet.", "Other assays for GSSP4 polypeptide activity in vitro and in vivo are also provided (Examples 2-13, for example), and equivalent assays can be designed by those with skill in the art.", "The term “obesity-related” or “metabolic-related” activity as used herein refers to at least one, and preferably all, of the activities described herein for GSSP4 polypeptides.", "Assays for the determination of these activities are provided herein, and equivalent assays can be designed by those with ordinary skill in the art.", "The term “metabolic-related activity” as used herein refers to at least one, and preferably all, of the activities described herein for GSSP4 polypeptides.", "Assays for the determination of these activities are provided herein, known in the art, or can be designed by those with ordinary skill in the art.", "Optionally, “metabolic-related activity” can be selected from the group consisting of control of blood glucose, partitioning of glucose, glucose metabolism, lipid partitioning, lipid metabolism, and insulin-like activity, or an activity within one of these categories.", "Optionally, “obesity-related” activity can be selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity, or an activity within one of these categories.", "By “lipid partitioning” activity is meant the ability to effect the location of dietary lipids among the major tissue groups including, adipose tissue, liver, and muscle.", "The inventors have shown that GSSP4 polypeptides of the invention play a role in the partitioning of lipids to the muscle, liver or adipose tissue.", "By “lipid metabolism” activity is meant the ability to influence the metabolism of lipids.", "The inventors have shown that GSSP4 polypeptides of the invention have the ability to affect the level of free fatty acids in the plasma as well as to increase the metabolism of lipids in the muscle through free fatty acid oxidation experiments (Examples 4, 8, 10, 11, and 12) and to transiently affect the levels of triglycerides in the plasma and the muscle (Examples 7, 10, and 13).", "By “insulin-like” activity is meant the ability of GSSP4 polypeptides to modulate the levels of glucose in the plasma.", "The inventors have found that GSSP4 polypeptides do not significantly impact insulin levels but do impact glucose levels similarly to the effects of insulin (Examples 9 & 10).", "These effects are not seen in the presence of the intact (full-length) GSSP4 polypeptide or are significantly greater in the presence of the GSSP4 polypeptides compared with the full-length GSSP4 polypeptide.", "By “intact” or “full-length” GSSP4 polypeptide as used herein is meant the full length polypeptide sequence of the GSSP4 polypeptide of SEQ ID NO:3, from the N-terminal methionine to the C-terminal stop codon.", "Preferred GSSP4 polypeptides have a biological activity described herein, and as they would be useful in making antibodies, diagnostic assays, etc.", "As a further preferred embodiment, GSSP4 polypeptides which allow a rise in plasma glucose to not more than 190 mg/dl, particularly following consumption of food, or which prevent a drop in serum glucose to not less than 70 mg/dl, particularly following consumption of food.", "As a further preferred embodiment, GSSP4 polypeptides which allow a rise in plasma fatty acids to not more than 420 mg/dl, particularly following consumption of food, or which prevent a drop in serum fatty acids to not less than 190 mg/dl, particularly following consumption of food.", "By “significantly” as used herein is meant statistically significant as it is typically determined by those with ordinary skill in the art.", "For example, data are typically calculated as a mean±SEM, and a p-value≦0.05 is considered statistically significant.", "Statistical analysis is typically done using either the unpaired Student's t test or the paired Student's t test, as appropriate in each study.", "Representative “metabolic-related assays” are provided in Examples below.", "These assays include, but are not limited to, in vivo and in vitro methods of measuring the postprandial response, methods of measuring glucose uptake, glucose oxidation, glucose concentration, lipid concentration, free fatty acid levels, fatty acid oxidation and methods of measuring weight modulation.", "In preferred embodiments, the post-prandial response is measured in non-human animals, preferably rodents.", "In preferred embodiments physiologic parameters are measured including, but not limited to, levels of glucose, fatty acids, insulin, and leptin.", "In other preferred embodiments, free fatty acid oxidation is measured in cells in vitro or ex vivo, preferably in muscle cells or tissue of non-human animals, preferably rodents.", "In yet other preferred embodiments weight modulation is measured in human or non-human animals, preferably rodents (rats or mice), primates, canines, felines or procines on a high fat/carbohydrate diet.", "Optionally, “metabolic-related activity” includes other activities not specifically identified herein.", "In general, “measurable parameters” relating to obesity and the field of metabolic research can be selected from the group consisting of free fatty acid levels, free fatty acid oxidation, triglyceride levels, glucose levels, insulin levels, leptin levels, food intake, and body weight.", "In these metabolic-related assays, preferred GSSP4 polypeptides or polynucleotides or both would cause a significant change in at least one of the measurable parameters selected from the group consisting of post-prandial lipemia, free fatty acid levels, triglyceride levels, glucose levels, glucose oxidation, glucose uptake, free fatty acid oxidation, and weight.", "Alternatively, preferred GSSP4 polypeptides or polynucleotides or both would have a significant change in at least one of the measurable parameters selected from the group consisting of an increase in blood fatty acid levels (FFA), an decrease in blood glucose levels, a decrease in insulin levels, an increase in glucose oxidation and an increase in FFA oxidation.", "The invention is drawn, inter alia, to isolated, purified or recombinant GSSP4 polypeptides.", "GSSP4 polypeptides of the invention are useful for treating or preventing insulin resistance, and reducing body weight or increasing body weight (using antagonists of GSSP4 polypeptides) either as a cosmetic treatment or for treatment or prevention of metabolic-related diseases and disorders.", "GSSP4 polypeptides are also useful inter alia in screening assays for agonists or antagonists of GSSP4 polypeptide activity, for raising GSSP4 polypeptide-specific antibodies, and in diagnostic assays.", "The GSSP4 polypeptides of the present invention are preferably provided in an isolated form, and may be partially or substantially purified.", "A recombinantly produced version of any one of the GSSP4 polypeptides can be substantially purified by the one-step method described by Smith et al.", "((1988) Gene 67(1):31-40) or by the methods described herein or known in the art.", "Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies directed against the polypeptides of the invention by methods known in the art of protein purification.", "Preparations of GSSP4 polypeptides of the invention involving a partial purification of or selection for the GSSP4 polypeptides are also specifically contemplated.", "These crude preparations are envisioned to be the result of the concentration of cells expressing GSSP4 polypeptides with perhaps a few additional purification steps, but prior to complete purification of the polypeptides.", "The cells expressing GSSP4 polypeptides are present in a pellet, they are lysed, or the crude polypeptide is lyophilized, for example.", "GSSP4 polypeptides can be any integer in length from at least 6 consecutive amino acids to 1 amino acids less than a full length GSSP4 polypeptide of SEQ ID NO:4.Thus, for SEQ ID NO:4, a GSSP4 polypeptide can be: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 consecutive amino acids.", "Each GSSP4 polypeptide as described above can be further specified in terms of its N-terminal and C-terminal positions.", "For example, every combination of a N-terminal and C-terminal position that polypeptides of from 6 contiguous amino acids to 1 amino acids less than the full length GSSP4 polypeptide could occupy, on any given intact and contiguous full length GSSP4 polypeptide sequence are included in the present invention.", "Thus, a 6 consecutive amino acid fragment could occupy positions selected from the group consisting of 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20, 16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30, 26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40, 36-41, 37-42, 38-43, 39-44, 40-45, 41-46, 42-47, 43-48, 44-49, 45-50, 46-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-60, 56-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70, 66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, 75-80, 76-81, 77-82, 78-83, 79-84, 80-85, 81-86, 82-87, 83-88, 84-89, 85-90, 86-91, 87-92, 88-93, 89-94, 90-95, 91-96, 92-97, 93-98, 94-99, 95-100, 96-101, 97-102, 98-103, 99-104, and 100-105, of a 105 consecutive amino acid fragment.", "Similarly, the positions occupied by all the other fragments of sizes between 6 amino acids and 105 amino acids of SEQ ID NO:3 are included in the present invention and can also be immediately envisaged based on these two examples and therefore, are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "Furthermore, the positions occupied by fragments of 6 to 105 consecutive amino acids of SEQ ID NO:3 are included in the present invention and can also be immediately envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "In addition, the positions occupied by fragments of 6 consecutive amino acids to 1 amino acid less than any other full length GSSP4 polypeptide can also be envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "The GSSP4 polypeptides of the present invention may alternatively be described by the formula “n to c” (inclusive); where “n” equals the N-terminal most amino acid position (as defined by the sequence listing) and “c” equals the C-terminal most amino acid position (as defined by the sequence listing) of the polypeptide; and further where “n” equals an integer between 1 and 99; and where “c” equals an integer between 7 and 105, the number of amino acids of the full length polypeptide sequence; and where “n” is an integer smaller then “c” by at least 6.Therefore, for the sequences provided in SEQ ID NO:3, “n” is any integer selected from the list consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99; and “c” is any integer selected from the group consisting of: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, and 105.Every combination of “n” and “c” positions are included as specific embodiments of the invention.", "Moreover, the formula “n” to “c” may be modified as “n1-n2” to “c1-c2”, wherein “n1-n2” and “c1-c2” represent positional ranges selected from any two integers above which represent amino acid positions of the sequence listing.", "Alternative formulas include “n1-n2” to “c” and “n” to “c1-c2”.", "These specific embodiments, and other polypeptide and polynucleotide fragment embodiments described herein may be modified as being “at least”, “equal to”, “equal to or less than”, “less than”, “at least ______ but not greater than ______” or “from ______ to ______”.", "a specified size or specified N-terminal and/or C-terminal positions.", "It is noted that all ranges used to describe any embodiment of the present invention are inclusive unless specifically set forth otherwise.", "The present invention also provides for the exclusion of any individual fragment specified by N-terminal and C-terminal positions or of any fragment specified by size in amino acid residues as described above.", "In addition, any number of fragments specified by N-terminal and C-terminal positions or by size in amino acid residues as described above may be excluded as individual species.", "Further, any number of fragments specified by N-terminal and C-terminal positions or by size in amino acid residues as described above may make up a polypeptide fragment in any combination and may optionally include non-GSSP4 polypeptide sequence as well.", "The term “GSSP4 fragment” as used herein refers to fragments of a full-length GSSP4 polypeptides that comprise at least 6 and any other integer number of amino acids up to 104 of the full-length GSSP4 polypeptide (defined above).", "GSSP4 polypeptides of the invention include variants, fragments, analogs and derivatives of the GSSP4 polypeptides described above, including modified GSSP4 polypeptides.", "Variants It will be recognized by one of ordinary skill in the art that some amino acids of the GSSP4 polypeptide sequences of the present invention can be varied without significant effect on the structure or function of the proteins; there will be critical amino acids in the sequence that determine activity.", "Thus, the invention further includes variants of GSSP4 polypeptides that have metabolic-related activity as described above.", "Such variants include GSSP4 polypeptide sequences with one or more amino acid deletions, insertions, inversions, repeats, and substitutions either from natural mutations or human manipulation selected according to general rules known in the art so as to have little effect on activity.", "Guidance concerning how to make phenotypically silent amino acid substitutions is provided below.", "There are two main approaches for studying the tolerance of an amino acid sequence to change (see, Bowie, et al.", "(1990) Science, 247, 1306-10).", "The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection.", "The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality.", "These studies have revealed that proteins are surprisingly tolerant of amino acid substitutions and indicate which amino acid changes are likely to be permissive at a certain position of the protein.", "For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.", "Other such phenotypically silent substitutions are described by Bowie et al.", "(supra) and the references cited therein.", "In the case of an amino acid substitution in the amino acid sequence of a polypeptide according to the invention, one or several amino acids can be replaced by “equivalent” amino acids.", "The expression “equivalent” amino acid is used herein to designate any amino acid that may be substituted for one of the amino acids having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.", "In particular embodiments, conservative substitutions of interest are shown in Table 1 under the heading of preferred substitutions.", "If such substitutions result in a change in biological activity, then more substantial changes, or as further described below in reference to amino acid classes, are introduced and the products screened.", "TABLE 1 Original Residue Exemplary Substitutions Preferred Substitutions Ala (A) val; leu; ile val Arg (R) lys; gin; asn lys Asn (N) gin; his; lys; arg gin Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gin; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu Substantial modifications in function or immunological identity of the GSSP4 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.", "Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.", "Non-conservative substitutions will entail exchanging a member of one of these classes for another class.", "Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.", "The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.", "Site-directed mutagenesis [Carter et al., Nucl.", "Acids Res., 13:4331 (1986); Zoller et al., Nucl.", "Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos.", "Trans.", "R. Soc.", "London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the GSSP4 variant DNA.", "Amino acids in the GSSP4 polypeptide sequences of the invention that are essential for function can also be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham, et al.", "(1989) Science 244(4908):1081-5).", "The latter procedure introduces single alanine mutations at every residue in the molecule.", "The resulting mutant molecules are then tested for metabolic-related activity using assays as described above.", "Of special interest are substitutions of charged amino acids with other charged or neutral amino acids that may produce proteins with highly desirable improved characteristics, such as less aggregation.", "Aggregation may not only reduce activity but also be problematic when preparing pharmaceutical or physiologically acceptable formulations, because aggregates can be immunogenic (see, e.g., Pinckard, et al., (1967) Clin.", "Exp.", "Immunol 2:331-340; Robbins, et al., (1987) Diabetes July;36(7):838-41; and Cleland, et al., (1993) Crit Rev Ther Drug Carrier Syst.", "10(4):307-77).", "Thus, the fragment, derivative, analog, or homolog of the GSSP4 polypeptides of the present invention may be, for example: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code (i.e.", "may be a non-naturally occurring amino acid); or (ii) one in which one or more of the amino acid residues includes a substituent group; or (iii) one in which the GSSP4 polypeptide is fused with another compound, such as a compound to increase the half-life of the fragment (for example, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the above form of the fragment, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the fragment or a pro-protein sequence.", "Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.", "A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of GSSP4 polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, not more than 40 conservative amino acid substitutions, not more than 30 conservative amino acid substitutions, and not more than 20 conservative amino acid substitutions.", "Also provided are polypeptides which comprise the amino acid sequence of a GSSP4 fragment, having at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.", "Another specific embodiment of a modified GSSP4 polypeptide of the invention is a polypeptide that is resistant to proteolysis, for example a GSSP4 polypeptide in which a —CONH— peptide bond is modified and replaced by one or more of the following: a (CH2NH) reduced bond; a (NHCO) retro inverso bond; a (CH2—O) methylene-oxy bond; a (CH2—S) thiomethylene bond; a (CH2CH2) carba bond; a (CO—CH2) cetomethylene bond; a (CHOH—CH2) hydroxyethylene bond); a (N—N) bound; a E-alcene bond; or a —CH═CH— bond.", "Thus, the invention also encompasses a GSSP4 polypeptide or a variant thereof in which at least one peptide bond has been modified as described above.", "In addition, amino acids have chirality within the body of either L or D. In some embodiments it is preferable to alter the chirality of the amino acids in the GSSP4 polypeptides of the invention in order to extend half-life within the body.", "Thus, in some embodiments, one or more of the amino acids are preferably in the L configuration.", "In other embodiments, one or more of the amino acids are preferably in the D configuration.", "Percent Identity The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 50% identical, at least 60% identical, or 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a GSSP4 polypeptide as described above.", "By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a GSSP4 polypeptide amino acid sequence is meant that the amino acid sequence is identical to the GSSP4 polypeptide sequence except that it may include up to five amino acid alterations per each 100 amino acids of the GSSP4 polypeptide amino acid sequence.", "The reference sequence is the GSSP4 polypeptide with a sequence corresponding to the sequences provided in SEQ ID NO:3.Thus, to obtain a polypeptide having an amino acid sequence at least 95% identical to a GSSP4 polypeptide amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the sequence may be inserted, deleted, or substituted with another amino acid compared with the GSSP4 polypeptide sequence.", "These alterations may occur at the amino or carboxy termini or anywhere between those terminal positions, interspersed either individually among residues in the sequence or in one or more contiguous groups within the sequence.", "As a practical matter, whether any particular polypeptide is a percentage identical to a GSSP4 polypeptide can be determined conventionally using known computer programs.", "Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, (1988) Proc Natl Acad Sci USA April;85(8):2444-8; Altschul et al., (1990) J. Mol.", "Biol.", "215(3):403-410; Thompson et al., (1994) Nucleic Acids Res.", "22(2):4673-4680; Higgins et al., (1996) Meth.", "Enzymol.", "266:383-402; Altschul et al., (1997) Nuc.", "Acids Res.", "25:3389-3402; Altschul et al., (1993) Nature Genetics 3:266-272).", "In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”), which is well known in the art (See, e.g., Karlin and Altschul (1990) Proc Natl Acad Sci USA March;87(6):2264-8; Altschul et al., 1990, 1993, 1997, all supra).", "In particular, five specific BLAST programs are used to perform the following tasks: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.", "The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.", "High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.", "Preferably, the scoring matrix used is the BLOSUM62 matrix (see, Gonnet et al., (1992) Science June 5;256(5062): 1443-5; Henikoff and Henikoff (1993) Proteins September;17(1):49-61).", "Less preferably, the PAM or PAM250 matrices may also be used (See, e.g., Schwartz and Dayhoff, eds, (1978) Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).", "The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology.", "Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (See, e.g., Karlin and Altschul, (1990) Proc Natl Acad Sci USA March;87(6):2264-8).", "The BLAST programs may be used with the default parameters or with modified parameters provided by the user.", "Preferably, the parameters are default parameters.", "A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al.", "(1990) Comp.", "App.", "Biosci.", "6:237-245.In a sequence alignment the query and subject sequences are both amino acid sequences.", "The result of said global sequence alignment is in percent identity.", "Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group=25 Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=247 or the length of the subject amino acid sequence, whichever is shorter.", "If the subject sequence is shorter than the query sequence due to N-or C-terminal deletions, not because of internal deletions, the results, in percent identity, must be manually corrected because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity.", "For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, that are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.", "Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.", "This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.", "This final percent identity score is what is used for the purposes of the present invention.", "Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score.", "That is, only query amino acid residues outside the farthest N- and C-terminal residues of the subject sequence.", "For example, a 90 amino acid residue subject sequence is aligned with a 100-residue query sequence to determine percent identity.", "The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not match/align with the first residues at the N-terminus.", "The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program.", "If the remaining 90 residues are perfectly matched the final percent identity would be 90%.", "In another example, a 90-residue subject sequence is compared with a 100-residue query sequence.", "This time the deletions are internal so there are no residues at the N- or C-termini of the subject sequence, which are not matched/aligned with the query.", "In this case, the percent identity calculated by FASTDB is not manually corrected.", "Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected.", "No other manual corrections are made for the purposes of the present invention.", "Production Note, throughout the disclosure, wherever GSSP4 polypeptides are discussed, GSSP4 fragments are specifically intended to be included as a preferred subset of GSSP4 polypeptides.", "Specific production methods are addressed in detail in sections III, IV, V, and VI of the present specification.", "In brief, GSSP4 polypeptides are preferably isolated from mammalian tissue samples, preferably human samples, or expressed from mammalian genes, preferably human genes, in mammalian cells, preferably human cells.", "The GSSP4 polypeptides of the invention can be made using expression methods known in the art.", "The polynucleotides encoding the desired polypeptides of the invention, including fragments thereof, are ligated into an expression vector suitable for the particular host used.", "Both eukaryotic and prokaryotic host systems can be used in forming recombinant polypeptides.", "The polypeptides are isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use.", "In addition, shorter protein fragments may be produced by chemical synthesis.", "Purification is by techniques known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like.", "Nucleotides comprising the coding sequence of the polynucleotides of the invention, or fragments thereof, are cloned into expression vectors by anyone skilled in the art.", "Preferred expression vectors include eukaryotic and prokaryotic expression vectors.", "Further preferred is the pGEX-3X expression vector.", "Purification of GSSP4 polypeptides may be facilitated by recombinant fusion of a heterologous peptide to the C-or N-terminus of the GSSP4 polypeptides.", "Preferred fusion polypeptides expressed include heterologous polypeptides containing a His-tag added to the C-terminus, a glutathione-S-transferase (GST) tag and a factor Xa protease digestion site.", "A preferred method of making the polypeptides of the invention includes a method comprising the following steps: Escherichia coli cells, preferably BL21, and preferably comprising the pGEX-3X vector containing polynucleotides of the invention containing a His tag added to the C-terminus, GST tag and Xa protease site, are grown to subconfluence, preferably absorbance of 0.8, and induced with isopropyl beta-D-thiogalactoside.", "Cells are pelleted, washed and lysed in buffer, preferably in buffer containing guanidine hydrochloride.", "Polypeptides are allowed to bind to nickel, preferably Ni-NTA containing beads, and are washed with buffers, preferably buffers containing urea.", "Polypeptide bound to nickel are equilibrated with buffer, preferably buffer containing sodium chloride and calcium chloride, preferably at pH 7.5.Polypeptides bound to Nickel are digested, ie.", "treated with buffer comprising protease, preferably protease factor Xa.", "Digestion is preferably carried out at room temperature for 12 to 20 hours.", "The cleaved GST tag, if any, is washed away with buffer, preferably buffer with urea, preferably at pH 5.9.Polypeptides of the invention are eluted with buffer, preferably buffer with urea, preferably at pH 4.5.Polypeptides of the invention are refolded, preferably by methods comprising the following steps: Polypeptides are diluted in buffer common in the art, preferably to a concentration of 100 microgram/ml, preferably in buffer containing urea at pH 4.5.Polypeptides are dialyzed against buffer, preferably dialysis buffer comprising 4M urea, 5 mM cysteine, 0.02% Tween-20, 10% glycerol, 10 mM Tris, 150 mM sodium chloride, and 100 mM NaH2PO4, preferably at pH 8.3.Dialysis buffer comprising 2 M urea is used to replace the initial dialysis buffer, and dialysis buffer is replaced at least 1, 2, or 3 times over at least 1, 2, 3, 4, 5, or 6 days.", "The refolded polypeptide is desalted by any method known in the art, preferably using a spin column.", "Polypeptides of the invention are further purified by methods known in the art.", "Preferably polpeptides are purified with reverse phase HPLC.", "Polypeptides are preferably eluted with 0.08% trifluoroacetic acid and a 10-50% acetonitrile gradient, and elution is monitored preferably at 206 nm.", "Acetonitrile and trifluoroacetic acid are removed for the compositions comprising the purified polypeptide, preferably by evaporation by lyophilization.", "Further examples of methods useful for the expression or purification of polypeptides of the invention are found described in Methods in Enzymology.", "The nucleic acid encoding a GSSP4 fragment can be obtained by PCR from a vector containing the GSSP4 nucleotide sequence using oligonucleotide primers complementary to the desired GSSP4 cDNA and containing restriction endonuclease sequences.", "Transfection of a GSSP4 fragment-expressing vector into mouse NIH 3T3 cells is one embodiment of introducing polynucleotides into host cells.", "Introduction of a polynucleotide encoding a polypeptide into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods.", "Such methods are described in many standard laboratory manuals, such as Davis et al.", "((1986) Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., Amsterdam).", "It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.", "A polypeptide of this invention (i.e.", "a GSSP4 fragment) can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.", "Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.", "Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.", "Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.", "Preferably the polypeptides of the invention are non-glycosylated.", "In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.", "In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides.", "For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, see, e.g., U.S. Pat.", "No.", "5,641,670, issued Jun.", "24, 1997; International Publication No.", "WO 96/29411, published Sep. 26, 1996; International Publication No.", "WO 94/12650, published Aug. 4, 1994; Koller et al., (1989) Proc Natl Acad Sci USA November;86(22):8932-5; Koller et al., (1989) Proc Natl Acad Sci USA November;86(22):8927-31; and Zijlstra et al.", "(1989) Nature November 23;342(6248):435-8; the disclosures of each of which are incorporated by reference in their entireties).", "Modifications In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (See, e.g., Creighton, 1983 Proteins.", "New York, N.Y.: W. H. Freeman and Company; and Hunkapiller et al., (1984) Nature July 12-18;310(5973): 105-11).", "For example, a relative short fragment of the invention can be synthesized by use of a peptide synthesizer.", "Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the fragment sequence.", "Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.", "Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).", "The invention encompasses polypeptide fragments which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.", "Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.", "Additional post-translational modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.", "The polypeptide fragments may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.", "Also provided by the invention are chemically modified derivatives of the polypeptides of the invention that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity.", "See U.S. Pat.", "No.", "4,179,337.The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.", "The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.", "The polymer may be of any molecular weight, and may be branched or unbranched.", "For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.", "Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).", "The polyethylene glycol molecules (or other chemical moieties) should be attached to the polypeptide with consideration of effects on functional or antigenic domains of the polypeptide.", "There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al.", "(1992) Exp Hematol.", "September;20(8):1028-35, reporting pegylation of GM-CSF using tresyl chloride).", "Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.", "Multimers The polypeptide fragments of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers).", "Accordingly, the present invention relates to monomers and multimers of the polypeptide fragments of the invention, their preparation, and compositions (preferably, pharmaceutical or physiologically acceptable compositions) containing them.", "In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers.", "In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.", "Multimers encompassed by the invention may be homomers or heteromers.", "As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the GSSP4 polypeptides of the invention (including polypeptide fragments, variants, splice variants, and fusion proteins corresponding to these polypeptide fragments as described herein).", "These homomers may contain polypeptide fragments having identical or different amino acid sequences.", "In a specific embodiment, a homomer of the invention is a multimer containing only polypeptide fragments having an identical amino acid sequence.", "In another specific embodiment, a homomer of the invention is a multimer containing polypeptide fragments having different amino acid sequences.", "In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptide fragments having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptide fragments having identical and/or different amino acid sequences).", "In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.", "As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., corresponding to different proteins or polypeptide fragments thereof) in addition to the polypeptides of the invention.", "In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.", "In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.", "Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.", "Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution.", "In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.", "In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention.", "Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone).", "In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences, which interact in the native (i.e., naturally occurring) polypeptide.", "In another instance, the covalent associations are the consequence of chemical or recombinant manipulation.", "Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.", "In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat.", "No.", "5,478,925).", "In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein).", "In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety).", "In another embodiment, two or more polypeptides of the invention are joined through peptide linkers.", "Examples include those peptide linkers described in U.S. Pat.", "No.", "5,073,627 (hereby incorporated by reference).", "Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.", "Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence.", "Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference.", "Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.", "Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity.", "Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers.", "One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al.", "FEBS Letters (1994) May 16;344(2-3):191-5.and in U.S. patent application Ser.", "No.", "08/446,922, hereby incorporated by reference.", "Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.", "In another example, proteins of the invention are associated by interactions between Flag® & polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence.", "In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti Flag® antibody.", "The multimers of the invention may be generated using chemical techniques known in the art.", "For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "Additionally, at least 30 techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art.", "In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hyrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (See, e.g., U.S. Pat.", "No.", "5,478,925, which is herein incorporated by reference in its entirety).", "II.", "GSSP4 Polynucleotides of the Invention Preferred polynucleotides are those that encode GSSP4 polypeptides of the invention.", "The recombinant polynucleotides encoding GSSP4 polypeptides can be used in a variety of ways, including, but not limited to, expressing the polypeptides in recombinant cells for use in screening assays for antagonists and agonists of its activity as well as to facilitate its purification for use in a variety of ways including, but not limited to screening assays for agonists and antagonists of its activity, diagnostic screens, and raising antibodies, as well as treatment and/or prevention of metabolic-related diseases and disorders and/or to reduce body mass.", "The invention relates to the polynucleotides encoding GSSP4 polypeptides and variant polypeptide fragments thereof as described herein.", "These polynucleotides may be purified, isolated, and/or recombinant.", "In all cases, the desired GSSP4 polynucleotides of the invention are those that encode GSSP4 polypeptides of the invention having metabolic-related activity as described and discussed herein.", "Fragments A polynucleotide fragment is a polynucleotide having a sequence that entirely is the same as part, but not all, of the full length GSSP4 polypeptide or a specified GSSP4 polypeptide nucleotide sequence.", "Such fragments may be “free-standing”, i.e.", "not part of or fused to other polynucleotides, or they may be comprised within another non-GSSP4 (heterologous) polynucleotide of which they form a part or region.", "However, several GSSP4 polynucleotide fragments may be comprised within a single polynucleotide.", "The GSSP4 polynucleotides of the invention comprise from 18 consecutive bases to 18 consecutive bases less than the full length polynucleotide sequences encoding the intact GSSP4 polypeptides, for example the GSSP4 polynucleotide sequences in SEQ ID NO:1 or 2.In one aspect of this embodiment, the polynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, or 648 consecutive nucleotides of a polynucleotide of the present invention.", "In addition to the above preferred nucleic acid sizes, further preferred nucleic acids comprise at least 18 nucleotides, wherein “at least 18” is defined as any integer between 18 and the integer representing 18 nucleotides less than the 3′ most nucleotide position of the intact GSSP4 polypeptides cDNA as set forth in SEQ ID NO: 2, or elsewhere herein.", "Further included as preferred polynucleotides of the present invention are nucleic acid fragments at least 18 nucleotides in length, as described above, that are further specified in terms of their 5′ and 3′ position set forth in the sequence listing below.", "For allelic and degenerate and other variants, position 1 is defined as the 5′ most nucleotide of the ORF, i.e., the nucleotide “A” of the start codon (ATG) with the remaining nucleotides numbered consecutively.", "Therefore, every combination of a 5′ and 3′ nucleotide position that a polynucleotide fragment invention, at least 18 contiguous nucleotides in length, could occupy on an intact GSSP4 polypeptide polynucleotide of the present invention is included in the invention as an individual species.", "The polynucleotide fragments specified by 5′ and 3′ positions can be immediately envisaged and are therefore not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "It is noted that the above species of polynucleotide fragments of the present invention may alternatively be described by the formula “x to y”; where “x” equals the 5′ most nucleotide position and “y” equals the 3′ most nucleotide position of the polynucleotide; and further where “x” equals an integer between 1 and the number of nucleotides of the polynucleotide sequence of the present invention minus 18, and where “y” equals an integer between 19 and the number of nucleotides of the polynucleotide sequence of the present invention minus 18 nucleotides; and where “x” is an integer smaller then “y” by at least 18.The GSSP4 polynucleotide fragments of the invention comprise from 18 consecutive bases to the full length polynucleotide sequence encoding the GSSP4 fragments described in Section II of the Preferred Embodiments of the Invention.", "In one aspect of this embodiment, the polynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 740, 770, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100 or 2200 consecutive nucleotides of a polynucleotide of the present invention.", "In addition to the above preferred nucleic acid sizes, further preferred nucleic acids comprise at least 18 nucleotides, wherein “at least 18” is defined as any integer between 18 and the integer corresponding to the 3′ most nucleotide position of a GSSP4 fragment cDNA herein.", "Further included as preferred polynucleotides of the present invention are nucleic acid fragments at least 18 nucleotides in length, as described above, that are further specified in terms of their 5′ and 3′ position.", "The 5′ and 3′ positions are represented by the position numbers set forth in the sequence listing below.", "For allelic and degenerate and other variants, position 1 is defined as the 5′ most nucleotide of the open reading frame (ORF), i.e., the nucleotide “A” of the start codon (ATG) with the remaining nucleotides numbered consecutively.", "Therefore, every combination of a 5′ and 3′ nucleotide position that a polynucleotide fragment invention, at least 18 contiguous nucleotides in length, could occupy on a GSSP4 fragment polynucleotide of the present invention is included in the invention as an individual species.", "The polynucleotide fragments specified by 5′ and 3′ positions can be immediately envisaged and are therefore not individually listed solely for the purpose of not unnecessarily lengthening the specification.", "It is noted that the above species of polynucleotide fragments of the present invention may alternatively be described by the formula “x to y”; where “x” equals the 5′ most nucleotide position and “y” equals the 3′ most nucleotide position of the polynucleotide; and further where “x” equals an integer between 1 and the number of nucleotides of the GSSP4 polynucleotide sequences of the present invention minus 18, and where “y” equals an integer between 9 and the number of nucleotides of the GSSP4 polynucleotide sequences of the present invention; and where “x” is an integer smaller than “y” by at least 18.Every combination of “x” and ‘y’ positions are included as specific embodiments of the invention.", "Moreover, the formula “x” to “y” may be modified as “‘x1-x2” to “y1-y2’”, wherein “x1-x2” and “y1-y2” represent positional ranges selected from any two nucleotide positions of the sequence listing.", "Alternative formulas include “‘x1-x2” to “y’” and “‘x” to “y1-y2’”.", "These specific embodiments, and other polynucleotide fragment embodiments described herein may be modified as being “at least”, “equal to”, “equal to or less than”, “less than”, “at least ______ but not greater than ______”or “from ______ to ______”.", "a specified size or specified 5′ and/or 3′ positions.", "The present invention also provides for the exclusion of any species of polynucleotide fragments of the present invention specified by 5′ and 3′ positions or polynucleotides specified by size in nucleotides as described above.", "Any number of fragments specified by 5′ and 3′ positions or by size in nucleotides, as described above, may be excluded.", "Variants In other preferred embodiments, variants of GSSP4 polynucleotides encoding GSSP4 polypeptides are envisioned.", "Variants of polynucleotides, as the term is used herein, are polynucleotides whose sequence differs from a reference polynucleotide.", "A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.", "Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.", "Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.", "Polynucleotide variants that comprise a sequence substantially different from those described above but that, due to the degeneracy of the genetic code, still encode GSSP4 polypeptides of the present invention are also specifically envisioned.", "It would also be routine for one skilled in the art to generate the degenerate variants described above, for instance, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by other mammalian or bacterial host cells).", "As stated above, variant polynucleotides may occur naturally, such as a natural allelic variant, or by recombinant methods.", "By an “allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (See, e.g., B. Lewin, (1990) Genes IV, Oxford University Press, New York).", "Non-naturally occurring variants may be produced using art-known mutagenesis techniques.", "Such nucleic acid variants include those produced by nucleotide substitutions, deletions, or additions.", "The substitutions, deletions, or additions may involve one or more nucleotides.", "Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.", "Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of GSSP4 polypeptides of the invention.", "Also preferred in this regard are conservative substitutions.", "Nucleotide changes present in a variant polynucleotide are preferably silent, which means that they do not alter the amino acids encoded by the polynucleotide.", "However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence.", "In cases where the nucleotide substitutions result in one or more amino acid changes, preferred GSSP4 polypeptides include those that retain one or more metabolic-related activity as described in Section I of the Preferred Embodiments of the Invention.", "By “retain the same activities” is meant that the activity measured using the polypeptide encoded by the variant GSSP4 polynucleotide in assays is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, and not more than 101%, 102%, 103%, 104%, 105%, 110%, 115%, 120% or 125% of the activity measured using a GSSP4 polypeptide described in the Examples Section herein.", "By the activity being “increased” is meant that the activity measured using the polypeptide encoded by the variant GSSP4 polynucleotide in assays is at least 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 450%, or 500% of the activity measured using a GSSP4 polypeptide described in the Examples Section herein.", "By the activity being “decrease” is meant that the activity measured using the polypeptide encoded by the variant GSSP4 polynucleotide in assays is decreased by at least 25%, 30%, 35%, 40%, 45%, or 50% of the activity measured using a GSSP4 polypeptide described in the Examples Section herein.", "Percent Identity The present invention is further directed to nucleic acid molecules having sequences at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences of SEQ ID NO:1, or 2 or fragments thereof that encode a polypeptide having metabolic-related activity as described in Section I of the Preferred Embodiments of the Invention.", "Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences shown in SEQ ID NO:1, or 2 or fragments thereof will encode a polypeptide having biological activity.", "In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.", "It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having biological activity.", "This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described previously in Section I of the Preferred Embodiments of the Invention.", "By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the GSSP4 polypeptide.", "In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted, inserted, or substituted with another nucleotide.", "The query sequence may be an entire sequence or any fragment specified as described herein.", "The methods of determining and defining whether any particular nucleic acid molecule or polypeptide is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be done by using known computer programs.", "A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., ((1990) Comput Appl Biosci.", "July;6(3):237-45).", "In a sequence alignment the query and subject sequences are both DNA sequences.", "An RNA sequence can be compared by first converting U's to T's.", "The result of said global sequence alignment is in percent identity.", "Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.", "If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results.", "This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity.", "For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence.", "Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.", "This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.", "This corrected score is what is used for the purposes of the present invention.", "Only nucleotides outside the 5′ and 3′ nucleotides of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.", "No other manual corrections are made for the purposes of the present invention.", "Fusions Further included in the present invention are polynucleotides encoding the polypeptides of the present invention that are fused in frame to the coding sequences for additional heterologous amino acid sequences.", "Also included in the present invention are nucleic acids encoding polypeptides of the present invention together with additional, non-coding sequences, including for example, but not limited to non-coding 5′ and 3′ sequences, vector sequence, sequences used for purification, probing, or priming.", "For example, heterologous sequences include transcribed, non-translated sequences that may play a role in transcription, and mRNA processing, for example, ribosome binding and stability of mRNA.", "The heterologous sequences may alternatively comprise additional coding sequences that provide additional functionalities.", "Thus, a nucleotide sequence encoding a polypeptide may be fused to a tag sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide.", "In certain preferred embodiments of this aspect of the invention, the tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available.", "For instance, hexa-histidine provides for convenient purification of the fusion protein (See, Gentz et al., (1989) Proc Natl Acad Sci USA February;86(3):821-4).", "The “HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein (See, Wilson et al., (1984) Cell 37(3):767-78).", "As discussed above, other such fusion proteins include GSSP4 fragment cDNA fused to Fc at the N- or C-terminus.", "III.", "Recombinant Vectors of the Invention The term “vector” is used herein to designate either a circular or a linear DNA or RNA molecule, that is either double-stranded or single-stranded, and that comprises at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism.", "The present invention relates to recombinant vectors comprising any one of the polynucleotides described herein.", "The present invention encompasses a family of recombinant vectors that comprise polynucleotides encoding GSSP4 polypeptides of the invention.", "In a first preferred embodiment, a recombinant vector of the invention is used to amplify the inserted polynucleotide in a suitable cell host, this polynucleotide being amplified every time that the recombinant vector replicates.", "The inserted polynucleotide can be one that encodes GSSP4 polypeptides of the invention.", "A second preferred embodiment of the recombinant vectors according to the invention consists of expression vectors comprising polynucleotides encoding GSSP4 polypeptides of the invention.", "Within certain embodiments, expression vectors are employed to express a GSSP4 polypeptide of the invention, preferably a modified GSSP4 fragment described in the present invention, which can be then purified and, for example, be used as a treatment for metabolic-related diseases, or simply to reduce body mass of individuals.", "Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources, that drive expression of the genes of interest in host cells.", "Dominant drug selection markers for establishing permanent, stable, cell clones expressing the products are generally included in the expression vectors of the invention, as they are elements that link expression of the drug selection markers to expression of the polypeptide.", "More particularly, the present invention relates to expression vectors which include nucleic acids encoding a GSSP4 polypeptide of the invention, or a modified GSSP4 fragment as described herein, or variants or fragments thereof, under the control of a regulatory sequence selected among GSSP4 polypeptides, or alternatively under the control of an exogenous regulatory sequence.", "Consequently, preferred expression vectors of the invention are selected from the group consisting of: (a) a GSSP4 fragmentregulatory sequence and driving the expression of a coding polynucleotide operably linked thereto; and (b) a GSSP4 fragment coding sequence of the invention, operably linked to regulatory sequences allowing its expression in a suitable cell host and/or host organism.", "Some of the elements which can be found in the vectors of the present invention are described in further detail in the following sections.", "1) General Features of the Expression Vectors of the Invention: A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid, or even a linear DNA molecule which may consist of a chromosomal, non-chromosomal, semi-synthetic or synthetic DNA.", "Such a recombinant vector can comprise a transcriptional unit comprising an assembly of: (1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers.", "Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription; (2) a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in (1); and (3) appropriate transcription initiation and termination sequences.", "Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.", "Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include a N-terminal residue.", "This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.", "Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.", "The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium.", "In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5′-flanking non-transcribed sequences.", "DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.", "2) Regulatory Elements Promoters The suitable promoter regions used in the expression vectors of the present invention are chosen taking into account the cell host in which the heterologous gene is expressed.", "The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.", "Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter.", "A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed.", "Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.", "Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.", "Preferred bacterial promoters are the LacI, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trp promoters (EP 0036776), the polyhedrin promoter, or the p10 protein promoter from baculovirus (Kit Novagen) (Smith et al., (1983) Mol Cell Biol December;3(12):2156-65; O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.", "Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L.", "In addition, promoters specific for a particular cell type may be chosen, such as those facilitating expression in adipose tissue, muscle tissue, or liver.", "Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.", "The choice of a promoter is well within the ability of a person skilled in the field of genetic engineering.", "For example, one may refer to Sambrook et al.", "(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, Vol.", "1, 2, 3 (1989), or also to the procedures described by Fuller et al.", "(1996) Immunology in Current Protocols in Molecular Biology.", "Other Regulatory Elements Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.", "The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.", "Also contemplated as an element of the expression cassette is a terminator.", "These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.", "3) Selectable Markers Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.", "The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRP 1 for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli, or levan saccharase for mycobacteria, this latter marker being a negative selection marker.", "4) Preferred Vectors Bacterial Vectors As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017).", "Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and pGEM1 (Promega Biotec, Madison, Wis., USA).", "Large numbers of other suitable vectors are known to those of skill in the art, and are commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).", "Baculovirus Vectors A suitable vector for the expression of polypeptides of the invention is a baculovirus vector that can be propagated in insect cells and in insect cell lines.", "A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC NoCRL 1711) which is derived from Spodoptera frugiperda.", "Other suitable vectors for the expression of GSSP4 polypeptides in a baculovirus expression system include those described by Chai et al.", "(1993; Biotechnol Appl Biochem.", "December; 18 (Pt 3):259-73); Vlasak et al.", "(1983; Eur J Biochem September 1;135(1):123-6); and Lenhard et al.", "(1996; Gene March 9;169(2):187-90).", "Viral Vectors In one specific embodiment, the vector is derived from an adenovirus.", "Preferred adenovirus vectors according to the invention are those described by Feldman and Steg (1996; Semin Interv Cardiol September;1(3):203-8) or Ohno et al.", "(1994; Science August 5;265(5173):781-4).", "Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin (French patent application No.", "FR-93.05954).", "Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans.", "These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.", "Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vivo gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus.", "Particularly preferred Murine Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298).", "Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728).", "Other preferred retroviral vectors are those described in Roth et al.", "(1996), PCT Application No WO 93/25234, PCT Application No WO 94/06920, Roux et al., ((1989) Proc Natl Acad Sci USA December;86(23):9079-83), Julan et al., (1992) J. Gen. Virol.", "3:3251-3255 and Neda et al., ((1991) J Biol Chem August 5;266(22):14143-6).", "Yet another viral vector system that is contemplated by the invention consists of the adeno-associated virus (AAV).", "The adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., (1992) Curr Top Microbiol Immunol;158:97-129).", "It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al., (1992) Am J Respir Cell Mol Biol September;7(3):349-56; Samulski et al., (1989) J Virol September;63(9):3822-8; McLaughlin et al., (1989) Am.", "J. Hum.", "Genet.", "59:561-569).", "One advantageous feature of AAV derives from its reduced efficacy for transducing primary cells relative to transformed cells.", "5) Delivery of the Recombinant Vectors In order to effect expression of the polynucleotides of the invention, these constructs must be delivered into a cell.", "This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain disease states.", "One mechanism is viral infection where the expression construct is encapsulated in an infectious viral particle.", "Several non-viral methods for the transfer of polynucleotides into cultured mammalian cells are also contemplated by the present invention, and include, without being limited to, calcium phosphate precipitation (Graham et al., (1973) Virology August;54(2):536-9; Chen et al., (1987) Mol Cell Biol August;7(8):2745-52), DEAE-dextran (Gopal, (1985) Mol Cell Biol May;5(5): 1188-90), electroporation (Tur-Kaspa et al., (1986) Mol Cell Biol February;6(2):716-8; Potter et al., (1984) Proc Natl Acad Sci USA November;81(22):7161-5.", "), direct microinjection (Harland et al., (1985) J Cell Biol September;101(3):1094-9), DNA-loaded liposomes (Nicolau et al., (1982) Biochim Biophys Acta October 11;721(2):185-90; Fraley et al., (1979) Proc Natl Acad Sci USA July;76(7):3348-52), and receptor-mediated transfection (Wu and Wu, (1987) J Biol Chem April 5;262(10):4429-32; Wu and Wu (1988) Biochemistry February 9;27(3):887-92).", "Some of these techniques may be successfully adapted for in vivo or ex vivo use.", "Once the expression polynucleotide has been delivered into the cell, it may be stably integrated into the genome of the recipient cell.", "This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non specific location (gene augmentation).", "In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.", "Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.", "One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect.", "This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.", "Compositions for use in vitro and in vivo comprising a “naked” polynucleotide are described in PCT application No.", "WO 90/11092 (Vical Inc.) and also in PCT application No.", "WO 95/11307 (Institut Pasteur, INSERM, Universit{acute over (e )} d'Ottawa) as well as in the articles of Tascon et al.", "(1996) Nature Medicine.", "2(8):888-892 and of Huygen et al.", "((1996) Nat Med August;2(8):893-8).", "In still another embodiment of the invention, the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al.", "((1990) Curr Genet February;17(2):97-103).", "In a further embodiment, the polynucleotide of the invention may be entrapped in a liposome (Ghosh and Bacchawat, (1991) Targeted Diagn Ther;4:87-103; Wong et al., (1980) Gene 10:87-94; Nicolau et al., (1987) Methods Enzymol.;149:157-76).", "These liposomes may further be targeted to cells expressing LSR by incorporating leptin, triglycerides, ACRP30, or other known LSR ligands into the liposome membrane.", "In a specific embodiment, the invention provides a composition for the in vivo production of an GSSP4 polypeptides described herein.", "It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells of the tissue to express the said polypeptide.", "The amount of vector to be injected to the desired host organism varies according to the site of injection.", "As an indicative dose, it will be injected between 0.1 and 100 μg of the vector in an animal body, preferably a mammal body, for example a mouse body.", "In another embodiment of the vector according to the invention, it may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell.", "In a subsequent step, the cell that has been transformed with the vector coding for the desired GSSP4 polypeptides or the desired fragment thereof is reintroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.", "IV.", "Recombinant Cells of the Invention Another object of the invention consists of host cells recombinant for, i.e., that have been transformed or transfected with one of the polynucleotides described herein, and more precisely a polynucleotide comprising a polynucleotide encoding a GSSP4 polypeptide of the invention such as any one of those described in “Polynucleotides of the Invention”.", "These polynucleotides can be present in cells as a result of transient or stable transfection.", "The invention includes host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as any one of those described in “Recombinant Vectors of the Invention”.", "Generally, a recombinant host cell of the invention comprises at least one of the polynucleotides or the recombinant vectors of the invention that are described herein.", "Preferred host cells used as recipients for the recombinant vectors of the invention are the following: a) Prokaryotic host cells: Escherichia coli strains (I.E.", "DH5-α strain), Bacillus subtilis, Salmonella typhimurium, and strains from species like Pseudomonas, Streptomyces and Staphylococcus, and b) Eukaryotic host cells: HeLa cells (ATCC NoCCL2; NoCCL2.1; NoCCL2.2), Cv 1 cells (ATCC NoCCL70), COS cells (ATCC NoCRL1650; NoCRL1651), Sf-9 cells (ATCC NoCRL1711), C127 cells (ATCC No CRL-1804), 3T3 (ATCC No CRL-6361), CHO (ATCC No CCL-61), human kidney 293 (ATCC No 45504; No CRL-1573), BHK (ECACC No 84100501; No 84111301), PLC cells, HepG2, and Hep3B.", "The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.", "Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.", "Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.", "Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.", "Such methods are well known by the skilled artisan.", "Further, according to the invention, these recombinant cells can be created in vitro or in vivo in an animal, preferably a mammal, most preferably selected from the group consisting of mice, rats, dogs, pigs, sheep, cattle, and primates, not to include humans.", "Recombinant cells created in vitro can also be later surgically implanted in an animal, for example.", "Methods to create recombinant cells in vivo in animals are well-known in the art.", "The present invention also encompasses primary, secondary, and immortalized homologously recombinant host cells of vertebrate origin, preferably mammalian origin and particularly human origin, that have been engineered to: a) insert exogenous (heterologous) polynucleotides into the endogenous chromosomal DNA of a targeted gene, b) delete endogenous chromosomal DNA, and/or c) replace endogenous chromosomal DNA with exogenous polynucleotides.", "Insertions, deletions, and/or replacements of polynucleotide sequences may be to the coding sequences of the targeted gene and/or to regulatory regions, such as promoter and enhancer sequences, operably associated with the targeted gene.", "The present invention further relates to a method of making a homologously recombinant host cell in vitro or in vivo, wherein the expression of a targeted gene not normally expressed in the cell is altered.", "Preferably the alteration causes expression of the targeted gene under normal growth conditions or under conditions suitable for producing the polypeptide encoded by the targeted gene.", "The method comprises the steps of: (a) transfecting the cell in vitro or in vivo with a polynucleotide construct, the polynucleotide construct comprising; (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; and (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination.", "The present invention further relates to a method of altering the expression of a targeted gene in a cell in vitro or in vivo wherein the gene is not normally expressed in the cell, comprising the steps of: (a) transfecting the cell in vitro or in vivo with a polynucleotide construct, the polynucleotide construct comprising: (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; and (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell; and (c) maintaining the homologously recombinant cell in vitro or in vivo under conditions appropriate for expression of the gene.", "The present invention further relates to a method of making a polypeptide of the present invention by altering the expression of a targeted endogenous gene in a cell in vitro or in vivo wherein the gene is not normally expressed in the cell, comprising the steps of: a) transfecting the cell in vitro with a polynucleotide construct, the polynucleotide construct comprising: (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell; and c) maintaining the homologously recombinant cell in vitro or in vivo under conditions appropriate for expression of the gene thereby making the polypeptide.", "The present invention further relates to a polynucleotide construct that alters the expression of a targeted gene in a cell type in which the gene is not normally expressed.", "This occurs when a polynucleotide construct is inserted into the chromosomal DNA of the target cell, wherein the polynucleotide construct comprises: a) a targeting sequence; b) a regulatory sequence and/or coding sequence; and c) an unpaired splice-donor site, if necessary.", "Further included are polynucleotide constructs, as described above, wherein the construct further comprises a polynucleotide which encodes a polypeptide and is in-frame with the targeted endogenous gene after homologous recombination with chromosomal DNA.", "The compositions may be produced, and methods performed, by techniques known in the art, such as those described in U.S. Pat.", "Nos.", "6,054,288; 6,048,729; 6,048,724; 6,048,524; 5,994,127; 5,968,502; 5,965,125; 5,869,239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580,734; International Publication Nos:WO96/29411, WO 94/12650; and scientific articles described by Koller et al., (1994) Annu.", "Rev.", "Immunol.", "10:705-730; the disclosures of each of which are incorporated by reference in their entireties).", "The expression of GSSP4s in mammalian, and typically human, cells may be rendered defective, or alternatively it may be enhanced, with the insertion of a GSSP4 genomic or cDNA sequence with the replacement of the GSSP4 gene counterpart in the genome of an animal cell by a GSSP4 polynucleotide according to the invention.", "These genetic alterations may be generated by homologous recombination events using specific DNA constructs that have been previously described.", "One kind of host cell that may be used are mammalian zygotes, such as murine zygotes.", "For example, murine zygotes may undergo microinjection with a purified DNA molecule of interest, for example a purified DNA molecule that has previously been adjusted to a concentration range from 1 ng/ml—for BAC inserts—3 ng/μl —for P1 bacteriophage inserts—in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine, and 70 μM spermidine.", "When the DNA to be microinjected has a large size, polyamines and high salt concentrations can be used in order to avoid mechanical breakage of this DNA, as described by Schedl et al ((1993) Nature March 18;362(6417):258-61).", "Any one of the polynucleotides of the invention, including the DNA constructs described herein, may be introduced in an embryonic stem (ES) cell line, preferably a mouse ES cell line.", "ES cell lines are derived from pluripotent, uncommitted cells of the inner cell mass of pre-implantation blastocysts.", "Preferred ES cell lines are the following: ES-E14TG2a (ATCC No.CRL-1821), ES-D3 (ATCC No.CRL1934 and No.", "CRL-11632), YS001 (ATCC No.", "CRL-11776), 36.5 (ATCC No.", "CRL-11116).", "To maintain ES cells in an uncommitted state, they are cultured in the presence of growth inhibited feeder cells which provide the appropriate signals to preserve this embryonic phenotype and serve as a matrix for ES cell adherence.", "Preferred feeder cells are primary embryonic fibroblasts that are established from tissue of day 13-day 14 embryos of virtually any mouse strain, that are maintained in culture, such as described by Abbondanzo et al.", "(1993; Methods Enzymol;225:803-23) and are inhibited in growth by irradiation, such as described by Robertson ((1987) Embryo-derived stem cell lines.", "In: E. J. Robertson Ed.", "Teratocarcinomas and embrionic stem cells: a practical approach.", "IRL Press, Oxford), or by the presence of an inhibitory concentration of LIF, such as described by Pease and Williams (1990; Exp Cell Res.", "October;190(2):209-11).", "The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.", "Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.", "Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.", "Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.", "Such methods are well known by the skilled artisan.", "V. Transgenic Animals The present invention also provides methods and compositions for the generation of non-human animals and plants that express recombinant GSSP4 polypeptides, i.e.", "recombinant GSSP4 fragments or full-length GSSP4 polypeptides.", "The animals or plants can be transgenic, i.e.", "each of their cells contains a gene encoding a GSSP4 polypeptide, or, alternatively, a polynucleotide encoding a GSSP4 polypeptide can be introduced into somatic cells of the animal or plant, e.g.", "into mammary secretory epithelial cells of a mammal.", "In preferred embodiments, the non-human animal is a mammal such as a cow, sheep, goat, pig, or rabbit.", "Methods of making transgenic animals such as mammals are well known to those of skill in the art, and any such method can be used in the present invention.", "Briefly, transgenic mammals can be produced, e.g., by transfecting a pluripotential stem cell such as an ES cell with a polynucleotide encoding a polypeptide of interest.", "Successfully transformed ES cells can then be introduced into an early stage embryo which is then implanted into the uterus of a mammal of the same species.", "In certain cases, the transformed (“transgenic”) cells will comprise part of the germ line of the resulting animal, and adult animals comprising the transgenic cells in the germ line can then be mated to other animals, thereby eventually producing a population of transgenic animals that have the transgene in each of their cells, and which can stably transmit the transgene to each of their offspring.", "Other methods of introducing the polynucleotide can be used, for example introducing the polynucleotide encoding the polypeptide of interest into a fertilized egg or early stage embryo via microinjection.", "Alternatively, the transgene may be introduced into an animal by infection of zygotes with a retrovirus containing the transgene (Jaenisch, R. (1976) Proc.", "Natl.", "Acad.", "Sci.", "USA 73, 1260-1264).", "Methods of making transgenic mammals are described, e.g., in Wall et al.", "(1992) J Cell Biochem 1992 June;49(2): 113-20; Hogan, et al.", "(1986) in Manipulating the mouse embryo.", "A Laboratory Manual.", "Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; in WO 91/08216, or in U.S. Pat.", "No.", "4,736,866.In a preferred method, the polynucleotides are microinjected into the fertilized oocyte.", "Typically, fertilized oocytes are microinjected using standard techniques, and then cultured in vitrountil a “pre-implantation embryo” is obtained.", "Such pre-implantation embryos preferably contain approximately 16 to 150 cells.", "Methods for culturing fertilized oocytes to the pre-implantation stage are described, e.g., by Gordon et al.", "((1984) Methods in Enzymology, 101, 414); Hogan et al.", "((1986) in Manipulating the mouse embryo.", "A Laboratory Manual.", "Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y) (for the mouse embryo); Hammer et al.", "((1985) Nature, 315, 680) (for rabbit and porcine embryos); Gandolfi et al.", "((1987) J. Reprod.", "Fert.", "81, 23-28); Rexroad et al.", "((1988) J. Anim.", "Sci.", "66, 947-953) (for ovine embryos); and Eyestone et al.", "((1989) J. Reprod.", "Fert.", "85, 715-720); Camous et al.", "((1984) J. Reprod.", "Fert.", "72, 779-785); and Heyman et al.", "((1987) Theriogenology 27, 5968) (for bovine embryos); the disclosures of each of which are incorporated herein in their entireties.", "Pre-implantation embryos are then transferred to an appropriate female by standard methods to permit the birth of a transgenic or chimeric animal, depending upon the stage of development when the transgene is introduced.", "As the frequency of transgene incorporation is often low, the detection of transgene integration in pre-implantation embryos is often desirable using any of the herein-described methods.", "Any of a number of methods can be used to detect the presence of a transgene in a pre-implantation embryo.", "For example, one or more cells may be removed from the pre-implantation embryo, and the presence or absence of the transgene in the removed cell or cells can be detected using any standard method e.g.", "PCR.", "Alternatively, the presence of a transgene can be detected in utero or post partum using standard methods.", "In a particularly preferred embodiment of the present invention, transgenic mammals are generated that secrete recombinant GSSP4 polypeptides in their milk.", "As the mammary gland is a highly efficient protein-producing organ, such methods can be used to produce protein concentrations in the gram per liter range, and often significantly more.", "Preferably, expression in the mammary gland is accomplished by operably linking the polynucleotide encoding the GSSP4 polypeptide to a mammary gland specific promoter and, optionally, other regulatory elements.", "Suitable promoters and other elements include, but are not limited to, those derived from mammalian short and long WAP, alpha, beta, and kappa, casein, alpha and beta lactoglobulin, beta-CN 5′ genes, as well as the the mouse mammary tumor virus (MMTV) promoter.", "Such promoters and other elements may be derived from any mammal, including, but not limited to, cows, goats, sheep, pigs, mice, rabbits, and guinea pigs.", "Promoter and other regulatory sequences, vectors, and other relevant teachings are provided, e.g., by Clark (1998) J Mammary Gland Biol Neoplasia 3:337-50; Jost et al.", "(1999) Nat.", "Biotechnol 17:160-4; U.S. Pat.", "Nos.", "5,994,616; 6,140,552; 6,013,857; Sohn et al.", "(1999) DNA Cell Biol.", "18:845-52; Kim et al.", "(1999) J. Biochem.", "(Japan) 126:320-5; Soulier et al.", "(1999) Euro.", "J. Biochem.", "260:533-9; Zhang et al.", "(1997) Chin.", "J. Biotech.", "13:271-6; Rijnkels et al.", "(1998) Transgen.", "Res.", "7:5-14; Korhonen et al.", "(1997) Euro.", "J. Biochem.", "245:482-9; Uusi-Oukari et al.", "(1997) Transgen.", "Res.", "6:75-84; Hitchin et al.", "(1996) Prot.", "Expr.", "Purif.", "7:247-52; Platenburg et al.", "(1994) Transgen.", "Res.", "3:99-108; Heng-Cherl et al.", "(1993) Animal Biotech 4:89-107; and Christa et al.", "(2000) Euro.", "J. Biochem.", "267:1665-71; the entire disclosures of each of which is herein incorporated by reference.", "In another embodiment, the polypeptides of the invention can be produced in milk by introducing polynucleotides encoding the polypeptides into somatic cells of the mammary gland in vivo, e.g.", "mammary secreting epithelial cells.", "For example, plasmid DNA can be infused through the nipple canal, e.g.", "in association with DEAE-dextran (see, e.g., Hens et al.", "(2000) Biochim.", "Biophys.", "Acta 1523:161-171), in association with a ligand that can lead to receptor-mediated endocytosis of the construct (see, e.g., Sobolev et al.", "(1998) 273:7928-33), or in a viral vector such as a retroviral vector, e.g.", "the Gibbon ape leukemia virus (see, e.g., Archer et al.", "(1994) PNAS 91:6840-6844).", "In any of these embodiments, the polynucleotide may be operably linked to a mammary gland specific promoter, as described above, or, alternatively, any strongly expressing promoter such as CMV or MoMLV LTR.", "The suitability of any vector, promoter, regulatory element, etc.", "for use in the present invention can be assessed beforehand by transfecting cells such as mammary epithelial cells, e.g.", "MacT cells (bovine mammary epithelial cells) or GME cells (goat mammary epithelial cells), in vitro and assessing the efficiency of transfection and expression of the transgene in the cells.", "For in vivo administration, the polynucleotides can be administered in any suitable formulation, at any of a range of concentrations (e.g.", "1-500 μg/ml, preferably 50-100 μg/ml), at any volume (e.g.", "1-100 ml, preferably 1 to 20 ml), and can be administered any number of times (e.g.", "1, 2, 3, 5, or 10 times), at any frequency (e.g.", "every 1, 2, 3, 5, 10, or any number of days).", "Suitable concentrations, frequencies, modes of administration, etc.", "will depend upon the particular polynucleotide, vector, animal, etc., and can readily be determined by one of skill in the art.", "In a preferred embodiment, a retroviral vector such as as Gibbon ape leukemia viral vector is used, as described in Archer et al.", "((1994) PNAS 91:6840-6844).", "As retroviral infection typically requires cell division, cell division in the mammary glands can be stimulated in conjunction with the administration of the vector, e.g.", "using a factor such as estrodiol benzoate, progesterone, reserpine, or dexamethasone.", "Further, retroviral and other methods of infection can be facilitated using accessory compounds such as polybrene.", "In any of the herein-described methods for obtaining GSSP4 polypeptides from milk, the quantity of milk obtained, and thus the quantity of GSSP4 polypeptides produced, can be enhanced using any standard method of lactation induction, e.g.", "using hexestrol, estrogen, and/or progesterone.", "The polynucleotides used in such embodiments can either encode a full-length GSSP4 polypeptide or a GSSP4 fragment.", "Typically, the encoded polypeptide will include a signal sequence to ensure the secretion of the protein into the milk.", "Where a full length GSSP4 sequence is used, the full length protein can, e.g., be isolated from milk and cleaved in vitro using a suitable protease.", "Alternatively, a second, protease-encoding polynucleotide can be introduced into the animal or into the mammary gland cells, whereby expression of the protease results in the cleavage of the GSSP4 polypeptide in vivo, thereby allowing the direct isolation of GSSP4 fragments from milk.", "VI.", "Pharmaceutical or Physiologically Acceptable Compositions of the Invention The GSSP4 polypeptides of the invention can be administered to non-human animals and/or humans, alone or in pharmaceutical or physiologically acceptable compositions where they are mixed with suitable carriers or excipient(s).", "The pharmaceutical or physiologically acceptable composition is then provided at a therapeutically effective dose.", "A therapeutically effective dose refers to that amount of a GSSP4 polypeptide sufficient to result in prevention or amelioration of symptoms or physiological status of metabolic-related diseases or disorders as determined by the methods described herein.", "A therapeutically effective dose can also refer to the amount of a GSSP4 polypeptide necessary for a reduction in weight or a prevention of an increase in weight or prevention of an increase in the rate of weight gain in persons desiring this affect for cosmetic reasons.", "A therapeutically effective dosage of a GSSP4 polypeptide of the invention is that dosage that is adequate to promote weight loss or weight gain with continued periodic use or administration.", "Techniques for formulation and administration of GSSP4 polypeptides may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.", "Other diseases or disorders that GSSP4 polypeptides of the invention could be used to treat or prevent include, but are not limited to, obesity and metabolic-related diseases and disorders such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.", "Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.", "The GSSP4 polypeptides may also be used to enhance physical performance during work or exercise or enhance a feeling of general well-being.", "Physical performance activities include walking, running, jumping, lifting and/or climbing.", "The GSSP4 polypeptides or antagonists thereof may also be used to treat dyslexia, attention-deficit disorder (ADD), attention-deficit/hyperactivity disorder (ADHD), and psychiatric disorders such as schizophrenia by modulating fatty acid metabolism, more specifically, the production of certain long-chain polyunsaturated fatty acids.", "It is expressly considered that the GSSP4 polypeptides of the invention may be provided alone or in combination with other pharmaceutically or physiologically acceptable compounds.", "Other compounds useful for the treatment of obesity and other diseases and disorders are currently well-known in the art.", "In a preferred embodiment, the GSSP4 polypeptides are useful for, and used in, the treatment of insulin resistance and diabetes using methods described herein and known in the art.", "More particularly, a preferred embodiments relates to process for the therapeutic modification and regulation of glucose metabolism in an animal or human subject, which comprises administering to a subject in need of treatment (alternatively on a timed daily basis) GSSP4 polypeptide (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.", "Further preferred embodiments relate to methods for the prophylaxis or treatment of diabetes comprising administering to a subject in need of treatment (alternatively on a timed daily basis) a GSSP4 polypeptide (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.", "Routes of Administration.", "Suitable routes of administration include oral, nasal, rectal, transmucosal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocular injections using methods known in the art.", "A particularly useful method of administering compounds for promoting weight loss involves surgical implantation, for example into the abdominal cavity of the recipient, of a device for delivering GSSP4 polypeptidesover an extended period of time.", "Other particularly preferred routes of administration are aerosol and depot formulation.", "Sustained release formulations, particularly depot of the invented medicaments are expressly contemplated.", "Composition/Formulation Pharmaceutical or physiologically acceptable compositions and medicaments for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries.", "Proper formulation is dependent upon the route of administration chosen.", "Certain of the medicaments described herein will include a pharmaceutically or physiologically acceptable acceptable carrier and at least one polypeptide that is a GSSP4 polypeptide of the invention.", "For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer such as a phosphate or bicarbonate buffer.", "For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.", "Such penetrants are generally known in the art.", "Pharmaceutical or physiologically acceptable preparations that can be taken orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.", "The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.", "In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.", "In addition, stabilizers may be added.", "All formulations for oral administration should be in dosages suitable for such administration.", "For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.", "For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable gaseous propellant, e.g.", "carbon dioxide.", "In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.", "Capsules and cartridges of, e.g.", "gelatin, for use in an inhaler or insufflator, may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.", "The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.", "Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.", "The compositions may take such forms as suspensions, solutions or emulsions in aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.", "Pharmaceutical or physiologically acceptable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.", "Aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.", "Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.", "Alternatively, the active ingredient may be in powder or lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.", "In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.", "Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.", "Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.", "Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.", "Various sustained release materials have been established and are well known by those skilled in the art.", "Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.", "Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.", "The pharmaceutical or physiologically acceptable compositions also may comprise suitable solid or gel phase carriers or excipients.", "Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.", "Effective Dosage.", "Pharmaceutical or physiologically acceptable compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose.", "More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.", "Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.", "For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays.", "For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range shown to increase leptin or lipoprotein uptake or binding in an in vitro system.", "Such information can be used to more accurately determine useful doses in humans.", "A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient.", "Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50, (the dose lethal to 50% of the test population) and the ED50 (the dose therapeutically effective in 50% of the population).", "The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD5O and ED5O.", "Compounds that exhibit high therapeutic indices are preferred.", "The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.", "The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50, with little or no toxicity.", "The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.", "The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.", "(See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.", "1).", "Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain or prevent weight loss or gain, depending on the particular situation.", "Dosages necessary to achieve these effects will depend on individual characteristics and route of administration.", "Dosage intervals can also be determined using the value for the minimum effective concentration.", "Compounds should be administered using a regimen that maintains plasma levels above the minimum effective concentration for 10-90% of the time, preferably between 30-90%; and most preferably between 50-90%.", "In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.", "The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.", "A preferred dosage range for the amount of a GSSP4 polypeptide of the invention, which can be administered on a daily or regular basis to achieve desired results, including a reduction in levels of circulating plasma triglyceride-rich lipoproteins, range from 0.01-0.5 mg/kg body mass.", "A more preferred dosage range is from 0.05-0.1 mg/kg.", "Of course, these daily dosages can be delivered or administered in small amounts periodically during the course of a day.", "It is noted that these dosage ranges are only preferred ranges and are not meant to be limiting to the invention.", "VII.", "Methods of Treatment The invention is drawn inter alia to methods of preventing or treating metabolic-related diseases and disorders comprising providing an individual in need of such treatment with a GSSP4 polypeptide of the invention.", "Preferably, the GSSP4 polypeptide has metabolic-related activity either in vitro or in vivo.", "Preferably the GSSP4 polypeptide is provided to the individual in a pharmaceutical composition that is preferably taken orally.", "Preferably the individual is a mammal, and most preferably a human.", "In preferred embodiments, the metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "In highly preferred embodiments, GSSP4 polypeptides in pharmaceutical compositions are used to modulate body weight in healthy individuals for cosmetic reasons.", "The invention also features a method of preventing or treating metabolic-related diseases and disorders comprising providing an individual in need of such treatment with a compound identified by assays of the invention (described in Section VI of the Preferred Embodiments of the Invention and in the Examples).", "Preferably these compounds antagonize or agonize effects of GSSP4 polypeptides in cells in vitro, muscles ex vivo, or in animal models.", "Alternatively, these compounds agonize or antagonize the effects of GSSP4 polypeptides on glucose metabolism, fatty acid metabolism, or lipid metabolism.", "Preferably, the compound is provided to the individual in a pharmaceutical composition that is preferably taken orally.", "Preferably the individual is a mammal, and most preferably a human.", "In preferred embodiments, the metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.", "Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.", "In highly preferred embodiments, the pharmaceutical compositions are used to modulate glucose levels.", "In highly preferred embodiments, the pharmaceutical compositions are used to modulate body weight for cosmetic reasons.", "In a further preferred embodiment, NIDDM patients are often treated with oral insulin secretagogues, such as 1,1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate (BTS67582) or sulfonylureas including tolbutamide, tolazamide, chlorpropamide, glibendamide, glimepiride, glipizide and glidazide, or with insulin sensitising agents including metformin, ciglitazone, trogitazone and pioglitazone.", "A further use of the present invention is in therapy of NIDDM patients to improve their weight and glucose control, comprising a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in combination with an oral insulin secretagogue or an insulin sensitising agent.", "Preferably, the oral insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.", "Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.", "The present invention further provides a method of improving the weight and glucose control of NIDDM patients comprising the administration of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect alone, without an oral insulin secretagogue or an insulin sensitising agent.", "In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with the oral insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent).", "Accordingly, the present invention further provides a product containing a composition of said a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect and an oral insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of weight and glucose control in NIDDM patients.", "The ratio of the present composition to the oral insulin secretagogue or insulin sensitising agent is such that the quantity of each active ingredient employed will be such as to provide a therapeutically effective level, but will not be larger than the quantity recommended as safe for administration.", "The action of reducing insulin resistance by the present invention indicates that compounds of such invention may be useful in the manufacture of a medicament which can be used as an insulin sensitiser.", "Accordingly, the present invention further provides for the use in the manufacture of a medicament which is an insulin sensitiser.", "In further embodiments, some patients who are diagnosed with Insulin Dependent Diabetes Mellitus (IDDM, Type I) can also show a certain amount of insulin resistance.", "Therefore, there may be benefits in treating these patients with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in order to reduce their insulin resistance.", "This would mean that these patients would require a lower dosage of insulin in order to maintain similar or better control of their diabetes since the insulin dose would be associated with a greater blood glucose lowering efficacy.", "Such therapy would provide long-term benefits in terms of reducing the detrimental effects which can be caused by prolonged high-dosage of insulin treatment.", "Additionally, some Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II) patients are also treated with insulin and have insulin resistance.", "Accordingly the present invention further provides a method for, and the use thereof in the manufacture of the medicament for, reducing the amount of insulin required daily by a human having NIDDM.", "The present invention also provides a method for, and the use of the composition in the manufacture of a medicament for, the prophylaxis of long-term detrimental effects caused by prolonged high dosage of insulin in humans having IDDM.", "A consequence of the resistance is that glucose concentrations rise.", "This leads, in turn, to an increased release of insulin.", "Hyperinsulinemia, both in the fasting and postprandial states, is a hallmark of insulin resistance.", "Hyperinsulinemia is also caused by stimulation of gluconeogenesis.", "Epidemilogical studies have shown that hyperinsulinemia is a risk factor for morbidity and mortality in cardiovascular disease (Smith U.", "(1994) Am.", "J. Clin.", "Nutr.", "59, suppl.", "686S).", "Accordingly, the invention also provides therapeutics and methods for reducing or preventing hypersecretion of insulin and disorders or conditions resulting therefrom.", "NIDDM is associated with various complications.", "As defined herein, “complications of NIDDM” is referred to as cardiovascular complications or several of the metabolic and circulatory disturbances that are associated with hyperglycemia, e.g., insulin resistance, hyperinsulinemia and/or hyperproinsulinemia, delayed insulin release, dyslipidemia, retinopathy, peripheral neuropathy, hypertension, and other coronary artery diseases (CADs).", "CAD is a major cause of morbidity and mortality in patients with NIDDM.", "Thus, by providing therapeutics and methods for reducing glucose levels, the invention provides therapeutics and methods for treating and preventing NIDDM and consequences thereof.", "The invention also provides therapeutics and methods for treating and preventing having impaired glucose tolerance (IGT).", "The usual meaning of impaired glucose tolerance is that it is a condition associated with insulin-resistance which is intermediate between frank, NIDDM and normal glucose tolerance (NGT).", "A high percentage of the IGT population is known to progress to NIDDM relative to persons with normal glucose tolerance (Sad, et al., New Engl.", "J. Med.", "1988; 319:1500-6).", "Thus, by providing therapeutics and methods for reducing or preventing IGT, i.e., for normalizing insulin resistance, the progression to NIDDM can be delayed or prevented.", "IGT is diagnosed by a procedure wherein an affected person's postprandial glucose response is determined to be abnormal as assessed by 2-hour postprandial plasma glucose levels.", "In this test, a measured amount of glucose is given to the patient and blood glucose levels measured regular intervals, usually every half hour for the first two hours and every hour thereafter.", "In a “normal” or non-IGT individual glucose levels rise during the first two hours to level less than 140 mg/dl and then drop rapidly.", "In an IGT individual, the blood glucose levels are higher and the drop-off level is at a slower rate.", "Resistance to insulin-stimulated glucose uptake in individuals who do not become frankly hyperglycemic nevertheless increases the likelihood of these individuals to develop numerous other diseases.", "In particular, an attempt to compensate for insulin resistance sets in motion a series of events that play an important role in the development of both hypertension and coronary artery disease (CAD), such as premature atherosclerotic vascular disease.", "This cluster of abnormalities is commonly called the “Metabolic Syndrome”, or the “Insulin-Resistance Syndrome” or “Syndrome X”.", "Increased plasma triglyceride and decreased HDL-cholesterol concentrations, conditions which are known to be associated with CAD, have also been reported to be associated with insulin resistance.", "Thus, by providing therapeutics and methods for reducing or preventing insulin resistance, the invention provides methods for reducing and/or preventing the appearance of insulin-resistance syndrome.", "Yet other diseases are associated with insulin resistance and, thus, be treated or prevented according to the methods of the invention.", "For example, obesity, which is the result of an imbalance between caloric intake and energy expenditure is highly correlated with insulin resistance and diabetes (Hotamisligil, Spiegelman et al., Science, 1993, 259:87-91).", "In humans obesity can be defined as a body weight exceeding 20% of the desirable body weight for individuals of the same sex, height and frame (Slans, L. B., in Endocrinology & Metabolism, 2d Ed., McGraw-Hill, New York 1987, pp.", "1203-1244; see also, R H. Williams, Textbook of Endocrinology, 1974, pp.", "904-916).", "In other animals (or also in humans) obesity can be determined by body weight patterns correlated with prolactin profiles given that members of a species that are young, lean and “healthy” (i.e., free of any disorders, not just metabolic disorders) have daily plasma prolactin level profiles that follow a regular pattern that is highly reproducible with a small standard deviation.", "Obesity, or excess fat deposits, correlate with and may trigger the onset of various lipid metabolism disorders, e.g.", "hypertension, Type II diabetes (NIDDM), atherosclerosis, cardiovascular disease, etc.", "Even in the absence of clinical obesity (according to the above definition) the reduction of body fat stores (notably visceral fat stores) in man especially on a long-term or permanent basis would be of significant benefit, both cosmetically and physiologically.", "Thus, by preventing or treating obesity, the methods of the invention will allow an individual to have a more comfortable life and avoid the onset of various diseases triggered by obesity.", "In yet another embodiment, the invention provides a method for treating a subject having polycystic ovary syndrome (PCOS).", "PCOS is among the most common disorders of premenopausal women, affecting 5-10% of this population.", "It is a syndrome of unknown Etiology characterized by hyperandrogenism, chronic anovulation, defects in insulin action, insulin secretion, ovarian steroidogenesis and fibrinolysis.", "Women with PCOS frequently are insulin resistant and at increased risk to develop glucose intolerance or NIDDM in the third and fourth decades of life (Dunaif et al.", "(1996) J. Clin.", "Endocrinol.", "Metab.", "81:3299).", "Hyperandrogenism also is a feature of a variety of diverse insulin-resistant states, from the type A syndrome, through leprechaunism and lipoatrophic diabetes, to the type B syndrome, when these conditions occur in premenopausal women.", "It has been suggested that hyperinsulinemia per se causes hyperandrogenism.", "Insulin-sensitizing agents, e.g., troglitazone, have been shown to be effective in PCOS and that, in particular, the defects in insulin action, insulin secretion, ovarian steroidogenosis and fibrinolysis are improved (Ehrman et al.", "(1997) J. Clin.", "Invest.", "100:1230), such as in insulin-resistant humans.", "Accordingly, the invention provides methods for reducing insulin resistance, normalizing blood glucose thus treating and/or preventing PCOS.", "Insulin resistance is also often associated with infections and cancer.", "Thus, prevention or reducing insulin resistance according to the methods of the invention may prevent or reduce infections and cancer.", "Insulin resistance can be diagnosed by various methods, such as by the intravenous glucose tolerance test or by measuring the fasting insulin level.", "It is well known that there is an excellent correlation between the height of the fasting insulin level and the degree of insulin resistance.", "Therefore, one could use elevated fasting insulin levels as a surrogate marker for insulin resistance for the purpose of identifying which normal glucose tolerance (NGT) individuals have insulin resistance.", "Another way to do this is to follow the approach as disclosed in The New England Journal of Medicine, No.", "3, pp.", "1188 (1995), i.e.", "to select obese subjects as an initial criteria for entry into the treatment group.", "Some obese subjects have impaired glucose tolerance (IGT) while others have normal glucose tolerance (NGT).", "Since essentially all obese subjects are insulin resistant, i.e.", "even the NGT obese subjects are insulin resistant, they have fasting hyperinsulinemia.", "Therefore, the target of the treatment according to the present invention can be defined as NGT individuals who are obese or who have fasting hyperinsulinemia, or who have both.", "Insulin resistance can also be diagnosed by the euglycemic glucose clamp test.", "This test involves the simultaneous administration of a constant insulin infusion and a variable rate glucose infusion.", "During the test, which lasts 3-4 hours, the plasma glucose concentration is kept constant at euglycemic levels by measuring the glucose level every 5-10 minutes and then adjusting the variable rate glucose infusion to keep the plasma glucose level unchanged.", "Under these circumstances, the rate of glucose entry into the bloodstream is equal to the overall rate of glucose disposal in the body.", "The difference between the rate of glucose disposal in the basal state (no insulin infusion) and the insulin infused state, represents insulin mediated glucose uptake.", "In normal individuals, insulin causes brisk and large increase in overall body glucose disposal, whereas in NIDDM subjects, this effect of insulin is greatly blunted, and is only 20-30% of normal.", "In insulin resistant subjects with either IGT or NGT, the rate of insulin stimulated glucose disposal is about half way between normal and NIDDM.", "For example, at a steady state plasma insulin concentration of about 100 uU/ml (a physiologic level) the glucose disposal rate in normal subjects is about 7 mg/kg/min.", "In NIDDM subjects, it is about 2.5 mg/.kg/min., and in patients with IGT (or insulin resistant subjects with NGT) it is about 4-5 mg/kg/min.", "This is a highly reproducible and precise test, and can distinguish patients within these categories.", "It is also known, that as subjects become more insulin resistant, the fasting insulin level rises.", "There is an excellent positive correlation between the height of the fasting insulin level and the magnitude of the insulin resistance as measured by euglycemic glucose clamp tests and, therefore, this provides the rationale for using fasting insulin levels as a surrogate measure of insulin resistance.", "Thus, any of the above-described tests or other tests known in the art can be used to determine that a subject is insulin-resistant, which patient can then be treated according to the methods of the invention to reduce or cure the insulin-resistance.", "Alternatively, the methods of the invention can also be used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.", "More generally, the instant invention is drawn to treatment with GSSP4 polypeptides where an individual is shown to have a particular genotype for GSSP4 marker.", "Treatment comprises providing pharmaceutically acceptable GSSP4 polypeptides to the individual.", "The exact amount of GSSP4 polypeptide provided would be determined through clinical trials under the guidance of qualified physicians, but would be expected to be in the range of 5-7 mg per individual per day.", "In general, a preferred range would be from 0.5 to 14 mg per individual per day, with a highly preferred range being between 1 and 10 mg per individual per day.", "Individuals who could benefit from treatment with GSSP4 polypeptides could be identified through genotyping.", "GSSP4 Genotyping The methods treatment using genotyping to identify individuals that would benefit from treatments of the invention are based on the finding that single nucleotide polymorphisms (SNPs) in GSSP4 have been identified that show an association in obese adolescents with free fatty acid (FFA) and respiratory quotient levels, others that show an association with the relationship between BMI and leptin, and still others that show an association with glucose levels.", "Further, a combination of GSSP4 SNPs associated with FFA and leptin metabolism may also predict people who will be seriously overweight.", "Briefly, the term “genotype” as used herein refers to the identity of the alleles present in an individual or a sample.", "The term “genotyping” a sample or an individual for a biallelic marker consists of determining the specific allele or the specific nucleotide carried by an individual at a biallelic marker.", "Biallelic markers generally consist of a polymorphism at one single base position.", "Each biallelic marker therefore corresponds to two forms of a polynucleotide sequence which, when compared with one another, present a nucleotide modification at one position.", "Usually, the nucleotide modification involves the substitution of one nucleotide for another, optionally either the original or the alternative allele of the biallelic markers disclosed in SEQ ID NO:1.Optionally either the original or the alternative allele of these biallelic markers may be specified as being present.", "Preferred polynucleotides may consist of, consist essentially of, or comprise a contiguous span of nucleotides upstream and down-stream of the alternate allele position noted in SEQ ID No:1 as well as sequences which are complementary thereto.", "The “contiguous span” may be at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID.", "Methods of genotyping comprise determining the identity of a nucleotide at GSSP4 biallelic marker site by any method known in the art.", "Preferably, microsequencing is used.", "The genotype is used to determine whether an individual should be treated with GSSP4 polypeptides.", "Thus, these genotyping methods are performed on nucleic acid samples derived from a single individual.", "These methods are well-known in the art, and discussed fully in the applications referenced briefly below.", "Any method known in the art can be used to identify the nucleotide present at a biallelic marker site.", "Since the biallelic marker allele to be detected has been identified and specified in the present invention, detection will prove simple for one of ordinary skill in the art by employing any of a number of techniques.", "Many genotyping methods require the previous amplification of the DNA region carrying the biallelic marker of interest.", "While the amplification of target or signal is often preferred at present, ultrasensitive detection methods that do not require amplification are also encompassed by the present genotyping methods.", "Methods well-known to those skilled in the art that can be used to detect biallelic polymorphisms include methods such as conventional dot blot analysis, single strand conformational polymorphism analysis (SSCP; Orita et al.", "(1989) Proc Natl Acad Sci USA April;86(8):2766-70), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other conventional techniques as described in Sheffield et al.", "(1991; Am J Hum Genet October;49(4):699-706); White et al.", "(1992), Grompe et al.", "((1989) Proc Natl Acad Sci USA August;86(15):5888-92; (1993) Nat Genet.", "October;5(2):111-7).", "Another method for determining the identity of the nucleotide present at a particular polymorphic site employs a specialized exonuclease-resistant nucleotide derivative as described in U.S. Pat.", "No.", "4,656,127.Preferred methods involve directly determining the identity of the nucleotide present at a biallelic marker site by sequencing assay, allele-specific amplification assay, or hybridization assay.", "The following is a description of some preferred methods.", "A highly preferred method is the microsequencing technique.", "The term “sequencing” is used herein to refer to polymerase extension of duplex primer/template complexes and includes both traditional sequencing and microsequencing.", "Preferred biallelic markers, as shown in SEQ ID NO:1 and corresponding 47 mers as shown in SEQ ID NOs:4-18, include but are not limited to: 1.Biallelic marker 1: position base 148, alternate alleles A/G 2.Biallelic marker 2: position base 2551, alternate alleles G/A 3.Biallelic marker 3: position base 4417, alternate alleles A/C 4.Biallelic marker 4: position base 6322, alternate alleles A/G (amino acid change isoleucine to valine) 5.Biallelic marker 7: position base 816, alternate alleles G/A 6.Biallelic marker 8: position base 924, alternate alleles G/A 7.Biallelic marker 9: position base 1206, alternate alleles C/A 8.Biallelic marker 10: position base 1851, alternate alleles T/C 9.Biallelic marker 11: position base 3124, alternate alleles C/T 10.Biallelic marker 12: position base 3563, alternate alleles G/A 11.Biallelic marker 13: position base 3792, alternate alleles G/A 12.Biallelic marker 14: position base 5757, alternate alleles T/C 1) Sequencing Assays The nucleotide present at a polymorphic site can be determined by sequencing methods.", "In a preferred embodiment, DNA samples are subjected to PCR amplification before sequencing using any method known in the art.", "Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol.", "Sequence analysis allows the identification of the base present at the biallelic marker site.", "2) Microsequencing Assays In microsequencing methods, the nucleotide at a polymorphic site in a target DNA is detected by a single nucleotide primer extension reaction.", "This method involves appropriate microsequencing primers that hybridize just upstream of the polymorphic base of interest in the target nucleic acid.", "A polymerase is used to specifically extend the 3′ end of the primer with one single ddNTP (chain terminator) complementary to the nucleotide at the polymorphic site.", "The identity of the incorporated nucleotide is then determined in any suitable way.", "Preferred microsequencing primers are described in SEQ ID NO:1.Typically, microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the incorporated nucleotide as described in EP 412 883.Alternatively capillary electrophoresis can be used in order to process a higher number of assays simultaneously.", "Different approaches can be used for the labeling and detection of ddNTPs.", "A homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok ((1997) Nucleic Acids Res.", "January 15;25(2):347-53) and Chen et al.", "((1997) Proc Natl Acad Sci USA September 30;94(20):10756-61).", "In this method, amplified genomic DNA fragments containing polymorphic sites are incubated with a 5′-fluorescein-labeled primer in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq polymerase.", "The dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template.", "At the end of the genotyping reaction, the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification.", "All these steps can be performed in the same tube and the fluorescence changes can be monitored in real time.", "Alternatively, the extended primer may be analyzed by MALDI-TOF Mass Spectrometry.", "The base at the polymorphic site is identified by the mass added onto the microsequencing primer (see Haff and Smirnov, (1997) Nucleic Acids Res.", "September 15;25(18):3749-50; (1997) Genome Res.", "April;7(4):378-88).", "Microsequencing may be achieved by the established microsequencing method or by developments or derivatives thereof.", "Alternative methods include several solid-phase microsequencing techniques.", "The basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogeneous phase assay, in which the primer or the target molecule is immobilized or captured onto a solid support.", "To simplify the primer separation and the terminal nucleotide addition analysis, oligonucleotides are attached to solid supports or are modified in such ways that permit affinity separation as well as polymerase extension.", "The 5′ ends and internal nucleotides of synthetic oligonucleotides can be modified in a number of different ways to permit different affinity separation approaches, e.g., biotinylation.", "If a single affinity group is used on the oligonucleotides, the oligonucleotides can be separated from the incorporated terminator regent.", "This eliminates the need of physical or size separation.", "More than one oligonucleotide can be separated from the terminator reagent and analyzed simultaneously if more than one affinity group is used.", "This permits the analysis of several nucleic acid species or more nucleic acid sequence information per extension reaction.", "The affinity group need not be on the priming oligonucleotide but could alternatively be present on the template.", "For example, immobilization can be carried out via an interaction between biotinylated DNA and streptavidin-coated microtitration wells or avidin-coated polystyrene particles.", "In the same manner, oligonucleotides or templates may be attached to a solid support in a high-density format.", "In such solid phase microsequencing reactions, incorporated ddNTPs can be radiolabeled (Syvänen, (1994) Clin Chim Acta.", "May;226(2):225-36) or linked to fluorescein (Livak and Hainer, (1994) Hum Mutat.;3(4):379-85).", "The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques.", "The detection of fluorescein-linked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such as p-nitrophenyl phosphate).", "Other possible reporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., (1993) Clin Chem.", "November;39(11 Pt 1):2282-7) or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712).", "As yet another alternative solid-phase microsequencing procedure, Nyren et al.", "((1993) Anal Biochem.", "January;208(1):171-5).", "described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA).", "Pastinen et al.", "((1997) Genome Res.", "June;7(6):606-14) describe a method for multiplex detection of single nucleotide polymorphism in which the solid phase minisequencing principle is applied to an oligonucleotide array format.", "High-density arrays of DNA probes attached to a solid support (DNA chips) are further described below.", "It will be appreciated that any primer having a 3′ end immediately adjacent to the polymorphic nucleotide may be used.", "Similarly, it will be appreciated that microsequencing analysis may be performed for any biallelic marker or any combination of biallelic markers of the present invention.", "3) Allele-Specific Amplification Assay Methods Discrimination between the two alleles of a biallelic marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without, or at a much higher rate than, amplification of the other allele.", "This is accomplished by placing the polymorphic base at the 3′ end of one of the amplification primers.", "Because the extension forms from the 3′ end of the primer, a mismatch at or near this position has an inhibitory effect on amplification.", "Therefore, under appropriate amplification conditions, these primers only direct amplification on their complementary allele.", "Determining the precise location of the mismatch and the corresponding assay conditions are well with the ordinary skill in the art.", "The “Oligonucleotide Ligation Assay” (OLA) uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules.", "One of the oligonucleotides is biotinylated, and the other is detectably labeled.", "If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected.", "OLA is capable of detecting single nucleotide polymorphisms and may be advantageously combined with PCR as described by Nickerson et al.", "((1990) Proc Natl Acad Sci USA November;87(22):8923-7).", "In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.", "Other amplification methods which are particularly suited for the detection of single nucleotide polymorphism include LCR (ligase chain reaction) and Gap LCR (GLCR).", "LCR uses two pairs of probes to exponentially amplify a specific target.", "The sequences of each pair of oligonucleotides are selected to permit the pair to hybridize to abutting sequences of the same strand of the target.", "Such hybridization forms a substrate for a template-dependant ligase.", "In accordance with the present invention, LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallelic marker site.", "In one embodiment, either oligonucleotide will be designed to include the biallelic marker site.", "In such an embodiment, the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the biallelic marker on the oligonucleotide.", "In an alternative embodiment, the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a “gap” is created as described in WO 90/01069.This gap is then “filled” with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides.", "Thus at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential allele-specific amplification of the desired sequence is obtained.", "Ligase/Polymerase-mediated Genetic Bit Analysis™ is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271).", "This method involves the incorporation of a nucleoside triphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a primer molecule, and their subsequent ligation to a second oligonucleotide.", "The reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution.", "4) Hybridization Assay Methods A preferred method of determining the identity of the nucleotide present at a biallelic marker site involves nucleic acid hybridization.", "The hybridization probes, which can be conveniently used in such reactions, preferably include probes specific for GSSP4 cDNA surrounding GSSP4 biallelic markers.", "Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybridization and solid-phase hybridization (see Sambrook et al., supra).", "Hybridization refers to the formation of a duplex structure by two single stranded nucleic acids due to complementary base pairing.", "Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch.", "Specific probes can be designed that hybridize to one form of a biallelic marker and not to the other and therefore are able to discriminate between different allelic forms.", "Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele.", "Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles.", "Stringent, sequence specific hybridization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., supra).", "Stringent conditions are sequence dependent and will be different in different circumstances.", "Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.", "Although such hybridizations can be performed in solution, it is preferred to employ a solid-phase hybridization assay.", "The target DNA comprising a biallelic marker of the present invention may be amplified prior to the hybridization reaction.", "The presence of a specific allele in the sample is determined by detecting the presence or the absence of stable hybrid duplexes formed between the probe and the target DNA.", "The detection of hybrid duplexes can be carried out by a number of methods.", "Various detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybrid duplexes.", "Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected.", "Those skilled in the art will recognize that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate.", "Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the primers and probes.", "Two recently developed assays allow hybridization-based allele discrimination with no need for separations or washes (see Landegren U. et al., (1998) Genome Res.", "August;8(8):769-76).", "The TaqMan assay takes advantage of the 5′ nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product.", "TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence resonance energy transfer (FRET).", "Cleavage of the TaqMan probe by the advancing polymerase during amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence.", "All reagents necessary to detect two allelic variants can be assembled at the beginning of the reaction and the results are monitored in real time (see Livak et al., 1995).", "In an alternative homogeneous hybridization based procedure, molecular beacons are used for allele discriminations.", "Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions.", "When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., (1998) Nat Biotechnol.", "January;16(1):49-53).", "The polynucleotides provided herein can be used to produce probes which can be used in hybridization assays for the detection of biallelic marker alleles in biological samples.", "These probes are characterized in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising a biallelic marker of the present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide variation.", "A particularly preferred probe is 25 nucleotides in length.", "Preferably the biallelic marker is within 4 nucleotides of the center of the polynucleotide probe.", "In particularly preferred probes, the biallelic marker is at the center of said polynucleotide.", "In preferred embodiments the polymorphic base is within 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.", "Preferably the probes of the present invention are labeled or immobilized on a solid support.", "By assaying the hybridization to an allele specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample.", "High-Throughput parallel hybridizations in array format are specifically encompassed within “hybridization assays” and are described below.", "5) Hybridization To Addressable Arrays Of Oligonucleotides Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence variants.", "Efficient access to polymorphism information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (e.g., the chip) at selected positions.", "Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime.", "The chip technology has already been applied with success in numerous cases.", "For example, the screening of mutations has been undertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and in the protease gene of HIV-1 virus (Hacia et al., (1996) Nat Genet.", "December; 14(4):441-7; Shoemaker et al., (1996) Nat Genet December; 14(4):450-6; Kozal et al., (1996) Nat Med.", "July;2(7):753-9).", "Chips of various formats for use in detecting biallelic polymorphisms can be produced on a customized basis by Affymetrix (GeneChip™), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.", "In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual, which target sequences include a polymorphic marker.", "EP 785280 describes a tiling strategy for the detection of single nucleotide polymorphisms.", "Briefly, arrays may generally be “tiled” for a large number of specific polymorphisms.", "By “tiling” is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of monomers, i.e.", "nucleotides.", "Tiling strategies are further described in PCT application No.", "WO 95/11995.In a particular aspect, arrays are tiled for a number of specific, identified biallelic marker sequences.", "In particular, the array is tiled to include a number of detection blocks, each detection block being specific for a specific biallelic marker or a set of biallelic markers.", "For example, a detection block may be tiled to include a number of probes, which span the sequence segment that includes a specific polymorphism.", "To ensure probes that are complementary to each allele, the probes are synthesized in pairs differing at the biallelic marker.", "In addition to the probes differing at the polymorphic base, monosubstituted probes are also generally tiled within the detection block.", "These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymorphism, substituted with the remaining nucleotides (selected from A, T, G, C and U).", "Typically the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the biallelic marker.", "The monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artefactual cross-hybridization.", "Upon completion of hybridization with the target sequence and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes.", "The hybridization data from the scanned array is then analyzed to identify which allele or alleles of the biallelic marker are present in the sample.", "Hybridization and scanning may be carried out as described in PCT application No.", "WO 92/10092 and WO 95/11995 and U.S. Pat.", "No.", "5,424,186.Thus, in some embodiments, the chips may comprise an array of nucleic acid sequences of fragments of about 15 nucleotides in length.", "In preferred embodiments the polymorphic base is within 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.", "In some embodiments, the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides.", "6) Integrated Systems Another technique, which may be used to analyze polymorphisms, includes multicomponent integrated systems, which miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device.", "An example of such technique is disclosed in U.S. Pat.", "No.", "5,589,136, which describes the integration of PCR amplification and capillary electrophoresis in chips.", "Integrated systems can be envisaged mainly when microfluidic systems are used.", "These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip.", "The movements of the samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts.", "Varying the voltage controls the liquid flow at intersections between the micro-machined channels and changes the liquid flow rate for pumping across different sections of the microchip.", "For genotyping biallelic markers, the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser-induced fluorescence detection.", "In a first step, the DNA samples are amplified, preferably by PCR.", "Then, the amplification products are subjected to automated microsequencing reactions using ddNTPs (specific fluorescence for each ddNTP) and the appropriate oligonucleotide microsequencing primers which hybridize just upstream of the targeted polymorphic base.", "Once the extension at the 3′ end is completed, the primers are separated from the unincorporated fluorescent ddNTPs by capillary electrophoresis.", "The separation medium used in capillary electrophoresis can for example be polyacrylamide, polyethyleneglycol or dextran.", "The incorporated ddNTPs in the single-nucleotide primer extension products are identified by fluorescence detection.", "This microchip can be used to process at least 96 to 384 samples in parallel.", "It can use the usual four color laser induced fluorescence detection of the ddNTPs.", "GSSP4 Association Studies Association studies focus on population frequencies and rely on the phenomenon of linkage disequilibrium.", "Linkage disequilibrium is the deviation from random of the occurrence of pairs of specific alleles at different loci on the same chromosome.", "If a specific allele in a given gene is directly associated with a particular trait, its frequency will be statistically increased in an affected (trait positive) population, when compared to the frequency in a trait negative population or in a random control population.", "As a consequence of the existence of linkage disequilibrium, the frequency of all other alleles present in the haplotype carrying the trait-causing allele will also be increased in trait positive individuals compared to trait negative individuals or random controls.", "Therefore, association between the trait and any allele (specifically a biallelic marker allele) in linkage disequilibrium with the trait-causing allele will suffice to suggest the presence of a trait-related gene in that particular region.", "Case-control populations can be genotyped for biallelic markers to identify associations that narrowly locate a trait causing allele, as any marker in linkage disequilibrium with one given marker associated with a trait will be associated with the trait.", "Linkage disequilibrium allows the relative frequencies in case-control populations of a limited number of genetic polymorphisms (specifically biallelic markers) to be analyzed as an alternative to screening all possible functional polymorphisms in order to find trait-causing alleles.", "Association studies compare the frequency of marker alleles in unrelated case-control populations, and represent powerful tools for the dissection of complex traits.", "Case-Control Populations (Inclusion Criteria) Population-based association studies do not concern familial inheritance, but compare the prevalence of a particular genetic marker, or a set of markers, in case-control populations.", "They are case-control studies based on comparison of unrelated case (affected or trait positive) individuals and unrelated control (unaffected, trait negative or random) individuals.", "Preferably, the control group is composed of unaffected or trait negative individuals.", "Further, the control group is ethnically matched to the case population.", "Moreover, the control group is preferably matched to the case-population for the main known confusion factor for the trait under study (for example age-matched for an age-dependent trait).", "Ideally, individuals in the two samples are paired in such a way that they are expected to differ only in their disease status.", "The terms “trait positive population”, “case population” and “affected population” are used interchangeably herein.", "An important step in the dissection of complex traits using association studies is the choice of case-control populations (see, Lander and Schork, (1994) Science, September 30;265(5181):2037-48).", "A major step in the choice of case-control populations is the clinical definition of a given trait or phenotype.", "Any genetic trait may be analyzed by the association method proposed here by carefully selecting the individuals to be included in the trait positive and trait negative phenotypic groups.", "Four criteria are often useful: clinical phenotype, age at onset, family history and severity.", "The selection procedure for continuous or quantitative traits (such as blood pressure for example) involves selecting individuals at opposite ends of the phenotype distribution of the trait under study, so as to include in these trait positive and trait negative populations individuals with non-overlapping phenotypes.", "Preferably, case-control populations consist of phenotypically homogeneous populations.", "Trait positive and trait negative populations consist of phenotypically uniform populations of individuals representing each between 1 and 98%, preferably between 1 and 80%, more preferably between 1 and 50%, and more preferably between 1 and 30%, most preferably between 1 and 20% of the total population under study, and preferably selected among individuals exhibiting non-overlapping phenotypes.", "The clearer the difference between the two trait phenotypes, the greater the probability of detecting an association with biallelic markers.", "The selection of those drastically different but relatively uniform phenotypes enables efficient comparisons in association studies and the possible detection of marked differences at the genetic level, provided that the sample sizes of the populations under study are significant enough.", "In preferred embodiments, a first group of between 50 and 300 trait positive individuals, preferably about 100 individuals, are recruited according to their phenotypes.", "A similar number of trait negative individuals are included in such studies.", "In the present invention, typical examples of inclusion criteria include obesity and disorders related to obesity as well as physiologic parameters associated with obesity, such as free fatty acid levels, glucose levels, insulin levels, leptin levels, triglyceride levels, free fatty acid oxidation levels, and weight loss.", "Association Analysis The general strategy to perform association studies using biallelic markers derived from a region carrying a candidate gene is to scan two groups of individuals (case-control populations) in order to measure and statistically compare the allele frequencies of the biallelic markers of the present invention in both groups.", "If a statistically significant association with a trait is identified for at least one or more of the analyzed biallelic markers, one can assume that: either the associated allele is directly responsible for causing the trait (i.e.", "the associated allele is the trait causing allele), or more likely the associated allele is in linkage disequilibrium with the trait causing allele.", "The specific characteristics of the associated allele with respect to the candidate gene function usually give further insight into the relationship between the associated allele and the trait (causal or in linkage disequilibrium).", "If the evidence indicates that the associated allele within the candidate gene is most probably not the trait-causing allele but is in linkage disequilibrium with the real trait-causing allele, then the trait-causing allele can be found by sequencing the vicinity of the associated marker, and performing further association studies with the polymorphisms that are revealed in an iterative manner.", "Association studies are usually run in two successive steps.", "In a first phase, the frequencies of a reduced number of biallelic markers from the candidate gene are determined in the trait positive and trait negative populations.", "In a second phase of the analysis, the position of the genetic loci responsible for the given trait is further refined using a higher density of markers from the relevant region.", "However, if the candidate gene under study is relatively small in length a single phase may be sufficient to establish significant associations.", "Haplotype Analysis As described above, when a chromosome carrying a disease allele first appears in a population as a result of either mutation or migration, the mutant allele necessarily resides on a chromosome having a set of linked markers: the ancestral haplotype.", "This haplotype can be tracked through populations and its statistical association with a given trait can be analyzed.", "Complementing single point (allelic) association studies with multi-point association studies also called haplotype studies increases the statistical power of association studies.", "Thus, a haplotype association study allows one to define the frequency and the type of the ancestral carrier haplotype.", "A haplotype analysis is important in that it increases the statistical power of an analysis involving individual markers.", "In a first stage of a haplotype frequency analysis, the frequency of the possible haplotypes based on various combinations of the identified biallelic markers of the invention is determined.", "The haplotype frequency is then compared for distinct populations of trait positive and control individuals.", "The number of trait positive individuals, which should be, subjected to this analysis to obtain statistically significant results usually ranges between 30 and 300, with a preferred number of individuals ranging between 50 and 150.The same considerations apply to the number of unaffected individuals (or random control) used in the study.", "The results of this first analysis provide haplotype frequencies in case-control populations, for each evaluated haplotype frequency a p-value and an odds ratio are calculated.", "If a statistically significant association is found the relative risk for an individual carrying the given haplotype of being affected with the trait under study can be approximated.", "Interaction Analysis The biallelic markers of the present invention may also be used to identify patterns of biallelic markers associated with detectable traits resulting from polygenic interactions.", "The analysis of genetic interaction between alleles at unlinked loci requires individual genotyping using the techniques described herein.", "The analysis of allelic interaction among a selected set of biallelic markers with appropriate level of statistical significance can be considered as a haplotype analysis.", "Interaction analysis consists in stratifying the case-control populations with respect to a given haplotype for the first loci and performing a haplotype analysis with the second loci with each subpopulation.", "VIII.", "Assays for Identifying Modulators of GSSP4 Polypeptide Activity The invention features methods of screening for one or more compounds that modulate the activity of GSSP4s in cells, which includes providing potential compounds to be tested to the cells.", "Exemplary assays that may be used are described in the Examples 2, 5-7,9-11.To these assays would be added compounds to be tested for their inhibitory or stimulatory activity as compared to the effects of GSSP4 polypeptides alone.", "Other assays in which an effect is observed based on the addition of GSSP4 polypeptides can also be used to screen for modulators of GSSP4 polypeptide activity or effects of the presence of GSSP4 polypeptides on cells.", "The essential step is to apply an unknown compound and then to monitor an assay for a change from what is seen when only GSSP4 polypeptides are applied to the cell.", "A change is defined as something that is significantly different in the presence of the compound plus GSSP4 polypeptide compared to GSSP4 polypeptide alone.", "In this case, significantly different would be an “increase” or a “decrease” in a measurable effect of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.", "The term “modulation” as used herein refers to a measurable change in an activity.", "Examples include, but are not limited to, lipolysis stimulated receptor (LSR) modulation, leptin modulation, lipoprotein modulation, plasma FFA levels, FFA oxidation, TG levels, glucose levels, and weight.", "These effects can be in vitro or preferably in vivo.", "Modulation of an activity can be either an increase or a decrease in the activity.", "Thus, LSR activity can be increased or decreased, leptin activity can be increased or decreased, and lipoprotein activity can be increased or decreased.", "Similarly, FFA, TG, and glucose levels (and weight) can be increased or decreased in vivo Free Fatty Acid oxidation can be increased or decreased in vivo or ex vivo.", "By “LSR” activity is meant expression of LSR on the surface of the cell, or in a particular conformation, as well as its ability to bind, uptake, and degrade leptin and lipoprotein.", "By “leptin” activity is meant its binding, uptake and degradation by LSR, as well as its transport across a blood brain barrier, and potentially these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.", "Similarly, by “lipoprotein” activity is meant its binding, uptake and degradation by LSR, as well as these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.", "Exemplary assays are provided in Examples 2, 5-7,9-11.These assay and other comparable assays can be used to determine/identify compounds that modulate GSSP4 polypeptide activity.", "In some cases it may be important to identify compounds that modulate some but not all of the GSSP4 polypeptide activities, although preferably all activities are modified.", "The term “increasing” as used herein refers to the ability of a compound to increase the activity of GSSP4 polypeptides in some measurable way compared to the effect of GSSP4 polypeptides in its absence.", "As a result of the presence of the compound leptin binding and/or uptake might increase, for example, as compared to controls in the presence of the GSSP4 polypeptide alone.", "Preferably, an increase in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% compared to the level of activity in the presence of the GSSP4 polypeptide.", "Similarly, the term “decreasing” as used herein refers to the ability of a compound to decrease an activity in some measurable way compared to the effect of a GSSP4 polypeptide in its absence.", "For example, the presence of the compound decreases the plasma concentrations of FFA, TG, and glucose in mice.", "Also as a result of the presence of a compound leptin binding and/or uptake might decrease, for example, as compared to controls in the presence of the GSSP4 polypeptide alone.", "Preferably, an decrease in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% as compared to the level of activity in the presence of the GSSP4 polypeptide alone.", "The invention features a method for identifying a potential compound to modulate body mass in individuals in need of modulating body mass comprising: a) contacting a cell with a GSSP4 polypeptide and a candidate compound; b) detecting a result selected from the group consisting of LSR modulation, leptin modulation, lipoprotein modulation; FFA oxidation modulation; and c) wherein said result identifies said potential compound if said result differs from said result when said cell is contacted with the GSSP4 polypeptide alone.", "In preferred embodiments, said contacting further comprises a ligand of said LSR.", "Preferably said ligand is selected from the group consisting of cytokine, lipoprotein, free fatty acids, and C1q, and more preferably said cytokine is leptin, and most preferably said leptin is a leptin polypeptide fragment as described in U.S.", "Provisional application No.", "60/155,506 hereby incorporated by reference herein in its entirety including any figures, drawings, or tables.", "In other preferred embodiments, said GSSP4 polypeptide is mouse or is human.", "In other preferred embodiments, said cell is selected from the group consisting of PLC, CHO-K1, Hep3B, and HepG2.In yet other preferred embodiments, said lipoprotein modulation is selected from the group consisting of binding, uptake, and degradation.", "Preferably, said modulation is an increase in said binding, uptake, or degradation.", "Alternatively, said modulation is a decrease in said binding, uptake, or degradation.", "In other preferred embodiments, leptin modulation is selected from the group consisting of binding, uptake, degradation, and transport.", "Preferably, said modulation is an increase in said binding, uptake, degradation, or transport.", "Alternatively, said modulation is a decrease in said binding, uptake, degradation, or transport.", "Preferably, said transport is across a blood-brain barrier.", "In yet other preferred embodiments, said LSR modulation is expression on the surface of said cell.", "Preferably, said detecting comprises FACS, more preferably said detecting further comprises antibodies that bind specifically to said LSR, and most preferably said antibodies bind specifically to the carboxy terminus of said LSR.", "In still other preferred embodiments, said potential compound is selected from the group consisting of peptides, peptide libraries, non-peptide libraries, peptoids, fatty acids, lipoproteins, medicaments, antibodies, small molecules, and proteases.", "IX.", "Epitopes and Antibody Fusions A preferred embodiment of the present invention is directed to eiptope-bearing polypeptides and epitope-bearing polypeptide fragments.", "These epitopes may be “antigenic epitopes” or both an “antigenic epitope” and an “immunogenic epitope”.", "An “immunogenic epitope” is defined as a part of a protein that elicits an antibody response in vivo when the polypeptide is the immunogen.", "On the other hand, a region of polypeptide to which an antibody binds is defined as an “antigenic determinant” or “antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes.", "See, e.g., Geysen, et al.", "(1983) Proc.", "Natl.", "Acad.", "Sci.", "USA 81:39984002.It is particularly noted that although a particular epitope may not be immunogenic, it is nonetheless useful since antibodies can be made in vitro to any epitope.", "An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope.", "Generally an epitope consists of at least 6 such amino acids, and more often at least 8-10 such amino acids.", "In preferred embodiment, antigenic epitopes comprise a number of amino acids that is any integer between 3 and 50.Fragments which function as epitopes may be produced by any conventional means.", "See, e.g., Houghten, R. A., Proc.", "Natl.", "Acad.", "Sci.", "USA 82:5131-5135 (1985), further described in U.S. Pat.", "No.", "4,631,211.Methods for determining the amino acids which make up an immunogenic epitope include x-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping, e.g., the Pepscan method described by H. Mario Geysen et al.", "(1984); Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 81:3998-4002; PCT Publication No.", "WO 84/03564; and PCT Publication No.", "WO 84/03506.Another example is the algorithm of Jameson and Wolf, Comp.", "Appl.", "Biosci.", "4:181-186 (1988) (said references incorporated by reference in their entireties).", "The Jameson-Wolf antigenic analysis, for example, may be performed using the computer program PROTEAN, using default parameters (Version 4.0 Windows, DNASTAR, Inc., 1228 South Park Street Madison, Wis.).", "The epitope-bearing fragments of the present invention preferably comprises 6 to 50 amino acids (i.e.", "any integer between 6 and 50, inclusive) of a polypeptide of the present invention.", "Also, included in the present invention are antigenic fragments between the integers of 6 and the full length sequence of the sequence listing.", "All combinations of sequences between the integers of 6 and the full-length sequence of a polypeptide of the present invention are included.", "The epitope-bearing fragments may be specified by either the number of contiguous amino acid residues (as a sub-genus) or by specific N-terminal and C-terminal positions (as species) as described above for the polypeptide fragments of the present invention.", "Any number of epitope-bearing fragments of the present invention may also be excluded in the same manner.", "Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies that specifically bind the epitope (See, Wilson et al., 1984; and Sutcliffe, J. G. et al., 1983).", "The antibodies are then used in various techniques such as diagnostic and tissue/cell identification techniques, as described herein, and in purification methods.", "Similarly, immunogenic epitopes can be used to induce antibodies according to methods well known in the art (See, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al.", ";(1985) and Bittle, F. J. et al., (1985).", "A preferred immunogenic epitope includes the polypeptides of the sequence listing.", "The immunogenic epitopes may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) if nessary.", "Immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.).", "Epitope-bearing polypeptides of the present invention are used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods (See, e.g., Sutcliffe, et al., supra; Wilson, et al., supra, and Bittle, et al., 1985).", "If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.", "For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as—maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.", "Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μgs of peptide or carrier protein and Freund's adjuvant.", "Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody, which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.", "The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.", "As one of skill in the art will appreciate, and discussed above, the polypeptides of the present invention including, but not limited to, polypeptides comprising an immunogenic or antigenic epitope can be fused to heterologous polypeptide sequences.", "For example, the polypeptides of the present invention may be fused with the constant region comprising portions of immunoglobulins (IgA, IgE, IgG, IgM), or portions of the constant region (CH1, CH2, CH3, any combination thereof including both entire domains and portions thereof) resulting in chimeric polypeptides.", "These fusion proteins facilitate purification, and show an increased half-life in vivo.", "This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (See, e.g., EPA 0,394,827; and Traunecker et al., 1988).", "Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion can also be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone (See, e.g., Fountoulakis et al., 1995).", "Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag to aid in detection and purification of the expressed polypeptide.", "Additonal fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, or codon-shuffling (collectively referred to as “DNA shuffling”).", "DNA shuffling may be employed to modulate the activities of polypeptides of the present invention thereby effectively generating agonists and antagonists of the polypeptides.", "See, for example, U.S. Pat.", "Nos.", "5,605,793; 5,811,238; 5,834,252; 5,837,458; and Patten, P. A., et al., (1997); Harayama, S., (1998); Hansson, L. O., et al (1999); and Lorenzo, M. M. and Blasco, R., (1998).", "(Each of these documents are hereby incorporated by reference).", "In one embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of coding polynucleotides of the invention, or the polypeptides encoded thereby may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc.", "of one or more heterologous molecules.", "Antibodies: The present invention further relates to antibodies and T-cell antigen receptors (TCR), which specifically bind the polypeptides, and more specifically, the epitopes of the polyepeptides of the present invention.", "The antibodies of the present invention include IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY.", "As used herein, the term “antibody” (Ab) is meant to include whole antibodies, including single-chain whole antibodies, and antigen binding fragments thereof.", "In a preferred embodiment the antibodies are human antigen binding antibody fragments of the present invention include, but are not limited to, Fab, Fab′ F(ab)2 and F(ab)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.", "The antibodies may be from any animal origin including birds and mammals.", "Preferably, the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken.", "Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire or partial of the following: hinge region, CH1, CH2, and CH3 domains.", "Also included in the invention are any combinations of variable region(s) and hinge region, CH1, CH2, and CH3 domains.", "The present invention further includes chimeric, humanized, and human monoclonal and polyclonal antibodies, which specifically bind the polypeptides of the present invention.", "The present invention further includes antibodies that are anti-idiotypic to the antibodies of the present invention.", "The antibodies of the present invention may be monospecific, bispecific, and trispecific or have greater multispecificity.", "Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or solid support material.", "See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al.", "(1991); U.S. Pat.", "Nos.", "5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; Kostelny, S. A. et al.", "(1992).", "Antibodies of the present invention may be described or specified in terms of the epitope(s) or epitope-bearing portion(s) of a polypeptide of the present invention, which are recognized or specifically bound by the antibody.", "In the case of proteins of the present invention secreted proteins, the antibodies may specifically bind a full-length protein encoded by a nucleic acid of the present invention, a mature protein (i.e., the protein generated by cleavage of the signal peptide) encoded by a nucleic acid of the present invention, a signal peptide encoded by a nucleic acid of the present invention, or any other polypeptide of the present invention.", "Therefore, the epitope(s) or epitope bearing polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or otherwise described herein (including the sequence listing).", "Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded as individual species.", "Therefore, the present invention includes antibodies that specifically bind specified polypeptides of the present invention, and allows for the exclusion of the same.", "Antibodies of the present invention may also be described or specified in terms of their cross-reactivity.", "Antibodies that do not specifically bind any other analog, ortholog, or homolog of the polypeptides of the present invention are included.", "Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein, eg., using FASTDB and the parameters set forth herein) to a polypeptide of the present invention are also included in the present invention.", "Further included in the present invention are antibodies, which only bind polypeptides encoded by polynucleotides, which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein).", "Antibodies of the present invention may also be described or specified in terms of their binding affinity.", "Preferred binding affinities include those with a dissociation constant or Kd value less than 5×10−6M, 10−6M, 5×10−7M, 10−7M, 5×10−8M, 10−8M, 5×10−9M, 10−9M, 5×10−10M, 10−10M, 5×10−11M, 10−11M, 5×10−12M, 10−12M, 5×10−13M, 10−13M, 5×10−14M, 10−14M, 5×10−15M, and 10−15M.", "Antibodies of the present invention have uses that include, but are not limited to, methods known in the art to purify, detect, and target the polypeptides of the present invention including both in vitro and in vivo diagnostic and therapeutic methods.", "For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples (See, e.g., Harlow et al., 1988).", "The antibodies of the present invention may be used either alone or in combination with other compositions.", "The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.", "For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins.", "See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.", "No.", "5,314,995; and EP 0 396 387.The antibodies of the present invention may be prepared by any suitable method known in the art.", "For example, a polypeptide of the present invention or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.", "The term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.", "The term “antibody” refers to a polypeptide or group of polypeptides which are comprised of at least one binding domain, where a binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen, which allows an immunological reaction with the antigen.", "The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.", "Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technology.", "Hybridoma techniques include those known in the art (See, e.g., Harlow et al.", "1988); Hammerling, et al, 1981).", "(Said references incorporated by reference in their entireties).", "Fab and F(ab′)2 fragments may be produced, for example, from hybridoma-produced antibodies by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments).", "Alternatively, antibodies of the present invention can be produced through the application of recombinant DNA technology or through synthetic chemistry using methods known in the art.", "For example, the antibodies of the present invention can be prepared using various phage display methods known in the art.", "In phage display methods, functional antibody domains are displayed on the surface of a phage particle, which carries polynucleotide sequences encoding them.", "Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g.", "human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead.", "Phage used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.", "Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman U. et al.", "(1995); Ames, R. S. et al.", "(1995); Kettleborough, C. A. et al.", "(1994); Persic, L. et al.", "(1997); Burton, D. R. et al.", "(1994); PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat.", "Nos.", "5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743.As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria.", "For example, techniques to recombinantly produce Fab, Fab′ F(ab)2 and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax, R. L. et al.", "(1992); and Sawai, H. et al.", "(1995); and Better, M. et al.", "(1988).", "Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat.", "Nos.", "4,946,778 and 5,258,498; Huston et al.", "(1991); Shu, L. et al.", "(1993); and Skerra, A. et al.", "(1988).", "For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies.", "Methods for producing chimeric antibodies are known in the art.", "See e.g., Morrison, (1985); Oi et al., (1986); Gillies, S. D. et al.", "(1989); and U.S. Pat.", "No.", "5,807,715.Antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat.", "Nos.", "5,530,101; and 5,585,089), veneering or resurfacing, (EP 0 592 106; EP 0 519 596; Padlan E. A., 1991; Studnicka G. M. et al., 1994; Roguska M. A. et al., 1994), and chain shuffling (U.S. Pat.", "No.", "5,565,332).", "Human antibodies can be made by a variety of methods known in the art including phage display methods described above.", "See also, U.S. Pat.", "Nos.", "4,444,887,4,716,111, 5,545,806, and 5,814,318; WO 98/46645; WO 98/50433; WO 98/24893; WO 96/34096; WO 96/33735; and WO 91/10741.Further included in the present invention are antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide of the present invention.", "The antibodies may be specific for antigens other than polypeptides of the present invention.", "For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.", "Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art (See e.g., Harbor et al.", "supra; WO 93/21232; EP 0 439 095; Naramura, M. et al.", "1994; U.S. Pat.", "No.", "5,474,981; Gillies, S. O. et al., 1992; Fell, H. P. et al., 1991).", "The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.", "For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.", "The antibody portion fused to a polypeptide of the present invention may comprise the hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.", "The polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half-life of the polypeptides or for use in immunoassays using methods known in the art.", "The polypeptides may also be fused or conjugated to the above antibody portions to form multimers.", "For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.", "Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.", "Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art.", "See e.g., U.S. Pat.", "Nos.", "5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. et al.", "(1991); Zheng, X. X. et al.", "(1995); and Vil, H. et al.", "(1992).", "The invention further relates to antibodies that act as agonists or antagonists of the polypeptides of the present invention.", "For example, the present invention includes antibodies that disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.", "Included are both receptor-specific antibodies and ligand-specific antibodies.", "Included are receptor-specific antibodies, which do not prevent ligand binding but prevent receptor activation.", "Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art.", "Also include are receptor-specific antibodies which both prevent ligand binding and receptor activation.", "Likewise, included are neutralizing antibodies that bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies that bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.", "Further included are antibodies that activate the receptor.", "These antibodies may act as agonists for either all or less than all of the biological activities affected by ligand-mediated receptor activation.", "The antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein.", "The above antibody agonists can be made using methods known in the art.", "See e.g., WO 96/40281; U.S. Pat.", "No.", "5,811,097; Deng, B. et al.", "(1998); Chen, Z. et al.", "(1998); Harrop, J.", "A. et al.", "(1998); Zhu, Z. et al.", "(1998); Yoon, D. Y. et al.", "(1998); Prat, M. et al.", "(1998) J.; Pitard, V. et al.", "(1997); Liautard, J. et al.", "(1997); Carlson, N. G. et al.", "(1997) J.; Taryman, R. E. et al.", "(1995); Muller, Y.", "A. et al.", "(1998); Bartunek, P. et al.", "(1996).", "As discussed above, antibodies of the polypeptides of the invention can, in turn, be utilized to generate anti-idiotypic antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art (See, e.g.", "Greenspan and Bona (1989); and Nissinoff (1991).", "For example, antibodies which bind to and competitively inhibit polypeptide multimerization or binding of a polypeptide of the invention to ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization or binding domain and, as a consequence, bind to and neutralize polypeptide or its ligand.", "Such neutralization anti-idiotypic antibodies can be used to bind a polypeptide of the invention or to bind its ligands/receptors, and therby block its biological activity, The invention also concerns a purified or isolated antibody capable of specifically binding to a mutated full length or mature polypeptide of the present invention or to a fragment or variant thereof comprising an epitope of the mutated polypeptide.", "In another preferred embodiment, the present invention concerns an antibody capable of binding to a polypeptide comprising at least 10 consecutive amino acids of a polypeptide of the present invention and including at least one of the amino acids which can be encoded by the trait causing mutations.", "Non-human animals or mammals, whether wild-type or transgenic, which express a different species of a polypeptide of the present invention than the one to which antibody binding is desired, and animals which do not express a polypeptide of the present invention (i.e.", "a knock out animal) are particularly useful for preparing antibodies.", "Gene knock out animals will recognize all or most of the exposed regions of a polypeptide of the present invention as foreign antigens, and therefore produce antibodies with a wider array of epitopes.", "Moreover, smaller polypeptides with only 10 to 30 amino acids may be useful in obtaining specific binding to any one of the polypeptides of the present invention.", "In addition, the humoral immune system of animals which produce a species of a polypeptide of the present invention that resembles the antigenic sequence will preferentially recognize the differences between the animal's native polypeptide species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence.", "Such a technique will be particularly useful in obtaining antibodies that specifically bind to any one of the polypeptides of the present invention.", "Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.", "The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.", "The antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known in the art.", "Consequently, the invention is also directed to a method for detecting specifically the presence of a polypeptide of the present invention according to the invention in a biological sample, said method comprising the following steps: a) bringing into contact the biological sample with a polyclonal or monoclonal antibody that specifically binds a polypeptide of the present invention; and b) detecting the antigen-antibody complex formed.", "The invention also concerns a diagnostic kit for detecting in vitro the presence of a polypeptide of the present invention in a biological sample, wherein said kit comprises: a) a polyclonal or monoclonal antibody that specifically binds a polypeptide of the present invention, optionally labeled; b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.", "The antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known in the art.", "EXAMPLES The following Examples are provided for illustrative purposes and not as a means of limitation to support the finding that GSSP4 polypeptides have efficacy in reducing insulin resistance and improving glucose and lipid metabolism and may have an insulin sensitising action.", "One of ordinary skill in the art would be able to design equivalent assays and methods based on the disclosure herein all of which form part of the instant invention.", "Example 1 Northern Analysis of GSSP4 RNA Analysis of GSSP4 expression in different human tissues (adult and fetal) and cell lines, as well as mouse embryos in different stages of development, is accomplished by using poly A+ RNA blots purchased from Clontech (e.g.", "#7780-1, 7757-1, 7756-1, 7768-1and 7763-1).", "Labeling of RNA probes is performed using the RNA Strip-EZ kit from Ambion as per manufacture's instructions.", "Hybridization of RNA probes to RNA blots is performed Ultrahyb hybridization solution (Ambion).", "Briefly, blots are prehybridized for 30 min at 58° C. (low-strigency) or 65° C. (high stringency).", "After adding the labeled probe (2×106 cpm/ml), blots are hybridized overnight (14-24 hrs), and is washed 2×20 min at 50° C. with 2× SSC/0.1% SDS (low stringency), 2×20 min at 58° C. with 1× SSC/0.1% SDS (medium stringency) and 2×20 min at 65° C. with 1× SSC/0.1% SDS (high stringency).", "After washings are completed blots are exposed on the phosphoimager (Molecular Dynamics) for 1-3 days.", "Example 2 In Vitro Tests of Metabolic-Related Activity The activity of various preparations of GSSP4 polypeptides are assessed using various in vitro assays including those provided below.", "These assays are also exemplary of those that can be used to develop GSSP4 polypeptide antagonists and agonists.", "To do that, the effect of GSSP4 polypeptides in the above assays, e.g.", "on glucose uptake and fatty acid oxidation and partitioning in the presence of the candidate molecules would be compared with the effect of GSSP4 polypeptides in the assays in the absence of the candidate molecules.", "In Vitro Muscle Cells Glucose Uptake L6 Muscle cells are obtained from the European Culture Collection (Porton Down) and are used at passages 7-11.Cells are maintained in standard tissue culture medium DMEM, and glucose uptake is assessed using [.sup.3H]-2-deoxyglucose (2DG) with or without GSSP4 polypeptides in the presence or absence of insulin (10.sup.-8 M) as has been previously described (Walker P S et al, Glucose transport activity in L6 muscle cells is regulated by the coordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription, JBC, 1990;265(3), 1516-1523, and Kilp A et al, Stimulation of hexose transport by metformin in L6 muscle cells in culture, Endocrinology,1992;130(5), 2535-2544).", "Uptake of 2DG is expressed as the percentage change compared with control (no added insulin or GSSP4).", "Values are presented as mean .+−.SEM of sets of 4 wells per experiment.", "Differences between sets of wells are evaluated by Student's t test, probability values p<0.05 are considered to be significant.", "Effect on Muscle Cell Fatty Acid Oxidation C2C12 cells are differentiated in the presence or absence of 2 μg/mL GSSP4 protein for 4 days.", "On day 4, oleate oxidation rates are determined by measuring conversion of 1-14C-oleate (0.2 mM) to 14CO2 for 90 min This experiment can be used to screen for active fragments and peptides as well as agonists and antagonists or activators and inhibitors of GSSP4 polypeptides.", "The effect of polypeptides on the rate of oleate oxidation can be compared in differentiated C2C12 cells (murine skeletal muscle cells; ATCC, Manassas, Va. CRL-1772) and in a hepatocyte cell line (Hepa1-6; ATCC, Manassas, Va. CRL-1830).", "Cultured cells are maintained according to manufacturer's instructions.", "The oleate oxidation assay is performed as previously described (Muoio et al (1999) Biochem J 338;783-791).", "Briefly, nearly confluent myocytes are kept in low serum differentiation media (DMEM, 2.5% Horse serum) for 4 days, at which time formation of myotubes became maximal.", "Hepatocytes are kept in the same DMEM medium supplemented with 10% FCS for 2 days.", "One hour prior to the experiment the media is removed and 1 mL of preincubation media (MEM, 2.5% Horse serum, 3 mM glucose, 4 mM Glutamine, 25 mM Hepes, 1% FFA free BSA, 0.25 mM Oleate, 5 μg/mL gentamycin) is added.", "At the start of the oxidation experiment 14C-Oleic acid (1 μCi/mL, American Radiolabeled Chemical Inc., St. Louis, Mo.)", "is added and cells are incubated for 90 min at 37° C. in the absence/presence of 2.5 μg/mL GSSP4 polypeptides.", "After the incubation period 0.75 mL of the media is removed and assayed for 14C-oxidation products as described below for the muscle FFA oxidation experiment.", "Triglyceride and Protein Partitioning following Oleate Oxidaiton in Cultured Cells Following transfer of media for oleate oxidation assay, cells are placed on ice.", "To determine triglyceride and protein content, cells are washed with 1 mL of 1×PBS to remove residual media.", "To each well 300 μL of cell dissociation solution (Sigma) is added and incubated at 37° C. for 10 min.", "Plates are tapped to loosen cells, and 0.5 mL of 1×PBS is added.", "The cell suspension is transferred to an eppendorf tube, each well is rinsed with an additional 0.5 mL of 1×PBS, and is transferred to appropriate eppendorf tube.", "Samples are centrifuged at 1000 rpm for 10 minutes at room temperature.", "Supernatant is discarded and 750 μL of 1×PBS/2% chaps is added to cell pellet.", "Cell suspension is vortexed and placed on ice for 1 hour.", "Samples are then centrifuged at 13000 rpm for 20 min at 4° C. Supernatants are transferred to new tube and frozen at −20° C. until analyzed.", "Quantitative measure of triglyceride level in each sample is determined using Sigma Diagnostics GPO-TRINDER enzymatic kit.", "The procedure outlined in the manual is adhered to, with the following exceptions: assay is performed in 48 well plate, 350 μL of sample volume is assayed, control blank consisted of 350 μL PBS/2% chaps, and standard contained 10 μL standard provide in kit plus 690 μL PBS/2% chaps.", "Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm.", "Protein analysis is carried out on 25 μL of each supernatant sample using the BCA protein assay (Pierce) following manufacturer's instructions.", "Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm.", "Cellular Binding and Uptake of GSSP4 Polypeptides as Detected by Fluorescence Microscopy Fluorecein isothiocyanate (FITC) conjugation of GSSP4 polypeptides: Purified GSSP4 proteins at 1 mg/mL concentration are labeled with FITC using Sigma's FluoroTag FITC conjugation kit (Stock No.", "FITC-1).", "Protocol outlined in the Sigma Handbook for small scale conjugation is followed for GSSP4 protein labeling.", "Cell Culture: C2C12 mouse skeletal muscle cells (ATCC, Manassas, Va. CRL-1772) and Hepa-1-6 mouse hepatocytes (ATCC, Manassas, Va. CRL-1830) are seeded into 6 well plates at a cell density of 2×105 cells per well.", "C2C12 and Hepa-1-6 cells are cultured according to repository's instructions for 24-48 hours prior to analysis.", "Assay is performed when cells are 80% confluent.", "FITC labeled GSSP4 proteincellular binding and uptake using microscopy: C2C12 and Hepa 1-6 cells are incubated in the presence/absence of antibody directed against human LSR (81B: N-terminal sequence of human LSR; does not cross react with mouse LSR and 93A: c-terminal sequence, cross reacts with mouse LSR) or an antiserum directed against gC1qr (953) for 1 hour at 37° C., 5% CO2.LSR antibodies are added to the media at a concentration of 2 μg/mL.", "The anti-gC1qr antiserum is added to the media at a volume of 2.5 μL undiluted serum (high concentration) or 1:100 dilution (low concentration).", "Following incubation with specified antibody, FITC-GSSP4 polypeptide (50 nM/mL) is added to each cell culture well.", "Cells are again incubated for 1 hour at 37° C., 5% CO2.Cells are washed 2× with PBS, cells are scraped from well into 1 mL of PBS.", "Cell suspension is transferred to an eppendorf tube and centrifuged at 1000 rpm for 2 minutes.", "Supernatant is removed and cells resuspended in 200 μL of PBS.", "Binding and uptake of FITC-GSSP4 polypeptide is analyzed by fluorescence microscopy under 40× magnification.", "This assay may be useful for identifying agents that facilitate or prevent the uptake and/or binding of GSSP4 polypeptides to cells.", "Example 3 In Vivo Tests for Metabolic-Related Activity in Rodent Diabetes Models As metabolic profiles differ among various animal models of obesity and diabetes, analysis of multiple models is undertaken to separate the effects GSSP4 polypeptides on hyperglycemia, hyperinsulinemia, hyperlipidemia and obesity.", "Mutation in colonies of laboratory animals and different sensitivities to dietary regimens have made the development of animal models with non-insulin dependent diabetes associated with obesity and insulin resistance possible.", "Genetic models such as db/db and ob/ob (See Diabetes, (1982) 31(1): 1-6) in mice and fa/fa in zucker rats have been developed by the various laboratories for understanding the pathophysiology of disease and testing the efficacy of new antidiabetic compounds (Diabetes, (1983) 32: 830-838; Annu.", "Rep. Sankyo Res.", "Lab.", "(1994).", "46: 1-57).", "The homozygous animals, C57 BL/KsJ-db/db mice developed by Jackson Laboratory, US, are obese, hyperglycemic, hyperinsulinemic and insulin resistant (J. Clin.", "Invest., (1990) 85: 962-967), whereas heterozygous are lean and normoglycemic.", "In db/db model, mouse progressively develops insulinopenia with age, a feature commonly observed in late stages of human type II diabetes when blood sugar levels are insufficiently controlled.", "The state of pancreas and its course vary according to the models.", "Since this model resembles that of type II diabetes mellitus, the compounds of the present invention are tested for blood sugar and triglycerides lowering activities.", "Zucker (fa/fa) rats are severely obese, hyperinsulinemic, and insulin resistant (Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds.", "(Elsevier Science Publishing Co., Inc., New York, ed.", "4, 1990), pp.", "299-340), and the fa/fa mutation may be the rat equivalent of the murine db mutation (Friedman et al., Cell 69:217-220, 1992; Truett et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 88:7806, 1991).", "Tubby (tub/tub) mice are characterized by obesity, moderate insulin resistance and hyperinsulinemia without significant hyperglycemia (Coleman et al., J. Heredity 81:424, 1990).", "Previously, leptin was reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al.", "Nature 401: 73-76 (1999).", "Leptin is found to be less effective in a different lipodystrophic mouse model of lipoatrophic diabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporated herein in its entirety including any drawings, figures, or tables).", "The streptozotocin (STZ) model for chemically-induced diabetes is tested to examine the effects of hyperglycemia in the absence of obesity.", "STZ-treated animals are deficient in insulin and severely hyperglycemic (Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds.", "(Elsevier Science Publishing Co., Inc., New York, ed.", "4, 1990), pp.", "299-340).", "The monosodium glutamate (MSG) model for chemically-induced obesity (Olney, Science 164:719, 1969; Cameron et al., Cli.", "Exp.", "Pharmacol.", "Physiol.", "5:41, 1978), in which obesity is less severe than in the genetic models and develops without hyperphagia, hyperinsulinemia and insulin resistance, is also examined.", "Finally, a non-chemical, non-genetic model for induction of obesity includes feeding rodents a high fat/high carbohydrate (cafeteria diet) diet ad libitum.", "The instant invention encompasses the use of GSSP4 polypeptides for reducing the insulin resistance and hyperglycemia in any or all of the above rodent diabetes models or in humans with Type I or Type II diabetes or other prefered metabolic diseases described previously or models based on other mammals.", "In the compositions of the present invention the GSSP4 polypeptides may, if desired, be associated with other compatible pharmacologically-active antidiabetic agents such as insulin, leptin (U.S. provisional application No.", "60/155,506), or troglitazone, either alone or in combination.", "Assays include that described previously in Gavrilova et al.", "((2000) Diabetes November;49(11): 1910-6; (2000) Nature February 24;403(6772):850) using A-ZIP/F-1 mice, except that GSSP4 polypeptides are administered intraperotineally, subcutaneously, intramuscularly or intravenously.", "The glucose and insulin levels of the mice would be tested, and the food intake and liver weight monitored, as well as other factors, such as leptin, FFA, and TG levels, typically measured in our experiments.", "In Vivo Assay for Anti-Hyperglycemic Activity of GSSP4 Polypeptides Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old) are housed (7-9 mice/cage) under standard laboratory conditions at 22.degree.", "C. and 50% relative humidity, and maintained on a diet of Purina rodent chow and water ad libitum.", "Prior to treatment, blood is collected from the tail vein of each animal and blood glucose concentrations are determined using One Touch BasicGlucose Monitor System (Lifescan).", "Mice that have plasma glucose levels between 250 to 500 mg/dl are used.", "Each treatment group consists of seven mice that are distributed so that the mean glucose levels are equivalent in each group at the start of the study.", "db/db mice are dosed by micro-osmotic pumps, inserted using isoflurane anesthesia, to provide GSSP4 polypeptides, saline, and an irrelevant peptide to the mice subcutaneously (s.c.).", "Blood is sampled from the tail vein hourly for 4 hours and at 24, 30 h post-dosing and analyzed for blood glucose concentrations.", "Food is withdrawn from 0-4 h post dosing and reintroduced thereafter.", "Individual body weights and mean food consumption (each cage) are also measured after 24 h. Significant differences between groups (comparing GSSP4 treated to saline-treated) are evaluated using Student t-test.", "In Vivo Insulin Sensitivity Assay In vivo insulin sensitivity is examined by utilizing two-step hyperinsulinemic-euglycemic clamps according to the following protocol.", "Rodents from any or all of the various models described in Example 2 are housed for at least a week prior to experimental procedures.", "Surgeries for the placement of jugular vein and carotid artery catheters are performed under sterile conditions using ketamine and xylazine (i.m.)", "anesthesia.", "After surgery, all rodents are allowed to regain consciousness and placed in individual cages.", "GSSP4 polypeptides or vehicle is administered through the jugular vein after complete recovery and for the following two days.", "Sixteen hours after the last treatment, hyperinsulinemic-euglycemic clamps are performed.", "Rodents are placed in restrainers and a bolus of 4 .mu Ci [3-.sup.3 H] glucose (NEN) is administered, followed by a continuous infusion of the tracer at a dose of 0.2 mu.Ci/min (20 mu.1/min).", "Two hours after the start of the tracer infusion, 3 blood samples (0.3 ml each) are collected at 10 minute intervals (−20-0 min) for basal measurements.", "An insulin infusion is then started (5 mU/kg/min), and 100 .mu.1 blood samples are taken every 10 min.", "to monitor plasma glucose.", "A 30% glucose solution is infused using a second pump based on the plasma glucose levels in order to reach and maintain euglycemia.", "Once a steady state is established at 5 mU/kg/min insulin (stable glucose infusion rate and plasma glucose), 3 additional blood samples (0.3 ml each) are obtained for measurements of glucose, [3-.sup.3H] glucose and insulin (100-120 min.).", "A higher dose of insulin (25 mU/kg/min.)", "is then administered and glucose infusion rates are adjusted for the second euglycemic clamp and blood samples are taken at min.", "220-240.Glucose specific activity is determined in deproteinized plasma and the calculations of Rd and hepatic glucose output (HGO) are made, as described (Lang et al., Endocrinology 130:43, 1992).", "Plasma insulin levels at basal period and after 5 and 25 mU/kg/min.", "infusions are then determined and compared between GSSP4 treated and vehicle treated rodents.", "Insulin regulation of glucose homeostasis has two major components; stimulation of peripheral glucose uptake and suppression of hepatic glucose output.", "Using tracer studies in the glucose clamps, it is possible to determine which portion of the insulin response is affected by the GSSP4 polypeptides.", "Example 4 Effect of GSSP4 Polypeptides on Mice Fed a High-Fat Diet Experiments are performed using approximately 6 week old C57B1/6 mice (8 per group).", "All mice are housed individually.", "The mice are maintained on a high fat diet throughout each experiment.", "The high fat diet (cafeteria diet; D12331 from Research Diets, Inc.) has the following composition: protein kcal % 16, carbohydrate kcal % 26, and fat kcal % 58.The fat is primarily composed of coconut oil, hydrogenated.", "After the mice are fed a high fat diet for 6 days, micro-osmotic pumps are inserted using isoflurane anesthesia, and are used to provide full-length GSSP4 polypeptides, GSSP4 polypeptide fragments, saline, and an irrelevant peptide to the mice subcutaneously (s.c.) for 18 days.", "GSSP4 polypeptides are provided at doses of 100, 50, 25, and 2.5 μg/day and the irrelevant peptide is provided at 10 μg/day.", "Body weight is measured on the first, third and fifth day of the high fat diet, and then daily after the start of treatment.", "Final body weight and final blood samples are taken by cardiac puncture and are used to determine triglyceride (TG), total cholesterol (TC), glucose, leptin, and insulin levels.", "The amount of food consumed per day is also determined for each group.", "Plasma glucose is determined by a glucose oxidase procedure (Analox GM7) and plasma insulin determined by radioimmunoassay (Amerlex, Amersham).", "Example 5 Effect of GSSP4 Polypeptides on Plasma Free Fatty Acid in C57 BL/6 Mice The effect of GSSP4 polypeptides on postprandial lipemia (PPL) in normal C57BL6/J mice is tested.", "The mice used in this experiment are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.", "All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point).", "At time 0 (8:30 AM), a standard high fat meal (6 g butter, 6 g sunflower oil, 10 g nonfat dry milk, 10 g sucrose, 12 mL distilled water prepared fresh following Nb#6, JF, pg.1) is given by gavage (vol.=1% of body weight) to all animals.", "Immediately following the high fat meal, 25 μg a GSSP4 polypeptide is injected i.p.", "in 100 μL saline.", "The same dose (25 μg/mL in 100 μL) is again injected at 45 min and at 1 hr 45 min.", "Control animals are injected with saline (3×100 μL).", "Untreated and treated animals are handled in an alternating mode.", "Blood samples are taken in hourly intervals, and are immediately put on ice.", "Plasma is prepared by centrifugation following each time point.", "Plasma is kept at −20° C. and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).", "Due to the limited amount of plasma available, glucose is determined in duplicate using pooled samples.", "For each time point, equal volumes of plasma from all 8 animals per treatment group are pooled.", "Example 6 Effect of GSSP4 Polypeptides on Plasma Leptin and Insulin in C57 BL/6 Mice The effect of GSSP4 polypeptides on plasma leptin and insulin levels during postprandial lipemia (PPL) in normal C57BL6/J mice is tested.", "The experimental procedure is the same as previously described, except that blood is drawn only at 0, 2 and 4 hours to allow for greater blood samples needed for the determination of leptin and insulin by RIA.", "Briefly, 16 mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.", "All blood samples are taken from the tail using EDTA coated capillary tubes (100 μL each time point).", "At time 0 (9:00 AM), a standard high fat meal is given by gavage (vol.=1% of body weight) to all animals.", "Immediately following the high fat meal, 25 μg of a GSSP4 polypeptide is injected i.p.", "in 100 μL saline.", "The same dose (25 μg in 100 μL) is again injected at 45 min and at 1 hr 45 min (treated group).", "Control animals are injected with saline (3×100 μL).", "Untreated and treated animals are handled in an alternating mode.", "Blood samples are immediately put on ice and plasma is prepared by centrifugation following each time point.", "Plasma is kept at −20° C. and free fatty acids (FFA) are determined within 24 hours using a standard test kit (Wako).", "Leptin and Insulin are determined by RIA (ML-82K and SRI-13K, LINCO Research, Inc., St. Charles, Mo.)", "following the manufacturer's protocol.", "However, only 20 μL plasma is used.", "Each determination is done in duplicate.", "Due to the limited amount of plasma available, leptin and insulin are determined in 4 pools of 2 animals each in both treatment groups.", "Example 7 Effect of GSSP4 Polypeptides on Plasma FFA, TG and Glucose in C57 BL/6 Mice The effect of GSSP4 polypeptides on plasma FFA, TG, glucose, leptin and insulin levels during postprandial lipemia (PPL) in normal C57BL6/J mice has been described.", "Weight loss resulting from GSSP4 polypeptides (2.5 μg/day) given to normal C57BL6/J mice on a high fat diet is shown.", "The experimental procedure is similar to described previously.", "Briefly, 14 mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.", "All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point).", "At time 0 (9:00 AM), a standard high fat meal is given by gavage (vol.=1% of body weight) to all animals.", "Immediately following the high fat meal, 4 mice are injected 25 μg of a GSSP4 polypeptide i.p.", "in 100 μL saline.", "The same dose (25 μg in 100 μL) is again injected at 45 min and at 1 hr 45 min.", "A second treatment group receives 3 times 50 μg GSSP4 polypeptide at the same intervals.", "Control animals are injected with saline (3×100 μL).", "Untreated and treated animals are handled in an alternating mode.", "Blood samples are immediately put on ice.", "Plasma is prepared by centrifugation following each time point.", "Plasma is kept at −20° C. and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).", "Example 8 Effect of GSSP4 Polypeptides on FFA Following Epinephrine Injection In mice, plasma free fatty acids increase after intragastric administration of a high fat/carbohydrate test meal.", "These free fatty acids are mostly produced by the activity of lipolytic enzymes i.e.", "lipoprotein lipase (LPL) and hepatic lipase (HL).", "In this species, these enzymes are found in significant amounts both bound to endothelium and freely circulating in plasma.", "Another source of plasma free fatty acids is hormone sensitive lipase (HSL) that releases free fatty acids from adipose tissue after β-adrenergic stimulation.", "To test whether GSSP4 polypeptides also regulate the metabolism of free fatty acid released by HSL, mice are injected with epinephrine.", "Two groups of mice are given epinephrine (5 μg) by intraperitoneal injection.", "A treated group is injected with a GSSP4 polypeptide (25 μg) one hour before and again together with epinephrine, while control animals receive saline.", "Plasma is isolated and free fatty acids and glucose are measured as described above.", "Example 9 Effect of GSSP4 Polypeptides on Muscle FFA Oxidation To investigate the effect of GSSP4 polypeptides on muscle free fatty acid oxidation, intact hind limb muscles from C57BL/6J mice are isolated and FFA oxidation is measured using oleate as substrate (Clee et al (2000) J Lipid Res 41:521-531; Muoio et al (1999) Am J Physiol 276:E913-921).", "Oleate oxidation in isolated muscle is measured as previously described (Cuendet et al (1976) J Clin Invest 58:1078-1088; Le Marchand-Brustel (1978) Am J Physiol 234:E348-E358).", "Briefly, mice are sacrificed by cervical dislocation and soleus and EDL muscles are rapidly isolated from the hind limbs.", "The distal tendon of each muscle is tied to a piece of suture to facilitate transfer among different media.", "All incubations are carried out at 30° C. in 1.5 mL of Krebs-Henseleit bicarbonate buffer (118.6 mM NaCl, 4.76 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgSO4, 2.54 mM CaCl2, 25 mM NaHCO3, 10 mM Hepes, pH 7.4) supplemented with 4% FFA free bovine serum albumin (fraction V, RIA grade, Sigma) and 5 mM glucose (Sigma).", "The total concentration of oleate (Sigma) throughout the experiment is 0.25 mM.", "All media are oxygenated (95% O2; 5% CO2) prior to incubation.", "The gas mixture is hydrated throughout the experiment by bubbling through a gas washer (Kontes Inc., Vineland, N.J.).", "Muscles are rinsed for 30 min in incubation media with oxygenation.", "The muscles are then transferred to fresh media (1.5 mL) and incubated at 30° C. in the presence of 1 μCi/mL [1-14C] oleic acid (American Radiolabeled Chemicals).", "The incubation vials containing this media are sealed with a rubber septum from which a center well carrying a piece of Whatman paper (1.5 cm×11.5 cm) is suspended.", "After an initial incubation period of 10 min with constant oxygenation, gas circulation is removed to close the system to the outside environment and the muscles are incubated for 90 min at 30° C. At the end of this period, 0.45 mL of Solvable (Packard Instruments, Meriden, Conn.) is injected onto the Whatman paper in the center well and oleate oxidation by the muscle is stopped by transferring the vial onto ice.", "After 5 min, the muscle is removed from the medium, and an aliquot of 0.5 mL medium is also removed.", "The vials are closed again and 1 mL of 35% perchloric acid is injected with a syringe into the media by piercing through the rubber septum.", "The CO2 released from the acidified media is collected by the Solvable in the center well.", "After a 90 min collection period at 30° C., the Whatman paper is removed from the center well and placed in scintillation vials containing 15 mL of scintillation fluid (HionicFlour, Packard Instruments, Meriden, Conn.).", "The amount of 14C radioactivity is quantitated by liquid scintillation counting.", "The rate of oleate oxidation is expressed as nmol oleate produced in 90 min/g muscle.", "To test the effect of gACRP30 or ACRP30 on oleate oxidation, these proteins are added to the media at a final concentration of 2.5 μg/mL and maintained in the media throughout the procedure.", "Example 10 Effect of GSSP4 Polypeptides on Triglyceride in Muscle & Liver Isolated from Mice To determine whether the increased FFA oxidation induced by GSSP4 polypeptides is also accompanied by increased FFA delivery into muscle or liver, the hindlimb muscle and liver triglyceride content is measured after the GSSP4 polypeptide treatment of mice.", "Hind limb muscles as well as liver samples are removed from treated and untreated animals and the triglyceride and free fatty acid concentration is determined following a standard lipid extraction method (Shimabukuro et al (1997) Proc Natl Acad Sci USA 94:4637-4641) followed by TG and FFA analysis using standard test kits.", "Example 11 Effect of GSSP4 Polypeptides on FFA following Intralipid Injection Two groups of mice are intravenously (tail vein) injected with 30 μL bolus of Intralipid-20% (Clintec) to generate a sudden rise in plasma FFAs, thus by-passing intestinal absorption.", "(Intralipid is an intravenous fat emulsion used in nutritional therapy).", "A treated group (GSSP4 polypeptide-treated) is injected with a GSSP4 polypeptide (25 μg) at 30 and 60 minutes before Intralipid is given, while control animals (▴ control) received saline.", "Plasma is isolated and FFAs are measured as described previously.", "The effect of GSSP4 polypeptides on the decay in plasma FFAs following the peak induced by Intralipid injection is then monitored.", "Example 12 Tests of Obesity-Related Activity in Humans Tests of the efficacy of in humans are performed in accordance with a physician's recommendations and with established guidelines.", "The parameters tested in mice are also tested in humans (e.g.", "food intake, weight, TG, TC, glucose, insulin, leptin, FFA).", "It is expected that the physiological factors would show changes over the short term.", "Changes in weight gain might require a longer period of time.", "In addition, the diet is carefully monitored.", "GSSP4 is given in daily doses of about 6 mg protein per 70 kg person or about 10 mg per day.", "Other doses are tested, for instance 1 mg or 5 mg per day up to 20 mg, 50 mg, or 100 mg per day.", "Example 13 Tests of Obesity-Related Activity in a Murine Lipoatrophic Diabetes Model Leptin was reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al.", "Nature 401: 73-76 (1999).", "Leptin is found to be less effective in a different lipodystrophic mouse model of lipoatrophic diabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporated herein in its entirety including any drawings, figures, or tables).", "The instant invention encompasses the use of GSSP4 or polypeptide fragments for reducing the insulin resistance and hyperglycaemia in this model either alone or in combination with leptin, the leptin peptide (U.S. provisional application No.", "60/155,506), or other compounds.", "Assays include that described previously in Gavrilova et al.", "((2000) Diabetes November;49(11): 1910-6; (2000) Nature February 24;403(6772):850) using A-ZIP/F-1 mice, except that would be administered using the methods previously described in Example 5 (or Examples 8-10).", "The glucose and insulin levels of the mice would be tested, and the food intake and liver weight monitored, as well as other factors, such as leptin, FFA, and TG levels, typically measured in our experiments (see Example 5, above, or Examples 8-10)." ] ]	Patent_10467554
[ [ "Method, module, device and server for voice recognition", "The invention concerns a voice recognition method implemented in at least a terminal, the voice recognition method using a language model, comprising the following steps: detecting at least a non-recognised expression in one of the terminals; recording in the terminal data representing the non-recognised expression; transmission by the terminal of the recorded data to a remote server; analysis, at the remote server, of said data and generation of data for correcting said language model taking into account at least part of the non-recognised expressions; and transmitting from the server to at least a terminal correction data, so as to enable subsequent recognition of at least some of the non-recognised expressions.", "The invention also concerns corresponding modules, devices and remote server." ], [ "1.Voice recognition process implemented in at least one terminal, the said voice recognition process using a language model, wherein it comprises the following steps: detection of at least one unrecognized expression in one of the said terminals; recording in the said terminal of data representative of the said unrecognized expression; transmission by the said terminal of the said recorded data to a remote server, via a first transmission channel; analysis, at the level of the said remote server, of the said data and generation of information for correcting the said language model taking account of at least one part of the said unrecognized expressions; and transmission via a second transmission channel from the said server to at least one terminal of the said correcting information, so as to allow future recognition of at least certain of the said unrecognized expressions.", "2.Process according to claim 1, wherein the said data representative of the said unrecognized expressions comprise a compressed voice recording representative of parameters descriptive of the acoustic signal.", "3.Process according to claim 1, wherein during the said step of transmission by the said terminal, the latter furthermore transmits to the said server at least one of the items of information forming part of the group comprising: information of context of use of the said voice recognition process when an expression has not been recognized; and information relating to the speaker who has uttered an unrecognized expression.", "4.Process according to claim 1, wherein it implements an encryption and/or a scrambling of the said recorded data and/or of the said correcting information.", "5.Voice recognition module using a language model, wherein it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the said recorded data to a remote server; and a receiver of correcting information allowing the correcting of the said language model transmitted to the said module allowing future recognition of at least certain of the said unrecognized expressions by the said module, the correcting information having been transmitted by the said remote server after analysis at the level of the said remote server of the said data, and after generation of information for correcting the said language model taking account of at least one part of the unrecognized expressions.", "6.Voice recognition device using a language model, wherein it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the said recorded data to a remote server; and a receiver of correcting information allowing the correcting of the said language model transmitted to the said device allowing future recognition of at least certain of the said unrecognized expressions by the said device, the correcting information having been transmitted by the said remote server after analysis at the level of the said remote server of the said data, and after generation of information for correcting the said language model taking account of at least one part of the unrecognized expressions.", "7.Voice recognition server, the said recognition being implemented in a set of at least one remote terminal, using a language model, wherein it comprises the following means: a receiver of data representative of at least one expression unrecognized by at least one terminal forming part of the said set and having detected the said unrecognized expression during a voice recognition operation; and a sender sending to the said set of at least one remote terminal correcting information obtained on the basis of an analysis of the said data received at the level of the said server, the said correcting information allowing the correcting by each of the terminals of the said set, of the said language model allowing future recognition of at least one part of the unrecognized expressions." ], [ "The present invention concerns the field of voice interfaces.", "More precisely, the invention relates to the optimization of language models and/or of phonetic units in terminals using voice recognition.", "Information or control systems are making ever increasing use of a voice interface to make interaction with the user faster and/or more intuitive.", "Since these systems are becoming ever more complex, the requirements in terms of voice recognition are ever more considerable, both as regards the extent of recognition (very large vocabulary) and the speed of recognition (real time).", "Voice recognition processes based on the use of language models (probability that a given word of the vocabulary of the application follows another word or group of words in the chronological order of the sentence) and of phonetic units are known in the state of the art.", "These techniques are in particular described in the work by Frederik Jelinek “Statistical methods for speech recognition” published by MIT Press in 1997.These techniques rely on language models and phonetic units which are produced from representative voice samples (emanating for example from a population of users of a terminal who are made to utter commands).", "In practice, the language models must take account of the speaking style ordinarily employed by a user of the system, and in particular of his “defects”: hesitations, false starts, change of mind, etc.", "The quality of a language model used greatly influences the reliability of the voice recognition.", "This quality is most often measured by an index referred to as the perplexity of the language model, and which schematically represents the number of choices which the system must make for each decoded word.", "The lower this perplexity, the better the quality.", "The language model is necessary to translate the voice signal into a textual string of words, a step often used by dialogue systems.", "It is then necessary to construct a comprehension logic which makes it possible to comprehend the query so as to reply to it.", "There are two standard methods for producing large vocabulary language models: The so-called N-gram statistical method, most often employing a bigram or trigram, consists in assuming that the probability of occurrence of a word in the sentence depends solely on the N words which precede it, independently of the rest of its context in the sentence.", "If one takes the example of the trigram for a vocabulary of 1000 words, it would be necessary to define 10003 probabilities to define the language model, this being impossible.", "The words are therefore grouped into sets which are either defined explicitly by the model designer, or deduced by self-organizing methods.", "This language model is therefore constructed from a text corpus automatically.", "This type of language model is used mainly for voice dictation systems whose ultimate functionality is to translate the voice signal into a text, without any comprehension phase being necessary.", "The second method consists in describing the syntax by means of a probabilistic grammar, typically a context-free grammar defined by virtue of a set of rules described in the so-called Backus Naur Form or BNF, or an extension of this form to contextual grammars.", "The rules describing grammars are most often handwritten.", "This type of language model is suitable for command and control applications, in which the recognition phase is followed by a phase of controlling an appliance or of searching for information in a database.", "The language model of an application describes the set of expressions (for example sentences) that the application will be required to recognize.", "A drawback of the prior art is that, if the language model is of poor quality, the recognition system, even if it performs extremely well at the acoustico-phonetic decoding level, will have mediocre performance for certain expressions.", "The stochastic type language models do not have, properly speaking, a clear definition of the expressions which are in the language model, and of those which are outside.", "Certain expressions simply have a higher a priori probability of occurrence than others.", "The language models of probabilistic grammar type show a clear difference between expressions belonging to the language models, and expressions external to the language model.", "In these models, expressions therefore exist which will never be able to be recognized, regardless of the quality of the phonetic models used.", "These are generally expressions having no meaning or that carry a meaning outside of the field of application of the system developed.", "It turns out that the language models of probabilistic type and their derivatives are more effective for command and control applications.", "These grammars are often written by hand, and one of the main difficulties of the development of dialogue systems is to offer a language model of good quality.", "In particular, as far as the models of grammar type are concerned, it is not possible to exhaustively define a language in particular if the latter is liable to be used by a large population (case for example of a remote control for mass-market appliances).", "It is not possible to take account of all the possible expressions and turns of phrase (from formal language to slang), and/or of errors of grammar, etc.", "The invention relates to a voice recognition process and system making it possible to modify and improve a language model remotely, on the basis of the recordings of expressions unrecognized by the system.", "More precisely, the subject of the invention is a voice recognition process implemented in at least one terminal, the said voice recognition process using a language model, characterized in that it comprises the following steps: detection of at least one unrecognized expression in one of the terminals; recording in the terminal of data representative of the unrecognized expression; transmission by the terminal of the recorded data to a remote server, via a first transmission channel; analysis, at the level of the remote server, of the data and generation of information for correcting the language model taking account of at least one part of the unrecognized expressions; and transmission via a second transmission channel from the server to at least one terminal of the correcting information, so as to allow future recognition of at least certain of the unrecognized expressions.", "Thus, the invention relies on an entirely novel and inventive approach to voice recognition, which makes it possible to update the various elements allowing voice recognition as a function of locally unrecognized expressions, a remote server being furnished with considerable resources (for example human and/or computational capabilities) generating correcting information.", "It is noted that here the language models comprise: language models in the strict sense (this being the case, for example, when the data, the subject of the recognition, are of purely textual type); models formed of one or more language models in the strict sense and of one or more sets of phonetic units (this corresponding in particular to the general case of voice recognition applied to voice samples).", "The invention goes well beyond the straightforward updating of a vocabulary.", "Specifically, it is possible that, although all the words of an expression feature in the vocabulary used by the language model of the terminal, this expression may not be recognized.", "Only the updating of the language model itself then makes it possible to have this expression recognized subsequently.", "The updating of the vocabulary, which is one of the items of information from which the language model is derived, is not sufficient.", "Here, the expressions are to be taken within the wide sense and relate to any vocal expression allowing interaction between a terminal and its user.", "Expressions (or utterances) comprise in particular sentences, phrases, isolated or non-isolated words, code words specific to the terminal, instructions, commands, etc.", "The correcting information may comprise in particular information allowing partial or complete modification of the language model and/or phonetic units present in each terminal by deleting, replacing or adding elements therein.", "The server can receive data from each terminal allowing it to improve the language model and/or the phonetic units present in the data sending terminal and also in all other terminals, each of the terminals benefiting from the shared experience acquired by the server from all the terminals.", "Thus, the invention makes it possible to take into account language styles or turns of phrase specific to certain users (for example, the expression “8 pm in the evening” (pleonasm which is hard to imagine a priori) instead of “8 pm” or “8 o'clock in the evening”) and for which provision had not been made in the course of the construction of the language model implemented.", "Furthermore, the invention takes into account the evolution of living languages (new turns of phrase or expressions, etc).", "It is noted that the invention applies equally well to language models of stochastic type and to language models of probabilistic grammar type.", "When the invention is applied to language models of stochastic type, there are generally very many correcting data for influencing recognition, whereas the correcting data for a model of probabilistic grammar type may be scanty and have an appreciable influence on the effectiveness and reliability of recognition.", "According to a particular characteristic, the process is noteworthy in that the data representative of the unrecognized expressions comprise a compressed voice recording representative of parameters descriptive of the acoustic signal.", "Thus, the invention advantageously makes it possible to take into account the voice data sent to the source for fine analysis at the server level, while limiting the volume of data transmitted to the remote server.", "According to a particular characteristic, the process is noteworthy in that during the step of transmission by the terminal, the latter furthermore transmits to the server at least one of the items of information forming part of the group comprising: information of context of use of the voice recognition process when an expression has not been recognized; and information relating to the speaker who has uttered an unrecognized expression.", "Thus, voice recognition of the expressions unrecognized by the terminal, which may be performed remotely, is facilitated.", "Furthermore, a check of the validity of the content of the unrecognized expressions may be performed as a function of the context.", "(For example, a command “record the transmission” has a meaning and is therefore valid when the terminal to which it is addressed is a video recorder and has no meaning in respect of a mobile telephone).", "According to a particular characteristic, the process is noteworthy in that it implements an encryption and/or a scrambling of the recorded data and/or of the correcting information.", "Thus, the data are made secure effectively and remain confidential.", "The information also relates to a voice recognition module using a language model, characterized in that it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the recorded data to a remote server; and a receiver of correcting information allowing the correcting of the language model transmitted to the module allowing future recognition of at least certain of the unrecognized expressions by the module, the correcting information having been transmitted by the remote server after analysis at the level of the remote server of the data, and after generation of information for correcting the language model taking account of at least one part of the unrecognized expressions.", "The invention also relates to a voice recognition device using a language model, characterized in that it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the recorded data to a remote server; and a receiver of correcting information allowing the correcting of the language model transmitted to the device allowing future recognition of at least certain of the unrecognized expressions by the device, the correcting information having been transmitted by the remote server after analysis at the level of the remote server of the data, and after generation of information for correcting the language model taking account of at least one part of the unrecognized expressions.", "The invention also relates to a voice recognition server, the recognition being implemented in a set of at least one remote terminal, using a language model, characterized in that it comprises the following means: a receiver of data representative of at least one expression unrecognized by at least one terminal forming part of the set of at least one remote terminal and having detected the unrecognized expression during a voice recognition operation; and a sender sending to the set of at least one remote terminal correcting information obtained on the basis of an analysis of the data received at the level of the server, the correcting information allowing the correcting, by each of the terminals of the set, of the language model allowing future recognition of at least one part of the unrecognized expressions.", "The particular characteristics and the advantages of the module, of the device and of the server for voice recognition being similar to those of the voice recognition process, they are not recalled here.", "Other characteristics and advantages of the invention will become more clearly apparent when reading the following description of a preferred embodiment given by way of straightforward non-limiting illustrative example, and of the appended drawings, among which: FIG.", "1 depicts a general schematic of a system comprising a voice-controlled box, in which the technique of the invention may be implemented; FIG.", "2 depicts a schematic of the voice recognition box of the system of FIG.", "1; FIG.", "3 describes an electronic diagram of a voice recognition box implementing the schematic of FIG.", "2; FIG.", "4 depicts a schematic of the server of the system of FIG.", "1; FIG.", "5 represents a flow chart of the process for testing an expression and for recording data relating to unrecognized expressions, as implemented by the recognition engine of FIG.", "2; FIG.", "6 represents a flow chart of the process for sending data relating to unrecognized expressions, as implemented by the rejection module of FIG.", "2; FIG.", "7 represents a flow chart of the process for receiving correcting data, as implemented by the module for loading the language models of FIG.", "2; and FIG.", "8 represents a flow chart of the process for receiving and for processing correcting data, as implemented within the remote server of FIG.", "4.The general principle of the invention therefore relies on voice recognition implemented in terminals, the voice recognition process using a language model and/or a set of phonetic units that may be updated by a remote server when, in particular, the latter deems it necessary.", "In a general manner each terminal can recognize expressions (for example sentence or command) formulated by a speaker and execute a corresponding action.", "Nevertheless, it is often found that certain expressions that are entirely comprehensible to a human being are not recognized by the device or the module implementing the voice recognition.", "The failure of recognition may have multiple reasons: vocabulary used by the speaker not forming part of the language model; particular pronunciation (with accent for example); particular turn of phrase not provided for by the voice recognition device or module; etc.", "Specifically, the language models and the sets of phonetic units are often constructed on the basis of statistical data which take into account samples of expressions customarily used by a typical population, certain words of vocabulary, pronunciations, and/or turns of phrase then not being (and unable to be) taken into account.", "The invention relies firstly on detecting expressions unrecognized by the voice recognition device or module.", "When an expression has not been recognized, the terminal records data representative of the signal corresponding to the unrecognized expressions (such as, for example, a voice digital recording of the expression), with a view to sending them to a remote server.", "At the level of the remote server centralizing the unrecognized expressions from a set of terminals, a human operator can then analyse the unrecognized expressions.", "Certain of them may prove to be incomprehensible and/or unutilizable and will be discarded.", "On the other hand, others will be entirely comprehensible to the operator who will be able (if he deems it useful) through a man/machine link up to “translate” these expressions hitherto unrecognized by the terminals into a code comprehensible to the server.", "The server can then take these expressions into account together with their translation so as to generate information for correcting the language model and/or the set of phonetic units.", "It will be noted that correction is understood here as: modification of the model; and/or supplementing the model.", "The server then sends the correcting information to each of the terminals which can update its language model and/or set of phonetic units which are enriched with numerous expressions unrecognized by itself or by other terminals.", "Thus, the voice recognition of each of the terminals is improved by benefitting from the experience shared by all the terminals.", "According to a particular mode of the invention, the analysis is not performed by an operator but by the server itself which may have much more considerable resources at its disposal than a straightforward terminal.", "According to particular embodiments, the terminals send the server context data (for example the time, the date, a control performed manually or vocally after the failure of a voice command, the location, the type of terminal, etc.)", "together with the data representative of the signal corresponding to the unrecognized expressions.", "This may facilitate the analysis work of the operator and/or of the server.", "A general schematic of a system comprising a voice-controlled box, in which the technique of the invention may be implemented, is depicted in conjunction with FIG.", "1.This system comprises in particular: a remote server 116 controlled by a human operator 122; and a plurality of user systems 114, 117 and 118.The remote server 116 is linked to each of the user systems 114, 117 and 118 via communication downlinks 115, 119 and 120 respectively.", "These links may be permanent or temporary and be of any type well known to the person skilled in the art.", "They may in particular be of broadcasting type and be based on RF, satellite or wire channels used by television or any other type such as, for example, an Internet type link.", "FIG.", "1 describes in particular the user system 114 which is linked via a communication uplink 121 to the server 116.This link can likewise be of any type well known to the person skilled in the art (in particular telephone, Internet, etc).", "The user system 114 comprises in particular: a voice source 100 which may in particular consist of a microphone intended to pick up a voice signal produced by a speaker; a voice recognition box 102; a control box 105 intended to drive an appliance 107; a controlled appliance 107, for example of television, video recorder or mobile communication terminal type; a unit 109 for storing the expressions detected as unrecognized; an interface 112 allowing upward and downward communications to the server 116.The source 100 is linked to the voice recognition box 102, via a link 101 which allows it to transmit an analogue source wave representative of a voice signal to the box 102.The box 102 can retrieve context information 104 (such as for example the type of appliance 107 which can be controlled by the control box 105 or the list of control codes) via a link 104 and send commands to the control box 105 via a link 103.The control box 105 sends commands via a link 106, for example infrared, to the appliance 107, as a function of the information which it recognizes according to its language model and its dictionary.", "The control box 105 detects the expressions which it does not recognize, and, instead of simply rejecting them, by sending a non-recognition signal, it performs a recording of these expressions to the storage unit 109 via a link 108.The unit 109 for storing the unrecognized expressions sends representative data to the interface 112 via a link 111, which relay them to the server 116 via the link 121.After correct transmission, the interface 110 can send a signal 110 to the storage unit 109 which can then erase the transmitted data.", "The control box 105 receives, moreover, correcting data from the interface 112 via a link 113, that the interface 112 has itself received from the remote server via the link 115.These correcting data are taken into account by the control box 105 for the updating of language models and/or of sets of phonetic units.", "According to the embodiment considered the source 100, the voice recognition box 102, the control box 105, the storage unit 109 and the interlace 112 form part of one and the same device and thus the links 101, 103, 104, 108, 111, 110 and 113 are links internal to the device.", "The link 106 is typically a wireless link.", "According to a first variant embodiment of the invention described in FIG.", "1, the elements 100, 102, 105, 109 and 112 are partly or completely separate and do not form part of one and the same device.", "In this case, the links 101, 103, 104, 108, 111, 110 and 113 are external wire or other links.", "According to a second variant, the source 100, the boxes 102 and 105, the storage unit 109 and the interface 112 as well as the appliance 107 form part of one and the same device and are interlinked by internal buses (links 101, 103, 104, 108, 111, 110, 113 and 106).", "This variant is particularly beneficial when the device is, for example, a mobile telephone or a portable communication terminal.", "FIG.", "2 depicts a schematic of a voice-controlled box such as the box 102 illustrated with regard to FIG.", "2.It is noted that the box 102 receives from outside the analogue source wave 101 which is processed by an Acoustico-Phonetic Decoder 200 or APD (also called the “front end”).", "The APD 200 samples the source wave 101 at regular intervals (typically every 10 ms) so as to produce real vectors or vectors belonging to code books, typically representing oral resonances that are sent via a link 201 to a recognition engine 203.The APD is for example based on a PLP (standing for “Perceptual Linear Prediction”) described in particular in the article “Perceptual Linear Prediction (PLP) analysis of speech” written by Hynek Hermansky and published in “Journal of the Acoustical Society of America”, Vol.", "97, No4, 1990 on pages 1738-1752.With the aid of a dictionary 202, the recognition engine 203 analyses the real vectors that it receives, using in particular hidden Markov models or HMMs and language models (which represent the probability of one word following another word).", "Recognition engines are in particular described in detail in the book “Statistical Methods for Speech Recognition” written by Frederick Jelinek, and published by MIT Press in 1997.The language model allows the recognition engine 203 (which may use in particular hidden Markov networks) to determine which words may follow a given word of any expression usable by the speaker in a given application, and to give the associated probability.", "The words in question belong to the vocabulary of the application, which may be, independently of the language model, of small size (typically from 10 to 300 words), or of large size (for example of size greater than 300 000 words).", "Patent application PCT/FR00/03329 dated 29 Nov. 1999 filed in the name of Thomson Multimedia describes a language model comprising a plurality of syntactic blocks.", "The use of the invention which is the subject of the present patent application is particularly advantageous in conjunction with this type of modular language model, since the modules may be updated separately, thereby avoiding the downloading of overly large volume files.", "The language models are transmitted by a language model loading module 207.The module 207 itself receives language models, updates or corrections of language models and/or of phonetic units transmitted from the server via the link 113.It is noted that the dictionary 202 belongs to the language model making reference to words from the dictionary.", "Thus, the dictionary 202 itself can be updated and/or corrected via a language model loaded by the module 207.After implementation of a recognition operation based on the use of a Viterbi algorithm, the recognition engine 203 supplies the rejection module 211 with an ordered list of sequences of words in accordance with the language model, which exhibits the best score for the expression uttered.", "The rejection module 211 works downstream of the recognition engine 203 and operates according to one or more of the following principles: Sometimes, for reasons specific to the Viterbi algorithm, the latter may not produce a consistent list because the scores are so low that the limit of acceptable accuracies of the machine in terms of arithmetic computation is overstepped.", "There is therefore no consistent complete proposal.", "Thus, when the rejection module 211 detects one or more scores below a predetermined acceptable limit, the expression is rejected.", "Each element of the list calculated by the Viterbi algorithm has been retained because the associated score was among the highest relative scores of all the possible expressions, according to the language model.", "Additionally, the Markov network associated with each of these expressions makes it possible to evaluate the intrinsic probability of the network in question producing the expression associated with the score observed.", "The rejection module 211 analyses this probability and if it is less than a predetermined threshold of acceptability of probability, the expression is rejected.", "According to another method, for the best proposals obtained via the Viterbi algorithm, the rejection module 211 performs a complementary processing of the expressions, using criteria which had not been taken into account in the course of the Viterbi development.", "For example, it checks that those parts of the signal that have to be voiced because they are associated with vowels, are actually so.", "If the expressions proposed do not fulfil these conditions, they are rejected.", "When the rejection module 211 rejects an expression, as illustrated previously, the expression is said to be unrecognized and a signal indicating the rejected expression is sent to the recognition engine 203.In parallel, the rejection module transmits a recording of the unrecognized expression to the storage unit 109 via the link 108.The recognition engine 203 is responsible for recognizing expressions emanating from the APD 200 in the form of phonetic samples.", "Thus, the recognition engine 203 uses: the phonetic units to construct the phonetic representation of a word in the form of a Markov model, each word of the dictionary 202 possibly possessing several “phonetizations”; and simultaneously the language model in the strict sense for recognizing expressions of greater or lesser complexity.", "The recognition engine 203 supplies expressions which have been recognized (that is to say not rejected by the module 211) and which it has identified on the basis of the vectors received to a means 205 for translating these expressions into commands that can be understood by the appliance 107.This means 205 uses an artificial intelligence translation process which itself takes into account a context 104 supplied by the control box 105 before sending one or more commands 103 to the control box 105.FIG.", "3 diagrammatically illustrates a voice recognition module or device 102 such as illustrated in conjunction with FIG.", "1, and implementing the schematic of FIG.", "2.The box 102 comprises interlinked by an address and data bus: a voice interface 301; an analogue-digital converter 302; a processor 304; a nonvolatile memory 305; a random access memory 306; a reception module 312; a transmission module 313; and an inputs/outputs interface 307.Each of the elements illustrated in FIG.", "3 is well known to the person skilled in the art.", "These commonplace elements are not described here.", "It is observed moreover that the word “register” used throughout the description designates in each of the memories mentioned, both a memory area of small capacity (a few data bits) and a memory area of large capacity (making it possible to store an entire program or the whole of a sequence of transaction data).", "The nonvolatile memory 305 (ROM) holds in particular the program for operating the processor 304 in a “prog” register 308.The random access memory 306 holds data, variables and intermediate results of processing in registers which for convenience possess the same names as the data that they hold and comprise in particular: a register 309 in which are held recordings of unrecognized expressions, “Exp_Not_Rec; a counter 310 of unrecognized sentences Nb_Exp_Not _Rec; and a language model in a register 311, Model_Language.", "It is noted furthermore that the receive module 312 and send module 313 are modules that allow the transmission of data respectively from or to the remote server 116.The wire-based or wireless techniques for receiving and for sending are well known to the person skilled in the art of telecommunications and will not be detailed further.", "FIG.", "4 depicts the server 116 of the system illustrated with regard to FIG.", "1.It is noted that the server 116 is controlled by a human operator 122 via any man/machine interface 404 (for example, of keyboard and screen type).", "The server 116 itself comprises in particular: a receiver 400; an analyser 401; a module 402 for construction of correction of a language model and/or a set of phonetic units; and a sender 403.The receiver 400 is compatible with the sender 313 of a terminal and can receive from each terminal in particular data (for example recording) representative of unrecognized expressions via the link 121 and possibly of complementary data (for example contextual data).", "The analyser 401 receives the set of data from the receiver 400 via a link 121 which it transmits to the operator 122 via an interface 404 which is for example a terminal furnished: with a screen and with a keyboard allowing dialogue with the server 116 and its control; with loudspeakers or an audio headset for listening to unrecognized recordings.", "This interface 404 also allows the analyser 401 to receive information from the operator 122 indicating: that an unrecognized expression not covered by the language model remains incomprehensible, has no meaning within the application to the terminal and/or does not relate to the terminal (it should therefore not be included in the language model), this expression then being ignored for the correcting of the language model and discarded by the analyser 401; that an unrecognized expression nevertheless belongs to the language model in the strict sense (this then involves a pure recognition problem); in this case, this involves modifying the phonetic units and not the language model in the strict sense; or a translation into for example the form of a control code, after identification of the content of an expression by the operator, the unrecognized expression not belonging to the language model and having a meaning for the terminal for which it was intended; this then involves correcting the language model in the strict sense.", "A combination of the second and third solutions is possible; in this case, this involves both modifying the phonetic units and the language model in the strict sense.", "This embodiment corresponds to a manual processing of the unrecognized expressions.", "According to this embodiment, the human operator 122 listens to the unrecognized expression and analyses the reasons for its rejection.", "The operator 122 determines in particular whether the expression belongs to the language model or not.", "In the case where the expression belongs to the language model, the operator analyses the expression to identify the intrinsic recognition problem (expression belonging to the language model and which ought to have been recognized and which was not for other reasons: noise, accent of the speaker, etc.).", "According to a first variant, the processing is automatic and the intervention of a human operator becomes nil.", "In this case, the server 116 and in particular the analyser 401 possess a relatively considerable computational power which may in particular be much larger than a terminal.", "According to this variant, the analyser 401 analyses each unrecognized expression in a more appropriate manner than could be done by a terminal, using, for example, a richer language model and/or more complex phonetic models.", "Not being subject to such strict real-time computational requirements as might be a terminal (which often requires a fast reaction time to a speaker command), the analyser 401 can also, for example, allow recognition demanding a longer processing time than in a terminal.", "According to a second variant, the processing is semi-automatic and the intervention of a human operator remains limited to the cases that cannot be solved by the analyser.", "The general structure of a server 116 is according to the preferred embodiment described here similar to that of a terminal such as described with regard to FIG.", "3 and comprises in particular interlinked by an address and data bus: a processor; a random access memory; a nonvolatile memory; a suitable transmission module; a reception module; and a man/machine linkup interface.", "According to FIG.", "5 representing a flow chart of the testing of an expression and of the recording of data relating to unrecognized expressions, as implemented by the recognition engine 203 of FIG.", "2, in the course of a first step of initialization 500, the microprocessor 304 commences the execution of the program 308 and initializes the variables of the random access memory 306.Then, in the course of a step 501 of waiting for an expression, it waits for and receives an expression sent by a speaker.", "Next, in the course of a test 502, after having executed an operation of voice recognition for the expression received, it determines whether the expression has or has not been recognized according to one or more criteria illustrated with regard to the description of the rejection module 211 of FIG.", "2.If it has, in the course of a control step 504, the terminal 102 takes into account the result of the voice recognition applied to the expression received and executes an appropriate action such as for example a command.", "If it has not, in the course of a step 503 of recording an expression, the unrecognized expression is compressed and recorded in the storage unit 109 awaiting a transmission to the remote server 116 as illustrated with regard to FIG.", "6.On completion of one of the steps 503 or 504, step 501 of waiting for an expression is repeated.", "FIG.", "6 representing a flow chart of the sending of data relating to unrecognized expressions, as implemented by the rejection module of FIG.", "2, in the course of a first step of initialization 600, the microprocessor 304 commences the execution of the program 308 and initializes the variables of the random access memory 306.Then, in the course of a step 601 of waiting for expressions unrecognized by the voice recognition module 102, the microprocessor 304 waits for and then receives recordings of unrecognized expressions.", "Then, in the course of a step 602, the terminal 114 connects up to the remote server 116 according to methods well known to the person skilled in the art of telecommunications.", "Next, in the course of a step 603, the recordings of unrecognized expressions are shaped and sent to the remote server 116.Then, in the course of a step 604 of disconnection, the terminal disconnects from the remote server 116 and a signal is sent between the interface 112 with the remote server and the unit 109 for storing the data corresponding to the unrecognized expressions indicating the transmission of the recordings of expressions.", "The data corresponding to these expressions are then erased from the storage unit 109.Next, step 601 is repeated.", "FIG.", "7 represents a flow chart of the receiving of correcting data, as implemented by the module 207 for loading the language models of FIG.", "2.After a first step 700 of initialization, in the course of a step) 701, the terminal places itself on standby awaiting correcting data broadcast by a server 1116 to a plurality of terminals.", "Next, during a step 702, the terminal takes into account the correcting data so as to update the language model and/or its set of phonetic units used by the voice recognition module.", "According to the nature of the correcting data, these data will be able in particular: to substitute for existing data in the language model and/or its set of phonetic units; to modify existing data; to supplement existing data; and/or to bring about the deletion of existing data.", "After the execution of step 702, step 703 is repeated.", "FIG.", "8 represents a flow chart of the receiving and processing of correcting data, as implemented within the remote server of FIG.", "4.After a first step 800 of initialization of the parameters and of instigation of a program for management of the server, the server 116 places itself on standby waiting for a connection request originating from a terminal (which executes a step 602 illustrated with regard to FIG.", "6) and establishes a connection with the terminal according to methods well known to the person skilled in the art of telecommunications.", "Then, in the course of a step 802, the server 116 receives data originating from the connected terminal which executes a step 603 described previously.", "These data contain in particular recordings of one or more expressions rejected by the terminal because they were not recognized by a voice recognition module implemented in the terminal.", "When all the data have been received, the connection between the terminal and the server 116 is broken.", "Next, in the course of a step 803 of processing the data received, the server 116 processes each of the received recordings of expressions either manually by the operator 122 or automatically or semi-automatically according to various alternatives illustrated with regard to FIG.", "4.Then, in the course of a test 804, the server 116 determines whether in particular one or more expressions received were comprehensible and are relevant in respect of the terminal which transmitted this or these expressions.", "An updating of the language models and/or of the phonetic units is then necessary.", "If not, then the waiting step 801 is repeated.", "In the converse case, the server 116 constructs a correction of the language model which may take several forms allowing a step 607 (illustrated previously) within the terminals after receipt of the correcting data.", "These correcting data comprise in particular: an indicator specifying the nature of the correction (in particular substitution, modification, supplement, or deletion); and the correcting data themselves as a function of the indicator.", "It is noted that if the language model comprises a plurality of syntactic blocks (case in particular of the language models such as described in patent PCT/FR00/03329 mentioned above), each module can be corrected separately.", "In this case, the correction data also comprise an indicator of the module or modules to be corrected.", "Then, in the course of a step 806, the server 116 broadcasts the correcting data to one or preferably to a set of terminals which will be able to update their language model and/or set of phonetic units according to a step 607.Step 801 is then repeated.", "The procedure is thus iterative and may be repeated several times.", "It also allows the application to be upgraded by adding new queries.", "Of course, the invention is not limited to the exemplary embodiments mentioned hereinabove.", "In particular, the person skilled in the art will be able to alter the definition of the terminals implementing the invention, the invention relating to any type of device and/or module using or capable of using a voice recognition process (being of the type for example of multimedia terminal, television, video recorder, a multimedia digital decoder (or set top box), audio or video equipment, fixed or mobile terminal, etc.).", "Likewise, the invention relates to any type of remote server (for example Internet servers, equipment coupled to television program broadcasters, equipment coupled to mobile communication networks, service provider equipment, etc.).", "Furthermore, according to the invention, the transmission channel for the data corresponding to the unrecognized sentences and the transmission channel for the data for correcting the language models and/or phonetic units are any whatsoever and include in particular: RF transmission pathways; satellite transmission pathways; the channels of television broadcasting networks; the channels of Internet type networks: the channels of telephone networks; the channels of mobile networks; and removable media.", "Moreover, it is noted that the invention relates not only to unrecognized sentences but relates to any type of vocal expression such as for example one or more sentences, an isolated or unisolated word, a phrase, a voice code allowing dialogue between a machine and its user.", "These oral expressions may be associated not only with commands, but with any type of data that can form the subject of a dialogue between a machine and its user, as, for example, information data that the user can transmit to the machine, configuration data, programming data etc.", "It is also noted that the method of updating the language models which is described by the patent applies not only to processes of voice recognition in the strict sense, but applies also to processes for recognition of textual inputs supporting orthographic mistakes and/or typing mistakes, and also based on Markovian models or language models in the strict sense, as described in the patent.", "It will be noted that the invention is not limited to a purely hardware installation but that it can also be implemented in the form of a sequence of instructions of a computer program or any form mixing a hardware part and a software part.", "In the case where the invention is installed partially or totally in software form, the corresponding sequence of instructions may be stored in a removable storage means (such as for example a diskette, a CD-ROM or a DVD-ROM) or otherwise, this means of storage being readable partially or totally by a computer or a microprocessor." ] ]	Patent_10467586
[ [ "Tissue transglutaminase", "The present invention relates to tissue transglutaminase (tTG), and more particularly to DNA coding for and polypeptides containing, the primary structural conformaion of one or more epitopes of tTG and to the use thereof in the detection of autoantibodies and/or lymphocytes produced in response to tTG." ], [ "1.A DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes,: which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.", "Ids 1, 3, 5, 7, 9, 11, 13, 15, 17, 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes.", "2.A DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.", "Ids 1, 3, 5, 7 and 9; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq.", "Ids 2 4, 6, 8 and 10, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes.", "3.A DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.", "Ids 11, 13, 15, 17 or 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.", "Ids 12, 14, 16 and 18, or one or more active fragments of amino acids as depicted in one or more of Seq.", "Ids 12, 14, 16 and 18, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes.", "4.A DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.", "Ids 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies.", "5.A DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.", "Ids 1, 3, 5, 7 or 9; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8 and 10, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies.", "6.A DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.", "Ids 11, 13, 15, 17 or 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.", "Ids 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies.", "7.A DNA sequence as depicted in any of Seq.", "Ids 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19, or a DNA sequence differing therefrom due to the degeneracy of the genetic code.", "8.A biologically functional plasmid or DNA vector including a DNA sequence as defined in claim 1.9.A host cell which is transformed or transfected with a DNA sequence as defined in claim 1.10.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "11.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "12.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes; with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "13.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "14.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "15.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "16.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact,under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "17.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8 and 10, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "18.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.", "Ids 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.", "Ids 12, 14, 16, 18 and 20, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "19.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "20.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq.", "Ids 2, 4, 6, 8 and 10, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "21.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.", "Ids 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.", "Ids 12, 14, 16, 18 and 20, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "22.A polypeptide as depicted in any of Seq.", "Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.23.A process of preparing a polypeptide as defined in claim 10, which process comprises: (i) providing a host cell which is transformed or transfected with a DNA sequence or a biologically functional plasmid or DNA vector; (ii) growing the host cell; and (iii) recovering a polypeptide as defined in claim 10.24.A method of screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing either (i) said sample of body fluid from said subject or (ii) lymphocytes isolated from said sample; (b) contacting said sample or isolated lymphocytes with a polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or said lymphocytes, in said sample, or isolated from said sample.", "25.A method according to claim 24, for screening for autoantibodies produced in response to tTG and present in said sample.", "26.A method of screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with a polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.", "27.A method according to claim 24, which comprises directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide as defined in any of claims 10 to 22.28.A method according to claim 24, which further comprises providing at least one competitor, whereby in step (b) a polypeptide as defined in any of claims 10 to 22, can interact with either the competitor or autoantibodies to tTG and present in said sample.", "29.A method of screening for autoantibodies to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with (i) full length tTG, and (ii) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies to tTG, so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (c) monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample.", "30.A kit for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide as defined in claim 10; (b) means for contacting either (i) a sample of body fluid obtained from said subject, or (ii) lymphocytes isolated from a sample of body fluid obtained from said subject, with said polypeptide as defined in claims 10, under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) means for monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or lymphocytes, in said sample, or isolated from said sample.", "31.A kit according to claim 30, for screening for autoantibodies produced in response to tTG in said sample of body fluid.", "32.A kit for screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide as defined in claim 10; (b) means for contacting a sample of body fluid obtained from said subject with said polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) means for monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.", "33.A kit according to claim 30, which comprises means for directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.", "34.A kit according to of claim 30 to 32, which further comprises at least one competitor, whereby a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG, can interact with either the competitor or autoantibodies to tTG present in said sample of body fluid being screened.", "35.A kit for screening for autoantibodies to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) full length tTG; (b) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide as defined in claim 10; (c) means for contacting said sample of body fluid from said subject, said full length tTG and said competitor, under conditions that allow interaction of tTG with autoantibodies to tTG, so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (d) means for monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample.", "36.An antibody produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG, or one or more fragments of an antibody produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG.", "37.An antibody according to claim 36, which is obtained by the Examples.", "38.An antibody according to claim 36, which is monoclonal, recombinant or polyclonal.", "39.An antibody according to claim 38, which is monoclonal.", "40.A host cell containing an antibody as defined in claims 36.41.A hybridoma capable of secreting a monoclonal antibody as defined in claim 36.42.For use in diagnosis, an antibody as defined in claim 36.43.Use according to claim 42, in the diagnosis of autoimmune disease associated with an immune reaction to tTG.", "44.For use in therapy, an antibody as defined in claim 36.45.Use according to claim 44, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.", "46.Use according to claim 43, wherein the autoimmune disease is coeliac disease.", "47.An antibody according to claim 36, in the manufacture of a medicament for the treatment of coeliac disease.", "48.For use in diagnosis, a polypeptide as defined in claim 10.49.Use according to claim 48, in the diagnosis of autoimmune disease associated with an immune reaction to tTG.", "50.For use in therapy, a polypeptide as defined in claim 10.51.Use according to claim 50, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.", "52.Use according to claim 49, wherein the autoimmune disease is coeliac disease.", "53.A pharmaceutical composition comprising a polypeptide as defined in claim 10, together with a pharmaceutically acceptable carrier, diluent or excipient therefor, wherein said polypeptide can interact with autoantibodies and/or lymphocytes produced in response to tTG.", "54.A polypeptide as defined in claim 10, for use in the manufacture of a medicament for the treatment of coeliac disease.", "55.A method of diagnosing the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in a subject suspected of suffering from, susceptible to, having or recovering from, autoimmune disease associated with an immune reaction to tTG, the method comprising detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid from the subject according to a method as defined in claim 24, and whereby the detected autoantibodies and/or lymphocytes can provide a diagnosis of the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in the subject 56.A method of delaying or preventing the onset of autoimmune disease associated with an immune reaction to tTG in an animal subject suspected of suffering from, susceptible to or recovering from autoimmune disease associated with an immune reaction to tTG, which method comprises initially detecting autoantibodies or lymphocytes indicative of the onset or presence of autoimmune disease associated with an immune reaction to tTG in a sample of body fluid obtained from the subject according to a method as defined in claim 24, thereby providing a diagnosis of the likely onset of autoimmune disease associated with an immune reaction to tTG in the subject, and thereafter therapeutically treating the subject so as to delay the onset and/or prevent autoimmune disease associated with an immune reaction to tTG.", "57.A method of monitoring dietary compliance by a subject having, suspected of suffering from, susceptible to or recovering from gluten sensitive enteropathy, which method comprises screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject according to a method as defined in claim 24, and whereby the detection of such autoantibodies and/or lymphocytes produced in response to tTG in the sample of body fluid provides an indication as to the dietary compliance of the subject to a gluten-free or substantially gluten free diet.", "58.A method of treating autoimmune disease associated with an immune reaction to tTG in a subject, which method comprises initially detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject according to a method as defined in claim 24, thereby providing a diagnosis of autoimmune disease in the subject, and administering to the subject a therapeutically effective amount of at least one therapeutic agent effective in the treatment of such autoimmune disease.", "59.A method of treating autoimmune disease associated with an immune reaction to tTG in a subject, which method comprises administering to the subject a therapeutically effective amount of a therapeutic agent identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 492 of tTG.", "60.A therapeutic agent identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 491 of tTG.", "61.A therapeutic agent according to claim 60, for use in the therapeutic treatment of an autoimmune disease associated with an immune reaction to tTG.", "62.A method of cloning lymphocytes produced in response to tTG, which method comprises: providing a source of lymphocytes; contacting the lymphocytes with a polypeptide as defined in claim 10, so as to effect proliferation of said lymphocytes; and isolating and cloning the proliferating lymphocytes.", "63.Use of lymphocytes prepared according to claim 62, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.", "64.A pharmaceutical composition comprising lymphocytes prepared according to claim 62, together with a pharmaceutically acceptable carrier, diluent or excipient therefor.", "65.Use of lymphocytes prepared according to claim 62, in the manufacture of a medicament for the treatment of autoimmune disease associated with an immune reaction to Ttg.", "66.Use of lymphocytes prepared according to claim 62, in the manufacture of a medicament for the treatment of coeliac disease.", "67.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 2.68.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 3 69.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 4.70.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 5.71.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 6.72.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 7.73.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 67.74.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 68.75.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 69.76.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 70.77.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 71.78.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 72." ], [ "The present invention relates to tissue transglutaminase (tTG), and more particularly to DNA coding for and polypeptides containing, the primary structural conformation of one or more epitopes of tTG and to the use thereof in the detection of autoantibodies and/or lymphocytes produced in response to tTG.", "tTG belongs to the class of transglutaminases (TGs).", "The TGs are enzymes catalysing an acyl transfer depending on Ca2+, the γ-carboxamide groups of peptide-bonded glutamine residues acting as acyl donors.", "Primarily, protein-bonded lysine residues function as acyl acceptors, so that the transfer results in an ε-(γ-glutamyl)lysine bond.", "The substrate specificity of the TGs with respect to the acyl donors is very high (depending on the amino acid sequence), whereas an exceptionally wide spectrum of acceptors is available.", "The covalent peptide bonds formed are highly stable and protease-resistant, resulting in an increased resistance of the crosslinked proteins to chemical, enzymatic or physical effects.", "There is a widespread occurrence of various TGs in miscellaneous organs, tissues, in plasma and interstitial body fluids, correlating with the occurrence of transglutaminase-modified proteins in blood clots, on cell membranes, in the horny layer of the epidermis, in hair, in nails, and in the extracellular matrix.", "The described TGs may be distinguished by their physical properties, their location in the body, and their primary structure.", "tTG is also referred to as cellular, erythrocyte, endothelial, cytoplasmatic, liver, or type II TG, and is a monomer having a molecular weight of 75-85 kDa.", "The complete amino acid sequence comprising 687 residues has been derived from cDNA.", "At the protein level, there is an 84% homology between the human enzyme and the enzyme from mouse macrophages and an 81% homology between the human and guinea pig enzymes.", "There are a number of diseases that may be associated with tTG and can include autoimmune diseases and inflammatory diseases.", "In particular, autoimmune diseases associated with an immune reaction to tTG can be autoimmune diseases arising due to gluten sensitive enteropathy, such as coeliac disease or sprue.", "Coeliac disease is a disease of the small intestine mucosa, the first manifestation predominantly occurring during the late infant and toddler ages.", "If the corresponding clinical picture does not occur before the adult age, it is generally termed non-tropical sprue.", "Thus, both of these terms describe the same disease, which is hereinafter referred to as coeliac disease.", "Coeliac disease is estimated to effect about 1 in 300 individuals in Western countries.", "The characteristic intolerance of dietary gluten present in wheat, barley, rye and related products results in an inflammatory disease of the upper small intestine in genetically susceptible individuals.", "Typical symptoms include chronic diarrhoea, abdominal distension and failure to thrive.", "In some patients symptoms of malabsorption such as anaemia and osteoporosis may manifest.", "Other symptoms include inflammatory lesions in the small bowel, for example, villous atrophy, hypertrophic crypts and increased number of inter-epithelial lymphocytes.", "Provided the disease is diagnosed in time the symptoms can generally be treated by adhering to a gluten-free diet.", "However malabsorption may give rise to severe disease symptoms if the disease is not diagnosed and treated.", "Ultimately, there is the increased risk of developing an intestinal lymphoma and also other gastrointestinal neoplasias.", "Diagnosis of coeliac disease is currently based on clinical symptoms, histological identification of gluten sensitive enteropathy, and serological tests for observing any response to withdrawal of gluten from an individual's diet.", "An example of a serological test used for diagnosis and monitoring of coeliac disease is an immunofluorescence test measuring endomysial antibody and measurement of autoantibodies to tTG.", "WO98/03872 discloses that tTG is an autoantigen assocaited with coeliac disease and specifically discloses an ELISA based on guinea pig tTG of about 60% purity.", "It would be advantageous to be able to non-invasively efficiently and reliably diagnose and monitor autoimmune diseases associated with tTG, such as coeliac disease as referred to above.", "For example, the above described immunofluorescence test measuring endomysial antibody requires special expertise and equipment and is dependent on subjective assessment of fluorescence patterns.", "It would, therefore, be advantageous to be able to replace the immunofluorescence test measuring endomysial antibody with a non-invasive sensitive and specific test for measuring autoantibodies to tTG.", "It is, therefore, an aim of the present invention to provide an assay system, which alleviates some of the aforementioned problems arising in the diagnosis and monitoring of autoimmune diseases associated with an immune reaction to tTG.", "It is a further aim of the present invention to provide an assay for measuring autoantibodies or lymphocytes produced in response to tTG, with improved sensitivity and specificity and also provide diagnostic kits for use in the simple and rapid detection of autoantibodies or lymphocytes produced in response to tTG substantially as referred to above.", "A better understanding of tTG, and epitopes thereof, would be beneficial in achieving the above described aims of the present invention.", "More particularly, we have now specifically identified that antigenic determinants (or epitopes) of tTG which interact with autoantibodies and/or lymphocytes produced in response to tTG, and present in sera from patients with coeliac disease, are located in the N-terminal and/or central part of the tTG molecule.", "In particular, our studies have shown that the N-terminal region (typically amino acids 1 to 89) and/or the central region (typically amino acids 401 to 494 or amino acids 401 to 491) of the tTG molecule are particularly important for interaction with autoantibodies and/or lymphocytes produced in response to tTG and present in sera from coeliac patients.", "There is provided by the present invention, therefore, a DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.", "7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes).", "There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.", "7, 9, 11, 13 or 15; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14 and 16, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14 and 16, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes).", "There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.", "17, 19, 21, 23 or 25; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.", "18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "18, 20, 22, 24 and 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes).", "There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.", "7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies).", "There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.", "7, 9, 11, 13 or 15; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14 and 16, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14 and 16, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies).", "There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.", "17, 19, 21, 23 or 25; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.", "18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies).", "A DNA sequence as provided by the present invention can be of human origin (as depicted in FIGS.", "7 and 77), of guinea pig origin (as depicted in FIGS.", "9 and 99), of bovine origin (as depicted in FIGS.", "11 and 11), of mouse origin (as depicted in FIGS.", "13 and 33), or of rat origin (as depicted in FIGS.", "15 and 55).", "In particular, the present invention provides a DNA sequence as depicted in any of FIGS.", "7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a DNA sequence differing therefrom due to the degeneracy of the genetic code.", "More particularly, a DNA sequence as provided by the present invention can encode part or all of the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or part or all of the primary structural conformation of amino acid numbers 401 to 494 of tTG or part or all of the primary structural conformation of amino acid numbers 401 to 491 of tTG substantially as hereinafter described in greater detail.", "A DNA sequence as provided by the present invention can be included in a biologically functional plasmid or DNA vector for expressing a polypeptide according to the present invention substantially as hereinafter described in greater detail and there is further provided by the present invention a biologically functional plasmid or DNA vector including a DNA sequence substantially as hereinbefore described.", "Furthermore, there is still further provided by the present invention a host cell which is transformed or transfected with a DNA sequence substantially as hereinbefore described, or a plasmid or vector substantially as hereinbefore described.", "A suitable host cell can be any prokaryote or eukaryote cell capable of replicating and expressing a DNA sequence substantially as hereinbefore described and suitable techniques are well known to one of ordinary skill in the art.", "Polypeptides according to the present invention substantially as herein described can be expressed in various systems generating recombinant proteins.", "For example, for expression in E coli, cDNA coding for the appropriate polypeptides according to the present invention can be cloned into a vector, such as pMEX8, pGEXcT or pQE-81L His or an equivalent.", "In the case of expression in yeast (for example Saccharomyces cerevisiae or Schizosaccharomyces pombe), vectors such as pYES2, pESP2 or pYES2/CT or an equivalent, can be employed.", "AcMNPV (Bac-N-Blue) vector or an equivalent can be used for expression in insect cells and pRC/CMV vector or an equivalent can be used for expression in mammalian cells, such as Chinese Hamster Ovary (CHO) cells.", "A polypeptide according to the present invention can be expressed as a discrete protein, or as a fusion protein linked to, for example, glutathione S transferase (GST) or poly histidine linker.", "For a discrete protein, affinity column chromatography purification using a mouse monoclonal antibody to the relevant part of tTG coupled to a Sepharose particle can be used.", "If a tTG fragment is fused to GST, glutathione Sepharose chromatography purification can be used to isolate the fusion protein.", "Specific proteases can be used to separate GST from tTG peptide and a second round of glutathione Sepharose chromatography can be used to separate GST from a tTG fragment.", "In the case of peptides linked to poly histidine linker, the purification can be carried out using immobilised metal affinity chromatography.", "There is also provided by the present invention a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length Ttg).", "There is also provided by the present invention a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).", "There is also provided by the present invention a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).", "There is also provided by the present invention a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).", "There is also provided by the present invention a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).", "There is also provided by the present invention a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).", "The present invention further provides a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).", "The present invention further provides a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14 and 16, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14 and 16, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).", "The present invention further provides a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.", "18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "18, 20, 22, 24 and 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).", "The present invention further provides a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).", "The present invention further provides a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14 and 16, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14 and 16, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).", "The present invention further provides a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.", "18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).", "A polypeptide as provided by the present invention can be of human origin (as depicted in FIGS.", "8 and 88), of guinea pig origin (as depicted in FIGS.", "10 and 20), of bovine origin (as depicted in FIGS.", "12 and 22), of mouse origin (as depicted in FIGS.", "14 and 44), or of rat origin (as depicted in FIGS.", "16 and 66).", "In particular, the present invention provides a polypeptide as depicted in any of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.Typically, a polypeptide according to the present invention is a synthetic polypeptide derived by recombinant techniques substantially as herein described and is most suitably derived from recombinant human tTG typically with at least 70% purity, more preferably at least 90% purity and even more preferably at least 95% purity.", "Purity may be assessed by SDS PAGE analysis and Coomassie Blue staining as hereinafter discussed in connection with FIG.", "1.The recombinant tTG (typically human) may be purified using affinity chromatography with tTG antibodies such as mouse tTG monoclonal antibody.", "A polypeptide according to the present invention is preferably obtained by, or is obtainable by, expression of a DNA sequence according to the present invention substantially as hereinbefore described.", "A polypeptide according to the present invention obtained by such expression can be advantageous in being free from association with other eukaryotic polypeptides or contaminants which might otherwise be associated with tTG in its natural environment.", "The present invention further provides a process of preparing a polypeptide substantially as hereinbefore described, which process comprises: (i) providing a host cell substantially as hereinbefore described; (ii) growing the host cell; and (iii) recovering a polypeptide according to the present invention therefrom.", "Recovery of a polypeptide according to the present invention can typically employ conventional isolation and purification techniques, such as chromatographic separations or immunological separations, known to one of ordinary skill in the art.", "It is of course appreciated that there is provided within the scope of the present invention, one or more amino acid sequences or fragments thereof structurally distinct from the specific amino acid sequences of a polypeptide described herein (for example by the addition, deletion, substitution or insertion of one or more amino acids) but having substantially the same functional binding properties as the specific amino acid sequences of a polypeptide described herein.", "In other words, the present invention clearly encompasses within its scope amino acid sequences or fragments thereof substantially homologous to specific amino acid sequences of a polypeptide described herein, or representing variants of specific amino acid sequences of a polypeptide described herein, but having substantially the same functional binding properties as the specific amino acid sequences of a polypeptide described herein.", "A polypeptide according to the present invention can in a first preferred embodiment of the present invention be a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide consists essentially of (or consists of) the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or more particularly consists essentially of (or consists of) the primary structural conformation of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, substantially as hereinbefore described.", "Alternatively, in a second preferred embodiment of the present invention there is provided a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide consists essentially of (or consists of) one or more active fragments of amino acid numbers 1 to 89 of tTG, and/or one or more active fragments of amino acid numbers 401 to 494 of tTG or one or more active fragments of amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), or more particularly consists essentially of (or consists of) the primary structural conformation of one or more active fragments of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) substantially as hereinbefore described.", "For example, a polypeptide consisting essentially of (or consisting of) amino acid numbers 1 to 85 of tTG, amino acid numbers 5 to 89 of tTG, or the like, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes).", "A still further alternative in a third preferred embodiment of the present invention is a polypeptide (in particular a synthetic polypeptide, such as a scaffold type polypeptide typically providing amino acids corresponding to the amino acids of tTG as described herein in a form specifically adapted for use in an assay method or kit according to the present invention substantially as hereinafter described in greater detail) with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which includes part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide includes amino acids comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, and/or one or more active fragments of amino acid numbers 401 to 494 of tTG or one or more active fragments of amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); or more particularly which includes amino acids comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.", "8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).", "According to a particularly preferred embodiment of the present invention, there is also provided a method of screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing either (i) said sample of body fluid from said subject or (ii) lymphocytes isolated from said sample; (b) contacting said sample or isolated lymphocytes with a polypeptide according to the present invention substantially as hereinbefore described (under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG) so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or said lymphocytes, in said sample, or isolated from said sample.", "Substantially as described above, a method according to the present invention is suitable for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject.", "A method according to the present invention can, however, be particularly adapted for use in screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject substantially as hereinafter described in greater detail.", "There is in particular provided by the present invention, therefore, a method of screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with a polypeptide according to the present invention substantially as hereinbefore described (under conditions that allow interaction of tTG with autoantibodies produced in response to tTG) so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.", "A method according to the present invention may typically employ a control, such as a sample of body fluid from a normal subject, in other words a subject known to be without autoimmune disease associated with an immune reaction to tTG.", "A method of screening for autoantibodies to tTG according to the present invention may comprise directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide, as provided by the present invention substantially as hereinbefore described, typically by employing non-competitive sandwich type assay techniques known in the art.", "Typically, in a method according to the present invention employing non-competitive techniques, monitoring of the degree of interaction of (i) autoantibodies to tTG present in the sample and (ii) a polypeptide according to the present invention substantially as hereinbefore described, can comprise providing labelling means either to a polypeptide according to the present invention substantially as hereinbefore described, or to a binding partner for autoantibodies to tTG, either of which technique would enable monitoring of the above described interaction.", "For example, a method according to the present invention may comprise directly or indirectly labelling a polypeptide according to the present invention substantially as hereinbefore described; contacting the thus labelled polypeptide with a sample of body fluid being screened for tTG autoantibodies so as to provide a mixture thereof; and adding to the mixture a binding partner for autoantibodies to tTG (such as an anti-IgG reagent) present in the sample of body fluid, so as to cause precipitation of any complexes of labelled polypeptide and tTG autoantibodies present in the mixture.", "Alternatively, it may be preferred that a method according to the present invention further comprises adding a labelled binding partner for tTG autoantibodies (such as a labelled anti-IgG reagent, for example protein A or anti-human IgG, or labelled full length tTG or an epitope thereof) to a mixture obtained by contacting (i) a polypeptide according to the present invention substantially as hereinbefore described immobilised to a support and (ii) a sample of body fluid being screened for autoantibodies to tTG.", "It may alternatively be preferred that a method of screening for autoantibodies to tTG in the sample of body fluid according to the present invention, utilises the principles employed in known competitive assays.", "For example, a method according to the present invention may employ at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.", "Typically, a competitor as employed in a competitive assay method according to the present invention may comprise one or more antibodies, which may be natural or partly or wholly synthetically produced.", "A competitor as employed in the present invention may alternatively comprise any other protein having a binding domain or region which is capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.", "Preferably, however, a competitor as employed in the present invention comprises a monoclonal, recombinant or polyclonal antibody (especially a monoclonal antibody), capable of competing with tTG autoantibodies in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.", "Typically, therefore, a competitive assay method according to the present invention may further comprise providing at least one competitor, such as a monoclonal or polyclonal antibody, whereby in step (b) of a method as herein described a polypeptide according to the present invention substantially as hereinbefore described can interact with either a competitor, such as a monoclonal or polyclonal antibody, or autoantibodies to tTG present in said sample.", "Typically monitoring in a competitive assay method according to the present invention comprises comparing: (i) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the absence of said sample of body fluid being screened (that is a suspected disease sample), optionally in the presence of a sample of body fluid from a normal subject, typically a subject known to be without autoimmune disease associated with an immune reaction to tTG; with (ii) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the presence of said sample of body fluid being screened.", "Typically, the comparison involves observing a decrease in interaction of a polypeptide according to the present invention substantially as hereinbefore described and the competitor in (ii) compared to (i) so as to provide an indication of the presence of autoantibodies to tTG in said sample.", "Typically, the decrease in interaction can be observed by directly or indirectly labelling the competitor and monitoring any change in the interaction of the thus labelled competitor with a polypeptide according to the present invention substantially as hereinbefore described in the absence and in the presence of a sample of body fluid being screened for autoantibodies to tTG.", "Suitably a polypeptide according to the present invention substantially as hereinbefore described may be immobilised to facilitate the above mentioned monitoring.", "Alternatively, there is also provided by the present invention a method of screening for autoantibodies to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with (i) full length tTG (typically recombinantly obtained full length tTG), and (ii) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described, (under conditions that allow interaction of tTG with autoantibodies to tTG), so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (c) monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample.", "The full length tTG can typically be of human, guinea pig, bovine, mouse or rat origin, preferably human, and more preferably human recombinantly obtained full length tTG.", "A competitor for use in such an assay typically comprises a monoclonal or polyclonal antibody (preferably monoclonal) substantially as hereinbefore described.", "Suitably a detectable label that can be employed in a method according to the present invention can be selected from the group consisting of enzymic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes and the like.", "In the case where an isotopic label (such as 125I, 14C, 3H or 35S) is employed, monitoring may therefore comprise measuring radioactivity dependent on binding of a polypeptide according to the present invention substantially as hereinbefore described.", "Radioactivity is generally measured using a gamma counter, or liquid scintillation counter.", "In the case of a method of screening for lymphocytes according to the present invention, it is generally preferred that lymphocytes are initially isolated from a sample of body fluid from a subject using techniques well known to one of ordinary skill in the art, followed by contact with a polypeptide according to the present invention so as to stimulate the proliferation of the isolated lymphocytes.", "Monitoring of the effect of interaction of a polypeptide according to the present invention and such proliferating lymphocytes, typically employs means known in the art for monitoring such proliferation of lymphocytes.", "According to a further particularly preferred embodiment of the present invention, there is provided a kit for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide according to the present invention substantially as hereinbefore described; (b) means for contacting either (i) a sample of body fluid obtained from said subject, or (ii) lymphocytes isolated from a sample of body fluid obtained from said subject, with said polypeptide according to the present invention substantially as hereinbefore described (under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG) so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) means for monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or lymphocytes, in said sample or isolated from said sample.", "Substantially as described above, a kit according to the present invention is suitable for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject.", "A kit according to the present invention can, however, be particularly adapted for use in screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject substantially as hereinafter described in greater detail.", "There is in particular provided by the present invention, therefore, a kit for screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide according to the present invention substantially as hereinbefore described; (b) means for contacting a sample of body fluid obtained from said subject with said polypeptide according to the present invention substantially as hereinbefore described (under conditions that allow interaction of tTG with autoantibodies produced in response to tTG) so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) means for monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.", "A kit according to the present invention may typically further comprise control means, such as means for providing a sample of body fluid from a normal subject, in other words a subject known to be without autoimmune disease associated with an immune reaction to tTG.", "A kit for screening for autoantibodies to tTG according to the present invention may comprise means for directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide, as provided by the present invention substantially as hereinbefore described, typically comprising non-competitive sandwich type assay means known in the art.", "Typically, in a kit according to the present invention comprising non-competitive assay means, means are provided for monitoring the degree of interaction of (i) autoantibodies to tTG present in the sample and (ii) a polypeptide according to the present invention substantially as hereinbefore described, and can comprise labelling means provided either to a polypeptide according to the present invention substantially as hereinbefore described, or to a binding partner for autoantibodies to tTG, either of which would enable monitoring of the above described interaction.", "For example, a kit according to the present invention may comprise means for directly or indirectly labelling a polypeptide according to the present invention substantially as hereinbefore described; means for contacting the thus labelled polypeptide with a sample of body fluid being screened for tTG autoantibodies so as to provide a mixture thereof; a binding partner for autoantibodies to tTG (such as an anti-IgG reagent) present in the sample of body fluid; and means for adding the binding partner to the mixture so as to cause precipitation of any complexes of labelled polypeptide and tTG autoantibodies present in the mixture.", "Alternatively, it may be preferred that a kit according to the present invention further comprises a labelled binding partner for tTG autoantibodies (such as a labelled anti-IgG reagent, for example protein A or anti-human IgG, or labelled full length tTG or an epitope thereof) and means for adding the labelled binding partner to a mixture obtained by contacting (i) a polypeptide according to the present invention substantially as hereinbefore described immobilised to a support and (ii) a sample of body fluid being screened for autoantibodies to tTG.", "It may alternatively be preferred that a kit for screening for autoantibodies to tTG in the sample of body fluid according to the present invention, comprises known competitive assay means.", "For example, a kit according to the present invention may further comprise at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.", "Typically, a competitor as employed in a competitive assay kit according to the present invention may comprise one or more antibodies, which may be natural or partly or wholly synthetically produced.", "A competitor as employed in the present invention may alternatively comprise any other protein having a binding domain or region which is capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.", "Preferably, however, a competitor as employed in the present invention comprises a monoclonal or polyclonal antibody (especially a monoclonal antibody), capable of competing with tTG autoantibodies in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.", "Typically, therefore, a competitive assay kit according to the present invention may further comprise at least one competitor, such as a monoclonal or polyclonal antibody, whereby a polypeptide according to the present invention substantially as hereinbefore described can interact with either a competitor, such as a monoclonal or polyclonal antibody, or autoantibodies to tTG present in a sample of body fluid being screened.", "Typically monitoring means in a competitive assay kit according to the present invention comprise means for comparing: (i) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the absence of said sample of body fluid being screened (that is a suspected disease sample), optionally in the presence of a sample of body fluid from a normal subject, typically a subject known to be without autoimmune disease associated with an immune reaction to tTG; with (ii) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the presence of said sample of body fluid being screened.", "Typically, the comparison involves observing a decrease in interaction of a polypeptide according to the present invention substantially as hereinbefore described and the competitor in (ii) compared to (i) so as to provide an indication of the presence of autoantibodies to tTG in said sample.", "Typically, the decrease in interaction can be observed by directly or indirectly labelling the competitor and monitoring any change in the interaction of the thus labelled competitor with a polypeptide according to the present invention substantially as hereinbefore described in the absence and in the presence of a sample of body fluid being screened for autoantibodies to tTG.", "Suitably a polypeptide according to the present invention substantially as hereinbefore described may be immobilised to facilitate the above mentioned monitoring.", "Alternatively, there is also provided by the present invention a kit for screening for autoantibodies to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) full length tTG (typically recombinantly obtained full length tTG); (b) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described, (c) means for contacting said sample of body fluid from said subject, said full length tTG and said competitor (under conditions that allow interaction of tTG with autoantibodies to tTG), so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (d) means for monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample.", "The full length tTG can typically be of human, guinea pig, bovine, mouse or rat origin, preferably human, and more preferably human recombinantly obtained full length tTG.", "A competitor for use in such an assay kit typically comprises a monoclonal or polyclonal antibody (preferably monoclonal) substantially as hereinbefore described.", "Suitably a detectable label that can be employed in a kit according to the present invention can be selected from the group consisting of enzymic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes and the like.", "In the case where an isotopic label (such as 125I, 14C, 3H or 35S) is employed, monitoring means may therefore comprise means for measuring radioactivity dependent on binding of a polypeptide according to the present invention substantially as hereinbefore described.", "Radioactivity is generally measured using a gamma counter, or liquid scintillation counter.", "In the case of a kit for screening for lymphocytes according to the present invention, it is generally preferred that means are provided for initially isolating lymphocytes from a sample of body fluid from a subject, using techniques well known to one of ordinary skill in the art, and means are also provided for contacting a polypeptide according to the present invention to the present invention with such isolated lymphocytes so as to stimulate proliferation of the latter by the former.", "Means (again known to one of ordinary skill in the art) for monitoring the effect of interaction of a polypeptide according to the present invention and such proliferating lymphocytes, are also provided in such a kit according to the present invention.", "There is still further provided by the present invention an antibody produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or part or all of the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG, or one or more fragments of an antibody (such as Fab fragments) produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or part or all of the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG.", "An antibody according to the present invention or fragment thereof can interact with a polypeptide according to the present invention substantially as hereinbefore described and preferably is obtainable by, or is obtained by, techniques described in the Examples.", "Suitably, an antibody provided by the present invention can be monoclonal (preferred), recombinant or polyclonal.", "Typically an antibody, such as a monoclonal antibody, as provided by the present invention is in substantially purified form.", "More specifically, a monoclonal antibody as provided by the present invention can comprise a monoclonal antibody Mab A3-6B5, or a monoclonal antibody Mab A5-4E6, or one or more active fragments thereof (such as Fab fragments), as described in the Examples.", "The present invention further provides a host cell containing an antibody substantially as described above and also a hybridoma capable of secreting a monoclonal antibody substantially as hereinbefore described.", "There is still further provided by the present invention an antibody substantially as hereinbefore described, or one or more fragments of an antibody (such as Fab fragments) substantially as hereinbefore described, for use in diagnosis or therapy, in particular diagnosis or therapy of autoimmune disease associated with an immune reaction to tTG substantially as hereinbefore described.", "Particularly the autoimmune disease can be coeliac disease (although other autoimmune diseases substantially as hereinafter described in greater detail can be diagnosed or treated by an antibody according to the present invention) and the present invention also provides an antibody substantially as hereinbefore described for use in the manufacture of a medicament for the treatment of coeliac disease.", "There is still further provided by the present invention a polypeptide according to the present invention substantially as hereinbefore described, for use in diagnosis or therapy, in particular diagnosis or therapy of autoimmune disease associated with an immune reaction to tTG substantially as hereinbefore described.", "In particular, autoimmune disease associated with an immune reaction to tTG as described herein comprises autoimmune disease arising due to gluten sensitive enteropathy in a subject, in particular a human subject, such as coeliac disease.", "Other autoimmune diseases associated with an immune reaction to tTG that can be diagnosed or treated by the present invention include any of the following—dermatitis herpetiformis, Addison's disease, A1 (A1-autoimmune) haemolytic anaemia, A1 thrombocytopenic purpura, A1 thyroid diseases, A1 hyperthyroidism (Graves' disease), A1 hypothyroidism (including Hashimoto's thyroiditis), pernicious anaemia, myasthenia gravis, primary biliary cirrhosis, rheumatoid arthritis, Sjogren's syndrome, SLE, type 1 (insulin-dependent) diabetes mellitus, diabetes mellitus of other types, Wegener granulomatosis, and vitiligo.", "Substantially as herein described autoimmune disease associated with an immune reaction to tTG can be screened for or diagnosed in a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG.", "The patient group that can be classed as susceptible to an autoimmune disease associated with an immune reaction to tTG can typically include relatives of subjects previously diagnosed as having an autoimmune disease associated with an immune reaction to tTG, or subjects previously diagnosed with a primary or first autoimmune disease associated with an immune reaction to tTG and as such could be considered to be at risk of or prone to be suffering from a secondary or second autoimmune disease associated with an immune reaction to tTG.", "For example, a subject known to be suffering from type 1 diabetes mellitus could be considered to be susceptible to suffering from coeliac disease.", "It will be appreciated from the foregoing description that the present invention provides assay methods and kits for detecting autoantibodies (in particular) or lymphocytes produced in response to tTG in a sample of body fluid substantially as hereinbefore described.", "The detection of such autoantibodies and/or lymphocytes produced in response to tTG in the sample of body fluid (or at least the level of such autoantibodies and/or lymphocytes in the sample) is indicative of the presence of autoimmune disease associated with an immune reaction to tTG in the subject from which the sample was obtained and can, therefore, enable the diagnosis of the likely onset or presence of autoimmune disease associated with an immune reaction to tTG.", "There is, therefore, further provided by the present invention a method of diagnosing the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from, autoimmune disease associated with an immune reaction to tTG, the method comprising detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid from the subject substantially as hereinbefore described, and whereby the detected autoantibodies and/or lymphocytes can provide a diagnosis of the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in the subject.", "There is still further provided by the present invention a method of delaying or preventing the onset of autoimmune disease associated with an immune reaction to tTG in an animal subject (in particular a human subject) suspected of suffering from, susceptible to or recovering from autoimmune disease associated with an immune reaction to tTG, which method comprises initially detecting autoantibodies or lymphocytes indicative of the onset or presence of autoimmune disease associated with an immune reaction to tTG in a sample of body fluid obtained from the subject substantially as hereinbefore described, thereby providing a diagnosis of the likely onset of autoimmune disease associated with an immune reaction to tTG in the subject, and thereafter therapeutically treating the subject so as to delay the onset and/or prevent autoimmune disease associated with an immune reaction to tTG.", "Typically, such therapeutic treatment can, in the case of gluten sensitive enteropathy giving rise to coeliac disease, comprise compliance with a gluten-free or substantially gluten free diet.", "The present invention can further provide a method of monitoring dietary compliance by a subject (in particular a human subject) having, suspected of suffering from, susceptible to or recovering from gluten sensitive enteropathy, which method comprises screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject substantially as hereinbefore described, and whereby the detection of such autoantibodies and/or lymphocytes produced in response to tTG in the sample of body fluid (or at least the level of such autoantibodies and/or lymphocytes in the sample) provides an indication as to the dietary compliance of the subject to a gluten-free or substantially gluten free diet.", "A polypeptide according to the present invention substantially as hereinbefore described is particularly suitable for use in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.", "For example, tolerance can be achieved by administering a polypeptide according to the present invention substantially as hereinbefore described to a subject (in particular a human subject) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG.", "There is, therefore, further provided by the present invention a pharmaceutical composition comprising a polypeptide according to the present invention substantially as hereinbefore described, together with a pharmaceutically acceptable carrier, diluent or excipient therefor, wherein the polypeptide can interact with autoantibodies and/or lymphocytes produced in response to tTG.", "The present invention further provides a polypeptide according to the present invention substantially as hereinbefore described for use in the manufacture of a medicament for the treatment of coeliac disease.", "Compositions or medicaments according to the present invention should contain a therapeutic or prophylactic amount of at least one polypeptide according to the present invention in a pharmaceutically-acceptable carrier.", "The pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivery of the polypeptides to the patient.", "Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier.", "Pharmaceutically-acceptable adjuvants, buffering agents, dispersing agents and the like, may also be incorporated into the pharmaceutical compositions.", "Such compositions can contain a single polypeptide or may contain two or more polypeptides according to the present invention.", "It may be desirable to couple a polypeptide according to the present invention to immunoglobulins, e.g.", "IgG, or to lymphoid cells from the patient being treated in order to promote tolerance.", "Such an approach is described in Bradley-Mullen, Activation of Distinct Subsets of T Suppressor Cells with Type III Pneumococcal Polysaccharide Coupled to Syngeneic Spleen Cells, in: IMMUNOLOGICAL TOLERANCE TO SELF AND NON-SELF, Buttisto et al., eds., Annals N.Y. Acad.", "Sci.", "Vol.", "392, pp 156-166, 1982.Alternatively, the polypeptides may be modified to maintain or enhance binding to the MHC while reducing or eliminating binding to the associated T-cell receptor.", "In this way, the modified polypeptides may compete with natural tTG to inhibit helper T-cell activation and thus inhibit the immune response.", "In all cases, care should be taken that administration of the pharmaceutical compositions of the present invention ameliorate but do not potentiate the autoimmune response.", "Pharmaceutical compositions according to the present invention are useful for parenteral administration.", "Preferably, the compositions will be administered parenterally, i.e.", "subcutaneously, intramuscularly, or intravenously.", "Thus, the invention provides compositions for parenteral administration to a patient, where the compositions comprise a solution or dispersion of the polypeptides in an acceptable carrier, as described above.", "The concentration of the polypeptides in the pharmaceutical composition can vary widely, i.e.", "from less than about 0.1% by weight, usually being at least about 1% by weight to as much as 20% by weight or more.", "Typical pharmaceutical compositions for intramuscular injection would be made up to contain, for example, 1 ml of sterile buffered water and 1 to 100 μg of a purified polypeptide of the present invention.", "A typical composition for intravenous infusion could be made up to contain 100 to 500 ml of sterile Ringer's solution and 100 to 500 mg of a purified polypeptide of the present invention.", "Actual methods for preparing parenterally administrable compositions are well known in the art and described in more detail in various sources, including, for example, Remington's Pharmaceutical Science, 15th Edition, Mack Publishing Company, Easton, Pa. (1980).", "In addition to using a polypeptide according to the present invention directly in pharmaceutical compositions, it is also possible to use a polypeptide according to the present invention to enhance tolerance to tTG in a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, employing the following principles.", "More particularly, peripheral blood lymphocytes can be collected from the subject in a conventional manner and stimulated by exposure to a polypeptide according to the present invention, as defined above.", "Usually, other mitogens and growth enhancers will be present, e.g., phytohemagglutinin, interleukin 2, and the like.", "Proliferating T-helper cells may be isolated and cloned, also under the stimulation of a polypeptide according to the present invention.", "Clones which continue to proliferate may then be used to prepare therapeutic compositions for the subject.", "The cloned T-cells may be attenuated, e.g.", "by exposure to radiation, and administered to the subject in order to induce tolerance.", "Alternatively, the T-cell receptor or portions thereof may be isolated by conventional protein purification methods from the cloned T-cells and administered to the individual.", "Such immunotherapy methods are described generally in Sinha et al.", "(1990) Science 248:1380-1388.In some cases, after a T-helper cell has been cloned as described above, it may be possible to develop therapeutic peptides from the T-cell receptor, where the peptides would be beneficial for treating a patient population suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG.", "In such cases, the T-cell receptor gene may be isolated and cloned by conventional techniques and peptides based on the receptor produced by recombinant techniques as described above.", "The recombinantly-produced peptides may then be incorporated in pharmaceutical compositions as described above.", "There is also provided by the present invention a method of cloning lymphocytes produced in response to tTG, which method comprises: providing a source of lymphocytes; contacting the lymphocytes with a polypeptide according to the present invention substantially as hereinbefore described, so as to effect proliferation of said lymphocytes; and isolating and cloning the proliferating lymphocytes.", "The present invention also provides the use of cloned lymphocytes prepared as above, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.", "There is provided, therefore, a pharmaceutical composition comprising cloned lymphocytes prepared as above, together with a pharmaceutically acceptable carrier, diluent or excipient therefor and the use of such cloned lymphocytes in the manufacture of a medicament for the treatment of autoimmune disease associated with an immune reaction to tTG, in particular coeliac disease.", "There is also provided by the present invention one or more therapeutic agents identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 491 of tTG, and the present invention further provides one or more therapeutic agents for use in therapeutically interacting with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG or amino acids 401 to 491 of Ttg and as such for use in the therapeutic treatment of an autoimmune disease associated with an immune reaction to tTG.", "There is, therefore, still further provided by the present invention a method of treating autoimmune disease associated with an immune reaction to tTG in a subject, which method comprises initially detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject substantially as hereinbefore described, thereby providing a diagnosis of autoimmune disease in the subject, and administering to the subject a therapeutically effective amount of at least one therapeutic agent effective in the treatment of such autoimmune disease, such as a polypeptide according to the present invention substantially as hereinbefore described.", "The present invention also provides a method of treating autoimmune disease associated with an immune reaction to tTG in a subject (in particular a human subject), which method comprises administering to the subject a therapeutically effective amount of a therapeutic agent identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 491 of tTG.", "The amount of therapeutic agent administered will depend on the specific autoimmune disease state being treated, possibly the age of the patient and will ultimately be at the discretion of an attendant physician.", "There is still further provided by the present invention, in combination, a kit substantially as hereinbefore described, together with a therapeutically effective amount of at least one therapeutic agent effective in the treatment of autoimmune disease associated with an immune reaction to tTG substantially as hereinbefore described.", "Substantially as hereinbefore described, the sample of body fluid being screened by the present invention will typically comprise blood samples or other fluid blood fractions, such as in particular serum samples or plasma samples, but the sample may in principle be another biological fluid, such as saliva or urine or solubilised tissue extracts.", "The present invention will now be illustrated with reference to the accompanying Figures and Examples, which do not limit the invention in any way.", "FIG.", "1 shows results of SDS PAGE (9%) analysis of purified recombinant human tTG for use in the method and kit according to the invention.", "FIG.", "2 is a table showing binding of autoantibodies to tTG in sera from coeliac patients with full length and modified 35S-tTG proteins.", "FIG.", "3 is a table showing monoclonal antibody (namely monoclonal antibodies A3-6B5 and A5-4E6) binding to full length and modified 35S-tTG proteins.", "FIG.", "4 shows a schematic example of the method of the present invention when an antibody is employed.", "FIG.", "5 is a graph showing binding (absorbance at 450 nm in the vertical axis) of tTG with autoantibodies to tTG in different patients sera (horizontal axis).", "The binding is measured in the presence or absence of mouse tTG monoclonal antibody A3-6B5.FIG.", "6 is a graph showing binding (absorbance at 450 nm in the vertical axis) of tTG with autoantibodies to tTG in different patients sera (horizontal axis).", "The binding is measured in the presence or absence of mouse tTG monoclonal antibody A5-4E6.FIG.", "7 depicts cDNA derived from human tTG, encoding amino acids 1 to 89 of human tTG.", "FIG.", "8 depicts amino acids 1 to 89 of human tTG.", "FIG.", "9 depicts cDNA derived from guinea pig tTG, encoding amino acids 1 to 89 of guinea pig tTG.", "FIG.", "10 depicts amino acids 1 to 89 of guinea pig tTG.", "FIG.", "11 depicts CDNA derived from bovine tTG, encoding amino acids 1 to 89 of bovine tTG.", "FIG.", "12 depicts amino acids 1 to 89 of bovine tTG.", "FIG.", "13 depicts cDNA derived from mouse tTG, encoding amino acids 1 to 89 of mouse tTG.", "FIG.", "14 depicts amino acids 1 to 89 of mouse tTG.", "FIG.", "15 depicts cDNA derived from rat tTG, encoding amino acids 1 to 89 of rat tTG.", "FIG.", "16 depicts amino acids 1 to 89 of rat tTG.", "FIG.", "17 depicts cDNA derived from human tTG, encoding amino acids 401 to 491 of human tTG.", "FIG.", "18 depicts amino acids 401 to 491 of human tTG.", "FIG.", "19 depicts cDNA derived from guinea pig tTG, encoding amino acids 401 to 494 of guinea pig tTG.", "FIG.", "20 depicts amino acids 401 to 494 of guinea pig tTG.", "FIG.", "21 depicts cDNA derived from bovine tTG, encoding amino acids 401 to 491 of bovine tTG.", "FIG.", "22 depicts amino acids 401 to 491 of bovine tTG.", "FIG.", "23 depicts cDNA derived from mouse tTG, encoding amino acids 401 to 491 of mouse tTG.", "FIG.", "24 depicts amino acids 401 to 491 of mouse tTG.", "FIG.", "25 depicts cDNA derived from rat tTG, encoding amino acids 401 to 491 of rat tTG.", "FIG.", "26 depicts amino acids 401 to 491 of rat tTG.", "FIG.", "27 depicts the alignment of tTG sequences (human, guinea pig, bovine, mouse and rat) at the amino acid level.", "Further illustration of the present invention is given in the following detailed worked Examples.", "EXAMPLES Identification of Regions of tTG which are Recognised by tTG Autoantibodies Full length human tTG cDNA (endothelial cell isoform) was cloned into pTZ18R (Pharmacia) under control of the SP6 promoter.", "This construct was used to produce a reference full length tTG protein.", "Various in-frame deletions and truncations of the tTG gene were carried out using either naturally occurring restriction endonuclease sites or PCR mediated modifications to the sequence.", "The modified tTG constructs were cloned into pTZ18R (as above).", "Full length and modified tTG proteins were expressed in an in vitro transcription/translation rabbit reticulocyte system (Promega) incorporating 35S-methionine (Amersham Pharmacia Biotech) and purified as described before (Colls J, et al.", "Clin.", "Chem.", "1995; 41/3:375-380).", "Table 1 shows the vectors used and the corresponding tTG amino acid residues expressed following the in vitro transcription/translation described above.", "TABLE 1 Non-expressed Vector Name Expressed residues (deleted) residues pTGM19 1-687 (full length) None pTGM25 1-628 629-687 pTGM22 1-491 492-687 pTGM24 1-400 401-687 pTGM26 90-687 1-89 pTGM27 172-687 1-171 pTGM28 207-687 1-206 pTGM29 281-687 1-280 pTGM30 356-687 1-355 pTGM31 399-687 1-398 Binding of tTG autoantibodies to the purified full length and modified tTG proteins was analysed using an immunoprecipitation assay (Nakachi K, et al Clin Chim Acta, Volume 304, pages 75 to 84, 2002).", "Briefly, 10 μL of undiluted serum (in duplicate) was incubated with 50 μL of 35S-tTG (approx 15,000 dpm diluted in 10 mmol/L Tris HCl pH 7.6, 150 mmol/L NaCl, 2 mmol/L EDTA, 0.1% Tween 20, 10 g/L bovine serum albumin, 0.2 g/L NaN3; Assay Buffer) in Millipore filtration plates overnight at 4° C. Anti-human IgA agarose (50 μl) was then added and the plates incubated for 4 hours at 4° C. After incubation the reaction mixtures in the wells were washed 3 times.", "The wells were then punched out into vials containing 1 mL of scintillation liquid and counted in a beta-counter.", "FIG.", "2 shows binding of tTG autoantibodies with different modified tTG proteins.", "The results are expressed as % binding relative to binding to the full length tTG reference protein.", "Serum Samples Serum from 34 patients with coeliac disease were used in the study.", "The diagnosis was based on typical clinical symptoms and was confirmed by positive duodenal biopsy.", "The sera tested positive for endomysial antibody and autoantibodies to tTG by standard ELISA.", "In addition sera from healthy blood donors (n=10) negative for autoantibodies to tTG were used.", "Production of Mouse Monoclonal Antibodies Both guinea pig tTG (Sigma, Poole, UK) and recombinant human tTG (rhtTG) were used as antigens to immunise mice.", "Monoclonal antibodies (Mabs) were cloned using standard techniques.", "Selected tTG Mabs were grown in culture and purified by protein A affinity chromatography.", "The location of the binding sites of the mouse monoclonal antibodies was determined by an immunoprecipitation assay using the modified 35S-labelled tTG proteins as described above, except that anti-mouse IgG was added for precipitation.", "When required, purified IgG was labelled with 125I using the chloramine T method.", "Analysis of the mouse Mabs by immunoprecipitation with modified 35S-labelled tTG proteins indicated that the epitope for A3-6B5 Mab was contained within aa 1-89 of the human tTG protein and the epitope for A5-4E6 Mab was contained within aa 401-491 of the human tTG protein (FIG.", "3).", "Inhibition of Autoantibodies to tTG Binding with tTG by Mouse tTG Mabs The inhibition of autoantibodies to tTG binding with tTG by mouse tTG Mabs was investigated in a modified version of an ELISA described in Nakachi K, et al.", "Clin Chim Acta (Volume 304, pages 75 to 84, 2002).", "To streptavidin coated plate wells 50 μL of tTG-biotin was added plus 10μL of Mab (200 μ/ml) or Assay Buffer and incubated for 30 mins.", "40 μL of patients' sera (typically diluted 1:100) was then added and incubated for 30 mins.", "After this step all wells were aspirated, washed four times and then 100 μL of anti-human IgA-horseradish peroxidase (HRP) was added and incubated for 30 mins.", "After aspiration and four washes, 100 μL of tetramethyl benzidine (TMB) solution was added and the plate was incubated for 15 mins in the dark.", "50 μL of stop solution was then added and absorbance read at 450 nm in a plate reader.", "All steps were performed at room temperature, all incubations except the final TMB incubation were done with 200 rpm shaking.", "tTG-biotin, Mabs and human sera were diluted in Assay Buffer.", "The results are shown in FIG.", "5 which shows binding of tTG with autoantibodies to tTG in different patients sera in the presence or absence of mouse tTG monoclonal antibody A3-6B5 and FIG.", "6 which shows binding of tTG with autoantibodies to tTG in different patients sera in the presence or absence of mouse tTG monoclonal antibody A5-4E6.Preparation of Purified Recombinant Human tTG Recombinant human tTG was expressed in the yeast Saccharomyces cerevisiae strain c13ABYS86 using an expression system and culture techniques described previously (Powell M, et al.", "Clin Chim Acta 1996; 256: 175-188).", "Recombinant human tTG extracted from the yeast was partially purified by anion exchange chromatography and preparations of tTG of greater than 95% purity were obtained by affinity chromatography using mouse tTG Mab (A5-6C7) coupled to CNBr Sepharose.", "The purity of the purified tTG was assessed by SDS PAGE analysis on a 9% acrylamide gel followed by staining with Coomassie Blue as shown in FIG.", "1.Assay for Screening a Sample of Body Fluid for Autoantibodies to tTG Based on the Inhibition of 125I-Mab Binding to Recombinant Human tTG An assay was set up based on the principles of the schematic example shown in FIG.", "4.4 mL Nunc Maxisorp Startubes (Life Technologies) were coated overnight at 4° C. with 250 μL of 10 μg/mL recombinant human tTG (rhtTG).", "Following aspiration and three washes with 500 μL Assay Buffer, tubes were incubated for 30 minutes at room temperature with 500 μL of Post-Coat Buffer containing bovine serum albumin.", "After two washes with Assay Buffer, tubes were incubated with 100 μL of serum sample diluted 1:5 in Assay Buffer for 60 minutes at room temperature on a shaker.", "Following aspiration and three washes with 500 μL of Assay Buffer, tubes were incubated on a shaker for 30 minutes at room temperature with 100 μL of 125I-labelled tTG Mab A3-6B5 (30,000 cpm; diluted in Assay Buffer).", "After a final aspiration and 3 washes with 500 μL of Assay Buffer, tubes were counted for 1 minute in a gamma counter.", "The results, measured in counts per minute (cpm), were used to calculate the % inhibition of 125I-Mab binding as follows: % ⁢ ⁢ inhibition = 100 - [ A B × 100 ] wherein, A=125I-Mab (cpm) bound to rhtTG in the presence of test sera as a % of total cpm of material added to the tube; and B=125I-Mab (cpm) bound to rhtTG in the presence of healthy blood donor sera (mean of 2 or 3 sera) as a % of total cpm of material added to the tube.", "Results and Discussion FIG.", "2 showed that C-terminal deletions of amino acids (aa) 492-687 of the tTG protein had no or little effect on tTG autoantibody binding.", "More extensive C-terminal deletion (aa 401-687) resulted in lower tTG autoantibody binding in 14/15 sera studied.", "N-terminal deletion of aa 1-89 of the tTG protein resulted in lower tTG autoantibody binding in all sera studied.", "Therefore, the studies with modified tTG proteins described herein showed that tTG autoantibody binding sites on tTG are heterogenous and are dependent on the aa sequences in the N-terminal and central part of the molecule.", "Furthermore, these studies showed that aa 1-89, and aa 401-491 of human tTG protein are important for binding of tTG autoantibodies present in the coeliac sera studied.", "(aa 401-491 of human tTG correspond to aa 401-494 of tTG in other species, such as guinea pig tTG.)", "Analysis of mouse Mabs by immunoprecipitation with modified 35S-labelled tTG proteins indicated that the epitope for A3-6B5 Mab was contained within aa 1-89 of the human tTG protein and the epitope for A5-4E6 Mab was contained within aa 401-491 of the human tTG protein (FIG.", "3).", "Furthermore, these Mabs, namely A3-6B5 Mab and A5-4E6 Mab, inhibited binding of tTG autoantibody positive sera with tTG in ELISA (see FIGS.", "5 and 6).", "Identification of amino acid sequences of the tTG molecule important for binding of autoantibodies to tTG tested, and production of mouse Mabs reactive with these amino acid sequences, allowed the development of a new assay to screen for the presence of autoantibodies to tTG in a sample of body fluid as described above.", "The results of the assay based on autoantibodies to tTG in test sera inhibiting 125I-Mab A3-6B5 binding with recombinant human tTG are shown in Table 2.TABLE 2 Results of assay based on the inhibition of 125I-Mab binding to recombinant human tTG % Inhibition of tTG 35S rhtTG Serum 125I-A3-6B5 Mab Autoantibodies Autoantibodies Number binding ELISA u/mL IPA Index Coeliac 1 79 1000 100 2 35 110 52 3 91 42 7.7 4 49 150 21 5 89 1000 44 6 88 51 107 7 89 1000 122 8 42 102 107 9 48 45 3.3 Healthy blood donors 1 −11 Neg Neg 2 13 Neg Neg 3 −1 Neg Neg 4 3 Neg Neg 5 −3 Neg Neg Nine sera from coeliac patients which tested positive for tTG autoantibodies by immunoprecipitation assay (tTG autoantibodies index from 3.3-107) and ELISA (42-1000 u/mL) were analysed.", "All these sera inhibited binding of 125I-Mab to rhtTG coated on the tube (35 to 91% inhibition).", "In contrast, sera negative for autoantibodies to tTG only had a small effect on the binding of 125I-Mab to rhtTG (−11 to 13% inhibition)." ] ]	Patent_10467587
[ [ "Dispensing device", "The invention relates to a dispensing device for dispensing particle suspensions (12), especially cell suspensions, comprising a reservoir (10) for receiving the particle suspension (12).", "A suspending agent (26) is provided in the reservoir (10) that influences the particle suspension (12).", "The suspending agent (26) serves to preserve homogenous particle distribution in the particle suspension (12).", "A microdispenser (22) is connected to the reservoir (10) by a fluid element (18).", "The microdispenser serves for delivering microdroplets on a titer plate." ], [ "1.A dispensing device for the dispensing of particle suspensions (12), particularly of cell suspensions, comprising a reservoir (10) for receiving the particle suspension (12), a suspension means (26) acting on the particle suspension (12) for maintaining a substantially homogeneous particle distribution in the particle suspension (12), a microdispenser (22) for dispensing microdroplets of a volume smaller than 5 μl, and a fluid element (18) connecting the reservoir (10) to the microdispenser (22) for continuous provision of particle suspension (12) at the microdispenser (22), the suspension means being provided as a stirrer (26).", "2.The dispensing device according to claim 1, characterized in that the suspension means (26) is configured to the effect that substantially no damage is caused to the particles, particularly the cells.", "3.The dispensing device according to claim 1 or 2, characterized in that the stirrer comprises at least three, preferably at least four stirring blades.", "4.The dispensing device according to claim 3, characterized in that the stirring blades include an angle of 50 to 85°, preferably 60 to 70° relative to a longitudinal axis of the stirrer (26).", "5.The dispensing device according to claim 3 or 4, characterized in that the stirring blades comprise mixing faces of a slightly concave shape.", "6.The dispensing device according to any one of claims 1-5, characterized in that the reservoir (10) is connected to a gas supply device for gas supply to the particle suspensions.", "7.The dispensing device according to any one of claims 1-6, characterized in that the microdispenser (22) is piezoelectrically activated.", "8.The dispensing device according to any one of claims 1-7, characterized in that the microdispenser comprises a pump chamber having a volume between 100 nl and 5 ml, more preferably between 100 nl and 10 μl and most preferably between 500 nl and 2 μl.", "9.The dispensing device according to any one of claims 1-8, characterized in that the microdispenser comprises a dispensing opening (24) having a diameter smaller than 500 μm, preferably smaller than 200 μm and most preferably smaller than 100 μm.", "10.The dispensing device according to any one of claims 1-9, characterized in that the fluid element (18) has a shut-off device (20) connected thereto for interrupting the supply of particle suspension (12) to the microdispenser (22).", "11.The dispensing device according to claim 10, characterized in that the shut-off device is provided as a tube squeeze valve (20).", "12.The dispensing device according to any one of claims 1-11, characterized in that the microdispenser (22) is connected, via a second fluid element (56), to a supply container (46) for rinsing liquid (48) for cleaning the microdispenser (22).", "13.The dispensing device according to claim 12, characterized in that the second fluid element (56) is connected to a shut-off device (58)." ], [ "The invention relates to a dispensing device for the dispensing of particle suspensions, particularly of cell suspensions.", "Particularly when conducting research in the field of active substances, a large number of active substances is tested nowadays by means of high-throughput screening.", "In doing so, a large number of samples is tested in a short time in an automated process.", "In high-throughput screening, the active substances are inserted into miniaturized microtitration plates or other suitable sample carriers with wells provided therein.", "Since the costs for the active substances, the storage of these substances in substance libraries and the disposal of the active substances after the test process are extremely expensive, the volumes of the individual wells are as small as possible.", "In modern titration plates, the volumes of the individual wells are often in the microliter and nanoliter range.", "Thus, it is required that minimum quantities be inserted into the wells by pipetting or dispensing.", "A suitable pipetting device for active substances without sensitive particles is described e.g.", "in WO 96/24040.Since the examination of particle suspensions, particularly of cell suspensions, has gained increased importance, it has become desirable to develop a device adapted to supply even minimum quantities of particle suspension into wells.", "In this regard, a particular problem resides in that e.g.", "cells and other particles which have low mechanical stability may be easily damaged when introduced into the wells.", "Especially, pipetting of liquid samples cannot be performed if the samples are particle suspensions since the particles have to pass the very narrow pipette opening both during the suctional intake into the pipette and during ejection from the pipette.", "In this regard, for instance, a considerable danger exists that the cells are affected by shearing forces and that the surface function of the cells is impaired.", "Thus, the pipetting device described in WO 96/24040 is not useful for particle suspensions.", "Further, a system for the dispensing of liquids is known from WO 98/06821.In this system, one individual particle or one individual cell is dispensed per dispensing process.", "This known device is extremely complex and expensive.", "Such a device is particularly ill-suited for high-throughput screening since the filling of the individual wells which shall take up a specific number of particles or cells, is too time-consuming to allow e.g.", "10,000 tests to be carried out per day in effective systems.", "It is an object of the invention to provide a dispensing device for the dispensation of particle suspensions, particularly of cell suspensions, which is adapted to dispense minimum quantities over periods of several hours.", "According to the invention, the above object is achieved by a dispensing device having the features mentioned in claim 1.The dispensing device according to the invention comprises a reservoir for receiving a particle suspension, particularly a cell suspension.", "The particle suspension can further comprise synthetic microparticles such as beads, particularly polymeric beads.", "Particularly when dispensing cells or particles, the problem exists that these are not distributed homogeneously in the liquid, thus precluding the possibility to fill e.g.", "the wells of a titration plate with a reproducible number of cells or particles with small variance because, due to the occurrence of sedimentation effects and the like reasons, the concentration of the cells or particles in the suspensions will be locally altered.", "Thereby, the number of the dosed cells or particles is changed in each dosing process to such an extent that the measurement results are rendered unreliable.", "According to the invention, for homogenizing the particle distribution in the particle suspension, the dispensing device is provided with a suspension means acting on the particle suspension.", "The suspension means effects a mixing of the particle suspension so that a substantially homogeneous particle distribution in the particle suspension will be obtained.", "The variance of the number of the individual particles or cells in the wells will thus be extremely small.", "Preferably, the variance between the individual wells is smaller than 15%, and more preferably smaller than 10%.", "Such a small variance can also be realized during continuous dispensation over a period of several hours.", "For instance, the cell concentration in the suspension is between 1×105 and 2×106 per ml.", "This will result in a number of 100-2,000 cells per well in case of a filling quantity of 1 μl per well.", "Even with such high cell concentrations, the above mentioned low variance can be obtained through the provision of a suspension means in the reservoir.", "Further, the dispensing device comprises a microdispenser for the dispensing of microdroplets.", "The microdroplets, being of a volume smaller than 5 μl, preferably smaller than 100 nl, more preferably smaller than 10 nl, still more preferably smaller than 3 nl and most preferably smaller than 0.5 nl, are dispensed by the microdispenser into the wells or recesses of a sample carrier.", "For this purpose, the microdispenser is connected to the reservoir via a fluid element for continuous supply of particle suspension to the microdispenser.", "The inventive dispensing device makes it possible, due to the provision of a suspension means, to dispense a particle suspension, particularly a cell suspension, over a period of several hours with only low variance of the number of particles or cells in the individual dispensing processes.", "Thus, with the inventive dispensing device, one can generate a uniform and reproducible number of particles or cells in dependence from the supplied volume.", "Particularly, the suspension means is of a suitable type to substantially preclude damage to the particles, especially the cells, during the mixing of the particle suspension.", "To obtain the above effect, use can be made of a rod-type stirrer, for instance.", "This rod-type stirrer, which can be e.g.", "a rod-type stirrer with triangular stirring element, is driven e.g.", "by magnetic forces generated by a motor arranged below the bottom of the reservoir.", "In this manner, an extremely effective suspension can be obtained in the particle suspension.", "Since, however, in such a rod-type stirrer, friction will occur between the stirring element and the bottom of the reservoir, the resultant high shear forces may cause destruction of the cells.", "Thus, the suspension means is preferably provided as a suspended stirring system, particularly a suspended rod-type stirrer.", "This stirrer is installed in the reservoir, if possible in a centered position, via a rotatable axis connected e.g.", "to the cover of the reservoir.", "The driving motion can be imparted by a motor connected to the axis and arranged externally of the reservoir.", "The motor can also be arranged below the bottom of the reservoir and drive the rod-type stirrer by magnetic forces.", "To allow for the best possible driving of the stirrer by use of magnetic forces, the stirrer preferably comprises rare-earth metals.", "By the provision of the rare-earth metals, the driving force can be imparted to the stirrer also through thick reservoir walls, particularly through thick reservoir bottoms.", "A rod-type stirrer arranged in free suspended attachment in the particle suspension offers the advantage that the stirring can be performed without the stirrer touching the bottom or a wall of the reservoir.", "This will minimize the mechanical stresses acting on the cells or other particles included in the particle suspension while obtaining a good homogeneity within the particle suspension.", "The stirrer of the preferably used type comprises at least two, preferably four stirring blades.", "These are arranged preferably at uniform distances on the circumference of the stirrer.", "The individual stirring blades are preferably arranged at an angle of 50 to 85°, and more preferably 60 to 70° relative to the longitudinal axis of the stirrer.", "This angle will be determined to the effect that the stirring blades are arranged at an angle of 40 to 5° and 30 to 20°, respectively, relative to a plane extending horizontally when the stirrer is arranged vertically.", "As compared to this horizontal plane, the inclination of the stirring blades is thus relatively flat.", "This has the advantage that the thrust between the particles in the particle suspension and the stirring blades is reduced.", "This thrust would be largest if the stirring blades were arranged in parallel to the longitudinal axis.", "Preferably, the stirring blades have rounded edges.", "To further reduce the thrust between the particles and the stirring blades, the mixing faces of the mixing blades are of a slightly convex shape.", "The mixing faces are defined as those faces of the stirring blades onto which the particles impinge during the mixing.", "The stirrer is preferably made from inert biocompatible materials or is coated with such materials.", "Preferably, the stirrer is provided with a PTFE coating.", "Further, the surface of the stirring blades is preferably as smooth as possible to avoid damage to the particles.", "Particularly in case of dispensing cell particles over a period of several hours, the gas supply to the cells, particularly the supply with oxygen and carbon dioxide, is critical.", "Further, the supply of carbon dioxide serves for the pH regulation of the liquid.", "According to a preferred embodiment, the dispensing device of the invention comprises, in addition to the suspension means which does already enhance the gas infeed to the cells or particles, with a gas supply device.", "For this purpose, the reservoir is connected to the a gas supply device e.g.", "via a gas conduit.", "Preferably, the gas supply device is of a type which can be controlled to supply gas to the reservoir in dependence from e.g.", "the particle type.", "To this end, the reservoir is preferably configured such that it is not filled completely with the particle suspension and a gas space is formed in the upper region of the reservoir.", "To further improve the supply of particles, especially the cells, with oxygen, carbon dioxide or other gases, the gas can also be introduced into the liquid so that the gas will form bubbles which will rise in the liquid, thus safeguarding the gas supply to the particles.", "However, it has been found that the introduction of gases into the liquid disadvantageously entails the risk of shear effects on the cells caused by the rising and possibly bursting gas bubbles.", "Preferably, in the inventive device, the gas to be supplied to the particles, especially the cells, is not introduced directly into the particle suspension but only into the reservoir so that the corresponding gases will be introduced into the space between the surface of the liquid and the cover.", "The movement of the particle suspension effected by the suspension means will bring about a sufficient intake of the required gases into the suspension.", "To guarantee the dispensing of the particle suspension over a period of several hours, the reservoir preferably has a volume of 1-500 ml, with the size of the reservoir being adapted to the volumes of the wells provided e.g.", "in titration plates.", "The microdispenser provided for the dispensation of the particle suspension is preferably activated piezoelectrically.", "This offers the advantage that very small microdroplets can be generated whose volume is smaller than 10 nl, preferably smaller than 3 nl and most preferably smaller than 0.5 nl.", "Further, the microdispenser can be configured to the effect that one piezoelement acts onto a flexible channel, which channel preferably is a round hose closed in itself, or the like.", "Thus, advantageously, the microdispenser can be cleaned in a simple manner and thereby kept in a sterile condition.", "The microdispenser is provided e.g.", "as a glass-silicon structure having a pump chamber arranged therein.", "The pump chamber comprises an elastic chamber wall which is connected to an electrically controllable actuator provided to generate pressure in the pump chamber and to reduce the chamber volume, respectively.", "When the actuator is suitably controlled, the microdispenser will dispense a droplet.", "The pump chamber is preferably delimited by an inlet opening and an outlet opening which forms the outlet opening of the microdispenser or is connected thereto.", "The inlet opening is connected to the reservoir via a fluid element such as a hose.", "By the provision of an inlet opening, it is prevented that the pressure generated by the actuator in the pump chamber causes a displacement of liquid in the direction of the reservoir instead of the direction of the outlet opening.", "Via the inlet opening, a continuous inflow of the to-be-dispensed liquid is safeguarded.", "The pump chamber has a volume preferably between 100 nl and 5 ml, more preferably between 100 nl and 10 μl and most preferably between 500 nl and 2 μl.", "The nozzle width of the outlet opening is preferably smaller than 500 μm, more preferably smaller than 200 μm and most preferably smaller than 100 μm.", "Thus, with the preferred dispensing device, one can generate a drop size of a volume smaller than 10 nl, preferably smaller than 3 nl and most preferred smaller than 0.5 nl.", "Further, the microdispenser can be a piston-type dispensing unit with valve and nozzle, or the like.", "By suitable control of the piston and the valve, it is likewise possible to generate very small droplets.", "In this arrangement, the piston space corresponding to the above pump chamber also has the above described dimensions so that droplet sizes corresponding to those mentioned above can be generated also here.", "According to a preferred embodiment, the fluid element arranged between the reservoir and the microdispenser is provided with a shut-off unit.", "The shut-off unit serves for the interruption of the supply of the particle suspension to the microdispenser.", "Thus, it is possible to clean the microdispenser.", "Cleaning is performed e.g.", "at fixed time intervals.", "Preferably, the shut-off unit is a component which does not immediately interfere with the fluid system.", "This has the advantage that the sterility demands required for the handling of particle suspensions are fulfilled.", "The provision of a contactless shut-off unit further has the advantage that the shut-off unit is biocompatible.", "This means that no valve material, especially no metal particles, can come into contact e.g.", "with the cell suspension and affect the same.", "A particularly suited shut-off unit is a tube squeeze valve.", "To facilitate the cleaning of the microdispenser, the microdispenser is preferably connected, via a second fluid element, to a supply container having a rinsing liquid provided therein.", "Thus, an automatic cleaning of the microdispenser can be performed.", "For this purpose, preferably, the second fluid element has a second shut-off unit connected thereto which can have the same configuration as the above described first shut-off unit.", "Also, a conventional valve can be provided in the second fluid unit or at the outlet of the supply container for the rinsing liquid because lower sterility demands have to be kept for the rinsing liquid.", "Preferably, the inner diameters of the fluid elements and the shut-off devices do not or only slightly differ from each other.", "Thereby, a sedimentation of the particles and thus a de-homogenizing of the particle suspension are avoided.", "The diameters of the whole fluid system which preferably do not or only slightly differ from each other, are preferably in the range between 45 mm and 500 μm.", "Most preferred are diameters between 1.5 mm and 200 μm.", "Preferably, the diameters of the individual channels provided in the device, particularly in dependence from the dispensed quantity of liquid, are selected to the effect that the dwelling time of the particle suspension, particularly the cell suspension, in the system is shorter than 10 min., preferably shorter than 5 min.", "and most preferably shorter than 2 min.", "Thus, sedimentation effects in the system are substantially avoided.", "These dwelling times are of course not valid for the reservoir in which the sedimentation is avoided by the inventive provision of a suspension agent.", "A preferred embodiment of the invention will be explained in greater detail hereunder with reference to the accompanying drawing.", "In the drawing: FIG.", "1 is a schematic representation of an embodiment of the dispensing device according to the invention.", "The dispensing device comprises a reservoir 10 in which the particle suspension 12 is received.", "The reservoir 10 has an outlet 16 provided with an outlet connector piece 14.The connector piece 14 has a hose 18 connected thereto as a fluid element.", "The hose is connected to a microdispenser 22 with a tube squeeze valve 20 interposed along the hose.", "The inner diameters of hose 18 as well as of outlet connector piece 14 are preferably identical.", "Thus, the contact element is free of a dead volume.", "An identical connection exists also to the tube squeeze valve 20.By the provision of substantially constant inner diameters of the fluid elements, the occurrence of restriction regions which would form a flow resistance to the particles of the suspensions, is prevented.", "In case of such resistance, shear effects would occur, and the number of particles transported in the particle suspension would vary over time.", "This would lead to the so-called keystone effect, causing an non-reproducible number of cells or particles in the wells.", "The microdispenser 22 has an outlet opening 24 provided to eject the microdroplets for filling the wells of a titration plate.", "The suspension means of reservoir 10 is arranged as rod-type stirrer 26 by which the particle distribution in particle suspension 12 is kept homogenous throughout an extended period of preferably more than 6 hours, more preferably more than 10 hours.", "Instead of the suspended rod-type stirrer 26 illustrated according to the instant embodiment, also a plurality of suspension means can be provided.", "Preferably, these are also located internally of reservoir 10 and therein are either supported on the reservoir bottom 28 or are mounted in a suspended arrangement within particle suspension 12.The driving of the suspension means can be performed, as in the case of the rod-type stirrer 26 in the illustrated embodiment, by means of a motor 30.Motor 30 is arranged externally of reservoir 10.In the illustrated embodiment, motor 30 is arranged externally on a cover 32 of reservoir 10.Further, it is possible to drive the suspension means in a contactless manner or through magnetic actuation, for instance.", "A further possibility for the intermixing of the particle suspension and thus for the maintenance of a homogeneous particle distribution resides in generating a flow within the reservoir 10.This can be accomplished e.g.", "by the provision of circulating pumps.", "For this purpose, preferably two reservoirs are provided between which the particle suspension is moved by a circulating pump.", "The reservoir 10 is connected to a gas supply device 34 for supplying gas to the particle suspension 12, particularly the cell suspension.", "By means of the gas supply device 34, gas, e.g.", "oxygen or carbon monoxide, is fed via conduits 36,38 into a gas chamber 40.The gas chamber 40 is arranged internally of reservoir 10 and is provided above the particle suspension 12.For control of the quantity of gas in the gas chamber 40 and of the different gas components, the gas supply device 34 is provided with a suitable control means.", "Further, a pressure control element 42 is arranged on the cover 32 for measuring the gas pressure in the gas chamber 40 and to control the pressure through the gas supply device 34.Preferably, for tempering the particle suspension, a tempering element 44 is provided which in the illustrated embodiment is arranged on an outer wall of reservoir 10.The tempering element 44 serves for the heating and/or cooling of the particle suspension 12.Further, the illustrated embodiment of the dispensing device of the invention comprises a supply container 46 containing a rinsing liquid 48.The pressure container 46 is also connected to a pressure control element 50 for pressure control.", "The pressure control element 50 makes it possible to generate an underpressure or overpressure in supply container 46.An outlet 52 of supply container 46 is provided with an outlet connecting piece 54 which is connected to a tube 56.Tube 56 is connected, via a second tube squeeze valve 58, also with microdispenser 22.In the illustrated embodiment, the connection is effected via a Y-type tube connection member 60 so that the two tubes 18,56 are joined upstream of the microdispenser 22 and only one tube is connected to microdispenser 22." ] ]	Patent_10467596
[ [ "Information processing apparatus and method", "When reproducing content, a client reads attribute information defined in the header of content data corresponding to a content ID specified by a user.", "When the read attribute information meets an attribute condition defined in a license stored in a storage unit defining the attribute condition, the encrypted content data is decrypted and output.", "After the content is distributed, a collected edition or a best edition can be newly released without creating new content by issuing the license having an attribute condition that limits, for example, a release data and an artist.", "Defining a license with a specific subscription ID as the attribute condition allows the user with the license to use new release content with the subscription ID without additional purchase of a license." ], [ "1.An information processing apparatus comprising: content receiving means for receiving content including encrypted content data and attribute information; content storage means for storing the content; license receiving means for receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; license storage means for storing the license; determining means for determining whether the attribute information on the piece of the content meets the attribute condition of the license stored in the license storage unit; decrypting means for decrypting the encrypted content data of the piece of the content based on the determination of the determining means that the attribute information on the piece of the content meets the attribute condition of the license; and outputting means for outputting the content data decrypted by the decrypting means.", "2.An information processing apparatus according to claim 1, wherein the content further includes a content key for decrypting the content data.", "3.An information processing apparatus according to claim 1, wherein the attribute information includes a combination of an attribute item and an attribute value.", "4.An information processing apparatus according to claim 1, wherein the attribute item includes information on a record company, an artist, a release date, a content provider, a genre, a subscription, and a label.", "5.An information processing apparatus according to claim 1, wherein the attribute condition includes a combination of an attribute item, an attribute value, and an operator.", "6.An information processing apparatus comprising: receiving means for receiving a license request including a license ID for uniquely identifying the license including the attribute condition defining the condition regarding the attribute information included in the content; storage means for storing the license along with the license ID; obtaining means for obtaining the license corresponding to the license ID included in the license request; signature means for adding a digital signature to the license; and sending means for sending the license with the signature added thereto by the signature means.", "7.An information processing apparatus according to claim 6, further comprising license processing means for attaching a terminal ID to the license obtained by the obtaining means.", "8.An information processing apparatus comprising: storing means for storing content including encrypted content data and attribute information; receiving means for receiving a content request including a content ID for uniquely identifying the content; and sending means for sending a piece of content corresponding to the content ID included in the content request, wherein: the attribute information included in the piece of the content is information used for determining whether an attribute condition of the license is met when the piece of the content is used; and the attribute condition of the license is information defining a condition regarding the attribute information on the piece of the content that can be used.", "9.An information processing method comprising: a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.", "10.A program for causing a computer to execute: a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.", "11.A program storage medium containing a program to cause a computer to execute: a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step." ], [ "<SOH> BACKGROUND ART <EOH>Recently, systems have been realized for exchanging music data for free among a plurality of users in which a user provides his/her own music data to other users via the Internet and receives other users' music data that the user does not have.", "Theoretically, only with the presence of one piece of music data and another piece of content, such systems allow the other users to take advantage of them.", "Because of this, many users do not purchase the content, causing a copyright holder to lose their chance to obtain royalties that the copyright holder deserves.", "Accordingly, there are systems in which the content to be distributed is encrypted and the license for using the content is separately provided, so that the encrypted content cannot be decrypted and reproduced without the license corresponding to the encrypted content.", "This enables the content to be freely distributed while the copyright of the copyright holder is protected.", "However, the above system has difficulties in flexibly establishing a correspondence between the license and the content, and difficulties in newly distributing the content to which the already-distributed license can be applied." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 is a block diagram showing a construction of a content providing system to which the present invention is applied.", "FIG.", "2 is a block diagram showing a construction of a client in FIG.", "1 .", "FIG.", "3 is a flowchart illustrating content download processing by the client in FIG.", "1 .", "FIG.", "4 is a flowchart illustrating content providing processing by a content server in FIG.", "1 .", "FIG.", "5 is a diagram showing an example data format.", "FIG.", "6 is a diagram illustrating types of attribute items.", "FIG.", "7 is a diagram showing a license structure.", "FIG.", "8 is a flowchart illustrating reproduction processing by the client.", "FIG.", "9 is a flowchart illustrating license acquisition processing.", "FIG.", "10 is a flowchart illustrating license acquisition processing.", "FIG.", "11 is a flowchart illustrating license acquisition processing.", "FIG.", "12 is a flowchart illustrating details of the license acquisition processing.", "FIG.", "13 is a flowchart illustrating content data obtaining processing.", "FIG.", "14 is a diagram illustrating a key structure.", "FIG.", "15 is a diagram illustrating a relationship between the key structure and a license.", "FIG.", "16 is a diagram illustrating license granting processing by a license server.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to information processing apparatuses and methods, and more particularly to an information processing apparatus and a method for preventing unauthorized copying and using of content that is not licensed from a copyright holder.", "BACKGROUND ART Recently, systems have been realized for exchanging music data for free among a plurality of users in which a user provides his/her own music data to other users via the Internet and receives other users' music data that the user does not have.", "Theoretically, only with the presence of one piece of music data and another piece of content, such systems allow the other users to take advantage of them.", "Because of this, many users do not purchase the content, causing a copyright holder to lose their chance to obtain royalties that the copyright holder deserves.", "Accordingly, there are systems in which the content to be distributed is encrypted and the license for using the content is separately provided, so that the encrypted content cannot be decrypted and reproduced without the license corresponding to the encrypted content.", "This enables the content to be freely distributed while the copyright of the copyright holder is protected.", "However, the above system has difficulties in flexibly establishing a correspondence between the license and the content, and difficulties in newly distributing the content to which the already-distributed license can be applied.", "DISCLOSURE OF INVENTION In view of such cases, the present invention is made in order to allow content to be freely distributed and circulated and in order to freely establish a set of pieces of content that can be used in accordance with a license.", "A first information processing apparatus of the present invention includes content receiving means for receiving content including encrypted content data and attribute information; content storage means for storing the content; license receiving means for receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; license storage means for storing the license; determining means for determining whether the attribute information on the piece of the content meets the attribute condition of the license stored in the license storage unit; decrypting means for decrypting the encrypted content data of the piece of the content based on the determination of the determining means that the attribute information on the piece of the content meets the attribute condition of the license; and outputting means for outputting the content data decrypted by the decrypting means.", "The content can further include a content key for decrypting the content data.", "The attribute information can include a combination of an attribute item and an attribute value.", "The attribute item can include information on a record company, an artist, a release date, a content provider, a genre, a subscription, and a label.", "The attribute condition can include a combination of an attribute item, an attribute value, and an operator.", "A second information processing apparatus of the present invention includes receiving means for receiving a license request including a license ID for uniquely identifying the license including the attribute condition defining the condition regarding the attribute information included in the content; storage means for storing the license along with the license ID; obtaining means for obtaining the license corresponding to the license ID included in the license request; signature means for adding a digital signature to the license; and sending means for sending the license with the signature added thereto by the signature means.", "The information processing apparatus can further include license processing means for attaching a terminal ID to the license obtained by the obtaining means.", "A third information processing apparatus of the present invention includes storing means for storing content including encrypted content data and attribute information; receiving means for receiving a content request including a content ID for uniquely identifying the content; and sending means for sending a piece of content corresponding to the content ID included in the content request, wherein the attribute information included in the piece of the content is information used for determining whether an attribute condition of the license is met when the piece of the content is used and the attribute condition of the license is information defining a condition regarding the attribute information on the piece of the content that can be used.", "An information processing method of the present invention includes a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.", "A program of the present invention is a program which causes a computer to execute a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.", "A program storage medium of the present invention contains a program to cause a computer to execute a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a block diagram showing a construction of a content providing system to which the present invention is applied.", "FIG.", "2 is a block diagram showing a construction of a client in FIG.", "1.FIG.", "3 is a flowchart illustrating content download processing by the client in FIG.", "1.FIG.", "4 is a flowchart illustrating content providing processing by a content server in FIG.", "1.FIG.", "5 is a diagram showing an example data format.", "FIG.", "6 is a diagram illustrating types of attribute items.", "FIG.", "7 is a diagram showing a license structure.", "FIG.", "8 is a flowchart illustrating reproduction processing by the client.", "FIG.", "9 is a flowchart illustrating license acquisition processing.", "FIG.", "10 is a flowchart illustrating license acquisition processing.", "FIG.", "11 is a flowchart illustrating license acquisition processing.", "FIG.", "12 is a flowchart illustrating details of the license acquisition processing.", "FIG.", "13 is a flowchart illustrating content data obtaining processing.", "FIG.", "14 is a diagram illustrating a key structure.", "FIG.", "15 is a diagram illustrating a relationship between the key structure and a license.", "FIG.", "16 is a diagram illustrating license granting processing by a license server.", "BEST MODE FOR CARRYING OUT THE INVENTION FIG.", "1 shows a construction of a content providing system to which the present invention is applied.", "Clients 1-1 and 1-2 (hereinafter, referred to simply as client 1 when each of the clients does not have to be distinguished from one another) are linked to the Internet 2.Although only two clients are shown in this example, an arbitrary number of clients are linked to the Internet 2.The Internet 2 is also linked to at least one content server 3 providing content to the client 1; at least one license server 4 granting the client 1 a license required for using the content provided from the content server 3; and at least one accounting server 5 performing an accounting process for the client 1 when the license is granted to the client 1.The numbers of these at least one content servers 3, at least one license servers 4, and at least one accounting servers 5 linked to the Internet 2 are also arbitrary.", "FIG.", "2 shows a construction of the client 1.In FIG.", "2, a CPU (Central Processing Unit) 21 executes various processes in accordance with the programs stored in a ROM (Read Only Memory) 22 or loaded from a storage unit 28 to a RAM (Random Access Memory) 23.A timer 20 performs clocking to provide time information to the CPU 21.The RAM 23 also appropriately stores data and the like required for causing the CPU 21 to execute various processes.", "An encryption/decryption unit 24 encrypts content data and decrypts already-encrypted content data.", "A codec unit 25 uses, for example, ATRAC (Adaptive Transform Acoustic Coding) 3 mode to encode the content data, which is provided via an input/output interface 32 to and stored in a semiconductor memory 44 that is connected to a drive 30.The semiconductor memory 44 includes, for example, a memory stick (Trade Mark).", "The CPU 21, ROM 22, RAM 23, encryption/decryption unit 24, and codec unit 25 are interconnected via a bus 31.The input/output interface 32 is also connected to this bus 31.The input/output interface 32 includes an input unit 26 including a keyboard and a mouse; an output unit 27 including a display such as a CRT and a LCD and a speaker; a storage unit 28 including a hard disk; and a communication unit 29 including a modem and a terminal adapter.", "The communication unit 29 performs a communication process via the Internet 2.The communication unit 29 also performs analog signal or digital signal communication processing with another client.", "If necessary, the input/output interface 32 is also connected to the drive 30 on which a magnetic disk 41, optical disk 42, magneto-optic disk 43, or semiconductor memory 44 is appropriately mounted.", "If necessary, a computer program read from them is installed in the storage unit 28.Although not shown, the content server 3, the license server 4, and the accounting server 5 each have primarily the same construction as that of the client 1 shown in FIG.", "2.Next, processing will be described with reference to the flowchart in FIG.", "3 in which the client 1 receives the content from the content server 3.When a user operates the input unit 26 to request access to the content server 3, the CPU 21 controls the communication unit 29 to have access to the content server 3 via the Internet 2.At step S2, when the user operates the input unit 26 to specify content to be provided, the CPU 21 receives specification information and sends the content ID of the specified content from the communication unit 29 to the content server 3 via the Internet 2.As described below with reference to the flowchart in FIG.", "4, the content server 3 that receives this sends the content including the encrypted content data corresponding to the sent content ID.", "Accordingly, the CPU 21 receives this content via the communication unit 29 at step S3, and the content is provided to the storage unit 28 including a hard disk where it is stored.", "Next, content providing processing by the content server 3 that is the counterpart of the above-described processing by the client 1 will be described with reference to the flowchart in FIG.", "4.The construction of the client 1 in FIG.", "2 is also applied to that of the content server 3.At step S21, the CPU 21 of the content server 3 waits for access from the client 1 via the communication unit 29 from the Internet 2.When determining the occurrence of the access, the CPU 21 proceeds to step S22 where the content ID specifying the content sent from the client 1 is obtained.", "The content specification information is the content ID sent at step S2 in FIG.", "3.At step S23, the CPU 21 of the content server 3 reads the content specified with the information obtained by the process at step S22 from among the pieces of content data stored in the storage unit 28.The CPU 21 provides the content data read from the storage unit 28 at step S24 to the encryption/decryption unit 24 where the content data is encrypted.", "Since the codec unit 25 has already encoded the content data stored in the storage unit 28 by means of the ATRAC3 mode, this encoded content data is to be encrypted.", "Alternatively, the content data that is encrypted in advance can be stored in the storage unit 28.In this case, the process at step 24 can be skipped.", "Next, at step S25, the CPU 21 of the content server 3 adds a key required for decrypting the encrypted content data and attribute information representing various information regarding the content to a header that includes a format for sending the encrypted content data.", "At step S26, the CPU 21 of the content server 3 sends the content, which is obtained by formatting the content data encrypted by the process at step S24 and the header having the attribute information and the digital signature attached thereto by the process at step S25, to the client 1 that has access via the Internet 2 from communication unit 29.FIG.", "5 shows the format configuration when the content server 3 provides the content to the client 1 in this manner.", "As shown in the figure, this format includes the header and data.", "In the header, there are disposed attribute information (attribute list), a digital signature which is added to the attribute information with the encryption key of the license server, an enabling key block (EKB), and a content key Kc (KR (Kc)) encrypted with a root key KR obtained by decrypting the EKB with the DNK.", "In the attribute information, there is a plurality of descriptions of attribute entries each including a combination of an attribute item and the corresponding attribute value.", "FIG.", "6 shows types of the attribute items.", "CID, RCID, CIID, AID, GID, and LID are IDs uniquely identifying content, a record company, a content issuer, an artist, a genre, and a label.", "RelDate represents the release date of the content; the subscription ID is an attribute item used for the subscription license described below.", "The URL represents address information that is accessed when the license is obtained to use the content.", "Specifically, in the case of the system in FIG.", "1, it is the address of the license server 4 required for receiving the license.", "The data includes an arbitrary number of encryption blocks; the encryption blocks each include an initial vector (IV (Initial vector)), a Seed, and data EK′c(data) obtained by encrypting the content data with a key K′c.", "The key K′c is a value computed by applying the content key Kc and a Seed value determined with a random number to a hash function, as shown in the following expression: K′c=Hash(Kc, Seed).", "The initial vector IV and the seed of each encryption block are determined to take different values.", "This encryption is performed by dividing the content data in units of eight bytes to encrypt the content data in units of eight bytes.", "Encryption of subsequent eight-byte data is performed using a CBC (Cypher Block Chaning) mode which is performed by utilizing the result of encryption of the previous eight-byte data.", "In the case of the CBC mode, when the first eight bytes of the content data are encrypted, there is no previous eight-byte data.", "The encryption is therefore performed using the initial vector IV as the initial value when the first eight bytes are encrypted.", "Even though one encryption block is broken, the effects are prevented from reaching other encryption blocks because of the encryption using the CBC mode.", "This encryption will be described below with reference to FIGS.", "14 and 15.Thus, the client 1 can receive the content from the content server 3 without charge and restriction.", "However, each client 1 needs to acquire the license when using the obtained content.", "Acquiring the license, the client 1 registers online or offline in advance at the license server to obtain service data.", "The service data includes a device node key (DNK) and a terminal ID, which serve to decrypt the EKB.", "The service data and the license obtained from the license server are securely stored in the storage unit 28 of the client 1.FIG.", "7 shows the configuration of the license.", "The license includes a license ID, a timestamp, an expiration date, an attribute condition, a usage rule, and the digital signature, whereby the digital signature is added to these with the secret key of the license server; the timestamp represents the day of issue of the license; the expiration date represents the time limit up to when the license can be used and after which the license is expired; the attribute condition is the attribute condition of the content that the client 1 can utilize with the corresponding license, attribute condition which is represented with a conditional expression including a combination of an attribute item, the value corresponding to the attribute item, a comparison operator, and a logic operator; the usage rule represents a rule description for using the content to which the license can be applied, and it includes the same terminal ID as the one included in the service data.", "Hereinafter, there will be shown an example license configuration that can be realized with a combination of the attribute information included in the content and the attribute condition included in the license.", "The attribute information of the content c1 is defined as follows: c1:cid={1}, aid={0,1}, reldate=year 2000 Nov. 10th This means that the content ID is 1, the artist IDs are 0 and 1 (i.e., a collaboration of the artists of the artist ID 0 and the artist ID 1), and the release date is year 2000, Nov. 10th.", "Likewise, the attribute information of content c2, c3, and c4 will be defined as follows: c2: cid={2}, aid={0}, reldate=year 2000 Dec. 20th c3: cid={3}, aid={0}, reldate=year 2001 Mar.", "1st c4: cid={4}, aid={0}, reldate=year 2001 Oct. 21st On the other hand, the attribute condition of the license 11 is defined as follows: 11: 1 ε cid v 2 ε cid The right of use r1 corresponds to the content c1 and c2.In other words, the terminal with the right of use r1 allows the content c1 and c2 to be used.", "The attribute condition of the license 12 is defined as follows: 12: 0 ε aid {circumflex over ( )} (year 2001 Jan. 1st<reldate<year 2001 Dec. 31st) This means a condition that represents the content released by the artist of the ID 0 during year 2001.The license 12 corresponds to the content c3 and c4.At this point in time, the content c3 and c4 can be used at the terminal acquiring the license 12.It is assumed that content c5 with the following attribute information is provided afterwards.", "c5: cid={5}, aid={0}, reldate={year 2001 Dec. 1st} When this content is obtained by means of download or the like from the content server 3, the client 1 that has already acquired the license 12 can use the content c5 without another access to the license server or the like.", "When a distributor attempts to newly provide a combination of the content c1, c2, and c5 as a best edition, which is followed by the distribution thereof, issuing the following license 13 accommodating this situation enables the best edition to be provided without the creation of new content and with direct use of the content already distributed or in circulation.", "13: 1 ε cid v 2 ε cid v 5 ε cid Thus, the right of use obtained by combining the content already distributed and the content in circulation can be newly provided with ease.", "For example, by issuing a license with an attribute condition that limits the release date and the artist, a collected edition including works during a specific period by a certain artist can be newly provided.", "By issuing a license with an attribute condition limiting the artist, a collected edition of a certain group (including works by the group and solo works of members of the group) can be newly provided.", "Next, there will be described an example that defines, as a subscription service, a license that permits additional use of some of the new releases every month.", "The attribute condition of the license 14 is defined as follows: 14: 3 ε cid v 4 ε cid v 1 ε sid For the client 1 acquiring this license 14, the content c3 and c4 that are already provided can be used.", "It is assumed that content c6 and c7 with the following attribute information are provided as new releases the next month.", "C6: cid={6}, sid={1} C7: cid={7}, sid={1} In this case, the client 1 with the license 14 can use the content c6 and c7 without the necessity of purchasing a new license.", "Likewise, by providing the content with the subscription ID of 1 each month, without purchasing another license, the client 1 with the license 14 can add the content that can be used.", "Thus, by representing the attribute condition as a combination of the attribute item, the attribute value, and the operator, such as the logic operator and a relational operator, a set of the content that can be used is able to be flexibly set.", "The operators included in the attribute condition are not limited to the ones described here, and thus various types of other operators can be used.", "Processing will be described with reference to FIG.", "8 when the client 1 reproduces the content.", "At step S41, the CPU 21 of the client 1 obtains the content ID that is specified by the user through the operation of the input unit 26.The CPU 21 reads the attribute information defined in the header of the content data corresponding to the obtained content ID.", "Next, the CPU 21 proceeds to step S42 where the CPU 21 determines whether any license whose attribute condition defined therein is satisfied with the attribute information read at step S41 is already acquired and stored in the storage unit 28 by the client 1.When such a license cannot be found, the CPU 21 proceeds to step S43 where the CPU 21 causes a message prompting the acquisition of the license to be shown via the output unit 27 on the display.", "When it is determined at step S42 that the license is already acquired, the CPU 21 proceeds to step S44 where the CPU 21 determines whether the acquired license is expired.", "Whether the license is expired is determined by comparing the expiration date specified as a description of the license and the present time clocked by the timer 20.When it is determined that the expiration date is already reached, the CPU 21 proceeds to step S45 where license update processing is performed.", "The details of this license update processing will be described with reference to the flowchart in FIG.", "8.When it is determined at step 44 that the license is still unexpired, or when the license is updated at step S45, the CPU 21 proceeds to step S45 where the CPU 21 verifies the digital signature included in the header of the content and the digital signature in the license with the public key of the license server 4.When the verification result of the digital signature proves that the digital signature is valid, the CPU 21 proceeds to step S46 where the CPU 21 reads the encrypted content data from the storage unit 28 and stores it in the RAM 23.At step S47, the CPU 21 provides the encryption block data stored in the RAM 23 in units of encryption blocks disposed in the Data in FIG.", "5 to the encryption/decryption unit 24 where the encryption block data is decrypted.", "Furthermore, the CPU 21 provides the content data decrypted by the encryption/decryption unit 24 at step S48 to the codec unit 25 where the decrypted data is decoded.", "The CPU 21 provides the data decoded by the codec unit 25 from the input/output interface 32 to the output unit 27 where the decoded data is D-A converted for output from a loudspeaker.", "Processing will be described with reference to FIGS.", "9 to 11 in which the client acquires the license from the license server 4.FIG.", "9 shows license acquisition processing when a user of the client 1 determines the content to be used.", "When issuing of a license list is requested to the license server 4 in response to the specification of the content through the operation of the input unit 26 by the user, the CPU 21 controls the communication unit 29 to send the request of the license list including the content ID of the specified content to the content server 3 via the Internet 2.When receiving the license list request, the license server 4 extracts the licenses that can be applied to the content corresponding to the content ID included in the received license list and sends the client 1 the license list in which the license ID, the license name, the condition of the content to be used, the list of content can be used currently, the working conditions of the content, and the like of each of the licenses are defined.", "When the client 1 receives the license list from the license server 4, the CPU 21 displays information on each license included in the license list on the output unit 27.When the user selects a desired license by referring to the information, the CPU 21 controls the communication unit 29 to establish a session by means of a two-way authentication, such as SSL.", "Subsequently, a license request including the license ID, the terminal ID, the user ID for accounting, and the password of the selected license is encrypted to be sent to the content server 3 via the Internet 2.When receiving the license request sent from the client 1, the license server 4 performs license issuing processing, described later, and sends the client 1 the license corresponding to the license ID included in the license request.", "When receiving the license sent from the license server 4, the client 1 encrypts the received license and so on to be stored in a secure state in the storage unit 28.As described above, the user can acquire the license for using the content which the client 1 already has obtained.", "Alternatively, when the user performs an operation of reproducing the content that the client obtains and when the license for reproducing the content is not acquired, the above license acquisition processing may be automatically started.", "Next, processing is shown in FIG.", "16 in which the user specifies various search conditions to search for the license and acquire it.", "Initially, in order to search for a user's desired license, the user operates the input unit 26 to specify the search condition, such as the license name, the license type, the title of the content to which the license can be applied, the album name, the genre, the artist name, and the release date.", "The CPU 21 controls the communication unit to send the content server 3 the license request including data obtained by formatting the input search condition.", "When receiving the license list request sent from the client 1, the content server searches for the licenses satisfying the search condition included in the license list request from the storage unit 28, and the license list including information on each of the licenses, such as the license ID, is sent to the client 1.When the client 1 receives the license list from the license server 4, the CPU 21 causes information on each of the licenses included in the license list to be shown on the output unit 27.When the user selects a desired license by referring to the information, the CPU 21 controls the communication unit 29 to establish a session by means of the two-way authentication, such as the SSL.", "Subsequently, the license request including the license ID, the terminal ID, the user ID for accounting, and the password of the selected license is encrypted to be sent via Internet 2 to the content server 3.Receiving the license request sent from the client 1, the license server 4 performs the license issuing processing, described later, and then sends the client 1 the license corresponding to the license ID included in the license request.", "When receiving the license sent from the license server 4, the client 1 encrypts the received license to be stored in a secure state in the storage unit 28.As described above, the user can search for the desired license and acquire it.", "Next, the license acquisition processing is shown in FIG.", "11 in a case in which the user knows the license ID of the desired license.", "When the user operates the input unit 26 to specify the license ID of the desired license, the CPU 21 controls the communication unit 29 to establish a session by means of the two-way authentication, such as SSL.", "Subsequently, the license request including the license ID, the terminal ID, the user ID for accounting, and the password of the selected license is encrypted and sent via the Internet 2 to the content server 3.When receiving the license request sent from the client 1, the license server 4 performs the license issuing processing, described later, and then sends the client 1 the license corresponding to the license ID included in the license request.", "When receiving the license sent from the license server 4, the client 1 encrypts the received license or the like to be stored in a secure state in the storage unit 28.The user knows the license ID from license advertisements inserted in magazines or the like and can acquire the desired license in the above-described manner by specifying the license ID.", "Alternatively, the license acquisition processing may start by allowing the user to perform selection or the like by clicking to URL link information on the license server including the license IDs inserted in HTML files of Web sites, e-mail, and the like.", "The details of the license issuing processing in FIGS.", "9 to 11 will be described with reference to the flowchart in FIG.", "12.In this case again, the construction of the client 1 in FIG.", "2 is applied to the construction of the license server 4.Initially, at step S102, the CPU 21 obtains the license ID, the terminal ID, the user ID, and the password included in the license request.", "The CPU 21 of the license server 4 has access to the accounting server 5 via the communication unit 29 to request authorization processing of the user corresponding to the user ID and the password.", "When receiving a request of the authorization processing via the Internet 2 from the license server 4, the accounting server 5 checks the track record of the past payments of the user corresponding to the user ID and the password to determine whether the user has a record of no payment for the value of the license in the past.", "When the user does not have such a record, the result of the authorization that permits the grant of the license is sent.", "When the user has the record of “no payment”, the result of the authorization that does not permit the grant of the license is sent.", "At step S104, the CPU 21 of the license server 4 determines whether the result of the authorization from the accounting server 5 permits the grant of the license.", "When the grant of the license is permitted, the CPU 21 proceeds to step S105 where the license corresponding to the license ID is acquired from a database.", "The terminal ID is inserted into the usage rule field of the license and the digital signature is generated with the private key of the license server 4 to be attached.", "At step S107, the CPU 21 of the license server 4 sends the license with the terminal ID and the digital signature attached from the communication unit 29 via the Internet 2 to the client 1.At step S108, the CPU 21 of the license server 4 stores the license just sent at step S107 in the storage unit 28 in such a manner that the license is associated with the user ID and the password obtained by the process at step S102.In addition, the CPU 21 executes the accounting processing at step S109.To be specific, the CPU 21 requests the accounting server 5 via the communication unit 29 to perform the accounting processing on the user corresponding to the user ID and the password.", "The accounting server 5 performs the accounting processing on the user based on this accounting request.", "As described above, when the user does not pay in response to this accounting processing, the user is not able to receive the license afterwards even though the grant of the license is requested.", "In other words, since the authorization result that does not permit the grant of the license is sent from the accounting server 5 in this case, the CPU 21 proceeds from step S104 to step S110 where error processing is performed.", "To be specific, the CPU 21 of the license server 4 displays a message notifying the client 1, controlling the communication unit 29 to have access, that the grant of the license is unable to be permitted, and then the process is terminated.", "In this case, as described above, being unable to receive the license, the client 1 cannot use the content.", "Next, processing will be described with a reference to FIG.", "13 in which the content data of the content to which the license can be applied is obtained.", "When the user operates the input unit 26 to select the license, the CPU controls the communication unit 29 to send the content list request including the license ID of the selected license to the content server 3 via the Internet 2.When receiving the content list request, the license server 4 obtains the license ID included in the content list request.", "The license server 4 can extract the content to which the corresponding license can be applied, with the license ID as the key, from a license database.", "Subsequently, the license server sends the client 1 the content list including content information, such as the content ID, the URL for downloading the content, the content name, the artist name, and the genre of each piece of the extracted content.", "Controlling the output unit to receive the content list, the client 1 displays the content information of each piece of the content included in the content list.", "When the user selects the content to be downloaded by referring to the displayed content information, the client 1 sends the content request to the content server 3 in accordance with the URL of the content.", "When receiving the content request, the content server sends the client 1 the content with the content ID included in the content request.", "The client 1 receives the content from the content server 3 and stores the received content in the storage unit 28.As described above, the user retrieves the content to which the license is capable of being applied and causes the client to download from the content server 3.FIG.", "14 shows a key constructing method when Broadcast Encryption is adopted as a key management system.", "As shown in FIG.", "14, keys take a hierarchical tree structure whose lowest layer leaves each correspond to devices.", "In an example in FIG.", "14, keys corresponding to sixteen devices, numbered 0 to 15, are produced.", "Keys are each specified so as to correspond to the node indicated with a circle in the figure.", "In this example, the root node at the top layer corresponds to a key KR, the nodes at the second layer correspond to keys K0 and K1, the nodes at the third layer correspond to keys K00 to K11, and the nodes at the fourth layer correspond to keys K000 to K111.The leaves(device nodes) which serve as the nodes at the bottom layer correspond to keys K0000 to K1111.Because of the hierarchical structure, for example, the key K001 is regarded as the upper-layer key of the keys K0010 and K0011, and the key K00 is regarded as the upper-layer key of the keys K000 and K001.Likewise, the K0 is regarded as the upper-layer key of the keys K00 and K01, and the key KR is regarded as the upper-layer key of the keys K0 and K1.The keys for utilizing the content are formed using the keys corresponding to the nodes of a path from a device node (leaf) at the bottom layer to the root node at the top layer.", "For example, the keys for utilizing the content of number 3 are formed using the keys of the path from the leaf to the root including the keys K0011, K0001, K00, K0, and KR.", "In the system of the present invention, as shown in, for example, FIG.", "15, the hierarchical tree structure including keys corresponding to 8×24×32 stages of nodes are used.", "In this key system, categories correspond to the keys corresponding to nodes down to the lower eight stages from the root node.", "The category here indicates the category, such as the category of devices using a semiconductor memory, for example the memory stick, and the category of devices receiving a digital broadcast.", "In the example in FIG.", "15, a system of the present invention is applied to one node among the nodes from the root node down to the ones at the eighth stage.", "The licenses correspond to the keys that correspond to another twenty-four stages of lower layer nodes.", "This enables approximately 16 mega (=224=approximately one million and six hundred thousand) of licenses to be defined.", "In addition, the lowest thirty-two stage layers enable approximately four Giga (=232=approximately four billion) users to be defined.", "Keys corresponding to the nodes of the lowest thirty-two stages constitute DNK.", "Each piece of the content corresponds to one of the paths constituted by nodes of sixty-four (=8+24+32) stages.", "In other words, encrypting each piece of the content uses the keys corresponding to the nodes constituting the assigned path.", "The key at an upper layer is encrypted using the key at a lower proximate layer and are disposed in the EKB in FIG.", "5.Not being disposed in the EKB, the DNK at the bottom stage is disposed in the service data obtained at registration of the client to the license server and is provided to the user's client 1 as shown in FIG.", "16.The client 1 uses the DNK defined in the service data to decrypt the key at an upper proximate layer defined in the EKB distributed along with the content data.", "The client 1 uses the key obtained through the description to decrypt a key at a further upper layer included in the EKB.", "By sequentially performing the above-described process, the client 1 can obtain every key belonging to the path of the content.", "The client 1 uses the KR obtained after the above EKB decryption processing to decrypt the encrypted content key KR(KC) and the content key KC can be obtained.", "Alternatively, the keys in the present invention can be formed using keys other than the key system employing the Broadcast Encryption as shown in FIGS.", "14 and 15.In place of so-called a personal computer, the client to which the present invention is applied can be a PDA (Personal Digital Assistants), a cellular phone, a game terminal, or the like.", "When a series of processes is performed using software, the programs that constitute the software are installed from a network or storage media to a computer incorporated in dedicated hardware, a universal personal computer that can execute various features by installing various programs thereon, etc.", "These storage media, which are distributed, as shown in FIG.", "2, for providing the programs to the user, are formed using not only package medium involving the magnetic disk 41 (including a floppy disk), the optical disk 42 (including a CD-ROM (Compact Disk-Read Only Memory) and a DVD (Digital Versatile Disk)), a magneto-optic disk 43 (including an MD (Mini-Disk)), and a semiconductor memory 44, which are independent of the device itself and store the programs, but also the ROM 22 and the hard disk included in the storage unit 28, which are incorporated in the device beforehand to be provided to the user and store the programs.", "In the present specification, steps defining the programs stored in the storage media, steps which are sequentially performed in accordance with the described order, may not be necessarily performed sequentially.", "The steps may include a process executed parallel or separately.", "The system in the present specification represents the entirety of an apparatus including a plurality of the devices.", "INDUSTRIAL APPLICABILITY As described above, an information processing apparatus, a method, a program storage medium, and a program according to the present invention format encrypted data and attribute information on content in accordance with a predetermined format to be output and causes a license to include an attribute condition.", "When the attribute information of the content meets the attribute condition of the license, since the encrypted data can be decrypted, the data is prevented from being used in an authorized manner while the license can be flexibly issued." ] ]	Patent_10467603
[ [ "Gonococcal proteins and nucleic acids", "The invention provides proteins from gonococcus (Neisseria gonorrhoeae), including amino acid sequences, the corresponding nucleotide sequences, expression data, and serological data.", "The proteins are useful antigens for vaccines, immunogenic compositions, and/or diagnostics.", "They are also useful for distinguishing between gonococcus and meningococcus and, in particular, between gonococcus and serogroup B meningococcus." ], [ "1.A protein comprising an amino acid sequence selected from the group consisting of SEQ IDs 26, 72, 230, 984, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314 and 316: 2.A protein having 50% or greater sequence identity to a protein according to claim 1.3.A protein comprising a fragment of an amino acid sequence selected from the group consisting of 26, 72, 230, 984, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206,208, 210, 212,214, 216, 218,220,222, 224, 226, 228, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314 and 316.4.An antibody which specifically binds to a protein according to any one of claims 1 to 3.5.A nucleic acid molecule which encodes a protein according to any one of claims 1 to 3.6.A nucleic acid molecule according to claim 5, comprising a nucleotide sequence selected from the group consisting of SEQ IDs 25, 71, 229, 983, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313 and 315.7.A nucleic acid molecule comprising a fragment of a nucleotide sequence selected from the group consisting of SEQ IDs 25, 71, 229, 983, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313 and 315.8.A nucleic acid molecule comprising a nucleotide sequence complementary to a nucleic acid molecule according to any one of claims 5 to 7.9.A nucleic acid molecule comprising a nucleotide sequences having 50% or greater sequence identity to a nucleic acid molecule according to any one of claims 5 to 8.10.A nucleic acid molecule which can hybridise to a nucleic acid molecule according to any one of claims 5 to 9 under high stringency conditions.", "11.A composition comprising a protein, a nucleic acid molecule, or an antibody according to any preceding claim.", "12.A composition according to claim 11 being a vaccine composition or a diagnostic composition.", "13.A composition according to claim 11 or claim 12 for use as a pharmaceutical.", "14.The use of a composition according to claim 13 in the manufacture of a medicament for the treatment or prevention of infection due to streptococcus bacteria, particularly N.gonorrhoeae.", "15.A process for distinguishing N.gonorrhoeae from N.meningitidis, comprising the steps of: (a) contacting a protein, a nucleic acid molecule, or an antibody according to any one of claims 1 to 10 with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes." ], [ "<SOH> BACKGROUND ART <EOH>Neisseria gonorrhoeae is a bacterial pathogen.", "There is currently no effective vaccine against N.gonorrhoeae infection.", "It is an object of the invention to provide proteins and nucleic acid useful in vaccine study and/or manufacture.", "N.gonorrhoeae is related to N.meningitidis.", "Sequence data are now available for serogroup B of meningococcus [e.g.", "WO99/24578; WO99/36544; WO99/57280; WO00/22430; WO00/66791; Tettelin et al.", "(2000) Science 287:1809-1815] and also for serogroup A [Parkhill et al.", "(2000) Nature 404:502-506].", "It is a further object of the invention to provide proteins and nucleic acid useful in distinguishing between gonococcus and meningococcus and, in particular, between gonococcus and serogroup B meningococcus." ], [ "<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>There are no drawings.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD This invention relates to proteins from the bacterium Neisseria gonorrhoeae, and more particularly to such proteins which do not have corresponding homologs or orthologs in serogroup B N.meningitidis.", "BACKGROUND ART Neisseria gonorrhoeae is a bacterial pathogen.", "There is currently no effective vaccine against N.gonorrhoeae infection.", "It is an object of the invention to provide proteins and nucleic acid useful in vaccine study and/or manufacture.", "N.gonorrhoeae is related to N.meningitidis.", "Sequence data are now available for serogroup B of meningococcus [e.g.", "WO99/24578; WO99/36544; WO99/57280; WO00/22430; WO00/66791; Tettelin et al.", "(2000) Science 287:1809-1815] and also for serogroup A [Parkhill et al.", "(2000) Nature 404:502-506].", "It is a further object of the invention to provide proteins and nucleic acid useful in distinguishing between gonococcus and meningococcus and, in particular, between gonococcus and serogroup B meningococcus.", "DISCLOSURE OF THE INVENTION The invention provides proteins comprising the N.gonorrhoeae amino acid sequences disclosed in the examples (the even-numbered SEQ IDS 2 to 8622).", "159 of these have no homolog in serogroup B meningococcus and these have been given a name in the form “NGSn”.", "It also provides proteins comprising amino acid sequences having sequence identity to the N.gonorrhoeae amino acid sequences disclosed in the examples.", "Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (e.g.", "60%, 70%, 80%, 90%, 95%, 99% or more).", "These proteins include homologs, orthologs, allelic variants and functional mutants.", "Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence.", "Identity between proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.The invention further provides proteins comprising fragmnents of the N.gonorrhoeae amino acid sequences disclosed in the examples.", "The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (e.g.", "8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more).", "Preferably the fragments comprise one or more epitopes from the sequence.", "Other preferred fragments are (a) the N-terminal signal peptides of the proteins disclosed in the examples, and (b) the proteins disclosed in the examples, but without their N-terminal signal peptides.", "The proteins of the invention can, of course, be prepared by various means (e.g.", "recombinant expression, purification from Neisseria, chemical synthesis etc.)", "and in various forms (e.g.", "native, fusions etc.).", "They are preferably prepared in substantially pure form (i.e.", "substantially free from other N.gonorrhoeae or host cell proteins).", "The proteins of the invention are preferably Neisserial proteins, more preferably N.gonorrhoeae proteins.", "The invention provides antibodies which bind to these proteins.", "These may be polyclonal or monoclonal and may be produced by any suitable means.", "The antibodies may include a detectable label.", "The invention provides nucleic acid comprising the N.gonorrhoeae nucleotide sequences disclosed in the examples.", "In addition, the invention provides nucleic acid comprising nucleotide sequences having sequence identity to the N.gonorrhoeae nucleotide sequences disclosed in the examples.", "Furthermore, the invention provides nucleic acid which can hybridise to the N.gonorrhoeae nucleic acid disclosed in the examples, preferably under “high stringency” conditions (e.g.", "65° C. in a 0.1×SSC, 0.5% SDS solution).", "Nucleic acid comprising fragments of these sequences are also provided.", "These should comprise at least n consecutive nucleotides from the N.gonorrhoeae sequences and, depending on the particular sequence, n is 10 or more (e.g.", "12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).", "The invention also provides nucleic acid encoding the proteins and protein fragments of the invention.", "The invention includes nucleic acid comprising sequences complementary to those described above (e.g.", "for antisense or probing purposes).", "Nucleic acid according to the invention can be prepared in many ways (e.g.", "by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.)", "and can take various forms (e.g.", "single stranded, double stranded, vectors, probes etc.).", "In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.", "The invention provides vectors comprising nucleotide sequences of the invention (e.g.", "cloning or expression vectors) and host cells transformed with such vectors.", "The invention provides compositions comprising protein, antibody, and/or nucleic acid according to the invention.", "These compositions may be suitable as immunogenic compositions, for instance, or as diagnostic reagents, or as vaccines.", "The invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments (e.g.", "as vaccines) or as diagnostic reagents.", "It also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of: (i) a medicament for treating or preventing infection due to Neisseria; (ii) a diagnostic reagent for detecting the presence of Neisseria or of antibodies raised against Neisseria; and/or (iii) a reagent which can raise antibodies against Neisseria.", "Said Neisseria may be any species, but is preferably N.gonorrhoeae.", "The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of nucleic acid, protein, and/or antibody of the invention.", "According to further aspects, the invention provides various processes.", "A process for producing proteins of the invention is provided, comprising the step of culturing a host cell of to the invention under conditions which induce protein expression.", "A process for producing protein or nucleic acid of the invention is provided, wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.", "A process for detecting polynucleotides of the invention is provided, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridising conditions to form duplexes; and (b) detecting said duplexes.", "A process for detecting proteins of the invention is provided, comprising the steps of: (a) contacting an antibody of the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.", "A process for distinguishing N.gonorrhoeae from N.meningitidis is provided, comprising the steps of: (a) contacting an antibody of the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.", "Alternatively, the steps may be (a) contacting nucleic acid of the invention with a biological sample under conditions suitable for the nucleic acid hybridisation; and (b) detecting any such hybridisation.", "Alternatively, the steps may be (a) contacting a protein of the invention with a biological sample (e.g.", "blood or serum) under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.", "A summary of standard techniques and procedures which may be employed in order to perform the invention (e.g.", "to utilise the disclosed sequences for vaccination or diagnostic purposes) follows.", "This summary is not a limitation on the invention, but gives examples that may be used, but are not required.", "General The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.", "Such techniques are explained fully in the literature eg.", "Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989) or Third Edition (2000); DNA Cloning, Volumes I and II (D. N Glover ed.", "1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.", "1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds.", "1984); Animal Cell Culture (R. I. Freshney ed.", "1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds.", "1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds.", "(1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).", "Standard abbreviations for nucleotides and amino acids are used in this specification.", "Definitions A composition containing X is “substantially free of” Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.", "The term “comprising” means “including” as well as “consisting” e.g.", "a composition “comprising” X may consist exclusively of X or may include something additional e.g.", "X+Y.", "The term “heterologous” refers to two biological components that are not found together in nature; The components may be host cells, genes, or regulatory regions, such as promoters.", "Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene.", "Another example is where a Neisseria sequence is heterologous to a mouse host cell.", "A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.", "An “origin of replication” is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector.", "The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control.", "An origin of replication may be needed for a vector to replicate in a particular host cell.", "With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell, Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.", "A “mutant” sequence is defined as DNA, RNA or amino acid sequence differing from but having sequence identity with the native or disclosed sequence.", "Depending on the particular sequence, the degree of sequence identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (eg.", "60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the Smith-Waterman algorithm as described above).", "As used herein, an “allelic variant” of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence.", "A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared.", "An allelic variant can also comprise an alteration in the 5′ or 3′ untranslated regions of the gene, such as in regulatory control regions (eg.", "see U.S. Pat.", "No.", "5,753,235).", "Expression Systems The Neisseria nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, plants, bacteria, and yeast.", "i. Mammalian Systems Mammalian expression systems are known in the art.", "A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg.", "structural gene) into mRNA.", "A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site.", "The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.", "A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box.", "An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al.", "(1989) “Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: A Laboratory Manual, 2nd ed.].", "Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences.", "Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter.", "In addition, sequences derived from non-viral genes, such as the murine metallotheionein gene, also provide useful promoter sequences.", "Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.", "The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels.", "An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or beterologous promoters, with synthesis beginning at the normal RNA start site.", "Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al.", "(1987) Science 236:1237; Alberts et al.", "(1989) Molecular Biology of the Cell, 2nd ed.].", "Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range.", "Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J.", "4:7611 and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al.", "(1982b) Proc.", "Natl.", "Acad.", "Sci.", "79:6777] and from human cytomegalovirus [Boshart et al.", "(1985) Cell 41:521].", "Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet.", "2:215; Maniatis et al.", "(1987) Science 236:1237].", "A DNA molecule may be expressed intracellularly in mammalian cells.", "A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon.", "If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.", "Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cel Whitelaw (1988) “Termination and 3′ end processing of eukaryotic RNA, In Transcription and splicing (ed.", "B. D. Hames and D. M. Glover); Proudfoot (1989) Trends Biochem.", "Sci.", "14:105].", "These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.", "Examples of transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) “Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning: A Laboratory Manual].", "Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs.", "Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired.", "Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg.", "plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria.", "Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate.", "For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen.", "Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus.", "Additionally, the replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification.", "Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al.", "(1989) Mol.", "Cell.", "Biol.", "9:946] and pHEBO [Shimizu et al.", "(1986) Mol.", "Cell.", "Biol.", "6:1074].", "The transformation procedure used depends upon the host to be transformed.", "Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.", "Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg.", "Hep G2), and a number of other cell lines.", "ii.", "Baculovirus Systems The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector.", "Vector construction employs techniques which are known in the art.", "Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.", "After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine.", "The packaged recombinant virus is expressed and recombinant plaques are identified and purified.", "Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit).", "These techniques are generally known to those skilled in the art and fully described in Summers & Smith, Texas Agricultural Experiment Station Bulletin No.1555(1987) (“Summers & Smith”).", "Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector).", "This may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements.", "Intermediate transplacement constructs are often maintained in a replicon, such as an extra-chromosomal element (e.g.", "plasmids) capable of stable maintenance in a host, such as a bacterium.", "The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.", "Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373.Many other vectors, known to those of skill in the art, have also been designed.", "These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamH1 cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989)17:31.The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al.", "(1988) Ann.", "Rev.", "Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.", "Baculovirus transfer vectors usually contain a baculovirus promoter.", "A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5′to 3′) transcription of a coding sequence (eg.", "structural gene) into mRNA.", "A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence.", "This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site.", "A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene.", "Expression may be either regulated or constitutive.", "Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences.", "Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) “The Regulation of Baculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses (ed.", "Walter Doerfler); EPO Publ.", "Nos.", "127 839 and 155 476; and the gene encoding the p10 protein, Vlak et al., (1988), J. Gen. Virol.", "69:765.DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al.", "(1988) Gene, 73:409).", "Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human α-interferon, Maeda et al., (1985), Nature 315:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec.", "Cell.", "Biol.", "8:3129; human IL-2, Smith et al., (1985) Proc.", "Nat'l Acad.", "Sci.", "USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al.", "(1988) DNA, 7:99, can also be used to provide for secretion in insects.", "A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted.", "Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal.", "If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.", "Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects.", "The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.", "After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus—usually by co-transfection.", "The promoter and transcription termination sequence of the construct will usually comprise a 2-5 kb section of the baculovirus genome.", "Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art.", "(See Summers & Smith supra; Ju et al.", "(1987); Smith et al., Mol.", "Cell.", "Biol.", "(1983) 3:2156; and Luckow and Summers (1989)).", "For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene.", "Miller et al., (1989), Bioessays 4:91.The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5′ and 3′ by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.", "The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus.", "Homologous recombination occurs at low frequency (between about 1% and about 5%); thus, the majority of the virus produced after cotransfection is still wild-type virus.", "Therefore, a method is necessary to identify recombinant viruses.", "An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished.", "The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection.", "Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles.", "These occlusion bodies, up to 15 μm in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope.", "Cells infected with recombinant viruses lack occlusion bodies.", "To distinguish recombinant virus from wild-type virus, the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art.", "Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies.", "“Current Protocols in Microbiology” Vol.", "2 (Ausubel et al.", "eds) at 16.8 (Supp.", "10, 1990); Summers & Smith, supra; Miller et al.", "(1989).", "Recombinant baculovirus expression vectors have been developed for infection into several insect cells.", "For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti , Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985) J. Virol.", "56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol.", "Cell.", "Biol.", "3:2156; and see generally, Fraser, et al.", "(1989) In Vitro Cell.", "Dev.", "Biol.", "25:225).", "Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art.", "See, eg.", "Summers & Smith supra.", "The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host.", "Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced.", "Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients.", "The product may be purified by such techniques as chromatography, eg.", "HPLC, affinity chromatography, ion exchange chromatography, etc.", "; electrophoresis; density gradient centrifugation; solvent extraction, etc.", "As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also present in the medium, so as to provide a product which is at least substantially free of host debris, eg.", "proteins, lipids and polysaccharides.", "In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence.", "These conditions will vary, dependent upon the host cell selected.", "However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.", "iii.", "Plant Systems There are many plant cell culture and whole plant genetic expression systems known in the art.", "Exemplary plant cellular genetic expression systems include those described in patents, such as: U.S. Pat.", "Nos.", "5,693,506; 5,659,122; and 5,608,143.Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30:3861-3863 (1991).", "Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al., Mol.", "Gen. Genet.", "209:3340 (1987); Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J. Biol.", "Chem.", "260:3731-3738 (1985); Rothstein et al., Gene 55:353-356 (1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992).", "A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R. L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology,.", "Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp.", "21-52.References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038(1990); Maas et al., EMBO J.", "9:3447-3452 (1990); Benkel and Hickey, Proc.", "Natl.", "Acad.", "Sci.", "84:1337-1339 (1987).", "Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants.", "The expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host.", "The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host.", "The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes.", "Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed.", "A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol.", "Biol.", "Reptr, 11(2):165-185.Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended.", "These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a beterologous expression cassette into a plant genome.", "Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline.", "Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.", "The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest.", "Usually, there will be only one expression cassette, although two or more are feasible.", "The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5′ untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence.", "Unique restriction enzyme sites at the 5′ and 3′ ends of the cassette allow for easy insertion into a pre-existing vector.", "A heterologous coding sequence may be for any protein relating to the present invention.", "The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane.", "Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest.", "In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested.", "Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed.", "While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.", "Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery.", "If so, site-directed mutagenesis of the “intron” region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41:95-105, 1985.The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA.", "Crossway, Mol.", "Gen. Genet, 202:179-185, 1985.The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982.Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327,70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley.", "Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 79,1859-1863, 1982.The vector may also be introduced into the plant cells by electroporation.", "(Fromm et al., Proc.", "Natl Acad.", "Sci.", "USA 82:5824, 1985).", "In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct.", "Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids.", "Electroporated plant protoplasts reform the cell wall, divide, and form plant callus, All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene.", "It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables.", "Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobryclis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucum is, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.", "Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided.", "Callus tissue is formed and shoots may be induced from callus and subsequently rooted.", "Alternatively, embryo formation can be induced from the protoplast suspension.", "These embryos germinate as natural embryos to form plants.", "The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins.", "It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa.", "Shoots and roots normally develop simultaneously.", "Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture.", "If these three variables are controlled, then regeneration is fully reproducible and repeatable.", "In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant.", "Where the desired protein of the invention is secreted into the medium, it may be collected.", "Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues.", "The mixture may be suspended in a buffer solution to retrieve soluble proteins.", "Conventional protein isolation and purification methods will be then used to purify the recombinant protein.", "Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein.", "iv.", "Bacterial Systems Bacterial expression techniques are known in the art.", "A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg.", "structural gene) into mRNA.", "A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence.", "This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site.", "A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins.", "The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene.", "Constitutive expression may occur in the absence of negative regulatory elements, such as the operator.", "In addition, positive regulation may be achieved bya gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence.", "An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al.", "(1984) Annu.", "Rev.", "Genet.", "18:173].", "Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.", "Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences.", "Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al.", "(1977) Nature 198:1056], and maltose.", "Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al.", "(1980) Nuc.", "Acids Res.", "8:4057; Yelverton et al.", "(1981) Nucl.", "Acids Res.", "9:731; U.S. Pat.", "No.", "4,738,921; EP-A-0036776 and EP-A-0121775].", "The g-laotamase (bla) promoter system [Weissmann (1981) “The cloning of interferon and other mistakes.” In Interferon 3 (ed.", "1.Gresser)], bacteriophage lambda P L [Shimatake et al.", "(1981) Nature 292:128] and T5 [U.S. Pat.", "No.", "4,689,406] promoter systems also provide useful promoter sequences.", "In addition, synthetic promoters which do not occur in nature also function as bacterial promoters.", "For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat.", "No.", "4,551,433].", "For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al.", "(1983) Gene 25:167; de Boer et al.", "(1983) Proc.", "Natl.", "Acad.", "Sci.", "80:21].", "Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.", "A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes.", "The bacteriophage T7 RNA polymeraselpromoter system is an example of a coupled promoter system [Studier et al.", "(1986) J. Mol.", "Biol.", "189:113; Tabor et al.", "(1985) Proc Natl.", "Acad.", "Sci.", "82:1074].", "In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).", "In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes.", "In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al.", "(1975) Nature 254:34].", "The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ and of E. coli 16S rRNA [Steitz et al.", "(1979) “Genetic signals and nucleotide sequences in messenger RNA.” In Biological Regulation and Development: Gene Expression (ed.", "R. F. Goldberger)].", "To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al.", "(1989) “Expression of cloned genes in Escherichia coli.” In Molecular Cloning: A Laboratory Manual].", "A DNA molecule may be expressed intracellularly.", "A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon.", "If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO-A-O 219 237).", "Fusion proteins provide an alternative to direct expression.", "Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences.", "Upon expression, this construct will provide a fusion of the two amino acid sequences.", "For example, the bacteriophage lambda cell gene can be linked at the 5′ terminus of a foreign gene and expressed in bacteria.", "The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al.", "(1984) Nature 309:810].", "Fusion proteins can also be made with sequences from the lacZ [Jia et al.", "(1987) Gene 60:197], trpE [Allen et al.", "(1987) J. Biotechnol.", "5:93; Makoff et al.", "(1989) J. Gen. Microbiol.", "135:11], and Chey [EP-A-0 324 647] genes.", "The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site.", "Another example is a ubiquitin fusion protein.", "Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg.", "ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein.", "Through this method, native foreign protein can be isolated [Miller et al.", "(1989) Bio/Technology 7:698].", "Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [U.S. Pat.", "No.", "4,336,336].", "The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.", "The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).", "Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.", "DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) [Masui et al.", "(1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al.", "(1984) EMBO J.", "3:2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al.", "(1985) Proc.", "Natl.", "Acad.", "Sci.", "82:7212].", "As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis [Palva et al.", "(1982) Proc.", "Natl.", "Acad.", "Sci.", "USA 79:5582; EP-A-0 244 042].", "Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence.", "These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.", "Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription.", "Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.", "Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs.", "Expression constructs are often maintained in a replicon, such as an extracbromosomal element (eg.", "plasmids) capable of stable maintenance in a host, such as bacteria.", "The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification.", "In addition, a replicon may be either a high or low copy number plasmid.", "A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150.A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids.", "Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.", "Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector.", "Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate.", "Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome.", "For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0 127 328).", "Integrating vectors may also be comprised of bacteriophage or transposon sequences.", "Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed.", "Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al.", "(1978) Annu.", "Rev.", "Microbiol.", "32:469].", "Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.", "Alternatively, some of the above described components can be put together in transformation vectors.", "Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.", "Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria.", "For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al.", "(1982) Proc.", "Natl.", "Acad.", "Sci.", "USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al.", "(1981) Nature 292:128; Amann et al.", "(1985) Gene 40:183; Studier et al.", "(1986) J. Mol.", "Biol.", "189:113; EP-A-0 036 776,EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al.", "(1988) Appl.", "Environ.", "Microbiol.", "54:655]; Streptococcus lividans [Powell et al.", "(1988) Appl.", "Environ.", "Microbiol.", "54:655], Streptomyces lividans [U.S. Pat.", "No.", "4,745,056].", "Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl2 or other agents, such as divalent cations and DMSO.", "DNA can also be introduced into bacterial cells by electroporation.", "Transformation procedures usually vary with the bacterial species to be transformed.", "See eg.", "[Masson et al.", "(1989) FEMS Microbiol.", "Lett.", "60:273; Palva et al.", "(1982) Proc.", "Natl.", "Acad.", "Sci.", "USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al.", "(1988) Proc.", "Natl.", "Acad.", "Sci.", "85:856; Wang et al.", "(1990) J. Bacteriol.", "172:949, Campylobacter], [Cohen et al.", "(1973) Proc.", "Natl.", "Acad.", "Sci.", "69:2110; Dower et al.", "(1988) Nucleic Acids Res.", "16:6127; Kushner (1978) “An improved method for transformation of Escherichia coli with ColE1-derived plasmids.", "In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds.", "H. W. Boyer and S. Nicosia); Mandel et al.", "(1970) J. Mol.", "Biol.", "53:159; Taketo (1988) Biochim.", "Biophys.", "Acta 949:318; Escherichia], [Chassy et al.", "(1987) FEMS Microbiol.", "Lett.", "44:173 Lactobacillus]; [Fiedler et al.", "(1988) Anal.", "Biochem 170:38, Pseudomonas]; [Augustin et al.", "(1990) FEMS Microbiol.", "Lett.", "66:203, Staphylococcus], [Barany et al.", "(1980) J. Bacterial.", "144:698; Harlander (1987) “Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed.", "J. Ferretti and R. Curtiss III); Perry et al.", "(1981) Infect.", "Immun.", "32:1295; Powell et al.", "(1988) Appl.", "Environ.", "Microbial.", "54:655; Somkuti et al.", "(1987) Proc.", "4th Evr.", "Cong.", "Biotechnology 1:412, Streptococcus].", "v. Yeast Expression Yeast expression systems are also known to one of ordinary skill in the art.", "A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg.", "structural gene) into mRNA.", "A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence.", "This transcription initiation region usually includes an RNA polymerase binding site (the “TATA Box”) and a transcription initiation site.", "A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene.", "The UAS permits regulated (inducible) expression.", "Constitutive expression occurs in the absence of a UAS.", "Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.", "Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences.", "Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203).", "The yeast PH05 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al.", "(1983) Proc.", "Natl.", "Acad.", "Sci.", "USA 80:1].", "In addition, synthetic promoters which do not occur in nature also function as yeast promoters.", "For example, UAS sequences of one yeast promoter maybe joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter.", "Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat.", "Nos.", "4,876,197 and 4,880,734).", "Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556).", "Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription.", "Examples of such promoters include, inter alia, [Cohen et al.", "(1980) Proc.", "Natl.", "Acad.", "Sci.", "USA 77:1078; Henikoff et al.", "(1981) Nature 283:835; Hollenberg et al.", "(1981) Curr.", "Topics Microbiol.", "Immunol.", "96:119; Hollenberg et al.", "(1979) “The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids of Medical, Environmental and Commercial Importance (eds.", "K. N. Timmis and A. Publer); Mercerau-Puigalon et al.", "(1980) Gene 11:163; Panthier et al.", "(1980) Curr.", "Genet.", "2:109;].", "A DNA molecule may be expressed intracellularly in yeast.", "A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon.", "If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.", "Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems.", "Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences.", "Upon expression, this construct will provide a fusion of the two amino acid sequences.", "For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5′ terminus of a foreign gene and expressed in yeast.", "The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site.", "See eg.", "EP-A-0 196 056.Another example is a ubiquitin fusion protein.", "Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg.", "ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein.", "Through this method, therefore, native foreign protein can be isolated (eg.", "WO88/024066).", "Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein.", "Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro.", "The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.", "DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP-A-0 012 873; JPO.", "62,096,086) and the A-factor gene (U.S. Pat.", "No.", "4,588,684).", "Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EP-A-0 060 057).", "A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a “pre” signal sequence, and a “pro” region.", "The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U.S. Pat.", "Nos.", "4,546,083 and 4,870,008; EP-A-0 324 274).", "Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor.", "(eg.", "see WO 89/02463.)", "Usually, transcription termination sequences recognized by yeast are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence.", "These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.", "Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.", "Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs.", "Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg.", "plasmids) capable of stable maintenance in a host, such as yeast or bacteria.", "The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification.", "Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al.", "(1979) Gene 8:17-24], pCl/1 [Brake et al.", "(1984) Proc.", "Natl.", "Acad.", "Sci USA 81:4642-4646], and YRp17 (Stinchcomb et al.", "(1982) J. Mol.", "Biol.", "158:157].", "In addition, a replicon may be either a high or low copy number plasmid.", "A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150.A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20.Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.", "See eg.", "Brake et al., supra.", "Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector.", "Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct.", "Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al.", "(1983) Methods in Enzymol.", "101:228-2451.An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector.", "See Orr-Weaver et al., supra.", "One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al.", "(1983) Proc.", "Natl.", "Acad.", "Sci.", "USA 80:6750].", "The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.", "Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed.", "Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively.", "In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal.", "For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al.", "(1987) Microbiol, Rev.", "51:351].", "Alternatively, some of the above described components can be put together into transformation vectors.", "Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.", "Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts.", "For example, expression vectors have been developed for, inter alia, the following yeasts:Candida albicans [Kurtz, et at.", "(1986) Mol.", "Cell.", "Biol.", "6:142], Candida maltosa [Kunze, et at.", "(1985) J.", "Basic Microbiol, 25:141].", "Hansenula polymorpha [Gleeson, et al.", "(1986) J. Gen. Microbiol.", "132:3459; Roggenkamp et al.", "(1986) Mol.", "Gen. Genet.", "202:302], Kluyveromyces fragilis [Das, et al.", "(1984) J. Bacteriol.", "158:1165], Kluyveromyces lactis [De Louvencourt et al.", "(1983) J. Bacteriol.", "154:737; Van den Berg et al.", "(1990) Bio/Technology 8:135], Pichia guillerimondii [Kunze et al.", "(1985) J.", "Basic Microbiol.", "25:141], Pichia pastoris [Cregg, et al.", "(1985) Mol.", "Cell.", "Biol.", "5:3376; U.S. Pat.", "Nos.", "4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al.", "(1978) Proc.", "Natl.", "Acad.", "Sci.", "USA 75:1929; Ito et al.", "(1983) J. Bacteriol.", "153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], and Yarrowia lipolytica [Davidow, et al.", "(1985) Curr.", "Genet.", "10:380471 Gaillardin, et al.", "(1985) Curr.", "Genet.", "10:49].", "Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations.", "Transformation procedures usually vary with the yeast species to be transformed.", "See eg.", "[Kurtz et al.", "(1986) Mol.", "Cell.", "Biol.", "6:142; Kunze et al.", "(1985) J.", "Basic Microbiol.", "25:141; Candida]; [Gleeson et al.", "(1986) J. Gen. Microbiol.", "132:3459; Roggenkamp et al.", "(1986) Mol.", "Gen. Genet.", "202:302; Hansenula]; [Das et al.", "(1984) J. Bacterial.", "158:1165; De Louvencourt et al.", "(1983) J. Bacteriol.", "154:1165; Van den Berg et al.", "(1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et at.", "(1985) Mol.", "Cell.", "Biol.", "5:3376; Kunze et al.", "(1985) J.", "Basic Microbiol.", "25:141; U.S. Pat.", "Nos.", "4,837,148 and 4,929,555; Pichia]; [Hinnen et al.", "(1978) Proc.", "Natl.", "Acad.", "Sci.", "USA 75;1929; Ito et al.", "(1983) J. Bacteriol.", "153:163 Saccharomyces]; [Beach and Nurse (1981) Nature 300:706; Schizosaccbaromyces]; [Davidow et al.", "(1985) Curr.", "Genet.", "10:39; Gaillardin et al.", "(1985) Curr.", "Genet.", "10:49; Yarrowia].", "Antibodies As used herein, the term “antibody” refers to a polypeptide or group of polypeptides composed of at least one antibody combining site.", "An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen.", "“Antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.", "Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishing/identifying Neisseria proteins.", "Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods.", "In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat.", "Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies.", "Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly).", "A dose of 50-200 μg/injection is typically sufficient.", "Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant.", "One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization.", "Polyclonal antisera is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25° C. for one hour, followed by incubating at 4° C. for 2-18 hours.", "The serum is recovered by centrifugation (eg.", "1,000g for 10 minutes).", "About 20-50 ml per bleed may be obtained from rabbits.", "Monoclonal antibodies are prepared using the standard method of Kohler & Milstein [Nature (1975) 256:495-96], or a modification thereof.", "Typically, a mouse or rat is immunized as described above.", "However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells.", "If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen.", "B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension.", "Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (eg.", "hypoxanthine, aminopterin, thymidine medium, “HAT”).", "The resulting hybridomas are plated by limiting dilution, and are assayed for production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens).", "The selected MAb-secreting hybridomas are then cultured either in vitro (eg.", "in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).", "If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques.", "Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32P and 125I), electron-dense reagents, enzymes, and ligands having specific binding partners.", "Enzymes are typically detected by their activity.", "For example, horseradish peroxidase is usually detected by its ability to convert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer.", "“Specific binding partner” refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor.", "Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art.", "It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes.", "For example, 125I may serve as a radioactive label or as an electron-dense reagent.", "HRP may serve as enzyme or as antigen for a MAb.", "Further, one may combine various labels for desired effect.", "For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with 125I, or with an anti-biotin MAb labeled with HRP.", "Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.", "Pharmaceutical Compositions Pharmaceutical compositions can comprise either polypeptides, antibodies, or nucleic acid of the invention.", "The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.", "The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect.", "The effect can be detected by, for example, chemical markers or antigen levels.", "Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature.", "The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration.", "Thus, it is not useful to specify an exact effective amount in advance.", "However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.", "For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.", "A pharmaceutical composition can also contain a pharmaceutically acceptable carrier.", "The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents.", "The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.", "Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.", "Such carriers are well known to those of ordinary skill in the art.", "Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.", "A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub.", "Co., N.J. 1991).", "Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol.", "Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.", "Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.", "Liposomes are included within the definition of a pharmaceutically acceptable carrier.", "Delivery Methods Once formulated, the compositions of the invention can be administered directly to the subject.", "The subjects to be treated can be animals; in particular, human subjects can be treated.", "Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue.", "The compositions can also be administered into a lesion.", "Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg.", "see WO98/20734), needles, and gene guns or hyposprays.", "Dosage treatment may be a single dose schedule or a multiple dose schedule.", "Vaccines Vaccines according to the invention may either be prophylactic (ie.", "to prevent infection) or therapeutic (ie.", "to treat disease after infection).", "Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with “pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.", "Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.", "Such carriers are well known to those of ordinary skill in the art.", "Additionally, these carriers may function as immunostimulating agents (“adjuvants”).", "Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc.", "pathogens.", "Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds.", "Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.", "), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.)", "containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.)", "may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (eg.", "IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.", "), interferons (eg.", "gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.", "Alum and MF59™ are preferred.", "As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuram yl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.", "The immunogenic compositions (eg.", "the immunising antigen/immunogen/polypeptide/protein/nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc.", "Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.", "Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.", "The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.", "Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed.", "By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention.", "This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg.", "nonhuman primate, primate, etc.", "), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors.", "It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.", "The immunogenic compositions are conventionally administered parenterally, eg.", "by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg.", "WO98/20734).", "Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications.", "Dosage treatment may be a single dose schedule or a multiple dose schedule.", "The vaccine may be administered in conjunction with other immunoregulatory agents.", "As an alternative to protein-based vaccines, DNA vaccination may be used [eg.", "Robinson & Torres (1997) Seminars in Immunol 9:271-283; Donnelly et al.", "(1997) Annu Rev Immunol 15:617-648; later herein].", "Gene Delivery Vehicles Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically.", "These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality.", "Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters.", "Expression of the coding sequence in vivo can be either constitutive or regulated.", "The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences.", "The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector.", "The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus viral vector.", "See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol.", "53:160) polytropic retroviruses eg.", "MCF and MCF-MLV (see Kelly (1983) J. Virol.", "45:291), spumaviruses and lentiviruses.", "See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.Portions of the retroviral gene therapy vector may be derived from different retroviruses.", "For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a TRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.", "These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S. Pat.", "No.", "5,591,624).", "Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see WO96/37626).", "It is preferable that the recombinant viral vector is a replication defective recombinant virus.", "Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO95/30763 and WO92/05266), and can be used to create producer cell lines (also termed vector cell lines or “VCLs”) for the production of recombinant vector particles.", "Preferably, the packaging cell lines are made from human parent cells (eg.", "HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.", "Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus.", "Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No.", "VR-999), Friend (ATCC No.", "VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No.", "VR-998) and Moloney Murine Leukemia Virus (ATCC No.", "VR-190).", "Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection (“ATCC”) in Rockville, Md.", "or isolated from known sources using commonly available techniques.", "Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat.", "Nos.", "5,219,740, 4,405,712, 4,861,719, 4,980,289, 4,777,127, 5,591,624.See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.Human adenoviral gene therapy vectors are also known in the art and employable in this invention, See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and WO93/07283, WO93/06223, and WO93/07282.Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in WO94/12649, WO93/03769, WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654.Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum.", "Gene Ther.", "3:147-154 may be employed.", "The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors.", "Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, WO93/09239.Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides.", "The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie.", "there is one sequence at each end) which are not involved in HP formation.", "The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position.", "Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262.Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol.", "61:3096).", "Another exemplary AAV vector is the Double-D ITR vector.", "Construction of the Double-D ITR vector is disclosed in U.S. Pat.", "No.", "5,478,745.Still other vectors are those disclosed in Carter U.S. Pat.", "No.", "4,797,368 and Muzyczka U.S. Pat.", "No.", "5,139,941, Chartejee U.S. Pat.", "No.", "5,474,935, and Kotin WO94/288157.Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver.", "Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470.Additional AAV gene therapy vectors are described in U.S. Pat.", "Nos.", "5,354,678, 5,173,414, 5,139,941, and 5,252,479.The gene therapy vectors of the invention also include herpes vectors.", "Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Pat.", "No.", "5,288,641 and EP0176170 (Roizman).", "Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in WO95/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in WO90/09441 and WO92/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC with accession numbers VR-977 and VR-260.Also contemplated are alpha virus gene therapy vectors that can be employed in this invention.", "Preferred alpha virus vectors are Sindbis viruses vectors.", "Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat.", "Nos.", "5,091,309, 5,217,879, and WO92/10578.More particularly, those alpha virus vectors described in U.S. Ser.", "No.", "08/405,627, filed Mar.", "15, 1995, WO94/21792, WO92/10578, WO95/07994, U.S. Pat.", "Nos.", "5,091,309 and 5,217,879 are employable.", "Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Md.", "or isolated from known sources using commonly available techniques.", "Preferably, alphavirus vectors with reduced cytotoxicity are used (see U.S. Ser.", "No.", "08/679640).", "DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention.", "See WO95/07994 for a detailed description of eukaryotic layered expression systems.", "Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.", "Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol.", "Standardization 1:115; rhinovirus, for example ATCC VR-11 10 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; in U.S. Pat.", "Nos.", "4,603,112 and 4,769,330 and WO89/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in U.S. Pat.", "No.", "5,166,057 and in Enami (1990) Proc Natl Acad Sci 87:3802-3805; Enami & Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NEI Med 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol.", "66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med 121:190.Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors.", "Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see U.S. Ser.", "No.", "08/366,787, filed Dec. 30, 1994 and Curiel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see U.S. Ser.", "No.", "08/240,030, filed May 9, 1994, and U.S. Ser.", "No.", "08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Pat.", "No.", "5,149,655, ionizing radiation as described in U.S. Pat.", "No.", "5,206,152 and in WO92/11033, nucleic charge neutralization or fusion with cell membranes.", "Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl Acad Sci 91:1581-1585.Particle mediated gene transfer may be employed, for example see U.S. Ser.", "No.", "60/023,867.Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu & Wu (1987) J. Biol.", "Chem.", "262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.", "Naked DNA may also be employed.", "Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat.", "No.", "5,580,859.Uptake efficiency may be improved using biodegradable latex beads.", "DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads.", "The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.", "Liposomes that can act as gene delivery vehicles are described in U.S. Pat.", "No.", "5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968.As described in U.S. Ser.", "No.", "60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin.", "Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters.", "Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc.", "Natl.", "Acad.", "Sci.", "USA 91(24):11581-11585.Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials.", "Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Pat.", "No.", "5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Pat.", "No.", "5,206,152 and WO92/11033.Exemplary liposome and polycationic gene delivery vehicles are those described in U.S. Pat.", "Nos.", "5,422,120 and 4,762,915; in WO 95/13796; WO94/23697; and WO91/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H.", "Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.A polynucleotide composition can comprises therapeutically effective amount of a gene therapy vehicle, as the term is defined above.", "For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.", "Delivery Methods Once formulated, the polynucleotide compositions of the invention can be administered (1) directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) in vitro for expression of recombinant proteins.", "The subjects to be treated can be mammals or birds.", "Also, human subjects can be treated.", "Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue.", "The compositions can also be administered into a lesion.", "Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg.", "see WO98/20734), needles, and gene guns or hyposprays.", "Dosage treatment may be a single dose schedule or a multiple dose schedule.", "Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in eg.", "WO93/14778.Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hem atopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.", "Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.", "Polynucleotide and Polypeptide Pharmaceutical Compositions In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.", "A. Polypeptides One example are polypeptides which include, without limitation: asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin.", "Viral antigens, such as envelope proteins, can also be used.", "Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein of plasmodium falciparum known as RII.", "B. Hormones, Vitamins, etc.", "Other groups that can be included are, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.", "C. Polyalkylenes, Polysaccharides, etc.", "Also, polyalkylene glycol can be included with the desired polynucleotides/polypeptides.", "In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol.", "In addition, mono-, di-, or polysaccharides can be included.", "In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran.", "Also, chitosan and poly(lactide-co-glycolide) D. Lipids, and Liposomes The desired polynucleotidelpolypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom.", "Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid.", "The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid.", "For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim.", "Biophys.", "Acta.", "1097:1-17; Straubinger (1983) Meth.", "Enzymol.", "101:512-527.Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations.", "Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner (1987) Proc.", "Natl.", "Acad.", "Sci.", "USA 84:7413-7416); mRNA (Malone (1989) Proc.", "Natl.", "Acad.", "Sci.", "USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol.", "Chem.", "265:10189-10192), in functional form.", "Cationic liposomes are readily available.", "For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner supra).", "Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger).", "Other cationic liposomes can be prepared from readily available materials using techniques well known in the art.", "See, eg.", "Szoka (1978) Proc.", "Natl.", "Acad.", "Sci.", "USA 75:4194-4198; WO90/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.", "Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials.", "Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others.", "These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios.", "Methods for making liposomes using these materials are well known in the art.", "The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs).", "The various liposome-nucleic acid complexes are prepared using methods known in the art.", "See eg.", "Straubinger (1983) Meth.", "Immunol.", "101:512-527; Szoka (1978) Proc.", "Natl.", "Acad.", "Sci.", "USA 75:4194-4198; Papahadjopoulos (1975) Biochim.", "Biophys.", "Acta 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim.", "Biophys.", "Acta 443:629; Ostro (1977) Biochem.", "Biophys.", "Res.", "Commun.", "76:836; Fraley (1979) Proc.", "Natl.", "Acad.", "Sci.", "USA 76:3348); Enoch & Strittmatter (1979) Proc.", "Natl.", "Acad.", "Sci.", "USA 76:145; Fraley (1980) J. Biol.", "Chem.", "(1980) 255:10431; Szoka & Papahadjopoulos (1978) Proc.", "Natl.", "Acad.", "Sci.", "USA 75:145; and Schaefer-Ridder (1982) Science 215:166.E.", "Lipoproteins In addition, lipoproteins can be included with the polynucleotide/polypeptide to be delivered.", "Examples of lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL, and VLDL.", "Mutants, fragments, or fusions of these proteins can also be used.", "Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL.", "These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors.", "Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.", "Naturally occurring lipoproteins comprise a lipid and a protein portion.", "The protein portion are known as apoproteins.", "At the present, apoproteins A, B, C, D, and E have been isolated and identified.", "At least two of these contain several proteins, designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.", "A lipoprotein can comprise more than one apoprotein.", "For example, naturally occurring chylomicrons comprises of A, B, C & E, over time these lipoproteins lose A and acquire C & E. VLDL comprises A, B, C & E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, & E. The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev.", "Biochem 54:699; Law (1986) Adv.", "Exp Med.", "Biol.", "151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phospholipids, The composition of the lipids varies in naturally occurring lipoproteins.", "For example, chylomicrons comprise mainly triglycerides.", "A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth.", "Enzymol.", "128 (1986).", "The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity.", "The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.", "Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance.", "Such methods are described in Meth.", "Enzymol.", "(supra); Pitas (1980) J. Biochem.", "255:5454-5460 and Mahey (1979) J Clin.", "Invest 64:743-750.Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell.", "See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 30: 443.Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Mass., USA.", "Further description of lipoproteins can be found in Zuckermann et al.", "PCT/US97/14465.F.", "Polycationic Agents Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.", "Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location.", "These agents have both in vitro, ex vivo, and in vivo applications.", "Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.", "The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine.", "Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents.", "Briefly, transcriptional factors such as C/CEBP, cjun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.", "Organic polycationic agents include: spermine, spermidine, and purtrescine.", "The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.", "Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene.", "Lipofectin™, and lipofectAMINE™ are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.", "Immunodiagnostic Assays Neisseria antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Neisseria antibodies can be used to detect antigen levels).", "Immunoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics methods.", "Antibodies to Neisseria proteins within biological samples, including for example, blood or serum samples, can be detected.", "Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art.", "Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays.", "Protocols may also, for example, use solid supports, or may be by immunoprecipitation.", "Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules.", "Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.", "Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the invention, in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc.)", "required for the conduct of the assay, as well as suitable set of assay instructions.", "Nucleic Acid Hybridisation “Hybridization” refers to the association of two nucleic acid sequences to one another by hydrogen bonding.", "Typically, one sequence will be fixed to a solid support and the other will be free in solution.", "Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding.", "Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization.", "See Sambrook et al.", "[supral Volume 2, chapter 9, pages 9.47 to 9.57.“Stringency” refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ.", "For example, the combination of temperature and salt concentration should be chosen that is approximately 120 to 200° C. below the calculated Tm of the hybrid under study.", "The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies.", "See Sambrook et al.", "at page 9.50.Variables to consider when performing, for example, a Southern blot are (1) the complexity of the DNA being blotted and (2) the homology between the probe and the sequences being detected.", "The total amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to lug for a plasmid or phage digest to 10−9 to 10−8 g for a single copy gene in a highly complex eukaryotic genome.", "For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes an be used.", "For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with 1 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 108 cpm/μg.", "For a single-copy mammalian gene a conservative approach would start with 10 μg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 108 cpm/μg, resulting in an exposure time of ˜24 hours.", "Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing.", "In many cases the probe is not 100% homologous to the fragment.", "Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and form amide content of the hybridization buffer.", "The effects of all of these factors can be approximated by a single equation: Tm=81+16.6(log10Ci)+0.4[%(G+C)]0.6(% formamide)−600/n-1.5(% mismatch).", "where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth & Wahl (1984) Anal.", "Biochem.", "138: 267-284).", "In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered.", "The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust.", "As the temperature of the hybridization increases (ie.", "stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases.", "If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase.", "The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner.", "The stringency of the washes is also increased with decreasing salt concentrations.", "In general, convenient hybridization temperatures in the presence of 50% formamide are 42° C. for a probe with is 95% to 100% homologous to the target fragment, 37° C for 90% to 95% homology, and 32° C. for 85% to 90% homology.", "For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above.", "If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent.", "If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed.", "If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.", "Nucleic Acid Probe Assays Methods such as PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determine the presence of cDNA or mRNA.", "A probe is said to “hybridize” with a sequence of the invention if it can form a duplex or double stranded complex, which is stable enough to be detected.", "The nucleic acid probes will hybridize to the Neisseria nucleotide sequences of the invention (including both sense and antisense strands).", "Though many different nucleotide sequences will encode the amino acid sequence, the native Neisseria sequence is preferred because it is the actual sequence present in cells.", "mRNA represents a coding sequence and so a probe should be complementary to the coding sequence; single-stranded cDNA is complementary to mRNA, and so a cDNA probe should be complementary to the non-coding sequence.", "The probe sequence need not be identical to the Neisseria sequence (or its complement)—some variation in the sequence and length can lead to increased assay sensitivity if the nucleic acid probe can form a duplex with target nucleotides, which can be detected.", "Also, the nucleic acid probe can include additional nucleotides to stabilize the formed duplex, Additional Neisseria sequence may also be helpful as a label to detect the formed duplex.", "For example, a non-complementary nucleotide sequence may be attached to the 5′ end of the probe, with the remainder of the probe sequence being complementary to a Neisseria sequence.", "Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a Neisseria sequence in order to hybridize therewith and thereby form a duplex which can be detected.", "The exact length and sequence of the probe will depend on the hybridization conditions (e.g.", "temperature, salt condition etc.).", "For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least 30 nucleotides, although it may be shorter than this.", "Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.", "Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al.", "[J.", "Am.", "Chem.", "Soc.", "(1981) 103:31853, or according to Urdea et al.", "[Proc.", "Natl.", "Acad.", "Sci.", "USA (1983) 80: 7461], or using commercially available automated oligonucleotide synthesizers.", "The chemical nature of the probe can be selected according to preference.", "For certain applications, DNA or RNA are appropriate.", "For other applications, modifications may be incorporated eg.", "backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc.", "[eg.", "see Agrawal & Jyer (1995) Curr Opin Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387]; analogues such as peptide nucleic acids may also be used [eg.", "see Corey (1997) TIBTECH 15:224-229; Buchardt et al.", "(1993) TIBTECH 11:384-386].", "Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acid.", "The assay is described in Mullis et al.", "[Meth.", "Enzymol.", "(1987) 155:335-350] & U.S. Pat.", "Nos.", "4,683,195 & 4,683,202.Two “primer” nucleotides hybridize with the target nucleic acids and are used to prime the reaction.", "The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site.", "Typically, such sequence will flank the desired Neisseria sequence.", "A thermostable polymerase creates copies of target nucleic acids from the primers using the original target nucleic acids as a template.", "After a threshold amount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots.", "When using the Southern blot method, the labelled probe will hybridize to the Neisseria sequence (or its complement).", "Also, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al [supra].", "mRNA, or cDNA generated from mRNA using a polymerase enzyme, can be purified and separated using gel electrophoresis.", "The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose.", "The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe.", "Next, the duplexes containing the labeled probe are detected.", "Typically, the probe is labelled with a radioactive moiety.", "BRIEF DESCRIPTION OF DRAWINGS There are no drawings.", "MODES FOR CARRYING OUT THE INVENTION The following examples describe nucleic acid sequences which have been identified in N.gonorrhoeae, along with their inferred translation products.", "The examples are generally in the following format: a nucleotide sequence which has been identified in N.gonorrhoeae.", "The strain used is FA1090 [Dempsey et al.", "(1991) J. Bacteriol.", "173:5476-5486] the inferred translation product of this sequence a computer analysis (e.g.", "PSORT output) of the translation product, indicating antigenicity homologous sequences (where relevant) results of expression and purification (where relevant) These examples typically include details of sequence homology between species and strains.", "Proteins that are similar in sequence are generally similar in both structure and function, and the homology often indicates a common evolutionary origin.", "Comparison with sequences of proteins of known function is widely used as a guide for the assignment of putative protein function to a new sequence and has proved particularly useful in whole-genome analyses.", "Open reading frames (ORFs) within nucleotide sequences were predicted using the GLIMMER program [Salzberg et al.", "(1998) Nucleic Acids Res 26:544-8).", "All predicted open-reading frames longer than 60 aa were screened against the meningococcus serotype B (‘MenB’) ORFs (accession NC002183) using the BLASTP algorithm [Altschul et al.", "(1990) J. Mol.", "Biol.", "215:403-410].", "ORFs were considered to be gonococcus-specific if they showed sequence identity to a MenB ORF lower than 60% over the whole protein length, or matching the MenB ORF over less than 30% of the length.", "Open reading frames are usually shown with a N-terminal methionine.", "Where this is not the case (e.g.", "SEQ IDs 12, 18, 20, 32, 54, 62, 66, 84, 98, 102, 104, 112, 116, 118, 126, 128, 130, 134, 136, 138, 146, 152, 162, 186, 228, 238, 240, 278, 280, 288, 290, 298, 300, 308, 314), a non-ATG start codon is present, but the N-terminus amino acid will be methionine when translated using this start codon.", "If an upstream start codon is used, however, the “native” amino acid will be translated (e.g.", "if the sequence is expressed with N-terminus fusion sequences).", "Even where the first amino acid is not shown as methionine, the invention encompasses sequences in which the first amino acid is methionine.", "Various tests can be used to assess the in vivo immunogenicity of the proteins identified in the examples.", "For example, the proteins can be expressed recombinantly and used to screen patient sera by immunoblot.", "A positive reaction between the protein and patient serum indicates that the patient has previously mounted an immune response to the protein in question ie.", "the protein is an immunogen.", "This method can also be used to identify immunodominant proteins.", "The recombinant protein can also be conveniently used to prepare antibodies e.g.", "in a mouse.", "These can be used for direct confirmation that a protein is located on the cell-surface.", "Labelled antibody (e.g.", "fluorescent labelling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein.", "For protein expression of 14 antigens, sequences were amplified using the following primers: Restriction Sequences site NGS5 Fwd CGCGGATCCCATATG-TGGGCAGAACAACCGGC NdeI Rev CCCGCTCGAG-GTTTTCAGCAGGGGGATTG XhoI NGS7 Fwd CGCGGATCCCATATG-GCCGGTAAAGAGCAATTTAC NdeI Rev CCCGCTCGAG-AGCCAAGAAGAACCCGTTAT XhoI NGS13 Fwd CGCGGATCCGCTAGCTGCGTTGCCGACCCCG NheI Rev CCCGCTCGAG-CATGTGCCGTGCGGCGT XhoI NGS36 Fwd CGCGGATCCGCTAGC-GACACCCCGAACAATACC NheI Rev CCCGCTCGAG-AAACCTGCCCTTGATGCC XhoI NGS37 Fwd CGCGGATCCCATATG-GTAGAAGTTAAAGGCGGGG NdeI Rev CCCGCTCGAG-TTTTTTCGCGCCGCCGAA XhoI NGS38 Fwd CGCGGATCCCATATG-GCCGACGAACGCCGCC NdeI Rev CCCGCTCGAG-AAACCGATATTTAAAACCCAACAGCC XhoI NGS39 Fwd CGCGGATCCGCTAGCAACCAAGAAGGGATTACCG NheI Rev CCCGCTCGAG-TTTTTGAGCATAATGACTTTTGCCCT XhoI NGS67 Fwd CGCGGATCCCATATG-CGTGCGCACGGACACG NdeI Rev CCCGCTCGAG-GGCGGCGAGTTTTTCGC XhoI NGS106 Fwd CGCGGATCCCATATG-GCAAACAGCGGAACGATAG NdeI Rev CCCGCTCGAG-AAAATCCTGCGGGATCGGT XhoI NGS115 Fwd CGCGGATCCCATATG-GGGGGCGGCTCCGGC NdeI Rev CCCGCTCGAG-TTCGGCCAACAATGCTTCC XhoI NGSΔG115 Fwd CGCGGATCCCATATG-GATGCCCAATCTTCACAAAG NdeI Rev CCCGCTCGAG-TTCGGCCAACAATGCTTCC XhoI NGS118 Fwd CGCGGATCCCATATG-ACCGCCCTTCCCTCTGA NdeI Rev CCCGCTCGAG-CGGCTGCCATTCGCGTT XhoI NGS122 Fwd CGCGGATCCCATATG-AACCCGAACGATGCGTTTT NdeI Rev CCCGCTCGAG-AGGGTAAAACTTATTCAAATCGGCAA XhoI NGS144 Fwd CGCGGATCCCATATGGCTTCTGAAAATTCTGTAGC NdeI Rev CCCGCTCGAG-GAACACGCTTTTCATTACACCCA XhoI NGS151 Fwd CGCGGATCCCATATGCACGGTATGCATAAGAGCA NdeI Rev CCCGCTCGAG-TTGCTGATGCGGCTTTATTCG XhoI EXAMPLE 1 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 1> which encodes amino acid sequence <SEQ ID 2; NGS1>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.47 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.72 threshold: 0.0 PERIPHERAL Likelihood = 4.72 modified ALOM score: −1.44 Rule: cytoplasmic protein * * * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.326(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp\|P45941\|LYQC_BACSU HYPOTHETICAL 21.5 KD PROTEIN IN CWLA-CISA INTERGENIC REGION pir\|\|E69949 hypothetical protein yqcF-Bacillus subtilis dbj\|BAA06963.1\| (D32216) ORF95 [Bacillus subtilis] dbj\|BAA12427.1\| (P84432) YqcF [Bacillus subtilis] emb\|CAB14528.1\| (Z99117) yqcF [Bacillus subtilis] Length = 192 Score = 35.5 bits (81), Expect = 0.45 Identities = 36/162 (22%), Positives 77/162 (47%), Gaps 5/162 (3%) Query: 19 DSGSQYKLNIAAIPSSPNRDLKTYITLGLSKHDLHYK---SRFEILFVCSLKYDENQIFP 75 D ++I ++ P + +Y TLGLS H +NY+ + I V +++ + Sbjct: 29 DDNKSSIDILSVSDQPQEGITSYSTLGLSDHSIEGTPLRIEIV~AAMESASDIYAN 88 Query: 76 FLRWLAETIIENKKILLRGQVVYLPRSIVNS-TKMDALYVSAPFYFDDDFQVCYGEHYNI 134 L A II + G + S+ + T M + PF +++D ++ + N+ Sbjct: 89 VLSTCAFNIINSNFTCAPGVIFKNVISMYDQETDMKHIMFVPPFLWEEDLELLEFSNKNV 148 Query: 135 VFPLLVPLYKQEAELVEKKGWNAFEQFLLDNEVGNLSDMNRK 176 + + +P+ + E ++ EK G + + Q LL+++ ++ D+ R+ Sbjct: 149 TWLMALPISEGELQVAEKHG-SDYLQDLLESKQIDIFDIKRE 189 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 2 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 3> which encodes amino acid sequence <SEQ ID 4; NGS2>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −7.2 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.89 threshold: 0.0 PERIPHERAL Likelihood = 5.89 modified ALOM score: −1.68 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.367(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >emb\|CAC01359.1\|(AL390975) hypothetical protein SCP8.21 [Streptomyces coelicolor A3(2)] Length = 198 Score = 37.2.bits (85), Expect = 0.15 Identities = 29/107 (27%), Positives = 51/107 (47%), Gaps = 3/107 (2%) Query: 73 ETPEHIETLAMLASASYPDQFQLGKNIGRPWVEQSSFRHFLISLPYPYGQELEY 130 +T + + LA+LA++ G ++++G P + F L++ P ++LE Sbjct: 88 DTDKVLRPLAVLAASPQVEGVIVAPGASLDVGEPLWPGAPFTSVLVAEPGGLVEDLELDA 147 Query: 131 -MDNRFFWLLPITQTERLFLNTHSVEELETKFDEAGIDYLDINRAS 176 +D VRF LLP+T E + H L+ ++ G D D +R S Sbjct: 148 PLDPVFLPLLPMTPNEAAWKRVHGAPALQERWLNHGTDLRDPSRRS 194 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 3 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 5> which encodes amino acid sequence <SEQ ID 6; NGS3>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.69 Possible cleavage site: 32 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 33 ALOM: Finding transmembrane regions (Klein et al.)", "count: 5 value: −10.56 threshold: 0.0 INTEGRAL Likelihood = −10.56 Transmembrane 182-198 (171-201) INTEGRAL Likelihood = −7.86 Transmembrane 251-267 (244-273) INTEGRAL Likelihood = −7.48 Transmembrane 142-158 (136-167) INTEGRAL Likelihood = −6.32 Transmembrane 55-71 (50-82) INTEGRAL Likelihood = −2.97 Transmembrane 100-116 (99-117) PERIPHERAL Likelihood = 4.72 modified ALOM score: 2.61 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.522(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp\|P19845\|NOSY_PSEST MEMBRANE PROTEIN NOSY PRECURSOR pir\|\|S13585 nosY protein precursor-Pseudomonas stutzeri emb\|CAA37717.1\|(X53676) nosY [Pseudomonas stutzeri] Length = 276 Score = 163 bits (413), Expect = 2e−39 Identities = 117/275 (42%), Positives = 174/275 (62%), Gaps = 2/275 (0%) Query: 1 MNPVWIITGKEARDSLRNRWVLAAVLLLAALALSLGFLGSSPTGSVKVDPLTVTVVSLSS 60 MN VW I KE D LRNRW+LA LL A LA+ + +LG++ +G + + T+ SL+S Sbjct: 1 MNQVWNIARKELSDGLRNRWLLAISLLFAVLAGIAWLGAAASGQLGFTSIPATIASLAS 60 Query: 61 LLSIFLIPLIAMLLSYDALIGEIERGTMALLLSYPIWRNQILAGKFVGHLIILALATTAGY 120 L+ FL+PLIA+LL+YDA++GE E GT+ LLL+YP+ R QIL GKFVGH +ILALA G+ Sbjct: 61 LATFLMPLIALLLAYDAIVGEDEGGTLMLLLTYPLGRGQILLGKFVGHGLILALAVLIGF 120 Query: 121 GLAGITLQLANGGFDIAA-WKPFALLAASVILGAAFLSMGYLISAKVKERGTAAGISIG 179 G A + + L G ++ + F + +S +LG FL+ Y++S KV E+ +AAG+++G Sbjct: 121 GCAALAIALLVEGVELGMLFWAFGRFHISSTLLGWVFLAFAYVLSGKNEKSSAAGLALG 180 Query: 180 VWLFFVVIFDMALLGILVADSKQVITAPVVETVLLFNPTDIYRLLNLTGYENTAMYAGMA 239 VW F V+ +L + S+ ++ +LL NPTDIYRL+NL+G+E + G+ Sbjct: 181 VW-FLFVLVFDLVLLALLVLSEGKFNPELLPWLLLLNPTDIYRLINLSGFEGSGSAMGVL 239 Query: 240 GLSGQIGLTVPVLLTAQVLWVIIPLVLAAGIFRKR 274 L + + VL + W+ + L+LA IFR+R Sbjct: 240 SLGADLPVPAAVLCLLAWIGVSLLLAYAIFRRR 274 A homolog (amino acids 226-276) was found in serogroup A N.meninigitidis but not in serogroup B, so NGS3 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 4 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 7> which encodes amino acid sequence <SEQ ID 8; NGS4>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 1.53 Possible cleavage site: 58 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 0.63 threshold: 0.0 PERIPHERAL Likelihood = 0.63 modified ALOM score: −0.63 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.103(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp\|Q59746\|NOSZ_RHIME NITROUS-OXIDE REDUCTASE PRECURSOR (N(2)OR) (N2O REDUCTASE) gb\|AAC44023.1\|(U47133) nitrous-oxide reductase [Sinorhizobium meliloti] prf\|\|2209347B nitrous-oxide reductase [Rhizobium meliloti] Length = 639 Score = 660 bits (1704), Expect = 0.0 Identities = 344/536 (64%), Positives = 407/536 (75%), Gaps = 23/536 (4%) Query: 1 MSDEKLEQNGLSRRSFLGTAA--ASGAGIAGAGLIGLAGCSKDGEQAAANASGAAPVAKA 58 MS+E+ + L+RR LGT A A+ + G L L+G G A A+A Sbjct: 1 MSNEETKMR-LNRRQMLGTTAFMAAAGAVGAGGALTLSG-------------GTATPARA 46 Query 59 QGESKPGQLSSEVGPGELDQYYGFLSGGQSGEMRLIGLPSMRELMRIPVFNMDSATGWGR 118 Q S S EV PGELD+YY F S GQSGE+R++G PSMRE+MRIPVFN SATGWG+ Sbjct: 47 QETSGS---SYEVKPGELDEYYVFFSSGQSGEIRIVGAPSMREMMRIPVFNRCSATGWGQ 103 Query: 119 TNESLKVLNGNITEETRKFLKDSGLRCYPNGDLHHPHLSFTDQTYDGRYAYANDKANNRV 178 TNES KV+ + ET +FLKD G Y NGDLHHPH SFTD TYDGRY YANDK+N+RV Sbjct: 104 TNESRKVMTEGLLPETVEFLKDQG-GLYLNGDLHHPHPSFTDGTYDGRYLYANDKSNSRV 162 Query: 179 CRVRLDVMKADIIDIPNDSGIHGIHGLRPQRYPKTGYVFANGEHITPVSGVGK-LDDAKTWN 237 CR+RLDVMK DKII +PN +HGLR Q+YPKTGYVF NGE PV GK + D ++ Sbjct: 163 CRIRLDVMKCDKIIQLPNQHTVHGLRVQKYPKTGYVFCNGEDAVPVPNDGKTMGDKNSYQ 222 Query: 238 AVYTAIDGETMEIAWQVLVDGNLDNGDADYQGKYSFATCYNSERALTVQGASSNEQDWCV 297 A++TA+DGETME+AWQV+VDGNLDN DADYQGKY FATCYNSE T+ ++EQDW V Sbjct: 223 AIFTAVDGETMEVAWQVMVDGNLDNVDADYQGKYCFATCYNSEEGFTLADMMASEQDWVV 282 Query: 298 VFDLKAIEEGIKAGDFKEVNGVLDGRABAKSKYTRYIPVFNSPHGCNASPDGKYIMPN 357 +F+LK IEE + GD+KE+ GV +LDGR S YTRY+PVPNSPHG N +PDG +++ N Sbjct: 283 IFNLKRIEEAVAKGDYKEIGGVPVLDGR--KGSPYTRYVPVPNSPHGINTAPDGIHVVAN 340 Query: 358 GKLPPTVTVLDVSKLDDLFAGKIKERDVVAEPQLGLGPLHTAFDGRGNAYTTLFIDSQM 417 GKL PTVTV DV K DDLF KI+ RD VVAEP+LGLGPLHTA+DG+GNAYTTLFIDSQ+ Sbjct: 341 GKLSPTVTVFDVRKFDDLFDDKIQARDTVVAEPELGLGPLHTAYDGKGNAYTTLFIDSQV 400 Query: 418 VKWNIDDAIKAYGEKIDPIKQKLDVHYQPGHNHTTMGETKEADGQWLVSLNKFSKDRFL 477 KWNI+DA +AY GEK+DPI+ KLDVHYQPGHNHT+MG+TKEADG+WL+SLNKFSKDR+L Sbjct: 401 CKWNIEDAKRAYAGEKVDPIRHKLDVHYQPGHTSMGQTKEADGKWLISLNKFSKDRYL 460 Query: 478 NAGPLKPECDQLIGISGDEMRLVHDNPTFAEPHDLCLVAASKLNPGKTWDRKDPWF 533 N GPLKPE DQLI ISGDEM LVHDNPTFAEPHD +V ASK+NP W+R DP+F Sbjct: 461 NVGPLKPENDQLIDISGDEMVLVHDNPTFAEPHDATIVHASKINPVHVWNRDDPFF 516 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 5 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 9> which encodes amino acid sequence <SEQ ID 10; NGS5>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 1.09 Possible cleavage site: 19 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 20 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.43 threshold: 0.0 PERIPHERAL Likelihood = 7.43 modified ALOM score: −1.99 Score for OM-PP discrimination: 4.97 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 4.97 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Outer membrane?", "score: 0.496525 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.781(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.138(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.", "The protein was expressed in E.coli as an insoluble 43.56 kDa His-fusion product and then purified.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 6 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 11> which encodes amino acid sequence <SEQ ID 12; NGS6>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.93 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 6.42 threshold: 0.0 PERIPHERAL Likelihood = 6.42 modified ALOM score: −1.78 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.447(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|F83173 outer membrane protein OprC PA3790 [imported]-Pseudomonas aeruginosa (strain PAO1) dbj\|BAA05664.1\|(D28119) outer membrane protein C [Pseudomonas aeruginosa] gb\|AAG07177.1\|AE004797_12 (AE004797) outer membrane protein OprC [Pseudomonas aeruginosa] Length = 723 Score = 77.9 bits (191), Expect = 1e−13 Identities = 58/188 (30%), Positives = 89/188 (46%), Gaps = 13/188 (6%) Query: 49 VKDLIIFDRAHGQSGTASKGGIITRNVDAILFTAQAYARYNFNPHWAAGIKAAYNYGHN 108 +TL, V+D I+F G G++++ NVDAR+ + A Y +W AY +G N Sbjct: 546 VQDFILFSYREGMMGSSTQ-----ATNVDARIMGGELGASYQLTGNWKTDASLAYAWGKN 600 Query: 109 ETDGRPPYQIRPFEAAVQADYKNYFAHGSYNIGAATRFVAKQTRGDFDMASGLGIDKREA 168 +D R QI P EA Y+ G ++ G+ R VA Q R D + +G D ++ Sbjct: 601 SSDDRALPQIPPLEARFGLTYE----EGDWSAGSLWRVVAPQNRIARDQGNVVGKDFDKS 656 Query: 169 AKGFTVADVYAGVNIKDKYGLRLGVNNVFNKKYVEYI--SGDHVLALSPS-VVYAPGRTY 225 A GF V + + L GV+N+F+K Y E++ +GD S + V PGRT+ Sbjct: 657 A-GFGVFSLNGAYRVTRNVKLSAGVDNLFDKDYTEHLNKAGDAGFGFSANETVPEPGRTF 715 Query: 226 WLSLHAAF 233 W + +F Sbjct: 716 WTKVDFSF 723 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 7 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 13> which encodes amino acid sequence <SEQ ID 14; NGS7>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Hejine) Signal Score (−7.5): 4.94 Possible cleavage site: 26 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 27 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 0.79 threshold: 0.0 PERIPHERAL Likelihood = 0.79 modified ALOM score: −0.66 Score for OM-PP discrimination: −18.85 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −18.85 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Periplasmic space?‘0 score: 1.8846 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.929(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.211(Affimative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|\|D72405 hypothetical protein-Thermotoga maritima (strain MSB8) gb\|AAD35294.1\|AE001705_5 (AE001705) hypothetical protein [Thermotoga maritima] Length = 300 Score = 81.8 bits (201), Expect = 1e−14 Identities = 72/289 (24%), Positives = 124/289 (41%), Gaps = 17/289 (5%) Query: 38 PAMPSVTIAVAALQGKLAKQADVSLKIWRSPDQLRAGVASGQFKVMMSPSNVGVNLRNQG 97 +TL, P P++ V + GK+ DV ++IW++P++ A + S + + P VG NL +G Sbjct: 24 PLGPALIPVVPIMDGKIP--TDVKIEIWKNPEEAVAKIVSKEVDFAVLPVTVGANLYGKG 81 Query: 98 QKVGMNILTNGITQLVCKGSAIASP-QDLVGKKILVPF-KNDMPDIVLQALLKKLKIDA 155 ++ +V + +LV A + L G+++ P + D++++ L K + Sbjct: 82 VRIKLVGVHEWKVFYLVASDDATFDGWESLRGQEVYTPHGRGQTVDVLMRYFLSKAGLTL 141 Query: 156 HK-VSITYAATPPEAVGLFPSKGYHAVILPEPMATASLLKGKTIGINVVHGFDLVKAWGQ 214 + V I YA P E V LF S LPEP + L +GK + D K WG+ Sbjct: 142 DRDVKILYAP-PQEIVALFKSGKVKYAALPEPFVSMCLDRGKVV-------LDFQKEWGK 193 Query: 215 AFDTKPLIPMAGIIANEEYFHAHKAQFDIFHQDLKNALNWILANRQNAAKIGKNYLPAPE 274 IP+AG+ E K + + L +++ W+ N ++ L P Sbjct: 194 ELGVPGRIPIAGLFVRE---GVDKETVEKVKALIDSIRWMKENLDETVQLSSEKLGIPA 250 Query: 275 PALVHGLDGAPLTVSKGSEVKNEILKFYEILMQFNPELLGGKLPDNGFF 323 L L+ + + E+ F + L + PE K+PD GF+ Sbjct: 251 KILKSSLERIEFEYVPVEKCREEVETFLKKLNELYPEGF-EKIPDEGFY 298 The protein was expressed in E.coli as an insoluble 32.89 kDa His-fusion product and then purified.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 8 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 15> which encodes amino acid sequence <SEQ ID 16; NGS8>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 2.39 Possible cleavage site: 15 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 16 ALOM: Finding transmembrane regions (Klein et al.)", "count: 4 value: −8.23 threshold: 0.0 INTEGRAL Likelihood = −8.23 Transmembrane 49-65 (41-73) INTEGRAL Likelihood = −7.38 Transmembrane 83-99 (75-106) INTEGRAL Likelihood = −7.06 Transmembrane 110-126 (100-133) INTEGRAL Likelihood = −4.41 Tranamembrane 164-180 (163-187) PERIPHERAL Likelihood = 5.89 modified ALOM score: 2.15 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.429(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp\|P38044\|NRTB_SYNP7 NITRATE TRANSPORT PERMEASE PROTEIN NRTB pir\|\|S30892 nitrate transport protein nrtB-Synechococcus sp.", "(strain PCC 7942) emb\|CAA43810.1\|(X61625) nitrate transporter [Synechococcus sp.]", "prf\|\|1908370A nitrate transporter [Synechococcus sp.]", "Length = 279 Score = 67.5 bits (164), Expect = 1e−10 Identities = 54/202 (26%), Positives = 96/202 (46%), Gaps = 7/202 (3%) Query: 4 VALWAWGSAVFGEFMLPAPVEFQKSL--DLLKHFQEN-----EIGISLWRSVVGISVAL 56 +A+W SA+ G+ LP P+ V + +++ F +N +G+ + S+ +++ Sbjct: 36 LAIWQVISAILGQDRLPGPINVVANTWMPYIVEPFFDNGGTSKGLGLQILISLQRVAIGY 95 Query: 57 IAGLAAGLVAGLVAGSFKTAMALLKPVITILLAMPPIIWVVMALFWFGFGNPSVLFTIIV 116 + G++ G V G K L PVI +L +PP+ W ++L F N S +F I + Sbjct: 96 LLAACTGILVGGVLGMSKFLGKGLDPVIQVLRTVPPLAWFPISLMVFQDANTSAIFVIFI 155 Query: 117 LVAPLTFASAAVGMASVNKQHEELFDAYKLGRLKKIRYLYIPHLTGYVISSVGVAVAMGV 176 + AVG+ + + + KL + I + IP YV + + +AV + Sbjct: 156 TAIWPIIINTAVGINQIPDDYNNVARVLKLSKKDYILNILIPSTVPYVFAGLRIAVGLAW 215 Query: 177 KAVIMAELLGASKGVGARIADA 198 A++ AE+L A G+G I DA Sbjct: 216 LAIVAAEMLKADGGIGYFIWDA 237 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 9 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 17> which encodes amino acid sequence <SEQ ID 18; NGS9>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.07 Possible cleavage site: 29 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −1.81 threshold: 0.0 INTEGRAL Likelihood = −1.81 Transmembrane 97-113 (96-113) PERIPHERAL Likelihood = 4.24 modified ALOM score: 0.86 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.172(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp\|P97027\|SSUB_BACSU PUTATIVE ALIPHATIC SULFONATES THANSPORT ATP-BINDING PROTEIN SSUB pir\|\|G69816 nitrate ABC transporter (binding protein) homolog ygaL- B. subtilis emb\|CAB07520.1\|(Z93102) hypothetical 30.6 kd protein [Bacillus subtilis] emb\|CAB12711.1\|(Z99108) similar to nitrate ABC transporter (binding protein) [Bacillus subtilis] Length = 274 Score = 99.5 bits (247), Expect = 3e−20 Identities = 68/181 (37%), Positives 102/181 (55%), Gaps = 9/181 (4%) Query: 4 LFGPSGCGKTTVLRLIAGLETPKSGTIRNTFH-------KTGFLFQENRLPENLTAMQNI 56 L GPSGCGK+T+L++IAGL++ G++ + GF+FQE+RL LT QNI Sbjct: 56 LIGPSGCGKSTLLKIIAGLDSEYDGSVEINGRSVTAPGIQQGFIFQEHRLFPWLTVEQNI 115 Query: 57 A--IFMDNPDEGEIVALAAKVGLTAGDLNKYPTELSGGMAKRVAFLRLLLCGCDLALLDE 114 A + + +P + V ++ G YP ELSGGM++RVA R LL ++ LLDE Sbjct: 116 AADLNLKDPKVKQKVDELIEIVRLKGSEKAYPRELSGGMSQRVAITRALLREPELLLDE 175 Query: 115 PFVGLDRDLRDILVAMLVEKIERQGMACILVTHDRFEAARLSHEIMLLSAKGMNVQNVIT 174 PF LD R L +L++ ++ ILVTHD E+ L +E+ +L AK + ++ Sbjct: 176 PFGALDAFTRKHLQDVLLDIWRKKTTNILVTHDIDESVYLGNELAILKAKPGKIHKLMP 235 Query: 175 L 175 + Sbjct: 236 I 236 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 10 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 19> which encodes amino acid sequence <SEQ ID 20; NGS10>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal score (−7.5): 2.27 Possible cleavage site: 26 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.14 threshold: 0.0 PERIPHERAL Likelihood = 5.14 modified ALOM score: −1.53 * Reasoning Step: 2 imb2 HYPID: 2 CFP: 0.1 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.100(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|\|A82615 surface protein XF1981 [imported]-Xylella fastidiosa (strain 9a5c) gb\|AAF84783.1\|AE004017_6 (AE004017) surface protein [Xylella fastidiosa] Length = 1190 Score = 50.2 bits (119), Expect = 2e−05 Identities = 59/210 (28%), Positives = 92/210 (43%), Gaps = 5/210 (2%) Query: 17 SIGTSAEANAPGAALGGSSEASKFSIAEGYLASSDGYGAIAIGSAAKI-KQLEKGTIN 75 ++G A+A GA A+G + AS K S A G A + G++A+G AK + + Sbjct: 876 AVGVGTLASAEGATAVGSGAAASGKGSTAIGRNAVASADGSVALGDGARGAESYTG 935 Query: 76 HIVGNDNKGLYVDADGNVTKITVRTESEKDILSRYGQTYGAVALGFRSSSHNLFA----S 131 G N + + G+ +K RT S L + N + Sbjct: 936 KYSGLQNNTVGTVSVGDASKGETRTVSNVADAKEATDAVNLRQLDRVAQDANRYVDNKIE 995 Query: 132 SFGAFSTATAIESLAVGDSSQSTGYRSATFGSHSRALAEESLALGYETRANYGSVALGA 191 S T + SL + + G + G + A +S+A+G + A+A +VA+G Sbjct: 996 SLSEGQTFVKVNSLNNSATPIAAGVDATAIGVGATASGADSIAMGNKASADNAVAIGN 1055 Query: 192 ESVANEENTVSVSSDTLKRKIVNVIADGTED 221 SVA+ NTVSV S +R++ NVA GT D Sbjct: 1056 HSVADRANTVSVGSAGSERQVTNVAAGTAD 1085 >sp\|P10858\|YADA_YERPS INVASIN PRECURSOR (OUTER MEMBRANE ADHESIN) pir\|\|S04534 invasin precursor-Yersinia pseudotuberculosis plasmid pIBI emb\|CAA32088.1\|(X13883) Yop1 preprotein (AA 1-434) [Yersinia pseudotuberculosis] prf\|\|1411295A invasin [Yersinia pseudotuberculosis[ Length = 434 Score = 42.1 bits (98), Expect = 0.006 Identities = 35/134 (26%), Positives = 68/134 (50%), Gaps = 28/134 (20%) Query: 116 AVALGFRSSSHLFASSFGFSTATAIESDSSQSTGYRSATFGSHSRA-------- 167 ++A+G + + A + G+ S AT + S+A+G S++ G + T+G+ S A Sbjct: 107 SIAIGATAEAAKPAAVAVGSGSIATGVNSVAIGPLSKALGDSAVTYGASSTAQKDGVAIG 166 Query: 168 ----LAEESLALGYETRANAYGSVALGA----------------ESVANEENTVSVSSDT 207 ++ +A+G+ ++ +A SVA+G S + EN+VS+ ++ Sbjct: 167 ASDTGVAVGFNSKDAQNSVAIGHSSHVAADHGYSIAIGDHSKTDRENSVSIGHES 226 Query: 208 LKRKIVNVADGTED 221 L R++ ++A GTED Sbjct: 227 LNRQLTHLAAGTED 240 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 11 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 21> which encodes amino acid sequence <SEQ ID 22; NGS11>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.16 Possible cleavage site: 60 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.67 threshold: 0.0 PERIPHERAL Likelihood = 4.67 modified ALOM score: −1.43 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.297(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp\|P10858\|YADA_YERPS INVASIN PRECURSOR (OUTER MEMBRANE ADHESIN) pir\|\|S04534 invasin precursor-Yersinia pseudotuberculosis plasmid pIBI emb\|CAA32088.1\|(X13883) Yop1 preprotein (AA 1-434) [Yersinia pseudotuberculosis] prf\|\|1411295A invasin [Yersinia pseudotuberculosis] Length = 434 Score = 41.3 bits (96), Expect = 0.007 Identities = 27/71 (38%), Positives = 48/71 (67%), Gaps = 4/71 (5%) Query: 16 QLNRLSKRTNRVGASAAALASL-KPAQLGKNDKFAFSLGFGSYKNAQAVAMGAVFKPAEN 74 +L++L KR ++ AS+AAL SL +P +GK + F+ G G Y+++QA+A+G+ ++ E+ Sbjct: 353 RLDKLDKRVDKGLASSAALNSLFQPYGVGKVN---FTAGVGGYRSSQALAIGSGYRVNES 409 Query: 75 VLLNVAGSFAG 85 V L ++AG Sbjct: 410 VALKAGVAYAG 420 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 12 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 23> which encodes amino acid sequence <SEQ ID 24; NGS12>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.29 Possible cleavage site: 61 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 62 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 6.36 threshold: 0.0 PERIPHERAL Likelihood = 6.36 modified ALOM score: −1.77 Score for OM-PP discrimination: 6.03 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 6.03 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Outer membrane?", "score: 0.602784 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.867(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.158(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 13 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 25> which encodes amino acid sequence <SEQ ID 26; NGS13>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.64 Possible cleavage site: 51 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 21 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −1.01 threshold: 0.0 INTEGRAL Likelihood = −1.01 Transmembrane 36-52 (36-52) PERIPHERAL Likelihood = 5.14 modified ALOM score: 0.70 Rule: inner or outer membrane protein Rule: inner or outer membrane protein Rule: cytoplasmic membrane protein * Reasoning Step: 2 Lipoprotein?", "Inner membrane?", "----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.742(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gb\|AAC33455.1\|(AF067083) outer membrane protein homolog [Vitreoscilla sp.]", "Length = 217 Score = 236 bits (602), Expect = 2e−61 Identities = 134/217 (61%), Positives = 166/217 (75%) Query: 1 MTFFKPSTVVLTASALALSGCVADPVTGQQSPNKSAMYGLGGAAVCGWGALTHSGKGAR 60 M +K +++ T +A+ALS C DP+TGQ N + + LGGAA CGIVGALTH KGAR Sbjct: 1 WKAWKKFSLMATVAAVALSACATDPMTGQVDRNNTVLGALGGAATCGIGALTHGSKGAR 60 Query: 61 NSALACGAIGAGVGGYMDYQEQRLRQNLAGTQIEIQRQGNQIRLVMPESVTFATGSAALG 120 NSALACGAIGAGVG YMD+QE++LRQ+LA TQ+E+ R G++IRLVMPES+TFATGS L Sbjct: 61 NSALACGAIGAGVGAYMDHQERQLRQSLANTQVEVNRVGDEILVMPESITFATGSYQLN 120 Query: 121 GSAQYALNTAAQTLVQYPDTTLTINGHTDNTGSDAVNNPLSQHRAQAVAYYLQTRGVAAS 180 SA +LN+ + L QY DTT+ I GHTD+TGSDA+N PLS++RA AVA YL +R VA++ Sbjct: 121 SSASTSLNSVSSVLAQYTDTTINIVGHTDSTGSDAINEPLSRNRASAVANYLVSRNVASN 180 Query: 181 RLTVYGYGSHMPVASNATVEGRAQNRRVEILINPDQR 217 R+T G G PVASN TV GRA+NRRVEI +NP QR Sbjct: 181 RITTVGAGCRQPVASNNTVAGRAENRRVEITVNPIQR 217 >gb\|AAD40344.1\|U88088_22 (U88088) OmpA [Pseudomonas alcaligenes] Length = 220 Score = 130 bits (328), Expect = 1e−29 Identities = 90/219 (41%), Positives = 127/219 (57%), Gaps = 6/219 (2%) Query 7 STVVLTASALALSGCVA---DPVTGQQSPNKSAMYGLGGAAVCGIVGALTHSGKGARNSA 63 S + + L+GC + + T + + A L GA ++G + +GA A Sbjct: 3 SVIAASLVIFTLTGCASIQNEDGTTKNTALYGAGGALAGAVAGALIGK-ENRAQGALIGA 61 Query: 64 LACGAIGAGVGGYMDYQEQRLRQNLAGTQIEIQRQGNQIRLVMPESVTFATGSALLLGGSA 123 G++GAG G Y D QE LR+ + G+ ++++RQG++I +VMP ++TFATG A + + Sbjct: 62 AVAGSLGAGYGYYADKQEAELREQMKGSGVQVERQGDEIVIVMPGAITFATGKAEIQPNF 121 Query: 124 QYALNTAAQTLVQYPDTTLTINGHTDNTGSDAVNNPLSQHRAQAVAYYLQTRGVAASRLT 183 LN A + YPD+ L + GHTD+ GS N LSQ RAQ+VA +L+ GV R+ Sbjct: 122 ANTLNQLAGSFPNYPDSRLIVTGHTDSVGSYEANELLSQRRAQSVAQFLRGNGVQTDRIE 181 Query: 184 VGYGHMPVASNATEGRAQNRRVEILINPDQRAVNAA 222 V G G + PVASNAT EGRAQNRRVEI + P RAV A Sbjct: 182 VIGAGPNQPVASNATAEGRAQNRRVEIKLAP--RAVQQA 218 The protein was expressed in E.coli as a soluble 22.55 kDa His-fusion product and then purified.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 14 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 27> which encodes amino acid sequence <SEQ ID 28; NOS14>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.32 Possible cleavage site: 40 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.39 threshold: 0.0 PERIPHERAL Likelihood = 3.39 modified ALOM score: −1.18 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.254(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 15 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 29> which encodes amino acid sequence <SEQ ID 30; NGS15>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.75 Possible cleavage site: 45 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.89 threshold: 0.0 PERIPHERAL Likelihood = 5.89 modified ALOM score: −1.68 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.232(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp\|P10487\|RCI1_ECOLI SHUFFLON-SPECIFIC DNA RECOMBINASE pir\|\|S03815 probable integrase-Escherichia coli dbj\|BAA77989.1\|(AB027308) shufflon-specific DNA recoinbinase [Plasmid R64] Length = 384 Score = 67.1 bits (163), Expect = 3e−10 Identities = 75/301 (24%), Positives = 125/301 (40%), Gaps = 34/301 (11%) Query: 68 KVKMMTLSEAMRKYLNETLGAGRSKKMGL---RFLMEFPIGGIGIDKLKRSDFAEHVMQR 124 +++ M+LS A+ KYL + + + PI +D++ D A + R Sbjct: 5 RIRKMSLSRALDKYLKTVSVHKKGHQQEFYRSNVIKRYPIALRNMDEITTVDIATYRDVR 64 Query: 125 RRGIPELDIAPIAASTALQELQYIRSVLKHAFYVWGLEIGWQELDFAANGLKRSNMVAKS 184 I PI +T EL + S+ A WG N ++ S Sbjct: 65 LAEINPRTGKPITGNTELELALLSSLFNIARVEWG--------TCRTNPVELVEKPKVS 116 Query: 185 AIRDRLPTTEELQTLTTYFLRQWQSRKSSIPMHLIMWLAIYTSRRQDEICRLLFDDWHKN 244 + RDR T+E + L+YF R+ ++ +++I LA+ T+ RQ EI L W Sbjct: 117 SGRDRRLTSSEBRRLSRYF------REKNLMLYVIFHLALETAMRQGEILAL---RWEHI 167 Query: 245 DCTRPVRDLKNPNGSTGNNKEFDILPMALPVIDELPEESVRKRMLANKGIADSLVPCNGK 304 D V L P G++++ + A + +P + ++ Sbjct: 168 DLRHGVAL--PETNGHSRDVPLSRRARNFLQMMP-----------VNLHGNVFDYTAS 214 Query: 305 SVSAAWTRACKTLGIKDLRFHULREEAATRMAEDG-FTIPQNQRVTLHDGWNSLQRYVSVR 364 AW A + L I+DL FHDLRHEA +R E G + ++ ++ H N L+RY +R Sbjct: 215 GFKNAWRIATQRLRIEDLHRHDLRHEAISRFFELGSLNVMEIAAISGHRSMNMLKRYTHLR 275 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 16 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 31> which encodes amino acid sequence <SEQ ID 32; NGS16>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.64 Possible cleavage site: 20 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.67 threshold: 0.0 PERIPHERAL Likelihood = 4.67 modified ALOM score: −1.43 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.262(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp\|P10484\|T1M1_ECOLI TYPE I RESTRICTION ENZYME ECOR124II M PROTEIN (M.ECOR124II) pir\|\|S02166 type I site-specific deoxyribonuclease (EC 3.1.21.3) EcoR124/3 chain hsdM-Escherichia coli plasmid R124/3 emb\|CAA31541.1\|(X13145) hsdM protein (AA 1-520) [Escherichia coli] Length = 520 Score = 44.4 bits (104), Expect = 0.002 Identities = 65/235 (27%), Positives = 99/235 (41%), Gaps = 55/235 (23%) Query: 107 NRKKAGGYAEYITGGSLRRLVAARRYCGEHPGVFDGAAGSG--------QLEQYIEPS 158 N K+GG E+ T + +L+A ++D AAGSG Q + +I Sbjct: 191 NAGKSGG--EFFTPQHVSKLIAQLAMHGQTHVNKIYDPAAGSGSLLLQAKKQFDNHIIEE 248 Query: 159 DFRAVEIQAEACRALLQNYPAAKVYNTSLFL--------------------YTDGEPQDC 198 F EI N+ + ++FL + D +P D Sbjct: 249 GFFGQEI----------NHTTYNLARMNMFLHNINYDKRDIKLGNTLTEPHFRDEKPFDA 298 Query: 199 TVMNPPFSIKLKDLSEDEKSRIAQEYPWKKSGV------ADEIFVLKGLE--NARRFGFF 250 V NPP+S+K + D+ + I E + +GV AD FVL L +A+ Sbjct: 299 IVSNPPYSVKW--IGSDDPTLINDER-FAPAGVLAPKSKADFAFVLHALNYLSAKGRAAI 355 Query: 251 ILFPGIAYR-KSEQRFRE-IIGNRLAE--LNRIQNAFEDTPIEVLLLVIDKDKTD 301 + FPGI YR +EQ+ R+ ++ N E ++ N F T I V +LV+ K KTD Sbjct: 356 VCFPGIFYRGGAEQKIRQYLVDNNYVETVISLAPNLFFGTTIAVNILVLSKHKTD 410 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 17 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 33> which encodes amino acid sequence <SEQ ID 34; NGS17>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.29 Possible cleavage site: 16 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.60 threshold: 0.0 PERIPHERAL Likelihood = 2.60 modified ALOM score: −1.02 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.284(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >ref\|NP_052389.1\|translocator YopD [Yersinia enterocolitica] sp\|P37132\|YOPD_YEREN YOPD PROTEIN gb\|AAD16812.1\|(AF102990) translocator YopD [Yersinia enterocolitica] Length = 306 Score = 32.1 bits (72), Expect = 8.2 Identities = 29/93 (31%), Positives = 43/93 (46%), Gaps = 17/93 (18%) Query: 13 MLAAKRAAKESTRQERAVKRAGTVRNVDRNRLSARSKAQKENIARMLSGAKVSEDEALTC 72 +L R A+E Q+R ++ T+ AQKE +A M+SGAK+ A+ Sbjct: 89 LLELARKAREMGLQQRDIENKATI------------SAQKEQVAEMVSGAKLMIANAVVS 136 Query: 73 GIMMRLSLQDMRYACNQELINFAEHIVKQVQRL 105 GIM S ++ +E+ IVKQ Q L Sbjct: 137 GIMAATSTVASAFSIAKEV-----KIVKQEQIL 164 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 18 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 35> which encodes amino acid sequence <SEQ ID 36; NGS18>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.56 Possible cleavage site: 38 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.56 threshold: 0.0 PERIPHERAL Likelihood = 4.56 modified ALOM score: −1.41 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.397(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 19 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 37> which encodes amino acid sequence <SEQ ID 38; NGS19>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.12 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 8.49 threshold: 0.0 PERIPHERAL Likelihood = 8.49 modified ALOM score: −2.20 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.250(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >ref\|NP_043483.1\|orf14 [Bacteriophage HP1] sp\|P51716\|YO14_BPHP1 HYPOTHETICAL 14.9 KD PROTEIN IN REP-HOL INTERGENIC REGION (ORF14) pir\|\|S69520 hypothetical protein 14-phage HP1 gb\|AAB09199.1\|(U24159) orf14 [Bacteriophage HP1] Length = 133 Score = 73.3 bits (179), Expect = 1e−12 Identities = 44/129 (34%), Positives = 74/129 (57%), Gaps = 6/129 (4%) Query: 1 MFIPAALHKDEHSAYGVTIPDLPGCFSCGDTVEEAVANARSAAYMHIDGMIEDGGFKNLA 60 M P + K + Y V++PD+PGCFS GDT+ EA+ NA+ A HI+GM+ED + L Sbjct: 1 NLYPICIEK-VNDGYVVSVPDVPGCFSAGDTLSEAMLNAKEAISFHIEGMLEDD--EELP 57 Query: 61 VSS-IADLSQEPDYHGATWVMIEIDPAKISRQQIRFNVSWPQYLLDRVDEY--TSANHET 117 S+ I +P+Y ++++D + + + N++ P LL R+D++ T ++ Sbjct: 58 KSNPIEQYINQPEYKDFIVTVVDVDLTHLMGKAEKINITVPALLLHRIDQFIATHPEYKN 117 Query: 118 RSGFLAKAA 126 RS FL++ A Sbjct: 118 RSNFLSQLA 126 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 20 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 39> which encodes amino acid sequence <SEQ ID 40; NGS20>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.1 Possible cleavage site: 19 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.58 threshold: 0.0 PERIPHERAL Likelihood = 7.58 modified ALOM score: −2.02 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.057(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 21 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 41> which encodes amino acid sequence <SEQ ID 42; NGS21>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.52 Possible cleavage site: 52 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.83 threshold: 0.0 PERIPHERAL Likelihood = 5.83 modified ALOM score: −1.67 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.311(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >ref\|NP_040628.1\|cI (repressor; 237) [bacteriophage lambda] ref\|NP_061378.1\|phage lambda repressor protein CI [Escherichia coil] sp\|P03034\|RPC1_LAMBD REPRESSOR PROTEIN CI pir\|\|RPBPL repressor protein cI-phage lambda emb\|CAA24991.1\|(X00166) coding sequence cI gene [bacteriophage lambda] gb\|AAA96581.1\|(J02459) cI (repressor; 237) [bacteriophage lambda] emb\|CAB96428.1\|(AJ277653) phage lambda repressor protein CI [Escherichia coli] Length = 237 Score = 62.5 bits (151), Expect = 5e−09 Identities = 36/85 (42%), Positives = 51/85 (59%) Query: 2 KKRELNEIETAECAELKRIFNSKKEELKLTQYKLAEAVGVTQSAVNHYLNGTNALNASIA 61 KK+ L + + + LK I+ KK EL L+Q +A+ +G+ QS V NG NALNA A Sbjct: 4 KKKPLTQEQLEDARRKKAIYEKKKELGLSQESVADKMGMGQSGVGALFNGINALNAYNA 63 Query: 62 SQFAKILQIPVSDFSLRLAEEISSM 86 + AKIL++ V +FS +A EI M Sbjct: 64 ALLAKILKVSVEEFSPSIAREIYEM 88 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 22 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 43> which encodes amino acid sequence <SEQ ID 44; NGS22>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.6 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.74 threshold: 0.0 PERIPHERAL Likelihood = 7.74 modified ALOM score: −2.05 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.072(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|\|S30432 hypothetical protein-Streptomyces clavuligerus plasmid pSCL Length = 307 Score = 43.6 bits (102), Expect = 0.002 Identities = 25/86 (29%), Positives = 49/86 (56%), Gaps = 2/86 (2%) Query: 6 MGMAFKT-GIPRGRFVLVLCDCANDDGLCYPSQETLAEDTGFAETAVRQHIKWLKDNN 64 MGM F G+ ++ +L+ + + G C+PS++ L +D G + + V++ +L N Sbjct: 1 MGMVFAAEGLDGSEKLLLLGYTNWTDPYGYCWPSEDRLVDDCGTSRSTVQRTKRKLVKKN 60 Query: 65 FIKSARRQRGR-ERKSDIYRINVALL 89 ++S RR+ + E S++ R+N+ LL Sbjct: 61 LLRSVRRKNSKGEPISNLSRVNLPLL 86 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 23 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 45> which encodes amino acid sequence <SEQ ID 46; NGS23>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.8 Possible cleavage site: 59 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 0.05 threshold: 0.0 PERIPHERAL Likelihood = 0.05 modified ALOM score: −0.51 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Result ----- bacterial cytoplasm --- Certainty= 0.195(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp\|P07905\|DNAC_ECOLI DNA REPLICATION PROTEIN DNAC pir\|\|XMECNC DNA replication protein dnaC-Escherichia coli (strain K-12) Length = 245 Score = 110 bits (275), Expect = 2e−23 Identities = 75/224 (33%), Positives = 116/224 (51%), Gaps = 23/224 (10%) Query: 50 EAADEMAAYAETLRRGAMRDA----------LEKRIGRSGIAPRFRNCRIENYAV--SDS 97 + +E+ A+ + +GA+R A +++ RSGI P +NC ENY V Sbjct: 24 KTGEELLAWQK--EQGAIRSAALERENRAMKMQRTFNRSGIRPLHQNCSFENYRVECEGQ 81 Query: 98 IPGMARAKAAAAEYAANFADVLQTGRSMIFSGRRGTGKNHLACGIAREVIAAGKSALVIT 157 + +++A+ E+ N A S IFSG+ GTGKNHLA I E++ GKS L+IT Sbjct: 82 MNALSKARQYVEEFDGNIA-------SFIFSGKPGTGKNHLAAAICNELLLRGKSVLIIT 134 Query: 158 VGDMLRTVKDSF--GGGGEAGAVGIFVKPDLLVLDEFGAGSLSETDGRILFSVVNARYER 215 V D++ +KD+F G E + DLLV+DE G + S+ + I+ +V+ R Sbjct: 135 VADIMSAMKDTFRNSGTSEEQLLNDLSNVDLLVIDEIGVQTESKYEKVIINQIVDRRSSS 194 Query: 216 LMPMLVLTNLTAEAFRENTDARIRDRLRDGGGKLIPFDWESTRA 259 P +LTN E + R+ DR+R G + F+W+SYR+ Sbjct: 195 KRPTGHLTNSNMEEMTKLLGERVMDRMRLGNSLWVIFNWDSYRS 238 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 24 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 47> which encodes amino acid sequence <SEQ ID 48; NGS24>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.76 Possible cleavage site: 26 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 1.43 threshold: 0.0 PERIPHERAL Likelihood = 1.43 modified ALOM score: −0.79 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.112(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >ref\|NP_053228.1\|pXO2-73 [Bacillus anthracis] gb\|AAF13678.1\|AF188935_76 (AF188935) pXO2-73 [Bacillus anthracis] Length = 541 Score = 125 bits (315), Expect = 9e−28 Identities = 139/535 (25%), Positives = 254/535 (46%), Gaps = 70/535 (13%) Query: 14 PVLFIGTGMSLRYLDNSYTWDGLLSKIAIDLFGDDREYLNIKSRYCEDGRFQYEEIAEEL 73 P LFIG+G S RYL N W GL+ K + +L + EY Y + E+AE + Sbjct: 19 PFLFIGSGFSKRYL-NLEDWAGMKKFS-NLMPYEFEY------YSSTANKDWAEVAELM 70 Query: 74 QSKFDKVL--ENDPDGEFKEINDKFFENMRAGNTLSRFKIYISTLLSQLNYK----DNSN 127 F + E + KE D R + S K+ ++ L+ + YK + ++ Sbjct: 71 AKDFHPIWWKEQQFENNRKEFKD------RISSKQSPLKVEVAKYIiNSIEYKYGLDEKND 124 Query: 128 TELSELKKARKNVGSIITTNYDKLAQDIFEFNPL---IGN-DILLSNPY--GSVYKIHGC 181 E°+ LKK + IITTN+D L + IFE + IG ++L S+P +YKIHGC Sbjct: 125 KEIAALKKIV--IDGIITTNWDLLLEQIFEEQEMQVYIGQKELLFSHPLEINEIYKIHGC 182 Query: 182 VDDPSKIIITKKDYEKFKEKYELIRAQLLSLFIHNPIIFLGYNVGDENIKEILKTIFTYV 241 P +++T DY+ + EK + A+LL++FI +P+IFLGY++ D+NI++ILK I + Sbjct: 183 SSIPDSLVLTTSDYKGYNEKNAYLAAKLLTVFIEHPVIFLGYSISDDNIQQIILKAITRCL 242 Query: 242 EQNSPSANKIRRNFLLVEYEPESNNEDIVEDIDIT-GFSTIRINKIKTDNFSQIYKALA 300 +Q++ K R L+ E ED E++ +T G T+ I ++KT+++ +IY ALA Sbjct: 243 DQDNIHKLKDR----LIFVERAGQEEDSFENNSSLTIGKITVPITRVKTNDYEKIYNALA 298 Query: 301 ELTLPISAMDVRKFQSIAKEIYTGGNIKVSF---TEDMDNLNNSDKVVAIGSTKTISYNF 357 + S +R+ +S E+ + + + D+ + + V+ +G K + Sbjct: 299 QNKRKFSMKMMRQMKSQIYELVKTNDPEEKIYVVDGEYDDTQDIEFVIGLG-VKNVVEEM 357 Query: 358 QTTSEMMSN----------------YFKIIEEENS----QLLKLIDKHSIASTQYFPI-- 395 Q+ E+ ++ + +++ +E ++K+ + S QY P+ Sbjct: 358 QSNHEISADKELSEHGYGGISDIELFNELLSDEPKYDYDSIVKISLPQILRSNQYVPLFR 417 Query: 396 YGFSRICSDIHKEAVLKRQQKEKLDHFIEEINRRCKNNHSSIQSILDDENISDTYKNDAI 455 Y D ++ +K + K + F+ E ++ N S + + + + + + Sbjct: 418 YVLESSVEDELLDSKIKNKLKMRYTDFIITETQKNIKLDWQFKNLDEVLKGFPDIKV 477 Query: 456 AWG----IWNNQLSEDEVENYL---KNFVNKKN----THYKRLLCMFDYKKYADT 499 A + L+ D+++++L FV +KN T +RL ++D+ KY + Sbjct: 478 AIEQIPLLGQKNLNNCDDLDFLIKNSKFVKEKNTPERTGIRRLFRIYDWLKYGQS 532 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 25 A DNA sequence was identified in N,gonorrhoeae <SEQ ED 49> which encodes amino acid sequence <SEQ ID 50; NGS25>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 4.76837e−07 Possible cleavage site: 56 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 21 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 6.68 threshold: 0.0 PERIPHERAL Likelihood = 6.68 modified ALOM score: −1.84 Rule: inner or outer membrane protein Rule: inner or outer membrane protein * Reasoning Step: 2 Lipoprotein?", "Inner membrane?", "----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.700(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|7433O05\|pir\|\|B70313 cytochrome-C peroxidase (EC 1.11.1.5)- Aquifex aeolicus gi\|2982865\|gb\|AAC06485.1\|(AE000675) cytochrome C peroxidase [Aquifex aeolicus] Length = 355 Score = 345 bits (885), Expect = 7e−94 Identities = 180/336 (53%), Positives = 237/336 (69%), Gaps = 12/336 (3%) Query: 59 EDQDLLKRAQGVFQPLPTVEEMQKIRPFTEEQVKLGHQLWYEPRLSKGNTVSCNSCHNLA 118 +D++LLK A+ F+PLP V E + P T E+VKLG L+Y+PRLSK +SCN+CHNLA Sbjct: 22 DDKELLKKMARQYFKPLPKVAENPQ-NPVTPEKVKLGKMLYYDPRLSKSGLISHCNTCHNLA 80 Query: 119 SAGVDNMPTSQGHKGQFGGRNSPTALNAALLGSQFWDGRAADVEEQAGGPLVNPVEMAND 178 GVDN+PTS GH+ G RN+PT NAA+ +QFWDGRA DVEEQA GP+VNP+EMAN Sbjct: 81 RYGVDNLPTSIGHRWAIGPRNAPTVYNAAIHIAQFWDGRAKDVEEQALGPIVNPIEMAN- 139 Query: 179 SQEAAAAKIAKVPEYQEFKAFP-EDGAVSFKNITTALGAFERTLLTPTKWDEYLKGNV 237 ++E A + +PEY E+FKKAFP E V ++NI A+GAFERTL+TP+++DE+LKGN Sbjct: 140 TEENAVKTLKSIPEYVELFKKAFPNEKDPVKYENIGKAIGAFERTLMTPSRFDEFLKGNT 199 Query: 238 NALSEQERKGRAFDNGCIACHNGNLGGTTFQKRGLVQGPYWK------FIEDP--KR 289 AL+EQE++G++ F++ GC+ACHNG +GG F KFG++ YWK + P K Sbjct: 200 KALTEQEKRGLKTFIEVGCVACHNGPGVGGNMFAKFGMIT-EYWKVTYPYVLVGKPAIKV 258 Query: 290 DKGEADVTKKTEDEFFFRVPGLRNVAKTYPFHNGSVWELDKAVTIMGKAQLGKDIPKED 349 D GR VTKK ED F F+VP LRN+ TYPYFH+GSVW L+ AV IM K QLGK++ + Sbjct: 259 DFGRFGVTKKEEDMFVFKVPSLRNIEHTYPYFHDGSVWSLEDAVRIMAKTQLGKELTDQQ 318 Query: 350 VDNIVVFLNALSGNVSESARTHPELPLTAPMESKPD 385 V +IV FL AL+G + + A +PELP + KP+ Sbjct: 319 VKDIVAFLKALTGKIPKHALEVPELPPSTDKTPKPE 354 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 26 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 51> which encodes amino acid sequence <SEQ ID 52; NGS26>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.610001 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.04 threshold: 0.0 PERIPHERAL Likelihood = 5.04 modified ALOM score: −1.51 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.127(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|\|T13296 hypothetical protein 8-Streptococcus phage phi-O1205 gb\|AAC79524.1\|(U88974) ORF8 [Streptococcus thermophilus temperate bacteriophage O1205] Length = 157 Score = 62.5 bits (151), Expect = 2e−09 Identities = 53/161 (32%), Positives = 86/161 (52%), Gaps = 8/161 (4%) Query: 5 TLYRCDVQAALDYYFDSETEREDTLEAV--IGQFEVKAQSVIAYIKNQEITEKMLEGH 62 TLY + + D ET + DTLEA+ +E K + + IK+ E + + Sbjct: 3 TLYELTDQLLEIYNMDVDDET-KLDTLEAIDWTTDYENKVEGYVKVIKSLEADIEARKNE 6 Query: 63 IRQMTGKLKAAKARNQSLKDYLARNMQAAGITEIKADDGTFKASFRKSEAVVILDEAQIP 122 +++ G K+ +++ LK LA +M G T + D FK FRKSEAVV+ +E ++P Sbjct: 62 KKRLDGLNKSDQSKIDKLKTALAVSMAETGQTRV--DTTLFKVGFRKSEAVVV-NEEKLP 118 Query: 123 AEFMREAVKTEPDKTAIRXAIESGRQVAGAKIEGRKNLQIR 163 E+ K PDK +++ ++SG+ + GA +E R+NL IR Sbjct: 119 KEYQIATYK--PDKKTLKLKSGKHIEGATLEERRNLNIR 157 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 27 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 53> which encodes amino acid sequence <SEQ ID 54; NGS27>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Hejine) Signal Score (−7.5): −5.45 Possible cleavage site: 49 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 1.80 threshold: 0.0 PERIPHERAL Likelihood = 1.80 modified ALOM score: −0.86 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty = 0.559 (Affirmative) < succ> motifs: Subtilase_Asp (S, T, A, I, V)X(L, I, V, M, F)(L, I, V, M)D(D, S, T, A)G(L, I, V, M, F, C)x) (A)x(L)(I)D(D)G(I)x{2}(D) 79: DDDFL AALIDDGIVFD V A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS27 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 28 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 55> which encodes amino acid sequence <SEQ ID 56; NGS28>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.19 Possible cleavage site: 61 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 62 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 0.69 threshold: 0.0 PERIPHERAL Likelihood = 0.69 modified ALOM score: −0.64 Score for OM-PP discrimination: −24.78 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −24.78 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Periplasmic space?", "score: 2.47798 Periplasmic space?", "score: 2.47798 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.916(Affirmative) < succ> The protein has homology with the following sequences in the databases: >prf\|\|1306286A mobilization protein B [Escherichia coli] Length = 529 Score = 34.7 bits (78), Expect = 2.4 Identities = 24/69 (34%), Positives = 31/69 (44%), Gaps = 12/69 (17%) Query: 344 QLRARQQEIPVDYARTAVCGRIPFRRHSRPTLRSRTLGAQRRRIVPNVGQAGGIRAD--- 400 +LRA Q++P D+ +T V P R R + GA GQ G IR D Sbjct: 440 RLRAAGQDLPADFVKTTVLDNTPIRWFYRAASQESRSGA---------GQTGEIRVDVER 490 Query: 401 RTPNTQRGT 409 R P +RGT Sbjct: 491 RGPAGRRGT 499 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS28 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 29 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 57> which encodes amino acid sequence <SEQ ID 58; NGS29>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.61 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.03 threshold: 0.0 PERIPHERAL Likelihood = 4.03 modified ALOM score: −1.31 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.106(Affirmative) < succ> The protein has homology with the following sequences in the databases: emb\|CAB83930.1\|(AL162753) hypothetical protein NMA0640 [Neisseria meningitidis Z2491] Length = 387 Score = 653 bits (1685), Expect = 0.0 Identities = 324/388 (83%), Positives = 351/388 (89%), Gaps = 1/388 (0%) Query: 1 MNITIAAPYCSLPSEPHFNRFWYLAELLSQSHDVLLITSNFKHYDKSFRRPEDAKAASQG 60 MNITI APYCSLPSEP+FNRFWYLAE LSQSHDVLLITS F+HYDKSFRR EDA A S G Sbjct: 1 MNITIVAPYCSLPSEPYFNRFWYLAERLSQSHDVLLITSRFRHYDKSFRRHEDAAATSNG 60 Query: 61 RLKVMLLEESGYSKNVSLGRVTSHHRFVKHFEKWLENCRPGEQDVVYSAYPLIATNLLLG 120 RL+V LL+E GY KNVSL RV SH FV++ +WL + + EQD+VYSAYPL+ATNLLLG Sbjct: 61 RLRVKLLDEPGYRKNVSLARVASHRVFVRNLARWLHSPQAAEQDIVYSAYPLMATNLLLG 120 Query: 121 KHKARLGYKLIVDVQDVWPESFSSVVPFLKKIPHNLLPFASRANRAYRYADALVAVSQTY 180 KHKARLGYKLIVDVQDVWPESFSSVVPFLKK+PH LLPFASRANRAYR ADAL+AVSQTY Sbjct: 121 KHKARLGYKLIVDVQDVWPESFSSVVPFLKKVPHKLLPFASRANRAYRCADALIAVSQTY 180 Query: 181 LDRAKEANPNVPGEVVYIGADFAAIAPPPRFRSKTVRFFYLGTLSYNYDVETVCKGVRKL 240 LDRAKEANPNVPGE VYIG DFAAIA PPRFRSKTVR FYLGTLSY+YDVETVCKGVRKL Sbjct: 181 LDRAKEANPNVPGETVYIGTDFAAIA-PPRFRSKTVRLFYLGTLSYSYDVETVCKGVRKL 239 Query: 241 LDDGENVELHIMGGGPDLDRLKQYACDGIKFYGYIPYAEMMSVAKGCDIAVNAIHSYAMQ 300 LDDGENVELHIMGGGPDL++LKQY IKFYGY+PY+EMMS+AK CDIAVNAIHS+AMQ Sbjct: 240 LDDGENVELHIMGGGPDLEKLKQYENRAIKFYGYLPYSEMMSIAKACDIAVNAIHSHAMQ 299 Query: 301 SITNKLSDYMALQKPILNSQVHDEVAEVLTLLPHENYRSGDVDGFVQAAKDILKRKNDPV 360 S+TNKLSDYMALQKPILNSQ + EV ++L LLPHENYRSGDVD FVQAAK+ILKRK+DPV Sbjct: 300 SVTNKLSDYMALQKPILNSQNNAEVLDLLNLLPHENYRSGDVDSFVQAAKNILKRKDDPV 359 Query: 361 QSDEIVRRFRHDISYRKIVNLIERLANE 388 QSDEIVRRFR DISYRKIVNLIERLA+E Sbjct: 360 QSDEIVRRFRRDISYRKIVNLIERLAHE 387 >emb\|CAB58324.1\|(AL121855) hypothetical protein SCF62.09 [Streptomyces coelicolor A3 (2)] Length = 407 Score = 54.7 bits (130), Expect = 2e-06 Identities = 57/243 (23%), Positives = 105/243 (42%), Gaps = 24/243 (9%) Query: 99 RPGEQDVVYSAYP---LIATNLLLGKHKARLGYKLIVDVQDVWPESFSSVVPFLKKIPHN 155 R G DVV++ P L L L R G + + D D+ PE + S K + + Sbjct: 81 RVGPVDVVHACNPPDLLFLPALWL----KRRGARFVFDQHDLIPELYLSRFGRGKDLLYR 136 Query: 156 LLPFASANRAYRYADALVAVSQTYLDRAKEANPNVPGEVVYIGA-----DFAAIAPPPR 210 + R YR AD ++A +++Y D A P +V ++ F + P P Sbjct: 137 AVCALERWT--YRAADVVLATNESYKDVAIRRGGRRPDDVFVVRSAPATDRFQPVPPEPE 194 Query: 211 F-RSKTVRFFYLGTLSYNYDVETVCKGVRKLLDDGENVELH--IMGGGPDLDRLKQYA-- 265 R K YLG + V+ + + KL D+ + H +G G D + + + Sbjct: 195 LKRGKPHLLCYLGVMGPQDGVDYALRALAKLRDEVGRTDWHAVFVGSGDAFDAMVELSRS 254 Query: 266 ---CDGIKFYGYIPYAEMMSVAKGCDIAVNAIHSYAMQSIT--NKLSDYMALQKPILNSQ 320 + ++F G IP A++++ D+ ++ + ++ NK+ +YMA+ +PI++ Sbjct: 255 LGLDEQVRFTGRIPDADLVRHLSTADVCLSPDPRNPLNDVSTMNKVLEYMAMGRPIVSFD 314 Query: 321 VHD 323 + + Sbjct: 315 LRE 317 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS29 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 30 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 59> which encodes amino acid sequence <SEQ ID 60; NGS30>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −4.8 Possible cleavage site: 46 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 1.54 threshold: 0.0 PERIPHERAL Likelihood = 1.54 modified ALOM score: −0.81 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.113(Affirmative) < succ> The protein has homology with the following sequences in the databases: fastidiosa (strain 9a5c) gb\|AAF84279.1\|AE003977_2 (AB003977) conserved hypothetical protein [Xylella fastidiosa] Length = 376 Score = 73.6 bits (179), Expect = 3e−12 Identities = 82/354 (23%), Positives = 143/354 (40%), Gaps = 35/354 (9%) Query: 1 MKIILTTSMSGLGGTETATVRLGRLLKRHGHDIILASSDG-PFVGEAQASGIRWQPVDFY 59 MKI+ T + +G GG E R ++ GH + L G P A+ +G+ ++ + Sbjct: 1 MKILHTEAATGCGGEEIYIYRHMLSMQAQGHHMALLCQPGAPLSTMARNAGLPVYHINMH 60 Query: 60 RGGLAGYLKSTFAYARMLRREQPDIIDCQMARVVPACALAAKIVSPKTKIICHSHGLDAA 119 G L +L+RE D+++ A AA++ +T++I S L A Sbjct: 61 --GPWRVLNGIHTVQHLLQRETFDVVNTTSHVDTLIAAAAARLT--RTRLIVRSRHLMAP 116 Query: 120 TYPKTAKLFDKLGAYIIGNCICHEREKLIRHGFPAGRIAYA---------YNTPPEFHFRK 170 K+ + L +I +H R+ LI+ G RI +T PE +++ Sbjct: 117 I--KSQLTYTYLPHRVITVSQHRDLLIKQGIQPTRIGIVPPITA-QPPWMDTDPEHAWQR 174 Query: 171 TEK-------------ECAVLGTLSRLDTRAVHLMLDILKKMVGRNIPVRLNMAGIGEE 217 ++ ++G ++ L + +LD + + N + L +AG GE Sbjct: 175 LQQTRHVVRTELGFNDNDIIVGCVAVLREAKGHRELLDAIAPLCQANPRLHLVIAGDGEP 234 Query: 218 -MDNLKAQAKRLGIEDKVTFLGGVRDLTGYFKEVDILVNTPHCVGDHGAGVGNNILEAGL 276 M +L A K L +E ++ LG D DI + G LEA Sbjct: 235 VMQHLLAHRKTLTLETQIHLLGYRHDAPRLMSGFDIFA-----LATQKEAAGTVFLEAAQ 289 Query: 277 YDTPVVTYNMAGISEMVITGQTGYCIPFGDDEAFIEAVDTLIKHPELRSQNGKA 330 P++ + G+ EM+ G + G+ A A+ TL+ + + R MG+A Sbjct: 290 AGIPIIATRVGGVPEMLQEGTNAILVTPGNQTALTNALHTLVTNNQQRHSMGRA 343 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS30 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 31 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 61> which encodes amino acid sequence <SEQ ID 62; NGS31>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.36 Possible cleavage site: 16 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.50 threshold: 0.0 PERIPHERAL Likelihood = 3.50 modified ALOM score: −1.20 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.299(Affirmative) < succ> The protein has homology with the following sequences in the databases: gb\|AAB49297.1\|(U84350) hypothetical hydroxylase a [Amycolatopsis orientalis] Length = 491 Score = 111 bits (278), Expect = 1e−23 Identities = 87/269 (32%), Positives = 123/269 (45%), Gaps = 15/269 (5%) Query: 1 LKNGAAFSWGSRYTEFDF----TDKFSDGPGTVYQVRRAVRDKILIEEAAKQGVEVRFGH 56 +K G F WG+R + F + K + YQV RA FD IL++ A +GV VR G Sbjct: 73 IKRGGTFRWGARPEPWTFHFGISAKMAGSTSHAYQVERAKFDDILLKNAKSKGVVVREGC 132 Query: 57 GVTAFDNSGDFARLNIETDT-GESYELTAKFVLDASGY-GRVLPRLLNLETPSHLPPRQT 114 V G+ TD G ++E++A+FV+DASG R+ ++ S Sbjct: 133 SVNDVVEDGERVTGARYTDADGNAHEVSARFVIDASGNKSRLYTKVNGRNYSEFFRSLA 192 Query: 115 HFTHIDDNITHPKFDRNKILITTHPQHRDVWIWLIPFGDNRCSVGVV---GTPKLAGES 171 F + + P+ IL W W I P D SVG V DK+ G+ Sbjct: 193 LFGYFEGGKRLPEPVSGNILSVAFDSG---WFWYIPLSDTLTSVGAVVRREDADKIQGDR 249 Query: 172 ETVLKKFVYECPHLSEILDKAVWENDFPFRSIQ---GYSANVKSLHGEHFALLGNAAEFL 228 E L + ECP++SE L A + ++ YS S L+G+AA F+ Sbjct: 250 EKALNTLIAECPLISEYLSNATRVTRYGELRVRKDYSYQQDSYWRPGMVLVGDAACFV 309 Query: 229 DPVFSSGVTIALHSAELAADLLTKQLKGE 257 DPVFSSGV +A +SA LAA + L G+ Sbjct: 310 DPVFSSGVHLATYSALLAARSINSVLAGD 338 A homolog (amino acids 280-341) was found in serogroup A N.meningitidis but not in serogroup B, so NGS31 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 32 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 63> which encodes amino acid sequence <SEQ ID 64; NGS32>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.49 Possible cleavage site: 38 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.80 threshold: 0.0 PERIPHERAL Likelihood = 7.80 modified ALOM score: −2.06 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.278(Affirmative) < succ> The protein has homology with the following sequences in the databases: fastidiosa (strain 9a5c) gb\|AAF83310.1\|AE003899_2 (AE003899) phage-related repressor protein [Xylella fastidiosa] Length = 143 Score = 87.0 bits (214), Expect = 2e−16 Identities = 40/71 (56%), Positives = 54/71 (75%) Query: 1 MFSGEQLGQAISEAIKRKNVSQKEVADHFGVKQPSVSGWIKNGRIDKKHLDKLIDYFSDV 60 M +GEQLG+AI +A++ K V+ ++A+HFGVK PSV GWIK GRI K+ L L YFSDV Sbjct: 1 MLTGEQLGRAIKQAMQLKGVTPTKMAEHFGVKAPSSVYGWIKEGRISKEKLPSLWSYFSDV 60 Query: 61 VTPSHFGIETF 71 V P+H+G+E + Sbjct: 61 VGPTHWGLEAW 71 >sp\|P18680\|RPC1_BPHK0 26 KD REPRESSOR PROTEIN (REGULATORY PROTEIN CI) emb\|CAA34222.1\|(X16093) cI gene product (AA 1-208) [Bacteriophage HK022] Length = 235 Score = 80.5 bits (197), Expect = 2e−14 Identities = 60/200 (30%), Positives = 99/200 (49%), Gaps = 15/200 (7%) Query: 22 QKEVADHFGVKQPSVSGWIKNGRIDKKHLDKLIDYFSDVVTPSHF--------GIETFRV 73 Q ++A V ++S W 1 +K DK+ S + T + + GI + Sbjct: 29 QADLARLKVTPKAISKFNGESIPRK--DESLASVLGTTAAYLHGYADDDGITVNHL 86 Query: 74 LKSNEQSSIRFPRLNAEATCGAGT-INDHYIEVVDYVTVAAAWAREKLGGNLNK-IQVIT 131 +SN+ R L+ +A+ G GT +++ +IE + + AR G + ++VIT Sbjct: 87 SRSNDY--YRVDVLDVQASAGPGTMVSNEFIEKIRAIEYTTEQARILFNGRPQESVKVIT 144 Query: 132 ARGDSMEPTIENGDVMFVDTAVEAFDGDGLYLLWYIDGLKAKRLQSTVGGGLMIISDNSS 191 RGDSME TI GD +FVD ++ FDGDG+Y+ Y + KRLQ L +ISDN++ Sbjct: 145 VRGDSMEGTINPGDEIFVDVSITCFDGDGIYVFVYGKTMHVKRLQMQ-KNRLAVISDNAA 203 Query: 192 YRTETVRGEDLNAVRIIGRI 211 Y + + + I+ ++ Sbjct: 204 YDRWYIEEGEEEQLHILAKV 223 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 33 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 65> which encodes amino acid sequence <SEQ ID 66; NGS33>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.87 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.88 threshold: 0.0 PERIPHERAL Likelihood = 4.88 modified ALOM score: −1.48 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.313(Affirmative) < succ> The protein has homology with the following sequences in the databases: gb\|AAF31132.1\|(AF069529) Gp54 [Bacteriophage HK97] Length = 273 Score = 47.4 bits (111), Expect = 3e−04 Identities = 33/123 (26%), Positives = 52/123 (41%), Gaps = 20/123 (16%) Query: 221 NGGLSGKPKNANVPRRRKTHGVPLQEIADLYNEVLGGRLPSVQVLNDTRKRAIANRWCEM 280 NGG G+ K P RRK + + + YN +G RLP +N+ RKR + + Sbjct: 160 NGGGDGQVK----PERRKAERIDYESFLNAYNTEVGDRPHAVAVNEKRKRRL-KKIIPQ 214 Query: 281 LGTAAPNGKVRFGDKETGIAFAGFFRKVA--MNPFWMGENQTGFAVGFDWIFKAGNFVK 338 L T +G F + R PF+ G+N TG+ FD++ + + Sbjct: 215 LKTPNVDG-------------FRAYVRAFVHQAKPFYFGDNDTGWTADFDYLLREDSLTG 261 Query: 339 ILE 341 + E Sbjct: 262 VRE 264 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 34 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 67> which encodes amino acid sequence <SEQ ID 68; NGS34>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.65 Possible cleavage site: 50 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.76 threshold: 0.0 PERIPHERAL Likelihood = 3.76 modified ALOM score: −1.25 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.310(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|\|H82649 hypothetical protein XF1674 XF1569 [imported]- Xylella fastidiosa (strain 9a5c) gb\|AAF84378.1\|AE003986_8 (AE003986) hypothetical protein [Xylella fastidiosa] gg\|AAF84483.1\|AE003993_2 (AE003993) hypothetical protein [Xylella fastidiosa] Length = 316 Score = 167 bits (424), Expect = 2e−40 Identities = 108/308 (35%), Positives = 152/308 (49%), Gaps = 30/308 (9%) Query: 10 ETSVIRSLSSASLYMFTRRMFYQRRGYVWQRANHHAPICNLERVFTIGETKRLIINIPPR 69 E +VI++ A FTR F QR+ ++ HH I ++ V G K ++IN+PP Sbjct: 10 EQAVIKARCEADHLFFTRYFFKQRQQLRFRVNWHHHVIAGVVDDVIAGRRKDVVINVPPG 69 Query: 70 YSKTEIAVVNFIAWAMGRVPDCEFIHASYSAALAVNNSVQIRNLVQHEEYRAIFP-DLAL 128 SKTE+ +N +A + P F+H SYS LA+ NS R +VQ +EYRA++P ++A Sbjct: 70 SSKTELVAINVMARGLALNPYARFLHISYSDDLALLSETAREIVQSDEYRALWPLEIAD 129 Query: 129 AGESGHHWKTT-----AGGVMYXXXXXXXXXXXXXXRHREGFGGCIIIDDPHKADEARSE 183 +S W AGGV Y G+ G IIIDDP K ++A S+ Sbjct: 130 DAKSKKRWVVDGKKAGGV-YAVSLGGQVTGFRAGHAPGWQGAIIIDDPLKVEDAYSK 188 Query: 184 VRRQNVIDWFQNTVESRKNSPDTPIILIMQRLHEKDLAGWLLDGGNGEEWEHLCLPAIQE 243 R +TV+SRK SPDTPII+IMQRL + D G++ GG WE + +PA+ + Sbjct: 189 TGRSKANRKLVSTKSRASPDTPIIVIMQRLAQDDPTGFIQSGGFPGAWECIEIPALID 248 Query: 244 DG-----------------------TALWPEKHDIETLRREQAAPWFAGQYLQKPAPP 280 D + WP K + L +E YVF+GQY Q+P+P Sbjct: 249 DAYVSRLPEHVQGQVVRDAQDQDGRYSYWPYKEPLAELLALEATDRYVFSGQYQQRPSPL 308 Query: 281 DGGTFKPD 288 GG K D Sbjct: 309 GGGIIKGD 316 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 35 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 69> which encodes amino acid sequence <SEQ ID 70; NGS35>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.07 Possible cleavage site: 40 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 1.64 threshold: 0.0 PERIPHERAL Likelihood = 1.64 modified ALOM score: −0.83 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.020(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref\|NP_047925.1\|gp34 [Bacteriophage phi-C31] emb\|CAA07104.1\|(AJ006589) gp34 [Bacteriophage phi-C31] Length = 457 Score = 59.7 bits (143), Expect = 1e−07 Identities = 68/272 (25%), Positives = 117/272 (43%), Gaps = 49/272 (18%) Query: 226 GYSPVEQIIMTVNIALKRQVHALEYYTAGSVPDALVGVPETWSADDIRRFQEYWDLLLSG 285 G SP+ +++ AL Q + +++ G++P A+V VP T S + + R +E W SG Sbjct: 192 GCSPISYARESIGLALAAQKYGSKFFANGGAMPGAVVEVPGTMSKEGLARAREAWRAANSG 251 Query: 286 -----------ETAQRRKMRFVPGELSRNFRETRQPPLKDVYDEWLARVVCFAFSVEPTP 334 E A+ K+ P E F +T+Q + ++ AR+ F V P Sbjct: 252 VDNAHRVALLTEGAKFSKVAMSPDEAQ--FLQTRQFQVPEI-----ARI----FGVPPH- 299 Query: 335 FVAQVNRSVAETS--REQSLSDGMGSLKNWVKALIDDVLARYMDMAA--YEFVWKGEESL 390 ++ S + S EQ+++ M SL+ W++ + A + FV + + Sbjct: 300 LISDATNSTSWGSGLAEQNIAFTMFSLRPWLERIEAGFNRLLFAETADRFRFVKFNLDEI 359 Query: 391 N---PKEQAEIYAIYKNAGILTADEIRAELGKEPLP-GQG--------------QPEPDK 432 PKE+ E++++ GI + DE+RA PLP G G +PEP+ Sbjct: 360 KRGAPKERMELWSLGLQNGIYSIDEVRAAEDMTPLPDGLGEKYRVPLNLGEVGEEPEPEP 419 Query: 433 QDG----RKPEEPPNQGAEKLGKSESPMSEDE 460 P E P++ E GK + + +E Sbjct: 420 APAPPAIEPPAEEPDEEPEPEGKPDDEGATEE 451 A homolog (amino acids 641-700) was found in serogroup A N.meningitidis but not in serogroup B, so NGS35 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 36 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 71> which encodes amino acid sequence <SEQ ID 72; NGS36>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 4.3 Possible cleavage site: 26 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 27 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 1.70 threshold: 0.0 PERIPHERAL Likelihood = 1.70 modified ALOM score: −0.84 Score for OM-PP discrimination: 0.02 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 0.02 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Outer membrane?", "score: 0.00213559 Outer membrane?", "score: 0.00213559 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.232(Affirmative) < succ> The protein has homology with the following sequences in the databases: >pir\|\|D82437 TonB receptor-related protein VCA0625 [imported]-Vibrio cholerae (group O1 strain N16961) gb\|AAF96526.1\|(AE004392) TonB receptor-related protein [Vibrio cholerae] Length = 784 Score = 103 bits (256), Expect = 2e−20 Identities = 104/427 (24%), Positives = 162/427 (37%), Gaps = 100/427 (23%) Query: 31 NTEQQKELNTIVVHGKRS-ADQKGADDVYYKNVSNAYVGKEYLERYRVQSAGDVLKGLNG 89 NTEQ + T+ VHG+ DQ+ D L++ R + D+ G+ Sbjct: 57 NTEQAVD-ETVTVHGQSILTDQRTRSD---------------LDkVRGIANADIFSGITS 100 Query: 90 VYNMNTRTAGGAITPNIRGITGKGRIPVTIDGTEQTIDVWMNNYGVGDRNYLDPALFRSI 149 V + N GA+ IRG+ G+GR+P+ IDG+ Q+ GV DR Y+D L S+ Sbjct: 101 VQSNNMHNEAGALDIGIRGVQGEGRVIFIDGSLQSTHTSRGYQGVSDRTYIDTDLLSSL 160 Query: 150 AVEKSPALTRG--VKSGVGGAMSIRTIEPSDIIPEGRNWGIEVKTEFSGNTVAQKNDLRQ 207 V K + VGG ++ T+ DII + + +G+ +K Sbjct: 161 TVNKGATIESSPYASGAVGGVVNATTLGIKDIIKDDQAFGVVLK---------------- 204 Query: 208 FLGRDYRTLSPIGATADGVSGMPDVLTGYTGKPSPTALLLDEGIADTKFSGGKSHTNFKD 267 A A+ + PDV Y+ + LDE + F G Sbjct: 205 -------------ARANNHNRTPDVSGDYSEQGQ---YALDERGEHSAFKHG-------- 240 Query: 268 DRQLMLSAAFKTDITDGLAAYSHRQKGNYYAGKRGYQSYLNNPI--YGADACYDQYPDKS 325 LML ++ + + + AYS R KGN++AGK+GY+ Y P+ G + + S Sbjct: 241 --SLMLGLGYQAESFTVLAYSKRSKGNHFAGKKGYEEY-QEPVVGQGQEVVNTSFESDS 297 Query: 326 WREKDILCKSASLVPNMAVLFRPGEEIMNSHTDTKILLLKNNWYLPDNQKISLQYMDNK 385 W K S N +R H +L WY Y D K Sbjct: 298 WLFK---LASDTGTAHNADFNYR--------HHAQKAGEVLMAYWYKSSEDWEGNPYPDGK 347 Query: 386 IGFGEINPLITAWILGFAEQSLNEPVQQAPGIGTKIDSKTYKIGYEWKPQNNKWIDLQAD 445 + W LG A+ + TY Y ++P ++ W++L A+ Sbjct: 348 -------DRMPQWGLGTAKVN------------------TYSANYYYQP-DHPWLNLNAN 381 Query: 446 MWRVKTD 452 W + D Sbjct: 382 FWYTEAD 388 Score = 94.7 bits (234), Expect = 5e−18 Identities = 80/290 (27%), Positives = 126/290 (42%), Gaps = 37/290 (12%) Query: 929 SYDLDNHLFARYASRFPSLYELTAATGSGGLYGSETVAEYS----LKPEKSTNWEV 984 +Y L + +LF + +R R PSLYE T S V Y+ +KPE++ N EV Sbjct: 514 TYALTPSTQLFLKSSRTYRMPSLYETTL---------SNEVFSYNPYNPIKPEQAWNNEV 564 Query: 985 GYNFNFAPHFAKLRQGDLRLTYYSNKIKNQIDTSN--EDGGMIQ---------YDKAVSK 1033 G F + + + +L ++Y+ N IK+ I + GM + YDK Sbjct: 565 GVQFMASNSVLQDDRLNLSVSYFRNSIKDFISGGRLAKTPGMSEWQANFTFTNYDKLQLS 624 Query: 1034 GVELQSRLDSGRFFASFGGTYRLKHMVCDKGIAFKFDYYLQRVPECLEGGFGLSRFFQSL 1093 G EL + + F T + +C A C GF + Sbjct: 625 GWELGAHYQYAWILYTHPAATLYSETKICSVQQA-----QYAESDTCNSLGFAWGLTPTRI 679 Query: 1094 QPKYSLTLDVGTRFFNEKLELGMRAIHHSKAERRNYDKLIADGAGQVYARNGKPYGWHAA 1153 PK +L L+VGT+FFN+ L+ G++ +HS + N +A A Y Sbjct: 680 PPKQNLYLNVGTKFFNDTLDSGVSYHSG--KSNPSDWLAGTAANPILEIPSDY----- 732 Query: 1154 TLLDAYARYRIGKHIDLNFSVTNLANRYYLDPMSSTPVPGPGRTITFGIK 1203 +D Y++Y + + L F++ N+ +RY + P S +P PGRTIT G + Sbjct: 733 -TIDLYSQYELNANTQIFFAINNVTDRYQVRPGSVVSMPDPGRTITLGFE 781 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 37 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 73> which encodes amino acid sequence <SEQ ID 74; NGS37>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 4.47 Possible cleavage site: 21 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid composition of Predicted Mature Form: calculated from 22 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.21 threshold: 0.0 PERIPHERAL Likelihood = 7.21 modified ALOM score: −1.94 Score for OM-PP discrimination: 16.42 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 16.42 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Outer membrane?", "score: 1.64214 Outer membrane?", "score: 1.64214 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.938(Affirmative) < succ> The protein has homology with the following sequences in the databases: >sp\|Q03155\|AIDA_ECOLI ADHESIN AIDA-I PRECURSOR pir\|\|S28634 adhesin AIDA-I precursor-Escherichia coli plasmid pIB6 emb\|CAA46156.1\|(X65022) AIDA-I [Escherichia coli] Length = 1286 Score = 35.8 bits (81), Expect = 0.67 Identities = 34/138 (24%), Positives = 62/138 (44%), Gaps = 16/138 (11%) Query: 3 ASQLTLAVLLAAAFGSAYAVEVKGGDSSKGQLIQAAESDFLPFGSGAADIKVSTGNGLSK 62 A L + + + G+A+AV + G SS G + + E+ + G G ++ V++G ++ Sbjct: 31 AKNTLLVLAWSTIGNAPAVNISGTVSS-GGTVSSGETQIVYSGRGNSNATVNSGG-TQ 87 Query: 63 SINLEAGPAQRIRNKYGNAPINGGNQNTNVNGAANSRYLQPGDINPIA--GWFSKTRLA- 119 +N N + G+QN +GA S + G I ++ G S T L+ Sbjct: 88 IVNNGGKTTATTVN-------SSGSQNVGTSGATISTIVNSGGIQRVSSGGVASATNLSG 140 Query: 120 ---QVWYEKRANNTEVFS 134 ++ A+NT +FS Sbjct: 141 GAQNIYNLGHASNTVIFS 158 The protein was expressed in E.coli as an insoluble 32.45 kDa His-fusion product and then purified.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 38 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 75> which encodes amino acid sequence <SEQ ID 76; NGS38>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.34 Possible cleavage site: 24 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 25 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.98 threshold: 0.0 PERIPHERAL Likelihood = 3.98 modified ALOM score: −1.30 Score for OM-PP discrimination: 2.87 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 2.87 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Outer membrane?", "score: 0.287446 Outer membrane?", "score: 0.287446 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.607(Affirmative) < succ> The protein has homology with the following sequences in the databases: >sp\|Q03155\|AIDA_ECOLI ADHESIN AIDA-I PRECURSOR pir\|\|S28634 adhesin AIDA-I precursor-Escherichia coli plasmid pIB6 emb\|CAA461S6.1\|(X65022) AIDA-I [Escherichia coli] Length = 1286 Score = 35.8 bits (81), Expect = 0.67 Identities = 34/138 (24%), Positives = 62/138 (44%), Gaps = 16/138 (11%) Query: 3 ASQLTLAVLLAAAFGSAYAVKGGDSSKGQLIQAAESDFLPFGSGAADIKVSTGNGLSK 62 A L + + + G+A+A+V + G SS G + + E+ + G G ++ V++G ++ Sbjct: 31 AKNTLLVLAVVSTIGNAFAVNISGTVSS-GGTVSSGETQIVYSGRGNSNATVNSGG--TQ 87 Query: 63 SINLEAGPAQRIRKYGNAPINQGNQNTNVNGAANSRYLQPGDINPIA--GWFSKRLA- 119 +N N + G+QN +GA S + G I ++ G S T L+ Sbjct: 88 IVNNGGKTTATTVN-------SSGSQNVGTSGATISTIVNSGGIQRVSSGGVASATNLSG 140 Query: 120 ----QVWYEKRANNTEVFS 134 ++ A+NT +FS Sbjct: 141 GAQNIYNLGHASNTVIFS 158 >pir\|\|G81213 conserved hypothetical protein NMB0313 [imported]-Neisseria meningitidis (group B strain MD58) gb\|AAF40758.1\|(AE002388) conserved hypothetical protein [Neisseria meningitidis MC58] Length = 488 Score = 84.3 bits (207), Expect = 3e−15 Identities = 111/498 (22%), Positives = 185/498 (36%), Gaps = 35/498 (7%) Query: 7 LLFLPLCTVCLAAPSNDAADERRRLLDEGSRQTQQYRESGW--LDTEQARGEVEENDGY 64 +L LPL S A+E R D SR + E+ +D E+ G+V E + Sbjct: 19 MLLLPLLA------SAAYAEETPREPDLRSRPEFRLHEAEKPIDREKPGQVREKGKVL 72 Query: 65 SIGGEIYQVGDTAEELESAIYHALNARQWHKVRQFAARYAKLPRHKPALIHLADALQKRD 124 I GE E L A+Y A+ + +R Y + + L A + + Sbjct: 73 QIDGETLL--KNPELLSRAMYSAVVSNNIAGIRVILPIYLQQAQQDKMLALYAQGILAQA 130 Query: 125 EGDFRAAGNSFQTALEAEPDNPRLLLEAGRFYAEDNQNKESAAAFEKVLKTDIPAETRPI 184 +G + A + ++ + A+PD P + + E+ QN+ +A F+++ ++P + Sbjct: 131 DGRVKEAISHYRELIAAQPDAPAVRMRLAAALFENRQNEAAADQFDRLKAENLPPQLMEQ 190 Query: 185 VENYLSELGKRRRWHGQISLGYGYNSNVNQGNGINQCVWEIAGMCLMERTLPAPTDSTFS 244 VE Y L +R W N+NQ Q + T P D T Sbjct: 191 VELYRKALRERDAWKVNGGFSVTREHNINQAPKRQQ---------YGKWTFPKQVDGTAV 241 Query: 245 SYSATAEKTVPLKGNHGVQVRGVLYGNRYTEKDKDSAAMPDYGYNGSLYAGYAYADARS 304 +Y AEK LK G + G Y K + + G +AD R Sbjct: 242 NYRLGAEKKWSLKNGWYTTAGGDVSGRVYPGNKK-------FNDMTAGVSGGIGFADRRK 294 Query: 305 SFSLLPYFEYDFRNRHTHYRAWGADADWSRTLSPHNRINSHAGAKKTGYGGQSKTYFADF 364 L + E + GA ++R +P W+ S A + G ++ +D Sbjct: 295 DAGLAVFHERRTYGNDAYSYTNGARLYFNRWQTPKWQTLSSA---EWGRLKNTRRARSDN 351 Query: 365 KQYELGAGAEFSITLKSGLLVNFDAAPKAYP-EKSSSSKEYTARLGAYRLFSGGTYLNAV 423 ++ F + + D R+ P ++ + Y R A+ GG+ L+++ Sbjct: 352 THLQISNSLVFYRNARQYMGGLDFYRERNPADRGDNFNRYGLRF-AWGQEWGGSGLSSL 410 Query: 424 LLY--RRSLYDAASFVSDNK--RRRDKQYIMMAAAGFPQWNIKGVYPELRFRRTIAHSNA 479 L + Y+ F S K RRRDK+ + + KG+ P L SN Sbjct: 411 LRLGAAKRHYEKPGFFSGFKGERRRDKELNTSLSLWHRALHFKGITPRLTLSHRETRSND 470 Query: 480 VYYRYRQNEWLLGFKYRF 497 V+ Y +N +F F Sbjct: 471 VFNEYEKNRAFVEFNKTF 488 >pir\|\|C81790 conserved hypothetical protein NMA2174 [imported]- Neisseria meningitidis (group A strain Z2491) emb\|CAB85386.1\|(AL162758) conserved hypothetical protein [Neisseria meningitidis Z2491] Length = 490 Score = 84.0 bits (206), Expect = 4e−15 Identities = 111/498 (22%), Positives = 185/498 (36%), Gaps = 35/498 (7%) Query: 7 LLFLPLCTVCLAAPSNDAADERRRELLDEGSRQTQQYRESGW--LDTEQARGEVEENDGYI 64 +L LPL S A+E R D SR + E+ +D E+ G+V E + Sbjct: 23 MLLLPLLA------SAAYAEETPREPDLRSRPEFRLHEAEVKPIDREKVPGQVREKGKVL 74 Query: 65 SIGGEIYQVGDTAEELESAIYHALNARQWHKVRQFAARYAKLPRHKPALIHLADALQKRD 124 I GE E L A+Y A+ + +R Y + + L A + + Sbjct: 75 QIDGETLL--KNPELLSRAMYSAVVSNNIAGIRVILPIYLQQAQQDKMLALYAQGILAQA 132 Query: 125 EGDFRAAGNSFQTALEAEPDNPRLLLEAGRFYAEDNQNKESAAAFEKVLKTDIPAETRPI 184 +G + A + ++ + A+PD P + + E+ QN+ +A F+++ ++P + Sbjct: 133 DGRVKEAISHYRELIVAQPDAPAVRMRLAAALFENRQNEAAADQFDRLKAENLPPQLMEQ 192 Query: 185 VENYLSELGKRRWHGQISLGYGYNSNVNQGNGINQCVWEIAGMCLMERTLPAPTDSTFS 244 VE Y L +R W N+NQ Q + T P D T Sbjct: 193 VELYRRKALRERDAWKVNGGFSVTREHNINQAPKRQQ---------YGKWTFPKQVDGTAV 243 Query: 245 SYSATAEKTVPIKGNHGVQVGVLYGNRYTEKSDYGYRNGSLYAGYAYADARS 304 +Y AEK LK G + G Y K + + G +AD R Sbjct: 244 NYRLGAEKKWSLKNGWYTTAGGDVSGRVYPGNKK-------FNDMTAGVSGGIGFADRRK 296 Query: 305 SFSLLPYFEYDFRNRHTHYRAWGADADWSRTLSPHRINSHAGAKKTGYGGQSKTYFADF 364 L + E + GA ++R +P W+ S A +G ++ +D Sbjct: 297 DAGLAVFHERRTYGNDAYSYTNGARLYFNRWQTPKWQTLSSA---EWGRLKNTRRARSDN 353 Query: 365 KQYELGAGAEFSITLKSGLLVNFDAARKAYP-EKSSSSKEYTARLGAYRLFSGGTYLNAV 423 ++ F + + D R+ P ++ + Y R A+ GG+ L+++ Sbjct: 354 THLQISNSLVFYRNARQYWMGGLDFYRERNPADRGDNFNRYGLRF-AWGQEWGGSGLSSL 412 Query: 424 LLY--RRSLYDAASFVSDNK--RRRDKQYIMMAAAGFPQWNIKGVYPELRFRRTIAHSNA 479 L + Y+ F S K RRRDK+ + + KG+ P L SN Sbjct: 413 LRLGAAKRHYEKPGFFSGFKGERRRDKELNTSLSLWHRALHFKGITPRLTLSHRETRSND 472 Query: 480 VYYRYRQNEWLLGFKYRF 497 V+ Y +N + F F Sbjct: 473 VFNEYEKNRAFVEFNKTF 490 The protein was expressed in E.coli as an insoluble 52.03 kDa His-fusion product and then purified.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 39 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 77> which encodes amino acid sequence <SEQ ID 78; NGS39>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.38 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.16 threshold: 0.0 PERIPHERAL Likelihood = 7.16 modified ALOM score: −1.93 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.325(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref\|NP_052685.1\|serine protease EspP [Eseherichia coli] pir\|\|T00317 probable serine proteinase espP, extracellular-Escherichia coli plasmid pO157 pir\|\|T42120 probable serine proteinase espP, extracellular-Escherichia coli plasmid pO157 emb\|CAA66144.1\|(X97542) putative exoprotein-precursor [Escherichia coli] dbj\|BAA31836.1\|(AB011549) serine protease EspP [Escherichia coli] gb\|AAC70088.1\|(AF074613) putative exoprotein-precursor [Escherichia coli O157:H7] Length = 1300 Score = 58.9 bits (141), Expect = 2e−07 Identities = 153/687 (22%), Positives = 248/687 (35%), Gaps = 106/687 (15%) Query: 194 DLTVENKTLSDA---EFGVYALNTSMVNLSSKDNNEVKSTQVGLYSQDGGSINVDR--- 247 D +NT +DA Y N ++ +LS D E + + G + +V R Sbjct: 595 DYVAGMQNTEADAVKQNGNAYKTNNAVSDLSQPDW-ETGTFRFGTLHLENSDFSVGRNAN 653 Query: 248 --------KDNIIEGDAVALVGKGGSQNIRAS----RTLISSKSLGIHAEQAAKIAITG 295 K NI GD A + +NI R N++ S G E IT Sbjct: 654 VIGDIQASKSNITIGDTTAYIDLHAGKITGDGFGFRQNIVRGNSQG---ETLFTGGITA 710 Query: 296 ASNTIHASNAAIRSLDKSEVKIDGQITIDSNVANLARQDGSIH---LNYKDDTRITGATV 352 +TI + A ++ + TI+ N A++ Q G ++ + +TG Sbjct: 711 EDSTIVIKDKAKALFSNYVYLLNTKATIE-NGADVTTQSGMFSTSDISISGNLSMTGNPD 769 Query: 353 SDKGLVAIKPLNNTNIVADTIHYKGDVLAVNKGKVELDF----TPNILLAGRLDNFSGLT 408 D LN+ + + + ++A NK V D + +I+ + S L+ Sbjct: 770 KDNKFEPSIYLNDASYLLTPDSAR--LVMASWGPIHSTKSASIMPGHDESPLSQLS 827 Query: 409 DSKHKNLFENYVANLDSKSAGEINFNLAKDAL----WTMTGQSWLDKLEGQGTIDFNNDA 464 D K L + D G +N A + W +TG S L L+ ++ + D+ Sbjct: 828 DRTSKGLALGLLGGFDVSYRGSVNAPSASATMNNTWWQLTGDSALKTLKSTNSMVYFTDS 887 Query: 465 KTSGR--ALHIGELAGANK-FLMHLNKDGIHSDMLYVKKGTSTPQEVVVKNLSEVLDSMN 521 + + L + ELA +N + M N SD L VKK S ++ L + L Sbjct: 888 ANNKKFHTLTVDELATSNSAYAMRTNLS--ESDKLEVKKHLSGENNIL---LVDFLQKPT 942 Query: 522 YGERLRFATVTNSKNEFVNGKKYIDDTHLMEDALTVEYSAHNGXXXXXXXXXXSFNGSEM 581 ++L V+ K+ N K T D V Sbjct: 943 PEKQLNIELVSAPKTNENFKASKQTIGFSDVTPV------------------------ 978 Query: 582 TAEKAGDPYVNKTYTDNRQNVYLVKQATGNPSRNVKNINDMFDSTAHYAFT--LDTYAKR 639 + DD + T++ N K+AT N + S + AF ++ KR Sbjct: 979 ITTRETDDKI--TWSLTGYNTVANKEATRNAAA--------LFSVPYKAFLNEVNNLNKR 1028 Query: 640 EGERAFSTLDKKEGDWIRLTHTRVIQSNAFRFHNNDFEIGYDRFSLNEQEKKRKWGISLD 699 G+ ++ + G W R+ S F + ++G D+ K G+ L Sbjct: 1029 MGD--LRDINGEAGAWARIMSGTGSASGGFSDNYTHVQVGVDK-------KHELDGLDLF 1079 Query: 700 YGHGRTSLWNTFGKD----KIRKYELALYNTTQYIDKEGDETGYIDNVLKIGKLRNRVIA 755 G T ++ D K + LY + + D YID + K N A Sbjct: 1080 TGFTVTHTDSSASAFSGKTKSVGAGLYASAMF-----DSGAYIDLIGKYVHHDNEYTA 1134 Query: 756 RNHMGQLWGKGKYSNTLFSISTEYGRRKFLDDDKLWRITPQVQLQYSYLRGTGYRI-DNG 814 G G YS + E G R + +D W I PQ +L Y + G + D G Sbjct: 1135 -TFAGL--GTRDYSTHSWYAGAEAGYRYHVTEDA-W-IEPQAELVYGSVSGKQFAWKDQG 1189 Query: 815 INVNLSHA--NSLIGRLGLDVVRKFDG 839 +++++ N LIGR G+DV + F G Sbjct: 1190 MHLSMKDKDYNPLIGRTGVDVGKSFSG 1216 Score = 36.6 bits (83), Expect = 1.2 Identities = 97/412 (23%), Positives = 164/412 (39%), Gaps = 83/412 (20%) Query: 63 DNIVTMKSGDADADYVNNSKVLTETPYYKSKRGSNGIFAYGDKSLVKLIGEIVK--SE 120 D V G + ++ SK Y + +G + A+ S V + +N + +E Sbjct: 163 DKFVVETRGATEGADISLSKQQALERYGVNYKGEKKLIAFRAGSGVVSVKKNGRITPFNE 222 Query: 121 ISEKSKALNGGFRHIGIYS-W---QNAKVE----LSSKSDN--------------IVQGG 158 +S K + LNG F HI +S W N + + ++++ D+ +V G Sbjct: 223 VSYKPEMLNGSFVHIDDWSGWLILTNNQFDEFNNIASQGDSGSALFVYDNQKKKWVVAGT 282 Query: 159 IWGLYS----NNSSISLKGKNNVISNPKYNVFAYKKAKVDLTVENKNTLSDAEFGVYALN 214 +WG+Y+ N + K I N K N ++Y VD++ T+ + + + Sbjct: 283 VWGIYNYANGKNHAAYSKWNQTTIDNLK-NKYSY---NVDMSGAQVATIENGK--LTGTG 336 Query: 215 TSMVNLSSKDNNEVKSTQVGLYSQ----DGGSINVDRKDNIIEGDAVALVGKG-----GS 265 + ++ +KD + L S GG + D+K + GD G G GS Sbjct: 337 SDTTDIKNKDLIFTGGGDILLKSSFDNGAGGLVFNDKKTYRVNGDDFTFKGAGVDTRNGS 396 Query: 266 Q---NIR-ASRTNL--ISSKSLGIHAEQAAK------IAITGASNTIHASNAAIRSLDKS 313 NIR ++ NL I +L + Q + I GA T +N I S D Sbjct: 397 TVEWNIRYDNKDNLHKIGDGTLDVKTQNTNLKTGEGLVILGAEKTF--NNIYITSGD-G 453 Query: 314 EVKIDGQITIDSNVAN---LARQDGSIHLN-YKDDTRITGATSDKGLV--------AIK 361 V+++ + + N A+ G++ LN Y AT D G V +I Sbjct: 454 TVRLNAENALSGGEYNGIFFAKNGGTLDLNGYNQSFNKIAAT--DSGAVITNTSTKKSIL 511 Query: 362 PLNNTNIVADTIHYKG-----DVLAVNKGKVELDFTPNILLAGRLDNFSGLT 408 LNNT AD I++ DVL ++ K E ++L G +D + ++ Sbjct: 512 SLNNT---ADYIYHGNINGNLDVLQHHETKKE---NRRLILDGGVDTTNDIS 557 The protein was expressed in E.coli as an insoluble 95.92 kDa His-fusion product and then purified.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 40 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 79> which encodes amino acid sequence <SEQ ID 80; NGS40>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.18 Possible cleavage site: 17 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.05 threshold: 0.0 PERIPHERAL Likelihood = 7.05 modified ALOM score: −1.91 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.108(Affirmative) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 41 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 81> which encodes amino acid sequence <SEQ ID 82; NGS41>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) signal Score (−7.5): −2.47 Possible cleavage site: 17 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 16 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.37 threshold: 0.0 PERIPHERAL Likelihood = 7.37 modified ALOM score: −1.97 Rule: inner or outer membrane protein Rule: inner or outer membrane protein * Reasoning Step: 2 Lipoprotein?", "Inner membrane?", "----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> The protein has no homology sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 42 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 83> which encodes amino acid sequence <SEQ ID 84; NGS42>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.2 Possible cleavage site: 14 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 6.58 threshold: 0.0 PERIPHERAL Likelihood = 6.58 modified ALOM score: −1.82 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.514(Affirmative) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 43 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 85> which encodes amino acid sequence <SEQ ID 86; NGS43>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.34 Possible cleavage site: 39 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 2 value: −4.78 threshold: 0.0 INTEGRAL Likelihood = −4.78 Transmembrane 1881-1897 (1876-1898) INTEGRAL Likelihood = −1.01 Transmembrane 1966-1982 (1966-1982) PERIPHERAL Likelihood = 1.91 modified ALOM score: 1.46 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.291(Affirmative) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 44 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 87> which encodes amino acid sequence <SEQ ID 88; NGS44>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.49 Possible cleavage site: 58 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −1.33 threshold: 0.0 INTEGRAL Likelihood = −1.33 Transmembrane 141-157 (140-157) PERIPHERAL Likelihood = 2.54 modified ALOM score: 0.77 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.153(Affirmative) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 45 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 89> which encodes amino acid sequence <SEQ ID 90; NGS45>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.07 Possible cleavage site: 46 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.07 threshold: 0.0 PERIPHERAL Likelihood = 2.07 modified ALOM score: −0.91 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.333(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref\|NP_049512.1\|putative portal protein [Bacteriophage 933W] ref\|NP_050550.1\|hypothetical protein [Bacteriophage VT2-Sa] gb\|AAD25457.1\|AF125520_52 (AF125520) putative portal protein [Bacteriophage 933W] dbj\|BAA84334.1\|(AP000363) hypothetical protein [Bacteriophage VT2-Sa] dbj\|BAA94158.1\|(AP000422) portal protein [Escherichia coli] O157:H7] Length = 714 Score = 314 bits (805), Expect = 2e−84 Identities = 213/658 (32%), Positives = 327/658 (49%), Gaps = 22/658 (3%) Query: 7 ETGVLPDKNGEPLTIG---EYRLFVGEMMNQPAWRAVADKEMDYADGRQLDNELLQKQR 62 ET + KN T + + ++ +QP WR A+K Y DG QL E+LQ + Sbjct: 4 ETNTMATKNDNGATPRFSQRQLQALCSDIDSQPKWRDAANKACAYYDGDQLPPEVLQVLK 63 Query: 63 ELGLPPAVENLITPTLLSVQGYEATIRTDWRVTADGETGGRD-VADALNFKLNRAERQSR 121 + G P + NLI PT+ V G EA RTD V +D + +A+A+N + A R Sbjct: 64 DRGQPMTIHNLIAPTVDGVLGMAKTRTDLVVMSDEPDDETEKLAEAINAEFADACRLGN 123 Query: 122 ADKACSDAFRGQIACGIGWVTRNPNPFEFPYECGVIHRNAIHWDMKSYKYDLSDARWL 181 +KA SDA+ QI G+ WVEV RN +PF ++ + RN + WD S + DLSD RWL Sbjct: 124 MNKARSDAYAEQIKAGLSWVEVRRNSDPFGPEFKVSTVSRNEVFWLSREADLSDCRWL 183 Query: 182 IRRWLLPERLAQFFPEYAGHFKAMGRGGSDWR-ISGELDGGGNTGLADAWGISGRNTV 240 +RRRW+ + FP G + + DWR + G +L AW Sbjct: 184 MRRRWMDTDEAKATFP---GMAQVIDYAIDDWRGFVDTTVTEOQPSPLMSAWEEYQSWDR 240 Query: 241 SEEFWFNETTRELAVAEVWYRRWVTADCLRDKKTGRTVEFDGANPNHREMAANGAV-LFA 299 + W R + + V+YR + + + GR V FD N A+G V + Sbjct: 241 QQNEWLQRERRRVLLQVVYYRTFERLPVI-ELSNQRVVAFDKNNLMQAVAVASGRVQVKV 299 Query: 300 ASVPRMRRAFVVGDLVVRDEPTPYPHQKFPYPFFGFREDNTGIPYVRNMKYAQDNLN 359 V R+R A+ VG + D P P FP VPF+G++R+D TG PYG + AQD +N Sbjct: 300 GRVSRIREAWFVGPHFIVDRPCSAPQGMFPLVPFWGYRKDKTGEPYGLISEAIPAQDEVN 359 Query: 360 STNSKLRWGLSAIRTVRTKGIVDMSDEQFRRNIARVDADIVLNKIEAAQPGAR--FDVSR 417 KL W L A R + + +SD I R D I LN + Q F V + Sbjct: 360 FRRIKLTWLLQAKRVIMDEDATQLSDNDLMEQIERPDGIIKLNPVRKNQKSVADVFRVEQ 419 Query: 418 DFELSAQHWQMLQDSRATIRQISGITPSVGNRGNATSGRQESIQVEQSNQSLGLVMDNF 477 DF++++Q +Q++Q+S I+ G+ +F+G ATSG S VEQ +L + DN+ Sbjct: 420 DFQVASQQFQVMQESEKLIQDTMGVYSAFLGQDSGATSGVAISNLVEQGAPLAEINDNY 479 Query: 478 RQSRSLVGELLLAMIIEDLGS-DEQTVVIEGDAVTQGRTVVINRPETDPVTGKAYLSNDL 536 + + VG LLLA +++DL VVI D + +T+V+N E D L+ND+ Sbjct: 480 QFACQQVGRLLLAYLLDDLKKRRNHAVVINRDDRQRRQTIVLN-AEGD----NGELTNDI 534 Query: 537 QNIRLKVALEDVPSTNSYRSQQLGAMSEAVKSLPPEYQAAVLPFMVSLMDIPFKDKVIEK 596 + +AL V T ++++Q MSE ++ LPP+ QA VL V+L+D+P K + +E+ Sbjct: 535 SRLNTHIALAPVQQTPAFKAQLAQHMSEVIQGLPPQVQAVVLDLWVNLLDVPQKQEFVER 594 Query: 597 IK-EVRVQETPEQI--EARIAQAVQDALAKSGNDIKRRELALKEQRTASEIKEIEARA 651 I+ + ++P+++ E + A Q AL + +++ RE+A + + ++ A A Sbjct: 595 IRAALGTPKSPDEMTPEEQEVAAQQQALQQQQAELQMREMAGRVAKLEADAARAHAAA 652 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 46 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 91> which encodes amino acid sequence <SEQ ID 92; NGS46>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.25 Possible cleavage site: 37 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.77 threshold: 0.0 PERIPHERAL Likelihood = 4.77 modified ALOM score: −1.45 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.281(Affirmative) < succ> The protein has homology with the following sequences in the databases: >sp\|P44184\|YE10_HAEIN HYPOTHETICAL PROTEIN HI1410 pir\|\|E64028 hypothetical protein HI1410-Haemophilus influenzae (strain Rd KW20) gb\|AAC23058.1\|(U32820) H. influenzae predicted coding region HI1410 [Haemophilus influenzae Rd] Length = 394 Score = 150 bits (379), Expect = 3e−35 Identities = 75/168 (44%), Positives = 114/168 (67%), Gaps = 2/168 (1%) Query: 57 REIQKSMRDSVHRLLKDIWAQLGLGHYEITDFEIRGANGTLFVFSGLQSHTVDSIKSFE 116 REIQKS+ DSV ++L D++ L L F+++ +I G NG+ F F+GL+++ + SIKS Sbjct: 3 REIQKSISDSVIQMLADQIEMLSLQAFVQKTQIIGQNGSRFTFAGLKTN-ITSSIKSMT 61 Query: 117 GIDIVWVEEGHGVSKKSWDVLTPTIRKEGSEIWITLWPDMETDETYRRFIAMPSEDTWLC 176 GID+VWVEEG VSK+SWD+L PTIR++GS+I ++NP D+TY+RF+ P E Sbjct: 62 GIDVVWVEEGENVSESWDILIPTIREDGSQIIVSFNPKNILDDTYQRFVIHPPERCKSV 121 Query: 177 EINWRDNPWFPEALNRERLKAQRSMNKEDYGNIWEGRPRNVSEGAVYR 224 +NW+DNP+FP+L E ++ R + E Y +++EG P S+ A+ + Sbjct: 122 LVNWQDNPYFPKEL-MEDMEQMRERDYELYRHVYEGEPVADSDLAIIK 168 >ref\|NP_050979.1\|P18 [Bacteriophage APSE-1] gb\|AAF03961.1\|AF157835_18 (AF157835) P18 [Bacteriophage APSE-1] Length = 469 Score = 117 bits (294), Expect = 2e−25 Identities = 72/233 (30%), Positives = 110/233 (46%), Gaps = 13/233 (5%) Query: 17 LFKPCRYKVMYXXXXXXXXXXXXXXXXXXXXQRPLRILCAREIQKSMRDSVHRLLKDKVA 76 +FKP R KV + R LC RE S+ DS H +L+ +V Sbjct: 1 MFKPKRIKVYFGGRGGMKTVSFAKIALITASMHKRRFLCLREFMNSIEDSGHAVLQAEVE 60 Query: 77 QLGLGHFYEITDEEIRGANGTLFVFSGLQSHTVDSIKSFEGIDIVWVEEGHGVSKKSWDV 136 LGL + + I + I G N ++F + L + + SIKS D+ WVEE VS+KS D Sbjct: 61 TLGLQNRFRILNTYIEGINDSIFKYGQL-ARNIASIKSKHDFDVAWVEEAETVSEKSLDS 119 Query: 137 LTPTIRKEGSEIWITLNPDMETDETYRRFIA-----------MPSEDTWLCEINWRDNPW 185 L PTIRK GSE+W + NP E Y+RF+ +D ++ ++++ DNPW Sbjct: 120 LIPTIRKPGSELWFSFNPAEEDGAVYKRFVKPYKELIDTQGYYEDDDLYVGKVSYLDNPW 179 Query: 186 FPEALNRERLKAQRSMNKEDYGNIWEGRPMVSEGAVYRHEIQDAFHSGRVTL 238 P L + K +R N + + +++ G E A+ + E +A + L Sbjct: 180 LPAELKNDAQKMKRE-NYKKWRHVYGGECDANYEDALIQPEWVEAAIDAHIKL 231 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 47 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 93> which encodes amino acid sequence <SEQ ID 94; NGS47>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.87 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.88 threshold: 0.0 PERIPHERAL Likelihood = 4.88 modified ALOM score: −1.48 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.313(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref\|NP_037739.1\|Gp54 [Bacteriophage HK97] gb\|AAF31132.1\|(AF069529) Gp54 [Bacteriophage HK97] Length = 273 Score = 47.4 bits (111), Expect = 3e−04 Identities = 33/123 (26%), Positives = 52/123 (41%), Gaps = 20/123 (16%) Query: 242 NGGLSGKPKNANVPRRRKTHGVPLQEIADLYNEVLGGRLPSVQVLNDTRKRAIANRWCEM 301 NGG G+ K P RRK + + + YN +G RLP +N+ RKR + + Sbjct: 160 NGGGDGQVK----PERRKAERIDYESFLNAYNTEVGDRLPHAVAVNEKRKRRL-KKIIPQ 214 Query: 302 LGTAAPNGKVRFGDKETGLAWFAGFFEKVA--MNPFHGENQTGPAVGFDWIFKAGNFVK 359 L T +G F + R PF+ G+N TG+ FD++ + ++ Sbjct: 215 LKTPNVDG-------------FRAYVRAFHQAKPFFGDNDTGWTADFDYLLREDSLTG 261 Query: 360 ILE 362 + E Sbjct: 262 VRE 264 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 48 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 95> which encodes amino acid sequence <SEQ ID 96; NGS48>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.85 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.16 threshold: 0.0 PERIPHERAL Likelihood = 7.16 modified ALOM score: −1.93 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.379(Affirmative) < succ> The protein has homology with the following sequences in the databases: >dbj\|BAA36059.1\|(D90754) Outer membrane protein P.69 precursor [Escherichia coli] Length = 762 Score = 64.7 bits (156), Expect = 1e−09 Identities = 79/292 (27%), Positives = 121/292 (41%), Gaps = 55/292 (18%) Query: 3 NGARWTVTNDSMLKELDLSEDAQVEFSDNNK----FVKVSVSKLKGDGGVFKMYGDIV-- 56 N + W VT++S L L LS V+F+ + F ++V L G+ F M D+V Sbjct: 289 NNSVWNVTSNSNLDTLALSHST-VDFASHGSTAGTFATLNVENLSGNS-TFIMRADVVGE 346 Query: 57 ----KGESDKLITRKGSEGTHIIEYMDDAKAKTTGREYLKLVENKGNQEDNKASNKASYK 112 + D L S G H++ + TTG E L +V+ D AS AS + Sbjct: 347 GNGVNNKGDLLNISGSSAGNHVLAIRNQGSEATTGNEVLTVVKTT----DGAASFSASSQ 402 Query: 113 LNVRCTEQGGWCFALGESG------ASKKVNISTDGKRDF-------YLYPD-------- 151 + E GG+ + + ++G AS V T + PD Sbjct: 403 V-----ELGGYLYDVRKNGTNWELYASGTVPEPTPNPEPTPAPAQPPIVNPDPTPEPAPT 457 Query: 152 ---TLTPGASSSVLFGEALYQLNAVSDETLVQRMGEIHADGMPQEDNNVWIKRVGGKFSG 208 T T A + L Y LN V +TL+QRMG++ +D N+W++ GG Sbjct: 458 PKPTTTADAGGNYL--NVGYLLNYVENRTLMQRMGDLRNQ---SKDGNIWLRSYGGSLDS 512 Query: 209 SRSDYRVGGYGNRYWGFAGGFNRTGFGDKWIHYKGLMLRHLQSSYASEDYVG 260 S ++ G+ Y G G ++ D Y GL ++ S++AS DY G Sbjct: 513 FASG-KLSGFDMGYSGIQFGGDKR-LSDVMPLYVGL---YIGSTHASPDYSG 559 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 49 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 97> which encodes amino acid sequence <SEQ ID 98; NGS49>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −8.37 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.93 threshold: 0.0 PERIPHERAL Likelihood = 4.93 modified ALOM score: −1.49 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.355(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: gi\|11282647\|pir\|H81959 patch repair protein (EC 3.1.-.-) NMA0429 [imported]-Neisseria meningitidis (group A strain Z2491) gi\|7379179\|emb\|CAB83728.1\|(AL162753) patch repair protein [Neisseria meningitidis Z2491] Length = 140 Score = 256 bits (628), Expect = 8e−68 Identities = 131/140 (93%), Positives = 132/140 (93%) Query: 1 MTDIFTPSKRSFVMSKIHSKETKPEVLVRKFLFSQGFRYRKNDKRYAGKPDIVLPKYKTV 60 MTDIFT SKRSFVM KIHSKETKPEVLVRKFLF QGFRYRKNDKRY GKPDIVL KYKTV Sbjct: 1 MTDIFTTSKRSFVMLKIHSKETKPEVLVRFLFFQGFRYRKNDKRYVGKPDIVLSKYKTV 60 Query: 61 VFIHGCFWHGHSCNKGHIPKSNMDFWLEKITKNRERDIKNETELEKIGFKVIVVWECELK 120 VFIHGCFW+GHSCNKGHIPKSN DFWLEKITKN ERDIKNETELEKIGFKVIVVWECELK Sbjct: 61 VFIHGCFWYGHSCNKGHIPKSNTDFWLEKITKNCERDIKNETELEKIGFKVIVVWECELK 120 Query: 121 NKAICRERLNRLVEEIKDAV 140 NKAICRERLNRLV EIKDAV Sbjct: 121 NKAICRERLNRLVREIKDAV 140 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 50 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 99> which encodes amino acid sequence <SEQ ID 100; NGS50>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.6 Possible cleavage site: 50 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.80 threshold: 0.0 PERIPHERAL Likelihood = 7.80 modified ALOM score: −2.06 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.398(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|11352963\|pir\|\|G81959 conserved hypothetical protein NMA0428 [imported]-Neisseria meningitidis (group A strain Z2491) gi\|7379178\|emb\|CAB83727.1\|(AL162753) conserved hypothetical protein [Neisseria meningitidis Z2491] Length = 548 Score = 371 bits (954), Expect = e−102 Identities = 189/197 (95%), Positives = 194/197 (97%) Query: 1 VKGESGVDIENWKNKLPEKEREPVEVILNRLEDSELTNKEQAEVISALHSIIPEYPYYHW 60 VKGESGVDIE+WKNKLPEKEREPVEVILNRLEDSELTNKEQAEVISALHSIIPEYPYYHW Sbjct: 350 VKGESGVDIEDWKNKLPEKEREPVEVILNRLEDSELTNKEQAEVISALHSIIPEYPYYHW 409 Query: 61 RHLHQDLHTACNDFYNEKKDYLSAAIEAVKVFEDKVQKQTGLHSIDGRELIEKAFGSKKS 120 RHLHQDLHTACNDFYNEKKDYLSAAIEAVKVFEDKVQKQTGLHSIDGRELIE+AFGSK S Sbjct: 410 RHLHQDLHTACNDFYNEKKDYLSAAIEAVKVFEDKVQKQTGLHSIDGRELIEQAFGSKKS 469 Query: 121 MLLLTNNKTQAEQNLEDGLEQLACGTWTGFRNPVQHELRANLSPSIFNDKDALDLISLVS 180 +LLLTNNKT+AEQNLEDGLEQLACGTWTGFRNPVQHELRANLSPSIFNDKDALDLISLVS Sbjct: 470 ILLLTNNKTKAEQNLEDGLEQLACGTWTGFRNPVQHELRANLSPSIFNDKDALDLISLVS 529 Query: 181 YLLRKVEQTKKRAKPTS 197 YLLRKVEQTKKR+K S Sbjct: 530 YLLRKVEQTKKRSKVVS 546 >gi\|10955124\|ref\|NP_059780.1\|ymh [Agrobacterium tumefaciens] gi\|5738274\|gb\|AAB91582.2\|(AF242881) ymh [Agrobacterium tumefaciens] Length = 266 Score = 58.7 bits (141), Expect = 5e−08 Identities = 40/127 (31%), Positives = 69/127 (53%), Gaps = 5/127 (3%) Query: 61 RHLHQDLHTACNDFYNEKKDYLSAAIEAVKVFEDKVQKQTGLHSIDGRELIEKAFGSKKS 120 R +H D+ C + +Y A +EAVK DK++++TGL + DG L+++AF Sbjct: 137 RGVHPDVLRFCREEL-LVDNYFHAVLEAVKSVADKIRQTGL-TDDGAVLVDRAFSGDAP 194 Query: 121 MLLLTNNTQAEQNEDGLEQLACGTWTGFRNPVQHELRANLSPSIFNDKDALDLISLVS 180 ML + ++++E+ + G L GT++ FRN H R + S +DA DL S+ S Sbjct: 195 MLAINELQSESEKGEQRGFSNLVKGTFSMFRNTTAHAPRIHWQMS---KEDAEDLFSMFS 251 Query: 181 YLLRKVE 187 + R+++ Sbjct: 252 LMHRRID 258 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS50 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 51 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 101> which encodes amino acid sequence <SEQ ID 102; NGS51>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.14 Possible cleavage site: 42 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.67 threshold: 0.0 PERIPHERAL Likelihood = 5.67 modified ALOM score: −1.63 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.145(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|\|G81959 conserved hypothetical protein NMA0428 [imported]-Neisseria meningitidis (group A strain Z2491) emb\|CAB83727.1\|(AL162753) conserved hypothetical protein [Neisseria meningitidis Z2491] Length = 548 Score = 532 bits (1371), Expect = e−150 Identities = 272/285 (95%), Positives = 280/285 (97%) Query: 1 MSEEKLKMSFEPTVIEHLGVKMYSHTVPAIAELIANAYDACATEVEVRLFDKPEHKIVIK 60 MSEEKLKMSFEPTVIEHLGVKMYSHTVPAIAELIANAYDACATEVEVRLFDKPEHKIVIK Sbjct: 1 MSEEKLKMSFEPTVIEHLGVKMYSHTVPAIAELIANAYDACATEVEVRLFDKPEHKIVIK 60 Query: 61 DNGIGMSFDEINDFYLRIGRNRREEKQASPCGRIPTGKKGLGKLALFRLGNKIEISTIQG 120 DNGIGMSFDEINDFYLRIGRNRREEKQASPCGRIPTGKKGLGKLALF LGNKIEISTIQG Sbjct: 61 DNGIGMSFDEINDFYLRIGRNRREEKQASPCGRIPTGKKGLGKLALFRLGNKIEISTIQG 120 Query: 121 NERVTFTLDYAEIKKSERIYQPEFQKESVKPNTENGTTITLTELTKKQGYPLDNYVGHLS 180 NERVTFTLDYAEI++S+ IYQPEF+KESV+ N E+GTTITLTELTKKQGYPLDNYV HLS Sbjct: 121 NERVTFTIDYAEIRRSKGIYQPEFRKESVESNIESGTTITLTELTKKQGYPLDNYVEHLS 180 Query: 181 RLFDFPAQDFKIKVSLNGSEPRIIDGNLKYNLVTPQFEWEYQDLATNISSLSSKFEQYEY 240 RLFDFPAQDFKIKVSLNGSEP+IIDGNLKY+LVTPQFEWEYQDLATNISSLSSKFEQYEY Sbjct: 181 RLFDFPAQDFKIKVSLNGSEPKIIDGNLKYDLVTPQFEWEYQDLATNISSLSSKFEQYEY 240 Query: 241 SGLIQGKFITTEKPLKNNMKGITLFANGRMVNMPEFFTDSESSHF 285 SGLIQGKFITTEKPLKNNMKGITLFANGRMVNMPEFFTDSESSHF Sbjct: 241 SGLIQGKFITTEKPLKNNMKGITLFANGRMVNMPEFFTDSESSHF 285 >emb\|CAC22276.1\|(AJ302030) putative heat shock protein [Listeria monocytogenes] Length = 181 Score = 70.2 bits (171), Expect = 2e−11 Identities = 57/173 (32%), Positives = 90/173 (51%) , Gaps = 10/173 (5%) Query: 1 MSEEKLKMSFEPTVIEHLGVKMYSHTVPAIAELIANAYDACATEVEVRLFDKPEHKIVIK 60 MSE++ + +P ++E LG +Y++ + ELIANAYDA A V V E+K++++ Sbjct: 1 MSEKEYNLDIDPRILELLGPHLYTNIYYILGELIANAYDADAKNVYVIDRIDEENKLIVE 60 Query: 61 DNGIGMSFD--EINDFYLRIGRNRREEKQASPC---GRIPTGKKGLGKLALFRLGNKIEI 115 D+G GMS++ ++ +F L + + R S R G+KG+GKLA + + I Sbjct: 61 DDGSGMSYENKDVKNF-LSVAKESRTNAINSYTKLNNRRKMGRKGVGKLASLSVSENVNI 119 Query: 116 STIQGNERVFTLDYAEI-KKSERIYQPEFQKESVKPNTENGTTITLTELTKK 167 TI+ E+ F L I KK E I + +K +GT I +T T K Sbjct: 120 KTIKDGEKSGFVLSRKVINKKLEAINEDTISFIKIK---NHGTAIEMTNPTYK 169 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS51 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 52 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 103> which encodes amino acid sequence <SEQ ID 104; NGS52>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.5 Possible cleavage site: 49 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.64 threshold: 0.0 PERIPHERAL Likelihood = 7.64 modified ALOM score: −2.03 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.213(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: 2.1.1.73) NMA0427 [imported]-Neisseria meningitidis (group A strain Z2491) gi\|7379177\|emb\|CAB83726.1\|(AL162753) modification methylase (cytosine- specific DNA methylase) [Neisseria meningitidis Z2491] Length = 351 Score = 310 bits (794), Expect = 8e−84 Identities = 152/154 (98%), Positives = 153/154 (98%) Query: 1 LGMENGFPKIMAGHQDETDFMHSCAGLSDINLKRLALIPKNGGNRLAFAHIPELQLECFI 60 LGMENGFPKI+AGHQDETDFMHSCAGLSDINLKRLALIPKNGGNRLAFAHIPELQLECFI Sbjct: 198 LGMENGFPKIIAGHQDETDFMHSCAGLSDINLKRLALIPKNGGNRLAFAHIPELQLECFI 257 Query: 61 GKDNSFKDTFGRLWWDKPAPTITTKFFSISNGRAFAHPEEDRALSLREGATLQSFPRNYVF 120 GKDNSFKDTFGRLWWDKPAPTITTKFFSISNGRAFAHPEEDRALSLREGATLQSFPRNYVF Sbjct: 258 GKDNSFKDTFGRLWWDKPAPTITTKFFSISNGRAFAHPEEDRALSLREGATLQSFPRNYVF 317 Query: 121 KAGSRDKIARLIGNAVPPMYTEKIGRAIVDNIEC 154 KAGSRDKIARLIGNAVPPMY EKIGRAIVDNIEC Sbjct: 318 KAGSRDKIARLIGNAVPPMYAEKIGRAIVDNIEC 351 >gi\|1274411\|sp\|P25265\|MTD2_HERAU MODIFICATION METHYLASE HGIDII (CYTOSINE-SPECIFIC METHYLTRANSFERASE HGIDII) (M.HGIDII) gi\|538661\|pir\|\|JT0594 site-specific DNA-methyltransferase (cytosine-specific) (EC 2.1.1.73)-Herpetosiphon aurantiacus gi\|487731\|emb\|CAA38941.1\|(X55141) methyltransferase [Herpetosiphon aurantiacus] Length = 354 Score = 95.6 bits (237), Expect = 3e−19 Identities = 62/142 (43%), Positives = 82/142 (57%), Gaps = 9/142 (6%) Query: 12 AGHQDETDFM HSCAGLSDINLKRLALIPKNGGNRLAFAHIP-ELQLECFIGKD-NSFKDT 69 +G E D MH+ + L DINL+R+ G +A P EL EC + S+ Sbjct: 200 SGGHWEGDSMHAASRLEDINLRRIQHSVPGG----TWADWPEELIAECHKKESGESYGSV 255 Query: 70 FGRLWWDKPAPTITTKFFSISNGEFAHPEEDRALSLREGATLQSFPRNYVFKAGSRDK-- 127 +GR+ WDK APTITT+ NGRF HPE+DRA+SLRE A LQ+FPR+Y F + K Sbjct: 256 YGRMEWDKVAPTITTQCNGYGNGRFGHPEQDRAISLRALLQTFPRSYQFAPEGQLKFK 315 Query: 128 -IARLIGNAVPPMYTEKIGRAI 148 ++R IGNAVP I ++I Sbjct: 316 TVSRQIGNAVPVALGRVIAKSI 337 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS52 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 53 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 105> which encodes amino acid sequence <SEQ ID 106; NGS53>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −7.56 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.36 threshold: 0.0 PERIPHERAL Likelihood = 5.36 modified ALOM score: −1.57 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.189(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|11256915\|pir\|\|F81959 site-specific DNA-methyltransferase (cytosine- specific) (EC2.1.1.73) NMA0427 [imported]-Neisseria meningitidis (group A strain Z2491) gi\|7379177\|emb\|CAB83726.1\|(AL162753) modification methylase (cytosine- specific DNA methylase) [Neisseria meningitidis Z2491] Length = 351 Score = 247 bits (606), Expect = 5e−65 Identities = 124/149 (83%), Positives = 127/149 (85%) Query: 1 LQPETLEKELGLKKNDDDLILIGCSPCQYWSVIQTDKRKSEKSKSLLLEFQRFVEYFNPG 60 LQPETLEKELGLKKNDDDLILIGCSPCQYWSVIQTDKRKSEKSKSLLLEFQRFVEYFNPG Sbjct: 59 LQPETLEKELGLKKNDDDLILIGCSPCQYWSVIQTDKRKSEKSKSLLLEFQRFVEYFNPG 118 Query: 61 YVVVENVPGILSRMKESGLDNFIKLLEEKGFTVHFGIHNTADYGIPQSRKRFTLIANRIT 120 YVVVENVPGILSRMKESGLDNFIKLLEEKGFTVHFGIHNTADYGIPQSRKRFTLIANRIT Sbjct: 119 YVVVENVPGILSRMKESGLDNFIKLLEEKGFTVHFGIHNTADYGIPQSRKRFTLIANRIT 178 Query: 121 KKSWNQSSIRANGLRYAMFWEWKTAFPKL 149 K+ L + FPK+ Sbjct: 179 KEKLEPVKYSGKRLTVRDVLGMENGFPKI 207 >gi\|127441\|sp\|P25265\|MTD2_HERAU MODIFICATION METHYLASE HGIDII (CYTOSINE- SPECIFIC METHYLTRANSFERASE HGIDII) (M.HGIDII) gi\|538661\|pir\|\|JT0594 site-specific DNA-methyltransferase (cytosine- specific) (EC 2.1.1.73)-Herpetosiphon aurantiacus gi\|48773\|emb\|CAA38941.1\|(X55141) methyltransferase [Herpetosiphon aurantiacus] Length = 354 Score = 71.9 bits (169), Expect = 4e−12 Identities = 39/105 (37%), Positives = 57/105 (54%), Gaps = 1/105 (0%) Query: 12 LKKNDDDLILIGCSPCQYWSVIQTDKRKSEKSKSLLLEFQRFVEYFNPGYVVVENVPGIL 71 L N+IL+GC+PCQ +S T K ++ LL EF R + P + +ENVP + Sbjct: 64 LYPNNQHKILVGCAPCQDFSQY-TKKSRTGTKWQLLTEFSRLIREIEPDIISMENVPEVR 122 Query: 72 SRMKESGLDNFIKLLEEKGFTHFGIHNTADYGIPQSSRKRFTLIA 116 + + +NFI+ LE+ G+ V + + DYGIPQ R R L A Sbjct: 123 TFNRGEVFNFIQSLEQLGYWSHSVVHCPDYGIPQQRDRLVLFA 167 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS53 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 54 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 107> which encodes amino acid sequence <SEQ ID 108; NGS54>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.82 Possible cleavage site: 50 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 6.89 threshold: 0.0 PERIPHERAL Likelihood = 6.89 modified ALOM score: −1.88 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.253(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: gi\|1074456\|pir\|\|D64155 hypothetical protein HI0597-Haemophilus influenzae (strain Rd KW20) gi\|1573586\|gb\|AAC22254.1\|(U32741) conserved hypothetical protein [Haemophilus influenzae Rd] Length = 272 Score = 188 bits (459), Expect = 2e−47 Identities = 95/100 (95%), Positives = 97/100 (97%) Query: 1 MNLPFRAMVSDLGGTLLTPEHLVGDLTIDTLRVLEQKGVDIILATGRNHTDMSSILGKIG 60 MNLPFRAMVSDL GTLLTPEHLVGDLTIDTLR LEQKGVDIILATGRNHTD+SSILGKIG Sbjct: 1 MNLPFRAMVSDLDGTLLTPEHLVGDLTIDTLRALEQKGVDIILATGRNHTDVSSILGKIG 60 Query: 61 AERAVMITSNGARVRDLQGNLLYSNSLPEELVLELYKTSY 100 AERAVMITSNGARVRDLQGNLLYSNSLPEELVLELYKT + Sbjct: 61 AERAVMITSNGARVRDLQGNLLYSNSLPEELVLELYKTPF 100 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS54 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 55 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 109> which encodes amino acid sequence <SEQ ID 110; NGS55>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.46 Possible cleavage site: 37 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.02 threshold: 0.0 PERIPHERAL Likelihood = 3.02 modified ALOM score: −1.10 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.311(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: aeruginosa (strain PAO1) gi\|9948791\|gb\|AAG06104.1\|AEOO4699_9 (AE004699) probable FMN oxidoreductase [Pseudomonas aeruginosa] Length = 411 Score = 279 bits (686), Expect = 2e−74 Identities = 157/375 (41%), Positives 228/375 (59%), Gaps 10/375 (2%) Query: 1 MEEQLAQNDQ-PSEKLVRLYGAWAEGGAGVLVTGNVMVAESGKGSINDVLISDDRALKLM 59 MEE +4 Q PSE4+L+RLY AWA+GGAG+L++GNVMV V++ DD LE Sbjct: 24 MEENMADAAQAPSERLMRLYQAWADGGAGLLISGNVMVDSRAMTGPGGVVLEDDAQLEKF 83 Query: 60 KKWAKARTQNDTLLIMQINKAGKQSPAVKTPLAPSAVPL--GMNGFINPPRELSADE 117 ++WA+ +QINH G+Q A + + APSAVPL GM+ P+ + Sbjct: 84 RRWARIGRSAGAGFWLGINHPGRQMQANLGQQAWAPSAVPLELGGMSRHFATPKAMDEAM 143 Query: 118 INGLIQQFVQTAKIAEQAGFSGVQIYAVHGYLISQFLSPHHNRRQDQWGGSLENRMRFLL 177 I +IQ+F ++ +AE+AGFSGV+I+A HGYL+SQFLSP NRR D WGGSLENR R LL Sbjct: 144 IAEVIQRFARSAGLAERAGFSGVEIHHGYLLSQFLSPLSNRRSDAWGGSLENRARLLL 203 Query: 178 ETYTAIRAAGKDFLVGVXLNSADFQKGGFDESESVQVVQKLSEMGIDFIEVSGGNYESP 237 E A+RA F V VKLNSADFQ+GGF ++ +VV+ L +G+D +E+SGG+YE+P Sbjct: 204 EIVRAVRAEVAPGFAVAVKLNSADFQRGGFSADDAREVVRMLDGLGVDLVELSGGSYEAP 263 Query: 238 QMLA-AKDS-TRKREAFFIDYAEKARAASQAPLIITGGFRSQTAMEDALSSGHLDLVGIA 295 M A+D T REA+F+++A RAA++ P+++TGG R + E L+SG +D+VGI Sbjct: 264 AMQGEARDGRTLAREAYFVEFARDIRAAARMPVMVTGGIRRRVAEQVLASG-VDMVGIG 322 Query: 296 RPFALVPDLANKMQNRTYQTVQADRIQTGVDKKAGAMLEMNWYMTQMDLIGQGKQSN 355 A+ P+L + Q I + +K ++ M Q+ + +G+ +N Sbject: 323 TALAIEPNLPRDWRAGKDSAPQLRPI----TWENKPLASLANMAVKFQLRKLSRGRATN 378 Query: 356 PKIVGVESIAENFAG 370 P++ ++ AG Sbjct: 379 PRVSPLCALLAQQAG 393 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 56 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 111> which encodes amino acid sequence <SEQ ID 112; NGS56>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.69 Possible cleavage site: 54 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.29 threshold: 0.0 PERIPHERAL Likelihood = 4.29 modified ALOM score: −1.36 * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.042(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|7444004\|pir\|\|D70029 transcription regulator ArsR family homolog yvbA- Bacillus subtilis gi\|2635892\|emb\|CAB15384.1\|(Z99121) similar to transcriptional regulator (ArsR family) [Bacillus subtilis] Length = 90 Score = 51.3 bits (118), Expect = 3e−06 Identities = 24/65 (36%), Positives = 42/65 (63%), Gaps = 1/65 (1%) Query: 15 IFTVLSDENRHQILHVLWKHGRMNVNELTEHLHLSRPAVSHHLKIMLQAGAVAVEQVGKE 74 +F +SD R +IL +L K G M ++ EH ++S+P++SHHL I+QA ++ + G+ Sbjct: 4 VFKAISDPTRRKILDLL-KGGDMTAGDIAEHFNISHPSISHHLNILKQAEVISDHRKGQF 62 Query: 75 RFYSI 79 +YS+ Sbjct: 63 IYYSL 67 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 57 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 113> which encodes amino acid sequence <SEQ ID 114; NGS57>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.57 Possible cleavage site: 55 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.67 threshold: 0.0 PERIPHERAL Likelihood = 5.67 modified ALOM score: −1.63 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.160(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|10444407\|gb\|AAG17897.1\|AF297971_1 (AF297971) restriction endonuclease R.NgoMIII [Neisseria gonorrhoeae] Length = 213 Score = 319 bits (818), Expect = 1e−86 Identities = 156/156 (100%), Positives = 156/156 (100%) Query: 1 LYKQYADWNRLSYNAPIYVGKAVPKGWRQARNSDNALNQSTELFHRLKEHSRSIAAVSDL 60 LYKQYADWNRLSYNAPIYVGKAVPKGWRQARNSDNALNQSTELFHRLKEHSRSIAAVSDL Sbjct: 58 LYKQYADWNRLSYNAPIYVGKAVPKGWRQARNSDNALNQSTELFHRLKEHSRSIAAVSDL 117 Query: 61 DPSDFMCRFVIFEGAGSDMIGTIEAALIKLHKPLWNSCDGFGNHDPGKGRYEQAKSDWD 120 DPSDFMCRFVIFEGAGSDMIGTIEAALIKLHKPLWNSCDGFGNHDPGKGRYEQAKSDWD Sbjct: 118 DPSDFMCRFVIFEGAGSDMIGTIEAALIKLHKPLWNSCDGFGNHDPGKGRYEQAKSDWD 177 Query: 121 VLHSGRVWADRLNGIPNSYESILENINTHLEIIKRK 156 VLHSGRVWADRLNGIPNSYESILENINTHLEIIKRK Sbjct: 178 VLHSGRVWADRLNGIPNSYESILENINTHLEIIKRK 213 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 58 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 115> which encodes amino acid sequence <SEQ ID 116; NGS58>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.92 Possible cleavage site: 16 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.41 threshold: 0.0 PERIPHERAL Likelihood = 5.41 modified ALOM score: −1.58 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.107(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|10444408\|gb\|AAG17898.1\|AF29797_2 (AF297971) DNA cytosine methyltransferase M.NgoMIII [Neisseria gonorrhoeae] Length = 377 Score = 759 bits (1960), Expect = 0.0 Identities = 377/377 (100%), Positives = 377/377 (100%) Query: 1 MKSLEIFSGAGGLAKGLELAGFQHASFIELNKDACNSLRSNFNPKLVYQGDVADFDLSSQ 60 MKSLEIFSGAGGLAKGLELAGFQHASFIELNKDACNSLRSNFNPKLVYQGDVADFDLSSQ Sbjct: 1 MKSLEIFSGAGGLAKGLELAGFQHASFIELNKDACNSLRSNFNPKLVYQGDVADFDLSSQ 60 Query: 61 EGIEVIAGGPPCQPFSLGGKHLAHEDRRDMFPHAVRYVEYYRPKAFIFENVKGLLRKSFA 120 EGIEVIAGGPPCQPFSLGGKHLAHEDRRDMFPHAVRYVEYYRPKAFIFENVKGLLRKSFA Sbjct: 61 EGIEVIAGGPPCQPFSLGGKHLAHEDRRDMFPHAVRYVEYYRPKAFIFENVKGLLRKSFA 120 Query: 121 DYFEYILLRLTYPNLGILQNEDWKGHLTRLKEIEFNLYKGIKYKVSYQLLNAADYGVPQK 180 DYFEYILLRLTYPNLGILQNEDWKGHLTRLKEIEFNLYKGIKYKVSYQLLNAADYGVPQK Sbjct: 121 DYFEYILLRLTYPNLGILQNEDWKGHLTRLKEIEFNLYKGIKYKVSYQLLNAADYGVPQK 180 Query: 181 RERVVIVGIRADLDIDWKFPKRTHSEDRLNWEKYVTGEYWEKHNEPKRFNKDIAEKLQKK 240 RERVVIVGIRADLDIDWKFPKRTHSEDRLNWEKYVTGEYWEKHNEPKRFNKDIAEKLQKK Sbjct: 181 RERVVIVGIRADLDIDWKFPKRTHSEDRLNWEKYVTGEYWEKHNEPKRFNKDIAEKLQKK 240 Query: 241 YGIFEPEKKPWQTVRDTLSDIPHPLGNHKITGHEYRDGARIYPGHTGSGIDEPSKTIKAG 300 YGIFEPEKKPWQTVRDTLSDIPHPLGNHKITGHEYRDGARIYPGHTGSGIDEPSKTIKAG Sbjct: 241 YGIFEPEKKPWQTVRDTLSDIPHPLGNHKITGHEYRDGARIYPGHTGSGIDEPSKTIKAG 300 Query: 301 GHGVPGGENMIRYDDGTVRYFTSYEAKLLQTFPEEFVISGAWGEAMRQIGNAVPVKLSEI 360 GHGVPGGENMIRYDDGTVRYFTSYEAKLLQTFPEEFVISGAWGEAMRQIGNAVPVKLSEI Sbjct: 301 GHGVPGGENMIRYDDGTVRYFTSYEAKLLQTFPEEFVISGAWGEAMRQIGNAVPVKLSEI 360 Query: 361 LGKHLMGVLSEKSSLHN 377 LGKHLMGVLSEKSSLHN Sbjct: 361 LGKHLMGVLSEKSSLHN 377 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 59 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 117> which encodes amino acid sequence <SEQ ID 118; NGS59>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.82 Possible cleavage site: 60 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.86 threshold: 0.0 PERIPHERAL Likelihood = 2.86 modified ALOM score: −1.07 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.197(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|11353338\|pir\|\|F81882 hypothetical protein NMA1155 [imported] Neisseria meningitidis (group A strain Z2491) gi\|7379848\|emb\|CABB4417.1\|(AL162755) hypothetical protein NMA1155 [Neisseria meningitidis Z2491] Length = 120 Score = 131 bits (329), Expect = 2e−30 Identities = 64/68 (94%), Positives = 67/68 (98%) Query: 1 LSDISASRAAYMDVQKQYPFETVAVCVLPNHIHAIWTLPPDDADYSLLRRLIKTKFSAYS 60 +S+ISASRAAYMDVQKQYPFETVAVCVLPNHIHAIWTLPPDDADYSLLRRLIKTKFSAYS Sbjct: 1 MSNISASRAAYMDVQKQYPFETVAVCVLPNHIHAIWTLPPDDADYSLLRRLIKTKFSAYS 60 Query: 61 PHTKNLGA 68 P+TKNL A Sbjct: 61 PYTKNLSA 68 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 60 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 119> which encodes amino acid sequence <SEQ ID 120; NGS60>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.14 Possible cleavage site: 16 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.76 threshold: 0.0 PERIPHERAL Likelihood = 2.76 modified ALOM score: −1.05 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.330(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|11281269\|pir\|\|D81804 hypothetical protein NMA1789 [imported]-Neisseria meningitidis (group A strain Z2491) gi\|7380430\|emb\|CAB85016.1\|(AL162757) hypothetical protein [Neisseria meningitidis Z2491] Length = 243 Score = 154 bits (389), Expect 5e−37 Identities = 82/85 (96%), Positives = 82/85 (96%) Query: 12 MNTKTELQKLLEEDISTLKETLIRVDALPPRYVRSIATPIVRRWLIDKQLNILAKEIGLT 71 MNTKTELQKLLEEDISTL ETLI DALPPRYVRSIATPIVRRWLIDKQLNILAKEIGLT Sbjct: 1 MNTKTELQKLLEEDISTLTETLICADALPPRYVRSIATPIVRRWLIDKQLNILAKEIGLT 60 Query: 72 IELPILDTSLVFEKLSTLENKVNFY 96 IELPILDTSLVFEKLSTLENKVNFY Sbjct: 61 IELPILDTSLVFEKLSTLENKVNFY 85 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS60 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 61 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 121> which encodes amino acid sequence <SEQ ID 122; NGS61>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.88 Possible cleavage site: 32 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.52 threshold: 0.0 PERIPHERAL Likelihood = 5.52 modified ALOM score: −1.60 Rule: cytoplasmic protein * Reasoning step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.300(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|11281269\|pir\|\|D81804 hypothetical protein NMA1789 [imported]-Neisseria meningitidis (group A strain Z2491) gi\|7380430\|emb\|CAB5016.1\|(AL162757) hypothetical protein [Neisseria meningitidis Z2491] Length = 243 Score = 193 bits (491), Expect = 5e−49 Identities = 96/101 (95%), Positives = 97/101 (95%) Query: 1 MAGGVYLGGKIISPIYHSSQEFSGEPIIYAETNIILCPAEKFLTLKRVFHNGNIFNMNQI 60 MAGGVYLGG+ IS IYHSSQEFSGEPIIYAETNIILCPAEKFLTLKRVFHNGNIFNMNQI Sbjct: 86 MAGGVYLGGEFISSIYHSSQEFSGEPIIYAEPNIILCPAEKFLTLKRVFHNGNIFNMNQI 145 Query: 61 ITFLSNKQGGVRFDKNYDKYKTWQVAIEKAANFLKLGNPYN 101 ITFLSNKQGGV FDKNYDKYKTWQVAIEKAANFLKLGNPYN Sbjct: 146 ITFLSNKQGGVHFDKNYDKYKTWQVAIEKAANFLKLGNPYN 186) As a homolog (amino acids 1-96) was found in serogroup A N.meningitidis but not in serogroup B, NGS61 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 62 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 123> which encodes amino acid sequence <SEQ ID 124; NGS62>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.43 Possible cleavage site: 44 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.72 threshold: 0.0 PERIPHERAL Likelihood = 4.72 modified ALOM score: −1.44 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.324(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> A homolog was found in serogroup A N.meninigitidis but not in serogroup B, so NGS62 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 63 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 125> which encodes amino acid sequence <SEQ ID 126; NGS63>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.74 Possible cleavage site: 24 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of predicted Mature Form: calculated from 25 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 10.72 threshold: 0.0 PERIPHERAL Likelihood = 10.72 modified ALOM score: −2.64 Score for OM-PP discrimination: −22.14 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −22.14 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Periplasmic space?", "Score: 2.21378 Periplasmic space?", "Score: 2.21378 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.931(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.237(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|11284146\|pir\|\|B81142 hypothetical protein NMB0912 [imported]-Neisseria meningitidis (group B strain MD5B) gi\|7226150\|gb\|AAF41320.1\|(AE002443) hypothetical protein [Neisseria meningitidis MC58] Length = 208 Score = 51.7 bits (119), expect = 3e−06 Identities = 30/72 (41%), Positives = 40/72 (54%) Query: 5 LLKNWKPLLILSAIAFFAVSWQLDRAAQYRRGYGAAVSEVSERLKAAAVEHAEHARKSSA 64 LLK WKP+ +L I +W DRA +YR G AA +E+S RLK +E A+ AR + Sbjct: 43 LLKYWKPVGVLLLIVLIFTAWHFDRAEKYRMGREAAAAEISNRLKDGYIEQAKQARSAEQ 102 Query: 65 AYQAQKAAEEEK 76 A A R+ K Sbjct: 103 KAAAAPAERQTK 114 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 64 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 127> which encodes amino acid sequence <SEQ ID 128; NGS64>.", "Analysis of this protein sequence reveals the following: McG: Examining signal sequence (McGeoch) Length of UR: 0 Peak Value of UR: 2.99 Net Charge of CR: 4 Discriminant Score: 5.35 GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.53 Possible cleavage site: 33 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.23 threshold: 0.0 PERIPHERAL Likelihood = 3.23 modified ALOM score: −1.15 * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.054(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 65 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 129> which encodes amino acid sequence <SEQ ID 130; NGS65>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.61 Possible cleavage site: 61 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.34 threshold: 0.0 PERIPHERAL Likelihood = 3.34 modified ALOM score: −1.17 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.236(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|1175791\|sp\|P44189\|YE18_HAEIN HYPOTHETICAL PROTEIN HI1418 gi\|1074769\|pir\|\|A64029 hypothetica1 protein HI1418-Haemophilus influenzae (strain Rd KW20) gi\|1574254\|gb\|AAC23068.1\|(U32821) H. influenzae predicted coding region HI1418 [Haemophilus influenzae Rd] Length = 201 Score = 104 bits (251), Expect = 1e−21 Identities = 58/119 (48%), Positives = 72/119 (59%), Gaps = 4/119 (3%) Query: 51 LKMQNTISFSFKSQNRTQI-LGAEPFCLGDVAEILQIQNAR---QLPLKDQGIQKSS 106 +K Q S F+FK VR + E WFC DV IL N+R Q K G+ K Sbjct: 14 MKNQIQFSTFFKDLPVRTILDPKGEFFCGTDVCHILGYTNSRKALQDHCKQGGVTKRY 73 Query: 107 VATKKGNQELLFINEPNLYRVIFRSRKAEAVFQDWIFEEVIPQIRKTGGYQITPKTTA 165 TK +QE+ FINEPNLYR+I +SRK EA F+ W+FEEV+PQIRKTG YQ+ P+ A Sbjct: 74 TPTKSADQEMTFINEPNLYRLIIKSRKPEAEPFEAWVFEEVLPQIRKTGKYQLQPQQLA 132 >gi\|11281012\|pir\|\|A81144 hypothetical protein NMB0900 [imported]-Neisseria meningitidis (group B strain MD58) gi\|7226137\|gb\|AAF41308.1\|(AE002442) hypothetical protein [Neisseria meningitidis MC58] Length = 305 Score = 104 bits (249), Expect = 2e−21 Identities = 73/137 (53%), Positives = 93/137 (67%), Gaps = 2/137 (1%) Query: 190 YSMIHQRFNVEAVEGIPADKLPEAVAYHALTLHTG-LAGEVPDREPLPAPQPALPISGN 248 +S + +F E +PA++ PE ++ + + + G L GEV DREPLPAPQPALPISGN Sbjct: 164 WSAVKSKFGCSYKE-VPAEQFPELSMGRVAVENGVLYGEVLDREPLPAPQPALPISGN 222 Query: 249 ALADIAAMVYYGTRHIELGKDVSAPLKQLGCKQAVTMWTVWHETRSILKRSVAALEVLRG 308 AL D+A V YG I++G+DVS PLKQLGCRQAVTMWTVW ETRS LK + ALE L Sbjct: 223 ALYDLAVAVRYGAWAIQMGRDVSLPLKQLGCKQAVTMWTVWAETRSRLKAAANALEALNA 282 Query: 309 YADKDASGRIAACLEGI 325 +AD + + +I L I Sbjct: 283 HADAEHAAKIRPMLPEI 299 >gi\|7460273\|pir\|\|T13267 hypothetical protein-Lactococcus lactis phage BK5-T gi\|928839\|gb\|AAA98590.1\|(L44593) ORF266; putative [Lactococcus phage BK5-T] Length = 266 Score = 75.9 bits (179), Expect = 6e−13 Identities = 42/111 (37%), Positives = 63/111 (55%), Gaps = 3/111 (2%) Query: 55 NTISVFSFKSQNVRTQILGAEPWFCLGDVAEILQIQNAR---QLPLKDQGIQKSSVATKK 111 N + F+F + VRT ++ EPWF DVA + +N R + +KD+ ++S + T Sbjct: 2 NELQNFNFNNLPVRTVLINDEPWFVGKDVAIAIGYKNFRDALKSHVKDKYKRESRITTPS 61 Query: 112 GNQELLFINEPNLYRVIFRSRKAEAVKFQDWIFEBVIPQIRKTGGYQITPK 162 G Q + I+EP LY++ S+ A FQDW++EEV+P IRK G Y K Sbjct: 62 GVQSVTVISEPGLYQLAGESKLPSAEPFQDWEELPTIRKHGAYMTDAK 112 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 66 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 131> which encodes amino acid sequence <SEQ ID 132; NGS66>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.12 Possible cleavage site: 53 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 8.96 threshold: 0.0 PERIPHERAL Likelihood = 8.96 modified ALOM score: −2.29 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.402(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 67 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 133> which encodes amino acid sequence <SEQ ID 134; NGS67>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 5.71 Possible cleavage site: 22 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 23 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.81 threshold: 0.0 PERIPHERAL Likelihood = 2.81 modified ALOM score: −1.06 Score for OM-PP discrimination: −32.34 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −32.34 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Periplasmic space?", "Score: 3.23391 Periplasmic space?", "Score: 3.23391 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.928(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.199(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|7475078\|pir\|\|H69834 hypothetical protein yhjQ-Bacillus subtilis gi\|2226189\|emb\|CAA74479.1\|(Y14081) hypothetical protein [Bacillus subtilis] gi\|2633396\|emb\|CAB12900.1\|(Z99109) yhjQ [Bacillus subtilis] Length = 108 Score = 32.9 bits (74), Expect = 2.1 Identities = 27/98 (27%), Positives = 44/98 (44%), Gaps = 4/98 (4%) Query: 54 CLDAGQVCLTHCLSLLTQGDTSMSDCAVAVRQMLALCGAVHDLAAQNSPLTRDAAKVCLE 113 C+ A C T CL Q +S C R+ +C +SP ++ +C+ Sbjct: 15 CMKACNHCFTKCLEESVQ--HHLSGCIRLDRECADICALAVKAMQTDSPFMKEICALCAD 72 Query: 114 ACKQCAKACKEHSAHHAECKACYESCLDCIKECEKLAA 151 C+ C C +H H C+AC ++C C ++C +AA Sbjct: 73 ICEACGTECGKHD--HDHCQACAKACFTCAEQCRSMAA 108 >gi\|7479923\|pir\|\|T36241 hypothetical protein SCE39.31c-Streptomyces coelicolor gi\|4582392\|emb\|CAB40339.1\|(AL049573) hypothetical protein [Streptomyces coelicolor A3(2)] Length = 136 Score = 30.9 bits (69), Expect = 7.7 Identities = 27/102 (26%), Positives = 43/102 (41%), Gaps = 6/102 (5%) Query: 54 CLDAGQVCLTHCLSLLTQGDTSMSDCAVAVRQMLALCGAVHDLAAQ----NSPLTRDAAK 109 C A C CLS T D ++C +C A + ++ ++ +TR + Sbjct: 34 CAQACTACADACLSEPTVAD--LTKCIRTDMDCADVCTATAAVLSRHTGYDANVTRAVLQ 91 Query: 110 VCLEACKQCAKACKEHSAHHAECKACYESCLDCIKECEKLAA 151 C C C C H+ H C+ C E+C C + C++L A Sbjct: 92 ACATVCAACGDECARHAGMHEHCRVCAEACRSCEQACQELLA 133 The protein was expressed in E.coli as a soluble 14.19 kDa His-fusion product and then purified.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 68 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 135> which encodes amino acid sequence <SEQ ID 136; NGS68>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.05 Possible cleavage site: 38 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.25 threshold: 0.0 PERIPHERAL Likelihood = 5.25 modified ALOM score: −1.55 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.220(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|11353493\|pir\|\|A81795 hypothetical protein NMA2214 [imported]-Neisseria meningitidis (group A strain Z2491) gi\|7380833\|emb\|CAB85425.1\|(AL162758) hypothetical protein [Neisseria meningitidis Z2491] Length = 208 Score = 263 bits (673), Expect = 3e−69 Identities = 140/145 (96%), Positives = 143/145 (98%) Query: 1 LDWRGNKPLGAAELADLKPLYKDFMYWERGLHMYKASAVVPTGYVRVGNTAPLCGEDTQR 60 LDWRGNKPLGAAELADLKPLYKDFMYWERGLHMYKASAVVPTGYVRVGNTAPL GEDTQR Sbjct: 44 LDWQGNKPLGAAELADLKPLYKDFMYWERGLHMYKASAVVPTGYVRVGNTAPLVGEDTQR 103 Query 63 YASFWGDGYDVYRQLRWRQIPEKQRKAFKKAAKSKNTVMFAGREYGISKQNLSDVWDDFE 120 YASFWGDGYDVYRQLRW+QIPEKQRKAFKKAAKSK TVMFAGREYGISKQNLSDVWDDFE Sbjct: 104 YASFWGDGYDVYRQLRWQQIPEKQRKAFKKAAKSKKTVMFAGREYGISKQNLSDVWDDFE 163 Query: 121 DAMELAKAFPCLSSLFLTKWHKNLYE 145 DAMELAKAFPCLSSLFLTKWHKNLY+ Sbjct: 164 DAMELAKAFPCLSSLFLTKWHKNLYD 188 >gi\|11280955\|pir\|B81219 hypothetical protein NMB0273 [imported]-Neisseria meningitidis (group B strain MD58) gi\|7225497\|gb\|AAF40727.1\|(AE002383) hypothetical protein [Neisseria meningitidis MC58] Length = 141 Score = 216 bits (550), Expect = 5e−55 Identities = 117/121 (96%), Positives = 119/121 (97%) Query: 25 MYWERGLHMYKASAVVPTGYVRVGNTAPLCGEDTQRYASFWGDGYDVYRQLRWRQIPEKQ 84 MYWERGLHMYKASAVVPTGYVRVGNTAP CGEDTQRYASFWGDGYDVYRQLRW+QIPEKQ Sbjct: 1 MYWERGLHMYKASAVVPTGYVRVGNTAPLVGEDTQRYASFWGDGYDVYRQLRWQQIPEKQ 60 Query: 85 RKAFKKAAKSKNTVMFAGREYGISKQNLSDVWDDFEDAMELKAFPCLSSLFLTKWHKNLY 144 RKAFKKAAKSK TVMFAGREYGISKQNLSDVWDDFEDAMELKAFPCLSSLFLTKWHKNLY Sbjct: 61 RKAFKKAAKSKKTVMFAGREYGISKQNLSDVWDDFEDAMELKAFPCLSSLFLTKWHKNLY 120 Query: 145 E 145 + Sbjct: 121 D 121 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 69 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 137> which encodes amino acid sequence <SEQ ID 138; NGS69>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.63 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 6.84 threshold: 0.0 PERIPHERAL Likelihood = 6.84 modified ALOM score: −1.87 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.361(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|7464552\|pir\|\|C64707 hypothetical protein HP1499-Helicobacter pylori (strain 26695) gi\|2314683\|gb\|AAD08545.1\|(AE000648) H. pylori predicted coding region HP1499 [Helicobacter pylori 26695] Length = 272 Score = 38.2 bits (88), Expect = 0.13 Identities = 34/123 (27%), Positives = 58/123 (46%), Gaps = 10/123 (8%) Query: 3 EFKPIFGQDFGLSKKEAIRKVLKWLPSHLKFTLMVAQGIQG------FHPKAVFWKNDKN 56 EF+ I G DF + ++IR +L ++ K + FHPK + N K Sbjct: 54 EFEIIVGLDFKTTDSKSIRFLLDLNKTYKKLRFYCYGDKENNKTDIVFHPKIYMFDNGK- 112 Query: 57 EYYALIGSSNLTHAAFNSNYEAN-ILTKISEQDFIKVKSWADEI--AMKSIPVSEDWLEE 113 E ++IGS+NLT +N+E N I T+ + ++ + + I A +E++L+ Sbjct: 113 EKTSIIGSTNLTKGGLENNFEVNTIFTEKKPLYYTQLNAIYNSIKYADSLFTPNEEYLQN 172 Query: 114 YQE 116 Y E Sbjct: 173 YNE 175 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS69 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 70 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 139> which encodes amino acid sequence <SEQ ID 140; NGS70>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.18 Possible cleavage site: 22 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 23 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.19 threshold: 0.0 PERIPHERAL Likelihood = 4.19 modified ALOM score: −1.34 Score for OM-PP discrimination: −5.89 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −5.89 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Periplasmic space?", "Score: 0.588927 Periplasmic space?", "Score: 0.588927 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.849(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.106(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|11353344\|pir\|\|A81886 hypothetical protein NMAl183 [imported]-Neisseria meningitidis (group A strain Z2491) gi\|7379875\|emb\|CAB84445.1\|(AL162755) hypothetical protein NMA1183 [Neisseria meningitidis Z2491] Length = 74 Score = 58.7 bits (141), Expect = 2e−08 Identities = 30/43 (69%), Positives = 32/43 (73%) Query: 62 IGGFGGVGGFGGLKPALVYRNFRIIATNRPAATRARPRQTTVA 104 +G\|\|\|G+\|G\|GGLKP\|LVY\|N\|\|IIATNRPAATRA\|PR\|TTVA Sbjct: 32 MGNIDGIDGSGGLKPTLVYWNHCIIATNRPAATRAHPRHTTVA 74 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 71 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 141> which encodes amino acid sequence <SEQ ID 142; NGS71>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.98 Possible cleavage site: 28 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 29 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.04 threshold: 0.0 PERIPHERAL Likelihood = 5.04 modified ALOM score: −1.51 Score for OM-PP discrimination: −9.17 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −9.17 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Periplasmic space?", "Score: 0.916744 Periplasmic space?", "Score: 0.916744 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.923(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.146(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 72 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 143> which encodes amino acid sequence <SEQ ID 144; NGS72>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.18 Possible cleavage site: 42 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 4 value: −8.76 threshold: 0.0 INTEGRAL Likelihood = −8.76 Transmembrane 11-27 (8-37) INTEGRAL Likelihood = −6.90 Transmembrane 80-96 (75-102) INTEGRAL Likelihood = −2.39 Transmembrane 98-114 (98-114) INTEGRAL Likelihood = −1.12 Transmembrane 47-63 (47-64) PERIPHERAL Likelihood = 3.55 modified ALOM score: 2.25 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.450(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|11354095\|pir\|\|H81995 probable transmembrane transport protein NMA0047 [imported]-Neisseria meningitidis (group A strain Z2491) gi\|7378822\|emb\|CAB83364.1\|(AL162752) putative transmembrane transport protein [Neisseria meningitidis Z2491] Length = 405 Score = 257 bits (658), Expect = 5e−68 Identities = 152/162 (93%), Positives = 154/162 (94%) Query: 1 MTHTASKTPKLWAVIAAAAFILLITIGMRMTLGLFVQPVVNTTELNIAQFSLIITVFQLM 60 MTHTASKTPK W I AAAFILLITIGMRMTLGLFVQPVVNTTEL+IAQFSLII VFQLM Sbjct: 1 MTHTASKTPKFWLTITAAAFILLITIGMRMTLGLFVQPVVNTTELNIAQFSLIITVFQLM 60 Query: 61 WGVLQPLSGALADRFGAFRVLSGGALLLVCACLIASNIPTYWGLMIAVGLLLAFGTGSGG 120 WGV QPLSGALADRFGAFRVLSGGA+LLVCACLIA NIPTYWGLMIAVGLLLAFGTGSGG Sbjct: 61 WGVSQPLSGALADRFGAFRVLSGGAVLLVCACLIAPNIPTYWGLMIAVGLLLAFGTGSGG 120 Query: 121 FSIIMGQVAAQVPTHKRGLASGLVNAGGSAGQFLFAPLVQGL 162 FSIIMGQVAAQVP HKRGLASGLVNAGGSAGQFLFAPLVQGL Sbjct: 121 FSIIMGQVAAQVPAHKRGLASGLVNAGGSAGQFLFAPLVQGL 162 >gi\|11351469\|pir\|\|F83484 probable MFS transporter PA1286 [imported]- Pseudomonas aeruginosa (strain PAO1) gi\|9947221\|gb\|AAG04675.1\|AE004558_4 (AE004558) probable MFS transporter [Pseudomonas aeruginosa] Length = 399 Score = 72.5 bits (177), Expect = 3e−12 Identities = 53/149 (35%), Positives = 81/149 (53%) Query: 14 VIAAAAFILLITIGMRMTLGLFVQPVVNTTELNIAQFSLIITVFQLMWGVLQPLSGALAD 73 ++ + A IL +++G+R GLF+ P+ F+ I + L+WG+ QP +GALAD Sbjct: 8 ILLSGALILALSLGVRHGFGLFLAPMSADFGWGREVFAFAIALQNLVWGLAQPFTGALAD 67 Query: 74 RFGAFRVLSGGALLLVCACLIASNIPTYWGIMIAVGLLLAPGTGSGGFSIIMGQVAAQVP 133 R+GA R + G LL ++ + GL ++ GLL+ G FS+I+G V VP Sbjct: 68 RYGAARAVLVGGLLYALGLVLMGLSQSASGLSLSAGLLIGLGLSGTSFSVILGAVGRAVP 127 Query: 134 THKRGLASGLVNAGGSAGQFLPAPLVQGL 162 +R +A G+ +A GS GQF P GL Sbjct: 128 AEQRSMAMGISSAAGSFGQFAMLPGTLGL 156 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS72 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 73 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 145> which encodes amino acid sequence <SEQ ID 146; NGS73>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.26 Possible cleavage site: 52 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 3 value: −3.72 threshold: 0.0 INTEGRAL Likelihood = −3.72 Transmembrane 172-188 (171-190) INTEGRAL Likelihood = −2.97 Transmembrane 119-135 (114-137) INTEGRAL Likelihood = −1.86 Transmembrane 209-225 (205-225) PERIPHERAL Likelihood = 4.88 modified ALOM score: 1.24 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.249(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi\|11354095\|pir\|\|H81995 probable transmembrane transport protein NMA0047 [imported]-Neisseria meningitidis (group A strain Z2491) gi\|7378822\|emb\|CAB83364.1\|(AL162752) putative transmembrane transport protein [Neisseria meningitidis Z2491] Length = 405 Score = 222 bits (567), Expect = 3e−57 Identities = 116/123 (94%), Positives = 117/123 (94%), Gaps = 4/123 (3%) Query: 103 QGLVVLPEVGWTGTFYVWGAIALLILPVSWWLAGGNNGGNNAAHTQHTQATHGQSLGEAV 162 QGLVVLPEVGWTGTFYVWGAIALL LPVSWWLA GGNNAAHTQH QATHGQSLGEAV Sbjct: 160 QGLVVLPEVGWTGTFYVWGAIALLTLPVSWWLA----GGNNAAHTQHAQATHGQSLGEAV 215 Query: 163 KTAFKTPSYILLHLSFFACGFHIAFLVTHLPTEVALCGLPATVASTSIAIIGLANIAGCV 222 KTAFKTPSYILLHLSFFACGFHIAFLVTHLPTEVALCGLPATVASTSIAIIGLANIAGC+ Sbjct: 216 KTAFKTPSYILLHLSFFACGFHIAFLVTHLPTEVALCGLPATVASTSIAIIGLANIAGCI 275 Query: 223 FSG 225 FSG Sbjct: 276 FSG 278 >gi\|11351469\|pir\|\|F83484 probable MFS transporter PA1286 [imported]-Pseudomonas aeruginosa (strain PAO1) gi\|9947221\|gb\|AAG04675.1\|AE004558_4 (AE004558) probable MFS transporter [Pseudomonas aeruginosa] Length = 399 Score = 54.4 bits (130), Expect = 1e−06 Identities = 37/115 (32%), Positives = 56/115 (48%), Gaps = 10/115 (8%) Query: 111 VGWTGTFYVWGAIALLILPVSWWLAGGNNGGNNAAHTQHTQATHGQSLGEAVKTAFKTPS 170 +GW+ G + LI+P++ + H QSLGEA++ A Sbjct: 160 LGWSSALLALGLLVALIVPLAGLM----------KDRPLPPQGHEQSLGEALREACAHSG 209 Query: 171 YILLHLSFFACGFHIAFLVTHLPTEVALCGLPATVASTSIAIIGLANIAGCVFSG 225 + LL L FF CGF + F+ HLP + LPA V +T +A++GL N+ G +G Sbjct: 210 FWLLALGFFVCGFQVVFIGVHLPAYLVDQHLPAQVGTTVLALVGLFNVFGTYIAG 264 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS73 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 74 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 147> which encodes amino acid sequence <SEQ ID 148; NGS74>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.41 Possible cleavage site: 30 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 2 value: −1.49 threshold: 0.0 INTEGRAL Likelihood = −1.49 Transmembrane 15-31 (15-31) INTEGRAL Likelihood = −1.28 Transmembrane 8-99 (83-99) PERIPHERAL Likelihood = 1.59 modified ALOM score: 0.80 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.160(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|\|H81995 probable transmembrane transport protein NMA0047 [imported]- Neisseria meningitidis (group A strain Z2491) emb\|CAB3364.1\|(AL162752) putative transmembrane transport protein [Neisseria meningitidis Z2491] Length = 405 Score = 148 bits (374), Expect = 2e−35 Identities = 97/106 (91%), Positives = 103/106 (96%) Query: 1 MVLIYIFSPKTDLNFYIFAAALGFTWLATVAPTAAVTGKLFGTRYLATLFGLVMLTHQIG 60 M+LIYIFSPKTDLNFYIFAAALGFTWLATV PTA++TGKLFGTRYLATLFGL ML+HQIG Sbjct: 300 MILIYIFSPKTDLNFYIFAAALGFTWLATVTPTASITGKLFGTRYLATLFGLTMLSHQIG 359 Query: 61 GFLGSYIGGIVITQFGDYGWMWYADAVLAGTAALLVLPVREPRTAA 106 GFLGSYIGGIVITQFGDYGWMWYADA+LAGTAALL LP+REPRTAA Sbjct: 360 GFLGSYIGGIVITQFGDYGWMWYADALLAGTAALLNLPIREPRTAA 405 >pir\|\|F83484 probable MFS transporter PA1286 [imported]-Pseudomonas aeruginosa (strain PAO1) gb\|AAGO4675.1\|AE004558_4 (AE004558) probable MFS transporter [Pseudomonas aeruginosa Length = 399 Score = 59.0 bits (142), Expect = 2e−08 Identities = 40/101 (39%), Positives = 61/101 (59%) Query: 1 MVLIYIFSPKTDLFYIFAAALGFTWLATVAPTAAVGKLFGTRYLATLFGLVMLTHQIG 60 +++++++ P + + Y F A+G WL+TV T LFG R L+ L G+V L HQ+G Sbjct: 286 VIVLFLWLPLSVYSAYAFGVAMGLLWLSTVPLTNGTTLFGVRNLSMLGGIVFLFHQLG 345 Query: 61 GFLGSYIGGIVITQFGDYGWMWYADAVLAGTAALLVLPVRE 101 FLG ++GG+V + G Y +W +L+ AALL PVRE Sbjct: 346 AFLGGWLGGVVYDRTGSYDLVWQLSILLSLLAALLNWPVRE 386 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS74 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 75 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 149> which encodes amino acid sequence <SEQ ID 150; NGS75>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.2 Possible cleavage site: 22 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.45 threshold: 0.0 PERIPHERAL Likelihood = 4.45 modified ALOM score: −1.39 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.237(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: pir\|\|S04419 type II site-specific deoxyribonuclease (EC 3.1.21.4) NgoPII- Neisseria gonorrhoeae emb\|CAA368B7.1\|(X52661) NgoPII restriction and modification [N. gonorrhoeae] prf\|\|1613419A NgoPII endonuclease [Neisseria gonorrhoeae] Length = 278 Score = 240 bits (614), Expect = 4e−63 Identities = 128/129 (99%), Positives = 128/129 (99%) Query: 1 MNIIDAIINLANNPVVGVNSHSQSNNRANQAGDALEEYVKDLFSGSFNLNETQRIARHAK 60 MNIIDAIINLANNPVVGV SHSQSNNRANQAGDALEEYVKDLFSGSFNLNETQRIARHAK Sbjct: 1 MNIIDAIINLANNPVVGVESHSQSNNRANQAGDALEEYVKDLFSGSFNLNETQRIARHAK 60 Query: 61 VFSYLGNNSNPPDAMLRNGDAIEVKKIESKDSALALNSSHPKSKLSVDDSMLTKACKDAE 120 VFSYLGNNSNPPDAMLRNGDAIEVKKIESKDSALALNSSHPKSKLSVDDSMLTKACKDAE Sbjct: 61 VFSYLGNNSNPPDAMLRNGDAIEVKKIESKDSALALNSSHPKSKLSVDDSMLTKACKDAE 120 Query: 121 KWEEKDIIY 129 KWEEKDIIY Sbjct: 121 KWEEKDIIY 129 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 76 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 151> which encodes amino acid sequence <SEQ ID 152; NGS76>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.73 Possible cleavage site: 13 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 9.39 threshold: 0.0 PERIPHERAL Likelihood = 9.39 modified ALOM score: −2.38 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.272(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: pir\|\|S04419 type II site-specific deoxyribonuclease (EC 3.1.21.4) NgoPII- Neisseria gonorrhoeae emb\|CAA36887.1\|(X52661) NgoPII restriction and modification [N. gonorrhoeae] prf\|\|1613419A NgoPII endonuclease [Neisseria gonorrhoeae] Length = 278 Score = 268 bits (687), Expect = 2e−71 Identities = 136/136 (100%), Positives = 136/136 (100%) Query: 1 LAMVYGIDYCADAECYLKIKNQIKEGIGNIGGIQFAETKELGRVNRIDPLNITYLRVRGM 60 LAMVYGIDYCADAECYLKIKNQIKEGIGNIGGIQFAETKELGRVNRIDPLNITYLRVRGM Sbjct: 143 LAMVYGIDYCADAECYLKIKNQIKEGIGNIGGIQFAETKELGRVNRIDPLNITYLRVRGM 202 Query: 61 WGIENPWFVFNYIYQRNMEKSFNFMAIINEDKWNSFNNTDKLLAIQDSKLAISDIKIKNP 120 WGIENPWFVFNYIYQRNMEKSFNFMAIINEDKWNSFNNTDKLLAIQDSKLAISDIKIKNP Sbjct: 203 WGIENPWFVFNYIYQRNMEKSFNFMAIINEDKWNSFNNTDKLLAIQDSKLAISDIKIKNP 262 Query: 121 NNPARLRNAKLITYHL 136 NNPARLRNAKLITYHL Sbjct: 263 NNPARLRNAKLITYHL 278 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 77 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 153> which encodes amino acid sequence <SEQ ID 154; NGS77>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.51 Possible cleavage site: 58 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.18 threshold: 0.0 PERIPHERAL Likelihood = 3.18 modified ALOM score: −1.14 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.127(Affirmative) <succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|\|\|CTNHP2 site-specific DNA-methyltransferase (cytosine-specific) (EC 2.1.1.73) NgoPII-Neisseria gonorrhoeae emb\|CAA30038.1\|(X06965) NgoPII methylase (AA 1-341) [Neisseria gonorrhoeae] emb\|CAA3688B.1\|(K52661) NgoPII restriction and modification [Neisseria gonorrhoeae] gb\|AAA170419.1\|(L14564) cytosine methylase [Neisseria gonorrhoeae] prf\|\|1613419B NgoPII methylase [Neisseria gonorrhoeae] Length = 341 Score = 682 bits (1761), Expect = 0.0 Identities = 341/341 (100%), Positives = 341/341 (100%) Query: 1 MQNSSPTTYNPMKIISLFSGCGGLDLGFEKAGFEIPAANEYDKTIWATFKANHPKTHLIE 60 MQNSSPTTYNPMKIISLFSGCGGLDLGFEKAGFEIPAANEYDKTIWATFKANHPKTHLIE Sbjct: 1 MQNSSPTTYNPMKIISLFSGCGGLDLGFEKAGFEIPAANEYDKTIWATFKANHPKTHLIE 60 Query: 61 GDIRKIKEEDFPEEIDGIIGGPPCQSWSEAGALRGIDDARGQLFFDYIRILKSKQPKFFL 120 GDIRKIKEEDFPEEIDGIIGGPPCQSWSEAGALRGIDDARGQLFFDYIRILKSKQPKFFL Sbjct: 61 GDIRKIKEEDFPEEIDGIIGGPPCQSWSEAGALRGIDDARGQLFFDYIRILKSKQPKFFL 120 Query: 121 AENVSGMLANRHNGAVQNLLKMFDGCGYDVTLTMANAKDYGVAQERKRVFYIGFRKDLEI 180 AENVSGMLANRHNGAVQNLLKMFDGCGYDVTLTMANAKDYGVAQERKRVFYIGFRKDLEI Sbjct: 121 AENVSGMLANRHNGAVQNLLKMFDGCGYDVTLTMANAKDYGVAQERKRVFYIGFRKDLEI 180 Query: 181 KFSFPKGSTVEDKDKITLKDVIWDLQDTAVPSAPQNKTNPDAVNNNEYFTGSFSPIFMSR 240 KFSFPKGSTVEDKDKITLKDVIWDLQDTAVPSAPQNKTNPDAVNNNEYFTGSFSPIFMSR Sbjct: 181 KFSFPKGSTVEDKDKITLKDVIWDLQDTAVPSAPQNKTNPDAVNNNEYFTGSFSPIFMSR 240 Query: 241 NRVKAWDEQGFTVQASGRQCQLHPQAPKMEKHGANDYRFAAGKETLYRRMTVREVARIQG 300 NRVKAWDEQGFTVQASGRQCQLHPQAPKMEKHGANDYRFAAGKETLYRRMTVREVARIQG Sbjct: 241 NRVKAWDEQGFTVQASGRQCQLHPQAPKMEKHGANDYRFAAGKETLYRRMTVREVARIQG 300 Query: 301 FPDNFKFIYQNVNDAYKMIGNAVPVNLAYEIAAAIKKTLER 341 FPDNFKFIYQNVNDAYKMIGNAVPVNLAYEIAAAIKKTLER Sbjct: 301 FPDNFKFIYQNVNDAYKMIGNAVPVNLAYEIAAAIKKTLER 341 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 78 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 155> which encodes amino acid sequence <SEQ ID 156; NGS78>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −8.33 Possible cleavage site: 24 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.17 threshold: 0.0 PERIPHERAL Likelihood = 2.17 modified ALOM score: −0.93 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.220(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|\|H82000 hypothetical protein NMA0089 [imported]-Neisseria meningitidis (group A strain Z2491) emb\|CAB83405.1\|(AL162752) hypothetical protein NMA0089 [Neisseria meningitidis Z2491] Length = 226 Score = 422 bits (1085), Expect = e−117 Identities = 217/228 (95%), Positives = 220/228 (96%), Gaps = 2/228 (0%) Query: 1 MERYKNAVRKDKAAELYLLNLSLSRELFHVVSIFEIVLRNKIDICFRQEFKDRNRLYDSI 60 MERYKNAV KDKAAELYLLNLSLSRELFHVVSIFEIVLRNKIDIC +Q FKD N LY+SI Sbjct: 1 MERYKNAVGKDKAAELYLLNLSLSRELFHVVSIFEIVLRNKIDICLQQAFKDGNWLYNSI 60 Query: 61 QPQTNPALKYQGCFLRNGTKESAELIKVALSKIQNNSGGKFDHNQLVAGLGFGFWRYLFA 120 QPQTNPALKYQGCFLRNGTKESAELIKVALSKIQNNSGGKFDHNQLVAGLGFGFWRYLFA Sbjct: 61 QPQTNPALKYQGCFLRNGTKESAELIKVALSKIQNNSGGKFDHNQLVAGLGFGFWRYLFA 120 Query: 121 GGKDAQFDAAGKVLMKVFPKKPKSTPSVQYNQKWIFRELSNINNFRNRLAHHEPICFSFK 180 GGKDAQFDA GKVLMKVFPKKPKSTPSVQ+NQKWIFRELSNINNFRNRLAHHEPIC FK Sbjct: 121 GGKDAQFDATGKVLMKVFPKKPKSTPSVQHNQKWIFRELSNINNFRNRLAHHEPIC--FK 178 Query: 181 GAIKDTGYARNIHQSIFELLNYMDVDTASVFSHFSDQVIAVCDEIDKL 228 GAIKDTGYARNIHQSIFELLNYMDVDTASVFSHFSDQVIAVCDEIDKL Sbjct: 179 GAIKDTGYARNIHQSIFELLNYMDVDTASVFSHFSDQVIAVCDEIDKL 226 >ref\|NP_053274.1\|Hypothetical gene [Agrobacterium tumefaciens] dbj\|BAA87659.1\|(AB016260) Hypothetical gene [Agrobacterium tumefaciens] Length = 380 Score = 43.6 bits (102), Expect = 0.002 Identities = 53/215 (24%), Positives = 86/215 (39%), Gaps = 42/215 (19%) Query: 5 KNAVRKDKAAELYLLNLSLSRELFHVVSIFEIVLRNKIDICFRQEFKDRNRLYDSIQPQT 64 K ++ A LYL N +++ + +++ E+ LRN +D F Sbjct: 55 KGGNHEEYAMALYLYNARVAKAFLYPLNVAEVTLRNAVDGILVARFG------------- 101 Query: 65 NPALKYQGCFLRNGTKESAELIKVALSKIQNNSGGKFDHNQLVAGLGFGFWRYLFAGGKD 124 A +Q R+ T L L K +G +Q+VA L F FW LF Sbjct: 102 --ANWHQDATFRDQTLTGNGL--ATLDKAIQRAGAGAARDQIVATLTFDFWSNLFR---- 153 Query: 125 AQFDAAGKVLMKVPKKPKSTPSVQYNQKWIFRELSN----INNFRNRLAHHEPICFSFK 180 ++ + + + + P +Q+ + +E+ N IN FRNR+AHHEP+ Sbjct: 154 PEYGGLWRTTVNI------AFPHLQHGESR--QEIQNINKPINFRNRVAHHEPVL---- 201 Query: 181 GAIKDTGYARNIHQSIFELLNYMDVDTASVFSSFS 215 D +IH I L+ +TA+ H S Sbjct: 202 ----DLNVT-DIHAKIVRLIELRCAETATWMKHHS 231 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS78 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 79 A DNA sequence was identified in N.gonorrhoeae <SEQ D 157> which encodes amino acid sequence <SEQ ID 158; NGS79>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 2.07 Possible cleavage site: 17 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 18 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 9.49 threshold: 0.0 PERIPHERAL Likelihood = 9.49 modified ALOM score: −2.40 Score for OM-PP discrimination: −11.72 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −11.72 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Periplasmic space?", "Score: 1.17242 Periplasmic space?", "Score: 1.17242 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.932(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.240(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> A homolog (amino acids 23-85) was found in serogroup A N.meningitidis but not in serogroup B, so NGS79 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 80 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 159> which encodes amino acid sequence <SEQ ID 160; NGS80>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −8.49 Possible cleavage site: 57 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.98 threshold: 0.0 PERIPHERAL Likelihood = 4.98 modified ALOM score: −1.50 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.428(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|\|E81080 conserved hypothetical protein NMB1466 [imported]-Neisseria meningitidis (group B strain MD58) gb\|AAF41823.1\|(AE002496) conserved hypothetical protein [Neisseria meningitidis MC58] Length = 243 Score = 148 bits (375), Expect = 7e−35 Identities = 82/189 (43%), Positives = 109/189 (57%) Query: 120 VDRMFNMAGNHFARLGISSGSGVHYWNARDFSEQAFHAEVGYRYRNSRLEWGFRPFVKQNR 179 + R N +HF GISG GVHYW+ +DFSEQ+ GY+ R+ +G PFV+QN Sbjct: 1 MSREINAGRHHFLYGGISGGGVHYWDNKDFSEQSLRLSFGYKNRSVTRSFGIVPFVEQNL 60 Query: 180 LGNNRYTANTGIVLDYSRRLNEKWRSTQSFQYGRKQYHDEYIAKRYNSKTISVSGTFSYY 239 LG +RY G D+S+RL+E+WR T + K Y ++ A RY+S T Y Sbjct: 61 LGGSRYNFVGGFNADFSQRLSERWRLTLNAGNMWKHYQEDRTAARYDSHMPLAGATLMYS 120 Query: 240 AMSAWQLYGGISGNFDNTVEKEQASBRYGVSLGTVKILDGGLGLKLGAGYTKRIFKAPAT 299 A W LYGG + T E EQAS R G+ +G VK DGGLGL+ YT+R+F AP T Sbjct: 121 APKDWLLYGGADWSHNITKEAEQASIRKGLRVGAVKTFDGGLGLRANLRYTRRMFDAPGT 180 Query: 300 LIYNFTRRD 308 ++Y F R+D Sbjct: 181 IVYRFPRKD 189 >gb\|AAD11779.1\|(AF118122) putative outer membrane protein OmpU [Neisseria meningitidis] Length = 488 Score = 72.1 bits (176), Expect = 7e−12 Identities = 71/300 (23%), Positives = 128/300 (42%), Gaps = 17/300 (5%) Query: 3 EAADLYRELLSERPDLVYPRFDLGVMLFEDKQYREALVQLHRAE-EVLPPDMRQLAREYI 61 EA YREL++ +PD R L LF+++Q A Q R + E LPP + + Y Sbjct: 136 EAISHYRELIAAQPDAPAVRMRLAAALFDNRQNEAAADQFDRLKAENLPPQLMEQVELYR 195 Query: 62 RQAEAVQAWHPSFNMNYEQTDNVNNASLSRDIVINGRKWIKSEDSLPKRANT--IRYELG 119 + AW + + + N+N A + KW + PK+ +G + Y LG Sbjct: 196 KALRERDAWKVNGGFSVTREHNINQAPKRQQR----GLW-----TFPKQVDGTAVNYRLG 246 Query: 120 VDRMFNMAGNHFARLGISGSGVHYWNARDFSEQAFHAEVGYRYRNSRLEWGFRPFVKQNR 179 ++ +++ + G SG Y + F++ G + + R + G F ++ Sbjct: 247 AEKKWSLKNGWYTTAGGDVSGRVYPGNKKFNDMTAGVSGGIGFADRRKDAGLAVFHERRT 306 Query: 180 LGNNRYTANTGIVLDYSRRLNEKWRSTQSFQYGRKQYHDEYLAKRYNSKTISVSGTFSYY 239 GN+ Y+ G L ++R KW++ S ++GR + R ++ + +S + +Y Sbjct: 307 YGNDAYSYTNGARLYFNRWQTPKWQTLSSAEWGRLK---NTRRARSDNTHLQISNSLVFY 363 Query: 240 AMSAWQLYGGISGMFD-NTVEKEQASRRYGVSLGTVKILDG-GLGLKLGAGYTKRIFKAP 297 + GG+ + N ++ RYG+ + G GL L G KR ++ P Sbjct: 364 RNARQYWMGGLDFYRERNPADRGDNFNRYGLRFAWGQEWGGSGLSSLLRLGAAKRHYEKP 423 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 81 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 161> which encodes amino acid sequence <SEQ ID 162; NGS81>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.25 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.05 threshold: 0.0 PERIPHERAL Likelihood = 7.05 modified ALOM score: −1.91 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.232(Affirmative) < succ> The protein has homology with the following sequences in the databases: gi\|10803654\|ref\|NP_046052.1\|putative ISH4 transposase [Halobacterium sp.", "NRC-1] gi\|7484109\|pir\|\|T08324 probable transposase H1306-Halobacterium sp.", "(strain NRC-1) insertion sequence ISH4 plasmid pNRC100 gi\|2822385\|gb\|AAC82891.1\|(AF016485) putative ISH4 transposase [Halobacterium sp.", "NRC-1] gi\|10580476\|gb\|AAG19350.1\|(AE005029) Vng0918h [Halobacterium sp.", "NRC-1] Length = 294 Score = 52.1 bits (124), Expect = 4e−06 Identities = 36/139 (25%), Positives = 63/139 (44%), Gaps = 7/139 (5%) Query: 31 CPHCQSTHFVKNGKDCGNQRFLCRDCKKSFVEQTGTILYNTQKDIEVWEKYIHCMIE-KY 89 CP C++ ++ G QR+LC+DC ++F +QTGT+ ++ + W ++ I Sbjct: 28 CPSCRAESVIRYGSYRVFQRYLCKDCDRTFNDQTGTVFEHSAVALRKWFLAVYTYIRLNT 87 Query: 90 PLRKCAEICKINLATAFTWRHKILDALQNMMNEVELDGIVQADETYSTISYKGHHKNFNL 149 +R+ ++ T + + L AL L+G V+ DE Y KG ++ Sbjct: 88 SIRQLDAEIDVSYKTVYRRVQRFLRALD--APRPHLEGPVEIDEFYVKAGLKGRERD--- 142 Query: 150 PRPAHKRGTRATKRGISKE 168 +P+ RG RG E Sbjct: 143 -QPSRSRGLSTRGRGTYAE 160 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 82 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 163> which encodes amino acid sequence <SEQ ID 164; NGS82>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.66 Possible cleavage site: 57 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −0.85 threshold: 0.0 INTEGRAL Likelihood = −0.85 Transmembrane 76-92 (76-92) PERIPHERAL Likelihood = 1.75 modified ALOM score: 0.67 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.134(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi\|586070\|sp\|Q07605\|T4BA_BACCO RESTRICTION ENZYME BGCI ALPHA SUBUNIT [INCLUDES: ADENINE-SPECIFIC METHYLTRANSFERASE ACTIVITY] gi\|1075788\|pir\|\|A53125 restriction enzyme BcgI alpha chain-Bacillus coagulans gi\|304140\|gb\|AAA16626.1\|(L17341) restriction endonuclease alpha subunit [Bacillus coagulans] Length = 637 Score = 91.4 bits (226), Expect = 1e−17 Identities = 78/256 (30%), Positives = 123/256 (47%), Gaps = 42/256 (16%) Query: 1 MFALAASNMILRGDGKANLHQSSCFMTDFQDLIKNPKPETGLKRPNVGFLNPPYAQSKSD 60 +F +A +NMILRGDGK+NL + +C F + I N G+ N +NPPY+Q+K+D Sbjct: 394 LFTIATTNMILRGDGKSNLIRDNCLT--FDNTIMN---GYGI---NKILMNPPYSQAKND 445 Query: 61 AELH--ELYFVKEMLKMLAEGGTGIAIIPVSCVIAPSK----AKSEIVKYHRLKAVMSMP 114 H EL F+++ L+ML GG AI+P S ++ ++ K +I+K H L+ V+++ Sbjct: 446 QTQHLSELSFIQQALEMLVVGGKLCAIVPQSTMVGKNRHDKARKKQILKQHTLETVITLN 505 Query: 115 SELFYPVGTVTCIVVFEAHKPHFQTVVIDPDTQEEISTKKACENTWFGYWRDDGFEKTKH 174 + F+ VG CIV+F+A H + ++ F + DDG KH Sbjct: 506 KDTFHGVGVNPCIVIFKAGIKHPEN-----------------KRVSFVNFEDDGHVVRKH 548 Query: 175 LGRIDLYDRWQGIKARWLEHYL-----NNEVHTGESVTAFVTDNDEWVAEAYLETDYSKI 229 +G + G + EH L + + T V + D DEW+ Y D Sbjct: 549 VGLVG-----DGTEKGKREHLLAVLAGDEDDGTDLIVKTAIKDTDEWLHSFYYFND-GIP 602 Query: 230 TRADFEQVVREFALFQ 245 + DF + V + FQ Sbjct: 603 SEDDFYKTVANYLTFQ 618 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 83 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 165> which encodes amino acid sequence <SEQ ID 166; NGS83>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −8.04 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −1.44 threshold: 0.0 INTEGRAL Likelihood = −1.44 Transmembrane 55-71 (55-71) PERIPHERAL Likelihood = 4.03 modified ALOM score: 0.79 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.157 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi\|586071\|sp\|Q07606\|T4BB_BACCO RESTRICTION ENZYNE BGCI BETA SUBUNIT gi\|1075789\|pir\|\|B53125 restriction enzyme BcgI beta chain-Bacillus coagulans gi\|304141\|gb\|AAA16627.1\|(L17341) restriction endonuclease beta subunit [Bacillus coagulans] Length = 341 Score = 44.0 bits (103), Expect = 0.002 Identities = 46/195 (23%), Positives = 79/195 (39%), Gaps = 23/195 (11%) Query: 4 LQEIFDVSYGSKLDLNKMSSFNPTINFVGRSGKNNGVTASVDLLKNTKPYPAGLLTVALG 63 + ++FDV G +D NK ++R NG +D K K Y L + +G Sbjct: 12 ISDLFDVVIGKTIDGNKAQRNENGTPYITRKATRNGFEFMIDGEKE-KLYSGKLPVITIG 70 Query: 64 GSVLSTFLQNKPFYTAQNVAVLNPKTEMTEQQKLFYCAAIFANAYRFSACGREANRT-LR 122 F+Q F+T V + PK ++ L Y + NA + + N T L+ Sbjct: 71 NETSKPFVQEFHFFTGTKVNICIPKLDLNRNH-LLYITTMIENATKMFSYSYTINSTRLK 129 Query: 123 QL--FVPSLDEIPSW--------------VESVNLNPSAGVTEPKLKESLDLPVVRQSKR 166 L +P E P W ++ ++ + GV++ + + L + Sbjct: 130 SLKILLPIKGEEPDWDYMNTYISKILSNMEKNFDVQQNDGVSDLRSLKDLSW----SQFK 185 Query: 167 LPEIFTIQNGIAATK 181 +DEIF+I +G+ TK Sbjct: 186 MDEIFSINSGVRLTK 200 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 84 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 167> which encodes amino acid sequence <SEQ ID 168; NGS84>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): 3.15 Possible cleavage site: 33 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 1.22 threshold: 0.0 PERIPHERAL Likelihood = 1.22 modified ALOM score: −0.74 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.072(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi\|2495432\|sp\|P55409\|Y4DJ_RHISN HYPOTHETICAL TRANSCRIPTIONAL REGULATOR Y4DJ gi\|7465604\|pir\|\|T02773 y4dJ protein-Rhizobium sp.", "plasmid pNGR234a gi\|2182353\|gb\|AAB91639.1\|(AE000069) Y4dJ [Rhizobium sp.", "NGR234] Length = 77 Score = 44.4 bits (104), Expect = 7e−04 Identities = 25/61 (40%), Positives = 36/61 (58%) Query: 92 KAGGETFVSLRKGFTQSELATAAGLPQPYLSRIENSKQSLQDKTVQKLANALGVSPLE 151 K G F LR +KG TQ E+ +G Q YLS +E +++ T+ +LA ALGVS +E Sbjct: 5 KLVGSNFARLREEKGLTQEEEARSGFSQQYLSSLERGRRNPTVITLYELAQALGVSHVE 64 Query: 152 V 152 + Sbjct: 65 L 65 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 85 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 169> which encodes amino acid sequence <SEQ ID 170; NGS85>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −6.09 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.92 threshold: 0.0 PERIPHERAL Likelihood = 2.92 modified ALOM score: −1.08 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.480(Affirmative) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 86 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 171> which encodes amino acid sequence <SEQ ID 172; NGS86>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −2.92 Possible cleavage site: 21 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −2.76 threshold: 0.0 INTEGRAL Likelihood = −2.76 Transmembrane 179-195 (179-195) PERIPHERAL Likelihood = 2.17 modified ALOM score: 1.05 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.210 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >sp\|Q05205\|PPB_LYSEN ALKALINE PHOSPHATASE PRECURSOR (APASE) pir\|\|A42467 alkaline phosphatase (EC 3.1.3.1) phoA precursor-Lysobacter enzymogenes emb\|CAA39978.1\|(X56656) alkaline phosphatase [Lysobacter enzymogenes] Length = 539 Score = 37.5 bits (86), Expect = 0.40 Identities = 28/82 (34%), Positives = 43/82 (52%), Gaps = 8/82 (9%) Query: 189 VALGLQAYWDVAGANNGATGQSPNIKTAQVPAKITRRNADGTTDTFGGGSARKSAAASVS 248 V GL A W+V+ A + AQV +++ R+ GT D + G+A A AS S Sbjct: 458 VLRGLMA-WNVSSA------AGKTLTGAQVKLQVSDRST-GTYDLYRAGAAWTEANASYS 509 Query: 249 GIEAGKKVTAVIPAVRGAVAYA 270 G+ G K+ +V+P+ GA + A Sbjct: 510 GVSLGSKIGSVVPSATGAQSIA 531 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 87 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 173> which encodes amino acid sequence <SEQ ID 174; NGS97>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): 0.18 Possible cleavage site: 35 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 1.70 threshold: 0.0 PERIPHERAL Likelihood = 1.70 modified ALOM score: −0.84 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.138(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi\|12514207\|gb\|AAG55499.1\|AE005289_17 (AE005289) unknown protein encoded by cryptic prophage CP-933M [Escherichia coli O157:H7] gi\|12514720\|gb\|AAG55907.1\|AE005324_10 (AE005324) unknown protein encoded by prophage CP-933N [Escherichia coli O157:H7] Length = 108 Score = 30.9 bits (69), Expect = 9.1 Identities = 21/55 (38%), Positives = 28/55 (50%), Gaps = 3/55 (5%) Query: 1 MAAPVSLEEFKQRIGVEHDRRDDFFLSVIDGVSAAAEAYIGRSLLAADYVGRYDG 55 M A ++LEE K + V+HD DD + + +A AYI S D V R DG Sbjct: 1 MTALLTLEEIKAHLRVDHDADDDMLMDKVRQATAVLLAYIQGS---RDKVIREDG 52 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 88 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 175> which encodes amino acid sequence <SEQ ID 176; NGS88>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.69 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 6.05 threshold: 0.0 PERIPHERAL Likelihood = 6.05 modified ALOM score: −1.71 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.227(Affirmative) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 89 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 177> which encodes amino acid sequence <SEQ ID 178; NGS89>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −4.77 Possible cleavage site: 26 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 1.38 threshold: 0.0 PERIPHERAL Likelihood = 1.38 modified ALOM score: −0.78 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.284(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi\|9634142\|ref\|NP_037684.1\|gp24 [Enterobacteria phage HK022] gi\|6863134\|gb\|AAF30375.1\|AF069308_23 (AF069308) gp24 [Enterobacteria phage HK022] Length = 1183 Score = 44.9 bits (102), Expect = 0.006 Identities = 38/127 (29%), Positives = 64/127 (49%), Gaps = 11/127 (8%) Query: 851 NKALRDKINLIDGNGAGSVNERVEAVRSTADGNAAAVQTHARSI---NG-LEAQYTVK-- 904 NKA + +N + + ++ + +T +GN +A+ T+A++I NG L A Y +K Sbjct: 989 NKASINSLNQTFSDYQQATATQINGITATVNGNTSAITTNAQAIANVNGDLSAMYNIKVG 1048 Query: 905 VDANGK--VAGFGLATTPKNGTPESKFIVNADRFGI-GAAGKADVFPFVVDTQKNRVGIN 961 V +NG+ AG G+ +S+ I ADRF + AAG + PFV+ Q + I Sbjct: 1049 VSSNGQYYAAGMGIGVENTPSGMQSQVIFLADRFAVTTAAGNSVALPFVI--QNGQTFIR 1106 Query: 962 GELVVNG 968 + +G Sbjct: 1107 ASFIQDG 1113 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 90 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 179> which encodes amino acid sequence <SEQ ID 180; NGS90>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −2.82 Possible cleavage site: 24 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 4 value: −9.66 threshold: 0.0 INTEGRAL Likelihood = −9.66 Transmembrane 321-337 (317-349) INTEGRAL Likelihood = −6.48 Transmembrane 351-367 (340-371) INTEGRAL Likelihood = −5.73 Transmembrane 907-923 (903-926) INTEGRAL Likelihood = −0.00 Transmembrane 430-446 (430-446) PERIPHERAL Likelihood = 2.17 modified ALOM score: 2.43 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.486 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi\|12514839\|gb\|AAG56002.1\|AE005332_9 (AE005332) putative tail component of prophage CP-933X [Escherichia coli O157:H7] Length = 1026 Score = 111 bits (279), Expect = 3e−23 Identities = 78/274 (28%), Positives = 146/274 (52%), Gaps = 10/274 (3%) Query: 69 AAGNQAQQASEKVRAEVGKIGSGLSGLTKLLAGLATADFAKSVLDTADAMQSINSQVRQV 128 AA + ++A ++ +++ +I + G+T AG A ++ AD S+N++++Q Sbjct: 45 AAAREQRRALAELHSQLTEIRASAVGMTGAFAG---AFATGHLISLADEWSSVNARLKQA 101 Query: 129 TSSETEYLAVQQQLLDTANRTRASLESTANLYVSTSRALKDYGYTQQEILKFTEAANNAM 188 + S E+ + Q+ L+D + RT + A L+ ++ ++++YGY+ ++LK TEA + + Sbjct: 102 SQSSDEFASSQKVLMDISQRTGTAFSDNAALFARSAASMREYGYSADDVLKVTEAISTGL 161 Query: 189 TIGGVGAQQQAAALMQLSQALGSGVLQGDEFKSISEAAPILLDTIAEYMGKSRDEIKKLG 248 I G + + + Q SQAL GVL+G+EF S++E+ ++ +A MG +R ++K + Sbjct: 162 KISGASTAEAGSVITQFSQALAQGVLRGEEFNSVNESGDRIVRALAAGMGVARKDLKAMA 221 Query: 249 SEGKLTADVIFKAISGASEKFGEQAAKMPVTMGQALTVFSNNWQSMVSKLLNDSGTMSGI 308 +GKLTAD + A+ ++ A MP T+ ++T N + + V G + Sbjct: 222 DDGKLTADKVVPALISQLGILRDEYAAMPETVSSSITKVENAFMAWV-------GGANEA 274 Query: 309 AAVIKLIADNLNLVVPIVAGFAVAVAAAVAPTLA 342 + V K ++ LN V + A AV A VA +A Sbjct: 275 SGVTKTLSGMLNGVAGQIDNVATAVGALVAVGVA 308 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 91 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 181> which encodes amino acid sequence <SEQ ID 182; NGS91>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −0.63 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 0.16 threshold: 0.0 PERIPHERAL Likelihood = 0.16 modified ALOM score: −0.53 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.250(Affirmative) < succ> The protein has homology with the following sequences in the databases: (AF237934) putative integrase/recombinase [Pasteurella multocida] Length = 329 Score = 449 (206.9 bits), Expect = 4.4e−91, Sum P(2) = 4.4e−91 Identities = 93/196 (47%), Positives = 129/196 (65%) Query: 56 IFADLIRRYLSEVTPSKRGAREESYRIGRALKTPLAKVRLADLRPQDFADWRDQRLQEVS 115 IF D+I RY +EV+ +K+GAR E R+ R L+ ++ + + DLR +DF +W RL EVS Sbjct: 55 IFRDVIERYQNEVSITKKGARNEIIRLNRFLRYDISNLYIRDLRKEDFEEWIRIRLTEVS 114 Query: 116 PTSVGRELTTLSAVCEHAMKEWGLLRENPVRKISKPKKSRARTRRPTEQEIADICAALLY 175 SV REL T+S+V A+ +WG + +P+ I KPK S R R +EQ+I I Y Sbjct: 115 DASVRRELVTISSVLTTAINKWGYISRHPMTGIEKPKNSAERKERYSEQDIKTILETARY 174 Query: 176 RPNEKPKMAVQRVAVAVLFAIETAMRAGEICGLKWADVNMRRRIAHLPITKNGDSRDVPL 235 ++ P QRVA+A+LFAIETAMRAGEI +KW +V + +RI HLP TKNG SRDVPL Sbjct: 175 CEDKLPITLKQRVAIAMLFAIETAMRAGEIASIKWDNVFLEKRIVHLPTTKNGHSRDVPL 234 Query: 236 SLRAAELIEQLRGIDD 251 S RA LI +++ +++ Sbjct: 235 SQRAVALILKMKEVEN 250 Score = 248 (114.3 bits), Expect = 4.4e−91, Sum P(2) = 4.4e−91 Identities = 48/76 (63%), Positives = 57/76 (75%) Query: 254 VFSLDAKSLDVLFRRARDNCGIQGLHFHDTRREALTRLSKKVPVEVLAKISGHRDLRILL 313 VF +SL FR + CG++ LHFHDTRREALTRLSKKV V LAKISGHRDLRIL Sbjct: 254 VFQTTPESLSTTFRVLKKECGLEHLHFHDTRREALTRLSKKVDVMTLAKISGHRDLRILQ 313 Query: 314 NVYYRPDMADIAKMLD 329 N YY P+M+++A +LD Sbjct: 314 NTYYAPNMSEVANLLD 329 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 92 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 183> which encodes amino acid sequence <SEQ ID 184; NGS92>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −7.85 Possible cleavage site: 25 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −8.33 threshold: 0.0 INTEGRAL Likelihood = −8.33 Transmembrane 6-22 (1-25) PERIPHERAL Likelihood = 5.99 modified ALOM score: 2.17 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.433 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi\|963205\|ref\|NP_048840.1\|A484L [Paramecium bursaria Chlorella virus 1] gi\|7461623\|pir\|T17986 hypothetical protein A484L-Chlorella virus PBCV-1 gi\|1620155\|gb\|AAC96851.1\|(U42580) A484L [Paramecium bursaria Chlorella virus 1] Length = 155 Score = 31.6 bits (70), Expect = 3.5 Identities = 20/72 (27%), Positives = 36/72 (49%) Frame = +1 Query: 52 LQINLKMLEKRIDFLVENIDKYYQQYGSYPNNFDFISTKTDFTTESYCDFWDKNIAGYGN 231 + +NLKM I F +DKY +QY +Y N F T+ + + ++ + +I N Sbjct: 23 IAVNLKMNGVSIPF----VDKYSKQYPTYTKNALFHVTRFNNAYQKTFEYKNISIDTINN 78 Query: 232 CYFVKNDKDYTI 267 + +++D Y I Sbjct: 79 LFSIRDDVLYNI 90 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 93 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 185> which encodes amino acid sequence <SEQ ID 186; NGS93>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.08 Possible cleavage site: 14 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 0.79 threshold: 0.0 PERIPHERAL Likelihood = 0.79 modified ALOM score: −0.66 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.320(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gb\|AAG22017.1\|AF288038_2 (AF288038) putative HsdR ]Streptococcus thermophilus] Length = 740 Score = 674 bits (1738), Expect = 0.0 Identities = 364/746 (48%), Positives = 489/746 (64%), Gaps = 40/746 (5%) Query: 14 NENSRVKIPAVLHLMRLGYDYLSLKNANW---DRQTNIFPEIFVDSLCRINPDLPPDDAR 70 +E +RV+IPA HLMRLGY YL D +TNI IF + + N D Sbjct: 8 SELTRVQIPAAFHLMRLGYTYLPHNGKEIMGRDPETNILISIFREQFLKFNNYATDLDVE 67 Query: 71 RLLADIRLELDNEDLGQKFYERLTNQSGGKKLIDFQNFDNNSFHVVTELPCINGDEAFRP 130 R L +I++ELD DLG+ FY R+ + SG +D++N +NN+FH+ E+ C NG + FRP Sbjct: 68 RELNNIKIELDQNDLGRAFYNRIVSDSG-PTYVDWENPENNTFHLALEVTCQNGGDEFRP 126 Query: 131 DIALLVNGMPLVFIEVKKPN----NKGGIGEERERMGKRAKNPKFRRFINITQFMIFSNN 186 DI + +NG+PL +IEVK+PN K I E+ R R +N +FRRF NITQ + FS+N Sbjct: 127 DIVIFINGLPLSYIEVKQPNAIRDGKTAIQSEQSRTAVRFENRRFRRFNNITQLISFSDN 186 Query: 187 MEYDDGATEPAQGAFYASSACGKPVFNYFREEHKXXXXXXXXXXXXXXXXXVLQDNNLPV 246 + Y G + QG+FY S+A K FN F+EE + VL+D N Sbjct: 187 LPYISGQGQQKQGSFYCSNAFSKTKFNAFKEEREEELIYSIRSLGEEEIDAVLKDVNRFA 246 Query: 247 IKHSPEFISNKSPDTPTNRILTSLLCRERLSFLLQHGLTYVK--ASQGLVQ--KHIMRYP 302 +K PEF +N+ P TP N ++SL ++RL FLL++GL YV+ + G +Q KH+MRYP Sbjct: 247 LKSQPEFKTNQDPSTPCNTFISSLYQKKRLLFLLRYGLAYVEEHSKDGTIQLQKHVMRYP 306 Query: 303 QLFATLAIEKHLANGGKKGVIWHTQGSGKTALAYYNTRYLTHYYAKQGIVPKFYFIVDRL 362 Q FAT AIE + G +KGVIWHTQGSGKTAL+Y+N RYLT+Y++KQGIVP+FYF+VDRL Sbjct: 307 QFFATKAIEDAIGKGVRKGVIWHTQGSGKTALSYFNIRYLTNYFSKQGIVPQFYFVVDRL 366 Query: 363 DLLKQAQREFTARDLVVHTIDSREAFAADIKSAQTLHNHAGKAEITVVNIQKFQDDPDVV 422 DL QA REFT R L V I+S Q L+ ++ VVNIQKF+D+ D+ Sbjct: 367 DLADQATREFTKRGLKVKRINS----------PQELNEKHDAYQVAVVNIQKFKDNSDLT 416 Query: 423 ARNDYDLAIQRVYFLDEVHRSYNPKGSFLANLNQSDVNAVKIGLTGTPLI-----GVTA- 476 + YDL Q +YF+DE HRSYN KGS+L NL +D NA+KI LTGTPLI G T Sbjct: 417 DHSGYDLNRQNIYFIDEAHRSYNEKGSYLPNLYNADKNAIKIALTGTPLITYKKDGKTKE 476 Query: 477 GNVNTRELFGDYIHKYYYNASIADGYTLRLIREEIGSRYKAQLQEALAQLEIEKGSFDRK 536 + TR++FGDYIHKYYYN SI DG+TLRL+RE+I + YK LQ EI +G + Sbjct: 477 SHATTRDIFGDYIHKYYYNQSIDDGFTLRLMREDIETSYKETLQTI--NEEILRGDLSKD 534 Query: 537 EIYAHPHFVHPMLDYILDDFAKFRKTN-QDESLGAMVVCDSAEQARQL---FEHFQTASD 592 +I+AHP +V PMLG+IL+DF + R D+S+G M+VCDS++QAR++ E ++ + Sbjct: 535 DIFAHPRYVSPMLDFILEDFNRARDVVFDDDSIGGMIVCDSSKQAREIEKQLEERRSRGE 594 Query: 593 HNFTAALILHDVGTKEERDQWVKDFKAGKIDILFVYNMLLTGFDAPRLKKLYLGRLIKAH 652 N T+ALILHD G KE + V+ ++ GKID++ VY+MLLTGFDAPRLK+LYLGR IKAH Sbjct: 595 TNITSALILHDEGDKEYKKDRVESYREGKIDLVIVYSMLLTGFDAPRLKRLYLGRKIKAH 654 Query: 653 NLLQTLTRVNRTYKSYRYGYVVDFADIEREFDKTNRAYWDELSEN-----LGDEIGS-YS 706 NLLQTLTRVNR YK Y++GYV+DFADI +EFDKTNRAY +EL+ E G+++ + + Sbjct: 655 NLLQTLTRVNRPYKDYQFGYVIDFADISKEFDKTNRAYLEELNQEYDPKNTGEDVENVFG 714 Query: 707 QLFKTAEEIEQEIADIKNALFDFDTE 732 LF +A+EI +++ + L ++ TE Sbjct: 715 SLFVSADEISKQLEKSETILMNPTE 740 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 94 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 187> which encodes amino acid sequence <SEQ ID 188; NGS94>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.19 Possible cleavage site: 35 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.73 threshold: 0.0 PERIPHERAL Likelihood = 5.73 modified ALOM score: −1.65 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.302(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi\|10717100\|gb\|AAG22014.1\|AF288037_3 (AF288037) putative HsdS [Streptococcus thermophilus] Length = 402 Score = 154 bits (389), Expect = 2e−36 Identities = 123/348 (35%), Positives = 168/348 (47%), Gaps = 48/348 (13%) Query: 73 GKTAFVDILDDGEVAFGSTEFIVLRAKNET--NPEFLYYFAISPDFRKRAIECMEGTSGR 130 GKT ++ DGE ++ IV +E+ +FLYYF + F G++ + Sbjct: 74 GKT----VIFDGEDSYFQDSNIVWIENDESKVTNQFLYYFLQTNPFIT-----TNGSTIK 124 Query: 131 QRVNENALKTLELPIPEPQIQQSIAAVLSALDKKIALNKQINARLEEMAKTLYDYWFVQF 190 + N+N T +P Q Q I +L LDKKI +N QIN LE MAKTLYDYWFVQF Sbjct: 125 RLYNDNLRDTKIPNVPSIQQQNQITDILGTLDKKIQINNQINQELEAMAKTLYDYWFVQF 184 Query: 191 DFPDANGKPYKSSGGDMVFDETLKREIPKGWGSIELQSCL---AKIPNTTKILNKDIKDF 247 DFPD NGKPYKSSGG MV++ LKREIP+GWG+ +L S L + N K N++ K + Sbjct: 185 DFPDQNGKPYKSSGGKMVYNPELKREIPEGWGAEKLSSLLKIGKETTNPKKFPNEEFKYY 244 Query: 248 --------GKYPVVD----QSQDFICGFTNKEKSILNPQDAHIIFGDHTRIVKLVNFQYA 295 G Y + +S F + S LNP +I+ + F Sbjct: 245 SIPEFDTTGTYSLERGESIKSNKFKFKVEKNDLLVSKLNPWFNRVIYNLEENAIASTEF--- 301 Query: 296 RGADGTQVILSNNERMPNYLFYQIINQIDLSSY------GYARHFK-----FLKEFKIIL 344 ++ R YQ+ + Y G + K + F+I Sbjct: 302 -------IVWKTFNRFEKNFLYQVATGKEFIEYCTRFATGTSNSHKRVSPDIMVGFQIPF 354 Query: 345 PSKDISQKYNEIANTFFVKVRNNLKQNHHLTQLRDFLLPMLMNGQVSV 392 I QK+ EI ++ +V N +QN LTQLRD++LPMLMNGQV V Sbjct: 355 EKTHI-QKFGEIIDSIRTQVLQNNEQNQELTQLRDWILPMLMNGQVKV 401 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 95 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 189> which encodes amino acid sequence <SEQ ID 190; NGS95>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.12 Possible cleavage site: 19 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 3 value: −10.51 threshold: 0.0 INTEGRAL Likelihood = −10.51 Transmembrane 112-128 (109-132) INTEGRAL Likelihood = −4.46 Transmembrane 50-66 (46-70) INTEGRAL Likelihood = −2.23 Transmembrane 7-23 (7-23) PERIPHERAL Likelihood = 4.19 modified ALOM score: 2.60 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.520 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >pir\|\|G69096 hypothetical protein MTH1717-Methanobacterium thermoautotrophicum (strain Delta H) gb\|AAB86189.1\|(AE000928) unknown [Methanothermobacter thermautotrophicus] Length = 557 Score = 35.4 bits (80), Expect = 0.50 Identities = 25/80 (31%), Positives = 47/80 (58%), Gaps = 5/80 (6%) Query: 52 LLFYFLIPFIATATVLWLSKYLGKDEFKQGEVKELEYVNDNFLPSYLGYFFVALSIPDNN 111 L+F+F+ P + TATVL + K + ++ F++ EV L + +PS++ ++ IP++ Sbjct: 92 LVFFFISPLLGTATVLVIYK-VARETFEREEVALLSAFLFSMVPSFVAR--TSVFIPESM 148 Query: 112 LFLLFVMYGIIFLLVSCSKS 131 LL GI+++LV K+ Sbjct: 149 GLLL--TSGILYMLVKYLKT 166 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 96 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 191> which encodes amino acid sequence <SEQ ID 192; NGS96>.", "Analysis of this protein sequence reveals the following: GyH: Examining signal sequence (von Heijne) Signal Score (−7.5): −7.76 Possible cleavage site: 28 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 6.15 threshold: 0.0 PERIPHERAL Likelihood = 6.15 modified ALOM score: −1.73 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.362(Affirmative) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 97 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 193> which encodes amino acid sequence <SEQ ID 194; NGS97>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.48 Possible cleavage site: 13 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 8.86 threshold: 0.0 PERIPHERAL Likelihood = 8.86 modified ALOM score: −2.27 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.127(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref\|NP_052265.1\|P2 J homolog; baseplate or base of tail fibre [Enterobacteria phage 186] gb\|AAC34162.1\|(U32222) P2 J homolog; baseplate or base of tail fibre [Enterobacteria phage 186] Length = 302 Score = 112 bits (280), Expect = 3e−24 Identities = 65/151 (43%), Positives = 85/151 (56%), Gaps = 1/151 (0%) Query: 1 MGNSRLSQLPAPAAIEETDFEGIFARKKAALTALCPESIRETVAQTLELESEPLTIDLQQ 60 M LS LP P +EE DFE I A + A L +L PE +E VA+TL LESEP+ LQ+ Sbjct: 1 MATVDLSLLPVPDVVEELDFETILAERIATLISLYPEDQQEAVARTLALESEPIVKLLQE 60 Query: 61 QAYQELLVRNRINEAVKANLLAYAQGSDLDHIAAQYGLSRKTIRXXXXXXXXXXXXEYET 120 AY+E++ R R+NEA +A +LAYA+ SDLD++ A + + R +R E E Sbjct: 61 NAYREVIWRQRVNEAARAGMLAYARDSDLDNLGANFNVERLVVRPADDTTIPPTPAEMEL 120 Query: 121 DDAFRARV-QAHPEKYAAGPRTAYEAHAIDA 150 D FR R+ QA AG AYE H A Sbjct: 121 DADFRLRIQQAFEGMSVAGSTGAYEFHGRSA 151 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 98 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 195> which encodes amino acid sequence <SEQ ID 196; NGS98>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.68 Possible cleavage site: 33 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.61 threshold: 0.0 PERIPHERAL Likelihood = 4.61 modified ALOM score: −1.42 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.182(Affirmative) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 99 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 197> which encodes amino acid sequence <SEQ ID 198; NGS99>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −4.87 Possible cleavage site: 19 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.93 threshold: 0.0 PERIPHERAL Likelihood = 4.93 modified ALOM score: −1.49 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.189(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi\|10172952\|dbj\|BAB04058.1\|(AP001508) BH0339-unknown conserved protein in others [Bacillus halodurans] Length = 283 Score = 83.7 bits (206), Expect = 1e−15 Identities = 59/156 (37%), Positives = 87/156 (54%), Gaps = 8/156 (5%) Query: 10 VRGPVQLAFAQSIDPIVPPEVSITRMAVTNEKDLEKERTMGRKYIVPYVVYRVHGFISAN 69 VRGPV + A SIDPI IT+ + D TMG K+ V + VY G I+ Sbjct: 129 VRGPVSIHTATSIDPIDIVSTQITKSVNSVTGDKRSSDTMGMKHRVDFGVYVFKGSINTQ 188 Query: 70 LAAKTGFSDDDLAKLWQALTLMFEHDRSAAR--GEMARKLVFKHDSALGSQPAHKLFD 127 LA KTGF+++D K+ +AL +FE+D S+AR G M K+ ++H S LG + K+ Sbjct: 189 LAEKTGFTNEDAEKIKRALITLFENDSSSARPDGSMEVHKVYWWEHSSKLGQYSSAKVHR 248 Query: 128 AVKVERVNGESGTPASGFGDYKISVVSDGLNGVSVE 163 ++K+E ++ TP S F DY + + L+G+ VE Sbjct: 249 SLKIE---SKTDTPKS-FDDYAVELYE--LDGLGVE 278 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 100 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 199> which encodes amino acid sequence <SEQ ID 200; NGS100>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.03 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 6.63 threshold: 0.0 PERIPHERAL Likelihood = 6.63 modified ALOM score: −1.83 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.185(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi\|1175791\|sp\|P44189\|YE18_HAEIN HYPOTHETICAL PROTEIN HI1418 gi\|1074769\|pir\|\|A64029 hypothetical protein HI1418-Haemophilus influenzae (strain Rd KW20) gi\|1574254\|gb\|AAC23068.1\|(U32821) H. influenzae predicted coding region HI1418 [Haemophilus influenzae Rd] Length = 201 Score = 144 bits (364), Expect = 1e−33 Identities = 71/109 (65%), Positives = 79/109 (72%) Query: 8 NFQQNSVRTVADNKGELWFLANDVCEILGYTNPRRTVDLHCKSRGVTKRYTPTTSGEQEM 67 NF+ VR + D KGE WF DVC ILGYTN R+ + HCK GVTKRYTPT S +QEM Sbjct: 24 NFKDLPVRVILDPKGEFWFCGTDVCHILGYTNSRKALQDHCKQGGVTKRYTPTKSADQEM 83 Query: 68 TYINEPNLYRLIIKSRKPAAEAFEEWVMETVLPAIRKTGGCQVGPKTTA 116 T+INEPNLYRLIIKSRKP AE FE WV E VLP IRKTG Q+ P+ A Sbjct: 84 TFINEPNLYRLIIKSEKPEAEPFEAWVFEEVLPQIRKTGKYQLQPQQLA 132 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 101 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 201> which encodes amino acid sequence <SEQ ID 202; NGS101>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −1.23 Possible cleavage site: 47 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.55 threshold: 0.0 PERIPHERAL Likelihood = 3.55 modified ALOM score: −1.21 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.126(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi\|9632520\|ref\|NP_049514.1\|hypothetical protein [Bacteriophage 933W] gi\|9633449\|ref\|NP_050552.1\|hypothetical protein [Bacteriophage VT2-Sa] gi\|4585431\|gb\|AAD25459.1\|AF125520_54 (AF125520) hypothetical protein [Bacteriophage 933W] gi\|5881645\|dbj\|BAA84336.1\|(AP000363) hypothetical protein [Bacteriophage VT2-Sa] gi\|7649882\|dbj\|BAA94l60.1\|(AP000422) hypothetical protein [Escherichia coli O157:H7] Length = 404 Score = 177 bits (449), Expect = 3e−43 Identities = 130/425 (30%), Positives = 204/425 (47%), Gaps = 27/425 (6%) Query: 7 TAYGDPQAMMKQAAGLFAMHMQRNSTLNRLAGKMPAGTA-GAEATLRKQTTQHMPVVRCQ 65 T QA LF + S +N L + A A + KQT+ PVVR Sbjct: 2 TTVTSAQANKLYQVALFTAANRNRSMVNILTEQQEAPKAVSPDKKSTKQTSAGAPVVRIT 61 Query: 66 DLTRGMGDEIRFNLVNPVSALPIMGDNTAEGRGVGMSLSEAGLRVNQARFPVDGGGTMTN 125 DL + GDE+ F++++ +S P MGD EGRG +S ++ L++NQ R VD GG M+ Sbjct: 62 DLNKQAGDEVTFSIMHKLSKRPTMGDERVEGRGEDLSHADFSLKINQGRHLVDAGGRMSQ 121 Query: 126 QRSPADYRALIRPAAQSLMDRYADQTLLVHMAGARGFHDNIEWGVPLAGDPKFNDYAVNP 185 QR+ + + R + + DQ +VH+AGARG + +P A P+F +N Sbjct: 122 QRTKFNLASSARTLLGTYFNDLQDQCAIVHLAGARGDFVADDTILPTAEHPEFKKIMIND 181 Query: 186 VKAPSKNRHFTASGDAVTGVGDNGGELKIASTDLFTMDTVDSMRTVLDQIPLPPPIVKFE 245 V P+ +RHF GD +I + D+F++ VD++ +D++ P V+ Sbjct: 182 VLPPTHDRHFFG--------GDATSFEQIEAADIFSIGLVDNLSLFIDEMAHPLQPVRLS 233 Query: 246 GDKAAGDSPLRVWLLSPAQYNRF---AADPKFRQLQASAIARASQANQNPLFLGDAGLWN 302 GD+ G+ P V ++P Q+N + + + Q+ A+ RA N +PLF G+ +W Sbjct: 234 GDELHGEDPYYVLYVTPRQWNDWYTSTSGKDWNQMMVRAVNRAKGFN-HPLFKGECAMWR 292 Query: 303 GFILVKMP-RPIRFYAGDEMKYCADKFSEAESGLKIPASFADKFAVDRSVILGGQAVLEA 361 ++ K PIRFY G ++ + + A +DR++++LG QA+ A Sbjct: 293 NILVRKYAGMPIRFYQGSKVLVSENNLTATTK------EVAAATNIDRAMLLGAQALANA 346 Query: 362 FANTGKHGGMPFFWSEKELDHGNRVETLVGTIRGVAKTRFAVDVGGGAKEITDYGVTVVD 421 + G+ G F EK+ D NR E + I G+ K RF G ++ D+GV VD Sbjct: 347 Y---GQKAGGHFNMVEKKTDMDNRTEIAISWINGLKKIRFPEKSG----KMQDHGVIAVD 399 Query: 422 TVVPL 426 T V L Sbjct: 400 TAVKL 404 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 102 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 203> which encodes amino acid sequence <SEQ ID 204; NGS 102>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −6.09 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.92 threshold: 0.0 PERIPHERAL Likelihood = 2.92 modified ALOM score: −1.08 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.480(Affirmative) < succ The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 103 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 205> which encodes amino acid sequence <SEQ ID 206; NGS 103>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −1.29 Possible cleavage site: 34 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −0.00 threshold: 0.0 INTEGRAL Likelihood = −0.00 Transmembrane 22-38 (22-38) PERIPHERAL Likelihood = 4.88 modified ALOM score: 0.50 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.100(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi\|11277848\|pir\|\|E81145 replicative DNA helicase NMB0885 [imported]-Neisseria meningitidis (group B strain MD58) gi\|7226124\|gb\|AAF41296.1\|(AE002441) replicative DNA helicase [Neisseria meningitidis MC58] Length = 468 Score = 233 bits (594), Expect = 5e−60 Identities = 158/456 (34%), Positives = 245/456 (53%), Gaps = 34/456 (7%) Query: 15 SVGAEQNILGGILIEPTAIARCA-ILTPEKFYQAQHRIIFRALLDMAAANEPIDIITLND 73 S+ AEQ++LGG+++E A R A +++ E FY+ +HR+IFR++ + + P D+IT+ + Sbjct: 23 SMEAEQSVLGGLMLENPAWDRIADVVSGEDFYRHEHRLIFRSIAKLINESRPADVITVQE 82 Query: 74 KLEARGEAENAGGLAYLIDLNQNTPSAKNISRYVGIVNDRFVERGLLKASAAIEKIAVSK 133 L+ E E AGG YLI L QNTPSA NI RY IV +R + R L + I + A + Sbjct: 83 DLQRNEELEAAGGFEYLITLAWNTPSAANIRRYAEIVRERSIMRQLAEVGTEIARSAYNP 142 Query: 134 DGGTVAEKLSKAADELAAVGKDAVKRETKTFGQTVEDLIGGLDKRLDGVR--------FG 185 G + L +A +++ + + K +K + DL+ + +R+D + G Sbjct: 143 QGRDAGQLLDEAENKVFQIAESTAK--SKQGFLEMPDLLKEVVQRIDMLYSRDNPDEVTG 200 Query: 186 LPTGLMKLDGMTGGLPDGNLIVIAARPSMGKTVLAENIARFALKQGK-AVHFQSYEMSAV 244 +PTG + LD T GL G+LI++A RPSMGKT + NIA +G+ V S EM Sbjct: 201 VPTGFIDLDKKTSGLQPGDLIIVAGRPSMGKTAFSINIAEHVAVEGRLPVAVFSMEMGGA 260 Query: 245 ELARRGMAAECNIPMQNLKTGNLTQSDYANM----------PIYVSQAKEWKFDVNCDLL 294 +L R + + + LKTG L + + P+Y+ + Sbjct: 261 QLVMRMLGSVGRLDQSVLKTGRLEDEHWGRLNEAVVKLSDAPVYIDETPGLTALELRARA 320 Query: 295 NVDELCFLAKEKKLTTGLDLLVVDHLHIMPRAGRDE--VAELGNISRRLKNLAAELNTPV 352 F K L L+V+D+L +M +GR + +ELG ISR LK LA EL P+ Sbjct: 321 RRLARQFNNK-------LGLIVIDYLQLMAGSGRSDNRASELGEISRSLKALAKELQVPI 373 Query: 353 VLVAQLNRGNTKQADKRPNMADIRGSGAIEQDANIIIMPHRESYYDGNENP--SIAELII 410 + ++QL+R + DKRP M+D+R SGAIEQDA++I+ +R+ YY+ ++P +AE II Sbjct: 374 IALSQLSRTVESRTDKRPMMSDLRESGAIEQDADLIMFLMYRDEYYN-QDSPMKGLAECII 432 Query: 411 AKNRDGEMGTVVCGWKGQFMKFEEEPDLAWQAPKHD 446 K+R+G +G + W GQF KF+ + +A D Sbjct: 433 GKHRNGPVGKIFLTWTGQFTKFDNAAYIPEEAKIED 468 >gi\|11277846\|pir\|\|E81876 probable replicative DNA helicase (EC 3.6.1.-) NMA1105 [imported]- Neisseria meningitidis (group A strain Z2491) gi\|7379799\|emb\|CAB84367.1\|(AL162755) putative replicative DNA helicase [Neisseria meningitidis Z2491] Length = 468 Score = 230 bits (588), Expect = 2e−59 Identities = 158/456 (34%), Positives = 244/456 (52%), Gaps = 34/456 (7%) Query: 15 SVGAEQNILGGILIEPTAIARCA-ILTPEKFYQAQHRIIFRALLDMAAANEPIDIITLND 73 S+ AEQ++LGG+++E A R A +++ E FY+ +HR+IFR++ + + P D+IT+ + Sbjct: 23 SMEAEQSVLGGLMLENPAWDRIADVVSGEDFYRHEHRLIFRSIAKLINESRPADVITVQE 82 Query: 74 KLEARGEAENAGGLAYLIDLNQNTPSAKNISRYVGIVNDRFVERGLLKASAAIEKIAVSK 133 L+ E E AGG YLI L QNTPSA NI RY IV +R + R L + I + A + Sbjct: 83 DLQRNEELEAAGGFEYLITLAQNTPSAANIRRYAEIVRERSIMQLAEVGTEIARSAYNP 142 Query: 134 DGGTVAEKLSKAADELAAVGKDAVKRETKTFGQTVEDLIGGLDKRLDGVR--------FG 185 G + L +A +++ + + K +K + DL+ + +R+D + G Sbjct: 143 QGRDAGQLLDEAENKVFQIAESTAK--SKQGFLEMPDLLKEVVQRIDMLYSRDNPDEVTG 200 Query: 186 LPTGLMKLDGMTGGLPDGNLIVIAARPSMGKTVLAENIARFALKQGK-AVHFQSYEMSAV 244 + TG + LD T GL G+LI++A RPSMGKT + NIA +GK V S EM Sbjct: 201 VSTGFIDLDKKTSGLQPGDLIIVAGRPSMGKTAFSINIAEHVAVEGKLPVAVFSMEMGGA 260 Query: 245 ELARRGMAAECNIPMQNLKTGNLTQSDYANM----------PIYVSQAKEWKFDVNCDLL 294 +L R + + + LKTG L + + P+Y+ + Sbjct: 261 QLVMRMLGSVGRLDQSVLKTGRLEDEHWGRLNEAVVKLSDAPVYIDETPGLTALELRARA 320 Query: 295 NVDELCFLAKEKKLTTGLDLLVVDHLHIMPRAGRDE--VAELGNISRRLKNLAAELNTPV 352 F K L L+V+D+L +M +GR + +ELG ISR LK LA EL P+ Sbjct: 321 RRLARQFNNK-------LGLIVIDYLQLMAGSGRSDNRASELGEISRSLKALAKELQVPI 373 Query: 353 VLVAQLNRGNTKQADKRPNMADIRGSGAIEQDANIIIMPHRESYYDGNENP--SIAELII 410 + ++QL+R + DKRP M+D+R SGAIEQDA++I+ +R+ YY+ ++P +AE II Sbjct: 374 IALSQLSRTVESRTDKRPMMSDLRESGAIEQDADLIMFMYRDEYYN-QDSPMKGLAECII 432 Query: 411 AKNRDGEMGTVVCGWKGQFMKFEEEPDLAWQAPKHD 446 K+R+G +G + W GQF KF+ + +A D Sbjct: 433 GKHRNGPVGKIFLTWTGQFTKFDNAAYIPEEAKIED 468 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 104 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 207> which encodes amino acid sequence <SEQ ID 208; NGS104>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −2.11 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.04 threshold: 0.0 PERIPHERAL Likelihood = 5.04 modified ALOM score: −1.51 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.220(Affirmative) < succ The protein has homology with the following sequences in the databases: >gi\|7515458\|pir\|\|T13296 hypothetical protein 8-Streptococcus phage phi- O1205 gi\|2444088\|gb\|AAC79524.1\|(U88974) ORF8 [Streptococcus thermophilus temperate bacteriophage O1205] Length = 157 Score = 62.1 bits (150), Expect = 3e−09 Identities = 53/161 (32%), Positives = 86/161 (52%), Gaps = 8/161 (4%) Query: 5 TLYRCAADVQAGLDYYFDSETEREDTLEAV--IGQFEVKAQSVIAYIKNQEITEKMLEGH 62 TLY + + D ET + DTLEA+ +E K + + IK+ E + + Sbjct: 3 TLYELTDQLLEIYNMDVDDET-KLDTLEAIDWTTDYENKVEGYVKVIKSLEADIEARKNE 61 Query: 63 IRQMTGKLKAAKARNQSLKDYLARNMQAAGITEIKADDGTFKASFRKSEAVVILDEAQIP 122 +++ G K+ +++ LK LA +M G T + D FK FRKSEAVV+ +E ++P Sbjct: 62 KKRLDGLNKSDQSKIDKLKTALAVSMAETGQTRV--DTTLFKVGFRKSEAVVV-NEEEKLP 118 Query: 123 AEFMREAVKTEPDKTAIRKAIESGRQVAGAKIEGRKNLQIR 163 E+ K PDK +++ ++SG+ + GA +E R+NL IR Sbjct: 119 KEYQIATYK--PDKKTLKBLLKSGKHIEGATLEERRNLNIR 157 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 105 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 209> which encodes amino acid sequence <SEQ ID 210; NGS 105>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −5.52 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.60 threshold: 0.0 PERIPHERAL Likelihood = 2.60 modified ALOM score: −1.02 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.135(Affirmative) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 106 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 211> which encodes amino acid sequence <SEQ ID 212; NGS 106>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): 4.8 Possible cleavage site: 26 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 27 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.80 threshold: 0.0 PERIPHERAL Likelihood = 7.80 modified ALOM score: −2.06 Score for OM-PP discrimination: 4.38 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 4.38 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Outer membrane?", "Score: 0.437687 Outer membrane?", "Score: 0.437687 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.768(Affirmative) < succ> The protein has no homology with sequences in the databases, although it is similar to HMW1 from Haemophilus influenzae.", "The protein was expressed in E.coli as an insoluble 43.56 kDa His-fusion product and then purified.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 107 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 213> which encodes amino acid sequence <SEQ ID 214; NGS 107>.", "Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.83 Possible cleavage site: 51 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.61 threshold: 0.0 PERIPHERAL Likelihood = 4.61 modified ALOM score: −1.42 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.146(Affirmative) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 108 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 215> which encodes amino acid sequence <SEQ ID 216; NGS108>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.14 Possible cleavage site: 19 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 8.43 threshold: 0.0 PERIPHERAL Likelihood = 8.43 modified ALOM score: −2.19 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.574(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|\|G81977 probable lipoprotein NMA0586 [imported]-Neisseria meningitidis (group A strain Z2491) emb\|CAB83877.1\|(AL162753) putative lipoprotein [Neisseria meningitidis Z2491] Length = 280 Score = 52.9 bits (126), Expect = 5e−06 Identities = 43/134 (32%), Positives = 63/134 (46%), Gaps = 23/134 (17%) Query: 174 LGDIRGVATDEDKLPKAGSFQYEGRAFGGNGVLSKESLDNHNGVFRYTIDFDRRKGSGSI 233 +GDI G T DKLP+ G Y G AFG D+ +G YTIDF ++G G I Sbjct: 156 IGDIAGEHTSFDKLPEGGRATYRGTAFGS---------DDASGKLTYTIDFAAKQGHGKI 206 Query: 234 EGMEQYGKIKLEEAAIERIPYRESGSSLGLKDRVSYFGVNEGVAMLEKDNEIKKYHLGIF 293 E ++ ++ ++ AA + P ++ + + ++L E Y LGIF Sbjct: 207 EHLKS-PELNVDLAASDIKPDKKRHAVI-------------SGSVLYNQAEKGSYSLGIF 252 Query: 294 GEAANEVAGAVSQE 307 G A EVAG+ E Sbjct: 253 GGQAQEVAGSAEVE 266 >pir\|\|D81032 hypothetical protein NMB1870 [imported]-Neisseria meningitidis (group B strain MD58) gb\|AAF42204.1\|(AE002537) hypothetical protein [Neisseria meningitidis MC58] Length = 320 Score = 50.6 bits (120), Expect = 3e−05 Identities = 50/168 (29%), Positives 76/168 (44%), Gaps = 28/168 (16%) Query: 136 VYEQPYSVVRGYFGYSRKDGNPIEGDGQNPEEIPFDLYLGDIRGVATDEDKLPKAGSFQY 195 VY+Q +S + + +D E G+ + F +GDI G T DKLP+ G Y Sbjct: 163 VYKQSHSALTAFQTEQIQDS---EHSGKMVAKRQFR--IGDIAGEHTSFDKLPEGGRATY 217 Query: 196 EGRAFGGNGVLSKESLDNHNGVFRYTIDFDRRKGSGSIEGMEQYGKIKLEEAAIERIPYR 255 G AFG D+ G YTIDF ++G+G IE ++ ++ ++ AA + P Sbjct: 218 RGTAFGS---------DDAGGKLTYTIDFAAKQGNGKIEHLKS-PELNVDLAAADIKPDG 267 Query: 256 ESGSSLGLKDRVSYFGVNEGVAMLEKDNEIKKYHLGIFGEAANEVAGA 303 + + + ++L E Y LGIFG A EVAG+ Sbjct: 268 KRHAVI-------------SGSVLYNQAEKGSYSLGIFGGKAQEVAGS 302 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 109 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 217> which encodes amino acid sequence <SEQ ID 218; NGS109>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.39 Possible cleavage site: 25 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.00 threshold: 0.0 PERIPHERAL Likelihood = 7.00 modified ALOM score: −1.90 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.353(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir\|\|A82012 hypothetical protein NMA0179 [imported]-Neisseria meningitidis (group A strain Z2491) emb\|CAB83494.1\|(AL162752) hypothetical protein NMA0179 [Neisseria meningitidis Z2491] Length = 97 Score = 183 bits (464), Expect = 1e−45 Identities = 92/97 (94%), Positives = 95/97 (97%) Query: 44 MKANDKLNRQIDVLQKQSAAIHNEAYIEMNTLLYRHREVVSIHNRKADYAEKGKERIALF 103 MK NDKLNRQIDVLQKQSAAIHNEAYIEMNTLLYRHREVVS+HNRKADYAEKGKE+IALF Sbjct: 1 MKTNDKLNRQIDVLQKQSAAIHNEAYIEMNTLLYRHREVVSVHNRKADYAEKGKEQIALF 60 Query: 104 PRGLNGITKLPAAVLLPERPYHFDMKEVLYIFSRIPR 140 PRGLNGITKLPAAVLLPERPYHFDMKEVL+IFS IPR Sbjct: 61 PRGLNGITKLPAAVLLPERPYHFDMKEVLHIFSWIPR 97 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS109 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 110 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 219> which encodes amino acid sequence <SEQ ID 220; NGS110>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.76 Possible cleavage site: 41 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −0.00 threshold: 0.0 INTEGRAL Likelihood = −0.00 Transmembrane 88-104 (88-104) PERIPHERAL Likelihood = 7.69 modified ALOM score: 0.50 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.100(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 111 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 221> which encodes amino acid sequence <SEQ ID 222; NGS111>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.89 Possible cleavage site: 21 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.44 threshold: 0.0 PERIPHERAL Likelihood = 2.44 modified ALOM score: −0.99 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.293(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gb\|AAC45840.1\|(AF001598) restriction endonuclease [Neisseria gonorrhoeae] Length = 374 Score = 539 bits (1390), Expect = e−152 Identities = 285/285 (100%), Positives = 285/285 (100%) Query: 1 MGFIEPFLSSYTPLSRDYVQARTNRKRQTLLSKIVYTHSGFQRSVTENSNIRQINFLIKT 60 MGFIEPFLSSYTPLSRDYVQARTNRKRQTLLSKIVYTHSGFQRSVTENSNIRQINFLIKT Sbjct: 90 MGFIEPFLSSYTPLSRDYVQARTNRKRQTLLSKIVYTHSGFQRSVTENSNIRQINFLIKT 149 Query: 61 LVEHPQGKLNKKEIAAMMLVDLKTFQQDYLTETELNDYFQQGIESGFIERKYNQISYLWN 120 LVEHPQGKLNKKEIAAMMLVDLKTFQQDYLTETELNDYFQQGIESGFIERKYNQISYLWN Sbjct: 150 LVEHPQGKLNKKEIAAMMLVDLKTFQQDYLTETELNDYFQQGIESGFIERKYNQISYLWN 209 Query: 121 LLDKLDDLKRVGDDLYFAEDAQRIFGNLDEITVRKRDPYLHRLYKNQLQEESEEHYGNVK 180 LLDKLDDLKRVGDDLYFAEDAQRIFGNLDEITVRKRDPYLHRLYKNQLQEESEEHYGNVK Sbjct: 210 LLDKLDDLKRVGDDLYFAEDAQRIFGNLDEITVRKRDPYLHRLYKNQLQEESEEHYGNVK 269 Query: 181 CMLEKLAYPVLIASHIKPFILSDDTEAYDPNNGLLLSRTLDSLFDLKYISFDDEGNMVKS 240 CMLEKLAYPVLIASHIKPFILSDDTEAYDPNNGLLLSRTLDSLFDLKYISFDDEGNMVKS Sbjct: 270 CMLEKLAYPVLIASHIKPFILSDDTEAYDPNNGLLLSRTLDSLFDLKYISFDDEGNMVKS 329 Query: 241 KRLSDDVWRRWCDVKLDNNLLNDKRKSYLAYHRELMLQEDQEFHI 285 KRLSDDVWRRWCDVKLDNNLLNDKRKSYLAYHRELMLQEDQEFHI Sbjct: 330 KRLSDDVWRRWCDVKLDNNLLNDKRKSYLAYHRELMLQEDQEFHI 374 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 112 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 223> which encodes amino acid sequence <SEQ ID 224; NGS112>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −9.08 Possible cleavage site: 54 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −1.22 threshold: 0.0 INTEGRAL Likelihood = −1.22 Transmembrane 160-176 (160-177) PERIPHERAL Likelihood = 0.58 modified ALOM score: 0.74 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.149(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_12644572 gi\|12644572\|sp\|Q50973\|T2B1_NEIGO TYPE II RESTRICTION ENZYME NGOBI (ENDONUCLEASE NGOBI) (R.NGOBI) (R.NGOI) gb\|AAB03207.2\|(U42459) NgoI restriction endonuclease R.NgoI [N. gonorrhoeae] Length = 350 Score = 694 bits (1791), Expect = 0.0 Identities = 349/350 (99%), Positives = 349/350 (99%) Query: 1 MTLEEQQAKEALDGIIKKSRVHLYKPIQIAEILYHDRCIKQLDFLNLDTYRNQSKRWRDE 60 MTLEEQQAKEALDGIIKKSRVHLYKPIQIAEILYHDRCIKQLDFLNLDTYRNQSKRWRDE Sbjct: 1 MTLEEQQAKEALDGIIKKSRVHLYKPIQIAEILYHDRCIKQLDFLNLDTYRNQSKRWRDE 60 Query: 61 ICRRFLGISTSSAKFQDNLFEKNAIPPEKLAVLGTLNRQSDGGVESTITKQFFNRFSQM 120 ICRRFLGISTSSAKFQDNLFEKNAIPPEKLAVLGTLNRQSDGGVESTITKQFFNRFSQM Sbjct: 61 ICRRFLGISTSSAKFQDNLFEKNAIPPEKLAVLGTLNRQSDGGVESTITKQFFNRFSQM 120 Query: 121 SEALAYVGNTDRYSFQLSEFLNLFWLEPGLKRSIDKIYEIVVYALFDALVSELGITVSID 180 SEALAYVGNTDRYSFQLSEFLNLFWLEPGLKRSIDKIYEIVVYALFDALVSELGITVSID Sbjct: 121 SEALAYVGNTDRYSFQLSEFLNLFWLEPGLKRSIDKIYEIVVYALFDALVSELGITVSID 180 Query: 181 FPKENLFLWEEYQDFAEKIITMPKNEHLKLPAKIHRVGVTNAADRGLDMWSNFGLAIQVK 240 FPKENLFLWEEYQDFAEKIITMPKNEHLKLPAKIHRVGVTNAADRGLDMWSNFGLAIQVK Sbjct: 181 FPKENLFLWEEYQDFAEKIITMPKNEHLKLPAKIHRVGVTNAADRGLDMWSNFGLAIQVK 240 Query: 241 HLSLDEELAEDIVSSISADRIVIVCKKAEQSVIVSLLTQIGWKSRIQNIVTEDDLISWYE 300 HLSLDEELAEDIVSSISADRIVIVCKKAEQSVIVSLLTQIGWKSRIQNIVTEDDLISWYE Sbjct: 241 HLSLDEELAEDIVSSISADRIVIVCKKAEQSVIVSLLTQIGWKSRIQNIVTEDDLISWYE 300 Query: 301 KALRQQYPIAEALLENIKTEIMREFPAVNEANEFLDFAQNRGYDITVTHF 350 KALRQQYPIAEALLENIKTEIMREFPAVNEANEFLDFAQNRGYDITVTHF Sbjct: 301 KALRQQYPIAEALLENIKTEIMREFPAVNEANEFLDFAQNRGYDITVTHF 350 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 113 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 225> which encodes amino acid sequence <SEQ ID 226; NGS113>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −1.7 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 4 value: −9.77 threshold: 0.0 INTEGRAL Likelihood = −9.77 Transmembrane 187-203 (183-208) INTEGRAL Likelihood = −7.22 Transmembrane 25-41 (19-46) INTEGRAL Likelihood = −4.14 Transmembrane 139-155 (138-155) INTEGRAL Likelihood = −2.87 Transmembrane 86-102 (85-102) PERIPHERAL Likelihood = 1.27 modified ALOM score: 2.45 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.491(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_15902668 gi\|15902668\|ref\|NP_358218.1\|\\(NC_003098) ABC transporter membrane-spanning permease-glutamine transport [Streptococcus pneumoniae R6] gb\|AAK99428.1\|(AE008440) ABC transporter membrane-spanning permease- glutamine transport [Streptococcus pneumoniae R6] Length = 226 Score = 218 bits (556), Expect = 7e−56 Identities = 113/218 (51%), Positives = 155/218 (70%) Query: 1 MNWPYLIDAVPKFADAAKLTLELSVYGVVLSLLFLPVAVVTAYRIRPFYALARAYIELS 60 M+W + +P + A LTL ++V+G++ S L GL V+++ YRI +A AYIEILS Sbjct: 1 MNWPYLIDAVPKFADAAKLTLELSVYGVVLSLLFGLPVAVVTAYRIRPFYALARAYIELS 60 RNTPLLIQLFFLY+GLP++GI C + LVFLG SYMAE+ R+G+ A+ + Q G Query: 61 RNTPLLIQLFFLYYGLPKMGIKWDGFTCGVIALVFLGASYMAEAVRAGILAVPKGQVGAG 120 RNTPLLIQLFFLY+GLP++GI C +LVFLG SYMAE+ R+G+ A+ + G G Sbjct: 61 RNTPLLIQLFFLYFGLPRIGIVLSSEVCATLGLVFLGGSYMAESFRSGLEAISQTQQEIG 120 Query: 121 KAIGLSRFQVFRYVELPQVWAVAVPAIGANILFLMKETSVVSTVGIAELLFVTKDVIGMD 180 AIGL+ QVFRYV LPQ AVA+P+ AN++FL+KETSV S V +A+L++V KD+IG+ Sbjct: 121 LAIGLTPLQVFRYVVLPQATAVALPSFSANVIFLIKETSVFSAVALADLMYVAKDLIGLY 180 Query: 181 YKTNEALFLLFAAYLIILLPVSLLARRIENRVRSAKYG 218 Y+T+ AL +L AYLI+LLP+SL+ IE R+R A +G Sbjct: 181 YETDIALAMLVVAYLIMLLPISLVFSWIERRIRHAGFG 218 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 114 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 227> which encodes amino acid sequence <SEQ ID 228; NGS 114>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.46 Possible cleavage site: 17 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 18 ALOM: Finding transmembrane regions (Klein et al.)", "count: 3 value: −5.36 threshold: 0.0 INTEGRAL Likelihood = −5.36 Transmembrane 50-66 (47-67) INTEGRAL Likelihood = −4.83 Transmembrane 183-199 (176-200) INTEGRAL Likelihood = −1.81 Transmembrane 72-88 (72-88) PERIPHERAL Likelihood = 0.26 modified ALOM score: 1.57 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.314(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_15902667 gi\|15902667\|ref\|NP_358217.1\|\\(NC_003098) ABC transporter membrane-spanning permease-glutamine transport [Streptococcus pneumoniae R6] gb\|AAK99427.1\|(AE008440) ABC transporter membrane-spanning permease- glutamine transport [Streptococcus pneumoniae R6] Length = 225 Score = 218 bits (555), Expect = 9e−56 Identities = 111/206 (53%), Positives = 151/206 (72%) Query: 3 EGLLLTAQISLISVAASCVLGTLFGLVLRSRNRLVRFVGRFYLETIRIVPILVWLFGLYF 62 +GL +T IS++SV S + GT+ G+++ S +R++RF+ R YLE IRI+P LV LF +YF Sbjct: 20 QGLGVTIGISILSVLLSMMFGTVMGIIMTSHSRIIRFLTRLYLEFIRIMPQLVLLFIVYF 79 Query: 63 GLSVWTGIHIGGFWVCVWVFSLWGVAEMGKLVRGALESIEKHQVESGLAPGLSRGQVFRC 122 GL+ I+I G + VF+LWG AEMGDLVRGA+ S+ KHQ ESG A GL+ Q++ Sbjct: 80 GLARNFNINISGETSAIIVFTLWGTAEMGDLVRGAITSLPKHQFESGQALGLTNVQLYYH 139 Query: 123 IELPQSIRRVLPGAVNLFTRMIKTSSLAWLIGVIEVVKVGQQIIENSLLTQPNASFWVYG 182 I +PQ +RR+LP A+NL TRMIKT+SL LIGV+EV KVGQQII+++ LT P ASFW+YG Sbjct: 140 IIIPQVLRRLLPQAINLVTRMIKTTSLVVLIGVVEVTKVGQQIIDSNRLTIPTASFWIYG 199 Query: 183 LIFMLYFFCCWPLSLLAAKLEQKWEH 208 I +LYF C+P+S L+ LE+ W + Sbjct: 200 TILVLYFAVCYPISKLSTHLEKHWRN 225 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 115 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 229> which encodes amino acid sequence <SEQ ID 230; NGS 115>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.639999 Possible cleavage site: 38 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 23 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.25 threshold: 0.0 PERIPHERAL Likelihood = 5.25 modified ALOM score: −1.55 Rule: inner or outer membrane protein Rule: inner or outer membrane protein * Reasoning Step: 2 Lipoprotein?", "Inner membrane?", "----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.700(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_4588485 gi\|4588485\|gb\|AAD26123.1\|\\(AF109148) antigenic protein [Actinobacillus pleuropneumoniae] Length = 278 Score = 407 bits (1045), Expect = e−112 Identities = 212/282 (75%), Positives = 242/282 (85%), Gaps = 7/282 (2%) Query: 1 MKLNAKLKALLASAAIAVGLTACGGGSGDAQSSQSSGAA-TVAAIKEKGVIRIGVFGDKP 59 MKL+ LK LLA+A A LTAC +A ++QSS A +VA IKEKGVIRIGVFGDKP Sbjct: 1 MKLSTTLKTLLATAITAFALTACD----NANNAQSSTAKDSVAQIKEKGVIRIGVFGDKP 56 Query: 60 PFGYVDANGKNQGFDVEIAKDLAKDLLGSPDKVEFVLTEAANRVEYVRSGKVDLILANFT 119 PFGYVDANGK+QGFDVEIAK++A DLLGS DKVEFVLTEAANRVEY++S KVDLILANFT Sbjct: 57 PFGYVDANGKSQGFDVEIAKEIANDLLGSSDKVEFVLTEAANRVEYLKSNKVDLILANFT 116 Query: 120 QTPERAEAVDFADPYMKVALGVVSPKNKPITDMAQLKDQTLLVNKGTTADAFFTKSHPEV 179 +TPERAE VDFA PYM VALGVVSPK + I+D+ QL+ +TLLVNKGTTADA+FTK+HPE+ Sbjct: 117 KTPERAEVVDFAAPYMNVALGVVSPKVRLISDLKQLEGKTLLVNKGTTADAYFTKNHPEI 176 Query: 180 KLLKFDQNTETFDALKDGRGVALAHDNALLWAWAKENPNFEVAIGNLGPAEFIAPAVQKG 239 LLKFDQNTETFDALKDGRGVALAHDNAL+WAWAKENP F+VAIG++GPAE IAPVQKG Sbjct: 177 NLLKFDQNTETFDALKDGRGVALAHDNALVWAWAKENTFDVAIGSVGPAEQIAPAVQKG 236 Query: 240 NADLLNWVNGEIAAMKKDGRLKAAYEKTLLPVYGEKVKPEAL 281 N LL+ +N EIA K +G+LKAAYEKTL+PVYG+ KPE L Sbjct: 237 NQALLDVINKEIAEFKTNGKLKAAYERKTLVPVYGD--KPELL 276 The protein was expressed in E.coli as a soluble 28.16 kDa His-fusion product, lacking its leader peptide and its poly-glycine sequence (GGGSG), and then purified.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 116 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 231> which encodes amino acid sequence <SEQ ID 232; NGS116>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −7.13 Possible cleavage site: 61 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −1.86 threshold: 0.0 INTEGRAL Likelihood = −1.86 Transmembrane 51-67 (51-67) PERIPHERAL Likelihood = 1.54 modified ALOM score: 0.87 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.174(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 117 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 233> which encodes amino acid sequence <SEQ ID 234; NGS117>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 0.25 Possible cleavage site: 40 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 41 ALOM: Finding transmembrane regions (Klein et al.)", "count: 2 value: −4.57 threshold: 0.0 INTEGRAL Likelihood = −4.57 Transmembrane 100-116 (99-118) INTEGRAL Likelihood = −1.59 Transmembrane 54-70 (54-70) PERIPHERAL Likelihood = 0.53 modified ALOM score: 1.41 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.283(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_15793413 gi\|15793413\|ref\|NP_283235.1\|\\(NC_003116) putative integral membrane protein [Neisseria meningitidis Z2491] pir\|\|C81957 probable integral membrane protein NMA0408 [imported]- Neisseria meningitidis (group A strain Z2491) emb\|CAB83707.1\|(AL162753) putative integral membrane protein [Neisseria meningitidis Z2491] Length = 550 Score = 1115 bits (2885), Expect = 0.0 Identities = 539/550 (98%), Positives = 545/550 (99%) Query: 1 MVAYAFLFLFVTAAVLLIVRSHYRWTYFFASALFVFLAGGMLMLTAQWQRALNFASVWFV 60 MVAY FLFLFVTAA++LI+RSHYRWTYFFASALFVFLAGGMLMLTAQWQRALNFASVWFV Sbjct: 1 MVAYVFLFLFVTAALVLIIRSHYRWTYFFASALFVFLAGGMLMLTAQWQRALNFASVWFV 60 Query: 61 VLILFHRLKIHYYKQPLLISDFLLIADWRNWETLFHYKEAVIGMAGLLALAGYAVFGWSG 120 VLILFHRLKIHYYKQPLLISDFLLIADWRNWETLFHYKEAVIGMAGLLALA YAVFGWSG Sbjct: 61 VLILFHRLKIHYYKQPLLISDFLLIADWRNWETLFHYKEAVIGMAGLLALAAYAVFGWSG 120 Query: 121 ADSLGMPWRWAGAVLFAAAFVSVRHFSKHPGAVKTWLDSLPDDGRDVFLNLPMSCRAVFF 180 ADSL +PWRWAGAVLFAAAFVS+RHFSKHPGAVKTWLDSLPDDGRDVFLNLPMSCRAVFF Sbjct: 121 ADSLDVPWRWAGAVLFAAAFVSMRHFSKHPGAVKTWLDSLPDDGRDVFLNLPMSCRAVFF 180 Query: 181 QVPVFEGDGEAFARQMPSETRPYGMSDEKPDIVVTLMESTLDPHCFDFAAAKIPDLKMFG 240 QVPVFEGDGEAFARQMPSETRP GMSDEKPDIVVTLMESTLDPHCFDFAAAKIPDLKMFG Sbjct: 181 QVPVFEGDGEAFARQMPSETRPCGMSDEKPDIVVTLMESTLDPHCFDFAAAKIPDLKMFG 240 Query: 241 RQEDTVFSSPLRVHTFGGATWKSEFAFLAGVPSTDFGALASGVFYSVVPHLQTGFVRNLR 300 RQEDTVFSSPLRVHTFGGATWKSEFAFLAGVPSTDFGALASGVFYSVVPHLQTGFVRNLR Sbjct: 241 RQEDTVFSSPLRVHTFGGATWKSEFAFLAGVPSTDFGALASGVFYSVVPHLQTGFVRNLR 300 Query: 301 EHGYFCVALSPFTKGNYNAKAAYDHFGFNLMFQPQDLGYPAPMGKNLWHISSEEMMQYAR 360 EHGYFCVALSPFTKGNYNAKAAYDHFGFNLMFQPQDLGYPAPMGKNLWHISSEEMMQYAR Sbjct: 301 EHGYFCVALSPFTKGNYNAKAAYDHFGFNLMFQPQDLGYPAPMGKNLWHISSEEMMQYAR 360 Query: 361 MILEKRHPDLENVRQPMFVYVLTMKEHGPYRTDTDNVFDLDAPDLNAKTVSALNDYIGRI 420 MILEKRHPDLENVRQPMFVYVLTMKEHGPYRTDTDNVFDLDAPDLNAKTVSALNDYIGRI Sbjct: 361 MILEKRHPDLENVRQPMFVYVLTMKEHGPYRTDTDNVFDLDAPDLNAKTVSALNDYIGRI 420 Query: 421 ADLDKAVESFDRYLHERGKPFVFGYFGDHQVPFEGVSVRKKWDYAQPDYVTQFAVRSNIA 480 ADLDKAVESFDRYLHERGKPFVFGYFGDHQVPFEGVSVRKKWDYAQPDYVTQFAVRSNIA Sbjct: 421 ADLDKAVESFDRYLHERGKPFVFGYFGDHQVPFEGVSVRKKWDYAQPDYVTQFAVRSNIA 480 Query: 481 GGFVQRQDFLDLAFAGGVLMEAAGLEAKDGFMRANMAMRGLCGGGLEDCPNRELVGNYRN 540 GGFVQRQ+FLDLAFAGGVLMEAAGLEAKDGFMRANMAMRGLCGGGLEDCPN ELVGNYRN Sbjct: 481 GGFVQRQNFLDLAFAGGVLMEAAGLEAKDGFMRANMAMRGLCGGGLEDCPNWELVGNYRN 540 Query: 541 YLYDVLKIAR 550 YLYDVLKIAR Sbjct: 541 YLYDVLKIAR 550 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS117 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 118 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 235> which encodes amino acid sequence <SEQ ID 236; NGS118>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 0.59 Possible cleavage site: 19 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 22 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 8.33 threshold: 0.0 PERIPHERAL Likelihood = 8.33 modified ALOM score: −2.17 Rule: inner or outer membrane protein Rule: inner or outer membrane protein * Reasoning Step: 2 Lipoprotein?", "Inner membrane?", "----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.700(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.", "The protein was expressed in E.coli as a soluble 12.98 kDa His-fusion product and then purified.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 119 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 237> which encodes amino acid sequence <SEQ ID 238; NGS119>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.75 Possible cleavage site: 47 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.69 threshold: 0.0 PERIPHERAL Likelihood = 7.69 modified ALOM score: −2.04 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.213(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_2625122 gi\|2625122\|gb\|AAB86635.1\|\\(AF031495) putative hemoglobin receptor component precursor HpuA [Neisseria gonorrhoeae] Length = 360 Score = 668 bits (1724), Expect = 0.0 Identities = 331/331 (100%), Positives = 331/331 (100%) Query: 1 VSIPTATPLPAGEVTLSSDNGNIENINTAGAGSASDAPSRSRRSLDAAPQNTSGISIRQR 60 VSIPTATPLPAGEVTLSSDNGNIENINTAGAGSASDAPSRSRRSLDAAPQNTSGISIRQR Sbjct: 30 VSIPTATPLPAGEVTLSSDNGNIENINTAGAGSASDAPSRSRRSLDAAPQNTSGISIRQR 89 Query: 61 EVEKDYFGYKSKETSFIFKTPGGAQYALSSYADPITVSYSSPKFKIPDRHAGQRLADGSR 120 EVEKDYFGYKSKETSFIFKTPGGAQYALSSYADPITVSYSSPKFKIPDRHAGQRLADGSR Sbjct: 90 EVEKDYFGYKSKETSFIFKTPGGAQYALSSYADPITVSYSSPKFKIPDRHAGQRLADGSR 149 Query: 121 IFICCSDSGATSYAEITKQDYMKFGAWIGPNGEIDLFAGGFPVGKTPPPAFSYGSSTPET 180 IFICCSDSGATSYAEITKQDYMKFGAWIGPNGEIDLFAGGFPVGKTPPPAFSYGSSTPET Sbjct: 150 IFICCSDSGATSYAEITKQDYMKFGAWIGPNGEIDLFAGGFPVGKTPPPAFSYGSSTPET 209 Query: 181 ALSKGKITYQVWGIRVRNGQFVTSSYTPPKSGSYYGTLANTPVSFITANFNSNTLAGKI 240 ALSKGKITYQVWGIRVRNGQFVTSSYTPPKSGSYYGTLANTPVSFITANFNSNTLAGKI Sbjct: 210 ALSKGKITYQVWGIRVRNGQFVTSSYTPPKSGSYYGTLANTPVSFITANFNSNTLAGKI 269 Query: 241 LGNSDYGPDVDIQNATITGPTFSGDATSGGKSGKLEGKFFGKFASTRSSEVSIGGKITFD 300 LGNSDYGPDVDIQNATITGPTFSGDATSGGKSGKLEGKFFGKFASTRSSEVSIGGKITFD Sbjct: 270 LGNSDYGPDVDIQNATITGPTFSGDATSGGKSGKLEGKFFGKFASTRSSEVSIGGKITFD 329 Query: 301 GDRSLDTVFGGVSYEKKLDDTSQDTNHLTKQ 331 GDRSLDTVFGGVSYEKKLDDTSQDTNHLTKQ Sbjct: 330 GDRSLDTVFGGVSYEKKLDDTSQDTNHLTKQ 360 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 120 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 239> which encodes amino acid sequence <SEQ ID 240; NGS120>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −7.24 Possible cleavage site: 38 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 6.42 threshold: 0.0 PERIPHERAL Likelihood = 6.42 modified ALOM score: −1.78 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.280(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gsa_AAR91313 N. gonorrhoeae glycosyltransferase LgtC \|WO9610086-A1\|09-JUL-1996 Length = 306 Score = 535 bits (1379), Expect = e−151 Identities = 252/253 (99%), Positives = 252/253 (99%) Query: 8 GGGNIRFIDVNPEDFAGFPLNIRHISITTYARLKLGEYIADCDKVLYLDTDVLVRDGLKP 67 GGGNIRFIDVNPEDFAGFPLNIRHISITTYARLKLGEYIADCDKVLYLDTDVLVRDGLKP Sbjct: 54 GGGNIRFIDVNPEDFAGFPLNIRHISITTYARLKLGEYIADCDKVLYLDTDVLVRDGLKP 113 Query: 68 LWDTDLGGNWVGACIDLFVERQEGYKQKIGMADGEYYFNAGVLLINLKKWRRHDIFKMSC 127 LWDTDLGGNWVGACIDLFVERQEGYKQKIGMADGEYYFNAGVLLINLKKWRRHDIFKMSC Sbjct: 114 LWDTDLGGNWVGACIDLFVERQEGYKQKIGMADGEYYFNAGVLLINLKKWRRHDIFKMSC 173 Query: 128 EWVEQYKDVMQYQDQDILNGLFKGGVCYANSRFNFMPTNYAFMANGFASRHTDPLYLDRT 187 EWVEQYKDVMQYQDQDILNGLFKGGVCYANSRFNFMPTNYAFMANGFASRHTDPLYLDRT Sbjct: 174 EWVEQYKDVMQYQDQDILNGLFKGGVCYANSRFNFMPTNYAFMANGFASRHTDPLYLDRT 233 Query: 188 NTAMPVAVSHYCGSAKPWHRDCTVWGAERFTELAGSLTTVPEEWRGKLAVPPTKRMLQRW 247 NTAMPVAVSHYCGSAKPWHRDCTVWGAERFTELAGSLTTVPEEWRGKLAVPPTKRMLQRW Sbjct: 234 NTAMPVAVSHYCGSAKPWHRDCTVWGAERFTELAGSLTTVPEEWRGKLAVPPTKRMLQRW 293 Query: 248 RKKLSARFLRKIY 260 RKKLSARFLRKIY Sbjct: 294 RKKLSARFLRKIY 306 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 121 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 241> which encodes amino acid sequence <SEQ ID 242; NGS121>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −6.22 Possible cleavage site: 37 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.23 threshold: 0.0 PERIPHERAL Likelihood = 3.23 modified ALOM score: −1.15 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.402(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_15281345 gi\|15281345\|dbj\|BAB63435.1\|\\(AB058945) DNA adenine methylase M.Ssu4109IB [Streptococcus suis] Length = 271 Score = 269 bits (687), Expect = 4e−71 Identities = 127/211 (60%), Positives = 158/211 (74%), Gaps = 1/211 (0%) Query: 1 MIFADPPYFLSNDGFSCQNGQMVSVNKGNWDKSKGMAADLEFYEEWLRLCYALLKPNGTI 60 MIFADPPYFLSN G S GQ+VSV+KG+WDK + EF +W+RL +LKPNGTI Sbjct: 44 MIFADPPYFLSNGGISNSGGQVVSVDKGDWDKVNSLEEKHEFNRKWIRLAKNVLKPNGTI 103 Query: 61 WVCGTFHNIYLIGYLMQTVGYHILNNITWEKPNPPPNLSCRFFTHSTETILWAKK-NKKA 119 W+ G+FHNIY +G ++ G+ ILNNITW+K NP PNLSCR+FTHSTETILWA+K +KKA Sbjct: 104 WISGSFHNIYSVGMALEQEGFKILNNITWQKTNPAPNLSCRYFTHSTETILWARKDDKKA 163 Query: 120 KHTFHYEMMKAQNNGKQMKCVWTFAPPNKTEKTFGKHPTQKPLPLLERCILSASNIGDLI 179 +H ++YE+MK N+GKQMK VW K+EK GKHPTQKP LLER IL+++ GD I Sbjct: 164 RHYYNYELMKELNDGKQMKDVWVGGLTKKSEKWAGKHPTQKPEYLLERIILASTREGDYI 223 Query: 180 FDPFMGSGTTGVAALKHGRRFCGCELEEDFL 210 DPF+GSGTTGV A + GR+F G + E D+L Sbjct: 224 LDPFVGSGTTGVVAKRLGRKFIGIDAERDYL 254 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 122 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 243> which encodes amino acid sequence <SEQ ID 244; NGS122>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −2.55 Possible cleavage site: 23 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 15 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 11.46 threshold: 0.0 PERIPHERAL Likelihood = 11.46 modified ALOM score: −2.79 Rule: inner or outer membrane protein Rule: inner or outer membrane protein * Reasoning Step: 2 Lipoprotein?", "Inner membrane?", "----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.700(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases: The protein was expressed in E.coli as an insoluble 14.85 kDa His-fusion product and then purified.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 123 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 245> which encodes amino acid sequence <SEQ ID 246; NGS123>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.65 Possible cleavage site: 20 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.24 threshold: 0.0 PERIPHERAL Likelihood = 4.24 modified ALOM score: −1.35 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.404(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to the sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 124 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 247> which encodes amino acid sequence <SEQ ID 248; NGS124>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −1.59 threshold: 0.0 INTEGRAL Likelihood = −1.59 Transmembrane 289-305 (289-305) PERIPHERAL Likelihood = 3.76 modified ALOM score: 0.82 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.164(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_1617515 gi\|1617515\|gb\|AAC82509.1\|\\(U365994) pilin gene inverting protein homolog PivNG [Neisseria gonorrhoeae] Length = 320 Score = 614 bits (1584), Expect = e−175 Identities = 311/320 (97%), Positivies = 316/320 (98%) Query: 1 MRNTVGLDISKLTFDATAMVGKTEHSAKFDNDSKGLDQFSDRLKSLGYQNLHICMEATGS 60 MRN VGLDISKLTF+A+AMVGKTEHSAKFDNDSKGLDQFSDRLKSLG QNLHICMEATG+ Sbjct: 1 MRNAVGLDISKLTFNASAMVGKTEHSAKFDNDSKGLDQFSDRLKSLGCQNLHICMEATGS 60 Query: 61 YYEEVADYFAQYYSVYVVNPLKISKYAESRFKRTKTDKQDAKLIAQYCRSAQESELVKRQ 120 YYEEVADYFAQYYSVYVVNPLKISKYAESRFKRTKTDKQDAKLIAQYCR A+ESELVKRQ Sbjct: 61 YYEEVADYFAQYYSVYVVNPLKISKYAESRFKRTKTDKQDAKLIAQYCRLAKESELVKRQ 120 Query: 121 KPTDEQYRLSRMTAAYAKIKSECAAMKNRHHAAKDEEAAKAYAEIIKAMNEQLEVLKEKI 180 KPTDEQYRL RMTAAYAKIKSECAAMKNRHHAAKDEEAAKAYA+IIKAMNEQLEVLKEKI Sbjct: 121 KPTDEQYRLLRMTAAYAKIKSECAAMKNRHHAAKDEEAAKAYAQIIKAMNEQLEVLKEKI 180 Query: 181 KEQTEKPNCKEGVKRLETIPAIGRMTAAVLFHHLTSSKFETSNKFAAFAGLSPQQKESGT 240 KEQTEKPNCKEGVKRLETIPAIGRMTAAVLFHHLTSSKFETSNKFAAFAGLSPQQKESGT Sbjct: 181 KEQTEKPNCKEGVKRLETIPAIGRMTAAVLFHHLTSSKFETSNKFAAFAGLSPQQKESGT 240 Query: 241 SVRGKGKLTKFGNRKLRAVLFMPAMVAVYRIRAFPDFIKRLEEKKKPKKVIIAALMRKLAV 300 SVRGKGKLTKFGNRKLRAVLFMPAMVAVYRIRAFPDFIKRLEEKKKPKKVIIAALMRKLAV Sbjct: 241 SVRGKGKLTKFGNRKLRAVLFMPAMVAVYRIRAFPDFIKRLEEKKKPKKVIIAALMRKLAV 300 Query: 301 IAYHVHKKGGDYDPSRYKSA 320 IAYHVHKKGGDYDPSRYKSA Sbjct: 301 IAYHVHKKGGDYDPSRYKSA 320 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 125 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 249> which encodes amino acid sequence <SEQ ID 250; NGS127>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.8 Possible cleavage site: 52 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 1.70 threshold: 0.0 PERIPHERAL Likelihood = 1.70 modified ALOM score: −0.84 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.383(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_1076012 gi\|1076012\|pir\|\|B55225 stress-sensitive restriction system protein 2-Corynebacterium glutamicum (ATCC 13032) gb\|AAC00044.1\|(U13922) This orf may encode a typeI or typeIII restriction endonuclease which is stress-sensitive and ATP-dependent.", "It contains a typical ATP binding region (Walker motif) [Corynebacterium glutamicum] Length = 632 Score = 298 bits (764), Expect = 2e−79 Identities = 199/633 (31%), Positives = 321/633 (50%), Gaps = 32/633 (5%) Query: 2 LRTYLNQLTP-PELADSVKNTVDGFMEKLSQTEPKIA-QNVLLLGNVQSGKTAQVLGVLS 59 L Y + +L + V TVD F + I+ Q VLL G+VQSGKT+ +LG++++ Sbjct: 7 LNNYITSLSDNADLREKVTATVDAFRHTVMDDFDYISDQQVLLYGDVQSGKTSHMLGIIA 66 Query: 60 ALADDGDHKVFLYLTTDSVDLQDQTVKRAKANLKNFIVLSEADDRSFMEVMKAENP--IL 117 D H + + LT+ + L QT R + +V F K+ P + Sbjct: 67 DCLDSTFHTIVI-LTSPNTRLVQQTYDRVAQAFPDTLVCDRDGYNDFRANQKSLTPRKSI 125 Query: 118 VVIKKNARVLKRWRNLFASQSSLKGYPLVIVDDEADAASLNTNSDKPKDASTINKLLND 177 VV+ K VL W +F +L G+P++I+DDEADA SLNT ++ D STIN L Sbjct: 126 VVVGKIPAVLGNWLRVFNDSGALSGHPVLIIDDEADATSLNTKVNQ--SDVSTINHQLTS 183 Query: 178 IKNSCCQSLPIQLTATPQSLLLQHEESDWQPEFIHFFEAGEKYIGGNFVFSDPPS-YIVR 236 I++ +++Q+T TPQ++LLQ ++S+W E + F GE YIGG FS+ + Y+ Sbjct: 184 IRDLATGCIYLQVTGTPQAVLLQSDDSNWAAEHVLHFAPGESYIGGQLFFSELNNPYLRL 243 Query: 237 FIDSELDDMKDESGEIAEGAKQALLSFLITCAEFALCDKANCNFALHPSYKIQDHQAFSK 296 F +++ D+ S A+ ++L+T A F L ++ C +HPS+ H+ F++ Sbjct: 244 FANTQFDEDSRFS--------DAIYTYLLTAALFLRGESLCTMLIHPSHTASSHRDFAQ 295 Query: 297 KIQAFLNDLVQAVNNGEDLAGSFKESYLDLQKTKPDIHHFDEIYEKLTALLENKQISTLV 356 + + L + + +F+ +Y L +T ++ +I L + ++ I + Sbjct: 296 EARLQLTFAFERFYEPM-IQHNFQRAYEQLAQTDSNLPPLRKILNILGGMEDDFSIH--I 352 Query: 357 VNSQTET-DFDLEKGFNIIIGGNVIGRGLTIPKLQTVYYSRTAKKPNADTFWQHRIFGY 415 VNS T + D G+NII+GGN +GRGLT LQTV+Y R +K+P ADT WQH+R+FGY Sbjct: 353 VNSDNPTVEEDWADGYNIIVGGNSLGRGLTFNNLQTVFYVRESKRPQADTLWQHARMFGY 412 Query: 416 DRDKSLLRLYIPFDVYYFFVQLNQANNLIIGQAKNSG--GNIQVIYPKNINPTRKNVLKF 473 R K +R+++P + F ++ N I Q + +I+VI + PTR NVL Sbjct: 413 KRHKDTMRVFMPATIAQTFQEVYLGNEAIKNQLDHGTHINDIRVILGDGVAPTRANVLDK 472 Query: 474 DSINQIVGGVNYFPLHPNEDNLSEINKILPSILKDEIQSDLYQIDIEDLFLVLDKLGRYV 533 + + GGVNYF P N+ ++K L + L + I + + +L+ Sbjct: 473 RKVGNLSGGVNYFAADPRIIKNVEALDKKLLAYLDKHGEDS--TIGMRAIITILNAF-TVD 529 Query: 534 PDDWNKEKFIAGVEALKAQRPSFKTYVLIKTGRKLSRATGTMLSEDDRKLGEKYPNDLFL 593 P+D + F A + + +P ++++T RK+++ TG +LS D+ L L Sbjct: 530 PNDLDLATFKAALLDFERNQPHLTARMVLRTNRKVNQGTGALLSPTDQALSRAEVAHPLL 589 Query: 594 TLYQVVGNKDKG-------WQGKDFWLPNIKLP 619 LY++ G D W W+PNIKLP Sbjct: 590 ILYRIEGVNDAAAQRGEPTWSSDPIWVPNIKLP 622 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 126 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 251> which encodes amino acid sequence <SEQ ID 252; NGS128>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.98 Possible cleavage site: 20 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.37 threshold: 0.0 PERIPHERAL Likelihood = 7.37 modified ALOM score: −1.97 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.225(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_11357195 gi\|11387l95\|sp\|Q50976\|T2F7_NEIGO TYPE II RESTRICTION ENZYME NGOFVII (ENDONUCLEASE NGOFVII) (R.NGOFVII) (R.NGOVII) pir\|\|T10166 restriction endonuclease (EC 3.1.21.-) NgoVII-N. gonorrhoeae gb\|AAA86271.1\|(U43736) R.NgoVII [Neisseria gonorrhoeae] Length = 326 Score = 651 bits (1679), Expect = 0.0 Identities = 317/326 (97%), Positives = 320/326 (97%) Query: 1 MNTVFSNIANAKITEKSLNAVWMDLFKSADEVLMATGYVSNDAVVELHKILELNDHIQKI 60 MNTVFSNIANAKITEKSLNAVWMDLFKSADEVLMATGYVSNDAVVELHKILELNDHIQKI Sbjct: 1 MNTVFSNIANAKITEKSLNAVWMDLFKSADEVLMATGYVSNDAVVELHKILELNDHIQKI 60 Query: 61 DLLVGMHYLEGFSHLQYDSLCKLNDFLRHEKRGAVYVSPFVKFHGKMYSFKNYQKINGLI 120 DLLVGMHYLEGFSHLQYDSLCKLNDFLRHEKRGAVYVSPFVKFHGKMYSFKNYQKINGLI Sbjct: 61 DLLVGMHYLEGFSHLQYDSLCKLNDFLRHEKRGAVYVSPFVKFHGKMYSFKNYQKINGLI 120 Query: 121 GSANLTCFWDSTERTYETMLHLNGKPAQILQADIQSTIHKLGKNIQEVERPSKFIEHNSH 180 GSANLTCFWDSTERTYETMLHLNGKPAQILQADIQSTIHKLGKNIQEVERPSKFIEHNSH Sbjct: 121 GSANLTCFWDSTERTYETMLHLNGKPAQILQADIQSTIHKLGKNIQEVERPSKFIEHNSH 180 Query: 181 LENCLGVQKIAPEQIRQLFAQTSEYHFSIPAKTEEKSNLNVFFGEGRRDKRGFVKPRPWY 240 LENCLGVQKIAPEQIRQLFAQTSEYHFSIPAKTEEKSNLNVFFGEGRRDKRGFVKPRPWY Sbjct: 181 LENCLGVQKIAPEQIRQLFAQTSEYHFSIPAKTEEKSNLNVFFGEGRRDKRGFVKPRPWY 240 Query: 241 EVELIVSKDITSQEGYPVLKSFTVITDDGWQFQCKTSGDYSKNFRSENDLKTLGKWIKGR 300 EVELIVSKDITSQEGYPVLKSFTVITDDGWQFQCKTSGDYSK + +LKTLGKWIKGR Sbjct: 241 EVELIVSKDITSQEGYPVLKSFTVITDDGWQFQCKTSGDYSKTSTQKMNLKTLGKWIKGR 300 Query: 301 LESHGCLQNNEKITHETLREYGNDHF 326 LESHGCLQNNEKITHETLREYGN+ F Sbjct: 301 LESHGCLQNNEKITHETLREYGNESF 326 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 127 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 253> which encodes amino acid sequence <SEQ ID 254; NGS 129>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.5 Possible cleavage site: 48 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 10.03 threshold: 0.0 PERIPHERAL Likelihood = 10.03 modified ALOM score: −2.51 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.545(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_15804186 gi\|15804186\|ref\|NP_290225.1\|\\(NC_002655) DNA-damage- inducible protein [Escherichia coli O157:H7 EDL933] ref\|NP_312547.1\|(NC_002695) DNA-damage-inducible protein [Escherichia coli O157:H7] gb\|AAG58789.1\|AE005591_13 (AE005591) DNA-damage-inducible protein [Escherichia coli O157:H7 EDL933] dbj\|BAB37943.1\|(AP002566) DNA-damage-inducible protein [Escherichia coli O157:H7] Length = 278 Score = 340 bits (872), Expect = 2e−92 Identities = 161/266 (60%), Positives = 197/266 (73%) Query: 1 MTTENNAFENAKHIDETGNEYWSARTLQQILEYSEWRNFQRAIDKAITACETSGNDKNHH 60 M + FE +H G E+WSAR L +L+Y +WRNFQ+ + +A ACE S + H Sbjct: 5 MNEHQPFEEIRHYGTEGQEFWSARELAPLLDYRDWRNFQKVLAPATQACEASNQAASDH 64 Query: 61 FVETNKMIALGKGGQREVADYRLSRYACYLIVQNGDPSKSVIAAGQTYFAVQARRQELQD 120 FVET KM+LG G QRE+ D LSRYACYL+VQNGDP+K VIAAGQTYFA+Q RRQEL D Sbjct: 65 FVETTKMVVLGSGAQRELEDVHLSRYACYLVVQNGDPAKPVIAAGQTYFAIQTRRQELAD 124 Query: 121 EAAFRSLGEDKQRLLLRRQLREHNTDLAAAAKDAGVEKPVEYAVFQNHGYRGLYGGLDKQ 180 + AF+ L ED++RL LR +L+EHN L AA+ A V ++A+FQNHGY+GLYGGLD++ Sbjct: 125 DEAFKQLREDEKRLFLRNELKEHNKQLVEAAQQAAVATATDFAIFQNHGYQGLYGGLDQK 184 Query: 181 GIHSPRGLKKSQRILDHMNASEPAANLFRATQTEEKLRRKNIQGKTQANRVHFEVGQKVR 240 IH KGLKKSQ+ILDHM ++E AANLFRATQTEEKL+R + K QAN HF+VG KVR Sbjct: 185 AIEQLKGLKKSQKILDHMGSTELAANLFRATQTEEKLKRDGVNSKQQANTTHFDVGSKVR 244 Query: 241 QTIEELGGIMPENQPVPEKSIKQLEN 266 QTI+ELGG MPE P P+ SIKQLEN Sbjct: 245 QTIQELGGTMPEELPTPQVSIKQLEN 270 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 128 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 255> which encodes amino acid sequence <SEQ ID 256; NGS130>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −3.68 Possible cleavage site: 14 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 3 value: −3.45 threshold: 0.0 INTEGRAL Likelihood = −3.45 Transmembrane 68-84 (68-92) INTEGRAL Likelihood = −1.59 Transmembrane 10-26 (10-26) INTEGRAL Likelihood = −1.44 Transmembrane 46-62 (45-62) PERIPHERAL Likelihood = 1.48 modified ALOM score: 1.19 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.238(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_17988861 gi\|17998861\|ref\|NP_541494.1\|\\(NC_003318) hypothetical protein [Brucella melitensis] gb\|AAL53758.1\|(AE009687) hypothetical protein [Brucella melitensis] Length = 99 Score = 108 bits (270), Expect = 3e−23 Identities = 59/91 (64%), Positives = 69/91 (74%) Query: 11 LLFSCMLAVTCPTRLIGFFALRNRTLSRRAQTVMEAAPGCVLISVIAPYFVSDKPHELIA 70 L M +VT TR+ G+ LRNRTLS RA VMEAAPGCVLISVIAP FVSDKP LIA Sbjct: 8 LTILAMASVTYLTRIGGYVLLRNRTLSNRAMAVMEAAPGCVLISVIAPDFVSDKPANLIA 67 Query: 71 IALTAFAACRFSMLFTVLIGVGSSGISGWLM 101 +A+T FAA RFSML TVLIG+G++ I +L+ Sbjct: 6 LAVTVFAATRFSMLPTVLIGMGAASICRYLI 98 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 129 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 257> which encodes amino acid sequence <SEQ ID 258; NGS131>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −1.65 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.05 threshold: 0.0 PERIPHERAL Likelihood = 7.05 modified ALOM score: −1.91 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.152(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_16760390 gi\|16760390\|ref NP_456007.1\|\\(NC_003198) hypothetical protein [Salmonella enterica subsp.", "enterica serovar Typhi] emb\|CAD01841.1\|(AL621270) hypothetical protein [Salmonella enterica subsp.", "enterica serovar Typhi] Length = 227 Score = 104 bits (259), Expect = 2e−21 Identities = 68/221 (30%), Positivies = 115/221 (51%), Gaps = 11/221 (4%) Query 2 DKEKVLDKIKKCLALGRSVNEHEAAQALRQAQALMEKYKVNAEDIALSKVSEQKAD--RK 59 D++K ++K+KK LAL S N HEAA ALR+A+ LM+ + + DIA+S + E + Sbjct: 3 DQDKHIEKLKKLLALAASGNPHEAALALRRARKLMDVHGITHSDIAMSDIDETISHYWPT 62 Query: 60 MAFKLAGWQWGVANMIADIFGCKSYQRGKT---MMFYGIGNRAETSAYAFDVVYRQISAD 116 + + + G+ N+I + FG S T + FYG RA +AY ++V+ RQ+ Sbjct: 63 GSLRPPRYMLGLMNIIREAFGVNSIIHPGTYPGVGFYGNRERAALAAYTWEVLARQLKKA 122 Query: 117 RRKFLKT-CRAGKPSHRTYLADPFCGGWIASAWETVKKFEMSDEEKAIMDGYKKKEYPDM 175 R++++ + K + RT D+F GW+ + ++ F ++D+E+ +M + + +YP Sbjct: 123 RQQYISAQNKRIKTATRTSRGDQFAEGWVLAVISEIQSFALTDDERELMQQWLEHKYPQT 182 Query: 176 AEARTRDAKSSILQGSKMEYEALTRGMESGKQVKLHYAVNG 216 R R S G Y G G+ V+LH V+G Sbjct: 183 QTTRARKPGRS-RNGDASRY----AGFREGQNVRLHRPVSG 218 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 130 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 259> which encodes amino acid sequence <SEQ ID 260; NGS132>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.06 Possible cleavage site: 30 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.49 threshold: 0.0 PERIPHERAL Likelihood = 2.49 modified ALOM score: −1.00 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.075(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 131 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 261> which encodes amino acid sequence <SEQ ID 262; NGS133>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 1.64 Possible cleavage site: 53 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.82 threshold: 0.0 PERIPHERAL Likelihood = 3.82 modified ALOM score: −1.26 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.068(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to the following sequences in the databases: Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 132 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 263> which encodes amino acid sequence <SEQ ID 264; NGS135>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.67 Possible cleavage site: 39 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 5.52 threshold: 0.0 PERIPHERAL Likelihood = 5.52 modified ALOM score: −1.60 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.457(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases: Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 133 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 265> which encodes amino acid sequence <SEQ ID 266; NGS136>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −3.97 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 11.35 threshold: 0.0 PERIPHERAL Likelihood = 11.35 modified ALOM score: −2.77 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.523(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases: Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 134 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 267> which encodes amino acid sequence <SEQ ID 268; NGS 137>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −8.52 Possible cleavage site: 51 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.81 threshold: 0.0 PERIPHERAL Likelihood = 2.81 modified ALOM score: −1.06 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.374(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 135 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 269> which encodes amino acid sequence <SEQ ID 270; NGS138>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −7 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 10.66 threshold: 0.0 PERIPHERAL Likelihood = 10.66 modified ALOM score: −2.63 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.415(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_13559865 gi\|13559865\|ref\|NP_112075.1\|\\(NC_002730) terminase small subunit [Bacteriophage HK620] gb\|AAK28890.1\|AF335538_42 (AF335538) terminase small subunit [Bacteriophage HK620] Length = 140 Score = 125 bits (313), Expect = 5e−28 Identities = 56/122 (45%), Positivies = 85/122 (68%) Query: 4 TKRKLGRPTDYTKDMADKICEKIANGRSLRSICAEDGVPPMKTIYRWLEANEEFRHQYAR 63 T+ K GRP+DY ++AD IC +++G SL +C G+P T++RWL +E+FR +YA+ Sbjct: 3 TEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWLAKHEDFRDKYAK 62 Query: 64 AREKQADYFAEEIIEIADSAQAESAAVSKAKLQIDARKWAASKIAPKKYGDKSELDVKSGDG 125 A E +AD EEI EIAD+A ++A V+KA+L++D RKWA +++ P+KYGDK ++ DG Sbjct: 63 ATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVTNELVGKDG 124 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 136 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 271> which encodes amino acid sequence <SEQ ID 272; NGS139>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −1.49 Possible cleavage site: 32 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 8.65 threshold: 0.0 PERIPHERAL Likelihood = 8.65 modified ALOM score: −2.23 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.301(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_16127009 gi\|16127009\|ref\|NP_421573.1\|\\(NC_002696) hypothetical protein [Caulobacter crescentus] gb\|AAK24741.1\|(AE005943) hypothetical protein [Caulobacter crescentus] Length = 184 Score = 59.7 bits (143), Expect = 4e−08 Identities = 50/164 (30%), Positives = 74/164 (44%), Gaps = 20/164 (12%) Query: 30 ASGREFRTAYYTYPQWRFSLSFEVLRTKASVNELEKLAGFFNARKGSFESFLYEDPAD-- 87 ASG E RT+ ++ + R+ ++ ++E+ +L FF AR+G F + DPAD Sbjct: 5 ASGHERRTSPWSQSRRRYLIA----TAPRPLDEIAELVAFFEARRGRLHGFRFRDPADFK 60 Query: 88 -------NAVTDQPVGNTVQGAR-YQLVRSMGGFIEPVSAVKERP-----AVKVGGTAL 134 A DQ +G T GV + +QL ++ G E V+ +P V V G L Sbjct: 61 SCAPSVQPAAGDQAIG-TGDGVRKKAFQLRKTYGAGGEAVARTIAKPVAGTVTVAVAGVVL 119 Query: 135 AYGRDYTVTDKGVLVFNTPQPPGRPITWTGGFYFRVRFTSDTVD 178 A G G++ NT P G +T F VRF D +D Sbjct: 120 APGAFAVDVTTGLITLNTAPPAGAAVTAGFAFDTPVRFDLDRLD 163 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 137 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 273> which encodes amino acid sequence <SEQ ID 274; NGS140>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −3.86 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −4.94 threshold: 0.0 INTEGRAL Likelihood = −4.94 Transmembrane 34-50 (31-54) PERIPHERAL Likelihood = 8.01 modified ALOM score: 1.49 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.297(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_17987625 gi\|17987625\|ref\|NP_540259.1\|\\(NC_003317) Hypothetical Phage Protein [Brucella melitensis] gb\|AAL52523.1\|(AE009572) Hypothetical Phage Protein [Brucella melitensis] Length = 144 Score = 72.4 bits (176), Expect = 5e−12 Identities = 43/119 (36%), Positives = 64/119 (53%), Gaps = 7/119 (5%) Query: 10 RIVEEARSWLGTPYHHHAMVKGAGVDCAMLLVAVYGAV-GLLPEGFDPRPYPQDWHLHRD 68 R++ EA W+GTPY H A G DC L+ ++A+ G+ PE +P Y DW Sbjct: 6 RVLAEAHRWIGTPYRHGASTLGVSCDCLGVRQIWRALYGVEPE--NPGVYAPDWAEVSQ 63 Query: 69 CERYLGFVTQFC--RETESPQAGDIAV--WRFGRSFSHGGILAGGGKVIHSYIGRGVVS 123 + L ++ RE +PQ GD+ W+ G + H GI+A G+ IH+Y G GV++ Sbjct: 64 GDPMLEAAVRYMVRREEHAPQPGDLLVFRWKPGFAAKHMGIMAREGRFIHAYQGHGVLA 122 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 138 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 275> which encodes amino acid sequence <SEQ ID 276; NGS141>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 5.35 Possible cleavage site: 28 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 29 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 8.86 threshold: 0.0 PERIPHERAL Likelihood = 8.86 modified ALOM score: −2.27 Score for OM-PP discrimination: 1.53 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 1.53 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Outer membrane?", "Score: 0.152929 Outer membrane?", "Score: 0.152929 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.512(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.320(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_5915870 gi\|5915870\|sp\|Q50940\|CAH_NEIGO Carbonic anhydrase precursor (Carbonate dehydratase) emb\|CAA72038.1\|(Y11152) carbonic anhydrase [Neisseria gonorrhoeae] Length = 252 Scare = 523 bits (1347), Expect = e−147 Identities = 252/252 (100%), Positives = 252/252 (100%) Query: 1 MPRFPRTLPRLTAVLLLACTAFSAAAHGNHTHWGYTGHDSPESWGNLSEEFRLCSTGKNQ 60 MPRFPRTLPRLTAVLLLACTAFSAAAHGNHTHWGYTGHDSPESWGNLSEEFRLCSTGKNQ Sbjct: 1 MPRFPRTLPRLTAVLLLACTAFSAAAHGNHTHWGYTGHDSPESWGNLSEEFRLCSTGKNQ 60 Query: 61 SPVNITETVSGKLPAIKVNYKPSMVDVENNGHTIQVNYPEGGNTLTVNGTYTLKQFHFH 120 SPVNITETVSGKLPAIKVNYKPSMVDVENNGHTIQVNYPEGGNTLTVNGTYTLKQFHFH Sbjct: 61 SPVNITETVSGKLPAIKVNYKPSMVDVENNGHTIQVNYPEGGNTLTVNGTYTLKQFHFH 120 Query: 121 VPSENQIKGRTFPMEAHFVHLDENKQPLVLAVLYEAGKTNGRLSSIWNVMPMTAGKVKLN 180 VPSENQIKGRTFPMEAHFVHLDENKQPLVLAVLYEAGKTNGRLSSIWNVMPMTAGKVKLN Sbjct: 121 VPSENQIKGRTFPMEAHFVHLDENKQPLVLAVLYEAGKTNGRLSSIWNVMPMTAGKVKLN 180 Query: 181 QPFDASTLLPKRLKYYRFAGSLTTPPCTEGVSWLVLKTYDHIDQAQAEKFTRAVGSENNR 240 QPFDASTLLPKRLKYYRFAGSLTTPPCTEGVSWLVLKTYDHIDQAQAEKFTRAVGSENNR Sbjct: 181 QPFDASTLLPKRLKYYRFAGSLTTPPCTEGVSWLVLKTYDHIDQAQAEKFTRAVGSENNR 240 Query: 241 PVQPLNARVVIE 252 PVQPLNARVVIE Sbjct: 241 PVQPLNARVVIE 252 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 139 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 277> which encodes amino acid sequence <SEQ ID 278; NGS142>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.49 Possible cleavage site: 22 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 1.22 threshold: 0.0 PERIPHERAL Likelihood = 1.22 modified ALOM score: −0.74 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.145(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_15794480 gi\|15794480\|ref\|NP_284302.1\|\\(NC_003116) hypothetical protein [Neisseria meningitidis Z2491] pir\|\|F81851 hypothetical protein NMA1587 [imported]-Neisseria meningitidis (group A strain Z2491) emb\|CAB84814.1\|(AL162756) hypothetical protein [Neisseria meningitidis Z2491] Length = 181 Score = 358 bits (919), Expect = 6e−98 Identities = 173/181 (95%), Positives = 178/181 (97%) Query: 1 LKTDTARMNNLIPEHLAAYAHSDNLQIEGGHRCFSLSCQGRDTFHIRYYGEPFDGLITDT 60 +KTDTA+MNNLIPEHLAAYAHSD+LQIEG HRCFSLSCQGRDTFHIRYYGEPFDGL+TDT Sbjct: 1 MKTDTAKMNNLIPEHLAAYAHSDSLQIEGVHRCFSLSCQGRDTFHIRYYGEPFDGLMTDT 60 Query: 61 DKAPVKIVAVEAVSGDEIVLFDGAEHGYNAMFCDKYSQNQKQNRTLTDLDEYTYRVPIHL 120 DKAPVKIVAVEAVSGDEIVLFDGAEHGYNAMFCDKYS NQKQNRTLTDLDEYTYRV IHL Sbjct: 61 DKAPVKIVAVEAVSGDEIVLFDGAEHGYNAMFCDKYSPNQKQNRTLTDLDEYTYRVLIHL 120 Query: 121 YYNIDYEDEYEDFVNSEGQVPLIDGRIISFDSLKRNGFDAISIDLIDEKHSVRELLNEELS 181 YYNIDYEDEYEDFVNSEGQVPLIDGRIISFDSLKRNGFDAIS+DLIDEKHSVRELLNEELS Sbjct: 121 YYNIDYEDEYEDFVNSEGQVPLIDGRIISFDSLKRNGFDAISVDLIDEKHSVRELLNEELS 181 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS142 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 140 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 279> which encodes amino acid sequence <SEQ ID 280; NGS143>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −2.51 Possible cleavage site: 57 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 4 value: −15.23 threshold: 0.0 INTEGRAL Likelihood = −15.23 Transmembrane 84-100 (79-107) INTEGRAL Likelihood = −8.12 Transmembrane 259-275 (250-281) INTEGRAL Likelihood = −4.14 Transmembrane 159-175 (153-176) INTEGRAL Likelihood = −3.88 Transmembrane 216-232 (216-235) PERIPHERAL Likelihood = 1.11 modified ALOM score: 3.55 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.709(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_5764059 gi\|5764059\|emb\|CAB53350.1\|\\(AJ010260) NosR protein [Paracoccus denitrificans] Length = 724 Score = 393 bits (1009), Expect = e−108 Identities = 191/379 (50%), Positives = 249/379 (65%), Gaps = 22/379 (5%) Query: 1 LMVQRVLSVNDKAFVTADLDYELPQAYYVDDPKAPPVEISAPVEAVPAAASDTASDGIAE 60 L+VQR + +K F T DL Y+LPQ Y AP A PAA +D Sbjct: 358 LLVQREVGPIEKVFHTFDLGYQLPQKYLRSIAPAPEA---------AAPAAQAD-------- 402 Query: 61 DASAENGVSNQLWKQIWAKQGQIVVVGIALTILLLVFLFQDWIVRYEKWYDRFRFAFLT 120 E+ QLWK+IW + +I + L +L VF FQ + RYE+ + FR A+LT Sbjct: 403 ----ESQAQAQLWKRIWLDSKPKIAGLAAMLLVLTGVFFFQSFTTRYERAFYVFRMAYLT 458 Query: 121 FTLFYIGWYAQAQLSVVNTLTLFSAILTEFHWEFFLMDPIVFILWLFTAATMLLWNRGTF 180 TL ++GWYA AQLSVVN + LF +++ F W+ FL+DP+ FILW AA +L W RG + Sbjct: 459 VTLVFLGWYANAQLSVVNLMALFGSLVNGFSWQAFLLDPLTFILWFAVAAALLFWGRGAY 518 Query: 181 CGWLCPFGSLQELTNRIAKKLGVKQITVPHMLHTRLNVIKYLILFGFLAISLYDLGTAEK 240 CGWLCPFG+LQELTN++A+KL + Q T+P LH RL +KY+I G +SL + AE Sbjct: 519 CGWLCPFGALQELTNQVARKLRIPQWTLPWGLHERLWPVKYMIFLGLFGVSLMSVEQAEH 578 Query: 241 FAEVEPFKTAIILKFMCDWWFVAPAVALLIAGLFIERFFCRYLCPLGAGIALPGRFRVFD 300 AEVEPFKTAIILKF+ W FVA+A ALLIAGLF+ERF+CRYLCPLGA +A+P R R+FD Sbjct: 579 LAEVEPFKTAIILKFIRAWPFVAYAAALLIAGLFVERFYCRYLCPLGAALAIPARMRMFD 638 Query: 301 WLRRYKMCGNPCQICTHBCPVQAIAPEGDIHPNECIQCLHCQVMYHHDTRCPQVVAENKK 360 WL+RY CGNPCQ C +CPVQ+I P G+I+PNECI CLHCQV+Y +T CP V+ KK Sbjct: 639 WLKRYHECGNPCQTCARQCPVQSIHPTGEINPNECINCLHCQVLYQSETTCPVVI---KK 695 Query: 361 KQKQAAAKSGELENVSKQP 379 +++ A +G + + + P Sbjct: 696 LKRREAVAAGSMPKLGQPP 714 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 141 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 281> which encodes amino acid sequence <SEQ ID 282; NGS144>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 1.23 Possible cleavage site: 21 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 20 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −0.37 threshold: 0.0 INTEGRAL Likelihood = −0.37 Transmembrane 90-106 (89-106) PERIPHERAL Likelihood = 10.82 modified ALOM score: 0.57 Rule: inner or outer membrane protein Rule: inner or outer membrane protein Rule: cytoplasmic membrane protein * Reasoning Step: 2 Lipoprotein?", "Inner membrane?", "----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.734(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology no to sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 142 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 283> which encodes amino acid sequence <SEQ ID 284; NGS145>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 1.5 Possible cleavage site: 19 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 20 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 8.70 threshold: 0.0 PERIPHERAL Likelihood = 8.70 modified ALOM score: −2.24 Score for OM-PP discrimination: −9.24 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −9.24 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Periplasmic space?", "Score: 0.924443 Periplasmic space?", "Score: 0.924443 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.931(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.231(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_5051426 gi\|5051426\|emb\|CAB45007.1\|\\(AJ242839) OpcA protein [Neisseria gonorrhoeae] Length = 263 Score = 531 bits (1369), Expect = e−150 Identities = 262/263 (99%), Positives = 263/263 (99%) Query: 1 MKKALLALTIAAISGTAMAQLPDFLGKGEYTVRTDISKQTLKNADLKEKHKVQKNIGFRA 60 MKKALLALTIAAISGTAMAQLPDFLGKGEYTVRTDISKQTLKNADLKEKHKVQKNIGFRA Sbjct: 1 MKKALLALTIAAISGTAMAQLPDFLGKGEYTVRTDISKQTLKNADLKEKHKVQKNIGFRA 60 Query: 61 DMPFDDIHHGMRFEVSHSRDKKDMYVVTESTTKPFGKDVEEKRTDVYAGYTYTQPISEAT 120 DMPFDDIHHGMRFEVSHSRDKKDMYVVTESTTKPFGKDV+EKRTDVYAGYTYTQPISEAT Sbjct: 61 DMPFDDIHHGMRFEVSHSRDKKDMYVVTESTTKPFGKDVKEKRTDVYAGYTYTQPISEAT 120 Query: 121 KLRAGLGLGYEKYKDAVANEKGTVSTEREAFYTKAHADLTSDLGGGWYLNPWAEVKVDLD 180 KLRAGLGLGYEKYKDAVANEKGTVSTEREAFYTKAHADLTSDLGGGWYLNPWAEVKVDLD Sbjct: 121 KLRAGLGLGYEKYKDAVANEKGTVSTEREAFYTKAHADLTSDLGGGWYLNPWAEVKVDLD 180 Query: 181 AKLKHNATVAGVSADINAKTRGWGVGVGANIGKQITDTVGIEAGPFYKHRHFKASGSFVL 240 AKLKHNATVAGVSADINAKTRGWGVGVGANIGKQITDTVGIEAGPFYKHRHFKASGSFVL Sbjct: 181 AKLKHNATVAGVSADINAKTRGWGVGVGANIGKQITDTVGIEAGPFYKHRHFKASGSFVL 240 Query: 241 DGGNIRVDPTKINEYGVRVGVKF 263 DGGNIRVDPTKINEYGVRVGVKF Sbjct: 241 DGGNIRVDPTKINEYGVRVGVKF 263 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 143 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 285> which encodes amino acid sequence <SEQ ID 286; NGS146>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 0.1 Possible cleavage site: 51 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 52 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.50 threshold: 0.0 PERIPHERAL Likelihood = 3.50 modified ALOM score: −1.20 Score for OM-PP discrimination: −15.70 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −15.70 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Periplasmic space?", "Score: 1.56979 Periplasmic space?", "Score: 1.56979 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.944(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.375(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_5051429 gi\|5051429\|emb\|CAB45013.1\|\\(AJ242839) hypothetical protein [Neisseria gonorrhoeae] Length = 109 Score = 216 bits (549), Expect = 2e−55 Identities = 109/109 (100%), Positives = 109/109 (100%) Query: 1 MFKRPEEIIVLILAVLWIAGTYFLAALFGADAYVLKITALTLLWSAASFLLWQKKPQPA 60 MFKRPEEIIVLILAVLWIAGTYFLAALFGADAYVLKITALTLLWSAASFLLWQKKPQPA Sbjct: 1 MFKRPEEIIVLILAVLWIAGTYFLAALFGADAYVLKITALTLLWSAASFLLWQKKPQPA 60 Query: 61 YLAAAARLPDHLLVAVASESIGRTRFFTLACIMDVQHNLSPDSRNRRLSV 109 YLAAAARLPDHLLVAVASESIGRTRFFTLACIMDVQHNLSPDSRNRRLSV Sbjct: 61 YLAAAARLPDHLLVAVASESIGRTRFFTLACIMDVQHNLSPDSRNRRLSV 109 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 144 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 287> which encodes amino acid sequence <SEQ ID 288; NGS147>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.09 Possible cleavage site: 40 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −2.28 threshold: 0.0 INTEGRAL Likelihood = −2.28 Transmembrane 36-52 (36-52) PERIPHERAL Likelihood = 5.20 modified ALOM score: 0.96 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.191(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_6606516 gi\|6606516\|gb\|AAF19189.1\|AF200716_2 \\(AF200716) trafficking protein B [Neisseria gonorrhoeae] Length = 139 Score = 274 bits (700), Expect = 7e−73 Identities = 139/139 (100%), Positivies = 139/139 (100%) Query: 2 MILLDTNVISEPLRPQPNERVVAWLDSLILEDVYLSAITVAELRLGVALLLNGKKKNVLH 61 MILLDTNVISEPLRPQPNERVVAWLDSLILEDVYLSAITVAELRLGVALLLNGKKKNVLH Sbjct: 1 MILLDTNVISEPLRPQPNERVVAWLDSLILEDVYLSAITVAELRLGVALLLNGKKKNVLH 60 Query: 62 ERLEQSILPLFAGRILPFDEPVAAIYAQIRSYAKTHGKEIAAADGYIAATAKQHSLTVAT 121 ERLEQSILPLFAGRILPFDEPVAAIYAQIRSYAKTHGKEIAAADGYIAATAKQHSLTVAT Sbjct: 61 ERLEQSILPLFAGRILPFDEPVAAIYAQIRSYAKTHGKEIAAADGYIAATAKQHSLTVAT 120 Query: 122 RDTGSFFAADVAVFNPWHD 140 RDTGSFFAADVAVFNPWHD Sbjct: 121 RDTGSFFAADVAVFNPWHD 139 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 145 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 289> which encodes amino acid sequence <SEQ ID 290; NGS148>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 0.86 Possible cleavage site: 47 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 48 ALOM: Finding transmembrane regions (Klein et al.)", "count: 5 value: −15.44 threshold: 0.0 INTEGRAL Likelihood = −15.44 Transmembrane 157-173 (142-181) INTEGRAL Likelihood = −12.15 Transmembrane 62-78 (56-83) INTEGRAL Likelihood = −6.32 Transmembrane 194-210 (191-212) INTEGRAL Likelihood = −4.30 Transmembrane 87-103 (85-104) INTEGRAL Likelihood = −2.60 Transmembrane 121-137 (121-142) PERIPHERAL Likelihood = 2.92 modified ALOM score: 3.59 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.718(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_15777859\|gi\|15777859\|gb\|AAL05955.1\|\\(AY048756) putative cadmium binding protein [Staphylococcus aureus] Length = 209 Score = 354 bits (908), Expect = 1e−96 Identities = 177/208 (85%), Positives = 194/208 (93%) Query: 14 MRCFMFSTVITAAVLYIATAVDLLVILLIFFARANTRKEYRDIYIGQYLGSVILILVSLF 73 MRC M TV+TAAVLYIATAVDLLVILLIFFARA TRKEYRDIY+GQYLGS+ILILVSLF Sbjct: 1 MRCIMIQTVVAAAVLYIATAVDLLVILLIFFARAKTRKEYRDIYVGQYLGSIILILVSLF 60 Query: 74 LAFVLNYVPEKWVLGLLGLIPIYLGIKVAIYDDCEGEKRAKKELDEKGLSKLVGIVALVT 133 LAFVLNYVPEKW+LGLLGLIPIYLGIKVAIYDDCEGEKRAKKEL+EKGLSKLVG VA+VT Sbjct: 61 LAFVLNYVPEKWILGLLGLIPIYLGIKVAIYDDCEGEKRAKKELNEKGLSKLVGTVAIVT 120 Query: 134 VASCGADNIGLFVPYFVTLDLVDLLVTLLVFLILIFVLVYTAQRLANISGVGEIVEKFSR 193 +ASCGADNIGLFVPYFVTL + +LL+TL VFLILIF LV+TAQ+LANI G+GEIVEKFSR Sbjct: 121 IASCGADNIGLFVPYFVTLSVTNLLLTLFVFLILIFFLVFTAQKLANIPGIGEIVEKFSR 180 Query: 194 WIMAVIYIGLGLFIIIENNTIRTIISII 221 WIMA+IYI LGLFIIIEN+TI+TI+ I Sbjct: 181 WIMAIIYIALGLFIIIENDTIQTILGFI 208 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 146 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 291> which encodes amino acid sequence <SEQ ID 292; NGS149>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.63 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 2.12 threshold: 0.0 PERIPHERAL Likelihood = 2.12 modified ALOM score: −0.92 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.122(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_15675455 gi\|15675455\|ref\|NP_269629.1\|\\(NC_002737) conserved hypothetical protein [Streptococcus pyogenes] [Streptococcus pyogenes M1 GAS] gb\|AAK34350.1\|(AE006588) conserved hypothetical protein [Streptococcus pyogenes M1 GAS] Length = 224 Score = 106 bits (264), Expect = 3e−22 Identities = 63/151 (41%), Positives = 85/151 (55%), Gaps = 12/151 (7%) Query: 20 LSALQHYAFCPRQCALIHNEQAWAENYLTAQGKALHERVDSDE-PETCKGVRFEWTVHVL 78 LS +QH+ FC RQ ALIH EQ W +N TA G+ LH + D+ E K + + + Sbjct: 11 LSGIQHFQFCKRQWALIHIEQQWLDNEATAHGQVLHTKADNPYIKEKRKELLVSRAMPIS 70 Query: 79 ADKLGISGILDLVE---------VDTKTGRLKP--VEYKRGKPKPDPGDEIQLCAQGLCL 127 + +LG+SGI+D+VE + K G+ P VEYKRGKPK D D +QL AQ +CL Sbjct: 71 SAELGLSGIMDVVEFYKDDQGVSLRGKRGKWLPKVVEYKRGKPKKDTRDIVQLVAQTMCL 130 Query: 128 EEMTGQTVSEGALWYMQTRHRVPVVFSDGLR 158 EE ++EG L+Y RV V + LR Sbjct: 131 EETLDCDINEGCLYYHSVNQRVIVPMTSALR 161 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 147 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 293> which encodes amino acid sequence <SEQ ID 294; NGS150>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.71 Possible cleavage site: 19 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −0.85 threshold: 0.0 INTEGRAL Likelihood = −0.85 Transmembrane 79-95 (79-96) PERIPHERAL Likelihood = 6.52 modified ALOM score: 0.67 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.134(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 148 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 295> which encodes amino acid sequence <SEQ ID 296; NGS151>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 3.47 Possible cleavage site: 23 >>> Seems to have a cleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 24 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 14.32 threshold: 0.0 PERIPHERAL Likelihood = 14.32 modified ALOM score: −3.36 Score for OM-PP discrimination: −32.29 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −32.29 Rule: outer membrane or periplasmic protein * Reasoning Step: 2 Periplasmic space?", "Score: 3.22889 Periplasmic space?", "Score: 3.22889 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.933(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.253(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gsa_AAY75310 Neisseria gonorrheae ORF 649 protein sequence SEQ ID N0:2094\|WO9957280-A2\|21-MAR-2000 Length = 103 Score = 35.4 bits (80), Expect = 0.32 Identities = 25/85 (29%), Positives = 38/85 (44%), Gaps = 5/85 (5%) Query: 7 ILTGILLATALPASAHGMHKSKPLAMDELPPIQQYFKRAETCYNKAGNKADFARN-NTK 65 + T T+ PA H H SK L P C++Y +R Y GN + N + Sbjct: 13 VSTTAAAGTSEPAHRHTKHISKA-NKQMLHPECRKYLERRAAWYRSQGNVQELRENKKAR 71 Query: 66 FLFQALPAADLGQRKQMCQIAMDSF 90 F+ LP A ++K C+ A ++F Sbjct: 72 KAFRTLPYA---EQKIQCRAAYEAF 93 The protein was expressed in E.coli as a soluble 9.35 kDa His-fusion product and then purified.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 149 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 297> which encodes amino acid sequence <SEQ ID 298; NGS152>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.46 Possible cleavage site: 18 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −3.19 threshold: 0.0 INTEGRAL Likelihood = −3.19 Transmembrane 368-384 (367-384) PERIPHERAL Likelihood = 0.53 modified ALOM score: 1.14 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.227(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gsa_AAY81609 Streptococcus pneumoniae type 4 protein sequence #109 \|WO200006737-A2\|24-MAY-2000 Length = 1237 Score = 48.1 bits (113), Expect = 4e−04 Identities = 80/312 (25%), Positives = 142/312 (44%), Gaps = 59/312 (18%) Query: 57 RRQARIRVGNLITDSLEHIRVKALLPLPL----KLPVKRI---NLPRNLPALPVRLRKTI 109 RRQ R + ++ L+H RV L P+ ++PV+++ +PR A RL++ + Sbjct: 941 RRQVR-QPQQVLVHQLQHQRVHRLRRQPVHQSQQVPVRQLPHQQVPRLQQAPVRRLQQVL 999 Query: 110 SPRQIGDALPILKLQRI--RLTLHLKPLPLHPQLGLLHIKRPVRIPLRHLAVQRTLVRLN 167 +P+ P+ + Q++ RL H + PL L +P R + L QR VRLN Sbjct: 1000 APQP--QPQPVRQPQQVSQRLNRHQRVRPLQQVLA----PQPQRQQVHRL--QRQRVRLN 1051 Query: 168 RRIKPPLLQHRLTVRRILRRSRRQPFPAQFPDRRIFIMFRHNPARRIKLCRRQLTVQGPR 227 R + LQ L A P R+ +H +R++ ++ L Q R Sbjct: 1052 RHQRVRPLQQVL---------------APQPQRQQVHRLQH---QRVRPLQQVLAPQPQR 1093 Query: 228 IRRSRPLIKLPLLRRQRIRPGRHQRTLRVKITHRLAAPIHIPVKSQRRRRPSARIRRARI 287 + R L+RQR+R +HQR + + H+L +H PV+ Q + R + ++++ + Sbjct: 1094 QQVHR-------LQRQRVRLSQHQRVRQPQQAHQL-LNLHQPVR-QPQHRQAPQLQQVPV 1144 Query: 288 APREIRPGPRIGGKRLIAARKP-QTGIRTPFESTRPAQPPRPILNIVTAQIHHIPITRR 345 + R R+ + + R+P Q +R P R + P+P+ LN H P+ R+ Sbjct: 1145 RQPQRRQVRRL---QQVPVRQPQQVPVRQP--QRRQVRRPQPVHLN------ RHQPV-RQ 1192 Query: 346 PGLIIRNGTPHR 357 P ++ + H+ Sbjct: 1193 PQQVLVHQLQHQ 1204 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 150 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 299> which encodes amino acid sequence <SEQ ID 300; NGS153>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.48 Possible cleavage site: 13 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 6.31 threshold: 0.0 PERIPHERAL Likelihood = 6.31 modified ALOM score: −1.76 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.150(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_15832758 gi\|15832758\|ref\|NP_311531.1\|\\(NC_002695) hypothetical protein [Escherichia coli O157:H7] dbj\|BAB36927.1\|(AP002562) hypothetical protein [Escherichia coli O157:H7] Length = 188 Score = 73.9 bits (180), Expect = 3e−12 Identities = 54/169 (31%), Positives = 79/169 (45%), Gaps = 15/169 (8%) Query: 12 LTQEVLKELLRYDDNTGKLYWAERPRKYFNSGLHYKSWNTGFSGKEVFLYKGRLGYLKLK 71 LT + + ELL +D +TG W + + S F GY + Sbjct: 16 LTVKRIFELLSFDKSTGVFRWKVPTQ----GRIALNSVAGAFDSN------------GYSMIM 62 Query: 72 IFKKQYNAHRLIWLFVYGKH-ASSIGHINRDKTDNRISNLRDVTHAENMKNRGKFKNNTS 130 I ++Y H L++ + + A I H+N +TDNR NLR+ EN +N KN+ S Sbjct: 63 IDGRRYKTHVLVFYITHNRWPAGQIDHNGIRTDNRPENLRECLPIENSRNIRIRKNSKS 122 Query: 131 GHTGVYFKPSKKWQARIWINRKNKILGLFEHIEDAA-KAREAASKDFG 178 G GV +HK KKW R+ + K+K G F+ +E A A EA K +G Sbjct: 123 GCRGVTWMKRQKKWNVRLGFHGKSKHFGCFDDLELAVLVAEEARDKYYG 171 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 151 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 301> which encodes amino acid sequence <SEQ ID 302; NGS154>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −6.98 Possible cleavage site: 28 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 8.12 threshold: 0.0 PERIPHERAL Likelihood = 8.12 modified ALOM score: −2.12 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.423(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_15830449 gi\|15830449\|ref\|NP_309222.1\|\\(NC_002695) hypothetical protein [Escherichia coli O157:H7] dbj\|BAA94132.1\|(AP000422) hypothetical protein [Escherichia coli O157:H7] dbj\|BAB346l8.1\|(AP002554) hypothetical protein [Escherichia coli O157:H7] Length = 148 Score = 42.7 bits (99), Expect = 0.003 Identities = 27/99 (27%), Positives = 49/99 (49%), Gaps = 10/99 (10%) Query: 37 IRPRKSKRSVEQNRRLWFLYREISEKVFIDGRRFSQDVWHE-----FLKRKFIGCIEHPN 91 + ++ KRS QN R+W + ++S +V G+R + + W + +LK K + +P Sbjct: 33 VHVKEPKRSKAQNDRMWPMLNDVSRQVLWHGQRLAPEDWKDLFTALWLKTKKLEQRSVPG 92 Query: 92 GQ----LMGISTTKLSVEEMSEYQEKIISWASNEHGVLW 126 ++G+ T+K+ M+E E I+ W E V W Sbjct: 93 IDGGVVMLGVRTSKMRKASMTELIE-IMFEFGSERNVRW 130 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 152 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 303> which encodes amino acid sequence <SEQ ID 304; NGS155>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4 Possible cleavage site: 27 >>> Seems to have an uncleavable N-term signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.98 threshold: 0.0 PERIPHERAL Likelihood = 4.98 modified ALOM score: −1.50 * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.046(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_15801502 gi\|15801502\|ref\|NP_287519.1\|\\(NC_002655) putative endonuclease of prophage CP-9330 [Escherichia coli O157:H7 EDL933] ref\|NP_309804.1\|(NC_002695) endonuclease [Escherichia coli O157:H7] gb\|AAG56131.1\|AE005344_7 (AE005344) putative endonuclease of prophage CP-933O [Escherichia coli O157:H7 EDL933] dbj\|BAB35200.1\|(AP002556) endonuclease [Escherichia coli O157:H7] Length = 119 Score = 47.4 bits (1111, Expect = 2e−04 Identities = 38/122 (31%), Positives = 54/122 (44%), Gaps = 8/122 (6%) Query: 71 LILPYPVSANRYWRIWRNRAVRSAEAAAYKETVRRIA-QGAGAMPSEGAVAVYVRLIPKA 129 L+LPYP + N YWR + S Y+ V I Q + G +A+ + P Sbjct: 5 LVLPYPPTVNTYWERRGSTYFVSKAGERYRRAVVLIVRQQRLKLSLSGRLAIKIIABP-- 62 Query: 130 NKDGGANKTVIDLDNLKVTLDALQGVAYHNDRQVRRIAAEYGGEPVTGGGLAVEVGELE 189 +K DLDN LK LDAL D + G+PV+GG L V++ ++E Sbjct: 63 -----PDKRRRDLDNILKAPLDALTHAGVLMDDEQFDEINIVRGQPVSGGRLGVKIYKIE 117 Query: 190 ME 191 E Sbjct: 118 SE 119 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 153 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 305> which encodes amino acid sequence <SEQ ID 306; NGS156>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −2.61 Possible cleavage site: 49 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.96 threshold: 0.0 PERIPHERAL Likelihood = 7.96 modified ALOM score: −2.09 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.307(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gsa_AAG90098 C glutamicum protein fragment SEQ ID NO: 3852 \|EP1108790-A2\|26-SEP-2001 Length = 148 Score = 102 bits (253), Expect = 7e-21 Identities = 60/147 (40%).", "Positives = 88/147 (59%), Gaps = 18/147 (12%) Query: 3 NAYDVADFFLSPFEEEDGEQISNLKLQKLLYYAQGYALAILNRPLFAENIEHWQHGPVVP 62 +A ++A++F++ +E D E +S LKLQKLLYY+QG +A R LF++ I WQHGPV P Sbjct: 5 SAREIAEWFVAWGDELDAE-VSPLKLQKLLYYSQGEHIAATGRKLFSDKILAWQHGPVTP 63 Query: 63 CIYRTYKKYGGSPLPAAHIEPDKYADEEL---------VVLNRVRKEQGCYTAWALRNKT 113 +Y K YG +P I+PD++ E L V ++ G Y+AWALR KT Sbjct: 64 GVYSDTKSYGRNP-----IDPDEFVSDEFNWDDYSDVSDELVTVWRKYGIYSAWALREKT 118 Query: 114 HQEAPWIQT-RQGEVIGI--AIMGEYF 137 H E+PW+ QG+ I I A + ++F Sbjct: 119 HSESPWLDAWAQGQNIEITDAALKDFF 145 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 154 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 307> which encodes amino acid sequence <SEQ ID 308; NGS157>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.65 Possible cleavage site: 42 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 3.98 threshold: 0.0 PERIPHERAL Likelihood = 3.98 modified ALOM score: −1.30 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.291(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} gbp_6274533 gi\|6274533\|gb\|AAF06681.1\|AF163663_6 \\(AF058689) Toul [Neisseria meningitidis] Length = 272 Score = 546 bits (1408), Expect e−154 Identities = 267/272 (98%), Positives = 271/272 (99%) Query: 19 MKGMDKLRYQRDFLNIRPIFTAGEQEYLTELSDRLPLSVLTDSVRNIEEIGIDVYSPAK 78 MKGMDKLRYQ+DFLNIRPIFTAGEQEYLTELSDRLPLSVLTDSVRNIEEIGIDVYS AK Sbjct: 1 MKGMDKLRYQQDFLNIRPIFTAGEQEYLTELSDRLPLSVLTDSVRNIEEIGIDVYSSAK 60 Query: 79 LEGNTYNQYDTQALLKLGQTAGGKLYSDAVMLINLRESYRHLLSGLDSPKPFDWLDFLKT 138 LEGNTYNQYDTQALLKLGQTAGGKLYSDAVMLINLRESYRHLLSGLDSP+PFDWLDFLKT Sbjct: 61 LEGNTYNQYDTQALLKLGQTAGGKLYSDAVMLINLRESYRHLLSGLDSPEPFDWLDFLKT 120 Query: 139 THSLISENLLEKGSGGVVRRDSVTISGTDYTPLSNPQSLDTELKWLLQEAPKIENPFDRA 198 THSLISENLLEKGSGGVVRRDSVTISGTDYTPLSNPQSLDTELKWLLQEAPKIENPFDRA Sbjct: 121 THSLISENLLEKGSGGVVRRDSVTISGTDYTPLSNPQSLDTELKWLLQEAPKIENPFDRA 180 Query: 199 VYLHNNLAYLRYFKDCNKRTARNCMTLSLMRSGFFPCVFSPDSYPAYAEAVVAYYETGDY 258 VYLHNNLAYL+YFKDCNKRTARNCMTLSLMRSGFFPCVFSPDSYPAYAEAVVAYYETGDY Sbjct: 181 VYLHNNLAYLQYFKDCNKRTARNCMTLSLMRSGFFPCVFSPDSYPAYAEAVVAYYETGDY 240 Query: 259 GLFKKYFISAYENTVNKYGPQPDVDIFRNFSI 290 GLFKKYFISAYENTVNKYGPQPDVDIFRNFS+ Sbjct: 241 GLFKKYFISAYENTVNKYGPQPDVDIFRNFSL 272 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS157 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 155 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 309> which encodes amino acid sequence <SEQ ID 310; NGS158>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −6.98 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 0.16 threshold: 0.0 PERIPHERAL Likelihood = 0.16 modified ALOM score: −0.53 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.185(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: > {circumflex over ( )}{circumflex over ( )} gbp_15791833 gi\|15791833\|ref\|NP_281656.1\|\\(NC_002163) amino-acid ABC transporter ATP-binding protein [Campylobacter jejuni] pir\|\|H81391 amino-acid ABC transporter ATP-binding protein Cj0469 [imported]-Campylobacter jejuni (strain NCTC 11168) emb\|CAB75107.1\|(AL139075) amino-acid ABC transporter ATP-binding protein [Campylobacter jejuni] Length = 253 Score = 301 bits (772), Expect = 6e−81 Identities = 153/244 (62%), Positives = 195/244 (79%), Gaps = 2/244 (0%) Query: 1 MALLSIRKLHKQYGSVAIQSLDLDLEKGEVIVLLGPSGCGKSTLLRCVNGLEPHQGGSI 60 M++L I L K YGS A++ ++L+++ EV+V+LGPSGCGKSTLLRC+NGLE G+I Sbjct: 1 MSILKIENLQKYYGSHHALKDINLEVKAKEVVVILGPSGCGKSTLLRCINGLEEIASGNI 60 Query: 61 VMDGVGEFGKDVS-WQTARQKVGMVFQSYELFAHMTVIENILLGPVKVQNRDRAEAEAQA 119 +D + KD W RQKVGMVFQSYELF H++V ENILLGP+KVQ R + E +A Sbjct: 61 YIDNE-KIDKDFKEWPRMRQKVGMVFQSYELFEHLSVEENILLGPMKVQKRKKDEVLKEA 119 Query: 120 GKLLERVGLLDRKNAYPRELSGGQKQRIAIVRALCLNPEVILLDEITAALDPEMVREVLE 179 LE+VGLL + +AYPRELSGGQKQRIAIVR+LC+NPE++L DE+TAALDPE+VREVLE Sbjct: 120 KIWLEKVGLLHKIHAYPRELSGGQKQRIAIVRSLCMNPELMLFDEVTAALDPEIVREVLE 179 Query: 180 VVLELAREGMSMLIVTHEMGFARKVADRIVFMDKGGIVESSDPETFFSAPKSERARQFLA 239 V+L LA+EGM+MLIVTHEMGFA+ VAD+I+FMD+G I+E +DP++FF PKSERA++FL Sbjct: 180 VMLNLAKEGMTMLIVTHEMGFAKAVADKIIFMDEGKIIEENDPKSFFKNPKSERAKKFLN 239 Query: 240 GMDY 243 DY Sbjct: 240 LFDY 243 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 156 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 311> which encodes amino acid sequence <SEQ ID 312; NGS159>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.16 Possible cleavage site: 13 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 6.47 threshold: 0.0 PERIPHERAL Likelihood = 6.47 modified ALOM score: −1.79 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.312(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: > {circumflex over ( )}{circumflex over ( )} gbp_15794799 gi\|1579479.9\|ref\|NP_284621.1\|\\(NC_003116) hypothetical protein [Neisseria meningitidis Z2491] pir\|\|B81819 hypothetical protein NMA1914 [imported]- Neisseria meningitidis (group A strain Z2491) emb\|CAB85135.1\|(AL162757) hypothetical protein [Neisseria meningitidis Z2491] Length = 206 Score = 265 bits (678), Expect = 8e−70 Identities = 131/146 (89%), Positives = 138/146 (93%) Query: 1 MTKLYAQIAKTEAQDDGTVKVWGYASSEAVDSDGEVVAAEAMKAAIPDYMKFGAVREMHG 60 MTKLYA+IAK E QDDGTVKVWGYASSE +DSDGEV+AA AMKAAIPDYMKFGA REMHG Sbjct: 1 MTKLYAEIAKMETQDDGTVKVWGYASSEEIDSDGEIAAAAMKAAIPDYMKFGAGREMHG 60 Query: 61 SNAAGTAIEINVEDDGRTFFGAHIVDPVAVTKVKTGVYKGFSIGGSVTARNDLNKSQITG 120 SNAAGTAIEINVEDDG TFFGAHI+DPV V+KVKTGVYKGFSIGGSVTAR+DLNKSQITG Sbjct: 61 SNAAGTAIEINVEDDGITFFGAHIIDPVVVSKVKTGVYKGFSIGGSVTARDDLNKSQITG 120 Query: 121 LKLTEISLVDRPANPDAVFTCFKADK 146 LKLTEISL+DRPANPDAV TCFKADK Sbjct: 121 LKLTEISLIDRPANPDAVSTCFKADK 146 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS159 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 157 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 313> which encodes amino acid sequence <SEQ ID 314; NGS160>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −8.3 Possible cleavage site: 33 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 7.85 threshold: 0.0 PERIPHERAL Likelihood = 7.85 modified ALOM score: −2.07 Rule: cytoplasmic protein * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.407(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: > {circumflex over ( )}{circumflex over ( )} gbp_2126352 gi\|2126352\|pir\|\|JC5218 type I site-specific deoxyribonuclease (EC 3.1.21.3) Hsd chain S [validated]-Pasteurella haemolytica gb\|AAC44667.1\|(U46781) HSDS [Mannheimia haemolytica] Length = 442 Score = 97.1 bits (240), Expect = 3e−19 Identities = 55/149 (36%), Positives = 81/149 (53%), Gaps = 3/149 (2%) Query: 26 EVAEYSKNRICSDKLNEHNYVGVDNLLQNREGKKLSGYVPSEGKMTEYIVNDILIGNIRP 85 ++E +I L + NY+ DN+L N G L+ +P+ + DIL NIR Sbjct: 10 DIVELISEKIKIKDLKKENYISTDNMLPNFGGITLAENLPNSASCNRFAKKDILFSNIRT 69 Query: 86 YLKKIWQADCTGGTNGDVLVIRV--TDEKVNPKYLYQVLADDKFFAFNMKHAKGAKMPRG 143 Y KK+W A+ +GG + DVLV+R TD +N +YL+ ++ D F F + A GAKMPRG Sbjct: 70 YFKKVWLAEFSGGCSPDVLVMRSKNTDILLN-EYLFLLIRSDDFINFTVISANGAKMPRG 128 Query: 144 SKAAIMQYKIPIPPLPEQEKIVAILGKFD 172 K A+ + IP + Q+K +A FD Sbjct: 129 DKNAMKGFIFNIPSIEYQKKCIANYFAFD 157 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 158 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 315> which encodes amino acid sequence <SEQ ID 316; NGS161>.", "Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.08 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 1 value: −1.59 threshold: 0.0 INTEGRAL Likelihood = −1.59 Transmembrane 302-318 (302-318) PERIPHERAL Likelihood = 3.76 modified ALOM score: 0.82 Rule: cytoplasmic membrane protein * Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.164(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: pir\|\|E81921 probable DNA-invertase NMA0772 [imported]-Neisseria meningitidis (group A strain Z2491) emb\|CAB84055.1\|(AL162754) putative DNA-invertase [Neisseria meningitidis Z2491] Length = 321 Score = 295 bits (755), Expect = 9e−79 Identities = 151/322 (46%), Positives = 216/322 (66%), Gaps = 3/322 (0%) Query: 14 LRNAVGLDISKLTFDATAIVGNAEYSKFDNDSKGLDQFSDRLKSLGCQNLHICMEATGN 73 +RNAVGLDIS TFD I+ KF ND +G + + + +++++CMEATGN Sbjct: 1 MRNAVGLDISAKTFDVVTIINGETDYRKFSNDEQGCKNLKEWISAKREKDIYVCMEATGN 60 Query: 74 YYEEVADYFAQYYSVYVVNPLKISKYAESRFKRTKTDKQDAKLIAQYCRSAQESELVKRQ 133 YYE+ AD A+ Y V V+NPLKI YA+ RF R K DKQDAKLIA++C++A EL KR+ Sbjct: 61 YYEQAADCLAEEYHVSVINPLKIKAYAQKRFSRVKNDKQDAKLIAEFCQTALIEELPKRE 120 Query: 134 KPTDEQYRLSRMTAAYAQIKSECAAMKNRHHAAKDEEAAKAYAEIIKAMNEQLEVLKEKI 193 KPT++QY L R+ + +Q+ + + KNR AAKD K + + +K + L +K+KI Sbjct: 121 KPTEQQYSLKRLLSLQSQLLEQQTSQKNRIRAAKDSFVQKIHEKQLKELENHLNAVKKKI 180 Query: 194 KEQTEKPN--CKEGVKRLETIPAIGRMTAAVLFHHLTSSKFETSNKFAAFAGLSPQQKES 251 +QT K + KE KRLETIP++G+ TA L +L +S FE + +FA +AGL+P Q S Sbjct: 181 -DQTIKSDKKMKELTKRLETIPSVGKTTAISLMSYLINSTFENAKQFTAYAGLNPHQNIS 239 Query: 252 GTSVRGKGKLTKFGNRKLRAVLFMPAMVAYRIRAFPDFIKRLEEKKKPKKVIIAALMRKL 311 GTSV K K+TK+GNR++R LFM A+VA++ FP F RL++ KKPK +II ALMRK+ Sbjct: 240 GTSVNKKSKMTKYGNRRIRGSLFMAALVAFKNNYFPAFTNRLKKAKKPKMLIIGALMRKI 299 Query: 312 AVIAYHVHKKGGDYDPSRYKSA 333 V+A++++K D+D +RY++A Sbjct: 300 LVVAFNLYKTETDFDKTRYQTA 321 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS161 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.", "Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 159 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 983> which encodes amino acid sequence <SEQ ID 984; NGS162>.", "Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.86 Possible cleavage site: 33 >>> Seems to have no N-terminal signal seq.", "Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)", "count: 0 value: 4.08 threshold: 0.0 PERIPHERAL Likelihood = 4.08 modified ALOM score: −1.32 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.032(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: ref\|NP_312507.1\|(NC_002695) hypothetical protein [Escherichia coli O157:H7] gb\|AAG5B749.1\|AE005587_7 (AE005587) putative adhesin [Escherichia coli O157:H7 EDL933] dbj\|BAB37903.1\|(AP002566) hypothetical protein [Escherichia coli O157:H7] Length = 1588 Score = 120 bits (302), Expect = 4e−26 Identities = 109/359 (30%), Positives = 170/359 (46%), Gaps = 65/359 (18%) Query: 22 AVALGSSSTASGEYSYASGYNSVASGNKSYAAGYASVASAEGSVVIGDSRQVKPEADQGV 81 + A+G + A G+YS A G + A G S A G ++++ + S+ +G S + + Sbjct: 93 STAVGYDAIAEGQYSSAIGSKTHAIGGASMAFGVSAISEGDRSIALGASSYSLGQYSMAL 152 Query: 82 AVGSKATVKNKAKQRVVVGSEAKVNAERGIAIGKEAKAGGKTTNTLLDGPAYYADAIAVG 141 SKA K + +G +K IA+G KA T + +IA+G Sbjct: 153 GRYSKAL----GKLSIAMGDSSKAEGANAIALGNATKA----TEIM---------SIALG 195 Query: 142 YQAEAGKGGAIALGKQAKATKQNGMALGVESEAAGDFSTAVGNESKAKGQGG-------- 193 A A K ++ALG + A+++N +A+G E+EAA + +TA+GN +KAKG Sbjct: 196 DTANASKAYSMALGASSVASEENAIAIGAETEAA-ENATAIGNNAKAKGTNSMAMGFGSL 254 Query: 194 ------VGLGNQSKAEADFAVAV--GNKAEATKE------------NSLVIGRYARANGN 233 + LGN S+A AD A+A+ GNKA+ N++ +G + A G+ Sbjct: 255 ADKVNTIALGNGSQALADNAIAIGQGNKADGVDAIALGNGSQSRGLNTIALGTASNATGD 314 Query: 234 HSVSLGSRSEIKDGVSNSVAPGYGSVASENNVVSVAYKETPQSTELSYRKIVGVDDGV-- 291 S++LGS S +G+ NSVA G S+A +N VSV RKIV V +G Sbjct: 315 KSLALGSNSS-ANGI-NSVALGADSIADLDNTVSVGNSSLK-------RKIVNVKNGAIK 365 Query: 292 -NDFDAVNVRQLKAMQGQNMAELFSVRSEVRGVAASSAALSALTPLSYDANNPTQFMVG 349 + +DA+N QL A+ SV + G AA +T +Y+ N ++ VG Sbjct: 366 SDSYDAINGSQLYAISD-------SVAKRLGGGAAVDVDDGTVTAPTYNLKNGSKNNVG 417 Score = 86.3 bits (212), Expect = 1e−15 Identities = 68/253 (26%), Positives = 118/253 (45%), Gaps = 39/253 (15%) Query: 28 SSTASGEYSYASGYNSVASGNKSYAAGYASVASAEGSVVIGDSRQVKPEADQGVAVGSKA 87 S+ +G + G + A + Y S ++ +G V IG G+KA Sbjct: 38 SALVAGGMLSSFGALANAGNDNGQGVDYGSGSAGDGWVAIGK--------------GAKA 83 Query: 88 -TVKNKAKQRVVVGSEAKVNAERGIAIGKEAKAGGKTTNTLLDGPAYYADAIAVGYQAEA 146 T N + VG +A + AIG + A G + +A G A + Sbjct: 84 NTFMNTSGSSTAVGYDAIAEGQYSSAIGSKTHAIGGAS-------------MAFGVSAIS 130 Query: 147 GKGGAIALGKQAKATKQNGMALGVESEAAGDFSTAVGNESKAKGQGGVGLGNQSKAEADF 206 +IALG + + Q MALG S+A G S A+G+ SKA+G + LGN +KA Sbjct: 131 EGDRSIALGASSYSLGQYSMALGRYSKALGKLSIAMGDSSKAEGANAIALGNATKATEIM 190 Query: 207 AVAVGNKAEATKENSLVIGRYARANGNHSVSLGSRSEIKDGV-----------SNSVAPG 255 ++A+G+ A A+K S+ +G + A+ +++++G+ +E + +NS+A G Sbjct: 191 SIALGDTANASKAYSMALGASSVASEENAIAIGAETEAAENATAIGNNAKAKGTNSMAMG 250 Query: 256 YGSVASENNVVSV 268 +GS+A + N +++ Sbjct: 251 FGSLADKVNTIAL 263 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.", "EXAMPLE 160 Further open reading frames were identified in gonococcus <SEQ IDs 317/318 to 8621/8622>.", "These polypeptide and nucleotide sequences are useful for studying gonococcus, for diagnostic purposes, as antibiotic targets, and as vaccine antigens.", "It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention." ] ]	Patent_10467657
[ [ "Dry shaver for variable heights of cut", "A dry shaver (11) has a single cutter blade (15) and a single cooperating shear blade (17).", "The shear blade (17) is an elongate flexible foil, the thickness of which is different at different regions (21, 22) along the elongate extent of the foil (17).", "The foil (17) is mounted (27) for displacement in its elongate direction so that a foil region (21, 22) of a particular thickness may be selectively aligned with the cutter blade (15).", "The spacing between a skin engaging surface of the shear blade (17) and the opposite face of the shear blade (17) which cooperates with the cutter blade (15) is thus selectively variable.", "In a further embodiment, the dry shaver is of the rotary type and has a plurality of interchangeable shear blades of different thicknesses.", "In a still further embodiment, a rotary dry shaver has a mechanism for varying the spacing between the outer surface of a cover member overlying a shear blade and a cooperating rotary cutter blade." ], [ "1.A dry shaver unit comprising: at least one cutter blade; and a cooperating shear blade; wherein a spacing between a skin-engaging surface of the shaver unit and regions of the shear blade which cooperate with the cutter blade can be varied.", "2.A dry shaver unit according to claim 1, wherein different spacings between the skin-engaging surface of the shaver unit and the regions of a shear blade which cooperate with a cutter blade are selectively available.", "3.A dry shaver according to claim 2, wherein the shear blade is an elongate flexible foil, the thickness of which is different at different regions along the elongate extent of the elongate flexible foil; and wherein the elongate flexible foil is mounted for displacement in its elongate direction so that a foil region of a particular thickness may be selectively aligned with the cutter blades whereby a spacing between a skin engaging surface of the shear blade and the opposite face of the shear blade which cooperates with the cutter blade is selectively variable.", "4.A dry shaver according to claim 1, wherein said at least one cutter blade is a rotary cutter blade, wherein the cooperating shear blade is located over the rotary cutter blade, and wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade can be varied.", "5.A dry shaver according to claim 4, wherein the shaver is provided with a plurality of shear blades and the spacing between the skin engaging surface of one of the shear blades and the opposite face thereof for cooperation with an associated cutter blade is different from the spacing between the corresponding surface and opposite face of at least one other of the plurality of shear blades.", "6.A dry shaver according to claim 5, comprising at least two rotary cutter blades, wherein each rotary cutter blade has a general axis of symmetry and the axis of symmetry of at least one rotary cutter blade is oriented differently from the axis of symmetry of at least one other rotary cutter blade with respect to a generally longitudinal axis of the dry shaver as whole, so that only one of the rotary cutter blades can be used for shaving purposes at one time.", "7.A dry shaver according to claim 5 further comprising: at least two cutting head assemblies, each head assembly having at least one cutter blade and a respective overlying cooperating shear blade, wherein a spacing between a skin engaging surface and an opposite face of the shear blade in at least one of the cutting head assemblies is different from a corresponding spacing in other head assemblies, and the head assemblies are interchangeably mountable on a body portion of the dry shaver.", "8.A dry shaver according to claim 4 further comprising a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.", "9.", "(canceled) 10.A dry shaver according to claim 1, wherein the cutter blade comprises at least one rotary cutter blade, and wherein the cooperating shear blade is located over the rotary cutter blade and comprises a shear blade and cutter blade assembly, and further comprising a cover member overlying the shear blade, and a displacement mechanism for effecting controlled movement of the shear blade and cutter blade assembly towards and away from the cover member, so that the spacing between a skin engaging surface of the cover member and the face of the shear blade which cooperates with the cutter blade is variable.", "11.A dry shaver according to claim 2, wherein said at least one cutter blade is a rotary cutter blade, wherein the cooperating shear blade is located over the rotary cutter blade, and wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade can be varied.", "12.A dry shaver according to claim 6 further comprising: at least two cutting head assemblies, each head assembly having at least one cutter blade and a respective overlying cooperating shear blade, wherein a spacing between a skin engaging surface and an opposite face of the shear blade in at least one of the cutting head assemblies is different from a corresponding spacing in other head assemblies, and the head assemblies are interchangeably mountable on a body portion of the dry shaver.", "13.A dry shaver according to claim 5 further comprising a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.", "14.A dry shaver according to claim 6 further comprising a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention This invention relates to dry shavers having a cutter blade and a cooperating shear blade arrangement.", "The invention relates at least both to electric dry shavers of the type having a reciprocating cutter and a shear blade arrangement in the form of a perforated foil with a plurality of perforations, and also to dry shavers of the type having a rotary cutter member or blade underlying an external shear member provided with apertures for entry of the hairs to be cut.", "The history of shaving to date has been the search for the ever closer shave.", "In effectuating a close shave, certain skin and hair types encounter the problem of an inflammation due to re-entry into the skin of too short cut (tangentially exiting) hairs.", "It is an aim of the present invention to prevent or protect against this by purposefully cutting the hair long at a sufficient distance from the skin surface so as to prevent any possible re-entry into the skin of the close cut hairs.", "The invention is thus directed at cutting the hairs long enough away from the skin surface to prevent their re-entry.", "In shaving close, certain skin and hair types thus experience or develop inflammation due to re-entry into the skin of the closely shaved hairs.", "It is therefore a purpose of the present invention both to allow for a normal close shave and to allow also for a cut of hair at some distance from the skin surface, the distance from the skin surface being sufficient so as to prevent re-entry of the cut hairs into the skin.", "2.Description of the Prior Art GB-A-1,569,096 describes a dry shaver having a flexible perforated shear foil supported in a frame and a reciprocatable cutter which cooperates with the shear foil.", "The shear foil is supported in an arched position between two side members of the frame and a cutter blade or shear head is pressed against the interior side of the shear foil by means of a spring.", "Similar such units are described in U.S. Pat.", "No.", "3,729,821 and in a diversity of other patent documents.", "The cutter blade may be mounted for reciprocation in a direction substantially parallel to the axis of curvature of the shear blade foil, or alternatively, the cutter blade may be driven in oscillatory motion about this axis.", "In a particular example of the shear foil type dry shaver, there is described in U.S. Pat.", "No.", "3,694,916 an arrangement in which a reciprocatable cutter cooperates with a shearing blade having at least two shearing fields of different perforations.", "The shearing blade or foil is movable to bring a selected shearing field into the cooperating shearing disposition with the cutter blade.", "The arrangement enables a field of perforations which is most suitable for a particular type of skin to be selected but makes no reference to cutting hair at different lengths.", "The selected field of perforations is changed, but not the depth or height of cut.", "A diversity of rotary cutter shavers are also known, and reference may be directed inter alia to U.S. Pat.", "No.", "5,007,168, EP-A1-0,241,082, and EP-A1-0,484,795, all of which embody the rotary principle already indicated above.", "In WO-A-97/45235, there is described a rotary shaving apparatus in which radially directed hair entry apertures are of either broad or narrow dimension in the circumferential direction.", "The thickness of the shear blade in which the hair entry apertures are defined varies, depending on the dimension of the hair entry apertures.", "The shear blade is of greater thickness in a region thereof containing broad hair entry apertures than it is in a region containing narrow hair entry apertures.", "Thus the blade has a wavy or undulating configuration in the circumferential direction, with the depth or thickness of the blade increasing or reducing as between broad hair aperture entry regions and narrow hair entry aperture regions.", "The purpose of the arrangement is said to be to enable both short and long hairs to be caught and severed effectively with the end result of a close shave, while avoiding, as far as possible, irritation of the skin." ], [ "<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>It is a general object of the invention to provide a dry shaver unit directed to overcoming the inflammation problems discussed hereinabove.", "According to the invention in a first and most general aspect, there is provided a dry shaver unit comprising at least one cutter blade and a cooperating shear blade arrangement in which provision is made for the spacing between a skin-engaging surface of the shaver unit and regions of the shear blade arrangement which cooperate with the cutter blade to be increased as compared with the spacing required to achieve a “close shave”.", "It is a particular object of the present invention to provide a dry shaver in which the depth of cut is variable, so that hair to be cut may be left at a variable length over certain areas of the skin as required, in order inter alia to avoid the development of a shaving rash due to re-entry into the skin of too-short tangentially cut hairs exiting the skin at an acute angle.", "According to the invention in a first specific aspect, there is therefore provided a dry shaver unit wherein different spacings between a skin-engaging surface of the shaver unit and regions of a shear blade arrangement which cooperate with a cutter blade are selectively available.", "In a particular embodiment of dry shaver according to the invention, a single cutter blade cooperates with a single shear blade and the spacing between a skin engaging surface of the shear blade and the opposite face of the shear blade which cooperates with the cutter blade (i.e.", "the cutter blade surface or side of the shear blade) is selectively variable.", "In a favoured construction, the shear blade is an elongate flexible foil, the thickness of which is different at different regions along the elongate extent of the foil, and the foil is mounted for displacement in its elongate direction so that a foil region of a particular thickness may be selectively aligned with the cutter blade.", "The foil may vary in thickness in a substantially continuous manner over its elongate extent from a minimum thickness portion to a maximum thickness portion, or alternatively the foil may comprise a sequence of foil portions, each of which has a different thickness, the foil portions being flexibly interconnected with one another.", "A control member is suitably attached to the foil, the control member being displaceable in selective manner in the elongate direction of the foil.", "In a preferred arrangement, the control member comprises a control stud extending through a slot defined in a wall portion of the shaver, an external region of the control stud being manually engageable to effect displacement of the foil in its elongate direction.", "The foil may be retained in a particular selected position such as by frictional interaction between control stud and slot.", "In an alternative arrangement, opposite elongate ends of the foil may be engaged at opposite ends of a rocking mechanism, displacement of the foil in its elongate direction being effected by rocking displacement of the rocking mechanism.", "In a further aspect of the invention, there is provided a dry shaver in which said at least one cutter blade is a rotary cutter blade, the or each cutter blade having an overlying cooperating shear blade, wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.", "In a particular rotary construction of dry shaver according to the invention, the shaver is provided with a plurality of shear blades and the spacing between the skin engaging surface of one of the shear blades and the opposite face thereof for cooperation with an associated cutter blade (i.e.", "the cutter blade surface or side of the shear blade) is different from the spacing between the corresponding surface and opposite face of at least one other of the plurality of shear blades.", "Thus, for example, one of the shear blades may provide for a close shave and another shear blade for a less close shave For a shaver having a head with two or three blades used simultaneously, the invention may provide two or more sets of blades for use on the head, one of the sets of blades providing for a close shave and another of the sets of blades enabling a less close shave.", "It will be appreciated that more than two such sets may be provided, i, a third set for example facilitating a shave intermediate between a close shave and a long shave.", "In a variant of this aspect of the invention, a dry shaver may comprise at least two rotary cutter blades, wherein each rotary cutter blade has a general axis of symmetry and the axis of symmetry of at least one rotary cutter blade is oriented differently from the axis of symmetry of at least one other rotary cutter blade with respect to a generally longitudinal axis of the dry shaver as whole, so that only one of at said least one rotary cutter blade and said at least one other rotary cutter blade can be used for shaving purposes at one time.", "Thus in this variant of the invention, the shaver is provided with two or more shaving head regions, each of which faces away from the general longitudinal axis of the unit in a different direction, so that only one of the head regions can be used at a time.", "Each head region may have two or more blades and as previously indicated, the shaver unit may be provided with multiple sets of blades so that one head region may be set up for a close shave and another head region set up for a less close shave.", "Because this embodiment of the invention has two or more head regions facing in different directions, the unit may be set up to enable the user to choose a close shave or a less close shave simply by applying the appropriate head region to the skin.", "In yet another variant of this aspect of the invention, the shaver unit may have three head regions, each of which faces in a different direction away from the general longitudinal axis of the unit, the three head regions or head assemblies being spaced apart by substantially one hundred and twenty degrees about this longitudinal axis.", "In any variant of dry shaver according to the rotary aspect of the invention, the shaver unit may be provided in combination with at least two cutting head assemblies, each head assembly having at least one cutter blade and the or each cutter blade having a respective overlying cooperating shear blade, wherein the or each shear blade of one of the head assemblies has a spacing between its skin engaging surface and its opposite face which is the same for the or all of the shear blade(s) of said one of the head assemblies but is different from the corresponding spacing for the or all of the shear blade(s) of the other of the head assemblies, and the head assemblies are interchangeably mountable on a body portion of the dry shaver.", "In addition to being provided with two or more interchangeable head assemblies, multiple sets of blades providing for different depths of cut may also be included in the shaver kit.", "Thus one head may be set up with blades suitable for a close shave, and another of the head assemblies set up for the less close shave.", "The user wishing to change from one class of shave to the other simply exchanges the heads appropriately.", "Thus the invention extends to a shaver kit including a shaver body unit and two or more head assemblies interchangeably associatable with the body unit which contains the drive features of the shaver, and the kit may optionally include multiple blade assemblies for individual assocation as required with any of the head assemblies.", "Extensive flexibility of use is thus available, either by interchanging a head assembly or set of blades as required, or, for the arrangement provided with multiple head regions or head assemblies disposed in different angular orientations, merely by applying the appropriate head region to the skin, with optional advance mounting of blades of an appropriate depth of cut on the selected head region before use, as may be required.", "The dry shaver of the invention, in any of the rotary embodiments, may be provided in combination with a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.", "Thus, as already indicated, in a dry shaver kit according to this aspect of the invention and comprising any of the foregoing features of the invention, the kit may include multiple sets of blade assemblies, interchangeably mountable on the dry shaver, so that the user may select an appropriate depth of cut by mounting a blade or blades suitable for that depth of cut on a head assembly of the dry shaver unit or kit, as required.", "According to yet another aspect of the invention in its rotary configuration, there is provided a dry shaver comprising at least one rotary cutter blade, wherein the spacing between a skin-engaging surface of the dry shaver and the cutter blade is selectively variable and a value may be selected for said spacing which is increased as compared with the spacing required to achieve a “close shave” Thus in this variant of the invention, depth of cut is available in an infinitely variable manner similar to that afforded by the foil embodiment of the invention, but without necessitating either individual blade assemblies to be exchanged or the head assembly as a whole to be swapped for an alternative head assembly.", "In this aspect, the invention thus offers the immediately selectively variable depth of cut of the foil embodiment combined with features of the rotary construction.", "In a particular construction of the invention in this additional aspect, the shaver comprises at least one rotary cutter blade, the or each of which has an overlying cooperating shear blade and a cover member overlying the shear blade, the spacing between a skin engaging surface of the cover member and the face of the shear blade which cooperates with the cutter blade being variable.", "In this arrangement, the shaver may include a displacement mechanism for effecting controlled movement of a shear blade and cutter blade assembly towards and away from the cover member.", "Thus in these foregoing embodiments, the advantages of immediate or direct, controlled substantially infinitely variable adjustment of the depth of cut within predetermined limits is achieved for a rotary shaving unit also.", "The invention also extends to a blade assembly for a dry shaver comprising a rotary cutter blade and a cooperating shear blade, wherein the spacing between a skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.", "The invention likewise additionally encompasses a head assembly for a dry shaver, comprising at least one rotary cutter blade, the or each cutter blade having an overlying cooperating shear blade, wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.", "Thus the invention embraces in addition blade assemblies and head assemblies incorporating the features of the invention as independent units, whether in combination with a dry shaver or as entirely separate assemblies.", "The invention likewise extends to any kit of parts comprising a dry shaver drive unit, one or more head assemblies, and/or one or more blade assemblies, provided in combination or in any commercial association, in so far as such units incorporate the features of the invention or together cooperate in defining equipment lying within the scope of the invention.", "The invention also extends to a dry shaver substantially as described herein with reference to and as shown in any one or more of the accompanying drawings." ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention This invention relates to dry shavers having a cutter blade and a cooperating shear blade arrangement.", "The invention relates at least both to electric dry shavers of the type having a reciprocating cutter and a shear blade arrangement in the form of a perforated foil with a plurality of perforations, and also to dry shavers of the type having a rotary cutter member or blade underlying an external shear member provided with apertures for entry of the hairs to be cut.", "The history of shaving to date has been the search for the ever closer shave.", "In effectuating a close shave, certain skin and hair types encounter the problem of an inflammation due to re-entry into the skin of too short cut (tangentially exiting) hairs.", "It is an aim of the present invention to prevent or protect against this by purposefully cutting the hair long at a sufficient distance from the skin surface so as to prevent any possible re-entry into the skin of the close cut hairs.", "The invention is thus directed at cutting the hairs long enough away from the skin surface to prevent their re-entry.", "In shaving close, certain skin and hair types thus experience or develop inflammation due to re-entry into the skin of the closely shaved hairs.", "It is therefore a purpose of the present invention both to allow for a normal close shave and to allow also for a cut of hair at some distance from the skin surface, the distance from the skin surface being sufficient so as to prevent re-entry of the cut hairs into the skin.", "2.Description of the Prior Art GB-A-1,569,096 describes a dry shaver having a flexible perforated shear foil supported in a frame and a reciprocatable cutter which cooperates with the shear foil.", "The shear foil is supported in an arched position between two side members of the frame and a cutter blade or shear head is pressed against the interior side of the shear foil by means of a spring.", "Similar such units are described in U.S. Pat.", "No.", "3,729,821 and in a diversity of other patent documents.", "The cutter blade may be mounted for reciprocation in a direction substantially parallel to the axis of curvature of the shear blade foil, or alternatively, the cutter blade may be driven in oscillatory motion about this axis.", "In a particular example of the shear foil type dry shaver, there is described in U.S. Pat.", "No.", "3,694,916 an arrangement in which a reciprocatable cutter cooperates with a shearing blade having at least two shearing fields of different perforations.", "The shearing blade or foil is movable to bring a selected shearing field into the cooperating shearing disposition with the cutter blade.", "The arrangement enables a field of perforations which is most suitable for a particular type of skin to be selected but makes no reference to cutting hair at different lengths.", "The selected field of perforations is changed, but not the depth or height of cut.", "A diversity of rotary cutter shavers are also known, and reference may be directed inter alia to U.S. Pat.", "No.", "5,007,168, EP-A1-0,241,082, and EP-A1-0,484,795, all of which embody the rotary principle already indicated above.", "In WO-A-97/45235, there is described a rotary shaving apparatus in which radially directed hair entry apertures are of either broad or narrow dimension in the circumferential direction.", "The thickness of the shear blade in which the hair entry apertures are defined varies, depending on the dimension of the hair entry apertures.", "The shear blade is of greater thickness in a region thereof containing broad hair entry apertures than it is in a region containing narrow hair entry apertures.", "Thus the blade has a wavy or undulating configuration in the circumferential direction, with the depth or thickness of the blade increasing or reducing as between broad hair aperture entry regions and narrow hair entry aperture regions.", "The purpose of the arrangement is said to be to enable both short and long hairs to be caught and severed effectively with the end result of a close shave, while avoiding, as far as possible, irritation of the skin.", "BRIEF SUMMARY OF THE INVENTION It is a general object of the invention to provide a dry shaver unit directed to overcoming the inflammation problems discussed hereinabove.", "According to the invention in a first and most general aspect, there is provided a dry shaver unit comprising at least one cutter blade and a cooperating shear blade arrangement in which provision is made for the spacing between a skin-engaging surface of the shaver unit and regions of the shear blade arrangement which cooperate with the cutter blade to be increased as compared with the spacing required to achieve a “close shave”.", "It is a particular object of the present invention to provide a dry shaver in which the depth of cut is variable, so that hair to be cut may be left at a variable length over certain areas of the skin as required, in order inter alia to avoid the development of a shaving rash due to re-entry into the skin of too-short tangentially cut hairs exiting the skin at an acute angle.", "According to the invention in a first specific aspect, there is therefore provided a dry shaver unit wherein different spacings between a skin-engaging surface of the shaver unit and regions of a shear blade arrangement which cooperate with a cutter blade are selectively available.", "In a particular embodiment of dry shaver according to the invention, a single cutter blade cooperates with a single shear blade and the spacing between a skin engaging surface of the shear blade and the opposite face of the shear blade which cooperates with the cutter blade (i.e.", "the cutter blade surface or side of the shear blade) is selectively variable.", "In a favoured construction, the shear blade is an elongate flexible foil, the thickness of which is different at different regions along the elongate extent of the foil, and the foil is mounted for displacement in its elongate direction so that a foil region of a particular thickness may be selectively aligned with the cutter blade.", "The foil may vary in thickness in a substantially continuous manner over its elongate extent from a minimum thickness portion to a maximum thickness portion, or alternatively the foil may comprise a sequence of foil portions, each of which has a different thickness, the foil portions being flexibly interconnected with one another.", "A control member is suitably attached to the foil, the control member being displaceable in selective manner in the elongate direction of the foil.", "In a preferred arrangement, the control member comprises a control stud extending through a slot defined in a wall portion of the shaver, an external region of the control stud being manually engageable to effect displacement of the foil in its elongate direction.", "The foil may be retained in a particular selected position such as by frictional interaction between control stud and slot.", "In an alternative arrangement, opposite elongate ends of the foil may be engaged at opposite ends of a rocking mechanism, displacement of the foil in its elongate direction being effected by rocking displacement of the rocking mechanism.", "In a further aspect of the invention, there is provided a dry shaver in which said at least one cutter blade is a rotary cutter blade, the or each cutter blade having an overlying cooperating shear blade, wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.", "In a particular rotary construction of dry shaver according to the invention, the shaver is provided with a plurality of shear blades and the spacing between the skin engaging surface of one of the shear blades and the opposite face thereof for cooperation with an associated cutter blade (i.e.", "the cutter blade surface or side of the shear blade) is different from the spacing between the corresponding surface and opposite face of at least one other of the plurality of shear blades.", "Thus, for example, one of the shear blades may provide for a close shave and another shear blade for a less close shave For a shaver having a head with two or three blades used simultaneously, the invention may provide two or more sets of blades for use on the head, one of the sets of blades providing for a close shave and another of the sets of blades enabling a less close shave.", "It will be appreciated that more than two such sets may be provided, i, a third set for example facilitating a shave intermediate between a close shave and a long shave.", "In a variant of this aspect of the invention, a dry shaver may comprise at least two rotary cutter blades, wherein each rotary cutter blade has a general axis of symmetry and the axis of symmetry of at least one rotary cutter blade is oriented differently from the axis of symmetry of at least one other rotary cutter blade with respect to a generally longitudinal axis of the dry shaver as whole, so that only one of at said least one rotary cutter blade and said at least one other rotary cutter blade can be used for shaving purposes at one time.", "Thus in this variant of the invention, the shaver is provided with two or more shaving head regions, each of which faces away from the general longitudinal axis of the unit in a different direction, so that only one of the head regions can be used at a time.", "Each head region may have two or more blades and as previously indicated, the shaver unit may be provided with multiple sets of blades so that one head region may be set up for a close shave and another head region set up for a less close shave.", "Because this embodiment of the invention has two or more head regions facing in different directions, the unit may be set up to enable the user to choose a close shave or a less close shave simply by applying the appropriate head region to the skin.", "In yet another variant of this aspect of the invention, the shaver unit may have three head regions, each of which faces in a different direction away from the general longitudinal axis of the unit, the three head regions or head assemblies being spaced apart by substantially one hundred and twenty degrees about this longitudinal axis.", "In any variant of dry shaver according to the rotary aspect of the invention, the shaver unit may be provided in combination with at least two cutting head assemblies, each head assembly having at least one cutter blade and the or each cutter blade having a respective overlying cooperating shear blade, wherein the or each shear blade of one of the head assemblies has a spacing between its skin engaging surface and its opposite face which is the same for the or all of the shear blade(s) of said one of the head assemblies but is different from the corresponding spacing for the or all of the shear blade(s) of the other of the head assemblies, and the head assemblies are interchangeably mountable on a body portion of the dry shaver.", "In addition to being provided with two or more interchangeable head assemblies, multiple sets of blades providing for different depths of cut may also be included in the shaver kit.", "Thus one head may be set up with blades suitable for a close shave, and another of the head assemblies set up for the less close shave.", "The user wishing to change from one class of shave to the other simply exchanges the heads appropriately.", "Thus the invention extends to a shaver kit including a shaver body unit and two or more head assemblies interchangeably associatable with the body unit which contains the drive features of the shaver, and the kit may optionally include multiple blade assemblies for individual assocation as required with any of the head assemblies.", "Extensive flexibility of use is thus available, either by interchanging a head assembly or set of blades as required, or, for the arrangement provided with multiple head regions or head assemblies disposed in different angular orientations, merely by applying the appropriate head region to the skin, with optional advance mounting of blades of an appropriate depth of cut on the selected head region before use, as may be required.", "The dry shaver of the invention, in any of the rotary embodiments, may be provided in combination with a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.", "Thus, as already indicated, in a dry shaver kit according to this aspect of the invention and comprising any of the foregoing features of the invention, the kit may include multiple sets of blade assemblies, interchangeably mountable on the dry shaver, so that the user may select an appropriate depth of cut by mounting a blade or blades suitable for that depth of cut on a head assembly of the dry shaver unit or kit, as required.", "According to yet another aspect of the invention in its rotary configuration, there is provided a dry shaver comprising at least one rotary cutter blade, wherein the spacing between a skin-engaging surface of the dry shaver and the cutter blade is selectively variable and a value may be selected for said spacing which is increased as compared with the spacing required to achieve a “close shave” Thus in this variant of the invention, depth of cut is available in an infinitely variable manner similar to that afforded by the foil embodiment of the invention, but without necessitating either individual blade assemblies to be exchanged or the head assembly as a whole to be swapped for an alternative head assembly.", "In this aspect, the invention thus offers the immediately selectively variable depth of cut of the foil embodiment combined with features of the rotary construction.", "In a particular construction of the invention in this additional aspect, the shaver comprises at least one rotary cutter blade, the or each of which has an overlying cooperating shear blade and a cover member overlying the shear blade, the spacing between a skin engaging surface of the cover member and the face of the shear blade which cooperates with the cutter blade being variable.", "In this arrangement, the shaver may include a displacement mechanism for effecting controlled movement of a shear blade and cutter blade assembly towards and away from the cover member.", "Thus in these foregoing embodiments, the advantages of immediate or direct, controlled substantially infinitely variable adjustment of the depth of cut within predetermined limits is achieved for a rotary shaving unit also.", "The invention also extends to a blade assembly for a dry shaver comprising a rotary cutter blade and a cooperating shear blade, wherein the spacing between a skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.", "The invention likewise additionally encompasses a head assembly for a dry shaver, comprising at least one rotary cutter blade, the or each cutter blade having an overlying cooperating shear blade, wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.", "Thus the invention embraces in addition blade assemblies and head assemblies incorporating the features of the invention as independent units, whether in combination with a dry shaver or as entirely separate assemblies.", "The invention likewise extends to any kit of parts comprising a dry shaver drive unit, one or more head assemblies, and/or one or more blade assemblies, provided in combination or in any commercial association, in so far as such units incorporate the features of the invention or together cooperate in defining equipment lying within the scope of the invention.", "The invention also extends to a dry shaver substantially as described herein with reference to and as shown in any one or more of the accompanying drawings.", "BRIEF DESCRIPTION OF THE DRAWINGS A number of embodiments of the invention will now be described having regard to the accompanying drawings in which: FIG.", "1 is a diagrammatic representation of a portion of human skin, in the region of the surface of the skin, in section, showing inner and outer skin layers and hair growth, and representing also a particular condition in which an individual's body hair is subject to growth in a manner more horizontal than vertical, FIG.", "2 is a side sectional view of an adjustable foil blade in a dry shaver in accordance with the present invention, FIG.", "3 is a pictorial representation of an embodiment of foil blade suitable for a dry shaver according to the invention, FIG.", "4 is a side sectional representation of an alternative manner of adjustable foil blade mounting for a dry shaver in accordance with the present invention, FIG.", "5 is a diagrammatic view in section through a shear blade of the kind illustrated in FIGS.", "2 to 4, showing a known variant in which a sharp cutting edge is defined by a downturn around the periphery of the hair entry openings in the foil, FIG.", "6 is a pictorial view of a rotary blade razor of known construction, to which the present invention may be applied in an alternative embodiment, FIG.", "7 is a simplified pictorial view of an alternative rotary blade razor, having two blades as compared with the three-bladed configuration of FIG.", "6, to which again the present invention may be applied in an alternative embodiment, FIG.", "8 is a side sectional view of a shear blade of conventional construction for use in the shaver of FIG.", "6 or that of FIG.", "7, FIG.", "9 is a side sectional view of a modified shear blade for rotary embodiment of dry shaver in accordance with the present invention, FIG.", "10 is a pictorial representation from beneath of a shaver head construction providing for adjustability of the spacing between the cutting blades and the skin surface in a twin-bladed embodiment of dry shaver of a kind similar to that shown in FIG.", "6, and FIG.", "11 is a longitudinal sectional view on the longitudinal centre line of the arrangement of FIG.", "10.DETAILED DESCRIPTION OF THE DRAWINGS FIG.", "1 is a representation in sectional view of a portion of the surface of the skin of an individual.", "As shown in FIG.", "1, the outer layer 1 of the skin is underlaid by an inner layer 2.Hairs 3 and 3a grow outwardly through the skin from respective root regions 4 and 4a and emerge through the outer layer 1 of the skin.", "In the normal way, as indicated by hair 3, a hair should emerge from the skin outer layer I and extend outwardly above the skin surface substantially at right angles to the skin surface.", "Depending on the region of the skin surface in question, certain hairs such as that designated by reference 3a in FIG.", "1 do not grow upwardly substantially perpendicularly or vertically with respect to the skin surface but run tangentially close to but underneath the skin surface.", "Eventually, such a hair emerges from the skin surface, as indicated at reference 3b, but in a highly tangential manner.", "If an individual suffering from such tangential hair growth wishes to effect a close shave and cuts the hair relatively close to the skin outer surface, a condition develops in which the sharp edge of the cut hair burrows back beneath the surface of the skin, as indicated at reference 3c, rather than emerging from the skin and growing externally in the normal manner.", "Such ingrowth occurs if the sharp, cut edge of the hair becomes engaged or caught beneath the surface of the outer skin layer 1.If growth then continues with the hair 3 running into and under the skin, the inevitable result is some kind of inflammation.", "It is therefore preferable to prevent the development of any such inflammation and the present invention addresses this problem.", "The concept underlying the avoidance of such skin conditions is to cut the hair slightly above the surface of the skin in areas of the body surface where the possibility of such ingrowing conditions arising is likely to be greatest.", "The advantages of its being possible to cut hair in accordance with the principles of the present invention are not however limited only to skin conditions such as that illustrated with reference to FIG.", "1, but the invention is applicable to all skin circumstances where shaving bumps or other skin eruptions or imperfections militate against an excessively close shave.", "The invention provides the user of a dry shaver with the ability to selectively cut body hair either tightly or to a greater length, depending on the region of the skin in question, i.e.", "the area currently being shaved.", "An embodiment of the invention applicable to a foil type dry shaver is shown in FIG.", "2.A removable shaver head portion 11 is mounted on a shaver body portion 12.The head 11 is seated on the body portion 12 to enclose the upper or drive portion 13 of the shaver body portion 12 in conventional manner.", "A generally conventional drive arrangement is applied such as by drive member 14 to an outwardly curved or convex cutter blade 15 to provide either reciprocating movement in the axial direction of the curved cutter blade 15, or alternatively oscillatory or circumferential motion about this axis.", "A spring arrangement such as a helical spring 16 urges the curved displaceable cutter blade 15 against the inner surface of a cooperating perforated foil blade 17, which serves as a substantially stationary shear member for the movable blade cutter 15 under shaving conditions, i.e.", "foil blade 17 remains substantially static with respect to cutter blade 15 during an actual shaving operation.", "The perforated foil blade 17 is received in a bent-over configuration within an open top region of the shaver head 11.The foil blade 17 is received and accommodated in curved guide grooves extending along opposite inner walls of the shaver head 11.One such guide groove is indicated by reference 27.It is a particular feature of the invention that this perforated foil which serves as the shearing blade 17 has a thickness which varies along the length of the foil, as shown in FIGS.", "2 and 3.It is also possible for the size, i.e.", "the width or breadth of the hair-entry apertures, also their configuration, to vary with the thickness of the foil, e.g.", "the apertures being narrow where the foil is thin and wider where the foil is thick.", "Wide and narrow apertures may also be intermingled.", "Referring now also to FIG.", "3 in particular, there is shown a favoured embodiment of the perforated foil blade or shear cutter 17, formed as an integral unit, in which the perforated central region of the blade 17, designated by cross-hatching, is surrounded by side 18 and end 19 members or blade regions, which are unperforated and define therefore lateral members and end or cross-members of the blade structure.", "The blade 17 has a thinner region 21 towards one axial end in its generally longitudinal direction and a thicker region 22 towards its other axial end.", "In the region of the thicker end 22, an aperture 23 is provided through which a fastener 24, FIG.", "2, enables the blade 17 according to the invention to be coupled to an operating or control button 25, which is displaceable on the exterior of the shaver head 11 in the longitudinal direction of the blade 17 and is guided by a guide slot 26 provided in the housing of the shaver head 11.In an alternative arrangement (not illustrated), the flexible foil blade may be supported in a separate frame structure having end and side members.", "The end edges of the foil blade are fastened to the end bars of the frame structure and guiding grooves provided in opposite inner walls of the shearing or shaver head housing again serve to guide the lateral or side members of the frame, which are flexible, in similar manner to the flexible side edges of the integral blade structure of FIG.", "3.The perforated foil blade again varies in thickness along its longitudinal extent from one end edge to the other.", "In either arrangement, integral or frame-mounted, a foil portion of the desired thickness is selectively brought into a cutting disposition with respect to the cutter blade 15 by controlled sliding movement of the foil blade 17 in its longitudinal direction This is facilitated by the external button arrangement 25 which runs in the guide slot 26 along the surface of the shaver unit head 11.In this manner, a required depth of cut may be readily chosen and speedily varied in controlled manner as required by the user.", "Retention of a particular selected position of the foil 17 may be achieved by cooperating frictional interaction between button 25 and slot 26, e.g.", "by button 25 and connection or fastener portion 24 defining a “nip” or light gripping engagement between inner and outer face regions of head 11 bordering slot 26.In order to provide guidance for the side edges 1a of the variable thickness shear blade 17, the guide grooves 27 in the shaver head have a dimension between the opposite side surfaces of the guiding groove 27 which may vary but is always appropriate for the thickness dimension of the blade 17 in the relevant region.", "In order to ensure that the thinner regions of the foil 17 are appropriately guided, the guide grooves 27 then may be provided, if necessary, with appropriate resilient means, such as leaf springs or the like, to urge the foil 17 towards an appropriate side face of the groove 27, and thereby take up as it were any possible intervening redundant space between a thinner portion of the foil 17 and the opposite side wall of the guiding groove 27.Alternatively, in either embodiment of the foil, the side edges 18 or the corresponding side frame members may be arranged to have a uniform thickness throughout the longitudinal extent of the shear blade or foil 17, so that the provision of resilient means in the guide grooves is not then required.", "The installed foil blade 17 is stretched as it were over the curved surface of cutter blade 15 at the cutting end of the shaver head unit 11.The foil blade 17 is tensioned in the outward direction by the spring-loading (16), underlying cutter blade 15, which also serves for holding it in place.", "In the embodiment as shown in FIGS.", "2 and 3 of a foil 17 suitable for use with the invention, variation in thickness is substantially continuous from one end of the foil 17 to the other, from a minimum thickness portion 21 at a first end, to a maximum thickness region 22 at the other end.", "In this way, a substantially infinite variation of thickness can be selected between the two extremes.", "The side edges of the foil 18 may however be of constant or uniform thickness over the entire longitudinal extent of the blade 17, depending on the properties of the material selected for the foil 17, as already discussed above, or the side frame members, in a framed embodiment, may be likewise of uniform thickness.", "In an alternative embodiment (not illustrated), the foil may have a number of sections, each of which is of a different thickness and configuration, and each section being connected to an adjacent section by for example a hinge portion or by any other suitable manner of interconnection.", "The sections are therefore joined for flexure, i.e.", "to be flexible when the continuous foil structure is formed from the individual sections, and define a flexible continuous foil.", "The sections may for example be joined by being bonded to one another in some suitable manner.", "In such an embodiment, the foil has therefore a number of discrete portions or sections, each of which has its own individual thickness, so that a corresponding depth of cut applies.", "Again, an appropriate depth of cut is readily selected in controlled manner by sliding movement of the foil to the requisite location.", "Variable aperture size and configuration and/or intermingled sizes and configurations of aperture in different regions of the foil may also be provided in this constructional variant.", "In an alternative mounting arrangement shown in FIG.", "4, the structure and arrangement of the shaver head housing 11′ and shaver body 12′, as well as the cutter 15′ structure, correspond to the arrangements of FIG.", "2, features similar to those of FIG.", "2 being designated by use of the same reference numerals with the addition of an apostrophe.", "However, in order to secure longitudinal displacement of the perforated foil blade 17′, the sliding button-controlled arrangement of FIG.", "2 is replaced by a pivotal structure, in which end edges of the foil blade 17′ are connected 34, 35 to respective ends of a pivotally mounted rocker mechanism 36 arranged 37 for rocking movement within the shaver head housing 11′.", "Any suitable means may be provided for connecting the foil blade 17′ to cross-bar portions of the rocker lever structure 36, such as fasteners 34, 35.Likewise, any suitable arrangement may be provided to secure controlled pivoting 37 of the rocking blade mounting frame or structure 36, for example a lever arm or knob at the end of a pivot axis mounting 37, or alternatively, displacement may be effected merely by sliding the perforated foil blade 17′ over the surface of the cutter blade 15′, using the fingers.", "In either arrangement, the rocker member 36 is suitably pivotally mounted by way of pins or a shaft 37, bearingly accommodated on the interior of the side walls of the head housing 11′.", "Retention of the foil blade 17′ in a particular longitudinal disposition with respect to the blade 15′ may again be achieved such as by suitable frictional cooperation or interaction between pivot mounting 37 and the bearing features by which pivot axis or shaft 37 is accommodated in the walls of the head housing 11′, or by any other suitable equivalent arrangement, such as for example a locking screw or the like associated with a pivot position adjusting knob.", "A multiplicity of possible arrangements will be apparent for achieving substantial locking of the foil 17′ in a particular selected shaving disposition by securing the rocker arm or lever 36 in a particular pivoted disposition following selective controlled displacement of the foil to align a designated region of the foil with the cutter blade.", "In any variant of the variable thickness foil embodiment of the invention, the regions of different foil thickness may be differentiated from one another such as by for example colour coding.", "FIG.", "5 shows detail of a feature by which an especially effective cutting or shearing action may be achieved for a foil shear blade.", "In the arrangement shown, in the formation of the hair entry openings 28 in the foil structure shear blade 17, the periphery of each entry opening 28 is turned slightly downwards, so that the peripheral region 29 surrounding the opening 28 protrudes below the general level of the remainder of the underside of the foil shear blade 17 to define, as it were, a lip.", "An especially effective shearing type action is then achieved when the cutter blade 15 interacts with this peripheral region 29 of the foil shear blade 17 surrounding the hair entry opening 28.Referring now to FIG.", "6, an embodiment of the invention applicable to a rotary type dry shaver will now be described.", "In a typical construction, a rotary dry shaver 41 may have a number of cutter units 42, typically three as shown in the drawing, or alternatively two units 42, in each case located at the cutting end face 43 of the shaver body 44.The cutting drive and mounting arrangements of this variant of the invention are generally in accordance with substantially conventional arrangements well-known in the industry and only the particular deviations from such constructions required by the present invention are now described in detail.", "In such constructions, each cutter unit has an external or shear blade, which has hair entry apertures or slits and is underlaid, in the assembled condition, by a rotating cutter blade, which severs hairs extending into the cutting region of the cutter blade through the slits or perforations (apertures) of the shear blade.", "According to the invention, each of the rotary blade assemblies 42 is equipped with a shearing blade unit having a particular thickness or depth dimension of the hair entry slits or perforations, so as to provide for a corresponding depth of cut.", "Preferably, each blade assembly provides the same depth of cut.", "In a favoured arrangement according to the invention, two or more interchangeable head assemblies may be provided, each having blades providing a particular depth of cut, e.g.", "“close” shave or “long” cut, so that a user can choose a head assembly providing for a tight cut or a head assembly providing for a less close shave.", "Thus the invention provides a shaving kit incorporating a dry shaver body unit which includes drive features, one or more head assemblies which are interchangeable with the drive portion or body of the shaver and two or more sets of blades which can be interchangeably associated with any of the head assemblies.", "In this manner, a user may set up one head assembly with each blade providing for a close shave and a second head assembly on which each blade allows a more generous cut.", "For regions of the skin where the close shave is desired, the user applies the close shave blades by mounting the appropriate head assembly on the drive portion of the shaver unit.", "Where it is necessary or desirable to apply a less close shave, the user then exchanges the close shave head assembly for that enabling the less close shave.", "Referring now to FIG.", "7, there is shown, in pictorial view, a rotary shaver having two cutter heads, each of which is angled away from the longitudinal axis of the cutter unit, so that only one head can be used at any given time.", "Each cutter head is located on an angled end face region of the shaver, so that each cutter can be engaged individually against the skin, without engagement of the other cutter, located on the other angled face.", "In a variant, each angled face could carry two blades, in a two-bladed cutter head assembly.", "The shaver would then have two twin-blade head assemblies, one on each angled cutter face.", "In a shaver of this kind in accordance with the invention, all cutter blades and each head assembly are interchangeable with other blade units or head assemblies respectively.", "As already indicated, the shaver unit in this embodiment also may be provided as a kit, with for example two or more detachable and interchangeable head assemblies and two or more sets of blades providing for different depths of cut.", "The user sets up one of the head assemblies with blades suitable for a close shave and the other head assembly for a less close shave.", "The two head assemblies can be mounted on the base portion of the shaver unit for use in the manner already described, namely close shave where feasible and less close shave where necessary.", "Additional head assemblies may also be included in the kit of parts as also may additional sets of blades providing for still different depths of cut.", "In a further variant of the arrangement provided in FIG.", "7, the base unit may accommodate three heads, each angled outwardly from the generally longitudinal axis of the base portion which also defines the gripping or handle portion of the unit, so that the three head regions or head assembly mounting portions are spaced equidistantly around this axis substantially at 120° from each other.", "The unit may then be set up to provide for three different depths of cut and the user applies the appropriate depth of cut by rotating the unit so that the selected head region can be applied to the skin.", "In this manner, particular ease of use is facilitated with maximum interchangeability and flexibility as regards depths of cut, by the diverse options available for interchange or exchange of head assemblies and blade assemblies.", "Referring now to FIG.", "8, which shows an external cutting member or shear blade 51 of generally conventional configuration, a head 42 equipped with this type of shear blade 51 may be regarded as providing a minimum depth of cut, for a tight shave.", "As shown in this drawing, the cutting member 51, which is in the general shape of a top-hat, or an inverted cup, has a series of generally radially-oriented, optionally skewed, hair entry slits or apertures 52, in an outwardly-directed generally disc-shaped region 55 of the blade 51, which is upwardly-oriented when mounted on the cutting unit 42.The outer surface of this disc-shaped region 55 of cutting member 51 comes into contact with the skin during a shaving operation.", "From the side of this disc-shaped region 55 opposite to the skin-engaging surface, at the periphery of the disc, a generally annular peripheral wall portion 56 extends (downwardly in the mounted condition on cutting unit 42) to terminate in an externally-directed holding flange 53, by which the shear blade is secured in position on the shaver cutting unit 42.Centrally of the generally disc-shaped region 55, radially inward of the slits 52, there is a provided a central dished portion 54, which is set back from the skin-engaging surface defined by the outer face of region 55 at the slots 52.In order to provide for a more generous shave, in other words a less tight or close shave leaving a longer beard length, at least one other cutter 61 is provided, in the form of a shear blade of greater thickness, as shown in FIG.", "9, so that the depth of cut is greater when that particular cutter is applied to the face.", "The general construction of cutter or shear blade 61 is similar to blade 51, so that blades 51 and 61 are interchangeable on the dry shaver of FIG.", "6 or that of FIG.", "7.In the shaver of FIG.", "6, preferably all of the blades provide for a close shave or all of the blades provide for a less close shave.", "However, in the two head shaver of FIG.", "7 or in a three head variant of this design, the blade or blades of one of the heads may provide for a close shave and the blades of the other or another of the heads provide for a less close shave, so that different regions of the face may be shaved according to need, either to provide a smooth close shave or a less tight cut.", "Thus in the FIG.", "7 variant, a tight shave or a less close shave may be achieved without requiring the user to change either a blade assembly or a head assembly, although this option may also be present within the scope of the invention.", "Again as already discussed, the invention in this aspect offers extensive flexibility of use, not only by selecting the appropriate head region to be applied to the skin in the two-headed arrangement of FIG.", "7 or the alternative three-headed variant discussed previously, but further flexibility and interchangeability is facilitated by the inclusion of additional head assemblies and/or additional blade assemblies in a shaver kit provided in accordance with the principles of the is invention.", "The only dimensional difference between the blades 51 and 61 is in the thickness of the blade in the outer skin-engaging portion.", "Slits 62, flange 63 and central dished region 64 correspond to portions 52, 53 and 54 of the standard cutter 51 of FIG.", "8, but the slits 62 are substantially greater in depth than the slits 52 of blade 51.Accordingly, when this shear blade 61 is applied to the face, the hairs are cut at a longer length than in the case of the blade 51.In use of this embodiment of the invention, the second cutter 61 (FIG.", "9) is to be applied to skin areas where the cut is not to be too tight, so as to avoid the dermatological and other skin problems previously identified, while the first cutter 51 (FIG.", "8) will be used to achieve a tight shave and good appearance where this does not create any skin hazard or potential damage.", "Yet another cutter unit may provide another thickness of shear blade, so that a still further depth of cut may be effected, as required.", "Thus, at least two possible depths of cut are selectively available to the user during operation of the dry shaver.", "In the three-blade shaver of FIG.", "6, completely separate head units 45 may be provided for, first of all, a standard cut, and secondly and alternatively, for situations where a less tight cut is required, this second alternative head having all of the shear blades dimensioned to provide greater spacing from the skin surface according to the arrangement of FIG.", "9, while the first head has shear blades in accordance with FIG.", "8.Referring again to FIG.", "6, head unit 45 is separable from the body 44 of the shaver unit, so that the entire head structure may thus be removed and exchanged for one providing for such a different depth of cut by virtue of the shear blades of all of the cutting units of the alternative head portion 45 being provided with shear blades of different slit or aperture depth from the depth applicable to a standard cut.", "Again in this variant, at least two possible depths of cut are then available to the user, simply by head interchange.", "Thus each head, standard and alternative, has shear blades of uniform cut capability for all blades of that head, but the depth of cut achievable varies as between the heads.", "It is known in the rotary shaver art to provide head and blade assemblies that can be readily dismantled for cleaning and maintenance.", "Any known construction of head or blade assembly of this kind may be applied to the improved blade arrangements and techniques provided by the present invention.", "In particular, such known constructions may be applied to the interchangeable head assemblies, and/or individual interchangeable blade units, as provided the present invention.", "According to the present invention, the dismantling capability of head and blade assemblies is further developed to provide extensive interchangeability on the drive portion of the shaver as between head assemblies on the drive base and blade assemblies on the head assemblies.", "Thus a rotary shaver in accordance with the invention having a base portion suitable for mounting a single head assembly may be provided as a kit including two head assemblies, each of which has blades enabling a different depth of cut.", "One head assembly may provide for a close shave and the other head assembly for a less close shave.", "In addition, further sets of blades or blade assemblies may be provided, so that still different depths of cut may also be enabled by mounting a further alternative set of blades on one of the heads.", "In a variant of the invention, the base drive unit may accommodate more than one head assembly at the same time.", "Both head assemblies can be driven simultaneously or selectively by the base drive unit.", "One of the head assemblies is provided with blades suitable for a close shave and the other with blades suitable for a less close shave.", "One or other of the head assemblies can then be applied to the skin, depending on the depth of cut required.", "Yet a further variant of this aspect of the invention provides for the single drive unit to have no less than three head assemblies, all capable of use without requiring demounting of a head assembly and its replacement by an alternative.", "In any of the variants of the invention, the system may be provided as a kit of parts, with a single base unit or drive portion and two or three or more head assemblies.", "Each head assembly may be pre-provided with a set of blades of a particular depth of cut.", "In addition or alternatively, further blade assemblies may be included in the kit of parts, for interchangeable placement or mounting on any of the head assemblies as may be required.", "Great flexibility and adaptability in use is thus available in accordance with the principles of the invention.", "All head assemblies are readily removable from the base drive portion and all blade assemblies are readily removable from the respective head assembly.", "All sub-assemblies of the system are fully interchangeable, so that any head assembly may be exchanged for any other head assembly and any head assembly may receive any blade assembly.", "Thus any head portion of the unit may receive mounted on it, blade units of any depth of cut.", "Thus in a kit of parts according to the invention, the shaver unit is interchangeably associatable with any one of a plurality or multiplicity of heads.", "Each head may receive a plurality or multiplicity of blades and any blade may be demountably associated with any head.", "Where angled heads are provided for selective optional use without necessarily exchanging a head for an alternative head unit, the principles of full interchangeability and exchangeability continue to prevail, for ease of advance selection of the particular depth of cut required on any of the angled head regions.", "In FIGS.", "10 and 11, there is shown an alternative arrangement in which the actual depth of cut for a pair of rotary cutters may be selectively varied during use.", "The arrangement illustrated is for a two head cutter rotary dry shaver.", "As shown, there is an inner shear blade structure 81, against the underside of which rotary cutters 85 exercise the cutting action.", "An outer “dummy” shear blade or external cutter cover arrangement 91 is fixedly mounted in the cutter head 71 for cooperation with the inner shear blade structure 81 to provide for variable spacing between shear blade 81 and outer “dummy” blade or cover 91.The rotary blades 85 are rotatably mounted in known manner and are driven in rotary motion by likewise known drive arrangements (not illustrated).", "According to the present invention, the outer “dummy” shear blade or external cutter cover arrangement is a fixed outer perforated cover 91 and is associated with the inner shear blade structure 81 for variable depth of shave or cut, the shear blade structure 81 being mounted to be displaceable towards and away from the outer cover 91.The outer surface 92 of this cover or “dummy” shear blade 91 engages the skin.", "The cover or spacer head 91 is suitably provided with a series of apertures 93 which may be of relatively large size compared with the slits 82 of the inner shear blade 81, through which apertures 93 the facial hair to be cut passes to be gripped by the perforations or slits 82 of the shear blade 81 and cut in the usual way by the rotary blades 85.The adjustable arrangement enables the shear blade structure 81 to be moved towards and away from the outer cover 91, so that the depth of cut may be selectively varied in controlled manner by the user, depending on the portion of the skin to be shaved.", "Any one of a diversity of cover-to-blade space-adjusting constructions may be provided to enable the requisite adjustment.", "A particularly favoured arrangement for securing this spacing adjustment is shown in FIGS.", "10 and 11.In accordance with this arrangement, the dummy external cutter or cover 91 provided with the apertured region 93, which generally overlies the slit region of the shear blade 81, also has a substantially solid central region 94.The general configuration of this additional cover or dummy cutter 91 is therefore broadly similar to that of the actual shear blade 81.Variants are however also possible, to the extent that the cover 91 may be of more open work type construction, with the apertures 93 being defined between a series of radial and circumferential bars, wire type members, or perforated plates, and the central bridging region of “dummy” shear blade 91 is also not an essential structural feature.", "In order to secure the inward and outward displaceability of shear blade structure 81 towards and away from the fixed cover 91, FIGS.", "10 and 11 provide a control member 95 which is in the form of a peripheral endless band slideable in an external slot 96 in the external side wall 72 of the shaver unit head region 71.At four locations on its endless path, the peripheral band 95 has camming members 97, 98 which cooperate with mating camming members 87, 88 provided on a support structure 89 which either comprises or carries the shear blade 87.Movement of the endless band 95 in the direction indicated by arrow “a” in FIG.", "8 causes the shear blade structure 81 to be pulled away from the fixed cover 91 and therefore increases the depth of shave or cut, while movement of the band 95 in the direction indicated by arrow “b” in FIG.", "8 produces the converse effect, namely moves the shear blade 81 closer to the fixed blade 91.A structure of this type is known in itself and is applied in an existing dry shaver arrangement in which the shear blades are mounted in a quasi-floating manner.", "The structure is such that the rotor blades remain at all times in fixed proximity to the shear blade and travel inwards and outwards with the shear blade, being flexibly coupled to the drive arrangements so that rotation of the cutter blades may be effected at all locations of the shear blade.", "In this manner therefore the principles of the invention may be embodied in a dry shaver of the rotary cutter blade type also.", "The invention in this embodiment therefore provides substantially infinitely variable selective choice of a preferred depth of cut for any region of the body to be shaved, by varying the displacement between the skin-engaging outer or upper surface 92 of the cover or dummy shear blade 91 and the inner face of the cooperating shear blade 81, where the rotary action of the underlying cutter blades 85 effects actual severance of the hair.", "Thus according to the present variant of the invention, there is again achieved a dry shaver unit in which a variable depth of cut is selectively available at the user's choice.", "The control arrangement 95 shown in FIG.", "10 is exemplary only.", "The arrangements described and proposals indicated herein for shear blade 81 positional control relative to the dummy blade or outer cover 91 are also exemplary only and in no way limit the scope of the invention as defined by the appended claims.", "In particular, alternative control or drive arrangements for displacing the cover portion relative to the cutter blade may be devised and provided, including, but not limited to, mechanical inversions of the arrangements described and illustrated.", "The words “comprises/comprising” and the word “having” when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof." ] ]	Patent_10467689
[ [ "Extracts derived from chenopodium plants and uses thereof", "The present invention relates to pesticides.", "More particularly, the present invention relates to botanical pesticides.", "In particular, the present invention relates to compositions and methods for controlling plant-infesting pests with plant extracts and notably with compositions comprising oil extracts derived from Chenopodium sp.", "plant material.", "The invention further relates to compositions comprising such extracts as pesticidal compositions and providing the advantages of minimal development of resistance thereto, minimal toxicity to mammals, minimal residual activity and environmental compatibility.", "The pesticidal compositions of the present invention comprises α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone." ], [ "1.An essential oil extract derived from a Chenopodium species, comprising: from at least 30% to at least 65% α-terpinene, from at least 8% to at least 26% ρ-cymene, from at least 5% to at least 24% limonene, from at least 0.04% to at least 1% carvacrol, from at least 0.1% to at least 2% carveol, from at least 0.1% to at least 2.0% nerol, from at least 0.04% to at least 2% thymol, and from at least 0.04% to at least 1% carvone, and wherein said extract is active against one or more pests chosen from insects, acari, and fungi.", "2.", "(Canceled) 3.", "(Canceled) 4.", "(Canceled) 5.The essential oil extract according to claim 1, wherein said species is Chenopodium ambrosioides.", "6.A pesticidal composition, comprising an effective amount of the essential oil extract of claim 1 and a suitable emulsifier, spreader or sticking agent, and carrier.", "7.The pesticidal composition according to claim 6, comprising between 0.125% to 10% (by volume) essential oil extract.", "8.The pesticidal composition according to claim 7, comprising between 0.25% to 5% (by volume) of said essential oil extract.", "9.The pesticidal composition according to claim 6, wherein said composition comprises between 5% to 50% (by volume) of said essential oil extract.", "10.The pesticidal composition according to claim 9, comprising between 10% to 25% (by volume) of said essential oil extract.", "11.The pesticidal composition according to claim 10, comprising between 1% to 15% (by volume) of a suitable emulsifier, and between 50% to 70% (by volume) of a suitable carrier or solvent.", "12.The pesticidal composition according to claim 11, comprising between 2% to 20% (by volume) of a suitable spreader or sticking agent.", "13.A pesticidal composition, comprising between 30% to 50% (by volume) of the essential oil extract of claim 1, between 0.5% to 25% (by volume) of a suitable emulsifier, and between 10% to 50% (by volume) water.", "14.A method for controlling one or more pests chosen from phytophagous acari, phytophagous insects, and phytophagous fungi, which comprises applying to a locus where control is desired a pesticidally-effective amount of the pesticidal composition of claim 6.15.", "(Canceled) 16.", "(Canceled) 17.The method according to claim 14, wherein said locus is soil.", "18.The method according to claim 14, wherein said locus is a plant.", "19.The essential oil extract according to claim 1, further comprising ascaridole at a proportion of 9.86% or less.", "20.The essential oil extract according to claim 1, further comprising ascaridole at a proportion of 1% or less.", "21.The essential oil extract according to claim 20, wherein said extract has trace or undetectable amounts of ascaridole.", "22.A formulation for indoor or outdoor fumigation, comprising: between 1% to 15% (by volume) of the essential oil extract of claim 1.23.A formulation for topical application, comprising: between 1% to 10% (by volume) of the essential oil extract of claim 1.24.The formulation of claim 23, wherein the formulation further comprises between 2% to 5% (by volume) of an emollient, between 0.1% to 1% (by volume) of a stabilizer and between 0.1% to 0.5% (by volume) of a preservative.", "25.The formulation of claim 23, wherein the formulation further comprises between 1% to 10% (by volume) of an emulsifier, between 2% to 5% (by volume) of a coat or skin conditioner, between 0.1% to 1% (by volume) of a stabilizer, between 0.1% to 1% (by volume) of a preservative and between 0.1% to 0.5% (by volume) of an antioxidant.", "26.A microemulsion formulation, comprising: between 30% to 50% (by volume) of the essential oil extract of claim 1, between 0.5% to 25% (by volume) of a suitable emulsifier, and between 10% to 50% (by volume) water.", "27.The formulation of claim 26, wherein said formulation is for soil delivery.", "28.A method for controlling phytophagous pests, which comprises applying to a locus where control is desired a pesticidally-effective amount of the formulation according to any one of claims 22, 23, or 26." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Plant feeding mites are among the most voracious phytophagous pests of crops.", "To combat these pests, synthetic pesticides have been developed.", "These synthetic chemical pesticides, however, often have detrimental environmental effects that are harmful to humans and other animals and therefore do not meet the guidelines developed by most Integrated Pest Management programs.", "Moreover, resistance to these products has been found to develop with many of the new products put on the market (Georghiou, 1990; Nauen et al., 2001).", "Although resistance follows a highly complex genetic and biochemical process, it can generally develop rapidly with synthetic products because their active ingredients rely on one or more molecules of the same class.", "The organism can therefore respond to the toxin by developing physiological, behavioral or morphological defense mechanisms to neutralize the effect of the molecule (Roush and MacKenzie, 1987).", "Acari, such as spider mites, in particular, are extremely difficult to control with pesticides.", "Tetranychus urticae (the two-spotted spider mite), for example, has accumulated a considerable number of genes conferring resistance to all major classes of acaricides.", "Resistance to many registered acaricides have been reported, for example, resistance has been reported to hexythiazox, abamectin, and clofentezine.", "Furthermore, many of these pesticides have been found to exacerbate pest infestation by destroying the natural predators of mites (U.S. Pat.", "No.", "5,839,224).", "Additionally, many synthetic insecticides have been found to stimulate mite reproduction.", "For example, it was found that mites reproduce many times faster when exposed to carbaryl, methyl parathion, or dimethoate in the laboratory than untreated populations (Flint, 1990).", "As a result, there are very few pesticides remaining that are effective against spider mites (Georghiou, 1990).", "In the Farm Chemical Handbook (Meister, 1999), for example, only 48 products out of a total of 2,050 listed acaricides and insecticides (or 2.4%), were identified as acaricides and only 69 of these products (or 3.4%) were identified as both acaricides and insecticides.", "The control of insect infestation has also proven to be difficult.", "For example, European chafer Rhizotrogus majalis Razoumowsky (Coleoptera: Scarabaeidae) insects are an exotic species to Canada and the United States and are commonly known as Scarab beetles.", "Throughout the northeast, where the beetle has become established, R. majalis causes major damage to turfgrass, consuming the roots of home lawns as well as urban recreational areas and golf courses.", "The larvae of this species are extremely difficult and expensive to control using conventional insecticides.", "In several areas of the U.S. insecticide resistance has already developed within populations.", "The control of plant fungi is another source for growing concern.", "Phytopathogenic fungi are of great economic importance since fungal growth on plants or on parts of plants inhibits production of root, stem, foliage, fruit or seed, and the overall quality of a cultivated crop.", "About 25 percent of all fungal diseases in agriculture and horticulture are caused by powdery mildew phytopathogens (U.S. Pat.", "Nos.", "5,882,689 and 5,496,568).", "In cucumber crops, for example, powdery mildew caused by the plant pathogens, Erysiphe cichoracearum and Sphaerotheca fuliginea , is one of the most problematic disease.", "The fungus responsible for Gray Mold, Botrytis cinerea, can attack more than 200 species of cultivated plants and especially those growing in a greenhouse environment.", "It is a saprophyte that attacks dead or senescent plant tissue.", "B. cinerea is especially damaging to stems (causing stem rot), when it enters scars left by pruning of lower leaves.", "Following attack, the plant dies, causing heavy economic losses to the grower.", "Control of stem rot is attempted by treating leaf scars after leaf pruning.", "Although benomyl is presently used, it will likely be removed from the market shortly.", "Moreover, iprodione (another pesticide), is less effective because of developing fungal resistance.", "In spite of their strong fungicidal effect, agricultural fungicides currently in use have been a source of problems, because the required amount is ever increasing as tolerance of target plant pathogens increases.", "Moreover, many fungicides are synthetic agents, which when used, pose possible drawbacks for humans, animals and the environment.", "Thus, effective commercial products (to combat fungal attacks on crops), which reduce the dose of synthetic chemicals spread into the environment, are in need; keeping in mind a regard for environmental and human safety issues.", "As an alternative to synthetic agents, botanical pesticides offer the advantage of being naturally derived compounds that are safe to both humans and the environment.", "Specifically, botanical pesticides offer such advantages as being inherently less toxic than conventional pesticides, generally affecting only the target pest and closely related organisms, and are often effective in very small quantities.", "In addition, botanical pesticides often decompose quickly and, therefore, are ideal for use as a component of Integrated Pest Management (IPM) programs.", "There are few published reports of the acaricidal properties of botanical pesticides.", "For example, U.S. Pat.", "No.", "4,933,371 describes the use of saponins extracted from various plants (i.e., yucca, quillaja, agave, tobacco and licorice) as acaricides.", "This patent also describes the use of linalool extracted from the oil of various plants such as Ceylon's cinnamon, sassafras, orange flower, bergamot, Artemisia balchanorum, ylang ylang, rosewood and other oil extracts as acaricides.", "These methods, however, require the extraction of one active substance from the plant which often does not meet desired levels of toxicity towards acari.", "Plant essential oils are a complex mixture of compounds of which many can be biologically active against insect and mite pests, the compounds acting individually or in synergy with each other, to either repel or kill the pests by contact.", "These components are plant secondary metabolites or allelochemicals produced by plants as a defense mechanism against plant feeding pests (Ceske and Kaufman, 1999).", "Because of the complexity of the mixture, it has been observed that pests do not easily develop resistance to these products as they can to synthetic pesticides or botanical pesticides comprising a single active compound.", "In this respect, Feng and Isman (1995) demonstrated that repeated treatments of pure azadirachtin, a major active constituent of neem oil, against the green peach aphid led to a 9-fold resistance after 40 generations.", "However, repeated exposure during 40 generations to crude neem extracts did not lead to resistance.", "There remains a need to provide new and effective pesticidal products which overcome the shortcomings of products known in the art.", "For example, there remains a need for broad spectrum compositions which are less likely to enable pests to develop resistance thereto.", "There also remains a need to provide a method to combat pests at any given locus, using a composition which is not toxic to animals, especially to mammals, nor to any beneficial predator/parasitoid insects." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides essential oil extracts derived from Chenopodium sp.", "comprising, α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, wherein these extracts have one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.", "One or more of these extracts can be formulated into compositions that enable their application into different environments such as plants, soil, animals and buildings.", "If accordance with another aspect of the invention there is provided composition comprising one or more Chenopodium sp.", "-derived essential oil extracts, wherein said extract comprises α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, in combination with an emulsifier, carrier, spreader and/or sticking agent, to enable application of the composition to a specific environment, wherein the composition has one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities." ], [ "FIELD OF INVENTION The present invention relates to the field of pesticides for controlling plant-infesting pests.", "BACKGROUND OF THE INVENTION Plant feeding mites are among the most voracious phytophagous pests of crops.", "To combat these pests, synthetic pesticides have been developed.", "These synthetic chemical pesticides, however, often have detrimental environmental effects that are harmful to humans and other animals and therefore do not meet the guidelines developed by most Integrated Pest Management programs.", "Moreover, resistance to these products has been found to develop with many of the new products put on the market (Georghiou, 1990; Nauen et al., 2001).", "Although resistance follows a highly complex genetic and biochemical process, it can generally develop rapidly with synthetic products because their active ingredients rely on one or more molecules of the same class.", "The organism can therefore respond to the toxin by developing physiological, behavioral or morphological defense mechanisms to neutralize the effect of the molecule (Roush and MacKenzie, 1987).", "Acari, such as spider mites, in particular, are extremely difficult to control with pesticides.", "Tetranychus urticae (the two-spotted spider mite), for example, has accumulated a considerable number of genes conferring resistance to all major classes of acaricides.", "Resistance to many registered acaricides have been reported, for example, resistance has been reported to hexythiazox, abamectin, and clofentezine.", "Furthermore, many of these pesticides have been found to exacerbate pest infestation by destroying the natural predators of mites (U.S. Pat.", "No.", "5,839,224).", "Additionally, many synthetic insecticides have been found to stimulate mite reproduction.", "For example, it was found that mites reproduce many times faster when exposed to carbaryl, methyl parathion, or dimethoate in the laboratory than untreated populations (Flint, 1990).", "As a result, there are very few pesticides remaining that are effective against spider mites (Georghiou, 1990).", "In the Farm Chemical Handbook (Meister, 1999), for example, only 48 products out of a total of 2,050 listed acaricides and insecticides (or 2.4%), were identified as acaricides and only 69 of these products (or 3.4%) were identified as both acaricides and insecticides.", "The control of insect infestation has also proven to be difficult.", "For example, European chafer Rhizotrogus majalis Razoumowsky (Coleoptera: Scarabaeidae) insects are an exotic species to Canada and the United States and are commonly known as Scarab beetles.", "Throughout the northeast, where the beetle has become established, R. majalis causes major damage to turfgrass, consuming the roots of home lawns as well as urban recreational areas and golf courses.", "The larvae of this species are extremely difficult and expensive to control using conventional insecticides.", "In several areas of the U.S. insecticide resistance has already developed within populations.", "The control of plant fungi is another source for growing concern.", "Phytopathogenic fungi are of great economic importance since fungal growth on plants or on parts of plants inhibits production of root, stem, foliage, fruit or seed, and the overall quality of a cultivated crop.", "About 25 percent of all fungal diseases in agriculture and horticulture are caused by powdery mildew phytopathogens (U.S. Pat.", "Nos.", "5,882,689 and 5,496,568).", "In cucumber crops, for example, powdery mildew caused by the plant pathogens, Erysiphe cichoracearum and Sphaerotheca fuliginea, is one of the most problematic disease.", "The fungus responsible for Gray Mold, Botrytis cinerea, can attack more than 200 species of cultivated plants and especially those growing in a greenhouse environment.", "It is a saprophyte that attacks dead or senescent plant tissue.", "B. cinerea is especially damaging to stems (causing stem rot), when it enters scars left by pruning of lower leaves.", "Following attack, the plant dies, causing heavy economic losses to the grower.", "Control of stem rot is attempted by treating leaf scars after leaf pruning.", "Although benomyl is presently used, it will likely be removed from the market shortly.", "Moreover, iprodione (another pesticide), is less effective because of developing fungal resistance.", "In spite of their strong fungicidal effect, agricultural fungicides currently in use have been a source of problems, because the required amount is ever increasing as tolerance of target plant pathogens increases.", "Moreover, many fungicides are synthetic agents, which when used, pose possible drawbacks for humans, animals and the environment.", "Thus, effective commercial products (to combat fungal attacks on crops), which reduce the dose of synthetic chemicals spread into the environment, are in need; keeping in mind a regard for environmental and human safety issues.", "As an alternative to synthetic agents, botanical pesticides offer the advantage of being naturally derived compounds that are safe to both humans and the environment.", "Specifically, botanical pesticides offer such advantages as being inherently less toxic than conventional pesticides, generally affecting only the target pest and closely related organisms, and are often effective in very small quantities.", "In addition, botanical pesticides often decompose quickly and, therefore, are ideal for use as a component of Integrated Pest Management (IPM) programs.", "There are few published reports of the acaricidal properties of botanical pesticides.", "For example, U.S. Pat.", "No.", "4,933,371 describes the use of saponins extracted from various plants (i.e., yucca, quillaja, agave, tobacco and licorice) as acaricides.", "This patent also describes the use of linalool extracted from the oil of various plants such as Ceylon's cinnamon, sassafras, orange flower, bergamot, Artemisia balchanorum, ylang ylang, rosewood and other oil extracts as acaricides.", "These methods, however, require the extraction of one active substance from the plant which often does not meet desired levels of toxicity towards acari.", "Plant essential oils are a complex mixture of compounds of which many can be biologically active against insect and mite pests, the compounds acting individually or in synergy with each other, to either repel or kill the pests by contact.", "These components are plant secondary metabolites or allelochemicals produced by plants as a defense mechanism against plant feeding pests (Ceske and Kaufman, 1999).", "Because of the complexity of the mixture, it has been observed that pests do not easily develop resistance to these products as they can to synthetic pesticides or botanical pesticides comprising a single active compound.", "In this respect, Feng and Isman (1995) demonstrated that repeated treatments of pure azadirachtin, a major active constituent of neem oil, against the green peach aphid led to a 9-fold resistance after 40 generations.", "However, repeated exposure during 40 generations to crude neem extracts did not lead to resistance.", "There remains a need to provide new and effective pesticidal products which overcome the shortcomings of products known in the art.", "For example, there remains a need for broad spectrum compositions which are less likely to enable pests to develop resistance thereto.", "There also remains a need to provide a method to combat pests at any given locus, using a composition which is not toxic to animals, especially to mammals, nor to any beneficial predator/parasitoid insects.", "SUMMARY OF THE INVENTION The invention provides essential oil extracts derived from Chenopodium sp.", "comprising, α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, wherein these extracts have one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.", "One or more of these extracts can be formulated into compositions that enable their application into different environments such as plants, soil, animals and buildings.", "If accordance with another aspect of the invention there is provided composition comprising one or more Chenopodium sp.", "-derived essential oil extracts, wherein said extract comprises α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, in combination with an emulsifier, carrier, spreader and/or sticking agent, to enable application of the composition to a specific environment, wherein the composition has one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 shows the chemical content of three lots or pools of oil samples extracted from whole plant parts above root (00MC-21P, 00MC-24P and 00M-29P).", "FIG.", "2 shows the average mortality (%) of the two-spotted spider mite (TSSM: Tetranychus urticae) when tested with solutions of individual compounds present in the essential oil of Chenopodium ambrosioides.", "Results adjusted for control mortality with Abbott's formula.", "FIG.", "3 shows the average mortality (%) of the greenhouse whitefly (GWF; Trialeurodes vaporaiorum) when tested with solutions of individual compounds present in the essential oil of Chenopodium ambrosioides.", "Results adjusted for control mortality with Abbott's formula.", "FIG.", "4 shows adult spider mite (Tetranychus urticae) mortality obtained with bioassays using the RTU formulation of Chenopodium ambrosioides and commercial preparations of natural and synthetic insecticides.", "FIG.", "5 shows spider mite egg (Tetranychus urticae) mortality, using the RTU formulation of Chenopodium ambrosioides oil.", "FIG.", "6 shows spider mite nymph (Tetranychus urticae) mortality, using the RTU Chenopodium extract formulation and commercial preparations of synthetic and natural products.", "FIG.", "7 shows the mortality of adult spider mites 48 h following introduction on faba bean leaves treated one hour previously with the RTU formulation and selected natural acaricides.", "FIG.", "8 shows red mite, Panonychus ulmi mortality, using the RTU formulation.", "FIG.", "9 shows insect mortality (%) obtained with bioassays using the RTU formulation of Chenopodium ambrosioides.", "FIG.", "10 shows mortality of adult female twospotted spider mites 48 hours following applications.", "FIG.", "11 shows mortality of adult female European red mite 24 hours following applications.", "FIG.", "12 shows egg hatch (%) of the twospotted spider mite, 10 days following applications.", "FIG.", "13 shows egg hatch (%) of European red mite 10 days following applications.", "FIG.", "14 shows mortality of adult female two-spotted spider mites 48 hours following introduction on leaf discs treated with EC25% and Dicofol one hour previously.", "FIG.", "15 shows mortality of green peach aphids (Myzus persicae (Sulz.))", "48 hours following application of 0.125, 0.25, 0.5, 1.0 and 2.0% concentrations of formulation EC25% and the commercially available bioinsecticides Neem Rose Defense® and Safer's Trounce® FIG.", "16 shows lethal concentrations (LC50 and LC90) in % of EC25% for the green peach aphid (Myzus persicae (Sulz.))", "calculated with 48 hour mortality data.", "FIG.", "17 shows average number of green peach aphids (Myzus persicae (Sulz.))", "per cm2 of treated Verbena speciosa shoot following application of 0.25, 0.50 and 1.0% concentrations of EC25% and the commercially available bioinsecticides Neem Rose Defense® and Safer's Trounce® FIG.", "18 shows mortality of Western flower thrips (Frankliniella occidentalis (Perg.))", "24 hours following application of six concentrations (0.05, 0.125, 0.18, 0.25, 0.5 and 1.0%) of formulation EC25% and the commercially available bioinsecticides Neem Rose Defense® and Safer's Trounce® FIG.", "19 shows lethal concentrations (LC50 and LC90) in mg/cm2 of EC25% for the Western flower thrips (Frankliniella occidentalis (Perg.))", "calculated with 24 hour mortality data.", "FIG.", "20 shows average number of Western flower thrips/cm2 (WFT: Frankliniella occidentalis (Perg.))", "per treatment as a percentage of thrips present on leaves treated with the control during a greenhouse bioassay using two concentrations (0.25 and 1.0%) of EC25% and two commercially available bioinsecticides Neem Rose Defense® and Safer's Trounce® FIG.", "21 shows mortality of greenhouse whiteflies (Trialeurodes vaporariorum (Westw.))", "20 hours following application of five concentrations (0.0625, 0.125, 0.25, 0.5 and 1%) of formulation EC25% and the commercially available insecticides Neem Rose Defense®, Safer's Trounce® and Thiodan® FIG.", "22 shows lethal concentrations (LC50 and LC90) in mg/cm2 of EC25% for the greenhouse whitefly (Trialeurodes vaporariorum (Westw.))", "calculated with 20 hour mortality data.", "FIG.", "23 shows mortality of Encarsia formosa 24 hours following application of four concentrations (0.0625, 0.125, 0.25, 0.5 and 1.0%) of formulation EC25% and the commercially available bioinsecticides, Neem Rose Defense® and Safer's Trounce® FIG.", "24 shows the effect of three concentrations of EC25% with the tomato canker caused by the fungus Botrytis cinerea.", "FIG.", "25 shows the percentage control of the tomato canker following treatment with a selection of products 1 and 8 days following inoculation with Botrytis cinerea.", "FIG.", "26 shows the percentage (%) infection among blocks with powdery mildew.", "FIG.", "27 shows the effect of EC25% and reference products on Powdery Mildew.", "FIG.", "28 shows daily mean and total emergence of adult thrips from soil treated with different volumes of EC25% at 0.5% concentration.", "FIG.", "29 shows the percent survival of R. majalis 7 days after treatment with EC25%.", "FIG.", "30 shows EC25% median lethal concentrations (LC50), standard error of log LC50, 95 percent confidence interval and slope/intercept equation for two 7 day trials with R. majalis.", "FIG.", "31 shows average soil percent organic matter and pH±standard error determined for each tray in both R. majalis 7 day trial.", "FIG.", "32 shows treatments tested with the European chafer, Rhizotrogus majalis.", "FIG.", "33 shows counts for live European chafers on turfgrass plots.", "FIG.", "34 shows treatments evaluated with the HCB on turfgrass plots.", "FIG.", "35 shows counts for live HCB on turfgrass plots.", "FIG.", "36 shows mean mortality (%) of Amblyseius fallacis adult females following the direct application of several concentrations of EC25% and commercially available insecticides.", "FIG.", "37 shows contact toxicity of EC25% oil formulation on adult females of Amblyseius fallacis.", "Probit analysis.", "FIG.", "38 shows mean percent mortality of Phytoseiulus persimilis adult females to different insecticide treatments.", "FIG.", "39 shows overall percent mean mortality of adult wasps Aphidius colemani following direct application with EC25% and commercially available insecticides.", "FIG.", "40 shows male and female mean mortality (%) of Aphidius colemani adult wasps following direct application with EC25% and commercially available insecticides.", "FIG.", "41 shows contact toxicity of EC25% oil formulation on adult wasps Aphidius colemani.", "Probit analysis.", "FIG.", "42 shows mortality of adult wasps Aphidius colemani following exposure to EC25% and commercially available insecticide residues.", "FIG.", "43 shows probit analysis of adult wasps Aphidius colemani 24 H and 48 H following exposure to EC25% residues.", "FIG.", "44 shows the effect of treatment on Aphidius colemani emergence from treated mummies.", "FIG.", "45 shows fecundity assessment of female Aphidius colemani following contact with EC25% residues.", "FIG.", "46 shows mean mortality of Orius insidiosus second instar nymphs following application with EC25% and commercially available insecticides.", "FIG.", "47 shows mean mortality of Orius insidiosus adults following EC25% and other insecticide treatments.", "FIG.", "48 shows fecundity of Orius insidiosus females surviving insecticide treatments.", "FIG.", "49 shows probit analysis of Orius insidosus second instar nymphs following application with EC25%.", "FIG.", "50 shows probit analysis of Orius insidiosus adults following application with EC25%.", "DETAILED DESCRIPTION OF THE INVENTION Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.", "“Locus” means a site which is infested or could be infested with acari, insects, fungi, or other pests and may include, but is not restricted to, domestic, urban, agricultural, horticultural, and forest environments.", "“Essential Oil Extract” means the volatile, aromatic oils obtained by steam or hydro-distillation of plant material and may include, but are not restricted to, being primarily composed of terpenes and their oxygenated derivatives.", "Essential oils can be obtained from, for example, plant parts including, for example, flowers, leaves, seeds, roots, stems, bark, wood, etc.", "“Active Constituents” means the constituents of the essential oil extract to which the pesticidal, acaricidal, insecticidal, and/or fungicidal activity is attributed.", "The essential oil extract of the present invention generally comprises the active constituents including: α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone.", "The term “partially purified”, when used in reference to an essential oil extract means that the extract is in a form that is relatively free of proteins, nucleic acids, lipids, carbohydrates or other materials with which it is naturally associated in a plant.", "As disclosed herein, an essential oil extract of the invention is considered to be partially purified.", "In addition, the individual components of the essential oil extract can be further purified using routine and well known methods as provided herein.", "Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (ed.", "Parker, S., 1985), McGraw-Hill, San Francisco, incorporated herein by reference.", "The present invention provides essential oil extracts derived from Chenopodium sp.", "comprising, α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone.", "These extracts have one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.", "The present invention also provides for the use of one or more of these essential oil extracts to prepare compositions that enable their application into different environments such as plants, soil, animals and buildings.", "1.Essential Oil Extract Plant Material Plant material that may be used in the present invention includes plant material derived from the genus Chenopodium sp.", "taken individually or in a group and may include, but is not restricted to, the leaf, flowers, roots, seeds, and stems.", "As is known by persons skilled in the art, the chemical composition and efficacy of an essential oil extract varies with the phenological age of the plant (Jackson et al., 1994), percent humidity of the harvested material (Chialva et al., 1983), the plant parts chosen for extraction (Jackson et al., 1994; and Chialva et al., 1983), and the method of extraction (Perez-Souto, 1992).", "Methods well-known in the art can be adapted by a person of ordinary skill in the art to achieve the desired yield and quality of the essential oil extract of the present invention.", "In one embodiment, the plant material is derived from Chenopodium ambrosioides.", "Pre-Treatment of Plant Material In addition to such parameters as the phenological age of the plant, the percent humidity of the harvested material, the plant parts chosen for extraction, and the method of extraction, the chemical composition and efficacy of an essential oil extract may be affected by pre-treatment of the plant material.", "For example, when a plant is stressed, several biochemical processes are activated and many compounds, in addition to those constitutively expressed, are synthesized as a response.", "In addition to pests, fungi, and other pathogenic attacks, stressors include drought, heat, water and mechanical wounding.", "Moreover, persons of skill in the art will also recognize that combinations of stressors may be used.", "For example, the effects of mechanical wounding can be increased by the addition of compounds that are naturally synthesized by plants when stressed.", "Such compounds include jasmonic acid (JA).", "In addition, analogs of oral secretions of insects can also be used in this way, to enhance the reaction of plants to stressors.", "In one embodiment, the essential oil extracts of the present invention are derived from plant material which has been pre-treated, for example by stressing the plant by chemical or mechanical wounding, drought, heat, or cold, or a combination thereof, before plant material collection and extraction.", "Harvesting the Plant Material for Extraction and Optional Storage Treatment The plant material may be used immediately after harvesting.", "In one embodiment the fresh plant material having a humidity level of >75% is used.", "Otherwise, it may be desirable to store the plant material for a period of time, prior to performing the extraction procedure(s).", "In another embodiment wilted plant material having a humidity level of 40 to 60% is used.", "In another embodiment dry plant material having a humidity level of <20% is used.", "In a further embodiment, the plant material is treated prior to storage.", "In such cases, the treatment may include drying, freezing, lyophilisizing, or some combination thereof.", "Extraction of the Essential Oil Extract and Validation of Constituents Essential oil extracts can be extracted from plant material by standard techniques known in the art.", "A variety of strategies are available for extracting essential oils from plant material, the choice of which depends on the ability of the method to extract the constituents in the extract of the present invention.", "Examples of suitable methods for extracting essential oil extracts include, but are not limited to, hydro-distillation, direct steam distillation (Durbeck 1997), solvent extraction, and Microwave Assisted Process (MAP™) (Belanger et al., 1991).", "In one embodiment, plant material is treated by boiling the plant material in water to release the volatile constituents into the water which can be recovered after distillation and cooling.", "In another embodiment, plant material is treated with steam to cause the essential oils within the cell membranes to diffuse out and form mixtures with the water vapor.", "The steam and volatiles can then be condensed and the oil collected.", "In another embodiment, organic solvents are used to extract organically soluble compounds found in essential oils.", "Non-limiting examples of such organic solvents include methanol, ethanol, hexane, and methylene chloride.", "In a further embodiment, microwaves are used to excite water molecules in the plant tissue which causes cells to rupture and release the essential oils trapped in the extracellular tissues of the plant material.", "To confirm the presence of the constituents of the present invention in the essential oil extract, a variety of analytical techniques well known to those of skill in the art may be employed.", "Such techniques include, for example, chromatographic separation of organic molecules (e.g., gas chromatography) or by other analytical techniques (e.g., mass spectroscopy) useful to identify molecules falling within the scope of the invention.", "The invention provides an essential oil extract derived from Chenopodium sp.", "comprising α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone.", "In one embodiment, the essential oil extract comprises at least 30% α-terpinene, 8% ρ-cymene, 5% limonene, trace carvacrol, 0.1% carveol, 0.1% nerol, trace thymol, and trace carvone.", "In one embodiment, the essential oil extract comprises at least one of each compound selected from the range for each compounds of at least 30%, 32%, 34%, 38%, 40%, 44%, 48%, 50%, 55%, 60 %, 65% α-terpinene; 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26% ρ-cymene, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%,22%,24% limonene, trace, 0.04%, 0.08%, 0.10%, 0.12%, 0.14%, 0.2%, 0.5%, 0.8%, 1.0% carvacrol, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.4%, 1.6%, 1.8%, 2.0% carveol, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.4%, 1.6%, 1.8%, 2.0% nerol, trace, 0.04%, 0.08%, 0.10%,0.12%, 0.14%, 0.2%, 0.5%, 0.8%, 1.0%, 1.4%, 1.6%, 1.8%, 2.0% thymol, and trace, 0.04%, 0.08%, 0.10%, 0.12%, 0.14%, 0.2%, 0.5%, 0.8%, 1.0% carvone.", "2.Activity of the Essential Oil Extract Following extraction of an essential oil extract of the invention, it may be desirable to test the efficacy of the extracts for acaricidal, pesticidal, insecticidal, and fungicidal activities.", "Any number of tests familiar to a worker skilled in the art may be used to test the activity of the extracts, compositions, and formulations of the invention.", "Deternination of Acaricidal Activity of the Essential Oil Extract Acaricidal activity of an essential oil extract may be evaluated by using a variety of bioassays known in the art (Ebeling and Pence, 1953; Ascher and Cwilich, 1960; Dittrich, 1962; Lippold, 1963; Foot and Boyce, 1966; and Busvine, 1980).", "a) Contact Efficacy with the Adult Stage One exemplary method that may be used tests the contact efficacy of the essential oil extract, or formulations thereof, with the adult stage of a mite species.", "For example, adult mites may be placed on their dorsum with a camel hair brush on a double-sided sticking tape glued to a 9 cm Petri dish.", "Essential oil extracts and/or formulations may then be applied to the test subjects by spraying with the spray nozzle of a Potter Spray Tower mounted on a stand and connected to a pressure gauge set at 3 psi.", "Mites that fail to respond to probing with a fine camel hair brush with movements of the legs, proboscis or abdomen are considered dead.", "In one embodiment, the contact efficacy of an essential oil extract, or formulations thereof, is determined using the two-spotted spider mite (Tetranychus urticae), at the adult stage, as a model test subject.", "A person skilled in the art, however, will readily understand that other species of acari can be used.", "b) Ovicidal Activity The ovicidal effect can be determined by treating mite eggs with concentrations of essential oil extracts, or formulations thereof.", "For example, adult female T. urticae may be transferred to 2 cm diameter leaf disks cut out of lima bean leaves and left for four hours for oviposition.", "When at least 20 eggs/disk are laid, adult mites may then be removed.", "Essential oil extracts and/or formulations may then be applied by spraying the test subjects.", "Egg hatch is assessed daily and for 10 days following treatment by counting the number of eggs remaining on the leaf disks and the number of live and dead nymphs present.", "Percent egg hatch is determined with live nymphs only.", "The nymphs are considered dead if no movement is observed after repeated gentle probing with a single-hair brush.", "In one embodiment the ovicidal activity of an essential oil extract, or formulations thereof, is determined with mite eggs of the two-spotted spider mite (Tetranychus urticae), as a model test subject.", "A person skilled in the art, however, will readily understand that other species of acari can be used.", "Determination of Insecticidal Activity of the Essential Oil Extract Similar bioassays can be conducted to evaluate the insecticidal activity of an essential oil extract, or formulations thereof, by utilizing an insect model.", "In one embodiment, the greenhouse whitefly (Trialeurodes vaporariorum (Westw.))", "is used as a model test subject in an insecticide bioassay.", "For example, Whitefly adults may be glued to a black 5 cm×7,5 cm plastic card sprayed with Tangle-Trap® (Gempler's Co.) to obtain at least 20 active adults per card.", "Each card is sprayed with the essential oil extract, composition, or formulation and allowed to dry.", "The cards are then placed sideways on a Styrofoam rack in a closed clear plastic container of 5 L with moistened foam on the bottom to keep humidity high (>90% R.H.).", "The plastic container is stored in a growth chamber at 24° C. and 16 L:8 D photoperiod.", "Mortality is evaluated 20 hours following treatment by gently probing the whitefly with a single-hair brush under the binocular microscope.", "Absence of movement (antennae, leg, wing) following probing is recorded as dead.", "A person skilled in the art, however, will readily understand that other insect species can be used.", "Determination of Fungicidal Activity of the Essential Oil Extract The fungicidal activity of an essential oil extract, or formulations thereof, can also be evaluated by utilizing a fungal model evaluated by using a variety of bioassays known in the art.", "Examples of such known bioassays include the following.", "Laboratory Tests The fungicidal efficacy of an essential oil can be done in the laboratory using several methods.", "One method incorporates the test samples in an agar overlay in a Petri dish.", "A second method would use a filter disk saturated with the test samples and placed on top of untreated agar.", "Both systems are challenged with fungal plugs cut from lawns of indicator organisms at the same stage of growth.", "The plates will be incubated at 30° C. for 5-10 days with visual observations and the zone of inhibition measured and recorded.", "A positive control, i.e.", "a commercially available fungicide and a negative control, i.e.", "water are tested in the same way.", "Greenhouse Tests Greenhouse tests may also be employed to evaluate fungicidal efficacy.", "For example, the effect of the essential oil extracts, or formulations thereof, may be tested on host plants infected by a disease organism such as, for example, Botrytis cinerea, Erysiphe cichoracearum or Sphaerotheca fuliginea, Rhizoctonia solani, and Phytophthora infestans, by observing the percent damage or presence of lesions on the host plant after treatment and against controls.", "Botrytis cinerea.", "Tomato plants are seeded and grown following current commercial practices for greenhouse tomato production.", "About 2 months following seeding, lesions are made on the leaves and the stem (5 lesions/plant) and inoculated with a suspension of 3×106 spores of B. cinerea, 2 ml per lesion.", "Treatments are then applied to the plants.", "A positive control, i.e.", "a commercially available fungicide and a negative control, i.e.", "water are also tested and all treatments are done in a randomized block design.", "The length of lesions are measured every two weeks over a period of 3 months, then the number of fruit, the total weight of fruit and the average weight of fruit are calculated during the entire production period of the plant.", "The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.", "Erysiphe cichoracearum or Sphaerotheca fuliginea.", "These disease organisms are obligatory parasites that do not have the capacity to survive in absence of its host.", "Therefore to provide the inoculum for the test, cucumber leaves are taken from an infested greenhouse.", "The conidia present on these leaves will transfer onto cucumber plants grown for the experiment one or two months previously.", "New plants are periodically infested in this manner in order to increase the inoculum.", "Treatments are then applied to the plants before or after inoculation depending on the type of fungicide used.", "A positive control, i.e.", "a commercially available fungicide and a negative control, i.e.", "water are also tested and all treatments are done in a randomized block design.", "The effect of the disease is evaluated on individual leaves of all plants using a index of infestation from 0 to 5 (0=absence of blemish and 5=80-100% of the leaf surface with blemishes).", "The degree of the infestation is evaluated 3, 7, and 14 days following inoculation and reported in averages per plant.", "The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.", "Rhizoctonia solani.", "An isolate of Rhizoctonia solani is produced on a culture media (PDA) 3 days before inoculation and a plug of the disease is then transferred to Erlenmeyer flasks filled with a YMG broth for 5 days.", "The mycelium is filtered, suspended in distilled water and blended.", "Seeds of tomato are used and sterilized on the surface using successive ethanol 70%, bleach and distilled water solutions.", "A suitable sterile potting soil mix is used in which 60 mg blended mycelium is inoculated per 100 g of potting soil.", "Tests are done in bedding boxes of 72 cells/box and 3 boxes are used per treatment.", "The boxes are spread out in a randomized arrangement in a controlled atmosphere growth chamber the following conditions: 20° C. during the day and 16° C at night, 16 hours of light, 162 umol of light intensity and 60% humidity.", "The boxes are incubated in the growing chambers during 3 weeks.", "Treatments are then applied to the young plants before or after inoculation depending on the type of fungicide used.", "A positive control, i.e.", "a commercially available fungicide and a negative control, i.e.", "water are also tested and all treatments are done in a randomized block design.", "Plants are examined each week and the incidence of the disease is measured as well as the degree of infestation on a scale of 0 to 5 (0=absence of infestation and 5=80-100% of the leaf surface attacked).", "The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.", "Phytophthora infestans.", "On tomato plants.", "Tomato plants are seeded and grown following current commercial practices for greenhouse tomato production.", "About 2 months following seeding, leaves and stems are inoculated with a suspension of 1×104 spores of P. Infestans until the plant surfaces are completely covered.", "Treatments are then applied.", "A positive control, i.e.", "a commercially available fungicide and a negative control, i.e.", "water are also tested and all treatments are done in a randomized block design.", "Percent damage or presence of lesions is evaluated every 3-4 days for a period of 2 weeks on leaves that had been identified previously (15-30 leaves per plant).", "The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.", "On potato plants.", "Potato tubers are sown and grown in pots of 6-8 inches.", "About 1,5 months after seeding, the leaves and stems of the plants are inoculated with a suspension of 1×104 spores of P. Infestans until the plant surfaces are completely covered.", "Treatments are then applied.", "A positive control, i.e.", "a commercially available fungicide and a negative control, i.e.", "water are also tested and all treatments are done in a randomized block design.", "Percent damage or presence of lesions is evaluated every 3-4 days for a period of 2 weeks on leaves that had been identified previously (15-30 leaves per plant).", "The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.", "3.Formulations of the Essential Oil Extract Formulations containing the essential oil extracts of the present invention can be prepared by known techniques to form emulsions, aerosols, sprays, or other liquid preparations, dusts, powders or solid preparations.", "These types of formulations can be prepared, for example, by combining with pesticide dispersible liquid carriers and/or dispersible solid carriers known in the art and optionally with carrier vehicle assistants, e.g., conventional pesticide surface-active agents, including emulsifying agents and/or dispersing agents.", "The choice of dispersing and emulsifying agents and the amount combined is determined by the nature of the formulation, the intended form of application of the formulation to a specific environment (e.g., plant, animal, soil, building), and the ability of the agent to facilitate the dispersion of the essential oil extract of the present invention while not significantly diminishing the pesticidal, acaricidal, insecticidal, and/or fungicidal activity of the essential oil extract.", "Non-limiting examples of conventional carriers include liquid carriers, including aerosol propellants which are gaseous at normal temperatures and pressures, such as Freon; inert dispersible liquid diluent carriers, including inert organic solvents, such as aromatic hydrocarbons (e.g., benzene, toluene, xylene, alkyl naphthalenes), halogenated especially chlorinated, aromatic hydrocarbons (e.g., chloro-benzenes), cycloalkanes (e.g., cyclohexane), paraffins (e.g., petroleum or mineral oil fractions), chlorinated aliphatic hydrocarbons (e.g., methylene chloride, chloroethylenes), alcohols (e.g., methanol, ethanol, propanol, butanol, glycol), as well as ethers and esters thereof (e.g., glycol monomethyl ether), amines (e.g., ethanolamine), amides (e.g., dimethyl sormamide), sulfoxides (e.g., dimethyl sulfoxide), acetonitrile, ketones (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone), and/or water; as well as inert dispersible finely divided solid carriers such as ground natural minerals (e.g., kaolins, clays, vermiculite, alumina, silica, chalk, i.e., calcium carbonate, talc, attapulgite, montmorillonite, kieselguhr), and ground synthetic minerals (e.g., highly dispersed silicic acid, silicates).", "Surface-active agents, i.e., conventional carrier vehicle assistants, that can be employed with the present invention include, without limitation, emulsifying agents, such as non-ionic and/or anionic emulsifying agents (e.g., polyethylene oxide esters of fatty acids, polyethylene oxide ethers of fatty alcohols, alkyl sulfates, alkyl sulfonates, aryl sulfonates, albumin hydrolyzates, and especially alkyl arylpolyglycol ethers, magnesium stearate, sodium oleate); and/or dispersing agents such as lignin, sulfite waste liquors, methyl cellulose.", "Emulsifiers that can be used to solubilize the essential oil extracts of the present invention in water include blends of anionic and non-ionic emulsifiers.", "Examples of commercial anionic emulsifiers that can be used include, but are not limited to: Rhodacal™ DS-10, Cafax™ DB-45, Stepanol™ DEA, Aerosol™ OT-75, Rhodacal™ A246L, Rhodafac™ RE-610, and Rhodapex™ CO-436, Rhodacal™ CA, Stepanol™ WAC.", "Examples of commercial non-ionic emulsifiers that can be used include, but are not limited to: Igepal™ CO-887, Macol™ NP-9.5, Igepal™ CO-430, Rhodasurf™ ON-870, Alkamuls™ EL-719, Alkamuls™ EL-620, Alkamide™ L9DE, Span™ 80, Tween™ 80, Alkamuls™ PSMO-5, Atlas™ G1086, and Tween™ 20, Igepal™ CA-630, Toximul™ R, Toximul™ S, Polystep™ A7 and Polystep™ B1.If desired, colourants such as inorganic pigments, for example, iron oxide, titanium oxide, and Prussian Blue, and organic dyestuffs, such as alizarin dyestuffs, azo dyestuffs or metal phthalocyanine dyestuffs, and trace elements, such as salts of iron, manganeses, boron, copper, cobalt, molybdenum and zinc may be used.", "Spreader and sticking agents, such as carboxymethyl cellulose, natural and synthetic polymers (e.g., gum arabic, polyvinyl alcohol, and polyvinyl acetate), can also be used in the formulations.", "Examples of commercial spreaders and sticking agents which can be used in the formulations include, but are not limited to, Schercoat™ P110, Pemulen™ TR2, and Carbose™ 514 H, Umbrella™, Toximul™858 and Latron™CS-7.Time-release formulations are also contemplated by the present invention.", "For example, formulations which have been encapsulated and/or pelletized.", "In one embodiment, the formulation is a sprayable ready-to-use (RTU) formulation suitable, for example, for delivery by fogging, aerosol spraying, and pump spraying methods for application in a variety of environments.", "In a further embodiment, the formulation can also be prepared as an emulsifiable concentrate (EC) which can be diluted before use.", "In one embodiment, the emulsifiable concentrate comprises between 5% to 50% (by volume) Chenopodium-derived essential oil extract in combination with a suitable emulsifier, carrier, and spreader and/or sticking agent to enable application of the formulation to a specific environment.", "In another embodiment, the emulsifiable concentrate comprises between 10% to 25% (by volume) Chenopodium-derived essential oil extract in combination with a suitable emulsifier, carrier, and spreader and/or sticking agent to enable application of the formulation to a specific environment.", "In a further embodiment, the emulsifiable concentrate comprises between 10% to 25% (by volume) Chenopodium-derived essential oil extract in combination with between 1% to 15% (by volume) of a suitable emulsifier, and between 50% to 70% (by volume) of a suitable carrier or solvent to enable application of the formulation to a specific environment.", "In another embodiment, the emulsifiable concentrate may also comprise 2% to 20% (by volume) of a suitable spreader and/or sticking agent.", "The person skilled in the art, however, will understand that these concentrations can be modified in accordance with particular needs so that the formulation is acaricidal, insecticidal, and/or fungicidal, but not phytotoxic.", "Non-limiting examples of suitable emulsifiers that can be used in preparing emulsifiable concentrates of the present invention include: Rhodapex™ CO-436, Rhodapex™ CO-433, Igepal™ CO-430, Igepal™ CA-630, Igepal™ CO-887, Isopropanol, canola oil, Alkamuls™ EL-719, Rhodacal™ DS-10, Macol™ NP-9.5, Tergitol™ TMN-3, Tergitol™ TMN-6, Tergitol™ TMN-10, Morwet™ D425, and Tween™ 80.Suitable carriers or solvents that may be used include, but are not limited to, Isopar™ M, THFA™, ethyl lactate, butyl lactate, Soygold™ 1000, M-Pyrol, Propylene glycol, Agsolex™ 12, Agsolex™ BLO, Light mineral oil, Polysolve™ TPM, and Finsolv™ TN.", "Examples of suitable spreaders and/or sticking agents include, but are not limited to, Latex emulsion, Umbrella™, Adsee™ 775, Witconol™ 14, Toximul™ 858, Latron™ B-1956, Latron™ CS-7, Latron™ AG-44M, T-Mulz™ AO-2, T-Mulz™ 1204, Silwet™ L-774.Formulations containing the essential oil extracts of the present invention can be prepared by known techniques to enable application to specific environments.", "Plant Formulations The formulation can be prepared for application to plants and plant environments, for example, household/domestic plants, greenhouse plants, agricultural plants, and horticultural plants.", "In one embodiment, the formulation contains a final concentration of between 0.125% to 10% (by volume) of the Chenopodium-derived essential oil extract in combination with a suitable emulsifier, carrier, and spreader and/or sticking agent to enable sprayable application to a plant.", "In another embodiment, the formulation contains between 0.25% to 5% (by volume) of the Chenopodium-derived essential oil extract in combination with a suitable carrier, emulsifier, and spreader and/or sticking agent, to enable sprayable application to a plant.", "Fumigant Formulations for Closed and/or Open Environments The formulation can also be prepared for application as a fumigant for both outdoor as well as indoor application, for example in closed environments, such as greenhouses, animal barns or sheds, human domiciles, and other buildings.", "Persons of skill in the art will appreciate the various methods for preparing such fumigants, for example, as fogging concentrates and smoke generators.", "A fogging concentrate is generally a liquid formulation for application through a fogging machine to create a fine mist that can be distributed throughout a closed and/or open environment.", "Such fogging concentrates can be prepared using known techniques to enable application through a fogging machine.", "For example, the formulation may have the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-15 Spreader/sticker 1-5 Organic solvent To 100% Smoke generators, which are generally a powder formulation which is burned to create a smoke fumigant.", "Such smoke generators can also be prepared using known techniques.", "For example, the formulation may have the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-10 Absorbent silica 3-5 Pyrotechnic ingredients 3-10 Free flow aid 1-5 Filler To 100% Animal Formulations The formulation can be prepared in a form suitable for application to animals and animal environments, such as barns or sheds.", "In one embodiment, the formulation is prepared in a form suitable for topical application on an animal for controlling insects, acari, pests and fungi.", "Persons of skill in the art will appreciate the various methods for preparing such topical formulations, for example as powders or sprayable formulations.", "In one embodiment, the topical formulation is an aerosol and may have the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-5 Emollient (coat conditioner) 2-5 Stabilizer 0.1-1 Preservative 0.1-0.5 Organic solvent To 100% In another embodiment, the formulation is a water-based pump spray formulation having the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-10 Emulsifier(s) 2-10 Coat conditioner 2-5 Stabilizer 0.1-1 Preservative 0.1-1 Antioxidant 0.1-0.5 Water To 100% In a further embodiment, the formulation is an aerosol formulation for application to an animal environment, such as a barn or shed, having the following general composition: Ingredient % Chenopodium extract (active ingredient) 3-10 Stabilizer 0.1-1 Preservative 0.1-0.5 Organic solvent To 100% Human Formulations The formulation can be prepared in a form suitable for topical application on humans, for example as a repellent.", "Persons of skill in the art will appreciate the various methods for preparing such topical formulations, for example as lotions or sprayable formulations.", "In one embodiment, the formulation is a solvent-based sprayable formulation having the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-5 Emollient (skin conditioner) 2-5 Stabilizer 0.1-1 Organic solvent To 100% In another embodiment, the formulation is a water-based sprayable formulation having the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-5 Emulsifier(s) 1-5 Skin conditioner 2-5 Stabilizer 0.1-1 Preservative 0.1-1 Antioxidant 0.1-0.5 Water To 100% In a further embodiment, the formulation is a topical lotion formulation having the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-10 Emulsifier(s) 1-10 Skin conditioner 2-10 Thickener(s) 0.2-5 Stabilizer 0.1-1 Preservative 0.1-1 Antioxidant 0.1-0.5 Water To 100% Soil Formulations In a further embodiment, the formulation of the present invention can be prepared as a microemulsion.", "Microemulsions are low-viscosity, optically transparent dispersions of two immiscible liquids which are stabilized by at least one ionic or nonionic surfactant.", "In the case of microemulsions, the particle diameters are in the range from about 5-100 nm suspended in a continuous phase.", "The interfacial tension between the two phases is extremely low.", "The viscosity of many microemulsions of the oil and water type (O/W) is comparable with that of water.", "In contrast to microemulsions, “macroemulsions” have high viscosities and their particle diameter is in the range from about 10 to 100 micrometers.", "Macroemulsions are milky white in color and, upon heating, tend toward phase separation or toward sedimentation of the dispersed substances.", "It is commonly believed that pesticidal microemulsions can provide superior efficacy relative to macroemulsion formulas having the same levels of active ingredients.", "It is believed that the small size of the emulsion droplets may allow for better transport of the pesticide through cell membranes (plant and insect) thereby resulting in enhanced efficacy.", "Microemulsions are considered to be infinitely stable, thereby providing improved stability over traditional macroemulsion systems.", "Accordingly, microemulsion formulations of the present invention may be particularly suitable for certain applications, for example soil delivery.", "Microemulsion formulations of the present invention can be prepared using various known methods known in the art.", "In one embodiment, the essential oil extract of the present invention can be combined with a combination of emulsifiers to create a microemulsion.", "For example, a microemulsion can be prepared in a three phase process.", "Phase 1: Selection of Emulsifiers A selection of potential primary emulsifiers is tested for miscibility with the active ingredient.", "Solubility is evaluated by visually assessing for a clear stable solution.", "Each pesticide/emulsifier mixture is mixed with the required volume of water and the quality of emulsion/microemulsion produced is recorded.", "Emulsifiers producing the best emulsions are taken through into phase 2.Emulsion/microemulsion quality is determined by visual assessment.", "Transparent microemulsions are the best, white emulsions with a bluish hue are second, plain white emulsions are next and any emulsion showing phase separation is last.", "Phase 2: Selection of Secondary and Tertiary Emulsifiers Upon completion of phase 1, the most promising primary emulsifier systems are blended with other emulsifiers in various ratios until an emulsifier blend is identified which provides the desired physical properties.", "The optimized system generally consists of one main emulsifier and two or more co-emulsifiers/co-solvents.", "These co-emulsifiers/co-solvents tend to broaden the thermal phase stability range of the microemulsion system.", "Ternary phase diagrams are used in this phase of development to assist in the selection of emulsifier ratios.", "The best formulation at this stage is often one containing one anionic emulsifier and two non-ionics of different types.", "The criteria for assessing the microemulsion properties are again physical appearance as a function of time and temperature.", "Phase 3: Optimization of Emulsifier Levels Having established the best emulsifier combination, the final phase is optimizing the levels and ratios of each emulsifier component in the system.", "In one embodiment, the microemulsion comprises between 30% to 50% (by volume) Chenopodium-derived essential oil extract, between 0.5% to 25% (by volume) of a suitable emulsifier, and between 10% to 50% water.", "Non-limiting examples of emulsifiers that can be used in preparing microemulsions of the present invention include: Igepal™ CA-630, Rhodasurf™ ON-870, Alkamuls™ EL-719, Tween™ 80, Alkamuls™ PSMO-5, Rhodapex™ CO-436, Rhodafac™ RE-610, Rhodacal™ CA, Ammonyx™ CO, Aeros™ OT-S, Ammonyx™ LO, Stepanol™ WAC, Soprophor™ BSU, and Rhodaca™1 IPAM.", "It is contemplated that the formulations of the essential oil extract can be combined with a controlled release delivery system in order to time-release the bioactive agents.", "Such controlled release delivery systems include methods, known in the art, of encapsulation, dissolution, or incorporation of the active ingredient.", "It is further contemplated that the formulations of the present invention can be prepared in combination with nutrients (fertilizers) or herbicides.", "Thus the skilled artisan will appreciate that the instant invention may be further formulated to provide various dissolution rates and/or be prepared in combination with nutrients (fertilizers) or herbicides.", "4.Use of Essential Oil Extract Formulations The essential oil extracts of the present invention can be used for controlling pests by applying a pesticidally effective amount of the essential oil extract and/or formulation of the present invention to the locus to be protected.", "The essential oil extract formulations can be applied in a suitable manner known in the art such as, for example, spraying, atomizing, vaporizing, scattering, dusting, watering, squirting, sprinkling, pouring, fumigating, and the like.", "Loci, for the application of essential oil extracts or formulations thereof include, but are not limited to, agricultural, horticultural, forest, plantation, orchard, nursery, organically grown crops, turfgrass and urban environments.", "Specifically, the essential oil extracts or formulations thereof may be applied to soil or plants/trees at one or several sites including the leaves, petioles, stems, seeds, roots, flower, cones, bark, wood or tubers.", "It is further contemplated that the essential oil extract may be formulated for seed treatment either as a pre-treatment for storage or sowing.", "For example, the seed may form part of a pelleted composition or, alternatively, may be soaked, sprayed, dusted or fumigated with a formulation of the present invention.", "The essential oil extract, and formulations thereof, can be applied to the surface of the soil to control soil surface or soil-inhabiting pests.", "The essential oil extract of the present invention can also be used as part of an Organic Production system and Integrated Pest Management program.", "For example, in conjunction with augmentation of beneficial insects and mites.", "It is further contemplated that the essential oil extract, and formulations thereof, can be applied to ‘fertigation’ i.e.", "fertilization via the irrigation system of plants in greenhouses.", "For example, an essential oil microemulsion can be added to the water in small concentrations (0.1-0.5% AI) and as water is being irrigated to individual plants, the latter would be treated for soil-inhibating pests (insects and disease pathogens).", "5.Effect of the Essential Oil Extract or Formulations on Beneficial Insects and Mites Natural enemies of phytophagous pests include both predators and parasitoid.", "Predators are generally as large, or larger than the prey they feed on.", "They are quite capable of moving around to search out their food, and they usually consume many pests during their lifetime.", "Parasitoids, or parasitic insects, are smaller than their prey.", "One or more parasitoids grow and develop in or on a single host.", "The host is slowly destroyed as the parasitic larva(e) feed and mature.", "Such beneficial insects and mites can help prevent or delay the development of pesticide resistance by reducing the number of pesticides required to control a pest.", "They will also feed on the resistant pests that survive a pesticide application.", "Integrated pest management (IPM) programs take advantage of the biological pest control provided by beneficial insects and mites by conserving or augmenting natural enemies.", "When chemical controls are necessary in an IPM program, pesticides recommended are those that have minimal impact on naturally occurring beneficials.", "Essential oil extracts of the present invention, and formulations thereof, may be tested for their effect on beneficial insects and mites, i.e., predators and parasitoids, by means of standardized IOBC (International Organization for Biologicial Control) testing methods as illustrated in Example XIV for integration into IPM programs.", "The invention now being generally described, it will be more readily understood by references to the following examples, which are included for purposes of illustration only and are not intended to limit the invention unless so stated.", "EXAMPLES Example I Phytochemical Profile of an Essential Oil Extract Derived from Chenopodium ambrosioides Whole plants of C. ambrosioides were harvested.", "Plant material used for extraction purposes comprised the whole plant above root.", "Essential oil extracts were extracted from the plant material by steam distillation, i.e., distillation in water (DW) and/or direct steam distillation (DSD).", "Distillation in water was carried out in a 380 L distillator with a capacity for processing ca.", "20 kg of plant material.", "During the process of DW, plant material was completely immersed in an appropriate volume of water which was then brought to a boil by the application of heat with a steam coil located at the base of the still body.", "In DSD, the plant material was supported within the still body and packed uniformly and loosely to provide for the smooth passage of steam through it.", "Steam was produced by an external generator and allowed to diffuse through the plant material from the bottom of the tank.", "The rate of entry of the steam was set at (300 ml/min).", "With both methods, the oil constituents are released from the plant material and with the water vapor are allowed to cool in a condenser to separate into two components, oil and water.", "The essential oil extracts were analyzed by capillary gas chromatography (GC) equipped with a flame ionization detector (FID).", "GC was carried out using a Varian 6000 series Vista and peak areas were computed by a Varian DS 654 integrator.", "SPB-1 (30 m×0.25 mm φ, 0.25 μm) and Supelcowax (30 m×0.25 mm φ, 0.25 μm) fused silca columns were used.", "Compounds in the sample come off the column at different times in minutes (Rt's or Retention Times) and these are compared to known standards and the compounds can thus be identified.", "When GC-FID gave ambiguous identification of certain compounds, Mass Spectrometry (MS) was used to compare the mass spectra of the compounds with a database of known spectra.", "The relative amount of each component of the essential oil extracts was determined for different lots of a variety of C. ambroisiodes.", "Each lot represents pooled extractions taken from a crop within one harvest date.", "FIG.", "1 shows the phytochemical profile of the essential oil extract taken from three different lots.", "Lot No.", "00MC-21P indicates an ascaridole content of 9.86%; Lot No.", "00MC-24P has an ascaridole content of 6.39% and 00MC-29P has an ascaridole content of 3.6%.", "The activity of the extract is not apparently affected by the variability in relative amount of ascaridole as results from bioassays with these lots suggest.", "Example II Determination of the Active Constituents of the Essential Oil Extract Extensive testing was done in order to determine the active constituents of the essential oil extract.", "All compounds present in the oil were tested except for trans-ρ-mentha-2,8dien-1-ol and cis-ρ-mentha-2,8-dien-1-ol because they were unavailable.", "All compounds tested were obtained commercially (Sigma-Aldrich) except for ascaridole and iso-ascaridole that were isolated from a sample of our extract by Laboratoires LaSeve, Chicoutimi Qc.", "Acaricidal Activity Tests with the Two-Spotted Spider Mite (TSSM: Tetranychus urticae) To test acaricidal activity, thirty adult female mites were placed on their dorsum with a camel hair brush on a double-sided sticking tape glued to a 9 cm Petri dish.", "Three dishes were prepared for each concentration of each compound tested and the control (e.g., water) for a total of 90 mites per treatment per treatment day.", "One (1) ml of each preparation and of microfiltered water as control was added with a Gilson Pipetman™ P-1000 to the reservoir of the spray nozzle of a Potter Spray Tower mounted on a stand and connected to a pressure gauge set at 3 P.S.I.", "Petri dishes were weighed before and immediately after each application to calculate the amount of oil deposited (mg/cm2) with each sample tested.", "The entire procedure was followed three times to give a total number of 270 mites tested with each treatment.", "Mite mortality was assessed 24 and 48 h after treatment.", "Mites that failed to respond to probing with a fine camel hair brush with movements of the legs, proboscis or abdomen were considered dead.", "Individual compounds were tested at 0.125, 0.50, 1.0 and 2.0% concentrations with the two-spotted spider mite (TSSM: Tetranychus urticae).", "Results are illustrated in FIG.", "2.Comparisons were made with mortality data obtained with the 1% concentration of each compound and it was observed that carvacrol is the most active compound (90% mortality of TSSM) followed by carveol (82% mortality), nerol (82% mortality), thymol (78% mortality), carvone (78% mortality) and α-terpineol (71% mortality).", "Other compounds gave less than 40% mortality.", "No mortality was recorded for ascaridole at 1% .", "Although 3% mortality was obtained with a solution of 0.125% ascaridole, we believe that this is an erroneous udependable result because too few individuals were tested (n=125) and the standard deviation is high (13), compared to the higher number of individuals tested at the higher concentrations of this compound (n=300 each at 0.5% and 1.0%) where no mortality was recorded.", "The results obtained with individual compounds, do not indicate that the compounds present in large quantities in the oil, i.e α-terpinene, ρ-cymene, limonene, ascaridole, iso-ascaridole, have a great impact on the biological activity of the extract.", "Mortality obtained with each of these compounds tested at 1% concentration was 17% or less.", "Ascaridole and iso-ascaridole at 1% concentration had no effect on the spider mite (0% mortality).", "Carvacrol, carveol, nerol, thymol and carvone on the other hand may have a much greater impact on the activity of the oil (>70% of TSSM at a 1% concentration) even though each of these compounds are present in relatively small quantities (<1%) Insecticidal Activity Tests with the Greenhouse Whitefly (GWF: Trialeurodes vaporariorum) Tests were also done using compounds that had demonstrated the higher degree of activity, i.e.", "carvacrol, nerol and thymol with the greenhouse whitefly (Trialeurodes vaporariorum) our model bioassay for insecticidal effect.", "Whitefly adults were glued to a black 5 cm×7,5 cm plastic card sprayed with Tangle-Trap® (Gempler's Co.) by placing cards directly in the greenhouse colony cage until at least 20 adults have alighted on each card.", "Cards were observed before spraying under the binocular scope to remove all dead and immobile whiteflies.", "Only active whiteflies were kept for the experiment.", "Four cards were used per treatment.", "Each card was sprayed at 6 psi with 300 μl of emulsion using a BADGER 100-F® (Omer DeSerres Co., Montréeal, Canada) paintbrush sprayer mounted on a frame at a distance of 14.5 cm from the spray nozzle in an exhaust chamber.", "Cards were weighed immediately before and after spraying to calculate the amount of active ingredient deposited in mg/cm2.Cards were allowed to dry under the exhaust chamber and then placed sideways on a Styrofoam rack in a closed clear plastic container of 5 L with moistened foam on the bottom to keep humidity high (>90% R.H.).", "The plastic container was stored in a growth chamber at 24° C. and 16 L:8 D photoperiod.", "This procedure was repeated three times.", "Mortality was evaluated 20 hours following treatment by gently probing the whitefly with a single-hair brush under the binocular microscope.", "Absence of movement (antennae, leg, wing) following probing was recorded as dead.", "Relative efficacy of the compounds were compared by transforming mortality data to arcsin{square root}p and then subjecting to an ANOVA analysis using SAS® software (SAS Institute 1988).", "Results with the GWF, shown in FIG.", "3, confirm the important biological activity of these three compounds.", "Example III Ready-To-Use Acaricidal Formulations A ready-to-use (RTU) sprayable insecticidal formulation having as the active ingredient an extract of Chenopodium was prepared.", "In one embodiment, this formulation contains between 0.125% and 10% (by volume) of the essential oil extract, an emulsifier, a spreader and sticking agent, and a carrier.", "Examples of basic RTU formulations are as follows.", "Ingredient Amount (%) Amount (%) Amount (%) Essential oil 1.00 1.00 1.00 extract Rodacal IPAM 0.50 0.83 0.83 Igepal CA-630 — 0.50 — Macol NP 9.5 — — 0.50 Water 98.5 97.67 97.67 Examples of formulations with added polymers for added residual effect are as follows.", "Ingredient Amount (%) Amount (%) Amount (%) Essential oil extract 1.00 1.00 1.00 Rhodacal IPAM 0.83 0.83 0.83 Igepal CA-630 0.50 0.50 0.50 Carboset 514H 2.00 — — Pemulen TR2 — 0.05 — Schercoat P110 — — 5.00 Propylene glycol — 2.00 — Water 95.67 95.62 92.67 Example IV Acaricidal Efficacy of the Essential Oil Extract (RTU Formulation) Efficacy trials were conducted using a Ready-to-use (RTU) formulation of the present invention comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water.", "Thirty adult female mites were placed on their dorsum with a camel hair brush on a double-sided adhesive tape glued to a 9 cm Petri dish.", "Three dishes were prepared for each concentration of each formulations or products tested and the control, (e.g.", "water), for a total of 90 mites per treatment per treatment day.", "One (1) ml of each preparation and of microfiltered water as control was added with a Gilson Pipetman™P-1000 to the reservoir of the spray nozzle of a Potter Spray Tower mounted on a stand and connected to a pressure gauge set at 3 P.S.I.", "Petri dishes were weighed before and immediately after each application to calculate the amount of oil deposited (mg/cm2) with each sample tested.", "The ready-to-use formulation was tested according to the method mentioned above to identify the minimum concentration needed for the desired mortality (>95%) at different concentrations (00.125, 0.25, 0.5, 0.75, and 1%) in order to compare the relative efficacy of this RTU formulation and other acaricidal products (synthetic and natural) presently on the market.", "The entire procedure was followed three times to give a total number of 270 mites tested with each treatment.", "Mite mortality was assessed 24 and 48 h after treatment.", "Mites that failed to respond to probing with a fine camel hair brush with movements of the legs, proboscis or abdomen were considered dead.", "In order to obtain LC50 values (Lethal Concentration in mg/cm2 is the amount of product needed to kill 50% of the test organism; therefore the lower the LC50 value the more toxic the product) results of the 48 h counts were subjected to Probit analysis using POLO computer program (LeOra Software, 1987).", "Mortalities were entered with corresponding weighed dose (mg/cm2) to take into consideration variability in the application rate.", "The results obtained with these bioassays are shown in FIG.", "4.Although the toxicity tests presented herein were performed with female mites, it will be clear to a person skilled in the art that those results show that the mortality that would have been observed for male mites would have been the same if not higher knowing that male mites are smaller than females.", "Example V Effect on the Egg and Nymphal Stages of the Spider Mite (RTU Formulation) The RTU formulation (comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water) was also tested on the egg and the nymphal stages of the spider mite.", "The ovicidal effect was determined with eggs of the twospotted spider mite following treatment with concentrations of the RTU formulation.", "Adult female T. urticae are transferred to 2 cm diameter leaf disks cut out of lima bean leaves and left for four hours for oviposition.", "When at least 20 eggs/disk are laid, adult mites are then removed.", "Leaf disks are moist and then sprayed and Petri dishes are weighed before and after treatment and stored after treatment.", "Egg hatch is assessed daily and for 10 days following treatment by counting the number of eggs remaining on the leaf disks and the number of live and dead nymphs present.", "Percent egg hatch is determined with live nymphs only.", "The nymphs are considered dead if no movement is observed after repeated gentle probing with a single-hair brush.", "Results of the test on the egg stage (FIG.", "5) indicate that the RTU formulation has some effect on the eggs with 30% mortality using a 0.5% solution of the oil.", "It is expected that a higher concentration of the oil should show greater efficacy on eggs.", "Similarly to the effect of the RTU formulation on the nymphal stage, even at the 0.5% concentration, the RTU gave higher results (95.8%) than the existing commercial preparations of either Avid (80.1%) or Safer Soap (61.7%) (FIG.", "6).", "Example VI Residual Effect of the RTU Formulations of the Present Invention and Comparison Thereof with Commercially Available Acaricidal Products The residual effect of the RTU formulation (comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water) was also tested with the spider mite and compared to natural and synthetic products already on the market, (i.e.", "Kelthane™, Avid™, Safer's™ Soap and Wilson's dormant oil).", "The procedure for this test involved the preparation of vials containing a nutrient solution in which individual faba bean leaves were placed.", "Eighteen leaves were prepared for each concentration tested and each were sprayed with the indicated concentration until run-off and allowed to dry.", "Ten spider mites were placed on nine of the leaves one hour after spraying and ten were placed on the other nine leaves one day following treatment.", "Mortality was observed 24 and 48 hr following mite introduction on the leaves.", "The entire procedure was repeated three times.", "The results of the residual effect of the different products when the mite is introduced on the plant one hour following treatment are shown in FIG.", "7.These results indicate that there is a residual effect of the RTU and that this effect is greater than in the Safer product.", "However, it is inferior to the residual effect of synthetic products such as Kelthane and Avid.", "These results show the RTU formulation's very low persistence in the environment (about 23 mortality of spider mites when the pest is introduced on the plant one hour after treatment with the product).", "The RTU formulation is therefore compatible with the recommendations of the Integrated Pest Management program which supports control methods that do not harm natural enemy populations and permit rapid re-entry of workers to the tested area and uninterrupted periods of harvest while assuring safety to workers and consumers.", "Example VII Acaricidal Activity of the Extracts on Other Acari (RTU Formulation) To confirm the efficacy of the formulations of the present invention on plant infesting acari in general, certain bioassays were performed on another plant infesting mite, the European red mite, Panonychus ulmi, a mite which shows a close taxonomical relationship with T. Urticae.", "The RTU formulation (comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water) was thus tested on the red mite Panonychus ulmi, a pest of apple orchards, following the same protocol described for contact efficacy on adult spider mites in order to confirm its broad effect as an acaricide.", "The results confirm the effectiveness of the essential oil extract as a contact acaricide (FIG.", "8) which is not exclusively active on T. Urticae.", "Example VIII Insecticidal Efficacy of the Essential Oil Extract (RTU Formulation) Similar efficacy tests were also performed on several insect species that are serious pests of cultivated plants.", "The species tested were the greenhouse whitefly, Trialeurodes vaporariorum; the Western flower thrips, Frankliniella occidentalis; the green peach aphid, Myzus persicae; and the silverleaf whitefly, Bermisia argentifolii following the same protocol described in Example XI (C) below.", "Results presented in FIG.", "9 indicate that the RTU product (comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water) is toxic to all organisms tested.", "LC50 could be calculated for the greenhouse whitefly and the green peach aphid and results (LC50 of 0.00131 mg/ cm2 and 0.0009 mg/cm respectively) show that the product is as or more effective to these insects as the spider mite.", "Example IX Emulsifiable Concentrate Formulation An emulsifiable concentrate formulation with an extract of Chenopodium ambrosioides was also prepared.", "The concentrate contains between 10 to 25% essential oil extract, emulsifiers, a spreader/sticker, and a carrier.", "Examples of emulsifiable concentrate formulations are as follows.", "Amount Amount Amount Amount Amount Amount Ingredient (%) (%) (%) (%) (%) (%) Essential 25 25 25 25 25 25 oil extract Rhodopex 5 2.5 — — 1.25 — CO-436 Rhodopex — — — — — — CO-433 Igepal CO- — 2.5 — — 1.25 2.5 430 Igepal CA- — — 5 2.5 — — 630 Igepal CO- — — — 2.5 — — 887 Isopropanol — — 10 — — — Isopar M — — 60 70 — — Macol NP — — — — — 2.5 95 THFA 70 70 — — 72.5 70 Example X Acaricidal Efficacy of the Essential Oil Extract (Emulsifiable Concentrate Formulation) Contact and residual bioassays were conducted in the laboratory to test the efficacy of the essential oil extract of the present invention.", "A 25% emulsifiable concentrate (EC25%) formulation of oil was tested against the adult and eggs of the twospotted spider mite and the European red mite.", "The EC25% formulation tested comprised: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA.", "The twospotted spider mite was reared on Lima bean plants (Phaseolus sp.)", "and the European red mite on apple leaves cv McIntosh (Malus domestica Borkhausen).", "Contact Efficacy with the Adult Stage The methodology used for adults was the same for both species.", "Twospotted spider mite adults were treated with four concentrations of oil of a North American herbaceous plant (0.125, 0.25, 0.5 and 1.0% active ingredient (AI) EC25%; Urgel Delisle et Associes, Saint-Charles-sur-Richelieu, QC, Canada), neem oil (Neem Rose Defense® EC 90%; Green Light, San Antonio Tex, USA) at 0.7% AI, insecticidal soap (Safer's Trounce® EC 20% potassium salts of fatty acids with 0.2% pyrethrins; Safer Ltd. Scaborough, ON, Canada) at 1% AI and a water control.", "European red mite.", "adults were treated with five concentrations (0.0312, 0.0625, 0.125, 0.25 and 0.5%) of EC25%, abamectin (Avid® EC1.9%; Novartis, Greensboro, N.C., USA) at 0.006% AI and a water control.", "Twenty-five mature female mites were deposited dorsally on a 1 cm2 piece of double-coated tape glued on a glass microscope slide.", "For each treatment period, four slides were prepared for each treatment or acaricide application as defined above.", "Solutions for each treatment were prepared on the treatment day and each slide was sprayed at a pressure of 0.42 kg/cm2 under an exhaust chamber with 250 μl of solution using a Badger 100-F® paint brush sprayer (Badger Air-Brush Co., Franklin Park, Ill., USA) mounted on a frame at a distance of 15 cm from the slide.", "The slides were weighed immediately before and after spraying to calculate the amount of active ingredient deposited per surface area (mg/cm2); this quantity varied less than 15% between slides.", "After spraying, the slides were placed on a styrofoam rack in a closed clear plastic container with a wet foam at the bottom to keep moisture high (90% R.H.).", "The container was stored in a growth chamber at 24° C. and 16 L:8 D photoperiod.", "This experimental procedure was repeated on three consecutive days in a complete block design where treatment period was considered a block.", "Mortality was assessed under a binocular microscope 48 (twospotted spider mite) and 24 hours (European red mite) following treatment.", "Because European red mite mortality in the control group at 48 hours was high, it was judged to be inadequate for statistical evaluation.", "Mites were considered dead if movement was imperceptible after repeated gentle probing with a single-hair brush.", "Data were transformed by arcsin{square root}p and subjected to an ANOVA statistical analysis using SAS® software (SAS Institute, 1988).", "The LC50 and LC90 (in mg/cm2 of AI) of EC25% were calculated with PROBIT analysis using POLO-PC® software (LeOra Software, 1987).", "EC25% at 1% concentration and insecticial soap at 1% were most effective at controlling the adult twospotted spider mites causing 99.2 and 100% mortality respectively (FIG.", "10).", "At 0.5, 0.25 and 0.125% EC25% resulted in 94.7, 76.8 and 68% mortality respectively.", "The least effective treatment was neem oil, which at the recommended dose caused only 22.1% mortality.", "The LC50 and LC90 of EC25% for the twospotted spider mite were 0.009 mg/cm2 (99% confidence interval 0.0082-0.0099 mg/cm2) and 0.0292 mg/cm2 (99% confidence interval 0.0268-0.0321 mg/cm2) respectively (significant at P=0.01).", "In comparison, the LC50 of insecticial soap had been determined by the manufacturer to be 0.016 mg/cm2.At 0.5% concentration, EC25% was significantly more toxic (97.1% mortality) to P. ulmi adults than abamectin (82.4%) (FIG.", "11).", "Treatments with EC25% at concentrations ranging from 0.0625 to 0.25% gave statistically the same control level as abamectin.", "The LC50 and LC90 of EC25% for the red spider mite were 0.0029 mg/cm2 (99% confidence interval 0.0019-0.0038 mg/cm2) and 0.014 mg/cm2 (99% confidence interval 0.0108-0.0203 mg/cm2).", "EC25% gave <80% control of the adult stage of the two mites species at low doses.", "Ovicidal Activity The ovicidal effect of the following products was determined with eggs of the twospotted spider mite and the European red mite: six concentrations of EC25% (0.0625, 0.125, 0.25, 0.5, 1 and 2%), neem oil at 0.7% AI, insecticidal soap at 1% AI and abamectin at 0.006% and a water control.", "Twenty adult female T. urticae were transferred to 2 cm diameter leaf disks cut out of lima bean leaves and left for four hours for oviposition.", "Female P. ulmi were left for 24 hours to lay their eggs on 2 cm diameter leaf disks of apple leaves.", "When at least 20 eggs/disk were laid, adult mites were then removed with a soft brush Leaf disks were kept on moist soft cotton swabs placed in small (4 cm diameter) plastic Petri dishes.", "Three leaf disks were prepared for each treatment or acaricide application.", "Leaf disks were sprayed and Petri dishes were weighed before treatment and stored after treatment as for the slides used in the bioassay with adults.", "This experimental procedure was repeated on three consecutive days in a complete block design where treatment period was considered a block.", "Egg hatch was assessed daily and for 10 days following treatment by counting the number of eggs remaining on the leaf disks and the number of live and dead nymphs present.", "Percent egg hatch was determined with live nymphs only.", "The nymphs were considered dead if no movement was observed after repeated gentle probing with a single-hair brush.", "All nymphs (alive and dead) were removed daily from the leaf disks.", "Percent egg hatch (number of nymphs/total number of eggs on leaf disk×100) were transformed with arcsin {square root} and subjected to an ANOVA statistical analysis using SAS® software (SAS Institute, 1988).", "Egg hatch for the twospotted spider mite was significantly reduced by abamectin (8.0% egg hatch) and neem oil (2.1%) (FIG.", "12).", "Egg hatch was reduced to 67 and 40% with 1.0 and 2.0% concentrations of EC25% respectively and to 61.3% with insecticial soap.", "Egg hatch for the European Red mite was significantly reduced compared to the control treatment with the recommended doses of insecticial soap (27.2% egg hatch), abamectin (11.0%) and neem oil (14.2%) (FIG.", "13).", "Residual Bioassay with Adult Twospotted Spider Mites Leaf discs measuring 2 cm in diameter of bean leaves were sprayed on both sides with a VEGA 2000 sprayer (Thayer & Chandler Co., Lake Bluff, Ill., USA) at 0.42 kg/cm2 to runoff with 6.25 ml of each the following solutions: 2, 4, 8, and 16% of 99B-245, the recommended dose of dicofol (Kelthane® 35WP, Rohm and Haas Co., Philadelphia, Pa., USA) at 0.037% AI and a water control.", "Each treatment consisted of eight discs.", "One hour after treatment, 10 spider mites were transferred to each disc.", "Mortality was evaluated 48 hours following transfer of mites to the leaf discs.", "The procedure was repeated three times on three subsequent days.", "EC25% at 2, 4, 8 and 16% concentrations caused 23.0, 18.3, 13.9 and 32.5% mortality respectively to the adult spider mites when mites were introduced on bean leaves, 1 hr after treatment (FIG.", "14).", "Dicofol's residual activity was significantly higher (99.5% mortality) than any of the EC25% concentrations.", "EC25% was as effective as the insecticidal soap and synthetic acaricide abamectin to control adult twospotted spider mite and the European red mite.", "EC25% decreased egg hatch, but not as effectively as abamectin or neem oil.", "It may be important however to continue these investigations to determine the viability of emerged nymphs treated with the essential oil product because some botanicals, such as neem mixtures have shown growth-inhibiting properties to various pests (Rembald, 1989) and pulegone decreased larval growth of southern armyworm, Spodoptera eridania (Grunderson et al., 1985).", "Furthermore we demonstrated that when adult mites are introduced one hour after treatment, the mortality rate was statistically comparable to that of the control (FIG.", "14).", "A botanical such as EC25% may be an alternative to the more toxic or incompatible products.", "A contact acaricide with low residual activity can be used for treatments of localized infestations, before scheduled introductions of natural enemy populations or in absence of the natural enemy, i.e.", "treating at night in absence of diurnal parasitoids or predators.", "Plant essential oils may be phytotoxic (Isman, 1999).", "The oil used for EC25% was evaluated on several edible and ornamental plants for its phytotoxic effects and results indicate that at the recommended dose, i.e.", "0.5%, there were no observable effects on the leaves and flowers of tested plants (H. Chiasson, unpublished results).", "Example XI Insecticidal Efficacy of the Essential Oil Extract (Emulsifiable Concentrate Formulation) Efficacy trials were conducted (laboratory and small-scale greenhouse trials) using the emulsifiable concentrate formulation of the present invention (EC at 25% of chenopodium oil comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) with the following organisms: the green peach aphid (Myzus persicae), the Western flower thrips (Frankliniella occidentalis), the greenhouse whitefly (Trialeurodes vaporariorium) as well as the parasitoid Encarsia formosa.", "All bioassays were conducted in the laboratory of the Horticultural Research and Development Center (HRDC) of Agriculture and Agri-food Canada in Saint-Jean-sur-Richelieu, Quebec, Canada.", "A.", "Contact bioassays in the laboratory and greenhouse using EC25% and commercially available bioinsecticides with the green peach aphid (Myzus persicae (Sulz.))", "Laboratory Bioassay Five concentrations (0.125, 0.25, 0.5, 1 and 2%) of formulation EC25% were compared to commercial preparations of Neem Rose Defense® at 0.5% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1% (EC 20% with 0.2% pyrethrin) and a water control.", "Each treatment was repeated 12 times and each replicate consisted of a 2 month old shoot (10-15 cm) of Verbena speciosa ‘Imagination’ placed in a plastic Aqua-Pick® (tube used by florist to keep stems of cut flowers wet) filled with 10 ml of water.", "Aqua Picks were secured on a block of Styrofoam placed on the bottom of a 1 l transparent plastic container modified with screened sides and top to permit aeration.", "Green peach aphids (Myzus persicae (Sulz.))", "were collected in plastic containers from a rearing cage maintained in a greenhouse colony.", "Ten adults were transferred to each Verbena shoot.", "The shoot was sprayed at 8 psi under an exhaust chamber for about 15 seconds (long enough to cover the whole shoot) with a VEGA 2000® paintbrush sprayer equipped with a 20 ml reservoir (Thayer & Chandler Co., Lake Bluff, Ill., USA).", "Each shoot and plastic container was then stored in a growth chamber at 24° C., 65% R.H. and 16 L:8 N photoperiod.", "The entire procedure was repeated four times.", "Mortality was evaluated 48 hours following treatment by probing the aphid for movement with a small brush; absence of movement was recorded as dead.", "To evaluate the relative efficacy of EC25%, Neem Rose Defense® and Safer's Trounce®, percentage mortality data were transformed to arcsin{square root}p and subjected to ANOVA analysis using SAS® software (SAS Institute 1988).", "LC50 and LC90 were calculated using mortality results by PROBIT analysis using POLO-PC® software (LaOra Software 1994).", "Product concentrations (%) were used because data on quantity of active material deposited were not available.", "Results show that EC25% at 2.0% concentration was more effective (92.3% mortality) at controlling the green peach aphid than EC25% at 1% concentration (71.7%) and Safer's Trounce® (55.2%) though not significantly (FIG.", "15 ).", "This lack of distinction between treatments may be due to the low number (n) of aphids tested.", "Treatments with EC25% at concentrations of 0.5% and less and with Neem Rose Defense® resulted in <50% mortality of the aphids and results were not significantly different to those obtained with the water control.", "The LC50 and LC90 of EC25% for the green peach aphid was 0.63 (in % concentration) (Confidence Interval 0.47%-0.79%) and 1.84% (Confidence Interval of 1.39%-2.95%) respectively (FIG.", "16).", "Greenhouse Bioassay Three concentrations (0.25, 0.5 and 1%) of formulation EC25%, Neem Rose Defense® at 0.5% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1% (EC 20% with 0.2% pyrethrin) and a water control were tested with the green peach aphid (Myzus persicae (Sulz.)).", "Fifteen plants (replicates) of two month old Verbena speciosa ‘Imagination’ (10-15 cm) grown in small plastic insertions cells (used for potting plants) filled with Pro-Mix BX® were used for each treatment.", "Each insertion cell was glued to the bottom of a 1 l transparent plastic container with screened sides and top, to permit aeration.", "Green peach aphids were collected in plastic containers from a rearing cage maintained in a HRDC greenhouse and ten adults were transferred to each plant.", "The whole plant was sprayed for 15 seconds on average, at 8 psi under an exhaust chamber with a VEGA 2000® paintbrush sprayer equipped with a 20 ml reservoir (Thayer & Chandler Co., Lake Bluff, Ill., USA).", "Spraying was done three times over the course of the experiment, i.e.", "on days 0, 7 and 14.Containers with the sprayed plants were kept in a greenhouse under shade for the duration of the experiment.", "Counts were done on days 7, 14 (prior to spraying) and on day 21 by dismantling five of the fifteen replicates in each treatment.", "Aphids were individually counted when numbers were small (<50).", "For larger numbers, plants were shaken over a clear 250 ml container filled with soapy water over a black and white grid to evaluate the number of aphids present.", "Plant leaf surface (cm2) was measured with an area meter LI-3100® (LI-COR Inc., Lincoln, Nebr., USA) and counts were averaged to number of aphids/cm2 for each treatment and transformed to square root (x+0.5) for ANOVA analysis with SAS® software to evaluate the efficacy of the different treatments.", "Counts within treatments did not differ significantly (P=0.3647) from one sampling day to the other, so results within treatments were pooled and averaged for the whole experiment.", "All concentrations of EC25% and Safer's Trounce® were more effective in controlling the aphids than the water control (FIG.", "17 ).", "EC25% at 0.5% and 1.0% and Safer's Trounce were significantly more effective in reducing the number of aphids/cm2 than Neem Rose Defense® and EC25% at 0.25%.", "Both 0.5% and 1.0% EC25% concentrations were more effective (0.5 aphids/cm2 and 0.0 aphids/cm2 respectively) than Safer's Trounce® (0.9 aphids/cm2) though not significantly.", "B.", "Contact bioassays in the laboratory and greenhouse with the western flower thrips (Frankliniella occidentalis (Perg.))", "using EC25% formulation and two commercially available bioinsecticides.", "Laboratory bioassay Six concentrations (0.05, 0.18, 0.125, 0.25, 0.5 and 1%) of formulation EC25%, Neem Rose Defensee at 0.7% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1% (EC 20% with 0.2% pyrethrin) and a water control were tested with the Western flower thrips (WFT: Frankliniella occidentalis (Perg.)).", "WFT were collected in plastic containers by tapping infested Lima bean leaves over white paper.", "Ten WFT (either adults or 3rd or 4th instar nymphs) were transferred to a closed 250 ml transparent plastic container.", "Wet dental cotton was inserted through the lid for use as a water source.", "Four replicates were prepared for each treatment.", "Containers were sprayed at 6 psi under an exhaust chamber for 15 seconds with a VEGA 2000® paintbrush sprayer equipped with a 20 ml reservoir (Thayer & Chandler Co., Lake Bluff, Ill., USA).", "Containers were weighed just before and after spraying to calculate the amount of active ingredient deposited in mg/cm2.Containers were then stored in a growth chamber at 24° C., 65% R.H. and 16 L:8 D photoperiod.", "The entire procedure was repeated four times.", "Mortality was evaluated 24 hours following treatment under a binocular scope by probing WFT with a small brush.", "Absence of movement was recorded as dead.", "The efficacy of EC25% was compared to Neem Rose Defense® and Safer's Trounce® and data were transformed by arcsin{square root}p and subjected to ANOVA analysis using SAS® software (SAS Institute 1988).", "The LC50 and LC90 (in mg/cm2 of active ingredients) were calculated mortality results by PROBIT analysis using POLO-PC® software (LaOra Software 1987).", "Formulation EC25% at 0.5% and 1.0% were significantly more effective (98.8% and 95.8% mortality respectively) in controlling the WFT than all other treatments except for Safer's Trounce® (82.7% mortality) (FIG.", "18).", "EC25% at 0.25% caused significantly more mortality (63.7%) than the control (10.8%) but all remaining treatments did not.", "The LC50 and LC90 of EC25% for thrips was determined as 0.0034 mg/cm2 (Confidence Interval: 0.0027-0.0039 mg/cm2) and 0.0079 mg/cm2 (Confidence Interval: 0.0067-0.0099 mg/cm2) respectively (FIG.", "19).", "Greenhouse Bioassay Two concentrations (0.25% and 1%) of formulation EC25%, Neem Rose Defense® at 0.7% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1% (EC 20% with 0.2% pyrethrin) and a water control were used to evaluate their relative efficacy in controlling the Western Flower thrips (WFT: Frankliniella occidentalis (Perg.))", "in a greenhouse setting.", "Ten 10 day-old Lima bean plants (Phaseolus sp.)", "were prepared for each treatment.", "One leaf and the cotyledons of each plant were removed to keep only one leaf per plant grown in Pro-Mix BX® in a plastic insertion cell (used for potting plants) glued to the bottom of a clear plastic container (1 l) with screened sides and top.", "WFT were collected in small plastic containers by tapping infested bean leaves over white paper and lifted with a small brush.", "Ten adult thrips (or 3rd or 4th instar larvae) were transferred on each single leaf of each plant/insertion cell which were sprayed to drip point at 6 psi under an exhaust chamber with a VEGA 2000® paintbrush sprayer equipped with a 20 ml reservoir (Thayer & Chandler Co., Lake Bluff, Ill., USA).", "Spraying was done on days 0, 8 and 14.Each replicate/plastic container was then kept in a greenhouse under shade for the duration of the experiment.", "Counts were made on days 8 and 14 (prior to spraying) and on days 21 and 28.All live stages present on the whole plant were counted under a binocular scope and the leaf surface was measured by comparing it to a series of pre-measured hand-made leaf-size patterns.", "On the last day of the experiment (day 28), the leaf was cut and its surface was measured with an area meter LI-3100® (LI-COR Inc., Lincoln, Nebr., USA).", "Counts were calculated as average number of thrips/cm2 per treatment.", "In order to compare treatments, average counts were then calculated as a percentage of thrips present on the control plants: (n/cm2 on treated plants/n/cm2 on control plants)×100 The control treatment therefore had a value of zero and other treatments had positive or negative values indicating that more or less thrips were present respectively in relation to the control treatment.", "At the end of the experiment on day 28, leaves treated with EC25% at a concentration of 1.0% had 69.3% less WFT than leaves treated with the control while leaves treated with Safer's Trounce® had 101.1% more WFT (FIG.", "20).", "Leaves treated with Neem Rose Defense® had slightly more thrips (19.3%) than the control on day 28.Leaves treated with EC25% at 0.25% concentration had 52.3% more thrips than the control on day 28.C.", "Contact bioassay in the laboratory with the greenhouse whitefly (Trialeurodes vaporariorium (Westw.))", "using EC25% and commercially available insecticides Laboratory Bioassay Five concentrations (0.0625, 0.125, 0.25, 0.5 and 1%) of formulation EC25%, Neem Rose Defense® at 0.7% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1.0% (EC 20% with 0.2% pyrethrin), Thiodan® at 0.044% (50 WP) and a water control were used to evaluate their relative efficacy in controlling the greenhouse whitefly (Trialeurodes vaporariorium (Westw.)).", "Whitefly adults were collected with an insect aspirator from HRDC greenhouses and glued to a black 5 cm×7,5 cm plastic card sprayed with Tangle-Trap® (Gempler's Co.) by emptying the aspirator over the card to obtain at least 20 adults per card.", "Cards were observed before spraying under the binocular scope to remove all dead and immobile whiteflies.", "Only active whiteflies were kept for the experiment.", "Four cards were used per treatment.", "Each card was sprayed at 6 psi with 300 μl of emulsion using a BADGER 100-F® (Omer DeSerres Co., Montreal, Canada) paintbrush sprayer mounted on a frame at a distance of 14.5 cm from the spray nozzle in an exhaust chamber.", "Cards were weighed immediately before and after spraying to calculate the amount of active ingredient deposited in mg/cm2.Cards were allowed to dry under the exhaust chamber and then placed sideways on a Styrofoam rack in a closed clear plastic container of 5 L with moistened foam on the bottom to keep humidity high (>90% R.H.).", "The plastic container was stored in a growth chamber at 24° C. and 16 L:8 D photoperiod.", "This procedure was repeated three times.", "Mortality was evaluated 20 hours following treatment by gently probing the whitefly with a single-hair brush under the binocular microscope.", "Absence of movement (antennae, leg, wing) following probing was recorded as dead.", "Relative efficacy of EC25% and the two commercially available bioinsecticides, Neem Rose Defense® and Safer's Trounce®, and the synthetic insecticide Thiodan®, were compared by transforming mortality data to arcsin{square root}p and then subjecting to an ANOVA analysis using SAS® software (SAS Institute 1988).", "LC50 and LC90 (in mg/cm2 of active ingredients) were calculated by PROBIT analysis using POLO-PC® software (LaOra Software 1987).", "Formulation EC25% at concentrations 0.5% and 1.0% were significantly more effective (98.9% and 100.0% mortality respectively) at controlling the greenhouse whitefly than all other treatments except for Safer's Trounce® (98.0% mortality) (FIG.", "21).", "Formulation EC25% at 0.125% concentration and Neem Rose Defense® were significantly more effective than the control treatment but significantly less effective than EC25% at 0.25, 0.5 and 1.0% concentrations and Safer's Trounce®.", "Thiodan and EC25% at 0.0625% concentration were as effective as the control treatment.", "LC50 and LC90 were 0.0066 mg/cm2 (conf.", "int: 0.0054-0.0076 mg/cm2) and 0.0141 mg/cm2 (conf.", "int: 0.0121-0.0172mg/cm2) respectively (FIG.", "22).", "D. Contact bioassay in the laboratory with the parasitoïd (Encarsia formosa) using EC25% and commercially available bioinsecticides Laboratory Bioassay Four concentrations (0.0625, 0.125, 0.25 and 0.5%) of formulation EC25%, Neem Rose Defense® at 0.7% (EC-90%), Safer's Trounce® at 1.0% (EC 20.2%) and a water control were tested with the parasitoïd Encarsia formosa (EF) (obtained from Koppert Co. Ltd).", "EF were kept in a growth chamber at 24° C., 16 L:8 N photoperiod and 65% R.H. until emergence.", "Sixty newly emerged adult EF were transferred with a mouth aspirator into plastic Solo® cups of 20 ml.", "Cups were sprayed at 6 psi under an exhaust chamber with 250 ml of solution with a Badger 100-F® paintbrush sprayer (Omer de Serre Co., Montreal, Canada) mounted on a frame at a fixed distance of 14.5 cm.", "Solo® cups were weighed just before and after spraying to calculate the amount of active ingredient deposited in mg/cm2.Once sprayed, the EF were gently transferred with a small brush from the Solo cups to small clear plastic Petri dishes (10 EF/Petri) lined with a filter paper wetted with a 5% sugar solution as a food source.", "Four replicates were prepared for each treatment.", "The Petri dishes were then placed in a tray and stored in a growth chamber at 24° C., 65% R.H. and 16 L:8 D photoperiod.", "The entire procedure was repeated three times.", "Mortality was evaluated 24 hours following treatment under a binocular scope by observing the EF.", "Absence of movement was recorded as dead.", "The effect of EC25% was compared to Neem Rose Defense® and Safer's Trounce® using mortality data transformed by arcsin{square root}p and subjected to ANOVA analysis using SAS® software (SAS Institute 1988).", "All EC25% formulations at concentrations ranging from 0.0625 to 0.5% were significantly less effective than Safer's Trounce® at 1% (71.9%) (FIG.", "23).", "Results from all concentrations of EC25% and Neem Rose Defense® formulations were not significantly different than the control.", "These results indicate that the recommended dose (0.5%) of EC25% can be safely used with the biological control agent, Encarsia formosa.", "Example XII Fungicidal Efficacy of the Essential Oil Extract (Emulsifiable Concentrate Formulation) A.", "Effect of EC on Gray Mold Caused by the Plant Pathogen, Botrytis cinerea on Greenhouse Tomatoes Tomatoes (cv Trust) were sown and grown according to practices generally used for the production of greenhouse tomatoes.", "About two months after sowing, lesions are made on the stem (5 lesions/plant) and inocuated with a suspension of 3×106 spores of B. cinerea, 1 ml per lesions.", "The following treatments were evaluated: 1) control (−) with water only; 2) control (+) with B. cinerea; 3) Nova® (0.3 g/l); 4) Iprodione® (Rovral 1.0 g/l), 5) EC25% (comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) at a concentration of 0.125%; 6) EC25% at a concentration of 0.50%; 7) EC25% at a concentration of 0.75%; 8) M. ochracea (P130A) (5×108 spores/ml); 9) Rootshield® (0.6 g/l).", "The experimental design was a randomized block design with 8 blocks.", "Treatments were applied 1 and 8 days following inoculation of lesions with B. cinerea.", "Disease symptoms were evaluated according to the following severity index: 1=absence of symptoms, 2=necrosis (browning of tissue) around the lesion, 3=advanced necrosis and presence of spores on necrotic zones, 4=advanced necrosis, presence of spores on necrotic zones and the start of canker formation, 5=well developed canker and death of plant.", "Severity of symptoms was estimated 3 and 5 weeks after plant inoculation.", "The experiment was repeated and the effect of treatment was analyzed with an analysis of variance (ANOVA) and means were compared with a Least Significant Difference test (LSD).", "Statistical analyses demonstrated that there was a significant difference in efficacy between treatments (FIG.", "24).", "Control plants demonstrated average severity of disease symptoms on 26 of the 35 control plants.", "Plants from all other treatments presented a significantly lower level of severity than the control plants one day after inoculation or the first day of treatment.", "The two microbial fungicides Rootshield and P1304 did not significantly control the disease with an average severity between 25.33 and 21.67 8 days after treatment (FIG.", "24).", "Plants treated with the botanical biopesticide EC25% was less effective in controlling the disease symptoms than the commercial products, Nova and Rovral.", "However, all three concentrations of EC25% (0.125, 0.25 and 0.5% AI) decreased disease symptoms with severity indexes being 60, 44 and 37% lower respectively on plants treated with EC25% than the control plants.", "Severity indexes were reduced by 38 and 32% with Nova and Rovral respectively (FIG.", "25).", "B.", "Effect of EC25% on Powdery Mildew Caused by the Plant Pathogens, Erysiphe cichoracearum and Sphaerotheca fuliginea on Greenhouse Cucumbers The fungus responsible for powdery mildew are obligatory parasites incapable of survival in the absence of the host.", "In order to produce inoculum necessary for inoculation, infested cucumber leaves are collected from a grower in July of 2002.Conidia present on these leaves were transferred on leaves of cucumber plants grown in an experimental greenhouse, one of to two months after sowing the cucumber plants (cv Straight 8).", "The following treatments were evaluated: 1) negative control treated with water; 2) positive control treated with the disease organisms, E. cichoracearum and S. fliginea; 3) Nova® (0.3 g/l); 4) Benlate®+Manzate® (1.2 g/l and 4.g/l); 5) EC25% (comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) at 0.125% AI; 6) EC25% at 0.50% AI; 7) EC25% at 0.750% AI; 8) Mineral oil.", "The experimental design was a complete randomized block design with 7 blocks.", "Since it is almost impossile to inoculate an obligatory fungal parasite in an known or measurable quantity of inoculum, there is always variability in the level of infection of the plants.", "A first evaluation of powdery mildew was made on all plants inoculated in the greenhouse.", "The percentage of infection was analyzed in order to group 8 plants presenting the same level of infection (SE <1.5, see FIG.", "26).", "Severity of powdery mildew was also evaluated on each individual leaf with the use of a severity index 7 days after treatment applications and reported as average severity for each plant.", "The entire experiment was repeated a few days later.", "The effect of treatment was analyzed with an analysis of variance (ANOVA) and means were compared with a least significant difference test (LSD).", "Statistical analysis demonstrate that there is a difference of effect between treatments.", "(FIG.", "27).", "In fact, an increase of 45 and 39% of disease incidence was observed on control plants (water and disease).", "All plants treated with the various products presented a significantly lower increase in percentage of disease severity compared to the control plants.", "The botanical biopesticide EC25% was as effective as the commercial fungicide Nova in controlling the disease.", "At concentrations of 0.50 and 0.75%, EC25% demonstrated better control than Nova, permitting an increase of disease symptoms of only 2 and 2.5% respectively for both concentrations compared to 4.5% for Nova.", "However phytotoxicity was observed on plants treated with EC25% at those two concentrations Example XIII Effect of Essential Oil Extract for Controlling Soil Inhabiting Pests A.", "Effect of Soil Treatments with EC25% at 0.5% on Soil Emergence of Adult Western Flower Thrips Lettuce plants were transplanted in plastic pots of 12 cm of diameter and grown for six weeks in a greenhouse.", "The plants were heavily infested with all stages of thrips Frankliniella occidentalis Pergande and were watered and fertilized as required by good management practices.", "Then, the lettuce plants were cut at the soil level and the pots were sprayed with a preparation of EC25% (comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) at 0.5 concentration of active ingredients.", "Five volumes of EC25% were applied, 25 ml, 16.7 ml, 13.3 ml, 10 ml, and 6.67 ml per pot representing volumes of 1500 l/ha, 1000 l/ha, 800 l/ha, 600 l/ha and 400 l/ha respectively.", "A total of 10 pots were sprayed per volume and control pots were sprayed with 25 ml of tap water.", "The application was made using a badger sprayer connected to an air outlet to provide the required air pressure for pesticide application.", "Immediately after treatments, the pots were covered with a plastic petri dish cover of 14.5 cm diameter.", "The inner surface of the cover was smeared with a thin layer of tanglefoot insect glue and the joints were completely sealed with parafilm to prevent the escape of emerging adults from the soil pot or the introduction of foreign into the dish.", "Treatments were applied on 7 July 2002 and counts of emerging adults thrips were made 3, 5, 7, 10 and 12 days after treatments.", "At each observation time, emerging adults were removed from the glue and the cover was replaced on the top of the plastic pot and sealed with parafilm.", "Counts of emerging adults over a period of 2 weeks after treatments were analysed using an ANOVA and means for each treatment were separated using a Fischer LSD test.", "Most of the adult thrips emerged during the first week following treatment and this corresponds with the duration of pupal life that varies between 2 and 8 days according to the ambient temperature (FIG.", "28).", "Effect of treatment on the total number of adult thrips emergence from the soil was statistically highly significant ((F=5.71; dl=5; P<0.0001).).", "All treatments significantly reduced thrips emergence compared to the control.", "At 3 days after treatment, an important decrease in the number of emerging adult thrips was observed in the 1500 l/ha treatment compared with all other treatments.", "At all other dates, the 1500 l/ha treatment was the best treatment and caused the greatest reduction in adult thrips emergence.", "B.", "Using EC25% and Microemulsion Formulations for Controlling Soil-Inhabiting Pests of Turf i) Laboratory Tests using EC25% with the European Chafer, Rhizotrogus maialis The formulation EC25% (comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) was tested with third instar R. majalis.", "The insects were collected from Arnprior ON, and were kept in containers with soil at 4° C. until trials were initiated.", "Grass was grown in plastic seedling trays divided into 6 compartments.", "Soil within each compartment consisted of 1 part potting soil: 1 part sand to a depth of approximately 5 cm.", "The grass seedling mix consisted of Kentucky Blue 65%, Annual Rye Grass 20% and Fescue 15% and was distributed at 1.5 g per compartment 2-3 weeks prior to the addition of the insects.", "Ten third instar R. majalis were placed into each compartment so for a total of 60 larvae per tray.", "Three replicate trays were used for two separate experiments.", "One to two days after the larvae were added to the trays, treatment was initiated.", "Each of the EC25% treatment concentrations; 0.0625, 0.125, 0.25, 0.5 and 1%, were prepared in 100 mL of water by the addition of 0.25, 0.5, 1, 2 and 4 mL of stock EC respectively.", "The control was an EC blank, 0.375 mL of formulation excluding essential oil.", "The trays were watered once per day for the seven day period after treatment.", "After one week each compartment was sampled to determine the survival of the larvae.", "Samples of the soil were taken from each compartment after both trials and analysed for both pH (ASTM, 1989) and organic matter content (ASTM, 1987).", "Probit analysis was used with the data from the two European chafer trials in order to determine the LC50 values for EC25%.", "Comparison of the LC50 values within and between each trial was conducted using a z-test.", "Ninety percent (90%) R. majalis larvae survived at the 0.0625% concentrations of EC25% and 10% survived at 1% EC25% (FIG.", "29).", "Percent mortality from both trials were used to calculate LC50 values (FIG.", "30).", "Two different values were calculated for each trial since not all of the larvae were accounted for at the end of the exposure period.", "The first LC50 for each trial is based on the mortality within the actual number of larvae found in each treatment replicate at the end of the test (FIG.", "30).", "The second LC50 is calculated based on the assumption that there were 10 larvae present at the beginning and end of the experiment within each treatment replicate but due to possible deterioration of the organism it was not identified or located.", "A comparison of the LC50 using a z-test indicated that within each trial there was no significant difference between the calculated LC50s (z<0.885, z critical=1.96) Between trials it was determined that there was no significant difference (z=1.587, z critical=1.96) when comparing the first LC50 (based on the actual number of larvae found).", "When the LC50s based upon 30 larvae per treatment were compared between trials a significant difference (z=2.233, critical z=1.96) was detected.", "A mean LC50 and standard error for the two trials was calculated as 0.466±0.122% EC25% by averaging the two LC50s from trial one and two and these were not significantly different.", "Soil Organic Matter Content and pH.", "The soil organic matter content was determined to range from 8.9 to 11 with an average of 9.1 in the first trial and 10.5 in the second (FIG.", "31).", "The pH of the soil ranged from 4.9 to 5.0 with a mean of 4.94 in the first and 5.02 in the second trial.", "These values were consistent with those measured in previous R. majalis trials.", "ii) Field Tests using Microemulsion Formulations with the European Chafer, Rhizotrogus majalis The objective of this study was to measure the effect of broadcast applications of two microemulsion formulations (MEF), MEF1 and MEF2 of an essential oil extract from Chenopodium ambrosioides, on the presence of the European chafer, Rhizotrogus majalis.", "The microemulsion formulations tested comprised the following: Ingredient MEF 1 MEF 2 Chenopodium extract 50 50 Rhodafac RE-610 10 10 Ammonyx LO 4 4 Stepanol WAC 1 — Water 35 35 The experiment was conducted on lawn at the Guelph Turfgrass Institute (GTI), Ontario on turfgrass that was mowed once a week and received irrigation.", "Experimental design was a randomized complete block design with 4 replications and 8 treatments.", "Plots were 3 m×3 m and treatments tested are listed in FIG.", "32.Treatments were applied on Aug. 27, 2002, immediately after pre-treatment assessment of the European chafer, with a sprayer in water at 0.25 L/m2.The European chafers were at the second instar when treatments were applied and at the third instar by the end of the experiment.", "The live European chafers were counted using a golf course hole-cutter (15.2 cm, ‘turf mender’).", "Five plugs were taken from each plot.", "Once removed, plugs were broken up and the number of chafers was recorded.", "The last chafer count was done using a sod-cutter: three turf strips (0.3 m×3m) across each plot.", "The turfgrass sod was removed and chafers near the soil surface were counted.", "Counts were made at 5, 15 and 25 days after treatment on 2, 15, and 23 Sep. 2002, respectively.", "Analysis of variance (ANOVA) on the number of total European chafes alive was parformed by the General Linear Model Procedure of SAS statistical software (SAS Institute 1990).", "Fisher's Least Significant Difference test (LSD) was used for comparison of treatment means when the F-test was significant (P=0.05).", "Number of European chafers was not significantly different between plots prior to treatment application.", "On September 23 (25 days after the application), all insecticide treatments significantly reduced the number of chafers compared to the untreated control except treatments MEF1 (10.0 ml/m2) and MEF2 (20.0 ml/m2) (F=4.25; P<0.00046) (FIG.", "33).", "European chafer counts for both treatments were significantly higher than counts on plots treated with SEVIN XLR PLUS.", "No significant difference in the number of chafers in turfgrass plots was observed on September 2 (5 days after application) and on September 15 (15 days after application).", "For both dates, all treatments reduced chafers compared to the untreated control lower, the statistical model was not significant mainly because of the fact that European chafers were counted on a smaller area at these two dates.", "iii) Field Tests with the Microemulsion Formulations MEF1 and MEF2 and the Hairy Chinch Bug, Blissus leucopterus The experiment was conducted on a commercial lawn in Guelph, Ontario on low maintenance turfgrass that was mowed once a week and received no irrigation.", "Experimental design was a randomized complete block design with 4 replications and 8 treatments.", "Plots were 2 m×3 m and treatments tested are listed in FIG.", "34.Treatments were applied on Jul.", "30, 2003, immediately after pre-treatment assessment of the HCB, with a sprayer in water at 1 L/m2.Most of the HCB population was at the third instar.", "HCB were counted using the flotation method.", "A cylinder (346 cm2) was inserted into the turf to a depth of about 3 or 4 inches and was flooded to the brim with water.", "After 5 minutes, live HCB floating to the surface were counted.", "Three counts per plot were made (by the same person) and the total HCB per plot was used for statistical analysis.", "HCB were counted 5, 15 and 25 days after treatment on August 4, 12 and 24, respectively.", "Analysis of variance (ANOVA) on the number of total live HCB was performed using the General Linear Model Procedure of SAS statistical software (SAS Institute, 1990).", "Fisher's Least Significant Difference test (LSD) was used for comparison of treatment means when the F-test was significant (P=0.05).", "Number of HCB was not significantly different between plots prior to treatment application.", "All insecticide treatments significantly reduced the number of HCB compared to the untreated control on August 12, 15 days after the application (F=6.13; P<0.0005) (FIG.", "35).", "There were no differences between results with the Diazinon treatment and results with the two formulations.", "At this date, treatment MEF2 (20.0 ml/m2) had significantly more insects than treatment MEF2 (10.0 ml/m2).", "No significant difference in the number of HCB in turfgrass plots was observed on August 4 (5 days after application) and on August 24 (25 days after application).", "For both dates, all treatments reduced HCB compared to the untreated control.", "Example XIV Effect of Essential Oil Extract on Beneficial Pests A.", "Direct Toxicity of the Essential Oil Extract on Predatory Mites Amblyseius fallacis and Phytoseiulus persimilis The purpose of this study was to evaluate the direct toxicity of the EC25%, a botanical biopesticide with two predaceous mites Amblyseius fallacis, a natural regulator of mites in integrated control orchards and Phytoseiulus persimilis, a known mite predator for the control of the twospotted mite in vegetable crops grown under glasshouses in Quebec and elsewhere.", "The suitability of EC25% as a primary tool in IPM of greenhouse crops would therefore be determined.", "Rearing of Tetranychus urticae and Amblyseius fallacis The phytophagous mite, Tetranychus urticae has been reared on common bean plants (Phaseolus vulgare) for several years at the Horticultural Research and Development Center, St. Jean-sur-Richelieu, Quebec.", "The beans were sown at high densities of 40 to 50 plants per tray (39 cm×30 cm).", "Colonies of T. urticae were kept in a growth chamber set at 25° C., 75% HR and 16 L photoperiod.", "The predaceous mite Amblyseius fallacis was maintained on Tetranychus urticae and kept in a greenhouse set at 25° C., 75 HR and 16 L photoperiod.", "A fan placed in front of the cage containing both Amblyseius fallacis and the twospotted spider mite provided continuous air flow to the colonies.", "Trays containing bean plants infested with the twospotted spider mites were added regularly to provide sufficient food to the predator colonies.", "Rearing of Phytoseiulus persimilis Colonies of Phytoseiulus persimilis were bought from Koppert Canada and reared in the laboratory in the same conditions as for A. fallacis.", "The colonies originating from the shipment were maintained and acclimatized in a growth chamber set at 25 C, 70-85% RH and 16:8 (light/darkness) for two weeks.", "Contact Toxicity Assay The bioassays were carried out in Petri dishes using a leaf disc method.", "A wet sponge was placed in a plastic Petri dish (14 cm diameter and 1.5 high) and rings of apple leaf (cv.", "McIntosh; 3.5 cm of diameter) were cut and placed upside down on the surface of a water-saturated sponge.", "Sufficient numbers of all stages of the twospotted spider mite Tetranychus urticae Koch were then brushed onto each leaf disc.", "A total of five leaf discs were put in a Petri dish and each Petri dish represented one replicate.", "Ten replicates per treatment were prepared over a period of three weeks.", "Gravid females of Amblyseius fallacies (5) or Phytoseiulus persimilis (9), were picked up at random under a stereomicroscope from leaves taken from plants used to rear the predator colonies.", "They were transferred individually with a fine camel brush to a small Petri dish (5.5 cm of diameter) containing a leaf piece of the common bean, Phaseolus vulgare.", "They were treated topically with 0.3 ml of pesticide solution at different dosages using a paintbrush sprayer (Vega 2000, Thayer & Chandler, Lake Bluff, Ill., USA) at 6 psi set at 14.5 cm above the treated area.", "The pesticide solutions were prepared on the day of application.", "Treated females were then transferred carefully and individually to each apple leaf disc.", "To avoid contamination, a new camel brush was used for each concentration to transfer the treated females to leaf discs.", "Petri dishes were put in a black tray and covered with transparent plastic covers and a strip of brown paper was placed on top to reduce glare and to keep the mites within the leaf disc area.", "Water was added to the tray to maintain high relative humidity.", "The trays were incubated in a growth chamber set at 25° C., 75% HR and 16 L Photoperiod.", "Mortality was recorded 24 h and 48 h after treatment.", "One and 2 replicates were set up per day respectively for A. fallacis and P. persimilis and only 11 treatments were evaluated for P. persimilis.", "Treatments EC25% is an EC formulation with 25% essential oil as an active ingredient.", "Seven concentrations of EC25% were prepared as follows.", "The 1% concentration was prepared by mixing 0.4 ml of the formulation and 9.6 ml of tap water and successive dilutions were made from the stock solution.", "The following commercially available insecticides were used at their recommended rates: Trounce® (20.2% of fatty acids and 0.2% pyrethrin) at the recommended concentration of 1%; the insect growth regulator Enstar® (s-kinoprene) at the concentration of 0.065%; and Avid® (abamectin 1.9% EC), at the concentrations of 0.0057% and 0.000855%.", "A water treatment was used as a control for a total of twelve treatments with A. fallacies and 11 with P. persimilis where the Enstar treatment was dropped.", "The test product EC25% was sprayed first starting from the lower to the higher concentrations.", "Then the control treatment was applied followed the reference products Avid, Trounce and Enstar.", "The spray apparatus was rinsed three times between treatments using successively ethanol 95%, acetone, hexane, distilled water.", "Statistical Analysis Mortality percentages were transformed to logit or probit to determine which analysis gave a better fit as recommended by Robertson and Preisler (1992).", "The analysis which presents the highest number of small individual Chi square (χ2) is chosen.", "Probit mortality were regressed on 1+log10 (dose) for EC25%.", "Concentration mortality regression lines were determined to estimate the lethal concentration to kill 50% of the predator population using the POLO-PC program (LeOra, 1987).", "Toxicity values of LC50, LC90 and LC99 are given as percent (%) of active ingredient.", "Data were transformed to arcsine before analysis of variance.", "Comparison between treatments were analysed using GLM procedure and means were separated by the Fisher test at 5% probability (SAS, 1996).", "Results Amblyseius fallacis A total of 667 adult females of Amblyseius fallacis was tested and only 12 females (1.79%) walked out of the leaf disc area; number of missing was subtracted from initial total.", "Mortality in the control was 5.56% at 24 h and remained unchanged at 48 h following treatment (FIG.", "36).", "There was a highly significant difference between treatments at 24 h (F=30.32, df=11, P<0.001) and at 48 h (F=31.64, df=11, P<0.001).", "There was no mortality after 48 h was with EC25% at the concentration of 0.125% and 3,1% 7% and 23% mortality with EC25% at 0.25%, Enstar and EC25% at 0.5% and these results were not significantly different from the control.", "Note that at the concentration of 0.5% the EC25% suggested commercial rate, mortality was 23.11% which is less than the 50% limit of the IOBC for harmless pesticides.", "Amongst the commercially available products, Trounce caused the highest mortality (85.11%) after 48 H. This was followed by the Avid treatments at concentrations of 0.0057% (94.8% mortality) and 0.000855% (81.5% mortality) and results did not differ significantly, demonstrating that both products are equally toxic to Amblyseius fallacis.", "LC50, LC90 and LC99 values at 48 h (FIG.", "37) are well above (1.01%, 3.91% and 4.12% respectively) the 0.5% effective dose used to control the spider mite pest, Tetranychus urticae)(Chiasson, unpublished results).", "These results indicate that EC25% might have low or no residual toxicity to Ambiyseius fallacis and most adult females that remained alive 24 hours after the EC25% treatments continued to reproduce and were observed laying eggs.", "Phytoseiulus persimilis A cohort of 555 adult females was used to evaluate the toxicity of EC25% and the commercially available Trounce and Avid with the mite predator, Phytoseiulus persimilis.", "In this bioassay, 7.35% and 13.17% of the total number of gravid females escaped from the leaf disc 24 h and 48 h respectively after treatments.", "They contributed to 13.06% and 18.35% of the total mortality recorded at 24 h and 48 h respectively.", "The highest number of predator escapees were observed in the control treatment and in the EC25% treatments at concentrations lower than 2%.", "We will discuss only mortality calculated over total number treated minus missing individuals (3rd column of FIG.", "38).", "Highest mortality were caused by Trounce (99,71%) followed by Avid at the concentration of 0.0057% (93.69%).The lowest mortality was observed in the treatment with EC25% at the 0.125% concentration (13.43%).", "Mortality with EC25% at 0.125%, 0.25 and 0.5% were not significantly different from the control treatment.", "When missing females were deducted from the initial number of adults tested, the LC50 of P. persimilis was 1.2% and 0.8% at 24 h and 48 h after treatments respectively (FIG.", "37).", "B.", "Direct Toxicity of the Essential Oil Extract on Aphid Endoparasitoids Aphidius colemani (Hymenoptera: Brachonidae, Aphidiinae) In the present study, adult Aphidius colemani wasps were exposed to a direct spray application of EC25% and remained in permanent contact with the biopesticide residues, which is considered worse case conditions, to test the potential side effects this biopesticide may have on beneficial hymenoptera such as Aphidius colemani Rearing of Aphidius colemani Aphidius colemani wasps were purchased from.Plant Product Quebec in lots of 250 mixed mummies and adults.", "The emerged wasps and the remaining mummies were directly transferred to a 5 litre plastic bag filled with air and the wasps were provided with a 10% solution of sucrose and honey (w/w) as food source and water.", "Direct Contact Bioassay Six to 14 adult parasitoids less than 48 h old were transferred into a large solo cup (500 ml ca.)", "using a mouth aspirator.", "The solo cup was lined with a filter paper (Rothmans #1) and had two large openings drilled on the side and one on the cover to provide ventilation and these openings were covered with a fine screen to prevent escape of adult wasps and condensation of the pesticide vapour.", "The filter paper was humidified with a 10% solution of sucrose and honey.", "The solo cup containing the wasps was weighed and the wasps were dragged down to the bottom of the solo cup by means of successive beats on the cover with a 15 cm long stick.", "They were treated with 0.3 ml of the insecticide solution using a paintbrush sprayer (Vega 2000, Thayer & Chandler, Lake Bluff, Ill., USA) at 6 psi and set at 14.5 cm above the treated area.", "The solo cup was then covered and re-weighed to determine weight of pesticide used.", "The treated wasps were then incubated in a growth chamber set at 18° C.-22° C. and 60-65% HR.", "Assessment of treatment effects were made at 24 h and 48 h following treatment.", "Residual Bioassay Ten to 20 adult wasps including at least 5 females were picked up and put in a glass Petri dish and covered.", "The cover had an opening covered with a screen to enable ventilation and to prevent condensation of the pesticide vapour.", "The Petri dishes were previously treated with a pesticide solution exactly in the same manner as for direct toxicity bioassay but dishes were left to dry for an hour before covering and exposing the wasps to the pesticide residues.", "On the cover, two small circular holes were drilled and used to provide the wasps with water and a solution of honey and sucrose.", "Mortality was recorded at 24 h and 48 h. Treatments The test product EC25%, a 25% essential oil EC formulation obtained from Codena Inc.", "Seven concentrations were prepared as follows: EC25% at 8% was prepared by mixing 3.2 ml of EC25% and 6.4 ml of tap water and successive dilutions of 4%, 2%, 1%, 0.5% and 0.125% were made from the stock solution.", "Commercially available insecticides were used at their respective recommended doses as positive controls: Trounce® (20.2% of fatty acids, Safer Ltd, Scarborough, Ont.)", "at the recommended concentration of 1%, the insect growth regulator Enstare (s-kinoprene) at the concentration of 0.065%; Avid® (abamectin 1.9% EC) at the concentrations of 0.0057% and 0.000855%, and Thiodan® (endosulfan 50 WP) at the concentration of 5%.", "The test product EC25% was used first, starting from the lowest to the highest concentration and followed by the water control and finally by Avid, Trounce, Enstar and Thiodan.", "The spray apparatus was rinsed three times between treatments using successively ethanol 95%, acetone, hexane, distilled water.", "Statistical Analysis Concentration was analysed as main effect and the weight of pesticide applied was tested as a covariate to correct for difference in quantity of applied pesticide.", "This covariate was deleted from the model when found not significant.", "Mortality regression lines were determined to estimate the lethal concentration to kill 10%, 50% and 90% of the parasitoid population using the POLO-PC program (LeOra, 1987).", "Toxicity values of LC50 are given as percent of active ingredient.", "Data were transformed to arcsine before analysis of variance but actual means were presented.", "Comparison between treatments were analysed using GLM procedure and means were separated by Fisher test at 5% probability (SAS, 1996).", "Effect of Treatments on Aphidius colemani Emergence from Mummies Myzus persicae mummies parasitized by Aphidius colemani females on leaves of cabbage (cv.", "Lennox) were used in this test.", "Portions of leaves bearing mummies were cut and placed in a Petri dish.", "The Petri dish was weighted and treated with a pesticide solution and immediately re-weighted to determine the amount of pesticide used.", "The treated Petri dish was then covered and sealed with parafilm.", "The cover of the Petri had a screened opening to enable ventilation and to prevent escape of emerging Aphidius adults.", "The incubation period lasted 7 days and all mummies that did not emergence as adult wasps were considered dead.", "Fecundity Assessment Females that survived the pesticide residual treatments were assessed for fecundity on wheat plants infested with aphids.", "Myzus persicae aphids reared on cabbage plants (c.v. Lennox) were brushed onto a pot containing 25 to 30 plants of wheat 6 days old.", "Soon after, the brushed aphids climbed the wheat plants and a density of at least 100 aphids per pot was required.", "Female wasps that survived the 48 h residual treatments were removed individually from the test arena by means of an aspirator and confined over pots of aphid-infested plants using ventilated transparent plastic cylinders for a period of 24 h. The females were then removed and the plant bearing parasitized aphids were incubated for a period of 10 days at 18° C. to 22° C. At the end of the incubation period, the wheat plant was cut and put in a Petri dish.", "The number of parasitized aphids were counted.", "Results Direct Contact Bioassay A total of 1174 adult wasps including 657 or 55.9% female parasitoids were tested.in the bioassay.", "The mean quantity of pesticide solutions applied was 4.58±1.36 mg/cm which was more than double the amount of 2.0±0.2 mg/cm recommended for the typical bioassay (Mead-Briggs et al., 2000).", "Mortality with EC25% at concentrations up to 1% was not significantly different than for the water control after 24 h. though at 48 h, results with EC25% treatments at the 0.5% and 1% concentrations significantly different from the control (FIG.", "39).", "At the 0.5% concentration of EC25%, recommended for field application, mortality varied from 18.6% to 35.2% at 24 h and 48 H after treatments respectively.", "Highest mortality was observed with the Avid treatments at concentrations of 0.0057% and 0.000855% and with the EC25% treatment at concentrations of 4% and 8%.", "Results in FIG.", "40 show that female wasps were relatively less sensitive to treatments than adult males.", "LC50 values for EC25% on A. colemani females (FIG.", "41) was equal to 1.28% which is more than twice the recommended concentration of 0.5% for field application.", "The LC50 for A. colemani males was lower at 0.77% but still above the 0.5% field recommended concentration of EC25%.", "However, the 95% confidence limits (CL 95%) of LD50% for both males and females were overlapping and therefore their LD50% were not differently significant (Robertson and Presisler, 1992).", "Residual Assay Results shown in FIGS.", "42 and 43.Effect of Treatments on Aphidius colemani Emergence from Treated Mummies FIG.", "44 showed that the effects of treatments on emergence of Aphidius colemani adults from treated mummies were significant (F=6.94, dl=16, P<0.0001).", "The emergence rate of A. colemani decreased steadily when EC25% concentration increased and there was no emergence at the concentration of 8%.", "At the recommended concentration for field application, i.e.", "0.5%, emergence was 86.4% and this result was not statistically different from that observed in the control.", "In the reference products tested, the highest emergence was observed in the Avid treatment with 96.1% and the lowest was Enstar at 35% emergence.", "Fecundity Assessment The results of FIG.", "45 indicated that females that survived the treatment were able to parasitize Myzus persicae hosts and that their reproductive functions did not seem to be affected.", "There was no enough surviving female to test for the EC25% concentration of 4% and 8%.", "The lowest fecundity rate was observed in the treatment of Avid with 9.1 mummies per plants compared to 23.9 mummies per plant recorded in the control treatment.", "The number of mummies produced from females treated with EC25% treatments at concentrations varying from 0.125 to 2% were not significantly different from the control.", "C. Direct Toxicity of the Essential Oil Extract on Predatory Minute Bug Orius insidiosus Say Various Orius species including Orius insidiosus Say (Heteroptera: Anthocoridae) are effective biological control agents of western flower thrips (WFT) Frankliniella occidentallis Pergrande (Thysanoptera: Thripidae) in sweet pepper, cucumber and other vegetable and ornamental crops (Veire de van et al., 1996).", "The present study was initiated to evaluate the side effects of EC25% on the predatory bug Orius insidiosus under laboratory conditions.", "Culture of Orius insidiosus Orius insidiosus stock culture was initiated with individuals obtained from a commercial supplier (Plant Prod Quebec, 3370 Le Corbusier, Laval, Quebec) and maintained in a laboratory growth chamber.", "Eggs of Ephestia spp were served as a food source and snaps beans of Phaseolus vulgaris as an oviposition substrate.", "The beans containing eggs were then incubated in folded brown paper until emergence.", "The folded paper was used to reduce cannibalism.", "Emerging nymphs were then transferred into one litre jars containing bean pods and fed with Ephestia eggs until the adult stage.", "The stock culture was renewed regularly.", "Direct Contact Bioassay The bioassays were carried out in small Petri dishes (5.5 cm in dia.)", "using a leaf disc method.", "A thin layer of agar 2% (2-3 mm) was poured into each Petri dish and a ring of apple leaf (cv.", "McIntosh, 3.5 cm in dia.)", "was cut and placed upside down on the surface of the agar.", "At least 10 Orius insidiosus 2nd nymph instar or adults were transferred carefully using an aspirator on the surface of the apple leaf disc.", "The Petri dish containing the nymphs or the adults bugs were dragged down to the bottom of the Petri dish by means of successive beats on the cover with a 15 cm long stick.", "The Petri dishes were weighted and immediately, they were treated immediately with 0.3 ml of pesticide solution at different concentrations using a paintbrush sprayer (Vega 2000, Thayer & chandler, Lake Bluff, Ill., USA) at 6 psi and set at 14.5 cm above the treated area.", "The Petri dishes were then re-weighted to determine the quantity of pesticide applied.", "The pesticide solutions were prepared on the day of treatment.", "The treated nymphs or adults were then transferred carefully to the surface of the apple leaf disc containing eggs of Ephestia spp as a source of food.", "To avoid contamination, a new camel brush is used for each concentration to transfer the treated nymphs or adults to the leaf discs.", "The Petri dishes were put in a tray and incubated in a growth chamber set at 25° C., 65% HR and 16 L Photoperiod.", "A fan was placed in front of the tray to provide continuous air flow.", "Mortality of nymphs was recorded at 1, 2, 5, 7 and 9 days after treatment when more than 80% of the nymphs became adults.", "Mortality of adult predators was recorded at 24 H and 48 H following treatment.", "Ten replicates were prepared per treatment and 12 treatments were evaluated on second instar nymphs and adults.", "Treatments The test product is EC25%, a 25% EC essential oil formulation obtained from Codena Inc.", "Seven concentrations were prepared as follow: EC25% at 8% was prepared by mixing 3.2 ml of EC25% and 6.4 ml of tap water and successive dilutions of 4%, 2%, 1%, 0.5% and 0.125% were made from the stock solution.", "EC25% was compared to the recommended doses of the following commercially available insecticides: Trounce® (20.2% potassium salts of fatty acids and 0.2% pyrethrins) at the recommended concentration of 1%; the insect growth regulator Enstar® (S-kinoprene), at the recommended concentration of 0.065% and Avide® (abamectin 1.9% EC) at the concentration of 0.000855%, Thiodane® (endosulfan 50 WP) at the concentration of 5% and Cygon® (dimethoate) at the concentration of 4%.", "Water was used as a negative control.", "The test product EC25% was sprayed first, starting from the lowest to the highest concentration followed by the water control treatment and finally by the reference products Avid, Cygon, Enstar, Thiodan and Trounce.", "The sprayer was rinsed three times between treatments using successively ethanol 95%, acetone, hexane and distilled water.", "Fecundity Assessment The potential sublethal effects of EC25% on Orius insidiosus female fecundity was monitored.", "Fecundity assessment was carried out on females that survived 48 h after the direct contact pesticide treatments.", "Surviving females were separated from males and put individually in a Petri dish filled with a 2 mm layer of agar used as a support and an apple ring (5.5 cm) placed upside down on the agar surface along with a 3 cm long pod of faba bean (Phaseolus vulgare).", "The apple leaf disc and the bean pod were used as oviposition substrates.", "The Petri dish was covered with the correspondent cover and sealed with parafilm.", "The Petri cover had an opening covered with fine muslin tissue for ventilation and air exchange.", "Females were left undisturbed for 48 H for oviposion and then were fed with sufficient numbers of Ephestia spp eggs.", "After the 48 h period, females were then transferred to another Petri dish for a second 48 H oviposition test.", "During both periods, the eggs laid were counted and left to hatch for 5 days.", "The eggs that do not hatch after 5 days were considered dead and not viable.", "Statistical Analysis LC50 values of EC25% were determined using probit analysis with POLO software (LeOra, 1987).", "Concentrations were analysed as main effects and the weight of pesticide applied was tested as a covariance to correct for difference in quantity of the applied pesticide.", "This covariance was deleted from the model when found not significant.", "Mortalities were analysed using General Linear model (GLM) procedure within SAS (SAS, 1996) and the number of individuals initially introduced were tested as a covariant.", "Means were adjusted for covariance when appropriate and separated using the Fisher test for means comparison.", "However, actual means were presented in the results section.", "Results Results show (FIG.", "46) that nine days following treatment application, with Orius nymphs, the most toxic treatments were in decreasing order, Trounce (99,5% mortality), Cygon (98% mortality), EC25% at 8% concentration (87.6% mortality), Avid (82.5% mortality) and EC25% at 4% concentration (79.6% mortality).", "All results were significantly different from that of the control treatment (3.6% mortality).", "Less than 50% mortality was obtained with the other treatments though only Thiodan (45.7%) and EC25% (35.1%) results were significantly different from the control.", "Results with EC25% at the recommended concentration for field application of 0.5% were not significantly different from results obtained with the control.", "Results show (FIG.", "47) that the effects of the 12 treatments expressed as percent mortality of adults was significantly different at 24 H after treatment (F=55.9, df=11, p<0.0001) and at 48 H after treatment (F=63.2, df=11, p<0.0001).", "The least toxic treatments of EC25% at concentration of 0.125% and 0.25% were not statistically different from the control treatment.", "The treatment of EC25% at the recommended field concentration of 0.5% was the least toxic of the remaining treatments causing a mortality of 28%.", "The most toxic group included Cygon (100% mortality), Trounce (98.9% mortality), EC25% at concentrations of 4 and 8% (94% and 94% respectively) and Avid (87.8%).", "Fecundity Assessment The results from the fedundity assessment assay (FIG.", "48) showed that almost all females tested had laid eggs.", "There were few surviving females to test for fecundity following treatments with Avid, Cygon, Trounce and EC25% at concentrations of 2, 4 and 8%.", "The mean number of eggs laid per female per day in the control treatment was 7.6 which was almost 4 times the minimum number of 2 eggs per female per day set by the IOBC standards for fecundity for Orius leavigatus, a closely related species of O. insidiosus.", "The lowest rate was 2.8 eggs per female per day obtained in the treatment with Thiodan followed by EC25% at 0.5% concentration with 3.6 eggs per female per day and both were significantly different from the rate obtained with the water control (7.6 eggs per female per day).", "The date rate of eggs laid in the EC25% treatments at concentrations of 0.25% (5 eggs) and 0.5% (5.4 eggs) were not significantly different from the number of eggs laid in the control.", "The eclosion rate varied from 28.5% in the Thiodan treatment to 53% in the control.", "There was 33.9% egg eclosion in the EC25% at 0.5% concentration treatment.", "LC50 values for Orius nymphs were 2.65%, 9 days following treatment with EC25% (FIG.", "49) and for adults were 1.14%, 2 days following treatment with EC25% (FIG.", "50).", "References: Ascher, K. R. S. and Cwilich, R. (1960) Laboratory Evaluation of Acaricides Against Tetranychus Telarius L. on Sugar Beet and on Beans.", "Israel J Agric.", "Res.", "10:159-163.ASTM.", "1987.Standard test methods for moisture, ash, and organic matter of peat and other organic soils.", "American Standards for Testing and Materials (ASTM) D 2974-87.ASTM.", "1989.Standard test method for pH of soils.", "American Standards for Testing and Materials (ASTM) D 4972-89.Bélanger et al., (1991) Extraction et Détermination de Composés Volatils de L'ail (Allium sativum), Riv.", "Ital.", "EPPOS 2:455-461.Busvine, J. R. (1980) Recommended Methods for Measurement of Pest Resistance to Pesticides.", "FAO Plant Production and Protection Paper.", "pp.", "49-53.Ceske, L. J. and P. B. Kaufman, (1999) Regulation of Metabolite Synthesis in Plants.", "In: Natural Products from Plants.", "Kaufman et al., eds.", "CRC Press.", "Boca Raton.", "pp.", "91-121.Chialva et al., (1983) Chemotaxonomy of Wormwood (Artemisia Absinthum L.).", "Z. Lebensm Unters Forsch 176:363-366.Dittrich, V. (1962) A Comparative Study of Toxicological Test Methods on a Population of the Two-spotted Spider Mite (Tetranychus telarius).", "J. Econ.", "Entomol.", "55:644-648.Duerbeck, K., et al., (1997) The Distillation of Essential Oils.", "Manufacturing and Plant Constuction Handbook.", "Protrade: Dept.", "of Foodstuffs and Agricultural Products.", "Eschborn, Germany.", "pp.", "21-25.Ebeling, W. and R. J. Pence.", "(1953) Pesticide Formulation: Influence of Formulation on Effectiveness.", "J. Agr.", "Food Chem.", "1:386-397.Feng, R. and M. B. Isman.", "(1995) Selection for resistance to azadirachtin in the green peach aphid, Myzus persicae, Experientia 51:831-833.Flint, M. L. (1990) Pests of the Garden and Small Farm.", "A Grower's Guide to Using Less Pesticide.", "Statewide IPM Project.", "U. of California.", "Publication 3332.pp.", "116-123.Foott, W. H. and H. R. Boyce.", "(1966) A Modification of the Leaf-Disc Technique for Acaricide Tests.", "Proc.", "Entomol.", "Soc.", "Ont.", "96:117-119.Georghiou, G. P. (1990) Overview of Insecticide Resistance.", "In: Managing Resistance to Agrochemicals.", "M. E. Green, H. M. Le Baron and W. K. Moberg, eds., ACS Symposium Series.", "Chapter 2.Vol.", "421:18-41.Gundersin et al., (1985).", "Effects of the Mint Monoterpene Pulegone on Spodoptera Eridania (Lepidoptera: Noctuidae) Environ.", "Entomol.", "14:859-863.Isman, M. (1999) Pesticides Based on Plant Essential Oils.", "Pesticide Outlook.", "pp.", "68-72.Jackson, S. A. L. and R. K. M. Hay.", "(1994) Characteristics of varieties of thyme (Thymus vulgaris L.) for use in the UK: Oil content, composition and related characters.", "J. Hort.", "Sci.", "69:275-281.LeOra Software.", "POLO-PC, (1994) Probit or Logit Analysis.", "LeOra Software, Berkeley Calif. pp.", "1-28 Lippold, P. (1963) Acaricidal Testing Techniques in the Two-spotted Spider Mite.", "Adv.", "Acarol.", "1:174-180.Mead-Briggs, et al., (2000) A laboratory test for evaluating the effects of plant protection products on the parasitic wasp, Aphidius rhopalosiphi (Destephani-Perez)(Hymenoptera: Braconidae).", "Pages 13-25.In.", "Guidelines to evaluate side effects of plants protection products to non target arthropods, IOBC, BART and EPPO Joint Initiative.", "Candolfi, M. P., Blumel, S., Forster, R. Bakker, F. M., Grimm, C., Hassan, S. A., Heimbach, U., Mead-Briggs, M. A., Reber, B, Schmuck, R., Vogt, H.", "(Edts.", "), Reinham, Germany.", "Meister, R. T. ed.", "(1999) Farm Chemicals Handbook.", "Vol.", "85.Meister Publishing Company, Willoughby, Ohio.", "Perez- Souto, N. et al., (1992) Use of high-performance liquid chromatographic peak deconvolution and peak labeling to identify antiparasitic components in plant extracts.", "J. of Chrom.", "593:209-215.Rembald, H. (1989) Azadirachtins.", "Their Structure and Mode of Action.", "In: Insecticides of Plant Origin.", "ACS Symposium Series No.", "387.pp.", "150-163.Roush, R. T. and J.", "A. McKenzie.", "(1987) Ecological Genetics of Insecticide and Acaricide Resistance.", "Ann.", "Rev.", "Entomol.", "32:361-380 The McGraw-Hill Dictionary of Chemical Terms (ed.", "Parker, S., 1985), McGraw-Hill, Sam Francisco.", "U.S. Pat.", "No.", "4,933,371 U.S. Pat.", "No.", "5,839,224" ] ]	Patent_10467696
[ [ "Method and apparatus for treating renal disease with hemodialysis utilizing pulsatile pump", "A method of removing toxins from blood from a patient in need of such toxin removal includes: providing a countercurrent dialysis filter (20) having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; conveying blood from the patient through the blood compartment of a countercurrent filter (20) an back to the patient; and drawing dialysate from a reservoir (32) through the dialysate compartment of the countercurrent filter (20).", "At least one of the blood or dialysate experiences pulsatile flow.", "The steps are carried out such that blood toxins are drawn from the blood compartment through the semi-permeable membrane into the dialysate compartment." ], [ "1.A method of removing toxins from blood from a patient in need of such toxin removal, comprising: providing a countercurrent dialysis filter having a blood compartment and a dialysate compartment separated from said blood compartment by a semi-permeable membrane; conveying blood from the patient through the blood compartment of a filter an back to the patient; and drawing dialysate from a reservoir through the dialysate compartment of the filter such that blood toxins are drawn from said blood compartment through the semi-permeable membrane into the dialysate compartment; wherein at least one of the conveying and drawing steps is carried out with pulsatile flow.", "2.The method defined in claim 1, wherein said drawing step comprises drawing the dialysate through the dialysate compartment with a pulsatile pump.", "3.The method defined in claim 2, wherein said drawing step comprises drawing the dialysate through the dialysate compartment at a pulsatile pressure of greater than about 10 mm Hg.", "4.The method defined in claim 1, wherein said drawing step comprises drawing the dialysate through the dialysate compartment at between 30 and 100 pulses per minute.", "5.The method defined in claim 1, wherein said drawing step further comprises drawing the dialysate through the dialysate compartment with a roller head pump.", "6.The method defined in claim 1, wherein said conveying step comprises conveying blood from the patient through the blood compartment with a constant flow pump.", "7.The method defined in claim 1, wherein said providing step comprises providing a dialysis filter with a dialysate compartment comprising a dialysate casing and a blood compartment comprising a plurality of tubules that reside within the dialysate casing.", "8.The method defined in claim 1, wherein said conveying step comprises conveying blood with a pulsatile pump.", "9.The method defined in claim 1, wherein said conveying step comprises conveying the blood with a pulse pressure of greater than about 30 mm Hg.", "10.An apparatus for performing hemodialysis on a subject in need of such treatment, comprising: a dialysis filter having a blood compartment and a dialysate compartment separated from said blood compartment by a semi-permeable membrane; a first pump fluidly connected with the blood compartment that conveys blood from the patient through a blood compartment of the filter and back to the subject; and a second pump fluidly connected to the dialysate compartment; wherein at least one of said first and second pumps is configured to induce pulsatile flow.", "11.The apparatus defined in claim 10, wherein said second pump comprises a pulsatile pump.", "12.The apparatus defined in claim 11, wherein said second pump is configured to induce a pulsatile pressure of greater than about 10 mm Hg in dialysate in the dialysate compartment.", "13.The apparatus defined in claim 10, wherein said dialysate compartment comprises a dialysate casing and said blood compartment comprises a plurality of blood tubules residing within said dialysate casing.", "14.The apparatus defined in claim 10, further comprising a third constant flow pump fluidly connected to said second pump.", "15.The apparatus defined in claim 10, wherein said second pump is configured to provide pulsatile flow at a rate of between about 30 and 100 cycles per minute.", "16.The apparatus defined in claim 10, wherein said first pump and said second pump are pulsatile pumps.", "15.The apparatus defined in claim 10, wherein the pulsatile pressure induced by said first or second pump is at least 30 mm Hg.", "16.The apparatus defined in claim 10, wherein the pulsatile pressure induced by said first pump is at least 30 mm Hg." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Hemodialysis is a well-known treatment technique for ESRD, a condition in which the patient's renal system has essentially ceased to remove waste products and contaminants from the blood.", "Hemodialysis is a process that involves removing blood from the vasculature of a patient (usually a shunt or vein), purifying it with dialysate (a fluid that helps to remove toxins and return electrolytes to the blood), and returning the blood to the patient (usually through another vein).", "Hemodialysis machines typically operate with separate compartments for blood and dialysate.", "In conventional systems, the compartments are separated by a semi-permeable membrane that allows selective diffusion; toxins are removed from the blood, and electrolytes are added to bring the electrolyte concentration of the blood to desired levels.", "In many systems, the blood and dialysate compartments are arranged in a countercurrent flow exchange layout, with the blood traveling in one direction and the dialysate traveling in the opposite direction.", "One arrangement employs a large tube within which reside many smaller diameter tubules.", "Ordinarily, the large tube carries dialysate, and the smaller tubules carry blood.", "Blood is typically pumped through the tubules with a positive pressure pump (exemplary is a roller head pump), and the dialysate is typically drawn through the large tube with a roller head pump.", "There are some shortcomings with such an arrangement.", "The relatively constant flow of the blood and dialysate can create “dead” regions (where flow essentially stops) and regions of laminar flow within the tube.", "In each of these regions the component exchange between the blood and the dialysate can be adversely affected, as fluid in the dead regions and the regions of laminar flow tends to have less surface area contact with the membrane, thereby reducing the efficiency of exchange.", "Also, the constant flow of dialysate can create “shunting” of dialysate in certain regions, which can lead to blood/dialysate mismatch.", "As such, it would be desirable to provide a hemodialysis system with improved efficiency of exchange." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention can address some of the shortcomings of prior systems by improving the efficiency of dialysis through the use of pulsatile flow.", "As a first aspect, the invention includes a method of removing toxins from blood from a patient in need of such toxin removal, comprising: providing a dialysis filter having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; conveying blood from the patient through the blood compartment of a filter and back to the patient; and drawing dialysate from a reservoir through the dialysate compartment of the countercurrent filter.", "At least one of the blood or dialysate experiences pulsatile flow.", "The steps are carried out such that blood toxins are drawn from the blood compartment through the semi-permeable membrane into the dialysate compartment.", "As a second aspect, the invention includes an apparatus for performing hemodialysis on a subject in need of such treatment.", "The apparatus comprises: a dialysis filter having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; a first pump fluidly connected with the blood compartment that conveys blood from the patient through a blood compartment of a filter and back to the subject; and a second pump fluidly connected to the dialysate compartment.", "At least one of the first and second pumps is configured to induce pulsatile flow.", "It has been discovered that the use of pulsatile flow in the blood or dialysate circuits during hemodialysis can provide a number of advantages.", "These include increased efficiency of transport, reduction in dead regions, and intermittent increased transmembrane pressure.", "Objects of the present invention will be appreciated by those of ordinary skill in the art from a reading of the figures and the detailed description of the preferred embodiments which follow, such description being merely illustrative of the present invention." ], [ "FIELD OF THE INVENTION The present invention is directed generally to treatment of renal disease, and more specifically to treatment of end stage renal disease (ESRD) with hemodialysis.", "BACKGROUND OF THE INVENTION Hemodialysis is a well-known treatment technique for ESRD, a condition in which the patient's renal system has essentially ceased to remove waste products and contaminants from the blood.", "Hemodialysis is a process that involves removing blood from the vasculature of a patient (usually a shunt or vein), purifying it with dialysate (a fluid that helps to remove toxins and return electrolytes to the blood), and returning the blood to the patient (usually through another vein).", "Hemodialysis machines typically operate with separate compartments for blood and dialysate.", "In conventional systems, the compartments are separated by a semi-permeable membrane that allows selective diffusion; toxins are removed from the blood, and electrolytes are added to bring the electrolyte concentration of the blood to desired levels.", "In many systems, the blood and dialysate compartments are arranged in a countercurrent flow exchange layout, with the blood traveling in one direction and the dialysate traveling in the opposite direction.", "One arrangement employs a large tube within which reside many smaller diameter tubules.", "Ordinarily, the large tube carries dialysate, and the smaller tubules carry blood.", "Blood is typically pumped through the tubules with a positive pressure pump (exemplary is a roller head pump), and the dialysate is typically drawn through the large tube with a roller head pump.", "There are some shortcomings with such an arrangement.", "The relatively constant flow of the blood and dialysate can create “dead” regions (where flow essentially stops) and regions of laminar flow within the tube.", "In each of these regions the component exchange between the blood and the dialysate can be adversely affected, as fluid in the dead regions and the regions of laminar flow tends to have less surface area contact with the membrane, thereby reducing the efficiency of exchange.", "Also, the constant flow of dialysate can create “shunting” of dialysate in certain regions, which can lead to blood/dialysate mismatch.", "As such, it would be desirable to provide a hemodialysis system with improved efficiency of exchange.", "SUMMARY OF THE INVENTION The present invention can address some of the shortcomings of prior systems by improving the efficiency of dialysis through the use of pulsatile flow.", "As a first aspect, the invention includes a method of removing toxins from blood from a patient in need of such toxin removal, comprising: providing a dialysis filter having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; conveying blood from the patient through the blood compartment of a filter and back to the patient; and drawing dialysate from a reservoir through the dialysate compartment of the countercurrent filter.", "At least one of the blood or dialysate experiences pulsatile flow.", "The steps are carried out such that blood toxins are drawn from the blood compartment through the semi-permeable membrane into the dialysate compartment.", "As a second aspect, the invention includes an apparatus for performing hemodialysis on a subject in need of such treatment.", "The apparatus comprises: a dialysis filter having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; a first pump fluidly connected with the blood compartment that conveys blood from the patient through a blood compartment of a filter and back to the subject; and a second pump fluidly connected to the dialysate compartment.", "At least one of the first and second pumps is configured to induce pulsatile flow.", "It has been discovered that the use of pulsatile flow in the blood or dialysate circuits during hemodialysis can provide a number of advantages.", "These include increased efficiency of transport, reduction in dead regions, and intermittent increased transmembrane pressure.", "Objects of the present invention will be appreciated by those of ordinary skill in the art from a reading of the figures and the detailed description of the preferred embodiments which follow, such description being merely illustrative of the present invention.", "BRIEF DESCRIPTION OF THE FIGURES The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain principles of the invention.", "FIG.", "1 is a schematic diagram of a hemodialysis apparatus of the present invention.", "FIG.", "2 is an enlarged schematic view of a hollow fiber artificial kidney (HFAK) included in the hemodialysis apparatus of FIG.", "1.FIG.", "2A is a greatly enlarged schematic view of two blood tubules of the HFAK of FIG.", "2.FIG.", "3 is a graph plotting urea concentration as a function of time in a two pool model of a dialysate.", "FIG.", "4 is a graph plotting creatinine concentration as a function of time collected in a dialysis study on dogs.", "FIG.", "5 is a graph plotting BUN concentration as a function of time collected in a dialysis study on a dog.", "DETAILED DESCRIPTION OF THE INVENTION The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown and described.", "This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.", "Like numbers refer to like components throughout.", "Referring now to FIG.", "1, a hemodialysis apparatus, designated broadly at 10, is illustrated schematically.", "The hemodialysis apparatus 10 comprises a blood subsystem 12 and a dialysate subsystem 30, each of which will be described in greater detail below.", "The blood subsystem 12, which as the name implies conveys blood through the hemodialysis apparatus 10, includes a blood entry conduit 14 that leads from a patient's artery (usually in the wrist), a roller pump 16 fluidly connected with the blood entry conduit 14, an HFAK 20 of conventional construction, and a blood exit conduit 18 leading from the HFAK 20 back to the patient's vein.", "A bubble trap 19 is located on the blood exit conduit 18 between the HFAK 20 and the vein of the patient to prevent bubbles created during the processing of the blood from entering the patient.", "During operation, blood exits the patient through the blood entry conduit 14.The roller pump 16 forces the blood to the HFAK 20, wherein dialysis occurs as described below.", "The dialyzed blood then exits the HFAK 20 in the blood exit conduit 18, passes through the bubble trap 19 and returns to the patient's body.", "The dialysate subsystem 30, which conveys dialysate into and from the HFAK 20, includes a dialysate reservoir 32 that contains dialysate, a dialysate entry conduit 34 leading from the dialysate reservoir 32 to the HVAK 20, a dialysate exit conduit 36 leading from the HFAK 20, and an in-line pulsatile pump 38 located on the dialysate exit conduit 36.In operation, dialysate is pumped from the dialysate reservoir 32 through the dialysate entry conduit 34 and into the HFAK 20, wherein countercurrent exchange of components of the blood and dialysate.", "The spent dialysate is then pumped via the pulsatile pump 38 back to the dialysate reservoir 32.An exemplary HVAK 20 is illustrated in FIG.", "2.The HVAK 20 includes a plurality of narrow blood tubules 22 that are enclosed within a dialysate casing 24.As the name implies, blood is conveyed through the blood entry conduit 14 into the blood tubules 22, and dialysate is conveyed from the dialysate entry conduit 34 into the dialysate casing 24.The blood tubules 22 are formed of a semi-permeable membrane material, such as polysulfone, that enables countercurrent exchange of components to occur between the blood and dialysate.", "The dialysate casing 24 is typically formed of an impervious material such as a plastic material.", "The dialysate utilized in the dialysate subsystem 30 can be any dialysate known to those skilled in this art as being suitable for use in hemodialysis.", "An exemplary dialysate is available under the trade name Neutralyte from Fresenius Medical Care, Lexington, Mass.", "The use of the pulsatile pump 38 (which can be a piston pump, a modified roller head pump, or the like) in the dialysate subsystem 30 can have the effect of producing pulsatile flow of dialysate within the dialysate subsystem 30.As used herein, “pulsatile flow” means flow that has a pulse pressure of 10 mm Hg or greater, and preferably when applied to blood means a pulse pressure of 30 mm Hg or greater.", "Preferably, the pulsatile flow is induced in the dialysate at a pulse rate of between about 30 and 100 cycles per minute, and more preferably at a rate of between about 50 and 80 cycles per minute.", "Those skilled in this art will recognize that the pulsatile pump may be used alone or in combination with a more constant flow pump.", "Such pulsatile flow within the dialysate subsystem 30 can generate significant turbulence within the dialysate casing 24, which can reduce or eliminate the number of “dead” regions, where flow stops and exchange is minimal.", "A typical flow pattern in blood tubules 22 and the blood casing 24 is shown in FIG.", "2A (darker areas represent regions of higher flow), with a dead region 25 being illustrated between two tubules 22.Turbulence can increase the exchange efficiency between the blood and dialysate by increasing the amount of surface area contact between the dialysate and the blood tubules 22.Also, the pulsation can produce bursts of increased transmembrane pressure, which additionally helps exchange.", "The increased energy introduced into the system can also enhance transport.", "Further, reduction of “dead” regions reduces the tendency of clotting, with secondary improvement in transport.", "As a result of these combined effects, the overall efficiency of hemodialysis (for example, increased clearance of urea and creatinine) can be significantly increased over systems that lack a pulsatile dialysate flow.", "It should also be noted that the pump 16 of the blood subsystem 12 may also be a pulsatile pump, with many of the advantages described above for the dialysate subsystem 30 also being achievable with pulsatile flow in the blood subsystem 12.If a hemodialysis apparatus includes pulsatile pumps in both the blood and dialysate subsystems, the pulsatile pumps may be synchronous, such that they pump at the same rate and with matching amplitudes, or they may be dissynchronous, such that they pump at different rates and/or with mismatched amplitudes.", "Given that reduction of dead regions in the HFAK 20 can improve transport, it may be preferred to employ dissynchronous pulsatile pumps, as doing so may increase turbulence.", "The invention and the advantages achievable therewith will now be described in greater detail in the following non-limiting examples.", "Example 1 In Vitro Analysis of Pulsatile Flow of Dialysate In vitro dialysis experiments were conducted using a pair of pools filled with liquid and performing dialysis on these liquids.", "Experimental blood and dialysate circuits were created that each included a two-liter reservoir connected to a roller head pump (Sarns 7400 MDX, available from Sarns, Inc., Ann Arbor, Mich.).", "The roller head pump was connected to a dialysis filter, which was in turn connected to the two liter reservoir to form a closed loop.", "All connections were made with ⅜ inch plastic tubing.", "For the control apparatus, the dialysis machine used was a Fresenius 2008H dialysis machine (available from Fresenius USA, Lexington, Mass.", "), which employs a relatively constant flow pump.", "The experimental system employed a Fresenius F7NR dialysis filter (available from Fresenius USA, Lexington, Mass.)", "and a Sarns roller head pump that operated at 50 cycles per minute and at a pulse pressure of 80 mm Hg.", "For each system, the two liter reservoir was filled with a mixture of tap water and urea (2 liters water to 9.8-10 g of urea) to be used as experimental blood.", "This mixture had a measured level of 95-100 mg/dL urea.", "Tap water was used as the experimental dialysate.", "Experimental blood and dialysate were then allowed to flow through the system at predetermined rates.", "Samples of fluid were drawn from the reservoir at 5 minute intervals for 35 minutes, and the urea level was measured with an Olympus AU640 instrument (available from Olympus Corp., Dallas, Tex.).", "The results of the procedures can be seen in Tables 1 and 2 below and FIG.", "3.TABLE 1 Dialysate Flow -500 cc/min Pulsatile Experimental Blood Compartment Only Blood Flow Urea (cc/min) Procedure Baseline 5 min 10 min 15 min 20 min 200 Control Dialysis 94 77 53 40 28 (18.1%) (43.6%) (57.4%) (70.2%) Variable Speed 97 65 39 23 14 Dialysis (33%) (59.8%) (76.3%) (85.6%) % Greater Efficiency 45 27 22 18 250 Control Dialysis 97 64 42 25 15 (34.0%) (56.7%) (74.2%) (84.5%) Variable Speed 95 57 32 18 9 Dialysis (40.0%) (66.3%) (81.1%) (6.6%) % Greater Efficiency 15 9.6 8.5 6.6 330 Control Dialysis 97 61 33 18 10 (37.0%) (65.9%) (81.4%) (89.7%) Variable Speed 94 56 27 14 7 Dialysis (40.0%) (71.3%) (85.1%) (92.6%) % Greater Efficiency 7.5 7.6 3.7 3.0 400 Control Dialysis 113 75 53 33 20 (33.6%) (53.1%) (70.8%) (82.3%) Variable Speed 114 55 30 14 7 Dialysis (51.8%) (73.7%) (87.7%) (93.9%) % Greater Efficiency 35.1 20.6 19.3 12.4 TABLE 2 Dialysate Flow-190 cc/min Pulsatile Experimental Dialysate Compartment Only Blood Flow Urea (cc/min) Procedure Baseline 5 min 10 min 15 min 215 Control Dialysis 97 80 62 38 (17.5%) (36.0%) (60.8%) Variable Speed 96 73 54 30 Dialysis (23.9%) (43.7%) (68.8%) % Greater 26.7 17.6 11.6 Efficiency As can be seen from Tables 1 and 2 and FIG.", "3, the efficiency of hemodialysis increases significantly (as much as 35 percent) with the employment of a variable speed pump for pumping blood or dialysate.", "Example 2 The experimental apparatus used in Example 1 was employed again to perform dialysis on a dog with the following changes.", "A HB 500 filter was employed with the Century System III dialysis machine (both available from Gambro Corp., Lakewood, Colo.).", "Also, rather than tap water, a standard 3K dialysate was used to hemodialyze the dog.", "Pulsatile flow was induced in the blood subsystem with a roller head pump operating at 50 cycles/minute.", "In addition, both blood urea nitrogen (BUN) and creatinine levels were measured (BUN was measured with a Olympus AU640 instrument and creatinine was measured by a Jaffe assay).", "The results of the procedure are shown in Tables 3 and 4 and FIGS.", "4 and 5.TABLE 3 BUN Level Procedure Baseline 60 min 120 min Post 30 min Control Dialysis 110 110 (0.0%) 80 (27.3%) 97 Variable Speed Dialysis 126 84 (33.3%) 69 (45.3%) 73 % Greater Efficiency 39.8% TABLE 4 Creatinine Level Procedure Baseline 60 min 120 min Post 30 min Control Dialysis 10.2 8.7 (14.7%) 7.3 (28.5%) 9.1 Variable Speed 11.8 7.7 (34.7%) 6.3 (44.7%) 7.4 Dialysis % Greater 56.7% 36.3% Efficiency These results again indicate that the variable speed dialysis procedure is significantly more efficient than the control apparatus.", "The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof.", "The invention is defined by the following claims, with equivalents of the claims to be included therein." ] ]	Patent_10467755
[ [ "Dynamic management of access rights lists in a portable electronic object", "Access rights lists, such as capacity or access control lists, are dynamically managed in a data processing element such as a smart card from an administrator server.", "To access an access rights list from the server, the list is signed in the server so that a signature can be transmitted to the card.", "The card compares the signature received from the server to signatures determined according to the access rights lists contained in the card and keys associated with those lists.", "Server access to an identified list is only authorized when it corresponds to a signature which is found among the determined signatures in the card and which is identical to the received signature." ], [ "1.A method for managing lists of rights of access between subjects and objects, stored in a data processing means from an external administrator entity, comprising the following steps: initially associating keys of administrator entities with access rights lists and storing a security algorithm in the data processing means, and subsequently accessing an access rights list from the entity by: signing the access rights list in the entity by applying determined data from the list and an associated key to the security algorithm in order to produce a signature, transmitting the signature from the entity to the data processing means, comparing the signature received in the data processing means with determined signatures according to applications of determined data in lists of access rights contained in the data processing means and of keys respectively associated with these lists to the security algorithm, and allowing access of the entity to an access rights list found only if its determined signature is identical to the received signature.", "2.A method according to claim 1, wherein the association of the keys with the access rights lists is performed in advance in the processing means prior to bringing the data processing means into service.", "3.A method according to claim 1, wherein the association of the keys with an access rights list to be added to the data processing means is performed by transmitting the list with the keys from the entity to the data processing means, and determination of signatures of the list received in the data processing means is performed by applying the determined data in the list received and the keys received to the algorithm, and validation of the recording of the list received in the data processing means occurs when one of the signatures determined is identical with the signature received.", "4.A method according to claim 1, wherein the signing step is replaced by the reception in the entity of a signature transmitted by another entity.", "5.A method according to claim 1, wherein delegation information is transmitted with the signature, and access of the entity to the list found is not authorised when the delegation information is detected in association with the list found in the data processing means.", "6.A method according to claim 1, wherein the signatures to be compared with the signature received are determined in the data processing means prior to the transmitting step.", "7.A method according to claim 1, further including the step before authorising access of the entity to the control list, of updating a duration of life parameter of the access rights list found in order to erase the access rights list found when the updated duration of life parameter exceeds a maximum limit and in order to authorise access to the access rights list found to the entity when the updated duration of life parameter is less than the maximum limit.", "8.A method according to claim 7, wherein the duration of life parameter is a cumulative duration of sessions of use of the data processing means or a cumulative duration of absolute time.", "9.A method according to claim 7, wherein the duration of life parameter is a number of sessions of use of the data processing means, or a number of sessions using the access rights list found.", "10.A method according to claim 7, wherein the duration of life parameter is a number of commands received by the data processing means.", "11.A method according to claim 7, wherein the duration of life parameter is a synchronisation value changed periodically in external administrator entities and transmitted with the signature of the access rights list.", "12.A method according to claim 1, wherein the signatures of the access list associated with the keys of a list are again determined in the data processing means if the access rights list found has been modified by the entity.", "13.A method according to claim 1, wherein the determined data of the list which are applied to the security algorithm depend on characteristics of at least one subject and/or at least one subject group and/or at least one object and/or at least one right of access of a subject to an object relating to the list.", "14.A data processing means storing access rights lists managed from at least one external administrator entity comprising: a means for storing keys of administrator entities in association with access rights lists, a means for implementing a security algorithm, a means for determining signatures according to applications of determined data of the lists of access rights and keys respectively associated with said lists to the algorithm, a means for comparing a received signature of an access rights list which results, in the entity, from the application of determined data of the list and of the key of the entity to the algorithm and which is transmitted by the entity, to said determined signatures, and a means for authorising access of the entity to an access rights list found only in correspondence with a signature found amongst the determined signatures and identical to the received signature.", "15.A data processing means according to claim 14, further including a means for storing a duration of life parameter and a maximum duration limit for each access rights list, and a means for updating the duration parameter of the list found in order to erase the access rights list found when the duration of life parameter updated exceeds the maximum limit and in order to authorise access to the access list found to the entity when the duration of life parameter updated is less than the maximum limit." ], [ "The present invention relates in general to terms the management of rights of access, by subjects constituting users or software modules of a data processing means, to objects such as applications implemented in the data processing means.", "More particularly, the invention relates to the administration of accesses to resources in a portable electronic object, such as a chip card, also known as a microcontroller card or integrated circuit card, constituting the data processing means, in particular when the chip card is a multiapplication card.", "Because of the greater and greater number of more and more complex applications introduced into a chip card, the management of the applications constituting the principal resources of the chip card are more and more difficult to manage.", "The difficulties in management are also due to the many partners participating in the allocation of access to the applications and whose interests sometimes diverge.", "These partners may be the manufacturer of the chip card, the distributor or operator of the chip card, and the developers of the applications in the chip card.", "Nevertheless, despite this complexity, access to the chip card resources must be controlled and made secure.", "At the present time, access to a processing resource, such as an application, is made by transmitting, from a terminal accepting the chip card, at least one command constituting an application protocol data unit (APDU) which contains data or a reference to data present and to be processed in the card.", "According to another variant, access to a resource in the card can be effected at a higher level by invoking a method of an object present in the card when the latter contains applications written in an object oriented high level programming language such as Java.", "The coexistence and cooperation of several applications within the same chip card raises many problems from the point of view of security.", "In particular, each application has its own data for which the developer of the application defines access rights peculiar to the application.", "The access conditions are means of connection between external accesses which may be users of the card or software modules, such as user interfaces, and accesses internal to the card such as applications, possibly by means of other applications or other application software elements in the card.", "The control of the access conditions is based on the identification of the subjects Su, such as the users, which are the “active” elements which manipulate information contained in objects Ob, such as applications, which are “passive” elements containing data.", "The conditions for the access of the subjects Su to the objects Ob are governed by access control rules between the subjects and the objects.", "Each rule comprises an access right, that is to say a link between a subject and an object in the form of an action which can be performed by the subject on the object.", "It is known how to represent the conditions for access of subjects Su to objects Ob by an access matrix MA whose columns correspond to subjects and whose rows correspond to objects, as shown in FIG.", "1.For example, the matrix MA relates to three subjects S1, S2 and S3, such as three users, and to three objects O1, O2 and O3, such as files and programs.", "Each box in the matrix at the intersection of a row and column contains access rights, that is to say privileged actions which can be performed by the respective subject on the respective object.", "The access rights may be positive in order to allow a predetermined action by a subject on an object, or may be negative in order to prevent a predetermined action by a subject or an object.", "For example, the subject S2 can read and execute the object O2 but cannot write in this object, and the subject S3 can read the object O1 but cannot record and write to the object O1.As is known, the access control rules are generally dealt with according to two approaches.", "The first approach consists of access control lists (ACL) corresponding to the rows in the access matrix MA and each specifying rights of access by subjects to the object associated with the row.", "By way of example, in a multiapplication chip card of the Windows (registered trade mark) type, access control lists ACL define accesses by users to files included in the card.", "Conversely, the second approach consists of capacities corresponding to the columns in the matrix MA and each specifying the access rights of the subject associated with the column over the objects.", "For example, the access control relates to applet methods for multiapplication smart cards of the JavaCard type in which programs in Java language have been written.", "The capacities are in the form of pointers making calls for accessing methods constituting objects, in predetermined applets constituting subjects.", "For more simplicity, reference will be made hereinafter to the management of access control lists although the invention also relates to the management of capacities.", "Access control lists and capacities are to be considered to be lists of rights of access between at least one subject and at least one object.", "With a present-day chip card, the modification of access control lists is reserved for only one card administration authority.", "After authentication of the administration authority by the card, the authority demands modifications to the access control lists, for example by adding or eliminating lists, adding or eliminating subjects in a list, or adding or eliminating access rights of a subject with respect to an object.", "This single administration authority must of course comply with the requirements of the various partners participating in the production and management of the various application resources in the chip card.", "The objective of the present invention is to make possible the dynamic management of access control lists or capacities in a portable electronic object of the chip card type in order to refine the management of such lists or capacities and thus permit an increase in the number of administrators authorised to act with regard to security on modifications to the access control lists or capacities.", "To achieve this objective, a method for managing lists of rights of access between subjects and objects, stored in a data processing means from an external administrator entity, is characterised in that it comprises the following steps: initially associating keys of administrator entities with access rights lists and supplying a security algorithm in the data processing means, and in order to access an access rights list from the entity: signing the access rights list in the entity by applying determined data from the list and the key to the security algorithm in order to produce a signature, transmitting the signature from the entity to the data processing means, and comparing the signature received in the data processing means with determined signatures according to applications of determined data in lists of access rights contained in the data processing means and of keys respectively associated with these lists to the security algorithm, and allowing access of the entity to an access rights list found only in correspondence with a signature found amongst the determined signatures in the data processing means and identical to the received signature.", "Thus, according to the invention, any authorised administrator accesses an access rights list constituting an access control list or a capacity in a data processing means consisting for example of the microcontroller of a portable electronic object.", "An access right authorises or prohibits one or more subjects from performing an action on a subject, or a subject from performing an action on one or more objects.", "This access control list or capacity is then managed dynamically by each administrator, by obliging it first to sign the access control list or the capacity to which it wishes to gain access, so as to be recognised in the electronic object by its signature of the access control list or of the capacity.", "Management of the access control lists is thus decentralised in administrator entities external to the data processing means.", "According to another characteristic of the invention, the duration of application of an access rights list is limited.", "More precisely, before authorising access of the entity to the control list, a duration of life parameter of the access rights list found is updated in order to erase the access rights list found when the duration of life parameter updated exceeds a maximum limit and in order to allow access to the access rights list found to the entity when the duration of life parameter updated is less than the maximum limit.", "The limited duration of life of an access rights list then prompts the subject or subjects affected by this list which has become out of date and is now erased to return to the administrator or administrators of this list in order to once again request access rights from them.", "The invention also relates to a data processing means in particular in a portable electronic object, storing access rights lists managed from at least one external administrator entity, implementing the method of the invention.", "It is characterised in that it comprises: a means for storing keys of administrator entities in association with access rights lists, a means for implementing a security algorithm, a means for determining signatures according to applications of determined data of the lists of access rights and keys respectively associated with these lists to the algorithm, a means for comparing a received signature of an access rights list which results, in the entity, from the application of determined data of the list and of the key of the entity to the algorithm and which is transmitted by the entity, to the said determined signatures, and a means for authorising access of the entity to an access rights list found only in correspondence with a signature found amongst the determined signatures and identical to the received signature.", "The data processing means can also comprise a means for storing a duration of life parameter and a maximum duration limit for each access rights list, and a means for updating the duration parameter of the list found in order to erase the access rights list found when the duration of life parameter updated exceeds the maximum limit and in order to authorise access to the access list found to the entity when the duration of life parameter updated is less than the maximum limit.", "Other characteristics and advantages of the present invention will emerge more clearly from a reading of the following description of several preferred embodiments of the invention with reference to the corresponding accompanying drawings, in which: FIG.", "1 is a diagram showing a control matrix between three subjects and three objects, already commented on according to the prior art; FIG.", "2 is a schematic block diagram of a telecommunications system between an administrator server and a portable electronic object of the chip card type for implementing the management method according to the invention; and FIG.", "3 is an algorithm of steps of the method of dynamic management of access rights lists according to the invention.", "With reference to FIG.", "2, the data processing means is a controller, such as the microcontroller of a chip card CA which contains several applications constituting objects to which subjects, such as users or units or software means, can gain access according to access rights.", "In a known manner, the microcontroller of the chip card CA comprises a microprocessor PR, a memory MO of the ROM type, a non-volatile memory MNV of the programmable and erasable type, such as a EEPROM memory, and a memory MA of the RAM type receiving in particular data from a terminal TE accepting the card.", "In the memory MO there are included in particular an operating system OS of the card, communication, authentication and service applications AP constituting objects Ob, as well as a security algorithm AS used for recognising administrator signatures and an algorithm for managing access rights lists AG according to the invention.", "The memory MNV contains data in particular relating to the possessor of the card and to the supplier of the card, as well as access control lists ACL(Ob), or capacities.", "Each access control list relating to a respective object Ob, to which reference is made below as an access rights list, contains, as shown in FIG.", "1, a list of subjects Su each associated with a list of positive access rights authorising and/or negative access rights prohibiting the performance of actions on the respective object Ob.", "The memory MNV thus contains a subject set ES, an object set EO, that is to say a set referring to applications, and an access rule set ER.", "The memory MNV preferably also contains a subject group set EG.", "Each group Gp contains subjects Su each having at least one predetermined access to a predetermined object Ob; thus a subject in a group Gp has all the access rights granted to this group, and a subject can belong to one or more groups.", "A rule R for access to an object Ob concerns an action, such as for example reading, writing, execution or recording, which is authorised or prohibited to a subject Su or to a subject group Gp on an object Ob.", "Consequently an access control list ACL(Ob) relates to access rules for predetermined subjects and/or predetermined groups on the respective object Ob.", "According to the invention, when the card is manufactured, or before the card CA is brought into service, that is to say before it is passed to the owner of the card, administrators of the lists ACL contained in the card are defined at an initial step ET01.Each administrator AD is defined by a respective administrator key KAD which is associated with one or more access control lists ACL to which the administrator AD has access.", "Each access control list ACL is associated with an administrator sub-list consisting of the corresponding administrator keys KAD.", "As will be seen hereinafter, the administrator key list KAD is preferably supplemented by an administrator signature list SGAD.", "A signature represents the access control list ACL signed by the respective administrator according to the security algorithm AS.", "Thus, according to the invention, the administrator keys KAD and preferably the administrator signatures SGAD are previously written in the non-volatile memory MNV of the chip card CA in correspondence with the access control lists ACL.", "FIG.", "2 also shows diagrammatically an administrator entity external to the card CA in the form of an administrator server SAD connected to the chip card through a telecommunication network RT and the accepting terminal TE.", "The telecommunication network RT designates any type of telecommunication network, or combination of networks, such as a radiotelephony network, a switched telephone network, an integrated service digital network ISDN, a high-rate network of the ATM type, the Internet, a packet transmission network, etc.", "The server SAD represents an administrator or several administrators.", "Other administrators can be situated at other distant servers (not shown).", "The administrator AD is for example the distributor of the chip card CA, or a partner commercially associated with this distributor, or the developer of one or more applications AP constituting objects Ob implemented in the chip card CA.", "As a variant, an administrator himself possesses a chip card which is housed in an additional reader of the server SAD so that the server SAD reads in the chip card the references of the administrator such as an administrator identifier IAD and an administrator key KAD.", "As shown schematically also in FIG.", "2, the accepting terminal TE is provided with a keypad CL and a reader LE for receiving the chip card CA and connecting thereto by means of an electrical-contact connection LI according to this embodiment.", "The accepting terminal TE is for example a chip card commissioning terminal, a bank terminal, or a point of sale terminal, or a mobile telephony terminal in which the chip card CA constitutes a removable subscriber identity module SIM or an additional chip card when the mobile terminal is provided with an additional card reader.", "After the initial step ET01, the management method according to the invention preferably comprises a second initial step ET02 during which administrator signatures SGAD are determined.", "Step ET02 is also implemented for a list ACL which has just been modified or introduced into the memory MNV in the card CA.", "Each signature SGAD of an administrator SAD depends on the key KAD of the administrator and determined data in an access control list ACL with which the key KAD is associated.", "The determined data in the access control list ACL depend essentially on the characteristics of the objects Ob and the subjects Su and/or groups of subjects Gp each having at least one right of access to the object Ob, as well as where applicable access rights characteristics.", "The list ACL and the key KAD are applied to the security algorithm AS, the result of which constitutes the administrator signature SGAD.", "The management method shown in FIG.", "3 comprises principally steps ET2 to ET14 triggered by a first step ET1 during which the administrator server SAD requests the establishment of a call with the accepting terminal TE containing the chip card CA through the telecommunication network RT, so as to initiate an access control list management session with the chip card CA.", "At the start of this session the card CA attempts to authenticate the administrator SAD at step ET2.The authentication is conventional and consists essentially in transmitting a random number by means of the chip card CA to the server SAD and comparing in the chip card CA the results of the application of this random number and of an authentication key prestored in the card CA and the server SAD, performed both in the card CA and the server SAD.", "Conversely, the server SAD authenticates the chip card CA.", "According to another variant, the authentication is mutual and comprises an authentication of the server SAD by the card CA and an authentication of the card CA by the server SAD.", "If the authentication or one of the authentications at step ET2 gives different results in the server SAD and the chip card CA, the call is broken off at a final step ET14.In a variant, the management method comprises no authentication.", "When the authentication has succeeded, the administrator server SAD selects an access control list ACL which it has under management locally in a table of access control lists, at the following step ET3, although the administrator can manage only one access control list ACL.", "The server SAD next signs the selected access control list ACL by applying the determined data of the list ACL and the key KAD of the administrator server SAD to the security algorithm AS so as to produce an administrator signature SGAD at step ET4.In a variant, instead of itself signing the access rights list ACL and producing the administrator signature SGAD, the server SAD receives this signature SGAD transmitted by another main administrator editor, such as a main administrator server connected to the telecommunication network RT, after the main administrator server has authenticated the server SAD.", "Then the signature SGAD constituting the selected access control list ACL signed is transmitted by the server SAD to the chip card CA through the network RT and the terminal TE in the form of an appropriate message at step ET5.This message can contain other data such as an administrator identifier IAD which designates a respective table of access control lists TACL in the chip card CA.", "The table TACL contains identifiers of access control lists to which the administrator server SAD has access, and preferably contains signatures of these access control lists signed by the administrator server SAD.", "At the following step ET6, in response to the administrator signature SGAD, the microprocessor PR in the chip card CA seeks an access control list which is able to be signed by the administrator server SAD.", "This search can consist in determining all the administrator signatures respectively associated with the access control lists which the chip card CA contains, and then comparing the signatures determined with the signature SGAD received by the card CA.", "However, as indicated at step ET02, the processor PR of the chip card CA has preferably, prior at least to step ET5, determined the signatures SGAD corresponding to all the administrators respectively associated with the access control lists ACL and has written them in EEPROM memory MNV.", "The processor PR then compares the signature SGAD received at step ET5 only with the signatures previously determined and classified in the table TACL.", "If the processor PR finds no administrator signature in memory MNV identical to the signature SGAD transmitted by the server SAD, the processor PR invites the accepting terminal TE to break off the communication at step ET14.In a variant, when the administrator identifiers IAD are provided in the memory MNV, the processor PR compares the received signature SGAD only with the signatures contained in the table TACL designated by the received identifier IAD and associated with the key KAD, that is to say only with the signatures associated with access control lists to which the administrator server SAD has access.", "Thus, after step ET6, an access control list ACL corresponding to the received administrator signature SGAD is found in order to process it by means of the server SAD, as indicated at step ET7.Although, according to a simple variant, step ET7 is followed by a step ET10 allowing access of the administrator server SAD to the access control list ACL previously found, the invention provides intermediate steps ET8 and ET9 during which a duration of life parameter pdv of the access rights list ACL found is updated and compared with a maximum limit PDV.", "The parameter pdv and the limit PDV are written in the memory MNV of the card.", "The limit PDV expresses the duration of life of the access control list ACL found at step ET7 and is prestored in the memory MNV at the initial step ET01, when the card CA is manufactured or brought into service, or when there is an addition of the list ACL, as will be seen subsequently.", "At the initial step ET01, the duration of life parameter pdv is zeroed so that it is incremented each time step ET8 is executed.", "The duration of life parameter pdv can be a cumulative duration of sessions of use of the chip card CA expressed in hours and minutes like the limit PDV.", "According to a second variant, the parameter pdv is a cumulative duration of absolute time expressed in terms of date and time, the limit PDV then designating a maximum predetermined duration or an expiry date of the determined list ACL.", "The updating of the duration can be carried out in the card CA by interrogating a trustworthy time stamping device, for example included in the terminal TE, or in the administrator server SAD.", "According to other variants, the duration of life parameter pdv is a number of sessions of use of the chip card CA, or a number of sessions using the list of access rights found ACL, the limit ACL being a maximum number of sessions.", "According to yet another variant, the duration of life parameter pdv is a number of commands APDU received by the chip card CA, the limit PDV then being a maximum number of commands.", "According to yet another variant, the duration of life parameter is a synchronisation value changed periodically in the administrator servers and transmitted to the card by any administrator server SAD in each message which contains the signature SGAD of the list of access rights sought ACL, at step ET5, the limit PDV being at the maximum number.", "Step ET8 thus increments the duration of life parameter according to its nature, the increment being in particular a period or a difference in date, or a unit, or a number of commands.", "If at step ET9 the duration of life parameter pdv exceeds the limit PDV, the processor PR erases the access control list ACL which was found at step ET7 and all the data associated with the list ACL at step ET13, and breaks off the communication with the server SAD at step ET14.In a variant, step ET13 only partially erases the list found, for example by erasing therein only the positive or negative access rights.", "If the duration of life is not yet reached at step ET9, the administrator server SAD is authorised to effectively gain access to the access control list ACL found, at step ET10.The server SAD then proceeds with a modification of the access control list found ACL.", "This modification consists for example in totally erasing the access control list ACL in the memory MNV, or modifying one of the data in the list found ACL, in particular eliminating or adding a subject Su in the access control list found with corresponding access rights R, or eliminating or adding at least one access right R relating to a subject Su included in the list found.", "The modification can also consist in adding a new administrator key to be associated with the list found and included in the message transmitted at step ET5.If at step ET11 the access control list found ACL has not been modified or has been erased, the method passes directly to the breaking off of the communication at step ET14.On the other hand, if the access list found ACL has been modified by the server SAD, this modified list being designated by ACLm, the processor PR determines, at step ET12, new administrator signatures SGADm resulting from the application of the determined data in the modified access control list ACLm and respectively administrator keys KAD associated with the list, to the security algorithm AS.", "The signatures SGADm replace the previous signatures SGAD so that, during a future list management session, the administrator server SAD can access the control list ACLm thus modified in the memory MNV of the card CA.", "Then the management method ends with the breaking off of the communication between the server SAD and the card CA at step ET14.When an access control list established in the administrator server SAD is to be added in the memory MNV of the chip card CA, steps ET1 to ET5 are executed, the message transmitted at step ET5 also containing the list ACL to be added with the administrator keys KAD associated with the list.", "Then, in place of steps ET6 to ET13, the processor PR determines and stores the signatures SGAD of the list received in the respective list tables TACL in the card CA.", "For each administrator associated with the list, the respective signature SGAD results from application of the determined data of the list and of the respective key KAD received to the security algorithm AS.", "The processor PR compares the signatures determined with the signature received, as shown at a step ET15 in dotted lines in FIG.", "3.The recording of the list received and of the keys and associated administrator signatures in the memory MNV is validated at a step ET16 when one of the signatures determined is identical to the signature received.", "Otherwise step ET14 breaks off communication between the server SAD and the card CA.", "According to a variant of the embodiment described above, the server SAD is an entity delegated by another administrator server which has transmitted to it delegation rights information IDR.", "Delegation rights information IDR was previously stored in the memory MNV of the chip card CA at the initial step ET01 and is transmitted in the message containing the administrator signature SGAD at step ET5.At step ET6, the chip card CA authorises access to the access control list ACL when both the signature SGAD is recognised in the table TACL associated with the key KAD of the server SAD and the delegation rights information IDR is detected in association with the list ACL recognised in the table TACL of the card.", "Otherwise communication is broken off at step ET14.Although the invention has been described in relation to a chip card containing access control lists associated respectively with signatures, the chip card can also contain access control lists which are not signed.", "Thus an administrator server SAD can decide to erase all the access control lists loaded in the chip card CA and associated with the key KAD of the server SAD, transmitting the corresponding signatures at step ET4.As already stated, everything which has been described above for access control lists in the chip card CA, or in any other portable electronic object such as a personal electronic assistant or organiser, an electronic purse, a token or a pocket calculator, is applicable to an access rights list making several subjects correspond to an object, such as a capacity." ] ]	Patent_10467763
[ [ "Use of type 4 phosphodiesterase inhibitors in myocardial diseases", "The invention relates to the use of type 4 phosphodiesterase inhibitors to treat myocardial diseases." ], [ "1.Use of a) compounds of formula I disclosed in EP 0763534 in which B is an aromatic heterocycle having 1 to 4 N, O and/or S atoms, bonded via N or C, which can be unsubstituted or mono-, di- or trisubstituted by HaI, A and/or OA, and can also be fused to a benzene or pyridine ring, Q is absent or is alkylene having 1-6 C atoms, X is CH2, S or O, R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5, HaI, methylenedioxy, —NO2, —NH2, —NHR5 or —NR5R6, R5 and R6 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I and their stereoisomers and physiologically acceptable, salts and solvates; b) compounds of formula I disclosed in WO 99/65880 in which B is a phenyl ring which is unsubstituted or mono- or polysubstituted by R3, Q is absent or is alkylene having 1-4 C atoms, R1,R2 each independently of one another are —OR4, —S—R4, —SO—R4, —SO2—R4 or HaI, R1 and R2 together are also —O—CH2—O—, R3 is R4, HaI, OH, OR4, OPh, NO2, NHR4, N(R4)2, NHCOR4, NHSO2R4 or NHCOOR4, R4 is A, cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 5-10 C atoms or alkenyl having 2-8 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; c) compounds of formula I disclosed in WO 00/26201 in which R1, R2 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5 or HaI, R1 and R2 together are also —O—CH2—O—, R3 is NH2, NHA, NAA′ or a saturated heterocycle having 1 to 4 N, O and/or S atoms which can be unsubstituted or mono-, di- or tri-substituted by HaI, A and/or OA, Q is absent or is branched or unbranched alkylene having 1-10 C atoms, R5 is A, cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A, A′ in each case independently of one another are alkyl which has 1 to 10 C atoms and which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and the physiologically acceptable salts and solvates thereof; d) compounds of formula I disclosed in WO 98/06704 in which B is A, OA, NH2, NHA, NAA′ or an unsaturated heterocycle which has 1 to 4 N, O and/or S atoms and which can be unsubstituted or mono-, di- or trisubstituted by HaI, A and/or OA, Q is absent or is alkylene having 1-6 C atoms, R1, R2 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5, HaI, —NO2, —NH2, —NHR5 or —NRR6, R1 and R2 together are also —O—CH2—O—, R3, R4 in each case independently of one another are H or A, R5, R6 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A, A′ in each case independently of one another are alkyl which has 1 to 10 C atoms and which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and the stereoisomers and physiologically acceptable salts and solvates thereof; e) compounds disclosed in WO 00/59890 1-(4-ureidobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-propoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-trifluoroacetamidobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-propoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-isopropoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-propoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine and 1-(4-acetamidobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine, and their physiologically acceptable salts and solvates; f) compounds of formula I disclosed in DE 19604388 in which R1, R2 in each case independently of one another are H or A, R3, R4 in each case independently of one another are —OH, OA, —S-A, —SO-A, —SO2-A, HaI, methylenedioxy, —NO2, —NH2, —NHA or —NAA′, A, A′ in each case independently of one another are alkyl having 1 to 10 C-atoms, and which can be substituted by 1 to 5 F and/or Cl atoms, cycloalkyl having 3-7 C atoms or methylenecycloalkyl having 4-8 C atoms, B is —Y—R5 oder —O—Y—R5, Q is absent or is alkylene having 1-4 C atoms, Y is absent or is alkylene having 1-10 C atoms, X is CH2or S, R5 is NH2, NHA, NAA′ or is a saturated 3-8 membered heterocycle having at least one N atom, and wherein other CH2 groups optionally may be replaced by NH, NA, S or O, which can be unsubstituted or monosubstituted by A or OH, HaI is F, Cl, Br oder I and the stereoisomers and physiologically acceptable salts and solvates thereof; g) compounds of formula I disclosed in DE 19932315 in which R1, R2 in each case independently of one another are H, OH, OA, SA, SOA, SO2A, F, Cl or A′2N—(CH2)n—O—, R1 and R2 together are also —O—CH2—O—, R3, R4 in each case independently of one another are H, A, HaI, OH, OA, NO2, NHA, NA2, CN, COOH, COOA, NHCOA, NHSO2A or NHCOOA, R5, R6 in each case independently of one another are H or alkyl having 1 to 6 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms, is cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 5-10 C atoms or alkenyl having 2-8 C atoms, A′ is alkyl having 1, 2, 3, 4, 5 or 6 C atoms, n is 1, 2, 3 or 4, HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; h) compounds of formula I disclosed in EP 0723962 in which R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, —OR10, —S—R10, —SO—R10, —SO2R10, HaI, methylenedioxy, —NO2, —NH2, —NHR10 or —NR10R11, R5 is a phenyl radical which is unsubstituted or mono- or disubstituted by R6 and/or R7, Q is absent or is alkylene having 1-6 C atoms, R6 and R7 in each case independently of one another are —NH2, —NR8R9, —NHR10, —NR10R11, —NO2, HaI, —CN, —OA, —COOH or —COOA, R8 and R9 in each case independently of one another are H, acyl having 1-8 C atoms which can be substituted by 1-5 F and/or Cl atoms, —COOA, —S-A, —SO-A, —SO2A, —CONH2, —CONHA, —CONA2, —CO—COOH, —CO—COOA, —CO—CONH2, —CO—CONHA or —CO—CONA2, A is alkyl having 1 to 6 C atoms which can be substituted by 1-5 F and/or Cl atoms, R10 and R11 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C-atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; i) compounds of formula I disclosed in EP 0738715 in which R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, —OR10, —S—R10, —SO—R10, —SO2R10, HaI, methylenedioxy, —NO2, —NH2, —NHR10 or —NR10R11, R5 is a phenyl radical which is unsubstituted or mono- or disubstituted by R6 and/or R7, Q is absent or is alkylene having 1-6 C atoms, R6 and R7 in each case independently of one another are —NH2, —NR8R9, —NHR10, —NR10R11, —NO2, HaI, —CN, —OA, —COOH or —COOA, R8 and R9 in each case independently of one another are H, acyl having 1-8 C atoms which can be substituted by 1-5 F and/or Cl atoms, —COOA, —SO-A, —SO2A, —CONH2, —CONHA, —CONA2, —CO—COOH, —CO—COOA, —CO—CONH2, —CO—CONHA or —CO—CONA2, A is alkyl having 1 to 6 C atoms which can be substituted by 1-5 F and/or Cl atoms, R10 and R11 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C-atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.", "2.Use according to claim 1 of a) compounds disclosed in EP 0763534: 2-(3-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(2-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(3-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(2-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-trifluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-difluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-fluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-difluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-trifluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-fluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-ethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-hydroxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(4-methylsulfonylphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(4-methyleneoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(3-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminophenethyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminophenethyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-trifluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-difluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-fluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-difluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-trifluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-fluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-ethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-hydroxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methysulfonylphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methyleneoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-trifluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-difluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-fluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-difluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-trifluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-fluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-ethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-hydroxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methylsulfonylphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methyleneoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 2-(3-nicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isonicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pyrazinecarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-(isoxazole-5-carbonylamino)benzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3,4,-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, hydrochloride, and their stereoisomers and physiologically acceptable, salts and solvates; b) compounds disclosed in WO 99/65880 N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)benzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3,4-dichlorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-trifluoromethylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-chlorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-fluorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-butoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-pentoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-ethoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3,4-dimethoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-methylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-methoxybenzoyl-3-carboxamide, and their physiologically acceptable salts and solvates; c) compounds disclosed in WO 00/26201 3-dimethylaminopropyl{4-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-{4-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{4-[3-(3-isopropoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-{3[3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-propyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{4-[3-(3,4-diethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-{4-[3-(3,4-diethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate 3-dimethylaminopropyl{4-[3-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, and the physiologically acceptable salts and solvates thereof; d) compounds disclosed in WO 98/06704 1-(4-nicotinoylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine hydrochloride, 1-(2-nicotinoylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3,4-methylene-dioxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-methoxy-4-methylsulfonylphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-trifluoro-methoxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxy-carbonylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(2-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-cyclo-pentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3-cyclo-pentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3,4-methylene-dioxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-methoxy-4-methylsulfonylphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-trifluoro-methoxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, and the stereoisomers and physiologically acceptable salts and solvates thereof; e) compounds disclosed in EP 0723962 3-(4-ethoxycarbonylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-ethoxycarbonylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, and their physiologically acceptable salts and solvates; f) compounds disclosed in EP 0738715 2-(4-butyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropiridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4, 5-tetrahydropyridazin-3-one; 2-(4-methoxalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.", "3.Use according to claim 1 of compounds selected from 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methoxybenzoyl-3-carboxamide, 1-(4-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.", "4.Use according to claim 1 for preparing a medicament for treating myocardial diseases, where said myocardial diseases show inflammatory and immunological characteristics.", "5.Use according to claim 1 for preparing a medicament for treating coronary artery disease, reversible or irreversible myocardial ischemia/reperfusion injury, acute or chronic heart failure and restenosis, including instent-restenosis and stent-in-stent-restenosis." ], [ "The invention relates to the use of type 4 phosphodiesterase inhibitors to treat myocardial diseases.", "Coronary artery disease is the most common cause of death in the western world.", "In the presence of a critical stenosed coronary artery, a decrease of blood flow may result in myocardial ischemia.", "Initiation of reperfusion results, depending on the severity of the preceding ischemic period, in a reversibly or irreversibly injured myocardium, which is characterized by a long-lasting depression or an irreversible loss of contractile function.", "Depending on the size of the affected myocardial area, an acute or a chronic heart failure may develop.", "A particular clinical problem in the above mentioned scenario is the development of restenosis after a primarily successful reperfusion intervention by PTCA, even after stent implantation, thrombolysis or coronary artery bypass grafting.", "From experimental animal and clinical studies evidence exists, that in the different above mentioned heart diseases, i. e. coronary artery disease itself, reversible or irreversible myocardial ischemia/reperfusion injury, acute or chronic heart failure and restenosis, including instent-restenosis and stent-in-stent-restenosis, inflammatory processes play a casual role.", "These inflammatory processes involve resident and invading macrophages as well as neutrophils and TH1 and TH2 helper cells.", "This leukocyte response produces the characteristic cytokine pattern, involving TNF-α, IL-1β, IL-2, and IL-6, as well as IL-10 and IL-13 (Pulkki K J: Cytokines and cardiomyocyte death.", "Ann.Med.", "1997 29: 339-343.Birks E J, Yacoub M H: The role of nitric oxide and cytokines in heart failure.", "Coron.Artery.Dis.", "1997 8: 389-402).", "The formation of these species has been demonstrated in human patients with myocardial ischemia.", "Animal models show that cytokine production correlates with the invasion of peripheral macrophages and neutrophils which can spread the damage into the still intact myocardium.", "The main player in the cytokine response, however, is TNF-a, which integrates inflammatory and pro-apoptotic responses and additionally has a direct negative ionotropic effect on cardiac myocytes (Ceconi C, Curello S, Bachetti T, Corti A, Ferrari R: Tumor necrosis factor in congestive heart failure: a mechanism of disease for the new millennium?", "Prog.Cardiovasc.Dis.", "1998 41: 25-30.Mann D L: The effect of tumor necrosis factor-alpha on cardiac structure and function: a tale of two cytokines.", "J.Card.Fail.", "1996 2: S165-S172.Squadrito F, Altavilla D, Zingarelli B, et al: Tumor necrosis factor involvement in myocardial ischaemia-reperfusion injury.", "Eur.J.Pharmacol.", "1993 237: 223-230).", "It has been shown in animal models of myocardial infarction, that TNF-α is rapidly released during the reperfusion phase (Herskowitz A, Choi S, Ansari A A, Wesselingh S: Cytokine mRNA expression in postischemic/reperfused myocardium.", "Am.J.Pathol.", "1995 146: 419-428) and that the protective effects of drugs such as dexamethasone (Arras M, Strasser R, Mohri M, et al: Tumor necrosis factor-alpha is expressed by monocytes/macrophages following cardiac microembolization and is antagonized by cyclosporine.", "Basic.Res.Cardiol.", "1998 93: 97-107), cyclosporin A (Arras M, Strasser R, Mohri M, et al: Tumor necrosis factor-alpha is expressed by monocytes/macrophages following cardiac microembolization and is antagonized by cyclosporine.", "Basic.Res.Cardiol.", "1998 93: 97-107.Squadrito F, Altavilla D, Squadrito G, et al: Cyclosporin-A reduces leukocyte accumulation and protects against myocardial ischaemia reperfusion injury in rats.", "Eur.J.Pharmacol.", "1999 364: 159-168) or clorichromene (Squadrito F. Altavilla D, Zingarelli B, et al: The effect of cloricromene, a coumarine derivative, on leukocyte accumulation, myocardial necrosis and TNF-alpha production in myocardial ischaemia-reperfusion injury.", "Life Sci.", "1993 53: 341-355)correspond with a reduction of circulating TNF-α.", "Preferred PDE4 inhibitors mentioned below are potent antagonists of macrophage and T-cell cytokine production.", "They also inhibit the proliferation of T cells.", "Consequently, PDE4 inhibition may have a beneficial effect in those myocardial diseases, which are causally linked to cytokine production and inflammatory processes.", "As compared with PDE3 inhibitors and the early PDE4 inhibitor Rolipram, preferred PDE4 inhibitors are devoid of hemodynamic side effects, which can be dose limiting for the treatment of most cardiovascular disorders.", "The invention was based on the object of discovering new uses of compounds having valuable properties, especially those which may be used to prepare medicaments.", "It has been found that the preferred PDE IV inhibitors and their salts combine very valuable pharmacological properties with good tolerability for the treatment of myocardial diseases.", "Accordingly, the invention provides in particular for the use of a) compounds of formula l disclosed in EP 0763534 in which B is an aromatic heterocycle having 1 to 4 N, O and/or S atoms, bonded via N or C, which can be unsubstituted or mono-, di- or trisubstituted by HaI, A and/or OA, and can also be fused to a benzene or pyridine ring, Q is absent or is alkylene having 1-6 C atoms, X is CH2, S or O, R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5, HaI, methylenedioxy, —NO2, —NH2, —NHR5 or —NR5R6, R5 and R6 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I and their stereoisomers and physiologically acceptable, salts and solvates; b) compounds of formula I disclosed in WO 99/65880 in which B is a phenyl ring which is unsubstituted or mono- or polysubstituted by R3, Q is absent or is alkylene having 1-4 C atoms, R1,R2 each independently of one another are —OR4, —S—R4, —SO—R4, —SO2—R4 or HaI, R1 and R2 together are also —O—CH2—O—, R3 is R4, HaI, OH, OR4, OPh, NO2, NHR4, N(R4)2, NHCOR4, NHSO2R4 or NHCOOR4, R4 is A, cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 5-10 C atoms or alkenyl having 2-8 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; c) compounds of formula I disclosed in WO 00/26201 in which R1, R2 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5 or HaI, R1 and R2 together are also —O—CH2—O—, R3 is NH2, NHA, NAA′ or a saturated heterocycle having 1 to 4 N, O and/or S atoms which can be unsubstituted or mono-, di- or tri-substituted by HaI, A and/or OA, Q is absent or is branched or unbranched alkylene having 1-10 C atoms, R5 is A, cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A, A′ in each case independently of one another are alkyl which has 1 to 10 C atoms and which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and the physiologically acceptable salts and solvates thereof; d) compounds of formula I disclosed in WO 98/06704 in which B is A, OA, NH2, NHA, NAA′ or an unsaturated heterocycle which has 1 to 4 N, O and/or S atoms and which can be unsubstituted or mono-, di- or trisubstituted by HaI, A and/or OA, Q is absent or is alkylene having 1-6 C atoms, R1, R2 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5, HaI, —NO2, —NH2, —NHR5 or —NR5R6, R1 and R2 together are also —O—CH2—O—, R3 R4 in each case independently of one another are H or A, R5, R6 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A, A′ in each case independently of one another are alkyl which has 1 to 10 C atoms and which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and the stereoisomers and physiologically acceptable salts and solvates thereof; e) compounds disclosed in WO 00/59890 1-(4-ureidobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-propoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-trifluoroacetamidobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-propoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-isopropoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-propoxycarbonylaminobenzoyl)-3-(3ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine and 1-(4-acetamidobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine, and their physiologically acceptable salts and solvates; f) compounds of formula I disclosed in DE 19604388 in which R1, R2 in each case independently of one another are H or A, R3, R4 in each case independently of one another are —OH, OA, —S-A, —SO-A, —SO2-A, HaI, methylenedioxy, —NO2, —NH2, —NHA or —NAA′, A, A′ in each case independently of one another are alkyl having 1 to 10 C-atoms, and which can be substituted by 1 to 5 F and/or Cl atoms, cycloalkyl having 3-7 C atoms or methylenecycloalkyl having 4-8 C atoms, B is —Y—R5 oder —O—Y—R5, Q is absent or is alkylene having 1-4 C atoms, Y is absent or is alkylene having 1-10 C atoms, X is CH2 or S. R5 is NH2, NHA, NAA′ or is a saturated 3-8 membered heterocycle having at least one N atom, and wherein other CH2 groups optionally may be replaced by NH, NA, S or O, which can be unsubstituted or monosubstituted by A or OH, HaI is F, Cl, Br oder I and the stereoisomers and physiologically acceptable salts and solvates thereof; g) compounds of formula I disclosed in DE 19932315 in which R1, R2 in each case independently of one another are H, OH, OA, SA, SOA, SO2A, F, Cl or A′2N—(CH2)n—O—, R1 and R2 together are also —O—CH2—O—, R3, R4 in each case independently of one another are H, A, HaI, OH, OA, NO2, NHA, NA2, CN, COOH, COOA, NHCOA, NHSO2A or NHCOOA, R5, R6 in each case independently of one another are H or alkyl having 1 to 6 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms, is cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 5-10 C atoms or alkenyl having 2-8 C atoms, A′ is alkyl having 1, 2, 3, 4, 5 or 6 C atoms, n is 1, 2, 3 or 4, HaI is F, Cl, Br or 1, and their physiologically acceptable salts and solvates; h) compounds of formula I disclosed in EP 0723962 in which R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, —OR10, —S—R10, —SO—R10, —SO2R1°, HaI, methylenedioxy, —NO2, —NH2, —NHR10 or —NR10R11, R5 is a phenyl radical which is unsubstituted or mono- or disubstituted by R6 and/or R7, Q is absent or is alkylene having 1-6 C atoms, R6 and R7 in each case independently of one another are —NH2, —NR8R9, —NHR10, —NR10R11, —NO2, HaI, —CN, —OA, —COOH or —COOA, R8 and R9 in each case independently of one another are H, acyl having 1-8 C atoms which can be substituted by 1-5 F and/or Cl atoms, —COOA, —S-A, —SO-A, —SO2A, —CONH2, —CONHA, —CONA2, —CO—COOH, —CO—COOA, —CO—CONH2, —CO—CONHA or —CO—CONA2.A is alkyl having 1 to 6 C atoms which can be substituted by 1-5 F and/or Cl atoms, R10 and R11 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C-atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; i) compounds of formula I disclosed in EP 0738715 in which R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, —OR10, —S—R10, —SO—R10, —SO2R10, HaI, methylenedioxy, —NO2, —NH2, —NHR10 or —NR10R11, R5 is a phenyl radical which is unsubstituted or mono- or disubstituted by R6 and/or R7, Q is absent or is alkylene having 1-6 C atoms, R6 and R7 in each case independently of one another are —NH2, —NR8R9, —NHR10, —NR10R11, —NO2, HaI, —CN, —OA, —COOH or —COOA, R8 and R9 in each case independently of one another are H, acyl having 1-8 C atoms which can be substituted by 1-5 F and/or Cl atoms, —COOA, —SO-A, —SO2A, —CONH2, —CONHA, —CONA2, —CO—COOH, —CO—COOA, —CO—CONH2, —CO—CONHA or —CO—CONA2, A is alkyl having 1 to 6 C atoms which can be substituted by 1-5 F and/or Cl atoms, R10 and R11 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C-atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.", "Preferably, the invention provides for the use of a) compounds disclosed in EP 0763534: 2-(3-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(2-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(3-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(2-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-trifluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-difluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-fluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-difluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-trifluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-fluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-ethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-4-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-hydroxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(4-methylsulfonylphenyl)-5ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(4-methyleneoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(3-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminophenethyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminophenethyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-trifluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-difluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyi)-5-(3-methoxy-4-fluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-difluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-trifluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-fluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-ethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-hydroxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methysulfonylphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methyleneoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-trifluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-difluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-fluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-difluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-trifluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-fluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-ethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-hydroxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methylsulfonylphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methyleneoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 2-(3-nicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isonicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pyrazinecarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-(isoxazole-5-carbonylamino)benzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3,4,-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, hydrochloride, and their stereoisomers and physiologically acceptable, salts and solvates; b) compounds disclosed in WO 99/65880 N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)benzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3,4-dichlorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-trifluoromethylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-chlorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-fluorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-butoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-pentoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-ethoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3,4-dimethoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-methylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-methoxybenzoyl-3-carboxamide, and their physiologically acceptable salts and solvates; c) compounds disclosed in WO 00/26201 3-dimethylaminopropyl {4-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-4-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl {4-[3-(3-isopropoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl {3-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-3[3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-propyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{4-[3-(3,4-diethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-{4-[3-(3,4-diethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate 3-dimethylaminopropyl{4-[3-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, and the physiologically acceptable salts and solvates thereof; d) compounds disclosed in WO 98/06704 1-(4-nicotinoylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine hydrochloride, 1-(2-nicotinoylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3,4-methylene-dioxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-methoxy-4-methylsulfonylphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-trifluoro-methoxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxy-carbonylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(2-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-cyclo-pentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3-cyclo-pentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3,4-methylene-dioxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-methoxy-4-methylsulfonylphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-trifluoro-methoxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, and the stereoisomers and physiologically acceptable salts and solvates thereof; e) compounds disclosed in EP 0723962 3-(4-ethoxycarbonylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-ethoxycarbonylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, and their physiologically acceptable salts and solvates; f) compounds disclosed in EP 0738715 2-(4-butyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2, 3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2, 3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropiridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one; 2-(4-methoxalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butyloarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.", "Most preferably, the invention provides for the use of the following compounds 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methoxybenzoyl-3-carboxamide, 1-(4-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.", "The preferred compounds show a selective inhibition of phosphodiesterase IV, which is associated with an intracellular increase in cAMP (N. Sommer et al., Nature Medicine, 1, 244-248 (1995)).", "The inhibition of PDE IV can be demonstrated, for example, analogously to C. W. Davis in Biochim.", "Biophys.", "Acta 797, 354-362 (1984).", "The affinity of the compounds of the invention for phosphodiesterase IV is measured by determining their IC50 values (the concentration of inhibitor required to achieve 50% inhibition of the enzyme activity).", "Preferably, the invention provides for the use of the compounds mentioned above for preparing a medicament for treating myocardial diseases, where said myocardial diseases show inflammatory and immunological characteristics.", "Most preferably, the invention provides for the use of the compounds mentioned above for preparing a medicament for treating coronary artery disease, reversible or irreversible myocardial ischemia/reperfusion injury, acute or chronic heart failure and restenosis, including instent-restenosis and stent-in-stent-restenosis.", "The preparations for the treatment of the mentioned diseases can be used as medicaments in human or veterinary medicine.", "Possible excipients are organic or inorganic substances which are suitable for enteral (e.g.", "oral) or parenteral administration or topical application and do not react with the novel compounds, for example water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycerol triacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc and petroleum jelly.", "In particular, tablets, pills, coated tablets, capsules, powders, granules, syrups, juices or drops are used for oral administration, suppositories are used for rectal administration, solutions, preferably oily or aqueous solutions, and furthermore suspensions, emulsions or implants, are used for parenteral administration, and ointments, creams or powders are used for topical application.", "The novel compounds can also be lyophilized and the lyophilizates obtained used, for example, for the production of injection preparations.", "The preparations indicated can be sterilized and/or can contain auxiliaries such as lubricants, preservatives, stabilizers and/or wetting agents, emulsifiers, salts for affecting the osmotic pressure, buffer substances, colourants, flavourings and/or one or more further active compounds, e.g.", "one or more vitamins.", "In these indications the substances are generally administered preferably in doses of between approximately 1 and 500 mg, in particular between 5 and 100 mg per dose unit.", "The daily dose is preferably between approximately 0.02 and 10 mg/kg of body weight.", "The specific dose for each patient depends, however, on a wide variety of factors, for example on the efficacy of the specific compound used, on age, body weight, general state of health, gender, on the diet, on the time and route of administration, on the excretion rate, medicament combination and severity of the particular disease to which the therapy is applied.", "Oral administration is preferred.", "EXAMPLE 1 Effect of PDE4 Inhibitors on T-Cell Proliferation Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy donors by the Lymphoprep gradient method.", "200000 PBMC/well were cultured in RPMI1640 culture medium containing 5% heat inactivated human serum (AB pool) for 5 days at 37° C. and 10% CO2 in 96 well flat bottom microtiter plates.", "The T cells within the PBMC preparation were selectively stimulated with an monoclonal antibody to CD3.Cultures were set up as triplicates including a control group receiving no treatment.", "PDE4 inhibitors were dissolved in DMSO at 10−2 M and diluted in culture medium.", "Control cultures were treated with DMSO equivalent to the inhibitor concentration.", "18 hrs before the end of the assay, 3H-thymidine was added to the cultures.", "The incorporation of radioactivity into the cells was then measured in a beta-counter.", "The data of at least three independent experiments were calculated as percent inhibition of the control (mean ±SEM) without inhibitor.", "From this data the IC-50 value was determined.", "Results: PDE4 inhibitors afforded a marked reduction of T-cell proliferation (see table 1).", "TABLE 1 Effect of PDE4 inhibitors on T-cells and macrophages IL2 IFN-γ TNF-α IL10 IL12 T-cell T-cell macrophage macrophage macrophage T-cell prol.", "IC50 [μM] IC50 [μM] IC50 [μM] EC50 [μM] IC50 [μM] IC50 [μM] Rolipram 0.7 1 0.6 0.05 >0.1 0.3 EMD 94360 0.3 0.05 0.03 0.001 0.006 0.02 EMD 95832 0.2 0.01 0.009 0.02 0.0005 0.05 EMD 125025 0.002 0.007 0.003 no effect <1E−13M 0.006 EMD 125059 0.06 0.008 0.03 0.008 <1E−13M 0.04 EMD 219901 0.003 0.005 0.002 0.001 <1E−13M 0.007 EMD 94360 (EP 0738715; Page 17, line 41): EMD 95832 (EP 0763534): EMD 125025 (WO 98/06704; page 13, lines 16-17): EMD 125059 (WO 98/06704; page 27, lines 2-3): EMD 219901 (WO 99/65880; page 14, lines 20-21) EXAMPLE 2 Effect of PDE4 Inhibitors on Cytokine Production in Human Peripheral Blood Monocytic Cells Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy donors by the Lymphoprep gradient method.", "200000 PBMC/well were cultured in RPMI1640 culture medium containing 5% heat inactivated human serum (AS pool).", "at 37° C. and 10% CO2 in 96 well flat bottom microtiter plates.", "Cultures were set up as triplicates including a control group.", "Solutions of PDE4 inhibitors were prepared in DMSO at 10−2 M and diluted in culture medium.", "Control cultures were treated with concentrations of DMSO equivalent to the inhibitor concentrations.", "The cytokine of interest was stimulated as indicated in Table 2 The culture supernatants of three independent experiments were pooled and cytokine activity in the supernatant was measured with commercially available ELISA test kits.", "TABLE 2 Activation of PBMC to form T-cell and macrophage specific cytokines cytokine activator incubation in culture IL-2 mab anti-CD3 24 h IL-10 LPS 48 h TNF-α LPS 48 h IFN-γ mab anti-CD3 48 h IL-12 SAC + IFNγ 48 h The data were calculated as percent inhibition/stimulation of the control without the compound and the IC50 value or EC50 value in case of stimulation was determined thereof.", "Result PDE4 inhibitors afforded a marked reduction in the release of IL-2, IFN-γ, TNF-α and IL-12.The immunosuppressant cytokine IL-10, however, was stimulated by most PDE4 inhibitors (see Table 1).", "EXAMPLE 3 Effect of PDE4 Inhibitors on Experimental Myocardial Infarction in Rats Compound 5, administered intraperitoneally with 1, 3, and 10 mg/kg, 1 hour before reversible occlusion of the left coronary artery in rats caused a significant dose dependent reduction of infarct size up to 38%.", "In correspondence with this protection, a reduction of plasma TNF-□ levels was observed, as measured by ELISA.", "EXAMPLE 4 Effect of PDE4 Inhibitors on Experimental Myocardial Infarction in Rabbits There was a cardioprotective effect by PDE4 inhibition in anaesthetised rabbits subjected to 30 minutes of coronary artery occlusion (side branch of the ramus circumflexus of the left coronary artery) followed by 120 minutes of reperfusion.", "PDE4 inhibitors applied prior to the coronary occlusion reduced infarct size as compared with placebo treatment.", "The areas at risk were comparable between verum and placebo groups.", "The cardioprotective effect cannot be attributed to favorable hemodynamic effects, since heart rate and mean aortic pressure remained constant throughout the experimental protocol." ] ]	Patent_10467793
[ [ "Substrate processing method and substrate processing apparatus", "In a substrate processing apparatus, a control electrode (131) separates a process space (11C) including a substrate to be processed and a plasma formation space (11B) not including the substrate.", "The control electrode includes a conductive member formed in a processing vessel and having a plurality of apertures (131a) for passing plasma.", "A surface of the control electrode is covered by an aluminum oxide or a conductive nitride.", "In the substrate processing apparatus, a gas containing He and N2 is supplied into the processing vessel.", "In the plasma formation space, there is formed plasma under a condition in which atomic state nitrogen N* are excited.", "The atomic state nitrogen N* are used to nitride a surface of the substrate." ], [ "1.A method of processing a substrate by using a substrate processing apparatus which has such a construction that a process space, in which a substrate to be processed is contained, is separated from plasma formation space, in which the substrate to be processed is not contained, by a control electrode in a processing vessel, characterized by the steps of: supplying a gas containing He and N2 to said processing vessel; forming plasma in said plasma formation space under a condition such that there is caused excitation of atomic state nitrogen N* in said plasma; and nitriding a surface of the substrate to be processed by said atomic state nitrogen N* in said process space.", "2.The substrate processing method as claimed in claim 1, characterized in that said step of forming plasma is conducted such that an intermediate excitation state of the energy of 23 to 25 eV is realized.", "3.The substrate processing method as claimed in claim 1, characterized in that said step of forming plasma comprises the step of supplying a microwave to said plasma formation space.", "4.The substrate processing method as claimed in claim 3, characterized in that said step of supplying microwave is conducted by driving a radial line slot antenna.", "5.The substrate processing method as claimed in claim 1, characterized in that said step of forming plasma comprises the step of forming an induction magnetic field in said plasma formation space.", "6.The substrate processing method as claimed in claim 5, characterized in that said step of forming an induction magnetic field comprises the step of driving an induction coil wound around said processing vessel by a high frequency electric power.", "7.The substrate processing method as claimed in claim 1, characterized in the said control electrode is grounded during said step of exciting plasma.", "8.The substrate processing method as claimed in claim 1, characterized in that a negative potential is supplied to said control electrode in said step of forming plasma.", "9.The substrate processing method as claimed in claim 1, wherein said gas supplied to said processing vessel further contains O2.10.A substrate processing apparatus, comprising: a processing vessel defined by an outer wall and having a stage for holding a substrate to be processed thereon; an evacuation system coupled to said processing vessel; a plasma gas supplying part supplying a plasma excitation gas and a process gas into said processing vessel; a microwave window provided on said processing vessel so as to face said substrate to be processed; and a control electrode provided between said substrate to be processed on said stage and said plasma gas supplying part so as to face said substrate to be procesed, and separating a plasma excitation space containing said microwave window and a process space containing said substrate to be processed, said control electrode comprising a conductive member having a plurality of apertures for passing plasma formed in said processing vessel therethrough, and a surface of said control electrode being covered by any of aluminum oxide or electrically conductive nitride.", "11.The substrate processing apparatus as claimed in claim 10, characterized in that said control electrode has a lattice-shaped form and is grounded.", "12.The substrate processing apparatus as claimed in claim 10, characterized in that said control electro has a form of a lattice-like shape and said substrate processing apparatus comprises a negative voltage source connected to said control electrode.", "13.The substrate processing apparatus as claimed in claim 10, characterized in that an inner wall of said processing vessel is covered with an insulation film in said plasma excitation space.", "14.The substrate processing apparatus as claimed in claim 10, characterized by further comprising a microwave antenna coupled to said microwave window at an outer side of said processing vessel.", "15.A substrate processing apparatus, characterized by: a processing vessel defined by a wall of quartz glass and having a stage for holding a substrate to be processed; an evacuation system coupled to said processing vessel; a plasma gas supplying part supplying a plasma excitation gas and a process gas to said processing vessel; a control electrode provided so as to face said substrate to be processed on said stage and dividing an interior of said processing vessel into a process space containing said substrate to be processed and a plasma excitation space; and an induction coil provided outside said quartz glass wall in correspondence to said plasma excitation space, said control electrode comprising a conductive member having a plurality of apertures passing therethrough plasma formed in said processing vessel, and a surface of said control electrode being covered with any of aluminum oxide or electrically conductive nitride.", "16.The substrate processing apparatus as claimed in claim 15, characterized in that said quartz glass wall defines a dome-like space.", "17.The substrate processing apparatus as claimed in claim 15, characterized in that said control electrode is grounded.", "18.The substrate processing apparatus as claimed in claim 15, characterized in that said control gate is connected to a negative voltage source." ], [ "<SOH> BACKGROUND ART <EOH>FIG.", "1 shows the schematic construction of a conventional induction-coupled plasma processing apparatus 1 .", "Referring to FIG.", "1 , the plasma processing apparatus 1 includes a processing vessel 2 of a quartz dome evacuated by an evacuation line 2 A, and there is provided a stage 3 in a process space 2 B defined by the processing vessel 2 such that the stage 2 is rotated by a rotating mechanism 3 A.", "Further, a substrate 4 is held on the stage 3 .", "Further, an inert gas such as Ar and a process gas such as oxygen or nitrogen are supplied to the process space 2 B via a process gas supply line 2 C. Further, there is provided a coil 5 around the top part of the processing vessel 2 at the outside thereof, and high-density plasma 2 D is inducted at the top part of the process space 2 B by driving the coil 5 by a d.c. power source.", "In the plasma processing apparatus 1 of FIG.", "1 , the radicals of the process gas formed with the high-density plasma 2 D reach the surface of the substrate 4 and the substrate processing such as oxidation or nitridation is achieved.", "In such a conventional induction-coupled plasma processing apparatus 1 , on the other hand, there exists a drawback in that the high-density plasma 2 D is localized at the top part of the processing vessel and there appears an extremely non-uniform distribution in the radicals that are formed with the plasma.", "Particularly, the non-uniformity of the radical concentration in the radial direction of the substrate is not resolved even when the stage 3 is rotated by the rotating mechanism 3 A.", "Thus, in the conventional induction-coupled plasma processing apparatus 1 , the plasma processing apparatus was designed such that the substrate 4 is separated from the region in which the high-density plasma 2 D is formed with a large distance for realizing as uniform radical concentration distribution as possible at the surface of the substrate 4 .", "As a result of such a construction, on the other hand, the overall size of the substrate processing apparatus 1 is increased inevitably.", "Further, the amount of the radicals reaching the substrate 4 is reduced.", "These problems become particularly serious in the technology of current trend of processing a large-diameter substrate.", "On the other hand, there is a proposal of a microwave plasma processing apparatus that uses high-density plasma induced, not by an induction magnetic field but by a microwave electric field.", "For example, there is proposed a plasma processing apparatus that uses a planar antenna (radial line slot antenna) having a large number of slots arranged so as to produce a uniform microwave, for emitting a microwave into a processing vessel.", "In this apparatus, the microwave electric field thus induced is used to excite plasma by causing ionization in the gas in the vacuum vessel.", "Reference should be made to Japanese Laid-Open Patent Application 9-63793.By using the microwave plasma excited according to such a process, it becomes possible to realize a high-plasma density over a wide area right underneath the antenna, and uniform plasma processing becomes possible with short time period.", "Further, the microwave plasma thus excited has an advantageous feature of low electron temperature as a result of excitation of the plasma by using a microwave, and it becomes possible to avoid the problem of damages or metal contamination caused in the substrate.", "Further, it becomes possible to excite uniform plasma over a substrate of large area, and thus, the plasma processing apparatus can easily handle the fabrication of semiconductor devices on a large-diameter semiconductor wafer or fabrication of large flat panel display devices.", "FIG.", "2 shows the construction of a microwave plasma processing apparatus 10 that uses such a radial line slot antenna as proposed before by the inventor of the present invention.", "Referring to FIG.", "2 , the microwave plasma processing apparatus 10 includes a processing chamber 11 evacuated at a plurality of evacuation ports 11 a , and there is provided a stage 13 inside the processing chamber 11 for supporting a substrate 12 to be processed.", "In order to achieve uniform evacuation of the processing chamber 11 , there is provided a ring-shaped space 11 A around the stage 13 , and the processing chamber 11 is evacuated uniformly via the space 11 A and further via the evacuation ports 11 a by arranging the evacuation ports 11 a communicating with the space 11 A in axial symmetry with respect to the substrate.", "On the processing chamber 11 , there is provided a plate-like shower plate 14 formed of a low-loss dielectric such as Al 2 O 3 or SiO 2 as a part of the outer wall of the processing chamber 11 at a location facing the substrate 12 held on the stage 13 , wherein the shower plate 14 is provided via a seal ring not illustrated and includes a number of apertures 14 A.", "Further, a cover plate 15 also of a low-loss dielectric such as Al 2 O 3 or SiO 2 is provided at the outer side of the shower plate 14 via another seal ring not illustrated.", "The shower plate 14 is provided with a gas passage 14 B at a top surface thereof, and each of the apertures 14 A are provided so as to communicate with the gas passage 14 B.", "Further, there is provided a gas supply passage 14 C in the interior of the shower plate 14 in communication with a gas supply port lip provided at an outer wall of the processing vessel 11 .", "Thus, the plasma-excitation gas such as Ar or Kr supplied to the gas supply port 11 p is forwarded to the apertures 11 A via the supply passage 14 C and further via the passage 14 B and is released to the process space 11 B right underneath the shower plate 14 inside the processing vessel 11 from the foregoing apertures 14 A.", "On the processing vessel 11 , there is further provided a radial line slot antenna 20 at the outer side of the cover plate 15 with a separation of 4-5 mm from the cover plate 15 .", "The radial line slot antenna 20 is connected to an external microwave source (not illustrated) via a coaxial waveguide 21 and causes excitation of the plasma-excitation gas released into the process space 11 B by the microwave from the microwave source.", "It should be noted that the cover plate 15 and the radiation surface of the radial line slot antenna are contacted closely, and there is provided a cooling block 19 on the antenna 20 for cooling the antenna.", "The cooling block 19 includes a cooling water passage 19 A.", "The radial line slot antenna 20 is formed of a flat, disk-shaped antenna body 17 connected to an outer waveguide tube 21 A of the coaxial waveguide 21 and a radiation plate 16 provided at the opening of the antenna body 17 , wherein the radiation plate 16 is formed with a number of slots and a retardation plate of a dielectric plate having a constant thickness is interposed between the antenna body 17 and the radiation plate 16 .", "In the radial line slot antenna 20 having such a construction, the microwave fed thereto from the coaxial waveguide 21 propagates along a path between the disk-shaped antenna body 17 and the radiation plate 16 in the radial direction, wherein the microwave thus propagating undergoes compression of wavelength as a result of the existence of the retardation plate 18 .", "Thus, by forming the slots concentrically in correspondence to the wavelength of the microwave thus propagating in the radial direction, and by forming the slots so as to form a perpendicular angle with each other, it becomes possible to emit a plane wave having a circular polarization from the radial line slot antenna 20 in the direction substantially perpendicular to the radiation plate 16 .", "By using such a radial line slot antenna 20 , there is formed uniform high-density plasma in the process space 11 B right underneath the shower plate 14 .", "The high-density plasma thus formed has a feature of low electron temperature and the occurrence of damages in the substrate 12 to be processed is avoided.", "Further, there occurs no metal contamination caused by sputtering of the chamber wall of the processing vessel 11 .", "Thus, by supplying a process gas, such as an O 2 gas, an NH 3 gas, or a mixed gas of an N 2 gas and an H 2 gas, to the gas inlet port 11 p of the substrate processing apparatus 10 of FIG.", "2 in addition to the plasma-excitation gas such as Ar or Kr, there is caused an excitation of active species such as atomic state oxygen O* or hydrogen nitride radicals NH* in the process space 11 B by the high-density plasma, and it becomes possible to conduct oxidation processing, nitridation processing or oxynitridation processing on the surface of the substrate 12 .", "Further, there is proposed a substrate processing apparatus 10 A shown in FIG.", "3 having a construction similar to the substrate processing apparatus 10 of FIG.", "2 except that there is provided a lower shower plate 31 at the lower side of the shower plate 14 .", "The lower shower plate 31 is provided with a process gas passage 31 A communicating with a process gas inlet port 11 r formed at the surface of the processing vessel 1 and a large number of process gas inlet nozzle openings 31 B are formed in communication with the process gas passage 31 A.", "Further, the lower shower plate 31 is provided with large apertures for passing the process gas radicals formed in the space 11 B.", "Thus, in the substrate processing apparatus 10 A of FIG.", "3 , there is defined another process space 11 C underneath the lower shower plate 31 .", "By forming the lower shower plate 31 by a conductive material such as a stainless steel having a passivation surface by aluminum oxide (Al 2 O 3 ) in such an apparatus, it becomes possible to block the penetration of microwave to the process space 11 C. Thereby, the excitation of plasma is limited in the process space 11 B right underneath the upper shower plate 14 , and the radicals Kr* of Kr or Ar* of Ar penetrate into the process space 11 C through the large apertures formed in the shower plate 31 after excitation in the space 11 B.", "The radicals Kr* or Ar* thus penetrated into the process space 11 C cause activation of the process gas released from the nozzle apertures 31 B, and the processing of the substrate 12 is achieved by the process gas radicals thus activated.", "In the substrate processing apparatus 10 A of FIG.", "3 , it should be noted that the microwave is expelled from the process space 11 C by forming the lower shower plate 31 by a conductive material, and the damaging of the substrate by microwave is avoided.", "In the substrate processing apparatus 10 A of FIG.", "3 , it is also possible to conduct a plasma CVD process by introducing a CVD source gas from the lower shower plate 31 .", "Further, it is possible to conduct a dry etching process by introducing a dry etching gas from the lower shower plate 31 and applying a high-frequency bias to the stage 13 .", "Thus, in the substrate processing apparatus of FIG.", "2 of FIG.", "3 , Kr radicals (Kr) of intermediate excitation state having an energy of about 10 eV are excited at the time of conducting an oxidation processing, by introducing a Kr gas and an oxygen gas into the process space 11 B.", "The Kr radicals thus excited cause efficient excitation of atomic state oxygen O according to the reaction in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "O 2 →O+O, in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "while the atomic state oxygen O* thus excited cause the desired oxidation of the surface of the substrate 12 .", "In the case of conducting a nitridation processing of the substrate 12 , a Kr gas and an ammonia gas, or a Kr gas and a nitrogen gas and a hydrogen gas are introduced.", "In this case, the excited Kr radicals (Kr) or Ar radicals (Ar) cause the excitation of hydrogen nitride radicals NH* according to the reaction in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "NH 3 →NH+2H+ e − , in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "or in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "N 2 +H 2 →NH+NH, in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "wherein the hydrogen nitride radicals thus excited cause the desired nitridation processing of the substrate of the surface 12 .", "Meanwhile, there are cases in which it is preferable to use atomic state nitrogen (N), free from hydrogen and having a strong nitriding power, at the time of the nitridation processing of the substrate.", "The atomic state nitrogen N are formed according to the reaction in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "N 2 →N+N, in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "wherein it should be noted that such a reaction requires the energy of 23-25 eV.", "This means that it is not possible to excite the atomic state nitrogen N* according to the foregoing reaction, as long as Kr or Ar plasma is used.", "As noted previously, the energy of the Kr radicals or Ar radicals obtained by the Kr or Ar plasma is merely in the order of 10 eV.", "Thus, even when there is made an attempt to supply a nitrogen gas in the substrate processing apparatus of FIG.", "2 or FIG.", "3 in place of the Kr gas or the Ar gas, merely the reaction in-line-formulae description=\"In-line Formulae\" end=\"lead\"?", "N 2 →N 2 + +e − , in-line-formulae description=\"In-line Formulae\" end=\"tail\"?", "is obtained, and there is caused no desired atomic state oxygen N.", "FIG.", "4 shows the relationship between the state density of the Kr plasma and the excitation energy of the atomic state nitrogen N, hydrogen nitride radicals NH* and nitrogen atoms N 2 + .", "Referring to FIG.", "4 , it can be seen that the state density of the Kr plasma is large at the low energy side, while the state density shows a rapid decrease with increase of the energy.", "Such a plasma cannot achieve efficient excitation of the desired nitrogen radicals." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 is a diagram showing the construction of a conventional induction coupled plasma processing apparatus; FIG.", "2 is a diagram showing the construction of a previously proposed microwave substrate processing apparatus; FIG.", "3 is a diagram showing the construction of another previously proposed microwave substrate processing apparatus; FIG.", "4 is a diagram explaining the characteristics of plasma excitation in the microwave substrate processing apparatus of FIG.", "2 or FIG.", "3 ; FIG.", "5 is a diagram showing the construction of a microwave substrate processing apparatus according to a first embodiment of the present invention; FIG.", "6 is a diagram showing a part of the microwave substrate processing apparatus of FIG.", "5 ; FIG.", "7 is a diagram showing the characteristics of plasma excitation in the microwave substrate processing apparatus of FIG.", "5 ; FIG.", "8 is a diagram showing a modification of the microwave plasma processing apparatus of FIG.", "5 ; FIG.", "9 is a diagram showing the construction of a microwave plasma processing apparatus according to a second embodiment of the present invention; and FIG.", "10 is a diagram showing the construction of an induction coupled plasma processing apparatus according to a third embodiment of the present invention.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention generally relates to plasma processing apparatuses and more particularly to a microwave plasma processing apparatus.", "Plasma process and plasma processing apparatus constitute indispensable technology for fabricating ultrafine semiconductor devices such as the one called deep submicron device or deep sub-quarter micron device having a gate length near 0.1 μm or less, or for fabricating high-resolution flat panel display device including a liquid crystal display device.", "Conventionally, various plasma excitation methods have been employed in the plasma processing apparatus used for fabricating semiconductor devices or liquid crystal display devices.", "Particularly, high-frequency plasma apparatuses of parallel plate type or induction-coupled type plasma apparatus are used commonly.", "However, such a conventional plasma processing apparatuses suffers from the problem of non-uniform plasma formation in that the region in which high electron density is achieved is substantially limited, and there has been a difficulty in conducting a uniform processing over the entire surface of the substrate with a large processing rate or throughput.", "This problem becomes particularly serious in the case of processing a substrate of large diameter.", "Further, such a conventional plasma processing apparatus has inherent problems, associated with its high electron temperature, in that damages are caused in the semiconductor devices formed on the substrate.", "Further, severe metal contamination may be caused as a result of sputtering of the chamber wall.", "Thus, it is becoming difficult with conventional plasma processing apparatuses to satisfy the stringent demand of further miniaturization and further improvement of productivity of semiconductor devices or flat display devices.", "Meanwhile, there has been a proposal of a microwave plasma processing apparatus that uses high-density plasma excited, not by d.c. magnetic field, but by a microwave electric field.", "For example, there is a proposal of a plasma processing apparatus that excites plasma by emitting a microwave into a processing vessel from a planar antenna (radial line slot antenna) having a number of slots arranged so as to produce a uniform microwave, for emitting a microwave into a processing vessel.", "In this plasma processing apparatus, the microwave electric field induces plasma by causing ionization in the gas in the vacuum vessel.", "Reference should be made to Japanese Laid-Open Patent Application 9-63793.By using the microwave plasma excited according to such a process, it becomes possible to realize a high-plasma density over a wide area right underneath the antenna, and uniform plasma processing becomes possible with short time period.", "Further, the microwave plasma thus excited has an advantageous feature of low electron temperature as a result of excitation of the plasma by using a microwave, and it becomes possible to avoid the problem of damages or metal contamination caused in the substrate.", "Further, it becomes possible to excite uniform plasma over a substrate of large area, and thus, the plasma processing apparatus can easily handle the fabrication of semiconductor devices on a large-diameter semiconductor wafer or fabrication of large flat panel display devices.", "BACKGROUND ART FIG.", "1 shows the schematic construction of a conventional induction-coupled plasma processing apparatus 1.Referring to FIG.", "1, the plasma processing apparatus 1 includes a processing vessel 2 of a quartz dome evacuated by an evacuation line 2A, and there is provided a stage 3 in a process space 2B defined by the processing vessel 2 such that the stage 2 is rotated by a rotating mechanism 3A.", "Further, a substrate 4 is held on the stage 3.Further, an inert gas such as Ar and a process gas such as oxygen or nitrogen are supplied to the process space 2B via a process gas supply line 2C.", "Further, there is provided a coil 5 around the top part of the processing vessel 2 at the outside thereof, and high-density plasma 2D is inducted at the top part of the process space 2B by driving the coil 5 by a d.c. power source.", "In the plasma processing apparatus 1 of FIG.", "1, the radicals of the process gas formed with the high-density plasma 2D reach the surface of the substrate 4 and the substrate processing such as oxidation or nitridation is achieved.", "In such a conventional induction-coupled plasma processing apparatus 1, on the other hand, there exists a drawback in that the high-density plasma 2D is localized at the top part of the processing vessel and there appears an extremely non-uniform distribution in the radicals that are formed with the plasma.", "Particularly, the non-uniformity of the radical concentration in the radial direction of the substrate is not resolved even when the stage 3 is rotated by the rotating mechanism 3A.", "Thus, in the conventional induction-coupled plasma processing apparatus 1, the plasma processing apparatus was designed such that the substrate 4 is separated from the region in which the high-density plasma 2D is formed with a large distance for realizing as uniform radical concentration distribution as possible at the surface of the substrate 4.As a result of such a construction, on the other hand, the overall size of the substrate processing apparatus 1 is increased inevitably.", "Further, the amount of the radicals reaching the substrate 4 is reduced.", "These problems become particularly serious in the technology of current trend of processing a large-diameter substrate.", "On the other hand, there is a proposal of a microwave plasma processing apparatus that uses high-density plasma induced, not by an induction magnetic field but by a microwave electric field.", "For example, there is proposed a plasma processing apparatus that uses a planar antenna (radial line slot antenna) having a large number of slots arranged so as to produce a uniform microwave, for emitting a microwave into a processing vessel.", "In this apparatus, the microwave electric field thus induced is used to excite plasma by causing ionization in the gas in the vacuum vessel.", "Reference should be made to Japanese Laid-Open Patent Application 9-63793.By using the microwave plasma excited according to such a process, it becomes possible to realize a high-plasma density over a wide area right underneath the antenna, and uniform plasma processing becomes possible with short time period.", "Further, the microwave plasma thus excited has an advantageous feature of low electron temperature as a result of excitation of the plasma by using a microwave, and it becomes possible to avoid the problem of damages or metal contamination caused in the substrate.", "Further, it becomes possible to excite uniform plasma over a substrate of large area, and thus, the plasma processing apparatus can easily handle the fabrication of semiconductor devices on a large-diameter semiconductor wafer or fabrication of large flat panel display devices.", "FIG.", "2 shows the construction of a microwave plasma processing apparatus 10 that uses such a radial line slot antenna as proposed before by the inventor of the present invention.", "Referring to FIG.", "2, the microwave plasma processing apparatus 10 includes a processing chamber 11 evacuated at a plurality of evacuation ports 11a, and there is provided a stage 13 inside the processing chamber 11 for supporting a substrate 12 to be processed.", "In order to achieve uniform evacuation of the processing chamber 11, there is provided a ring-shaped space 11A around the stage 13, and the processing chamber 11 is evacuated uniformly via the space 11A and further via the evacuation ports 11a by arranging the evacuation ports 11a communicating with the space 11A in axial symmetry with respect to the substrate.", "On the processing chamber 11, there is provided a plate-like shower plate 14 formed of a low-loss dielectric such as Al2O3 or SiO2 as a part of the outer wall of the processing chamber 11 at a location facing the substrate 12 held on the stage 13, wherein the shower plate 14 is provided via a seal ring not illustrated and includes a number of apertures 14A.", "Further, a cover plate 15 also of a low-loss dielectric such as Al2O3 or SiO2 is provided at the outer side of the shower plate 14 via another seal ring not illustrated.", "The shower plate 14 is provided with a gas passage 14B at a top surface thereof, and each of the apertures 14A are provided so as to communicate with the gas passage 14B.", "Further, there is provided a gas supply passage 14C in the interior of the shower plate 14 in communication with a gas supply port lip provided at an outer wall of the processing vessel 11.Thus, the plasma-excitation gas such as Ar or Kr supplied to the gas supply port 11p is forwarded to the apertures 11A via the supply passage 14C and further via the passage 14B and is released to the process space 11B right underneath the shower plate 14 inside the processing vessel 11 from the foregoing apertures 14A.", "On the processing vessel 11, there is further provided a radial line slot antenna 20 at the outer side of the cover plate 15 with a separation of 4-5 mm from the cover plate 15.The radial line slot antenna 20 is connected to an external microwave source (not illustrated) via a coaxial waveguide 21 and causes excitation of the plasma-excitation gas released into the process space 11B by the microwave from the microwave source.", "It should be noted that the cover plate 15 and the radiation surface of the radial line slot antenna are contacted closely, and there is provided a cooling block 19 on the antenna 20 for cooling the antenna.", "The cooling block 19 includes a cooling water passage 19A.", "The radial line slot antenna 20 is formed of a flat, disk-shaped antenna body 17 connected to an outer waveguide tube 21A of the coaxial waveguide 21 and a radiation plate 16 provided at the opening of the antenna body 17, wherein the radiation plate 16 is formed with a number of slots and a retardation plate of a dielectric plate having a constant thickness is interposed between the antenna body 17 and the radiation plate 16.In the radial line slot antenna 20 having such a construction, the microwave fed thereto from the coaxial waveguide 21 propagates along a path between the disk-shaped antenna body 17 and the radiation plate 16 in the radial direction, wherein the microwave thus propagating undergoes compression of wavelength as a result of the existence of the retardation plate 18.Thus, by forming the slots concentrically in correspondence to the wavelength of the microwave thus propagating in the radial direction, and by forming the slots so as to form a perpendicular angle with each other, it becomes possible to emit a plane wave having a circular polarization from the radial line slot antenna 20 in the direction substantially perpendicular to the radiation plate 16.By using such a radial line slot antenna 20, there is formed uniform high-density plasma in the process space 11B right underneath the shower plate 14.The high-density plasma thus formed has a feature of low electron temperature and the occurrence of damages in the substrate 12 to be processed is avoided.", "Further, there occurs no metal contamination caused by sputtering of the chamber wall of the processing vessel 11.Thus, by supplying a process gas, such as an O2 gas, an NH3 gas, or a mixed gas of an N2 gas and an H2 gas, to the gas inlet port 11p of the substrate processing apparatus 10 of FIG.", "2 in addition to the plasma-excitation gas such as Ar or Kr, there is caused an excitation of active species such as atomic state oxygen O* or hydrogen nitride radicals NH* in the process space 11B by the high-density plasma, and it becomes possible to conduct oxidation processing, nitridation processing or oxynitridation processing on the surface of the substrate 12.Further, there is proposed a substrate processing apparatus 10A shown in FIG.", "3 having a construction similar to the substrate processing apparatus 10 of FIG.", "2 except that there is provided a lower shower plate 31 at the lower side of the shower plate 14.The lower shower plate 31 is provided with a process gas passage 31A communicating with a process gas inlet port 11r formed at the surface of the processing vessel 1 and a large number of process gas inlet nozzle openings 31B are formed in communication with the process gas passage 31A.", "Further, the lower shower plate 31 is provided with large apertures for passing the process gas radicals formed in the space 11B.", "Thus, in the substrate processing apparatus 10A of FIG.", "3, there is defined another process space 11C underneath the lower shower plate 31.By forming the lower shower plate 31 by a conductive material such as a stainless steel having a passivation surface by aluminum oxide (Al2O3) in such an apparatus, it becomes possible to block the penetration of microwave to the process space 11C.", "Thereby, the excitation of plasma is limited in the process space 11B right underneath the upper shower plate 14, and the radicals Kr* of Kr or Ar* of Ar penetrate into the process space 11C through the large apertures formed in the shower plate 31 after excitation in the space 11B.", "The radicals Kr* or Ar* thus penetrated into the process space 11C cause activation of the process gas released from the nozzle apertures 31B, and the processing of the substrate 12 is achieved by the process gas radicals thus activated.", "In the substrate processing apparatus 10A of FIG.", "3, it should be noted that the microwave is expelled from the process space 11C by forming the lower shower plate 31 by a conductive material, and the damaging of the substrate by microwave is avoided.", "In the substrate processing apparatus 10A of FIG.", "3, it is also possible to conduct a plasma CVD process by introducing a CVD source gas from the lower shower plate 31.Further, it is possible to conduct a dry etching process by introducing a dry etching gas from the lower shower plate 31 and applying a high-frequency bias to the stage 13.Thus, in the substrate processing apparatus of FIG.", "2 of FIG.", "3, Kr radicals (Kr) of intermediate excitation state having an energy of about 10 eV are excited at the time of conducting an oxidation processing, by introducing a Kr gas and an oxygen gas into the process space 11B.", "The Kr radicals thus excited cause efficient excitation of atomic state oxygen O according to the reaction O2→O+O, while the atomic state oxygen O* thus excited cause the desired oxidation of the surface of the substrate 12.In the case of conducting a nitridation processing of the substrate 12, a Kr gas and an ammonia gas, or a Kr gas and a nitrogen gas and a hydrogen gas are introduced.", "In this case, the excited Kr radicals (Kr) or Ar radicals (Ar) cause the excitation of hydrogen nitride radicals NH* according to the reaction NH3→NH+2H+e−, or N2+H2→NH+NH, wherein the hydrogen nitride radicals thus excited cause the desired nitridation processing of the substrate of the surface 12.Meanwhile, there are cases in which it is preferable to use atomic state nitrogen (N), free from hydrogen and having a strong nitriding power, at the time of the nitridation processing of the substrate.", "The atomic state nitrogen N are formed according to the reaction N2→N+N, wherein it should be noted that such a reaction requires the energy of 23-25 eV.", "This means that it is not possible to excite the atomic state nitrogen N* according to the foregoing reaction, as long as Kr or Ar plasma is used.", "As noted previously, the energy of the Kr radicals or Ar radicals obtained by the Kr or Ar plasma is merely in the order of 10 eV.", "Thus, even when there is made an attempt to supply a nitrogen gas in the substrate processing apparatus of FIG.", "2 or FIG.", "3 in place of the Kr gas or the Ar gas, merely the reaction N2→N2++e−, is obtained, and there is caused no desired atomic state oxygen N.", "FIG.", "4 shows the relationship between the state density of the Kr plasma and the excitation energy of the atomic state nitrogen N, hydrogen nitride radicals NH* and nitrogen atoms N2+.", "Referring to FIG.", "4, it can be seen that the state density of the Kr plasma is large at the low energy side, while the state density shows a rapid decrease with increase of the energy.", "Such a plasma cannot achieve efficient excitation of the desired nitrogen radicals.", "DISCLOSURE OF THE INVENTION Accordingly, it is a general object of the present invention to provide a novel and useful substrate processing apparatus wherein the foregoing problems are eliminated.", "Another and more specific object of the present invention is to provide a substrate processing method and apparatus capable of forming nitrogen radicals N* efficiently.", "Another object of the present invention is to provide a method of processing a substrate by using a substrate processing apparatus which has such a construction that a process space, in which a substrate to be processed is contained, is separated from a plasma formation space, in which the substrate to be processed is not contained, by a control electrode in a processing vessel, characterized by the steps of: supplying a gas containing He and N2 to said processing vessel; forming plasma in said plasma formation space under a condition such that there is caused excitation of atomic state nitrogen N* in said plasma; and nitriding a surface of the substrate to be processed by said atomic state nitrogen N* in said process space.", "Another object of the present invention is.", "to provide a substrate processing apparatus, comprising: a processing vessel defined by an outer wall and having a stage for holding a substrate to be processed thereon; an evacuation system coupled to said processing vessel; a plasma gas supplying part supplying a plasma excitation gas and a process gas into said processing vessel; a microwave window provided on said processing vessel so as to face said substrate to be processed; and a control electrode provided between said substrate to be processed on said stage and said plasma gas supplying part so as to face said substrate to be processed and separating a plasma excitation space containing said microwave window and a process space containing said substrate to be processed, said control electrode comprising a conductive member having a plurality of apertures for passing plasma formed in said processing vessel therethrough, and a surface of said control electrode being covered by any of aluminum oxide or electrically conductive nitride.", "Another object of the present invention is to provide a substrate processing apparatus, characterized by: a processing vessel defined by a wall of quartz glass and having a stage for holding a substrate to be processed; an evacuation system coupled to said processing vessel; a plasma gas supplying part supplying a plasma excitation gas and a process gas to said processing vessel; a control electrode provided so as to face said substrate to be processed on said stage and dividing an interior of said processing vessel into a process space containing said substrate to be processed and a plasma excitation space; and an induction coil provided outside said quartz glass wall in correspondence to said plasma excitation space, said control electrode comprising a conductive member having a plurality of apertures passing therethrough plasma formed in said processing vessel, and a surface of said control electrode being covered with any of aluminum oxide or electrically conductive nitride.", "According to the present invention, it becomes possible to form plasma having the energy sufficient for causing excitation of atomic state nitrogen N* in the substrate processing apparatus by using He for the plasma excitation gas, and it becomes possible to conduct an efficient nitridation of the substrate by using the atomic state nitrogen N* thus excited.", "By separating the plasma excitation space in which the high-density plasma is excited from the process space in which the substrate is included by means of the control electrode, it becomes possible to reduce the plasma energy in the process space to the level suitable for substrate processing.", "Further, it becomes possible to trap the positive ions formed in the plasma excitation space.", "In the case of applying the present invention to the substrate processing apparatus that uses microwave-excited plasma, it becomes possible to avoid excessive increase of the plasma energy by conducting the plasma excitation by using a microwave having the frequency of about 28 GHz or more.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a diagram showing the construction of a conventional induction coupled plasma processing apparatus; FIG.", "2 is a diagram showing the construction of a previously proposed microwave substrate processing apparatus; FIG.", "3 is a diagram showing the construction of another previously proposed microwave substrate processing apparatus; FIG.", "4 is a diagram explaining the characteristics of plasma excitation in the microwave substrate processing apparatus of FIG.", "2 or FIG.", "3; FIG.", "5 is a diagram showing the construction of a microwave substrate processing apparatus according to a first embodiment of the present invention; FIG.", "6 is a diagram showing a part of the microwave substrate processing apparatus of FIG.", "5; FIG.", "7 is a diagram showing the characteristics of plasma excitation in the microwave substrate processing apparatus of FIG.", "5; FIG.", "8 is a diagram showing a modification of the microwave plasma processing apparatus of FIG.", "5; FIG.", "9 is a diagram showing the construction of a microwave plasma processing apparatus according to a second embodiment of the present invention; and FIG.", "10 is a diagram showing the construction of an induction coupled plasma processing apparatus according to a third embodiment of the present invention.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [First Embodiment] FIG.", "5 shows the construction of a substrate processing apparatus 100 according to a first embodiment of the present invention.", "In FIG.", "5, those parts corresponding to the parts described previously are designated by the same reference numerals and the description thereof will be omitted.", "Referring to FIG.", "5, the shower plate 14 is mounted on the processing vessel 11 via a seal 11s, and the cover plate 15 is mounted on the shower plate 14 via a seal 11t.", "Further, the radial line slot antenna 20 is mounted on the processing vessel 11 via a seal 11u.", "Further, in the substrate processing apparatus 100 of FIG.", "5, the interface between the emission plate 16 and the cover plate 15 is evacuated via a ring-shaped groove 11g formed at the top part of the processing vessel 11 in the region where the processing vessel makes an engagement with the emission plate and further via an evacuation port 11G communicating with the ring-shaped groove 11g.", "After evacuation, a He gas is introduced into the foregoing interface with a pressure of about 0.8 atmospheres as a thermal conducting medium.", "The He gas thus introduced is confined therein by closing the valve 11V.", "In the substrate processing apparatus 100 of FIG.", "5, it should be noted that the lower shower plate 31 used in the substrate processing apparatus 10A of FIG.", "3 is removed and a control electrode 131 of a conductive member is formed, wherein the control electrode 31 has a lattice shape as represented in FIG.", "6 and is formed so as to separate the plasma excitation space 11B and the process space 11C.", "Referring to FIG.", "6, the lattice-shaped control electrode 131 is formed with large number of apertures 132 having a size set such that there occurs free passage of the radicals excited in the plasma excitation spate 11B, and thus, the plasma excited in the plasma excitation space 11B cause diffusion freely into the process space 11C through the control electrode 131.In the construction of FIG.", "5, it should be noted that the lattice-shaped control electrode 131 is grounded, and thus, the microwave introduced into the plasma excitation space 11B from the radial line slot antenna 11B is reflected by the lattice shaped control electrode 131, and there is caused no invasion of the microwave into the process space 11C.", "Thus, the problem of the microwave causing damages in the substrate 12 is not caused in the substrate processing apparatus 100 of FIG.", "5.It should be noted that the lattice-shaped control electrode 131 can be formed by W, Ti, or the like, wherein it is possible to increase the resistance against plasma irradiation by forming a layer 131a of a conductive nitride such as WN or TiN on the surface thereof.", "Further, it is possible to form such a lattice-shaped control electrode 131 by using a quartz glass and provide the conductive nitride layer 131a on the surface thereof.", "Further, in the substrate processing apparatus 100, it should be noted that the sidewall surface of the processing vessel 11 is covered by a quartz liner 11D for the part corresponding to the plasma excitation space 11B In the substrate processing apparatus 100 of FIG.", "5, a He gas and an N2 gas are introduced to the process gas inlet port 11p, and a microwave of about 28 GHz is supplied to the radial line slot antenna.", "Typically, the process pressure in the processing vessel 11 is set to the range of 66.5-266 Pa (0.5-2 Torr), and nitridation processing or oxynitridation processing of the substrate 12 is conducted in the temperature range of 200-500° C. FIG.", "7 shows the state density of the plasma excited in the substrate processing apparatus 100 of FIG.", "5 for the case He is used for the plasma gas.", "Referring to FIG.", "7, it should be noted that the use of He having a characteristically small collision cross-section for the plasma gas causes significant acceleration in the excited He radicals He* with the microwave electric field, and as a result, there is caused significant increase of plasma energy to the level suitable for excitation of the atomic state nitrogen N.", "On the other hand, it can be seen that the efficiency of excitation of the hydrogen nitride radicals NH or nitrogen ions N2+, which are excited efficiently in the case Kr is used for the plasma gas, is reduced significantly.", "Thus, in the present invention, efficient excitation of the atomic state nitrogen N* is achieved in the substrate processing apparatus 100 at the high plasma energy of 23-25 eV by using He for the plasma gas.", "In order to avoid excessive increase of the electron temperature in the plasma, the present invention uses a microwave source 22 that produce a microwave of the frequency higher than the previously proposed frequency, such as about 28 GHz or more, for driving the radial line slot antenna 20.Thereby, it is possible to select the frequency of the microwave source from the frequencies such as about 2.4 GHz or about 8.3 GHz.", "Further, by separating the plasma excitation space 1lB and the process space 11C by the control electrode 131, it is possible to reduce the electron temperature and the plasma energy to a level suitable for substrate processing.", "Particularly, it should be noted that the control electrode is protected effectively from the high-energy plasma by forming a conductive nitride such as an Al2O3 passivation film on the surface of the control electrode 131 as explained already.", "Further, the problem of sputtering of the inner wall of the processing vessel by the high-energy plasma and the associated problem of contamination of the substrate are avoided by covering the inner wall of the processing vessel 11 by a quartz liner 11D for the part corresponding to the plasma excitation region 11B.", "FIG.", "8 shows the construction of a substrate processing apparatus 100A according to a modification of the present embodiment.", "Referring to FIG.", "8, it becomes possible in the substrate processing apparatus 100A to capture the nitrogen ions N2+ excited in the plasma excitation space 11B with the positive electric charge, by controlling the potential of the control electrode 31 to a suitable negative potential value.", "Thereby, penetration of the nitrogen ions N2+ into the process space 11C is avoided.", "In the substrate processing apparatus 100 or 100A of the present embodiment, it is possible to conduct an oxynitridation processing of the substrate 12 by supplying a He gas, an N2 gas and an O2 gas to the plasma gas supply port 11p.", "[Second Embodiment] FIG.", "9 shows the construction of a substrate processing apparatus 200 according to a second embodiment of the present invention.", "In FIG.", "9, those parts corresponding to the parts described previously are designated by the same reference numerals and the description thereof will be omitted.", "Referring to FIG.", "9, it should be noted that the shower plate 14 is removed in the present embodiment, and in place of this, there are provided a plurality of process gas inlet ports lip on the processing vessel 11 such that the process gas inlet ports 11P are disposed with a symmetric relationship with respect to the substrate 12.As a result, therefore, the cover plate 15 constituting the dielectric window is exposed at the top part of the plasma excitation space 11B.", "Further, the sidewall surface of the processing vessel is covered by the quartz liner 11D for the part corresponding to the plasma excitation space 11B similarly to the previous embodiment.", "According to the present embodiment, the construction of the substrate processing apparatus 11 is simplified, and it becomes possible to conduct the nitridation processing of the substrate 12 efficiently with low cost by using the atomic state nitrogen N, by supplying a He gas and an N2 gas to the plasma gas supplying port lip and by supplying the microwave of about 28 GHz to the radial line slot antenna 20.Further, it is possible to conduct an oxynitridation processing by supplying a He gas, an N2 gas and an O2 gas to the plasma gas supplying port 11p.", "[Third Embodiment] FIG.", "10 shows the construction of a substrate processing apparatus 300 according to a third embodiment of the present invention.", "In FIG.", "10, those parts corresponding to the parts described previously are designated by the same reference numerals and the description thereof will be omitted.", "Referring to FIG.", "10, the substrate processing apparatus 300 has a construction similar to the substrate processing apparatus 1 explained before with reference to FIG.", "1, except that a control electrode 6 similar to the control electrode 31 is provided in the quartz vessel 2, and the space inside the quartz vessel 2 is divided by the control electrode 6 into a plasma excitation space 2B1 in which the high-density plasma 2D is excited and a process space 2B2 that includes the substrate 4 to be processed.", "In the present embodiment, a He gas and an N2 gas are introduced into the plasma excitation space 2B1 via the process gas supply line 2C, and there is formed high-density plasma 2D having a high electron temperature and plasma energy sufficient for exciting atomic state nitrogen N in the plasma excitation space 2B1.The atomic state nitrogen N* thus formed cause diffusion into the process space 2C through the control electrode 6, and the surface of the substrate 4 undergoes nitridation.", "In such a construction, it should be noted that the plasma has a very high electron temperature and energy in the plasma excitation space 2B1, while the electron temperature and the energy of the plasma are reduced to the level suitable for processing the substrate 4 in the process space 2B2.In the present embodiment, too, it becomes possible to remove the low energy positive ions such as N2+ formed in the plasma excitation space 2B1 from the process space 2B2 by trapping the same, by controlling the potential of the control electrode 6 by the voltage source 6A.", "Further, it becomes possible to control the state of the high-density plasma 2D in the plasma excitation space 2B1 by controlling the potential of the control electrode 6.In the substrate processing apparatus 200 of the present embodiment, it is also possible to conduct an oxynitridation processing of the substrate 4 in the process space 2B2 by introducing a He gas and an N2 gas and an O2 gas from the process gas supply line 2C.", "Further, the present invention is not limited to the specific preferred embodiments described heretofore, but various variations and modifications may be made without departing from the scope of the invention recited in the claims.", "INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to form plasma having the energy sufficient for causing excitation of atomic state nitrogen N* in the substrate processing apparatus by using He for the plasma excitation gas, and it becomes possible to conduct an efficient nitridation of the substrate by using the atomic state nitrogen N* thus excited.", "By separating the plasma excitation space in which the high-density plasma is excited from the process space in which the substrate is included by means of the control electrode, it becomes possible to reduce the plasma energy in the process space to the level suitable for substrate processing.", "Further, it becomes possible to trap the positive ions formed in the plasma excitation space.", "In the case of applying the present invention to the substrate processing apparatus that uses microwave-excited plasma, it becomes possible to avoid excessive increase of the plasma energy by conducting the plasma excitation by using a microwave having the frequency of about 28 GHz or more." ] ]	Patent_10467820
[ [ "Immunogenic compositions comprising an antigen and a purified m protein from respiratory syncytial virus", "Methods and compositions for enhancing an immune response to an antigen in a host are provided.", "Immunogenic composition comprising an antigen and an amount of purified M protein from respiratory syncytial virus are provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.", "The antigen can be an antigen from Respiratory syncytial virus." ], [ "1.An immunogenic composition comprising an antigen and an amount of purified M protein from respiratory syncytial virus or at least one immunoeffective fragment thereof, wherein the amount of said M protein or immunoeffective fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.", "2.The immunogenic composition of claim 1 wherein said antigen is from a pathogen.", "3.The immunogenic composition of claim 1 formulated as a vaccine.", "4.The immunogenic composition of claim 1 wherein said antigen or at least one immunogenic fragment thereof, is a protein antigen selected from the group consisting of, a viral antigen, a bacterial antigen, a respiratory syncytial virus antigen, a respiratory syncytial virus G antigen substantially free from respiratory syncytial virus F antigen, respiratory syncytial virus F antigen substantially free from respiratory syncytial virus G antigen, or a respiratory syncytial virus M22 antigen.", "5.The immunogenic composition of claim 1 wherein said pre-existing respiratory syncytial virus M-specific immune response is a T-cell immune response.", "6.The immunogenic composition of claim 1 wherein the amount of purified M protein is about 0.1 μg to about 1,000 μg per dose.", "7.The immunogenic composition of claim 6 wherein the amount of purified M protein is about 1 μg to about 100 μg per dose.", "8.The immunogenic composition of claim 7 wherein the amount of purified M protein is about 1 μg to about 50 μg per dose.", "9.The immunogenic composition of claim 1 wherein the purified M protein is greater than 50% pure as measured by SDS-PAGE analysis or in an M-specific ELISA assay.", "10.The immunogenic composition of claim 9 wherein the purity is greater that 70%.", "11.The immunogenic composition of claim 10 wherein the purity is greater that 90%.", "12.A method of making a immunogenic composition comprising providing an antigen and an amount of purified M protein from from respiratory syncytial virus or at least one immunoeffective fragment thereof, wherein the amount of said M protein or immunoeffective fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.", "13.The method of claim 12 further formulated with a pharmaceutically acceptable carrier to provide a vaccine.", "14.A method of immunizing a host comprising administering the immunogenic composition of claim 1 to said host.", "15.A method of enhancing an immune response to an antigen in a host having a pre-existing immune response to respiratory syncytial virus M protein comprising the steps of: i) purifying M protein of respiratory syncytial virus; ii) mixing a pre-selected amount of said purified M protein with a different antigen; iii) formulating said mixture as a vaccine; and iv) administering said vaccine to a host.", "16.The immunogenic composition of claim 1 wherein the antigen is encoded by a nucleic acid vector comprising an antigen encoding portion and a promoter to effect expression of the antigen encoding portion in the host.", "17.The use of a pre-selected amount of purified M from respiratory syncytial virus or immunoeffective fragments thereof, to enhance the immune response to an antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Adjuvants have been used for many years to improve the host immune response to antigens of interest in vaccines, especially subunit or component vaccines comprised of recombinant proteins.", "Adjuvants are immunomodulators that are typically non-covalently linked to antigens and are formulated to enhance the host immune response.", "Examples include aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum).", "While little or no systemic toxicity is observed with alum, its use is associated with local reactions such as erythema, subcutaneous nodules, contact hypersensitivity and granulomatous inflammation.", "Such local reactions may be of particular concern in the context of frequent, e.g., annual immunizations.", "Adjuvants enhance the immunogenicity of an immunogen but are not necessarily immunogenic themselves.", "Adjuvants have been identified that enhance the immune response to antigens delivered parenterally.", "Some of these adjuvants are toxic, however, and can cause undesirable side-effects making them unsuitable for use in humans and many animals.", "Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines.", "To efficiently induce humoral immune responses (HIR) and cell-mediated immunity (CMI), immunogens (antigens) are often emulsified in adjuvants.", "Many adjuvants are toxic, inducing granulomas, acute and chronic inflammations (Freund's complete adjuvant, FCA), cytolysis (saponins and pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP).", "Although FCA is a potent adjuvant and widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.", "Human Respiratory Syncytial Virus (RSV) is a major cause of respiratory tract infections.", "Globally, 65 million infections occur every year resulting in 160,000 deaths (ref.", "1; a list of references appears at the end of the disclosure and each of the references in the list is incorporated herein by reference thereto.)", "In the USA alone 100,000 children, may require hospitalization for pneumonia and bronchiolitis caused by RSV in a single year (refs.", "2).", "Providing inpatient and ambulatory care for children with RSV infections costs in excess of $340 million annually in the USA.", "RSV is a major cause of serious lower respiratory illness in elderly and immunocompromised adults (refs.", "3).", "Outbreaks in nursing or retirement homes are well documented (ref.", "3) and a significant proportion of disease involving the lower respiratory tract in outbreaks were associated with mortality.", "Approximately 7% of hospitalized community acquired pneumonias have been attributed to RSV (ref.", "4).", "Mortality due to RSV may exceed that due to influenza by 60 to 80% (ref.", "5) The annual costs attributed to hospitalizations for RSV pneumonia in the elderly in the USA has been conservatively estimated at between $150 to $680 million (ref.", "6).", "An RSV vaccine could therefore play an important role in lessening morbidity and mortality in the elderly and decreasing health care costs.", "RSV is an enveloped RNA virus of the family paramyxoviridae and of the genus pneumovirus.", "The structure and composition of RSV has been elucidated and is described in detail in the textbook “Fields Virology”, Fields, B.N.", "Raven Press, N.Y. (1996), pp 1313-1351 “Respiratory Syncytial Virus” by Collins, P., McIntosh, K., and Chanock, R. M. (ref.", "7).", "Cross neutralization studies have shown that RSV isolates can be classified into two major antigenic groups, designated A and B.", "(ref.", "8) The G glycoprotein shows the greatest divergence between groups showing 53% amino acid homology between RSV A and B.", "(ref.", "9) The two major protective antigens of RSV are the envelope fusion (F) and the attachment (G) glycoproteins (ref.", "10).", "The F protein is synthesized as an about 68 kDa precursor molecule (Fo) which is proteolytically cleaved into disulfide-linked F1 (about 48 kDa) and F2 (about 20 kDa) polypeptide fragments (ref.", "11).", "The G protein (about 33 kDa) is heavily O-glycosylated giving rise to a glycoprotein of apparent molecular weight of about 90 kDa (ref.", "12).", "Two broad subtypes of RSV have been defined A and B (ref.", "13).", "The major antigenic differences between these subtypes are found in the G glycoprotein while the F glycoprotein is more conserved (refs.", "14).", "Antibodies directed against the F protein or against the G protein can neutralize the virus.", "Antibodies to the F protein block the spread of the virus between cells.", "In addition to the antibody response generated by the F and G glycoproteins, human cytotoxic T cells produced by RSV infection have been shown to recognize the RSV F protein, matrix protein (M), 22 k protein (M22) nucleoprotein (N), small hydrophobic protein (SH), and the nonstructural protein (lb.)", "(ref 15).", "The human RSV M gene coding for the matrix protein has been sequenced (ref 18).", "It would be desirable to enhance the immune response to an antigen, in particular an RSV antigen to provide a more efficacious vaccine." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides methods and compositions of enhancing the response to vaccines.", "Accordingly, in one aspect of the present invention there is provided an immunogenic composition comprising an antigen and an amount of purified M protein from respiratory syncytial virus or at least one immunogenic fragment thereof, wherein the amount of said M protein or immunogenic fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.", "In accordance with another aspect of the present invention, there is provided a method of making a immunogenic composition comprising providing an antigen and an amount of purified M protein from from respiratory syncytial virus or at least one immunogenic fragment thereof, wherein the amount of said M protein or immunogenic fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.", "In a further aspect of the present invention there is provided the use of a pre-selected amount of purified M from respiratory syncytial virus or immunogenic fragments thereof, to enhance the immune response to an antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.", "The hosts protected against disease caused by RSV include humans and the invention includes methods of immunization and protection of hosts against disease caused by infection by RSV by administering the immunogenic and preparations and vaccines as provided herein to susceptible hosts.", "The hosts may be elderly humans or other humans previously exposed to RSV M protein and immunologically primed to respond to the immunization." ], [ "FIELD OF THE INVENTION The present invention relates to the field of immunology and is particularly concerned with enhancing an immune response to an antigen.", "BACKGROUND OF THE INVENTION Adjuvants have been used for many years to improve the host immune response to antigens of interest in vaccines, especially subunit or component vaccines comprised of recombinant proteins.", "Adjuvants are immunomodulators that are typically non-covalently linked to antigens and are formulated to enhance the host immune response.", "Examples include aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum).", "While little or no systemic toxicity is observed with alum, its use is associated with local reactions such as erythema, subcutaneous nodules, contact hypersensitivity and granulomatous inflammation.", "Such local reactions may be of particular concern in the context of frequent, e.g., annual immunizations.", "Adjuvants enhance the immunogenicity of an immunogen but are not necessarily immunogenic themselves.", "Adjuvants have been identified that enhance the immune response to antigens delivered parenterally.", "Some of these adjuvants are toxic, however, and can cause undesirable side-effects making them unsuitable for use in humans and many animals.", "Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines.", "To efficiently induce humoral immune responses (HIR) and cell-mediated immunity (CMI), immunogens (antigens) are often emulsified in adjuvants.", "Many adjuvants are toxic, inducing granulomas, acute and chronic inflammations (Freund's complete adjuvant, FCA), cytolysis (saponins and pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP).", "Although FCA is a potent adjuvant and widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.", "Human Respiratory Syncytial Virus (RSV) is a major cause of respiratory tract infections.", "Globally, 65 million infections occur every year resulting in 160,000 deaths (ref.", "1; a list of references appears at the end of the disclosure and each of the references in the list is incorporated herein by reference thereto.)", "In the USA alone 100,000 children, may require hospitalization for pneumonia and bronchiolitis caused by RSV in a single year (refs.", "2).", "Providing inpatient and ambulatory care for children with RSV infections costs in excess of $340 million annually in the USA.", "RSV is a major cause of serious lower respiratory illness in elderly and immunocompromised adults (refs.", "3).", "Outbreaks in nursing or retirement homes are well documented (ref.", "3) and a significant proportion of disease involving the lower respiratory tract in outbreaks were associated with mortality.", "Approximately 7% of hospitalized community acquired pneumonias have been attributed to RSV (ref.", "4).", "Mortality due to RSV may exceed that due to influenza by 60 to 80% (ref.", "5) The annual costs attributed to hospitalizations for RSV pneumonia in the elderly in the USA has been conservatively estimated at between $150 to $680 million (ref.", "6).", "An RSV vaccine could therefore play an important role in lessening morbidity and mortality in the elderly and decreasing health care costs.", "RSV is an enveloped RNA virus of the family paramyxoviridae and of the genus pneumovirus.", "The structure and composition of RSV has been elucidated and is described in detail in the textbook “Fields Virology”, Fields, B.N.", "Raven Press, N.Y. (1996), pp 1313-1351 “Respiratory Syncytial Virus” by Collins, P., McIntosh, K., and Chanock, R. M. (ref.", "7).", "Cross neutralization studies have shown that RSV isolates can be classified into two major antigenic groups, designated A and B.", "(ref.", "8) The G glycoprotein shows the greatest divergence between groups showing 53% amino acid homology between RSV A and B.", "(ref.", "9) The two major protective antigens of RSV are the envelope fusion (F) and the attachment (G) glycoproteins (ref.", "10).", "The F protein is synthesized as an about 68 kDa precursor molecule (Fo) which is proteolytically cleaved into disulfide-linked F1 (about 48 kDa) and F2 (about 20 kDa) polypeptide fragments (ref.", "11).", "The G protein (about 33 kDa) is heavily O-glycosylated giving rise to a glycoprotein of apparent molecular weight of about 90 kDa (ref.", "12).", "Two broad subtypes of RSV have been defined A and B (ref.", "13).", "The major antigenic differences between these subtypes are found in the G glycoprotein while the F glycoprotein is more conserved (refs.", "14).", "Antibodies directed against the F protein or against the G protein can neutralize the virus.", "Antibodies to the F protein block the spread of the virus between cells.", "In addition to the antibody response generated by the F and G glycoproteins, human cytotoxic T cells produced by RSV infection have been shown to recognize the RSV F protein, matrix protein (M), 22 k protein (M22) nucleoprotein (N), small hydrophobic protein (SH), and the nonstructural protein (lb.)", "(ref 15).", "The human RSV M gene coding for the matrix protein has been sequenced (ref 18).", "It would be desirable to enhance the immune response to an antigen, in particular an RSV antigen to provide a more efficacious vaccine.", "SUMMARY OF THE INVENTION The present invention provides methods and compositions of enhancing the response to vaccines.", "Accordingly, in one aspect of the present invention there is provided an immunogenic composition comprising an antigen and an amount of purified M protein from respiratory syncytial virus or at least one immunogenic fragment thereof, wherein the amount of said M protein or immunogenic fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.", "In accordance with another aspect of the present invention, there is provided a method of making a immunogenic composition comprising providing an antigen and an amount of purified M protein from from respiratory syncytial virus or at least one immunogenic fragment thereof, wherein the amount of said M protein or immunogenic fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.", "In a further aspect of the present invention there is provided the use of a pre-selected amount of purified M from respiratory syncytial virus or immunogenic fragments thereof, to enhance the immune response to an antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.", "The hosts protected against disease caused by RSV include humans and the invention includes methods of immunization and protection of hosts against disease caused by infection by RSV by administering the immunogenic and preparations and vaccines as provided herein to susceptible hosts.", "The hosts may be elderly humans or other humans previously exposed to RSV M protein and immunologically primed to respond to the immunization.", "BRIEF DESCRIPTION OF DRAWINGS The present invention will be further understood from the following description with reference to the drawings, in which: FIG.", "1 is a graphical representation of the IFN-γ secretion from spleen cells of mice primed with live RSV and boosted with various RSV antigens encoded by a viral vector (NYVAC).", "FIG.", "2 is a graphical representation of the CTL response from the viral vectors of FIG.", "1, showing the percentage specific lysis of target cells.", "FIG.", "3 shows the T-cell response in mice to other RSV antigens when RSV M protein is co-administered.", "FIG.", "4 shows the enhanced antibody response in mice to other RSV antigens when RSV M protein is co-administered.", "GENERAL DESCRIPTION OF THE INVENTION As discussed above, the present invention provides an enhanced response to antigens in sub-unit vaccines by the use of respiratory syncytial virus M protein.", "While the inventors have not identified the exact mechanism of the enhanced immune response to an antigen due to the RSV M protein, it is thought that the M protein mobilizes T-cell help.", "In a preferred embodiment, proteins to be included in the sub-unit vaccines may include the RSV F, G and M22 proteins.", "The proteins can be isolated from RSV by, for example, immunoaffinity purification, ion-exchange or other biochemical procedures as described in, for example, International Patent Application No.", "WO 94/27636 of Hancock, published Dec. 8, 1994 or by the procedure described in U.S. Pat.", "No.", "5,194,595.", "(Each of the cited patent documents are incorporated herein by reference thereto) The RSV proteins and immunogenic fragments thereof can be isolated from recombinant organisms that express the proteins or immunogenic fragments.", "The gene encoding the F protein is described in ref.", "16.The gene encoding the G protein is described in ref.", "17 and the gene encoding the M protein is described in ref.", "18.The production of recombinant organisms expressing the RSV proteins or immunogenic fragments thereof and the identification and purification of the expressed gene products is described in, for example, U.S. Pat.", "No.", "5,223,254 (and incorporated herein by reference thereto).", "Such recombinants include any bacterial transformants, yeast transformants, cultured insect cells infected with recombinant baculoviruses or cultured mammalian cells as known in the art, for example, Chinese hamster ovary cells that express the RSV virus proteins or immunogenic fragments thereof.", "The RSV proteins and immunogenic fragments thereof can also be chemically synthesized.", "The fusion (F) protein may comprise multimeric fusion (F) proteins which may include, when analyzed under nonreducing conditions, heterodimers of molecular weight approximately 70 kDa and dimeric and trimeric forms thereof: The attachment (G) protein may comprise, when analyzed under non-reducing conditions, oligomeric G protein, G protein of molecular weight approximately 95 kDa and G protein of molecular weight approximately 55 kDa.", "The matrix (M) protein may comprise, when analyzed under non-reducing conditions, protein of molecular weight approximately 28 to 34 kDa The immunogenic compositions provided herein may be formulated as a vaccine for in vivo administration to a host, which may be a primate, most preferably a human host, to confer protection against disease caused by RSV.", "The immunogenic compositions and vaccines provided herein may comprise at least one further immunogenic material which may be an antigen from a pathogen other than RSV, such as a bacterial or viral antigen to provide a combination vaccine for protection against a plurality of diseases.", "The immunogenic compositions may be formulated as a vaccine wherein a non-RSV antigen is provided.", "The non-RSV antigen may be a bacterial or viral antigen such that the RSV M protein enhances the host immune response to the non-RSV antigen.", "In this specification RSV M antigen is defined as purified and isolated RSV M protein or immunoeffective fragments thereof, or vectors delivering RSV M protein.", "Immunoeffective fragments of RSV M protein includes fragments or peptides derived from RSV M protein that are capable of eliciting the immunologic enhancement to another antigen.", "Purified in this specification means isolated away from other RSV proteins such that the RSV M protein represents the dominant material as assayed by SDS-PAGE or M-specific ELISA assays for example.", "Preferably the M protein should be greater than about 50% pure, more preferably greater than about 70% pure and most preferably greater than about 90% pure.", "Pre-selected amounts of RSV M protein refers to defined amounts of the M protein, for example as measured by M-specific ELISA, that are added to another antigen such that there is an enhanced immune response to the other antigen in the host when compared to the same composition without the RSV M protein.", "Enhanced immune response can be measured by interferon gamma (IFN γ) secretion, a CTL response as determined by specific cell lysis, IL-5 secretion, or antibody responses as measured by specific ELISA.", "Immune response can also be measured by any other assay available to one skilled in the art and is not limited to those exemplified here.", "A further measure can be by enhanced efficacy against disease in a relevant model system.", "Pre-selected amounts may differ depending upon the host to which it is administered.", "Examples of RSV M amounts can be from about 0.1 μg to about 1,000 μg, or about 0.1 μg to about 100 μg.", "In other situations it may be preferable to administer between about 1 μg to about 50 μg of RSV M protein.", "Pre-existing RSV M-specific response is defined as any detectable immune response to RSV M protein in the host.", "This includes T-cell responses as measured by cytokine production, for example IFNγ from CD8+ T-cells, as detected by cytokine specific-ELISA assay or ELISPOT assay, both well known assays in the art.", "RSV M protein pre-priming induces cytokine responses to heterologous proteins including viral derived from RSV or any other virus, bacterial or tumor antigens, CTL responses to heterologous antigens delivered by a vector (bacterial, viral or nucleic acid, or antigens formulated to deliver antigens for class I presentation such as liposomes) and antibody responses in naive or previously RSVimmune population.", "The antigen to be used with the RSV M protein can be a purified protein antigen, isolated from the disease causing organism (pathogen), or can be recombinantly produced in any of the well known bacterial or eukaryotic expression systems.", "The antigen can also be delivered by a nucleic acid vector such as non-replicating plasmid expression vectors encoding the antigen of choice, and containing a promoter to effect expression of said antigen in the host.", "Alternatively the nucleic acid vector can be a viral vector.", "Examples of non-replicating in the host viral vectors used to effect expression of said antigen are the pox vectors NYVAC and ALVAC (Ref 20) Viral vectors can also be replicating viral vectors such as recombinant alphavirus vectors as described in U.S. Pat.", "No.", "6,224,879 and U.S. Pat.", "No.", "6,015,686.Vaccine Preparation and Use Immunogenic compositions, suitable to be used as vaccines, may be prepared from mixtures comprising M protein and an antigen.", "The immunogenic composition elicits an immune response which produces antibodies, and/or cell mediated responses such as cytotoxic T-cell response to the specific antigen.", "Immunogenic compositions including vaccines may be prepared as injectables, as liquid solutions, suspensions or emulsions.", "The active immunogenic ingredients may be mixed with pharmaceutically acceptable excipients which are compatible therewith.", "Such excipients may include water, saline, dextrose, glycerol, ethanol and combinations thereof.", "The immunogenic compositions and vaccines may further contain auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof.", "Immunogenic compositions and vaccines may be administered parentally, by injection subcutaneous, intradermal or intramuscularly injection.", "Alternatively, the immunogenic compositions formulated according to the present invention, may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces.", "Thus, the immunogenic composition may be administered to mucosal surfaces by, for example, the nasal or oral (intragastric) routes.", "Alternatively, other modes of administration including suppositories and oral formulations may be desirable.", "For suppositories, binders and carriers may include, for example, polyalkalene glycols or triglycerides.", "Such suppositories may be formed from mixtures containing the active immunogenic ingredient(s) in the range of about 10%, preferably about 1 to 2%.", "Oral formulations may include normally employed carriers, such as, pharmaceutical grades of saccharine, cellulose and magnesium carbonate.", "These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 1 to 95% of the active ingredients, preferably about 20 to 75%.", "The immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, immunogenic and protective.", "The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies, and, if needed, to produce a cell-mediated immune response.", "Precise amount of active ingredients required to be administered depend on the judgment of the practitioner.", "However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of micrograms to milligrams of the active ingredients) per vaccination.", "Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent booster administrations.", "The dosage may also depend on the route of administration and will vary according to the size of the host.", "The concentration of the active ingredients in an immunogenic composition according to the invention is in general about 1 to 95%.", "A vaccine which contains antigenic material of only one pathogen is a monovalent vaccine.", "EXAMPLES The above disclosure generally describes the present invention.", "A more complete understanding can be obtained by reference to the following specific Examples.", "These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention.", "Changes in form, and substitution of equivalents are contemplated as circumstances may suggest or render expedient.", "Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.", "Methods of determining tissue culture infectious dose50 (TCID50/mL), plaque and neutralization titres, not explicitly described in this disclosure are amply reported in the scientific literature and well within the scope of those skilled in the art.", "Protein concentrations were determined by the bicinchoninie acid (BCA) method as described in the Pierce Manual (23220, 23225; Pierce Chemical company, U.S.A.), incorporated herein by reference.", "CMRL 1969 culture medium was used for cell culture and virus growth.", "The cells used in this study are vaccine quality African green monkey kidney cells (VERO lot M6) obtained from Institut Merieux.", "The RS viruses used were the RS virus subtype A (Long and A2 strains) obtained from the American Type culture Collection (ATCC) for use in the virus neutralization assay and a recent subtype A clinical isolate for viral protein purification.", "Example 1 This example illustrates a method of purifying RSV M protein.", "An RSV concentrate was pelleted by centrifugation for 30 minutes at 5,000×g and the viral pellet was extracted with 2% Triton® X-100 in 1 mM sodium phosphate pH 6.8, 300 mM NaCl by stirring for 1 hour at room temperature.", "The growth and harvest conditions for RSV can be found in U.S. Pat.", "No.", "6,020,182.The extract was centrifuged for 30 minutes at 15,000×g and the supernatant was collected.", "The soluble supernatant was diluted two-fold with 1 mM sodium phosphate pH 6.8, 2% Triton® X-100 and then applied to a ceramic hydroxyapatite type II (Bio-Rad Laboratories) column equilibrated with 1 mM sodium phosphate, pH 6.8, 50 mM NaCl and 0.02% Triton® X-100.The column was washed with the same buffer and then an RSV M-rich fraction was collected by elution with the 1 mM sodium phosphate pH 6.8, 300 mM NaCl and 0.02% Triton® X-100.The elution pool was concentrated using an Amicon stirred-cell concentrator and a YM-10 ultrafiltration membrane and then dialyzed overnight against 20 mM Tris, pH 8.0, 600 mM NaCl, 0.02% Triton® X-100.The dialyzed concentrate was applied to a Sephacryl® S-200 size exclusion column equilibrated with 20 mM Tris, pH 8.0, 600 mM NaCl, 0.02% Triton® X-100 and the M-rich peak was collected.", "Trace amounts of RSV F and RSV G glycoproteins were removed by passing the M-rich pool consecutively over an anti-F affinity column and a jacalin-Sepharose® column.", "The purified RSV M was concentrated using an Amicon stirred-cell concentrator and a YM-10 ultrafiltration membrane and then frozen at −70° C. Example 2 This example illustrates the T-cell response in mice to other RSV antigens when delivered by viral vector immunization.", "Eight-week old BALB/c mice used in this study were primed with either PBS or 103 pfu of live RSV intranasally.", "They were boosted (i.m.)", "4 weeks later with 107 pfu of NYVAC-M22 or NYVAC-F or NYVAC with or without RSV-M protein at 1 μg/mouse in PBS as outlined in Table 1 below.", "Sera samples were collected to measure anti-RSV F titers.", "Spleens were collected 4 weeks after boosting to look for CTL activity and cytokine production.", "Spleen cells were restimulated in vitro with BC or BCH4 cells (persistantly infected with RSV).", "Supematants were collected at 72 h and tested for IFN-γ and IL-5.For IFN-γ detection in the culture supernatants, wells of Nunc-Immulon-Maxisorp plates were coated with 50 mL of rat anti-mouse rIFN-γ antibody (Biosource) at 2 mg/mL in 0.05 M Carbonate/Bicarbonate buffer (pH 9.6), overnight at room temperature.", "Wells were blocked with 200 mL of 10% FBS in PBS for 1 hour at room temperature, followed by 2 washes in 0.05% Tween 20 in PBS (washing buffer).", "Samples and reference standards were diluted in sample buffer (0.05% Tween 20 in RPMI 1640) and 100 mL of each sample was added to the wells.", "Plates were incubated at 37° C. for 2 hours, followed by 5 washes in washing buffer.", "One hundred mL of biotinylated rat anti-mouse rIFN-g antibody diluted in sample buffer to 2 mg/mL, were added to each well and plates were incubated at 37° C. for 1 hr followed by 5 washes with washing buffer.", "To each well, 100 mL of avidin-HRP (1/2000 dilution) were then added and plates were incubated for one hour at room temperature, followed by 5 washes with washing buffer.", "Color reaction was developed by adding 100 mL of tetra methyl benzidine (TMB) /H2O2 and the reaction was terminated after 15 minutes by adding 50 mL of H2SO4.The plates were read at 450 nm in a Multiscan plate reader and the results were calculated using the Titersoft data reduction program.", "For IL-5 detection, similar protocol was followed with appropriate antibodies and stanadards from Pharmingen and Biosource International.", "Splenocytes were restimulated with BC or BCH4 cells at day 7 and then tested for CTL activity after 12 days.", "CTL assays were carried out as follows.", "Splenocytes (2.5 to 3×106 cells/mL) were incubated in complete RPMI containing 10 U/mL IL-2 and 2.5-3×106 cells/mL γ-irradiated (3,000 rads) syngeneic splenocytes which had been infected with 1 pfu/cell RSV for 2 hr at 37° C. After 5 days of culture, cells were washed and incubated (4 hr/37° C.) at varying effector cell dilutions with 2×103 51Cr-labeled target cells (persistently RSV-infected BCH4 fibroblasts).", "Uninfected BALB/c fibroblasts (BC cells) served as a control.", "Spontaneous and total chromium releases were measured in supernatants of target cells suspended in medium or 2.5% Triton-X 100.The percent of specific lysis was calculated as (counts−spontaneous counts)/(total counts−spontaneous counts)×100.Results are reported as mean specific lysis from triplicate assays.", "Each experiment was performed 3 times.", "These experiments show that boosting with RSV M protein antigen enhances CD8.T-cell responses as measured by IFN-γ shown in FIG.", "1, and CTL activity shown in FIG.", "2.RSV M antigen can enhance the specific response to at least two RSV antigens tested, RSV F and M22.Example 3 This example illustrates the T-cell response in mice to other RSV antigens when RSV M protein is co-administered.", "Groups of BALB/c mice were primed with 103 pfu of live RSV intranassally as before.", "Four weeks after priming the mice were boosted with PBS, purified RSV F protein (50 ng) in aluminum phosphate or purified RSV F plus purified RSV M (1 μg) in aluminum phosphate.", "Four weeks after boosting the spleens were harvested as before, cultured and incubated in vitro with purified RSV F, G or M proteins (0.5 μg/ml final).", "The cells were plated at 3×106 cells/ml and were stimulated with the same number of syngeneic spleen cells y-irradiated at 3000 rads.", "Supematants were collected at 72 h and were assayed for IFN-γ as described in Example 2.The results of this assay are shown in FIG.", "3.No detectable levels of anti-F, G or M responses were found in mice that were not boosted (PBS).", "Mice that were boosted with F plus M (F+M) responded to F, M and also G. The anti-G response is attributable to trace amounts of G present in the purified F. The mice that received only F boost (F) did not produce significant IFN-γ to any of the proteins.", "The IFN-γ response to RSV F in the F+M group of mice was enhanced when compared to the RSV F only boosted group.", "Example 4 This example illustrates the antibody response to other RSV proteins in mice when RSV M is co-administered.", "Groups of BALB/c mice were primed with 103 pfu of live RSV intranassally as before.", "Four weeks later the mice were boosted with either PBS, or FI-RSV (formalin-inactivated) 5 ng of F with 1 μg of M in alum (aluminum phosphate) or 5 ng of F in alum.", "The animals were boosted again four weeks later and the sera samples were collected to determine anti-F responses by F-specific ELISA.", "The spleens were also collected and were restimulated with 500 ng/ml of F, M, G or 106PFU of RSV.", "The culture supemates were collected 72 hrs later for IFN-gamma and IL-5 determinations.", "The antibody results shown in FIG.", "4, show that the inclusion of the RSV M antigen enhances the antibody response (antibody titre) to RSV F antigen by two logs when compared to the RSV F only boost.", "TABLE 1 Groups Primary Boost 1 Total # of mice 1 RSV 103 pfu PBS 5 2 RSV 103 pfu M (1 ug) + NYVAC-F 5 3 RSV 103 pfu M (1 ug) + NYVAC-M22 5 4 RSV 103 pfu NYVAC-F 5 5 RSV 103 pfu NYVAC-M22 5 6 RSV 103 pfu M (1 ug) + NYVAC 5 7 PBS PBS 5 8 PBS M (1 ug) + NYVAC-F 5 9 PBS M (1 ug) + NYVAC-M22 5 10 PBS NYVAC-F 5 11 PBS NYVAC-M22 5 12 PBS M (1 ug) + NYVAC 5 Summary of the Disclosure In summary of this disclosure, the present invention provides compositions and methods for enhancing an immune response to an antigen, in particular RSV antigens.", "The methods provide formulating immunogenic preparations to contain purified RSV M protein and at least one antigen from RSV or another antigen from a different infectious organism, or at least one immunogenic fragment thereof, in pre-selected amounts to elicit said enhanced immune response.", "Modifications are possible within the scope of the invention.", "REFERENCES 1.Robbins, A., and Freeman, P. (1988).", "Sci.", "Am.", "259:126-133.2.Hall C B.", "Prospects for a respiratory syncytial virus vaccine.", "(1994) Science 265:1393-1394.3.Falsey A R, Cunningham C K, Barker W H, et al.", "Respiratory syncytial virus and influenza A infections in the hospitalized elderly.", "(1995) J. Infect.", "Dis.", "172:389-394.4.Nicholson K G, Kent J, Hammersley V, Cancio E. Acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden.", "(1997) BMJ 315:1060-1064.5.Nicholson K G. Impact of influenza and respiratory syncytial virus on mortality in England and Wales from January 1975 to December 1990.Epidemiol Infect 1996;116:51-63.6.Han L. L., Alexander J. P_, Anderson L. J.", "(1999) Vaccine 179:25-30.7.Collins, P., McIntosh, K., and Chanock, R. M., in “Fields of Virology” ed_ Fields, B. M., Knipe, D. M_ and Howley P. M., Lippincott-Raven Press New York, (1996) pp.", "1313-1351.8.Coates, H. N., et al.", "(1966).", "Am.", "J. Epidemeol.", "83259-313.9.Johnson, R R., et al.", "(1987).", "J. Virol.", "61:3163-3166.10.Walsh, E. E., Hall, C. B., Briselli, M., Braudiss, M. W., and Schlesinger, J. J.", "(1987).", "J. Infec.", "Dis.", "155:1198-1204.11.Walsh, E. E., Hruska, J.", "(1983).", "J. Virol.", "47—171-177.12.Levine, S., Kleiber-France, R, and Paradiso, P. R. (1987) J. Gen- Virol.", "69:2521-2524.13.Anderson, L. J., Hierholzer, J C., Tsou, C., Hendry, R. M., Femie, B. F., Stone Y_ and McIntosh, K. (1985) J. Infec.", "Dis.151:626-633.14.Wertz, G. W., Sullender, W. M. (1992) Biotech 20:151-176.15.Cherrie, A. H., Anderson, K., Wertz, G. W. and Openshaw, P. J. M. (1992) J. Virol.", "66:2102-2110.16.Collins, P. L., Huang, Y. T. and Wertz, G. W. (1984).", "Proc.", "Nat).", "Acad.", "Sci.", "81:7683-7687.17.Wertz, G. W., Collins P. L., Huang, Y., Gruber C., Levine S., and Ball, L. A.", "(1985) Proc.", "Natl.", "Acad.", "Sci.", "82:4075-4079.18.Satake, M. and Venkatesan S. (1984).", "J.Virol.", "50:92-99.19.Levine S, Diliman T R, and Montgomery P C (1989).", "Proc.", "Soc.", "Exp.", "Biol Med 190:349-356.20.Paoletti E, Tartaglia J, Taylor J.", "(1994) Dev Biol Stand 82:65-69." ] ]	Patent_10467828
[ [ "Sequences involved in phenomena of tumour suppression, tumour reversion, apoptosis and/or virus resistance and their use as medicines", "This invention is directed to sequences that are involved in the molecular pathways of tumor suppression, tumor revision, apoptosis and/or virus resistance.", "The use of the compounds having these sequences, or encoded by them, in treating cancer, neurodegenerative diseases or viral diseases, and screening methods for compounds having such therapeutic properties are also encompassed." ], [ "1.An isolated nucleotide sequence comprising a nucleotide sequence chosen from the group comprising: a) SEQ.", "ID NO.", "72, b) a nucleotide sequence of at least 15 consecutive nucleotides of a sequence as defined in a), c) a nucleotide sequence having a percentage identity of al least 80%, after optimal alignment, with a sequence defined in a) or b), d) a nucleotide sequence which hybridizes under high stringency conditions with a sequence defined in a) or b), and e) a complementary nucleotide sequence or the RNA sequence corresponding to a sequence as defined in a), b), c) or d).", "2-30.", "(canceled)" ], [ "The present invention relates to the identification of genes involved in the molecular pathways of tumour suppression, tumour reversion, apoptosis and/or resistance to viruses.", "The present invention was made possible by the isolation of cDNA corresponding to messenger RNAs expressed or repressed during tumour suppression, tumour reversion and/or during the process of apoptosis.", "In order to isolate the genes activated or inhibited during tumour reversion, overall screening for the expression of the genes in a malignant cell line (U937) and a derived cell line (US4) was carried out with suppression of the malignant phenotype.", "Comparison of the expressed genes (messenger RNAs expressed in both types of cell) made it possible to identify genes expressed differentially, that is to say expressed in one of the cells while they are not in the other (the genes may be activated or inhibited).", "It is easily deduced therefrom that these genes are at least involved in the cancerization process, in one case by their absence, and in the other case by their presence.", "For this differential study, the method used is the method described in 2000 by Brenner et al.", "(Proc.", "Natl.", "Acad.", "Sci.", "USA, 97 (4), 1665-70).", "In order to produce a model, the inventors made the following hypotheses: if it were possible to select, from a tumour which is sensitive to the cytopathic effect of the parvovirus H-1, cells which are resistant, then this resistance could be due to a change in their malignant phenotype.", "It has been possible to demonstrate this for US4 cells selected from the U937 cancer cells.", "Unlike the U937 parental line, the US4 clones (but also US3 clones which will not be dealt with in the present invention) are resistant to the cytopathic effect of the parvovirus H-1.At the molecular level, it has been possible to observe that this suppression of the malignant phenotype was accompanied by activation of the expression of the p21waf1 gene, this being independently of the expression of the p53 gene.", "The approach to the problem according to the present invention made it possible to isolate sequences directly linked to several precise functions.", "Accordingly, unlike the random sequencing of ESTs, the sequences are sequences whose function is known since they are involved in the process of suppression of the malignant phenotype, of tumour reversion, of apoptosis and/or in resistance to viruses.", "Tumour reversion is distinguishable from tumour suppression by the fact that it covers a broader domain than that of tumour suppressor genes.", "In other words, tumour reversion is achieved by the use of metabolic and/or molecular pathways which are not limited to the metabolic and molecular pathways in which the tumour suppressor genes are involved.", "Thus, the present invention relates in particular to novel sequences and to the use of these sequences in the diagnostic field and for carrying out methods for screening test compounds.", "The invention also relates to methods for the detection and/or assay of the sequences of the invention or of their product(s) of expression in a biological sample.", "The present invention first of all relates to an isolated nucleotide sequence comprising a nucleotide sequence chosen from the group comprising: a) SEQ ID No.", "1 to SEQ ID No.", "1020, preferably SEQ ID No.", "72, b) a nucleotide sequence of at least 15 consecutive nucleotides of a sequence as defined in a), c) a nucleotide sequence exhibiting a percentage identity of at least 80%, after optimum alignment, with a sequence defined in a) or b), d) a nucleotide sequence which hybridizes under high stringency conditions with a sequence defined in a) or b), and e) a complementary nucleotide sequence where the RNA sequence corresponding to a sequence as defined in a), b), c) or d).", "The nucleotide sequence according to the invention defined in c) exhibits a percentage identity of at least 80% after optimum alignment with a sequence as defined in a) or b) above, preferably of at least 90%, more preferably of at least 98%.", "In the context of the present invention, SEQ ID No.", "72 is the preferred nucleotide sequence and corresponds to the TPT1 gene also called TCTP.", "This gene is involved in particular in the phenomenon of tumour reversion and has been the subject of more thorough experiments on the part of the inventors as indicated below.", "The expressions nucleotide sequence, nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide and polynucleotide sequence, terms which will be used without distinction in the present description, are understood to mean a precise succession of nucleotides, modified or unmodified, which makes it possible to define a fragment or a region of a nucleic acid, containing, or otherwise, unnatural nucleotides, and being capable of corresponding at the same time to a double-stranded DNA, a single-stranded DNA and transcriptional products of the said DNAs.", "Thus, the nucleic sequences according to the invention also cover PNAs (Peptide Nucleic Acids), or the like.", "The fragments of the nucleotide sequences of the invention comprise at least 15 consecutive nucleotides.", "Preferably, they comprise at least 20 consecutive nucleotides and still more preferably they comprise at least 30 consecutive nucleotides.", "It should be understood that the present invention does not relate to the nucleotide sequences in their natural chromosomal environment, that is to say in the natural state.", "It involves sequences which have been isolated and/or purified, that is to say that they were collected directly or indirectly, for example by copying, their environment having been at least partially modified.", "The nucleic acids obtained by chemical synthesis are understood to also be thereby designated.", "The expression “percentage identity” between two nucleic acid or amino acid sequences for the purposes of the present invention is understood to mean a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, which is obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed and over their entire length.", "The expression “best alignment” or “optimum alignment” is understood to mean the alignment for which the percentage identity determined as below is the highest.", "Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, the said comparison being carried out per segment or per “comparison window” in order to identify and compare the local regions with sequence similarity.", "Optimum alignment of the sequences for comparison may be carried out, apart from manually, by means of the Smith and Waterman local homology algorithm (1981), by means of the Neddleman and Wunsch local homology algorithm (1970), by means of the Pearson and Lipman search for similarity methods (1988), by means of computer software packages using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.).", "In order to obtain optimum alignment, the BLAST program is preferably used, with the BLOSUM 62 matrix.", "The PAM or PAM 250 matrices can also be used.", "The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences aligned optimally, it being possible for the nucleic acid or amino acid sequence to be compared to comprise additions or deletions in relation to the reference sequence for an optimum alignment between these two sequences.", "The percentage identity is calculated by determining the number of identical positions for which the nucleotide or amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions compared and by multiplying the result obtained by 100 in order to obtain the percentage identity between these two sequences.", "The expression nucleic sequences exhibiting a percentage identity of at least 80%, preferably of at least 90%, more preferably of at least 98%, after optimum alignment with a reference sequence, is understood to mean the nucleic sequences exhibiting, in relation to the reference nucleic sequence, certain modifications such as in particular a deletion, truncation, alignment, chimeric fusion and/or substitution, in particular point substitution, and whose nucleic sequence exhibits at least 80%, preferably at least 90%, more preferably at least 98%, identity after optimum alignment with the reference nucleic sequence.", "Preferably, the specific or high stringency hybridization conditions will be such that they ensure at least 80%, preferably at least 90%, more preferably at least 98% identity after optimum alignment between one of the two sequences and the sequence complementary to the other.", "Hybridization under high stringency conditions means that the temperature and ionic strength conditions are chosen so that they allow hybridization to be maintained between two complementary nucleic acid fragments.", "By way of illustration, high stringency conditions of the hybridization step for the purposes of defining the nucleotide sequences described above, will be advantageously the following.", "The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42° C. for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5×SSC (1×SSC corresponds to a solution containing 0.15 M NaCl+0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulphate (SDS), 10×Denhardt's, 5% dextran sulphate and 1% salmon sperm DNA; (2) hybridization proper for 20 hours at a temperature dependent on the size of the probe (i.e.", ": 42° C., for a probe of size>100 nucleotides) followed by 2 washes of 20 minutes at 20° C. in 2×SSC+2% SDS, 1 wash of 20 minutes at 20° C. in 0.1×SSC+0.1% SDS.", "The final wash is performed in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. for a probe of size>100 nucleotides.", "The high stringency hybridization conditions described above for a polynucleotide of defined size may be adjusted by persons skilled in the art for larger- or smaller-size oligonucleotides, according to the teaching of Sambrook et al., 1989.Among the nucleotide sequences exhibiting a percentage identity of at least 80%, preferably of at least 90%, more preferably of at least 98%, after optimum alignment with the sequences according to the invention, preference is also given to the variant nucleic sequences of the sequences of the invention, or of fragments thereof, that is to say all the nucleic sequences corresponding to allelic variants, that is to say individual variations of the sequences of the invention.", "The expression “variant nucleotide sequence” is understood to mean any RNA or cDNA resulting from a mutation and/or variation of a splice site of the genomic DNA corresponding to the nucleotide sequences of the invention.", "The subject of the present invention is also a polypeptide encoded by a nucleotide sequence in accordance with the invention.", "The expression “polypeptide” is understood to mean, for the purposes of the present invention, either proteins or peptides.", "According to a particular embodiment, the polypeptides in accordance with the invention comprise a polypeptide chosen from: a) a polypeptide encoded by a nucleotide sequence in accordance with the invention, b) a polypeptide exhibiting at least 80% identity with a polypeptide as defined in a), c) a fragment of at least 5 amino acids of a polypeptide as defined in a) orb), d) a biologically active fragment of a polypeptide as defined in a), b) or c), and e) a modified polypeptide of a polypeptide as defined in a), b), c) or d).", "The percentage identity is understood to be evaluated after optimum alignment of the relevant sequences.", "The expression polypeptide whose amino acid sequence exhibits a percentage identity of at least 80%, preferably of at least 90%, more preferably of at least 98% after optimum alignment with a reference sequence, is understood to mean the polypeptides exhibiting certain modifications in relation to the reference polypeptide, such as in particular one or more deletions or truncations, an extension, a chimeric fusion and/or one or more substitutions.", "Among the polypeptides whose amino acid sequence exhibits a percentage identity of at least 80%, preferably of at least 90% and more preferably of at least 98%, after optimum alignment with a reference sequence such as a polypeptide in accordance with the present invention or with one of its fragments, preference is given to the variant polypeptides encoded by the variant nucleotide sequences as previously defined, in particular the polypeptides whose amino acid sequence exhibits at least one mutation corresponding in particular to a truncation, deletion, substitution and/or addition of at least one residue in relation to the polypeptide sequences of the invention or with one of their fragments.", "The present invention also relates to a cell cloning and/or expression vector, characterized in that it comprises a nucleotide sequence according to the invention or that it encodes a polypeptide according to the invention.", "Such a vector may also contain the elements necessary for the expression and possibly for the secretion of the polypeptide in a host cell.", "Such a host cell is also a subject of the present invention.", "The vectors comprising promoter and/or regulatory sequences also form part of the present invention.", "The said vectors preferably contain a promoter, signals for initiation and termination of translation, and appropriate regions for regulation of transcription.", "They should be able to be stably maintained in the cell and they may also possess particular signals so as to allow the secretion of the translated protein.", "These different control signals are chosen according to the cellular host used.", "To this effect, the nucleic acid sequences according to the invention may be inserted into vectors which replicate autonomously in the chosen host or into integrating vectors of the chosen host.", "Among the autonomously replicating systems, use is preferably made, according to the host cell, of the plasmid or viral type systems, it being possible for the viral vectors in particular to be adenoviruses (5), retroviruses, lentiviruses, poxviruses or herpesviruses (5a).", "Persons skilled in the art know the technologies which can be used for each of these systems.", "When the integration of the sequence into the chromosomes of the host cell is desired, it is possible to use, for example, systems of the plasmid or viral type; such viruses are, for example, retroviruses (6) or AAVs (7).", "Advantageously, the vectors in accordance with the invention contain a sequence allowing tissue-specific targeting and/or expression.", "Among the nonviral vectors, preference is given to naked polynucleotides such as naked DNA or naked RNA according to the technique developed by the company VICAL, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) for expression in yeast, mouse artificial chromosomes (MAC) for expression in murine cells and preferably human artificial chromosomes (HAC) for expression in human cells.", "Such vectors are prepared according to methods commonly used by persons skilled in the art, and the clones resulting therefrom may be introduced into an appropriate host by standard methods such as for example lipofection, electroporation, heat shock, transformation after chemical permeabilization of the membrane, cell fusion.", "The invention additionally comprises the host cells, in particular the eukaryotic and prokaryotic cells, transformed with the vectors according to the invention and the transgenic animals, preferably the mammals, except humans, comprising one of the said transformed cells according to the invention.", "These animals may be used as models, for studying the aetiology of inflammatory and/or immune diseases, and in particular inflammatory diseases of the digestive tube, or for studying cancers.", "Among the cells which can be used for the purposes of the present invention, there may be mentioned bacterial cells (8), but also yeast cells (9), as well as animal cells, in particular mammalian cell cultures (10), and in particular Chinese hamster ovary (CHO) cells.", "There may also be mentioned insect cells in which it is possible to use methods using for example baculoviruses (11).", "A preferred cellular host for the expression of the proteins of the invention consists of COS cells.", "Among the mammals according to the invention, preference is given to animals such as rodents, in particular mice, rats or rabbits, expressing a polypeptide according to the invention.", "These transgenic animals are obtained, for example, by homologous recombination on embryonic stem cells, transfer of these stem cells into embryos, selection of the chimeras affected at the level of the reproductive lines, and growth of the said chimeras.", "The transgenic animals according to the invention can thus overexpress the gene encoding the protein according to the invention, or their homologous gene, or may express the said gene into which a mutation is introduced.", "These transgenic animals, in particular mice, are obtained for example by transfection of a copy of this gene under the control of a strong promoter of a ubiquitous nature, or which is selective for a type of tissue, or after viral transcription.", "The cells and mammals according to the invention can be used in a method for producing a polypeptide according to the invention, as described below, and can likewise serve as a model for analysis.", "The transformed cells or mammals as described above may also be used as models in order to study the interactions between the polypeptides according to the invention and the chemical or protein compounds involved directly or indirectly in the activities of the polypeptides according to the invention, this being in order to study the different mechanisms and interactions involved.", "They can in particular be used for the selection of products interacting with the polypeptides according to the invention, or their [lacuna] as a cofactor, or as an inhibitor, in particular competitive, or having an agonist or antagonist activity on the activity of the polypeptides according to the invention.", "Preferably, the said transformed cells or transgenic animals are used as a model in particular for the selection of products which make it possible to control pathologies linked to abnormal expression of this gene.", "The subject of the present invention is also a monoclonal or polyclonal antibody, a fragment of this antibody or a chimeric antibody capable of specifically recognizing a polypeptide in accordance with the present invention.", "The specific monoclonal antibodies may be obtained according to the conventional hybridoma culture method well known to persons skilled in the art.", "The antibodies according to the invention are for example humanized antibodies, Fab or F(ab′)2 fragments.", "They may also be provided in the form of immunoconjugates or antibodies labelled so as to obtain a detectable and/or quantifiable signal.", "The antibodies in accordance with the invention, but also the immunoconjugates, are therefore capable of specifically recognizing a polypeptide according to the present invention.", "The specific polyclonal antibodies may be obtained from the serum of an animal immunized against the polypeptides of the invention, in particular produced by genetic recombination or by peptide synthesis, according to the customary procedures.", "The importance of antibodies specifically recognizing the polypeptides, their variants or their immunogenic fragments, according to the invention,: should be noted in particular.", "The subject of the invention is also the use of a nucleotide sequence in accordance with the present invention as a probe or a primer for the detection, identification, assay and/or amplification of nucleic acid sequences.", "According to the invention, the nucleotide sequences which may be used as a probe or as a primer in methods for the detection, identification, assay and/or amplification of a nucleic acid sequence have a minimum size of 15 bases, preferably of 20 bases, or even better from 25 to 30 bases.", "The probes and primers according to the invention may be directly or indirectly labelled with a radioactive or nonradioactive compound by methods well known to persons skilled in the art, so as to obtain a detectable and/or quantifiable signal.", "The nonlabelled nucleic sequences according to the invention may be used directly as a probe or a primer.", "The sequences are generally labelled in order to obtain sequences which can be used for numerous applications.", "The labelling of the primers or of the probes according to the invention is carried out with radioactive elements or with nonradioactive molecules.", "Among the radioactive isotopes used, there may be mentioned 32P, 33P, 35S, 3H or 125I.", "The nonradioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenin, haptens, colourings, luminescent agents such as radioluminescent, chemoluminescent, bioluminescent, fluorescent and phosphorescent agents.", "The nucleotide sequences according to the invention may thus be used as a primer and/or a probe in methods using in particular the PCR (polymerase chain reaction) technique (11a).", "This technique requires the choice of pairs of oligonucleotide primers flanking the fragment which has to be amplified.", "Reference may be made, for example, to the technique described in American Patent U.S. Pat.", "No.", "4,683,202.The amplified fragments may be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatography technique such as gel filtration or ion-exchange chromatography, and then sequenced.", "The specificity of the amplification may be checked, using as primers, the nucleotide sequences of the invention and, as templates, plasmids containing these sequences or the derived products of amplification.", "The amplified nucleotide fragments may be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid having a sequence complementary to that of the said amplified nucleotide fragments.", "The invention also relates to the nucleic acids which can be obtained by amplification using primers according to the invention.", "Other techniques for amplifying the target nucleic acid may be advantageously used as an alternative to PCR (PCR-like) using pairs of primers of nucleotide sequences according to the invention.", "The expression PCR-like is understood to mean any of the methods using direct or indirect reproductions of the nucleic acid sequences, or in which the labelling systems were amplified, these techniques are of course known.", "In general, they involve the amplification of DNA by a polymerase, when the original sample is an RNA, a reverse transcription should be carried out beforehand.", "A great number of methods allowing this amplification currently exist, such as for example the SDA (Strand Displacement Amplification) technique (12), the TAS (Transcription-based Amplification System) technique described by (13), the 3SR (Self-Sustained Sequence Replication) technique described by (14), the NASBA (Nucleic Acid Sequence Based Amplification) technique described by (15), the TMA (Transcription Mediated Amplification) technique, the LCR (Ligase Chain Reaction) technique described by (16), the RCR (Repair Chain Reaction) technique described by (17), the CPR (Cycling Probing Reaction) technique described by (18), the Q-beta-replicase amplification technique described by (19).", "Some of these techniques have since been improved.", "In the case where the target polynucleotide to be detected is an mRNA, an enzyme of the reverse transcriptase type is advantageously used, prior to the use of an amplification reaction with the aid of the primers according to the invention or to the use of a method of detection with the aid of the probes of the invention, in order to obtain a cDNA from the mRNA contained in the biological sample.", "The cDNA obtained will then serve as a target for the primers or probes used in the method of amplification or detection according to the invention.", "The probe hybridization technique may be carried out in various ways (20).", "The method most generally used consists in immobilizing the nucleic acid extracted from the cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) and in incubating, under well defined conditions, the immobilized target nucleic acid with the probe.", "After hybridization, the excess probe is removed and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, the fluorescence or the enzymatic activity linked to the probe).", "According to another embodiment of the nucleic probes according to the invention, they may be used as capture probes.", "In this case, a probe, called “capture probe”, is immobilized on a support and serves to capture, through specific hybridization, the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected by means of a second probe, called “detection probe”, labelled with an easily detectable element.", "The nucleotide sequences according to the invention may moreover be of interest when they are used as antisense nucleotides, that is to say whose structure brings about, through hybridization with the target sequence, inhibition of the expression of the corresponding product.", "They may also be used as sense nucleotides which, through interaction with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition, or an activation of this expression.", "The subject of the invention is also the use of a nucleotide sequence according to the present invention for the production or the synthesis of a recombinant polypeptide.", "The method of producing a polypeptide of the invention in recombinant form, which is itself included in the present invention, is characterized in that the transformed cells, in particular the cells or mammals of the present invention, are cultured under conditions allowing the expression of a recombinant polypeptide encoded by a nucleotide sequence according to the invention, and that the said recombinant polypeptide is recovered.", "The recombinant polypeptides, characterized in that they can be obtained by the said method of production, also form part of the invention.", "The recombinant polypeptides obtained as indicated above may be provided either in glycosylated or nonglycosylated form and may or may not have the natural tertiary structure.", "The sequences of the recombinant polypeptides may also be modified in order to improve their solubility, in particular in aqueous solvents.", "Such modifications are known to persons skilled in the art, such as for example the deletion of hydrophobic domains or the replacement of hydrophobic amino acids with hydrophilic amino acids.", "These polypeptides may be produced from the nucleic acid sequences defined above, according to techniques for producing recombinant polypeptides known to persons skilled in the art.", "In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression in a cellular host.", "An effective system for producing a recombinant polypeptide requires having a vector and a host cell according to the invention.", "These cells may be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, and then culturing the said cells under conditions allowing the replication and/or the expression of the transfected nucleotide sequence.", "The methods used for purifying a recombinant polypeptide are known to persons skilled in the art.", "The recombinant polypeptide may be purified from cell lysates and extracts, from the supernatant of the culture medium, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity methods using specific monoclonal or polyclonal antibodies, and the like.", "The polypeptides according to the present invention may also be obtained by chemical synthesis using one of the numerous known peptide syntheses, for example the techniques using solid phases (21) or techniques using partial solid phases, by condensation of fragments or by conventional synthesis in solution.", "The polypeptides obtained by chemical synthesis and which may contain corresponding unnatural amino acids are also included in the invention.", "The subject of the present invention is also a DNA chip, characterized in that it contains at least one nucleotide sequence in accordance with the present invention.", "Actually, the nucleotide sequences according to the invention which it is envisaged to use as probe or as primer for the detection, identification, assay and/or amplification of nucleic acid sequences may be covalently or noncovalently immobilized on a support, this support being a DNA chip or a high density filter.", "The expression “DNA chip” or “high density filter” is understood to mean a support to which DNA sequences are attached, it being possible for each of them to be located by its geographic localization.", "These chips or filters differ mainly in their size, the support material, and possibly the number of sequences attached thereto.", "In particular, it is possible to carry out a synthesis in situ by photochemical targeting or by ink jet targeting.", "Other techniques consist in carrying out a synthesis ex situ or in attaching the probes to the support of the DNA chip by ink jet, electronic or mechanical addressing.", "These different methods are well known to persons skilled in the art.", "The subject of the invention is also a protein chip comprising a polypeptide or antibody according to the invention.", "Such a protein chip allows the study of interactions between the polypeptides according to the invention and other proteins or chemical compounds and can thus be useful for screening compounds which interact with the polypeptides according to the invention.", "It may also be possible to use the protein chips according to the invention for detecting the presence of antibodies directed against the polypeptides according to the invention in the serum of patients to be tested.", "It may also be possible to use a protein chip comprising an antibody according to the invention, this time to detect the presence of polypeptides in the serum of patients which are capable of being recognized by the said antibodies.", "The subject of the present invention is also the use of a compound chosen from a nucleotide sequence, a polypeptide, a vector, a cell or an antibody according to the invention for the preparation of a medicament.", "The pathologies more specifically targeted are viral diseases and diseases characterized by the development of tumour cells or cellular degeneration such as Alzheimer's disease or schizophrenia.", "Thus, the abovementioned medicament is intended for the prevention and/or the treatment of these diseases.", "In particular, the targeted disease is cancer.", "One of the benefits of the present invention is that it has demonstrated the involvement of a large number of nucleotide sequences in the phenomena of tumour suppression, tumour reversion, apoptosis and/or viral resistance.", "These sequences are therefore expressed differentially when one of these abovementioned processes is triggered.", "Consequently, in the presence of a patient in whom the onset of one of these processes is suspected or for whom it is desired to check the absence of such an onset, it is useful to be able to determine, or even quantify, the expression of one or more sequences in accordance with the invention from a biological sample from the said patient.", "Optionally, analysis of the expression of one or more of the said sequences may be accompanied by a comparison with a reference level of expression corresponding to that of a healthy individual.", "Consequently, the invention therefore also comprises a method for the diagnosis and/or prognostic evaluation of a viral disease or a disease characterized by tumour development or cell degeneration comprising the analysis of the expression of at least one sequence of the invention from a biological sample from a patient to be tested.", "According to a preferred embodiment, the said method comprises the following steps: isolation of the messenger RNA from a biological sample obtained from a patient to be tested, preparation of the complementary cDNA from the said messenger RNA, optionally, amplification of a portion of the complementary DNA corresponding to at least one sequence of the invention, and detection of the optionally amplified complementary DNA.", "In particular, analysis of the expression of the sequence may be carried out by means of a DNA chip as described above.", "Among the sequences of the present invention, some exhibit the characteristic of being receptors expressed at the surface of cells and in order to understand the mechanism thereof in the context of the abovementioned processes, it is useful to search for compounds capable of interacting with this receptor, that is to say of interacting with a polypeptide in accordance with the invention, it is also necessary to predict the secreted protein.", "This also applies to the polypeptides in accordance with the invention corresponding to secreted proteins (at the surface or outside the cells), hormone-like proteins, and the like.", "Consequently, the subject of the present invention is also a method for screening compounds capable of binding to a peptide in accordance with the invention and comprising the following steps: bringing a polypeptide or a cell according to the invention into contact with a candidate compound, and detection of the formation of a complex between the said candidate compound and the said polypeptide or the said cell.", "A method for screening compounds may also be useful in relation to compounds capable of interacting with a nucleotide sequence according to the invention, or even with the sequences necessary for the expression or the regulation of these sequences.", "Indeed, the compounds are capable of interacting with the said sequences, the effect being to reduce, inhibit or on the contrary potentiate the expression of the sequences in question.", "Such a method comprises the following steps: bringing a nucleotide sequence or a cell according to the invention into contact with a candidate compound, and detection of the formation of a complex between the said candidate compound and the said nucleotide sequence or the said cell.", "For reasons mentioned above, it may therefore be useful to search for and/or assay, in a biological specimen or sample from a patient to be tested, the presence of a nucleotide sequence according to the invention.", "Such a method of detection and/or assay comprises the following steps: bringing a labelled nucleotide sequence according to the invention into contact with the biological sample to be tested, under the conditions necessary for the formation of a hybrid, and detection and/or assay of the hybrid which may be formed between the said nucleotide sequence and the nucleic acid present in the said biological sample.", "This method may additionally comprise a step for amplifying the nucleic acid of the said biological sample with the aid of primers chosen from the nucleotide sequences according to the invention.", "In particular, this method may be carried out by means of the DNA chip described above.", "Persons skilled in the art know how to carry out such a method and may in particular use a reagent kit comprising: a) a nucleotide sequence according to the invention, used as a probe, b) the reagents necessary for carrying out a hybridization reaction between the said probe and the nucleic acid of the biological sample, c) the reagents necessary for the detection and/or assay of the hybrid formed between the said probe and the nucleic acid of the biological sample.", "Such a kit may also contain positive or negative controls in order to ensure the quality of the results obtained.", "Likewise, the detection and/or the assay of a polypeptide according to the invention can also be envisaged in the context of the present invention and this method therefore comprises the following steps: bringing the biological sample into contact with a labelled antibody according to the invention, and detection and/or assay of the complex formed by the said antibody of polypeptide present in the said sample.", "Advantageously, this method may be carried out by means of the protein chip as described above.", "Here again, persons skilled in the art know how to carry out such a method and may in particular use a reagent kit comprising: a) a monoclonal or polyclonal antibody according to the invention; b) optionally reagents for preparing a medium favourable for the antigen/antibody reaction; c) the reagents allowing the detection of the antigen/antibody complex.", "Finally, the subject of the present invention is also a computer readable support or a computer support on which are recorded at least one nucleotide sequence according to the Claim 1 and/or at least one polypeptide sequence as defined in Claim 3 or 4.In particular, this support is chosen from the group comprising: a) a floppy disk, b) a hard disk, c) a random access memory (RAM), d) a read only memory (ROM), e) a CD ROM.", "The invention is not limited to the present description but encompasses on the contrary all the variants and will be understood more clearly in the light of the experimental data below.", "FIGURES FIG.", "1 represents a curve for tumour growth for the U937 and US4 groups in SCID mice.", "FIG.", "2 represents a curve for the body weight of mice carrying a U937 or US4 tumour.", "FIG.", "3 illustrates: a/ the analysis of the expression of the TPT1/TCTP gene in the U937/US4.2 lines and in the MCF7/MCF7+SIAH1 lines by Northern blotting.", "The quantities of total transcripts are checked by monitoring GAPDH; b/ Western blot analysis of the expression of the TCTP protein in the U937/US4.2 model, in the MCF7/MCF7+siah model and analysis of the expression of the murine homologue of TCTP, TCTP_MOUSE, in the M1/LTR6 system after 20 h of induction at 32° C.; c/ the analysis of the expression of TCTP in U937 cells transfected with the antisense TPT1/TCTP.", "The clones I and III are compared with the control, U937 cells with the vector alone.", "On the right hand panel, detection of the cleavage of PARP, a marker for apoptosis, in the clones I and III, U937 cells transfected with the antisense TPT1/TCTP.", "Jurkat cells treated with an anti-Fas (anti-CD95) antibody, an inducer of apoptosis, serve as a control; d/ characterization of the apoptotic cells by double labelling FITC-Annexin V and propidium iodide (PI).", "The U937 cells+control vector and the U937 cells+antisense TPT1/TCTP are simultaneously labelled with annexin V coupled to FITC (which binds to the phosphatidylserine of the apoptotic cells in the early phase) and PI (which only labels necrotic cells whose plasma membrane is damaged).", "Cytofluorometric analysis makes it possible to determine the percentage of apoptotic cells (Annexin+, PI−), necrotic cells (Annexin+, PI+ or Annexin−, PI+) and intact cells (Annexin−, PI−); e/ evaluation of apoptosis in the cells transfected with the antisense TPT1/TCTP, by in situ labelling of the free 3′ ends of the nuclear DNA: TUNEL (“Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labelling”) method.", "This labelling makes it possible to demonstrate in situ the fragmentation of nuclear DNA resulting from the activation of endonucleases during apoptosis.", "The apoptotic cells appear in green.", "f/ the study of the in vivo tumorigenicity of the U937 cells transfected with: the vector alone (top curve—91 tumours/100), an antisense presenilin 1 (second curve from the top—12 tumours/20), SIAH1 (middle curve—12 tumours/20) and an antisense TPT1/TCTP, clone I and III (the bottom 2 curves—8 tumours/20 and 4 tumours/20).", "10 million cells injected into each flank and into each shoulder.", "FIG.", "4 represents cells cultured on an extra-cellular tumour matrix (Matrigel).", "The 184B5 line, mammary epithelial cells transformed with benzo(a)pyrene, is used as a control for the formation of acini.", "The tumour cells T47D and MCF7 form irregular colonies.", "The MCF7 cells transfected with SIAH1 partially recover an organized structure.", "The MCF7 and T47D cells transfected with the antisense TPT1/TCTP result in a cellular reorganization comparable to the normal formation of the acini.", "The cells which appear green (light spots in the figure) are labelled with an anti-E_Cadherin antibody coupled to FITC and the nuclei labelled with PI appear red (darker spots).", "Western blot analysis of the expression of TCTP in the MCF7 and T47D cells transfected with the antisense TPT1/TCTP.", "1—DATA RELATING TO THE U937 and US4 CELLS As seen above, the present invention uses the parental cells U937 and the “derived” cells US4.Indeed, the US4 cells and the US3 cells (which are not involved in the context of the present invention) share certain characteristics.", "Their mode of production and their properties are presented below.", "Selection and Characterization of US3 and US4 Cells The U937 cells were subjected to two series of limiting dilution until a single clonal population is obtained.", "These cells were infected with the H-1 parvovirus.", "The cytopathic effect of the virus creates massive cell death which spares two resistant clones which are US3 and US4 after three months of continuous culture.", "The survival of the cells is defined as the relative number of viable cells in the culture infected with the H-1 virus compared with the untreated culture, as measured four days after reinfection.", "To measure the tumorigenicity, 107 U937, US3 and US4 cells were subcutaneously injected into scid/scid mice (4 or 5 weeks old).", "The tumorigenicity is expressed by the number of tumours developed by the mice within two months following the injection.", "The approach was as follows: using clonal populations of malignant cells, subclones were derived with a suppressed tumorigenic phenotype.", "This selection made by means of the H-1 parvovirus is carried out by the elimination of the tumour cells which are preferentially killed while sparing the normal cells.", "The selection of the cells resistant to the cytopathic effect of the H-1 parvovirus outside a sensitive tumour can give rise to cells which have a reduced malignant phenotype.", "On this basis, a clonal population of U937 cells was isolated, these cells are sensitive to the cytopathic effect of H-1 parvovirus and the US3 and US4 clones are, for their part, resistant to the virus.", "The US3 and US4 clones have a strong suppressed tumorigenic phenotype while the parental U937 cells develop tumours in 80% of cases among the scid/scid mice which were infected with the parvovirus, the US3 cells form only a single tumour and the US4 cells develop one tumour per 20 inoculations with 107 cells.", "The results are assembled in the table below.", "Resistance to the H-1 Parvovirus and Tumorigenicity of the U397, US3 and US4 Cells Survival of the Tumorigenicity cells to in infection with the scid/scid Cell lines the H-1 virus mice U937 0.4 16/20 US3 96 0/10 US4 89 1/20 2—MATERIALS AND METHODS The aim is to compare the growth of subcutaneous tumours in SCID mice induced by subcutaneous injection of transfected U937 and US4 human leukaemic cell lines.", "A. Leukaemic Cell Lines and Culture Conditions All the injected cells of the U937 (ATCC) and US4 cell lines were provided in the form of cell suspensions in bottles supplemented with RPMI-1640 culture medium supplemented with 2 mM L-glutamine, 10% foetal calf serum and gentamycin.", "The U937 cell line is a CD4+ human monocytic cell line derived from a patient having a diffuse histiocytic lymphoma (1).", "The cells were counted in a haemocytometer and their viability was tested with exclusions of trypan blue dyes at 0.25%.", "The viability was 95.5% and 90.5% for the U937 cells and the US4 cells, respectively.", "The U937 and US4 cells were centrifuged and then resuspended in an RPMI medium before being injected into SCID mice.", "B.", "The animals 10 female SCID mice in good health (CB17/IcrHsd), 31 weeks old and weighing between 20 and 25 g, were supplied by Harlan France (Gannat, France).", "The animals were observed for 7 days in a specific room belonging to the Applicant which is the unit for treating specific-pathogen-free animals before their treatment.", "The unit for treating animals (INRA, Dijon, France) is authorized by the French Ministers of Agriculture and Research (authorization No.", "A21100).", "The animal experiments are carried out according to the European Ethical Directives for the well-being of animals in the context of experimental neoplasia (3).", "B.1.Environment The animals were kept in rooms under controlled conditions of temperature (24±1° C.), humidity (55±1%), light period (12 h of light/12 h of darkness) and air renewal.", "The animals were kept under SPF conditions and the temperature and the humidity of the room were continuously monitored.", "The aeration system was programmed to give rise to 14 air renewals per hour without recirculation.", "Fresh air from outside passes inside a series of filters before being uniformly diffused into each room.", "A high pressure (2 mm) was maintained in the experimentation rooms in order to prevent contamination or diffusion of pathogens within a mouse colony.", "All the staff working under SPF conditions followed specific directives as regards hygiene and clothing when they were in the breeding area.", "B.2.Breeding The animals were housed in polycarbonate cages (UAR, Epinay sur Orge, France) which were equipped so as to provide them with food and water.", "The standard size of the cages used is 637 cm2 for 10 mice according to the standard internal operating procedures.", "The litter for animals and made up of sterile wood shavings (UAR) and is replaced twice per week.", "B.3.Food and Drink The animal food was bought from Extralabo (Provins, France).", "The food was provided ad libitum and was placed on the metal lid at the top of the cage.", "Water was also provided ad libitum from bottles of water equipped with rubber taps.", "The water bottles were washed, sterilized and replaced once per week.", "The water supply was sterilized by filtration with a 0.2 μm absolute filter.", "B.4.Identification of the Animal and the Cage After random distribution, the animals were identified with 2 different numbers engraved on the two ears.", "Each cage was marked with a specific code.", "C—Experimental Data and Treatments C.1.Induction of Tumours in SCID Mice Before cell injection, the SCID mice were randomly divided into two groups, in an amount of 5 mice per group.", "107 US4 or U937 tumour cells in 0.2 ml of RPMI medium were subcutaneously inoculated at time 0 for each site of injection into SCID mice.", "Each animal received 4 injections of tumour cells located in different areas, one in each flank and one in each shoulder.", "C.2.Collection of the Tumours When the tumours reached a volume of 1 500 mm3, the mice were killed and the tumours were collected, weighed and frozen in liquid nitrogen, stored at −80° C. and then specifically labelled.", "C.3.Monitoring of the Mice Isoflurane forene (Minerve, Bondouble, France) was used to anaesthetize the animals before injection of cells for sacrificing.", "After injection of tumour cells, the mice were observed for 5 hours.", "The viability, behaviour, body weight of the mice and the growth of the subcutaneous tumour were recorded twice per week.", "During the period of experimentation, the animals were killed under anaesthetic with isoflurane by cervical dislocation if one of the following signs appears: sign of suffering (cachexia, weakness, difficulty in moving or eating), tumour growth up to 10% of the body weight, tumour ulceration and persistently becoming nude, position of the tumour interfering with movement and/or feeding, loss of weight of 20% for three consecutive days.", "An autopsy was performed on each animal to detect the possible presence of metastases or morphological abnormalities.", "D—Presentation of the Data D.1.Monitoring Parameters The calculation for the median and mean survival time was expressed as follows: Mean survival time=S1/(S2−NT) With: S1=sum of the daily survivors from day 0 up to the end of the experiment (without the survivors “not taken into account”) S2=number of animals at the beginning NT=number of animals “not taken into consideration” *“not taken into consideration”: they are animals with tumours which are smaller than the predetermined limit considered as resulting from a defect in implantation of the tumour.", "D.2.System for Inhibition of the Tumour The tumour size was measured twice per week with a compass (and the tumour volume in mm3) will be estimated according to the formula: (length×width2/2 (4).", "The experiments were stopped when the tumour sizes in the mice reached 1 500 mm3.After sacrificing, the tumours were excised and weighed.", "The curve for tumour growth for the US4 and U937 groups was noted using the mean of the tumour volumes.", "The tumour doubling time for the US4 and U937 groups was defined as the period required to reach a mean tumour volume of 200% during the growth period.", "The specific growth period over one or two doubling times (DT) from injections of cells is defined as follows: specific growth period=(DT US4−DT U937)/DT U937.The growth period is calculated as the difference between the median growth time for the US4 group and the U937 group in order to reach the same tumour size.", "D.3.Statistical Tests All the statistical analyses were carried out with the StatView® software (Abacus Concept, Berkeley, USA).", "The statistical analyses of the changes in mean body weight, the tumour doubling time and the time to reach “V” were performed using the Bonferroni/Dunn test.", "A value p<0.05 was considered to be significant.", "All the groups were compared with each other.", "E—Results The curves for mean tumour volumes and body weights are shown in FIGS.", "1 and 2 respectively.", "No significant loss of body weight in SCID mice from the two groups was observed between day 8 and day 19.A significant difference is observed as regards the time to reach “V” between the 2 groups of SCID mice whereas no significant difference was observed for the change in mean body weight (day 19−day 8) and for the doubling time.", "The autopsies performed did not show the presence of metastases or of developments of suspicious nodosity.", "The tumours were collected on animals sacrificed after anaesthetic and cervical dislocation.", "The tumours were immediately placed in tubes, frozen in liquid nitrogen and stored at −80° C. The excised tumours had an ovoid shape, a moderate consistency and a pinkish colour.", "The interactions of the tumour with its environment (skin and muscle tissue) were limited and superficial.", "F—Conclusions The US4 cell line showed an uptake level significantly lower compared with the U937 cell line in the SCID mice.", "The retardation of growth between the US4 and U937 tumours was 23.5 days and the doubling time was equivalent.", "3—INVOLVEMENT OF TPT1/TCPT IN TUMOUR REVERSION In order to study the involvement of TPT1/TCPT (Translationally controlled Tumour Protein encoding the Histamine Release Factor) in tumour reversion, the expression of the gene (FIG.", "3a) and the expression of the protein (FIG.", "3b) in various cellular models of tumour reversion: U937/US4.2, MCF7/MCF7+SIAH1 and M1/LTR6, were analysed.", "The level of expression of TPT1/TCTP is highly reduced in the revertant cells compared with the tumour cells.", "To deal with the physiological aspect of this reduction, the U937 tumour cells were transfected with the antisense TPT1/TCTP.", "After having checked the drop in the expression of TPT1/TCTP (FIG.", "3c, left panel), the inventors detected its consequences in apoptosis by three different methods: detection of the cleavage of PARP, labelling with Annexin V and TUNEL method (FIG.", "3c, right panel; FIG.", "3d; FIG.", "3e, respectively).", "The reduction in the expression of TPT1/TCTP in tumour cells in culture increases apoptosis.", "The inventors then expressed in vivo the physiological consequences of this loss of TPT1/TCTP functions.", "We injected U937 cells transfected with antisense TPT1/TCTP into mice and observed a drastic reduction in the number of tumours formed when the expression of TPT1/TCTP is reduced (FIG.", "3f).", "In parallel with these results, the reduction of TPT1/TCTP, obtained by transfection of double-stranded RNA specific for TPT1/TCTP (siRNA), into MCF7 and T47D cells, cultured on an extracellular matrix, leads to a cellular reorganization of acini which is comparable to a normal structure (FIG.", "4).", "REFERENCES (1) Sundström C. and Nilsson K., Int.", "J.", "Cancer, 17, 565, 1976.", "(2) Principe d'éthique de l'expérimentation animale [Principle of ethics of animal experimentation], Directive No.", "86/609 EEC of 24 Nov. 1986, Decree No.", "87/848 of 19 Oct. 1987, Application Order of 19 Apr.", "1988.", "(3) United Kingdom co-ordinating committee on cancer research guidelines for welfare of animals in experimental neoplasia, Br.", "J.", "Cancer, 77: 1-10, 1998.", "(4) Bissery M. C. et al., Bull.", "Cancer, 78: 587, 1991.", "(5) Perricaudet et al.", "(1991).", "La Recherche 23: 471.", "(5a) Epstein (1992), Médecine/Sciences, 8, 902.", "(6) Temin (1986), Retrovirus vectors for gene transfer.", "In Kucherlapati R., ed.", "Gene.", "(7) Carter (1993), Curr.", "Op.", "Biotechnology 3, 533.", "(8) Olins and Lee (1993), Curr.", "Op.", "Biotechnology 4: 520.", "(9) Buckholz (1993), Curr.", "Op.", "Biotechnology 4, 538.", "(10) Edwards and Aruffo (1993), Curr.", "Op.", "Biotechnology 4, 558.", "(11) Luckow (1993), Curr.", "Op.", "Biotechnology 4, 564.", "(11a) Rolfs, A. et al.", "(1991), Berlin: Springer-Verlag.", "(12) Walker (1992), Nucleic Acids Res.", "20: 1691.", "(13) Kwoh et al.", "(1989), Proc.", "Natl.", "Acad.", "Sci.", "USA, 86, 1173.", "(14) Guatelli et al.", "(1990), Proc.", "Natl.", "Acad.", "Sci.", "USA, 87, 1874.", "(15) Kievitis et al.", "(1991), J. Virol.", "Methods, 35, 273.", "(16) Landegren et al.", "(1988) Science 241, 1077.", "(17) Segev (1992), Kessler C. Springer Verlag, Berlin, New York, 197-205.", "(18) Duck et al.", "(1990), Biotechniques, 9, 142.", "(19) Miele et al.", "(1983), J. Mol.", "Biol., 171, 281.", "(20) Matthews et al.", "(1988), Anal.", "Biochem., 169, 1-25.", "(21) Stewart and Yound (1984), Solid phase peptides synthesis, Pierce Chem.", "Company, Rockford, 111, 2nd ed.", "(1984)." ] ]	Patent_10467851
[ [ "System and method for an auction of multiple types of items", "An improved system and method for a computer-implemented auction in which multiple types of items are auctioned together without imposing a particular division of supply or demand among the individual types of items.", "In some embodiments the auction of the present invention provides a means or method for establishing prices for the types of items, wherein the prices maintain a relationship.", "In other embodiments, the present invention provides a means or method for implying prices from price parameters in the bids received form bidders, based on a relation among the prices for the types of items.", "Market clearing may be defined by the condition that the aggregate quantity bid for all types of items is less than or equal to the available quantity of all types of items.", "The division among the types of items within is thus determined flexibly, based on the bids at the associated prices.", "In other embodiments, market clearing is defined by the condition that the quantity bid for one selected type of item is less than or equal to the available quantity of the selected type of item.", "The quantities of the other types of items are thus determined flexibly, based on the bids at the associated prices." ], [ "1.A computer system for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined by the computer system based on the bids, comprising: establishing means for establishing prices for the plurality of types of items, wherein the prices maintain a relationship, receiving means for receiving bids associated with the established prices, and processing means for processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise enabling the establishing means to alter the prices established for the plurality of types of items.", "2.A system as recited in claim 1 which further comprises means for constraining the changes in a bid from a given bidder as the established prices are altered by considering, in the processing means, only bids which satisfy a constraint enforced by the means for constraining.", "3.A system as recited in claim 2 wherein the constraining means includes summation means for calculating a sum of quantity parameters for a group of types of items.", "4.A system as recited in claim 3 wherein the constraining means includes comparing means for comparing the calculated sum with a previous calculated sum for the same group of types of items.", "5.A system as recited in claim 1 wherein each received bid comprises a quantity parameter for at least one of the types of items in the auction.", "6.A system as recited in claim 5 wherein the establishing means establishes a price for a first type of item and then determines prices for each other type of item based on the price for the first type of item.", "7.A system as recited in claim 6 wherein the establishing means determines prices from a predetermined schedule.", "8.A system as recited in claim 7 wherein the establishing means includes interpolation means for interpolating prices among prices on the predetermined schedule.", "9.A system as recited in claim 6 wherein the processing means includes summation means for calculating a sum of the quantity parameters for the first type of item.", "10.A system as recited in claim 9 wherein the processing means includes comparing means for comparing the calculated sum with an available quantity parameter.", "11.A system as recited in claim 1 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods of at least some of the contracts have at least some overlap.", "12.A system as recited in claim 5 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods of at least some of the contracts have at least some overlap.", "13.A system as recited in claim 12 wherein the processing means includes summation means for calculating a sum of the quantity parameters for the overlapping contracts.", "14.A system as recited in claim 13 wherein the processing means includes comparing means for comparing the calculated sum with an available quantity parameter.", "15.A system as recited in claim 1 wherein the relationship of the prices established by the establishing means is determined by a schedule.", "16.A system as recited in claim 1 wherein the relationship of the prices established by the establishing means is determined by an algorithm.", "17.A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing prices for the plurality of types of items, wherein the prices maintain a relationship, receiving bids associated with the established prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for the plurality of types of items.", "18.A method as recited in claim 17 which further comprises constraining the changes in a bid from a given bidder as the established prices are altered by considering, in the processing, only bids which satisfy a constraint.", "19.A method as recited in claim 18 wherein the constraining includes calculating a sum of quantity parameters for a group of types of items.", "20.A method as recited in claim 19 wherein the constraining includes comparing the calculated sum with a previous calculated sum for the same group of types of items.", "21.A method as recited in claim 17 wherein each received bid comprises a quantity parameter for at least one of the types of items in the auction.", "22.A method as recited in claim 21 wherein a price is established for a first type of item and then prices for each other type of item are determined based on the price for the first type of item.", "23.A method as recited in claim 22 wherein the determined prices are determined from a predetermined schedule.", "24.A method as recited in claim 23 wherein establishing prices includes interpolating prices among prices included in the predetermined schedule.", "25.A method as recited in claim 22 wherein processing the received bids includes calculating a sum of the quantity parameters for the first type of item.", "26.A method as recited in claim 25 wherein processing the received bids includes comparing the calculated sum with an available quantity parameter.", "27.A method as recited in claim 17 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods of at least some of the contracts have at least some overlap.", "28.A method as recited in claim 21 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods of at least some of the contracts have at least some overlap.", "29.A method as recited in claim 28 wherein processing the received bids includes calculating a sum of the quantity parameters for the overlapping contracts.", "30.A method as recited in claim 29 wherein processing the received bids includes comparing the calculated sum with an available quantity parameter.", "31.A method as recited in claim 17 wherein the relationship of the prices is based on a schedule.", "32.A method as recited in claim 17 wherein the relationship of the prices is based on an algorithm.", "33.A computer system for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: receiving means for receiving bids comprising a quantity parameter for at least one of the types of items in the auction and a price parameter, and processing means for processing the received bids to determine an allocation of the items among the bidders that is consistent with the bids in the event that an allocation consistent with the bids is possible, wherein the processing means includes implying means for implying prices from price parameters in the received bids based on a fixed relation among the prices of the plurality of types of items.", "34.A system as recited in claim 33 which includes means for inviting additional bids in the event that an allocation of items consistent with the received bids is not possible, and means for applying the additional bids to the processing means for processing the additional bids to determine an allocation of the items among the bidders consistent with the additional bids in the event that an allocation consistent with the additional bids is possible.", "35.A computer system as recited in claim 34 which further comprises means for constraining the additional bids from a given bidder by limiting the processing to only additional bids which satisfy a constraint.", "36.A system as recited in claim 35 wherein the constraining means includes summation means for calculating a sum of quantity parameters for a group of types of items.", "37.A system as recited in claim, 36 wherein the constraining means includes comparing means for comparing the calculated sum with a previous calculated sum for the same group of types of items.", "38.A system as recited in claim 33 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods for at least some of the contracts have at least some overlap.", "39.A system as recited in claim 35 wherein the processing means includes summation means for calculating a sum of the quantity parameters for at least some of the overlapping contracts.", "40.A system as recited in claim 39 wherein the processing means includes comparing means for comparing the calculated sum with an available quantity parameter.", "41.A system as recited in claim 33 wherein the fixed relation of the implying means is based on a schedule.", "42.A system as recited in claim 33 wherein the fixed relation of the implying means is based on an algorithm.", "43.A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: receiving bids comprising a quantity parameter for at least one of the types of items in the auction and a price parameter, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, wherein the processing includes implying prices from price parameters in the received bids based on a fixed relation among the prices of the plurality of types of items.", "44.A method as recited in claim 43 which includes inviting additional bids in the event that an allocation of items consistent with the received bids is not possible, and processing the additional bids to determine an allocation of the items among the bidders consistent with the additional bids in the event that an allocation consistent with the additional bids is possible.", "45.A method as recited in claim 44 which further comprises constraining the additional bids from a given bidder by considering, in the processing, only additional bids which satisfy a constraint.", "46.A method as recited in claim 45 wherein the constraining includes calculating a sum of quantity parameters for a group of types of items.", "47.A method as recited in claim 46 wherein the constraining includes comparing the calculated sum with a previous calculated sum for the same group of types of items.", "48.A method as recited in claim 43 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods for at least some of the contracts have at least some overlap.", "49.A method as recited in claim 48 wherein the processing includes calculating a sum of the quantity parameters for at least some of the overlapping contracts.", "50.A method as recited in claim 49 wherein the processing includes comparing the calculated sum with an available quantity parameter.", "51.A method as recited in claim 43 wherein the fixed relation of the processing is determined by a schedule.", "52.A method as recited in claim 43 wherein the fixed relation of the processing is determined by an algorithm.", "53.A computer system for conducting an auction of at least one type of item among a plurality of bidders in a plurality of rounds, wherein bids are received and an allocation of the items among the bidders is determined by the computer system based on the bids, comprising: establishing means for establishing starting prices and ending prices for each type of item in a plurality of rounds, receiving means for receiving bids, wherein a bid comprises a quantity for at least one type of item and a price parameter signifying a percentage of the distance from the established starting prices to the established ending prices, and processing means for processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise enabling the establishing means to alter the prices established for at least one type of item.", "54.A system as recited in claim 53 where the establishing means includes means to maintain a relation among the established prices.", "55.A system as recited in claim 54 wherein the means to maintain a relation among the established prices employs a schedule of prices.", "56.A system as recited in claim 54 wherein the means to maintain a relation among the established prices employs an algorithm.", "57.A system as recited in claim 53 which further comprises means for constraining the changes in a bid from a given bidder as the established prices are altered by considering, in the processing means, only bids which satisfy a constraint enforced by the means for constraining.", "58.A system as recited in claim 58 wherein the constraining means includes summation means for calculating a sum of quantities bid for a group of types of items.", "59.A system as recited in claim 59 wherein the constraining means includes comparing means for comparing the calculated sum with a previous calculated sum for the same group of types of items.", "60.A computer implemented method for conducting an auction of at least one type of item among a plurality of bidders in a plurality of rounds, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing starting prices and ending prices for each type of item in a plurality of rounds, receiving bids, wherein a bid comprises a quantity for at least one type of item and a price parameter signifying a percentage of the distance from the established starting prices to the established ending prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for at least one type of item.", "61.A method as recited in claim 61 where the establishing includes maintaining a relation among the established prices.", "62.A method as recited in claim 62 wherein the relation among the established prices is determined by a schedule.", "63.A method as recited in claim 62 wherein the relation among the established prices is determined by an algorithm.", "64.A method as recited in claim 61 which further comprises constraining the changes in a bid from a given bidder as the established prices are altered by considering, in the processing, only bids which satisfy a constraint.", "65.A method as recited in claim 65 wherein the constraining includes calculating a sum of quantities bid for a group of types of items.", "66.A method as recited in claim 66 wherein the constraining includes comparing the calculated sum with a previous calculated sum for the same group of types of items." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Sellers and buyers of commodities, financial securities, and other goods and services are increasingly turning to auctions to assist in the sale or purchase of a wide range of items.", "Auctions are useful for selling a unique item (e.g., a painting), a single type of item available in multiple units (e.g., 10 tons of gold), and multiple types of items available in multiple units (e.g., 10 tons of gold and 20 tons of silver).", "And just as auctions are useful for efficiently selling items at maximum price, they are also useful for efficiently procuring items at minimum cost.", "Auction formats in the art tend generally to be of the sealed-bid or ascending-bid variety.", "In standard sealed-bid auctions, bidders—in one single bidding round—simultaneously and independently submit bids to the auctioneer, who then determines the auction outcome.", "In standard ascending-bid auctions, bidders—in a dynamic bidding process—submit bids in real time until no more bids are forthcoming.", "Sealed-bid auction formats offer the advantage of speed of the auction process.", "Ascending-bid auction formats offer the advantage that there is feedback among participants' bids, which tends to result in more aggressive bidding and in more efficient auction outcomes.", "When auction systems and methods in the art are used to sell (or buy) multiple types of items, the auctioneer generally specifies an available supply (or demand) for each type of item.", "For example, the auctioneer announces a supply of 10 tons of gold and 20 tons of silver, and then solicits bids, in a sealed-bid or ascending-bid procedure, in order to obtain market-clearing prices for each of the two types of commodities.", "However, consider a situation where the types of items are related, for example, contracts for provision of a commodity covering different time periods.", "For example, a government may wish to sell three types of financial securities: 3-month Treasury bills; 6-month Treasury bills; and 12-month Treasury bills.", "The government's only constraint may be that it needs to sell $50 billion worth of new debt securities at this auction, since this is the quantity of old debt securities that are maturing in the current calendar quarter.", "However, the government may have no preference as to how the required $50 billion is divided among 3-month bills, 6-month bills, and 12-month bills, respectively, and the government may simply wish to “let the market decide” the division among the various durations Auction systems and methods in the art provide effective ways for the government to sell bonds with a fixed division among the various durations.", "However, new and better systems and methods can be devised for “letting the market decide” the division among the various durations.", "The same problem also occurs in real life in the sale of contracts for electricity and all kinds of other commodities, as well as in the procurement of related items.", "In several preferred embodiments, the present invention provides a system and method for the government in the above example to simply auction $50 billion of Treasury bills at the lowest possible cost (relative to the market's yield curve), without imposing a particular division among the various durations." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is an improved system and method for a computer-implemented auction, particularly for a computer-implemented auction in which multiple types of items are auctioned together.", "In several embodiments, the present invention provides a system and method to conduct an auction of a group of related types of items, without imposing a particular division of supply or demand among the individual types of items within the group.", "In other embodiments, the present invention provides a system and method to conduct an auction of a group of related types of items, without imposing a particular division of supply or demand on some of the individual types of items within the group.", "Instead, in some embodiments, the present invention provides a means or method for establishing prices for the types of items within a group, wherein the prices maintain a relationship.", "In other embodiments, the present invention provides a means or method for implying prices from price parameters in the bids received from bidders, based on a relation among the prices for the types of items within a group.", "In some embodiments, market clearing is defined by the condition that the aggregate quantity bid for all types of items within a group is less than or equal to the available quantity of all types of items within the group.", "The division among the types of items within the group is thus determined flexibly, based on the bids at the associated prices.", "In other embodiments, market clearing is defined by the condition that the quantity bid for one selected type of item within a group is less than or equal to the available quantity of the selected type of item.", "The quantities of the other types of items within the group are thus determined flexibly, based on the bids at the associated prices.", "In a first preferred embodiment, the present invention is: A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing prices for the plurality of types of items, wherein the prices maintain a relationship, receiving bids associated with the established prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for the plurality of types of items.", "In a second preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the first preferred embodiment.", "The system includes an establishing means, a receiving means, and a processing means.", "In both the first and second preferred embodiments, the relationship or relation among the prices may involve a schedule or algorithm.", "In the event that the relationship or relation is based on a schedule, determining prices may involve looking up and possibly interpolating numbers that are contained in a table.", "Alternatively the relationship or relation may be represented as an algorithm or a formula that provides a relationship among the prices.", "The data table may reflect a market beyond the auction (for example, it may reflect price differentials in a separate commodities market or exchange) and the data table may change during the course of the auction (for example, if price differentials change in the separate commodities market or exchange, or if a significant news event occurs during the course of the auction).", "Similarly, the data set to which the algorithm or formula are applied may reflect a market beyond the auction and the data set may change during the course of the auction.", "In a third preferred embodiment, the present invention is: A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: receiving bids comprising a quantity parameter for at least one of the types of items in the auction and a price parameter, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, wherein the processing includes implying prices from price parameters in the received bids based on a fixed relation among the prices of the plurality of types of items.", "In a fourth preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the third preferred embodiment.", "The system includes a receiving means and a processing means.", "In both the third and fourth preferred embodiments, the “price parameters” in the received bids that are used for “implying prices” could be prices for one or more selected types of items.", "(For example, the price parameter could be an actual price for 3-month Treasury bills, and the prices for 6-month and 12-month Treasury bills could be implied, based on a fixed relation among the prices.)", "The “price parameters” could also be neutral parameters or indicators (e.g., in the nature of an index) of a price.", "Indeed, in a fifth preferred embodiment, bids include price parameters that may be a particularly useful form of neutral parameter.", "For example, an auctioneer may announce a starting price vector of (4.00, 4.50, 4.75) and an ending price vector of (8.00, 8.50, 8.75).", "If a bidder enters a price parameter of 25%, this may signify that the bidder is indicating a price which is 25% of the distance from the starting price to the ending price, i.e., an implied price vector of (5.00, 5.50, 5.75).", "In the fifth preferred embodiment, the present invention is: A computer implemented method for conducting an auction of at least one type of item among a plurality of bidders in a plurality of rounds, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing starting prices and ending prices for each type of item in a plurality of rounds, receiving bids, wherein a bid comprises a quantity for at least one type of item and a price parameter signifying a percentage of the distance from the established starting prices to the established ending prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for at least one type of item.", "In a sixth preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the fifth preferred embodiment.", "The system includes an establishing means, a receiving means, and a processing means.", "Various preferred embodiments of the present invention resolve a number of important limitations to the systems and methods for computer-implemented auctions in the art.", "Thus, the present invention offers the further technical effect of improving computer-implemented auctions, making them operate more efficiently.", "The present invention improves the ability of bidders to readily participate in the auction and to express their needs and preferences.", "The present invention improves the ability of the auctioneer to sell at higher prices the items being sold in the auction or to obtain at lower cost the items being procured in the auction.", "The present invention improves the efficiency of the auction process by enabling the auction to operate in shorter time, expending less computer time, fewer communications resources, and less bidder and auctioneer time.", "Finally, the present invention improves the allocative efficiency of the auction process by better allocating items to bidders who value them the highest (or, in a procurement auction, to bidders who can provide them at lowest cost).", "Certain constraints are desirable in order for the present invention to operate optimally and to reach an economically efficient outcome.", "In many auctions, it is desirable to impose constraints on bidders that require them to bid significantly on items early in the auction in order for them to be allowed to bid on items later in the auction.", "Otherwise, one may find oneself in a situation where bidders refrain from submitting serious bids until near the very end of the auction, defeating the purpose of a dynamic auction.", "One exemplary constraint is an activity rule which constrains a bidder not to increase his quantity, summed over all of the types of items within a group, from one round to the next.", "Another exemplary constraint is a more stringent activity rule which constrains a bidder not to increase his quantity, individually on each of the individual types of items, from one bid or one round to the next.", "A third exemplary constraint is a reduction rule which constrains a bidder not to decrease his quantity, for any single type of item, beyond the point where the sum of the quantities bid for this type of item by all bidders equals the sum of the quantities being auctioned.", "(If, in a given round, two or more bidders simultaneously attempt to decrease their quantities, for any single type of item, having the effect of reducing bids beyond the point where the sum of the quantities bid for this type of item by all bidders equals the sum of the quantities being auctioned, the auction procedure will resolve this discrepancy.", "For example, the auctioneer may honor these attempts to decrease in order of time priority, or may ration these simultaneous attempts to decrease in proportion to the attempted reductions.)", "The present invention includes treating the case of auctioning a set of items which includes two (or more) items that are neither identical nor perfect substitutes to one another.", "Henceforth, this will be described for short as a situation with “multiple types of items,” or simply “heterogeneous items” or “heterogeneous objects.” Often, but not always, the heterogeneous items auctioned together will bear some relationship to one another: for example, they may be securities issued by the same entity but with different durations to maturity; or contracts for the same goods or services but with different durations.", "One particularly salient use for the present invention is as follows.", "A national government or a private corporation may wish to simultaneously sell 3-month, 6-month and 12-month bonds or notes.", "More generally, these may be contracts for provision of any commodity; the contracts are for the same commodity, but covering different but possibly overlapping time periods.", "For the government's or corporation's purposes, the allocation of sales among the 3-month, 6-month and 12-month bonds or notes may not be critical; the main issue is that the total quantity of the bonds or notes of the three durations add up to the desired dollar figure.", "Meanwhile, the current “yield curve” on financial markets may have 6-month bonds or notes yielding interest rates 0.50% higher than 3-month bonds or notes, and the current “yield curve” on financial markets may have 12-month bonds or notes yielding interest rates 0.25% higher than 6-month bonds or notes.", "Under these circumstances, the government or corporation may utilize the present invention by beginning the auction with an (interest rate) vector of (6.00%, 6.50%, 6.75%) for the three respective durations.", "If the aggregate quantity of the bonds or notes desired by all the bidders at these interest rates is greater than the quantity of bonds or notes that the government or corporation wishes to sell, the government or corporation might then continue the auction by announcing a new vector of (5.99%, 6.49%, 6.74%) for the three respective durations.", "If the aggregate quantity of the bonds or notes desired by all the bidders at these interest rates is still greater than the quantity of bonds or notes that the government or corporation wishes to sell, the government or corporation might then continue the auction by announcing a new vector of (5.98%, 6.48%, 6.73%) for the three respective durations.", "The auction continues in this way, with the government or corporation always following the yield curve in setting.", "the relative interest rates for the various durations.", "Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the m types of items, from one round to the next.", "That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month bonds or notes that he demands, but his total quantity demanded of bonds or notes may not be increased as the interest rate goes down.", "A second use for the present invention is as follows.", "An electric utility may wish to sell contracts to provide a given quantity of electricity for 3-month, 6-month and 12-month durations.", "For the electric utility's purposes, the allocation of sales among the 3-month, 6-month and 12-month contracts may not be critical; the main issue may be that the total quantity of the contracts add up to a desired amount of electricity.", "Meanwhile, current energy prices may imply that a 6-month contract should be priced at $2 per-unit higher than a 3-month contract, and that a 12-month contract should be priced at $1 per unit higher than a 6-month contract.", "Under these circumstances, the electric utility may utilize the present invention by beginning the auction with a price vector of ($10.50, $12.50, $13.50) for the three respective durations.", "If the aggregate quantity of contracts desired by all the bidders at these prices is greater than the quantity of contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective durations.", "If the aggregate quantity of contracts desired by all the bidders at these prices is still greater than the quantity of contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.70, $12.70, $13.70) for the three respective durations.", "The auction continues in this way, with the electric utility always following the implied price differential in setting the relative prices for the various durations.", "Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the types of items within a group, from one round to the next.", "That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month contracts that he demands, but his total quantity demanded of contracts may not be increased as the price goes up.", "In addition, the present invention may also be used to auction two or more groups of types of items.", "For example, the previous electric utility example could be complicated by the presence of two groups of electricity contracts: base and peak.", "(Base-load quantity is produced all of the time while peak-load quantity is only produced at times of peak demand.)", "Within each group, there might be three durations of contracts.", "Thus, in total, there could be six contracts for sale: 3-month, 6-month and 12-month base contracts; and 3-month, 6-month and 12-month peak contracts.", "To continue the example, current energy prices may imply that a 6-month base contract should be priced at $2 per unit higher than a 3-month base contract, and that a 12-month base contract should be priced at $1 per unit higher than a 6-month base contract.", "Meanwhile, current energy prices may imply that a 6-month peak contract should be priced at $3 per unit higher than a 3-month peak contract, and that a 12-month peak contract should be priced at $2 per unit higher than a 6-month peak contract.", "Under these circumstances, the electric utility may utilize the present invention by beginning the auction with a price vector of ($10.50, $12.50, $13.50) for the three respective base durations and a price vector of ($26.50, $29.50, $31.50) for the three respective peak durations.", "If the aggregate quantity of base contracts desired by all the bidders at these prices is slightly greater than the quantity of base contracts that the electric utility wishes to sell, but the aggregate quantity of peak contracts desired by all the bidders at these prices is much greater than the quantity of peak contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective base durations and a price vector of ($29.20, $32.20, $34.20) for the three respective peak durations.", "If the aggregate quantity of base contracts desired by all the bidders at these prices is still slightly greater than the quantity of base contracts that the electric utility wishes to sell, and the aggregate quantity of peak contracts desired by all the bidders at these prices is still somewhat greater than the quantity of peak contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective base durations and a price vector of ($29.40, $32.40, $34.40) for the three respective peak durations.", "The auction continues in this way, with the electric utility always following the implied price differential in setting the relative prices for the various durations within a group.", "(However, between groups, the prices may go up at very different rates.)", "Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the types of items within a group, from one round to the next.", "That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month base contracts that he demands, but his total quantity demanded of base contracts may not be increased as the price goes up.", "Similarly, the bidder is allowed to change the mix of 3-month, 6-month and 12-month peak contracts that he demands, but his total quantity demanded of peak contracts may not be increased as the price goes up.", "However, in this exemplary embodiment, the bidder is not allowed to substitute any quantity between the base group and the peak group.", "In many of the preferred embodiments, the auction with two or more groups of types of items ends at the first moment in time that the aggregate quantity of items bid for by all the bidders within each group is less than or equal to the quantity of items being auctioned within each group.", "An auction in accordance with an embodiment of the present invention could also allow various improvements to the bidding rules.", "In the previous example of base contracts and peak contracts, it may be the case that even if the price of only one group goes up, a bidder may be permitted to reduce his demands in both product groups.", "For example, if the base price vector is held constant and the peak price vector goes up, a bidder may be permitted to reduce his quantity bid on both base and peak products.", "The reason for allowing this is that the base and peak product may be complements (loosely speaking, products that are useful together, such as left shoes and right shoes), so an increased price for one product could reduce a bidder's demand for both.", "Throughout this document, the terms “objects”, “items”, “units” and “goods” are used essentially interchangeably.", "The inventive system and method may be used both for tangible objects, such as real or personal property, and intangible items, such as telecommunications licenses or electric power.", "The inventive system and method may be used in auctions where the auctioneer is a seller, buyer or broker, the bidders are buyers, sellers or brokers, and for auction-like activities which cannot be interpreted as selling or buying.", "The inventive system and method may be used for items including, but not restricted to, the following: public-sector bonds, bills, notes, stocks, and other securities or derivatives; private-sector bonds, bills, notes, stocks, and other securities or derivatives; communication licenses and spectrum rights; clearing, relocation or other rights concerning encumbrances of spectrum licenses; electric power and other commodity items; rights for terminal, entry, exit or transmission capacities or other rights in gas pipeline systems; airport landing rights; emission allowances and pollution permits; and other goods, services, objects, items or other property, tangible or intangible.", "It may also be used for option contracts on any of the above.", "It may be used in initial public offerings, secondary offerings, and in secondary or resale markets.", "The present invention is useful for “reverse auctions” conducted by or for buyers to acquire various kinds of items or resources, “standard auctions” conducted by sellers in which items are offered for sale, and “exchanges” in which both buyers and sellers place bids.", "Although terms such as “items or quantities demanded” (by a bidder) and “demand curve” (of a bidder) are used to describe the present invention, the terms “items or quantities offered” (by a bidder) and “supply curve” (of a bidder) are equally applicable.", "In some cases, this is made explicit by the use of both terms, or by the use of the terms “items or quantities transacted” (by a bidder) and “transaction curve” (of a bidder).", "The term “items or quantities transacted” includes both “items or quantities demanded” and “items or quantities offered”.", "The term “bid” includes both offers to sell and offers to buy.", "The term “transaction curve” includes both “demand curve” and “supply curve”.", "Moreover, any references to “items or quantities being offered” includes both “items or quantities being sold” by the auctioneer, in the case this is a standard auction for selling items, as well as “items or quantities being bought or procured” by the auctioneer, in the case this is a reverse auction for buying items or procuring items.", "Moreover, while standard auctions to sell typically involve ascending prices and reverse auctions to buy typically involve descending prices, the present invention may utilize prices that ascend and/or descend.", "While an auction method following this description could be conducted manually, computerized implementation of the auction method on an auction system allows the auction to be conducted with all bidding information taken into account, while carefully controlling the degree to which the information itself is disclosed to the participants.", "In one embodiment, all bidding information is displayed to the bidders.", "In another embodiment, no bidding information is displayed to the bidders; only the results of the auction are displayed.", "A number of intermediate embodiments are also possible, in which some but not all bidding information is displayed to the bidders.", "For example, in one preferred embodiment, the auctioneer discloses only the aggregate quantity bid for each type of item in each round, as opposed to disclosing each individual bid.", "Computerized implementation of the auction also allows the auction to be conducted swiftly and reliably, even if bidders are not located on-site.", "The auctioneer can receive large numbers of bids, determine the auction outcome, and transmit the results to bidders with minimal labor and in a very short span of time.", "In particular, sophisticated dynamic auction methods become feasible with a computer implementation; it becomes practical to operate iterative auction procedures in which there may be ten or more auction rounds, yet bidders can participate from different parts of the globe and the entire process can conclude in a single day.", "The present invention comprises a computer that receives bids in a static or dynamic bidding process and assigns the items to bidders, and a method for receiving bids in a static or dynamic bidding process and assigning the items to bidders.", "In one embodiment, the invention comprises a Bidding Information Processor (BIP), a plurality of Bidding Terminals (BT's) communicatively coupled to the BIP, and an Auctioneer's Terminal (AT) communicatively coupled to the BIP.", "Bidders at the BT's enter bids in multiple rounds, and may observe displayed auction information.", "The BIP, BT's and AT communicate and process information in order to conduct an auction.", "The network used, if any, can be any system capable of providing the necessary communication to/from a Bidding Information Processor (BIP), a Bidding Terminal (BT), and an Auctioneer's Terminal (AT).", "The network may be a local or wide area network such as, for example, Ethernet, token ring, the Internet, the World Wide Web, the information superhighway, an intranet or a virtual private network, or alternatively a telephone system, either private or public, a facsimile system, an electronic mail system, or a wireless communications system, or combinations of the foregoing.", "The following patents and published applications are related to the present invention: Ausubel, Lawrence M., U.S. Pat.", "No.", "5,905,975, May 1999.Ausubel, Lawrence M., U.S. Pat.", "No.", "6,021,398, Feb. 2000.Ausubel, Lawrence M., U.S. Pat.", "No.", "6,026,383, Feb. 2000.Ausubel, Lawrence M., Application No.", "00304195.1 at the European Patent Office, May 2000.The following other references may be related to the present invention: Arrow, 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Weber, “A Theory of Auctions and Competitive Bidding, II.” Unpublished manuscript, Northwestern University, 1982.Nisan, Noam (1999), “Bidding and Allocation in Combinatorial Auctions,” working paper, www-cs-huji-ac-il.", "Onsale release, “Onsale Brings Thrill of Auctions and Bargain Hunting Online; Unique Internet retail service debuts with week-long charity auction for The Computer Museum in Boston”; Dialog Abstract: File 610, Acc#0489267; May 24, 1995.D.", "C. Parkes (1999), “iBundle: An Efficient Ascending Price Bundle Auction”, Proceedings ACM Conference on Electronic Commerce (EC-99), Denver, 148-157.Perry, Motty and Philip J. Reny (1999a), “An Ex-Post Efficient Auction,” Working Paper, Hebrew University and University of Chicago, September.", "Perry, Motty and Philip J. Reny (1999b), “An Ex-Post Efficient Multi-Unit Ascending Auction,” Working Paper, Hebrew University and University of Chicago, September.", "Plott, Charles (1997), “Laboratory Experimental Testbeds: Application to the PCS Auction,” Journal of Economics and Management Strategy, 6: 605-638.Rassenti, S. J., V. L. Smith, and R. L. Bulfin (1982), “A Combinatorial Auction Mechanism for Airport Time Slot Allocation,” Bell Journal of Economics 13: 402-417.M.", "H. Rothkopf, A. Pekec and R. M. Harstad (1998), “Computationally Manageable Combinatorial Auctions”, Management Science , Vol.", "44, No.", "8, 1131-1147.Rothkopf, Michael, Thomas Teisberg and Edward Kahn (1990), “Why Are Vickrey Auctions Rare?” Journal of Political Economy, 98: 94-109.Sholtz & Associates, LLC, “ACE Market Operations Guide,” Jan. 3, 1996, pp.", "1-5.Siegmann, “Nowhere to Go But Up.", "(Onsale CEO Jerry Kaplan) (PC Week Inside) (Inside People)” ; PC Week; v12 n42; pA5 (1); Oct. 23, 1995; Dialog: File 148, Acc#08222496.U.S.", "Department of the Treasury, U.S. Securities and Exchange Commission, and Board of Governors of the Federal Reserve System, Joint Report on the Government Securities Market , Washington, D.C.: U.S.G.P.O., January 1992.Vickrey, William (1961), “Counterspeculation, Auctions and Competitive Sealed Tenders,” Journal of Finance , March, 16(1), pp.", "8-37.Vickrey, William (1962), “Auctions and Bidding Games,” Recent Advances in Game Theory, Princeton: Princeton University Conference, 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"Provisional Patent Application, Ser.", "No.", "60/294,246 Filed: May 31, 2001.Ausubel, “SYSTEM AND METHOD FOR AN EFFICIENT DYNAMIC MULTI-UNIT AUCTION”, U.S. patent application, Ser.", "No.", "09/898,483 Filed: Jul.", "05, 2001.FIELD OF THE INVENTION The present invention relates to improving computer-implemented auctions and, more particularly, to computer implementation of an auction in which multiple types of items are auctioned together.", "BACKGROUND OF THE INVENTION Sellers and buyers of commodities, financial securities, and other goods and services are increasingly turning to auctions to assist in the sale or purchase of a wide range of items.", "Auctions are useful for selling a unique item (e.g., a painting), a single type of item available in multiple units (e.g., 10 tons of gold), and multiple types of items available in multiple units (e.g., 10 tons of gold and 20 tons of silver).", "And just as auctions are useful for efficiently selling items at maximum price, they are also useful for efficiently procuring items at minimum cost.", "Auction formats in the art tend generally to be of the sealed-bid or ascending-bid variety.", "In standard sealed-bid auctions, bidders—in one single bidding round—simultaneously and independently submit bids to the auctioneer, who then determines the auction outcome.", "In standard ascending-bid auctions, bidders—in a dynamic bidding process—submit bids in real time until no more bids are forthcoming.", "Sealed-bid auction formats offer the advantage of speed of the auction process.", "Ascending-bid auction formats offer the advantage that there is feedback among participants' bids, which tends to result in more aggressive bidding and in more efficient auction outcomes.", "When auction systems and methods in the art are used to sell (or buy) multiple types of items, the auctioneer generally specifies an available supply (or demand) for each type of item.", "For example, the auctioneer announces a supply of 10 tons of gold and 20 tons of silver, and then solicits bids, in a sealed-bid or ascending-bid procedure, in order to obtain market-clearing prices for each of the two types of commodities.", "However, consider a situation where the types of items are related, for example, contracts for provision of a commodity covering different time periods.", "For example, a government may wish to sell three types of financial securities: 3-month Treasury bills; 6-month Treasury bills; and 12-month Treasury bills.", "The government's only constraint may be that it needs to sell $50 billion worth of new debt securities at this auction, since this is the quantity of old debt securities that are maturing in the current calendar quarter.", "However, the government may have no preference as to how the required $50 billion is divided among 3-month bills, 6-month bills, and 12-month bills, respectively, and the government may simply wish to “let the market decide” the division among the various durations Auction systems and methods in the art provide effective ways for the government to sell bonds with a fixed division among the various durations.", "However, new and better systems and methods can be devised for “letting the market decide” the division among the various durations.", "The same problem also occurs in real life in the sale of contracts for electricity and all kinds of other commodities, as well as in the procurement of related items.", "In several preferred embodiments, the present invention provides a system and method for the government in the above example to simply auction $50 billion of Treasury bills at the lowest possible cost (relative to the market's yield curve), without imposing a particular division among the various durations.", "SUMMARY OF THE INVENTION The present invention is an improved system and method for a computer-implemented auction, particularly for a computer-implemented auction in which multiple types of items are auctioned together.", "In several embodiments, the present invention provides a system and method to conduct an auction of a group of related types of items, without imposing a particular division of supply or demand among the individual types of items within the group.", "In other embodiments, the present invention provides a system and method to conduct an auction of a group of related types of items, without imposing a particular division of supply or demand on some of the individual types of items within the group.", "Instead, in some embodiments, the present invention provides a means or method for establishing prices for the types of items within a group, wherein the prices maintain a relationship.", "In other embodiments, the present invention provides a means or method for implying prices from price parameters in the bids received from bidders, based on a relation among the prices for the types of items within a group.", "In some embodiments, market clearing is defined by the condition that the aggregate quantity bid for all types of items within a group is less than or equal to the available quantity of all types of items within the group.", "The division among the types of items within the group is thus determined flexibly, based on the bids at the associated prices.", "In other embodiments, market clearing is defined by the condition that the quantity bid for one selected type of item within a group is less than or equal to the available quantity of the selected type of item.", "The quantities of the other types of items within the group are thus determined flexibly, based on the bids at the associated prices.", "In a first preferred embodiment, the present invention is: A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing prices for the plurality of types of items, wherein the prices maintain a relationship, receiving bids associated with the established prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for the plurality of types of items.", "In a second preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the first preferred embodiment.", "The system includes an establishing means, a receiving means, and a processing means.", "In both the first and second preferred embodiments, the relationship or relation among the prices may involve a schedule or algorithm.", "In the event that the relationship or relation is based on a schedule, determining prices may involve looking up and possibly interpolating numbers that are contained in a table.", "Alternatively the relationship or relation may be represented as an algorithm or a formula that provides a relationship among the prices.", "The data table may reflect a market beyond the auction (for example, it may reflect price differentials in a separate commodities market or exchange) and the data table may change during the course of the auction (for example, if price differentials change in the separate commodities market or exchange, or if a significant news event occurs during the course of the auction).", "Similarly, the data set to which the algorithm or formula are applied may reflect a market beyond the auction and the data set may change during the course of the auction.", "In a third preferred embodiment, the present invention is: A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: receiving bids comprising a quantity parameter for at least one of the types of items in the auction and a price parameter, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, wherein the processing includes implying prices from price parameters in the received bids based on a fixed relation among the prices of the plurality of types of items.", "In a fourth preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the third preferred embodiment.", "The system includes a receiving means and a processing means.", "In both the third and fourth preferred embodiments, the “price parameters” in the received bids that are used for “implying prices” could be prices for one or more selected types of items.", "(For example, the price parameter could be an actual price for 3-month Treasury bills, and the prices for 6-month and 12-month Treasury bills could be implied, based on a fixed relation among the prices.)", "The “price parameters” could also be neutral parameters or indicators (e.g., in the nature of an index) of a price.", "Indeed, in a fifth preferred embodiment, bids include price parameters that may be a particularly useful form of neutral parameter.", "For example, an auctioneer may announce a starting price vector of (4.00, 4.50, 4.75) and an ending price vector of (8.00, 8.50, 8.75).", "If a bidder enters a price parameter of 25%, this may signify that the bidder is indicating a price which is 25% of the distance from the starting price to the ending price, i.e., an implied price vector of (5.00, 5.50, 5.75).", "In the fifth preferred embodiment, the present invention is: A computer implemented method for conducting an auction of at least one type of item among a plurality of bidders in a plurality of rounds, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing starting prices and ending prices for each type of item in a plurality of rounds, receiving bids, wherein a bid comprises a quantity for at least one type of item and a price parameter signifying a percentage of the distance from the established starting prices to the established ending prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for at least one type of item.", "In a sixth preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the fifth preferred embodiment.", "The system includes an establishing means, a receiving means, and a processing means.", "Various preferred embodiments of the present invention resolve a number of important limitations to the systems and methods for computer-implemented auctions in the art.", "Thus, the present invention offers the further technical effect of improving computer-implemented auctions, making them operate more efficiently.", "The present invention improves the ability of bidders to readily participate in the auction and to express their needs and preferences.", "The present invention improves the ability of the auctioneer to sell at higher prices the items being sold in the auction or to obtain at lower cost the items being procured in the auction.", "The present invention improves the efficiency of the auction process by enabling the auction to operate in shorter time, expending less computer time, fewer communications resources, and less bidder and auctioneer time.", "Finally, the present invention improves the allocative efficiency of the auction process by better allocating items to bidders who value them the highest (or, in a procurement auction, to bidders who can provide them at lowest cost).", "Certain constraints are desirable in order for the present invention to operate optimally and to reach an economically efficient outcome.", "In many auctions, it is desirable to impose constraints on bidders that require them to bid significantly on items early in the auction in order for them to be allowed to bid on items later in the auction.", "Otherwise, one may find oneself in a situation where bidders refrain from submitting serious bids until near the very end of the auction, defeating the purpose of a dynamic auction.", "One exemplary constraint is an activity rule which constrains a bidder not to increase his quantity, summed over all of the types of items within a group, from one round to the next.", "Another exemplary constraint is a more stringent activity rule which constrains a bidder not to increase his quantity, individually on each of the individual types of items, from one bid or one round to the next.", "A third exemplary constraint is a reduction rule which constrains a bidder not to decrease his quantity, for any single type of item, beyond the point where the sum of the quantities bid for this type of item by all bidders equals the sum of the quantities being auctioned.", "(If, in a given round, two or more bidders simultaneously attempt to decrease their quantities, for any single type of item, having the effect of reducing bids beyond the point where the sum of the quantities bid for this type of item by all bidders equals the sum of the quantities being auctioned, the auction procedure will resolve this discrepancy.", "For example, the auctioneer may honor these attempts to decrease in order of time priority, or may ration these simultaneous attempts to decrease in proportion to the attempted reductions.)", "The present invention includes treating the case of auctioning a set of items which includes two (or more) items that are neither identical nor perfect substitutes to one another.", "Henceforth, this will be described for short as a situation with “multiple types of items,” or simply “heterogeneous items” or “heterogeneous objects.” Often, but not always, the heterogeneous items auctioned together will bear some relationship to one another: for example, they may be securities issued by the same entity but with different durations to maturity; or contracts for the same goods or services but with different durations.", "One particularly salient use for the present invention is as follows.", "A national government or a private corporation may wish to simultaneously sell 3-month, 6-month and 12-month bonds or notes.", "More generally, these may be contracts for provision of any commodity; the contracts are for the same commodity, but covering different but possibly overlapping time periods.", "For the government's or corporation's purposes, the allocation of sales among the 3-month, 6-month and 12-month bonds or notes may not be critical; the main issue is that the total quantity of the bonds or notes of the three durations add up to the desired dollar figure.", "Meanwhile, the current “yield curve” on financial markets may have 6-month bonds or notes yielding interest rates 0.50% higher than 3-month bonds or notes, and the current “yield curve” on financial markets may have 12-month bonds or notes yielding interest rates 0.25% higher than 6-month bonds or notes.", "Under these circumstances, the government or corporation may utilize the present invention by beginning the auction with an (interest rate) vector of (6.00%, 6.50%, 6.75%) for the three respective durations.", "If the aggregate quantity of the bonds or notes desired by all the bidders at these interest rates is greater than the quantity of bonds or notes that the government or corporation wishes to sell, the government or corporation might then continue the auction by announcing a new vector of (5.99%, 6.49%, 6.74%) for the three respective durations.", "If the aggregate quantity of the bonds or notes desired by all the bidders at these interest rates is still greater than the quantity of bonds or notes that the government or corporation wishes to sell, the government or corporation might then continue the auction by announcing a new vector of (5.98%, 6.48%, 6.73%) for the three respective durations.", "The auction continues in this way, with the government or corporation always following the yield curve in setting.", "the relative interest rates for the various durations.", "Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the m types of items, from one round to the next.", "That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month bonds or notes that he demands, but his total quantity demanded of bonds or notes may not be increased as the interest rate goes down.", "A second use for the present invention is as follows.", "An electric utility may wish to sell contracts to provide a given quantity of electricity for 3-month, 6-month and 12-month durations.", "For the electric utility's purposes, the allocation of sales among the 3-month, 6-month and 12-month contracts may not be critical; the main issue may be that the total quantity of the contracts add up to a desired amount of electricity.", "Meanwhile, current energy prices may imply that a 6-month contract should be priced at $2 per-unit higher than a 3-month contract, and that a 12-month contract should be priced at $1 per unit higher than a 6-month contract.", "Under these circumstances, the electric utility may utilize the present invention by beginning the auction with a price vector of ($10.50, $12.50, $13.50) for the three respective durations.", "If the aggregate quantity of contracts desired by all the bidders at these prices is greater than the quantity of contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective durations.", "If the aggregate quantity of contracts desired by all the bidders at these prices is still greater than the quantity of contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.70, $12.70, $13.70) for the three respective durations.", "The auction continues in this way, with the electric utility always following the implied price differential in setting the relative prices for the various durations.", "Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the types of items within a group, from one round to the next.", "That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month contracts that he demands, but his total quantity demanded of contracts may not be increased as the price goes up.", "In addition, the present invention may also be used to auction two or more groups of types of items.", "For example, the previous electric utility example could be complicated by the presence of two groups of electricity contracts: base and peak.", "(Base-load quantity is produced all of the time while peak-load quantity is only produced at times of peak demand.)", "Within each group, there might be three durations of contracts.", "Thus, in total, there could be six contracts for sale: 3-month, 6-month and 12-month base contracts; and 3-month, 6-month and 12-month peak contracts.", "To continue the example, current energy prices may imply that a 6-month base contract should be priced at $2 per unit higher than a 3-month base contract, and that a 12-month base contract should be priced at $1 per unit higher than a 6-month base contract.", "Meanwhile, current energy prices may imply that a 6-month peak contract should be priced at $3 per unit higher than a 3-month peak contract, and that a 12-month peak contract should be priced at $2 per unit higher than a 6-month peak contract.", "Under these circumstances, the electric utility may utilize the present invention by beginning the auction with a price vector of ($10.50, $12.50, $13.50) for the three respective base durations and a price vector of ($26.50, $29.50, $31.50) for the three respective peak durations.", "If the aggregate quantity of base contracts desired by all the bidders at these prices is slightly greater than the quantity of base contracts that the electric utility wishes to sell, but the aggregate quantity of peak contracts desired by all the bidders at these prices is much greater than the quantity of peak contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective base durations and a price vector of ($29.20, $32.20, $34.20) for the three respective peak durations.", "If the aggregate quantity of base contracts desired by all the bidders at these prices is still slightly greater than the quantity of base contracts that the electric utility wishes to sell, and the aggregate quantity of peak contracts desired by all the bidders at these prices is still somewhat greater than the quantity of peak contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective base durations and a price vector of ($29.40, $32.40, $34.40) for the three respective peak durations.", "The auction continues in this way, with the electric utility always following the implied price differential in setting the relative prices for the various durations within a group.", "(However, between groups, the prices may go up at very different rates.)", "Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the types of items within a group, from one round to the next.", "That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month base contracts that he demands, but his total quantity demanded of base contracts may not be increased as the price goes up.", "Similarly, the bidder is allowed to change the mix of 3-month, 6-month and 12-month peak contracts that he demands, but his total quantity demanded of peak contracts may not be increased as the price goes up.", "However, in this exemplary embodiment, the bidder is not allowed to substitute any quantity between the base group and the peak group.", "In many of the preferred embodiments, the auction with two or more groups of types of items ends at the first moment in time that the aggregate quantity of items bid for by all the bidders within each group is less than or equal to the quantity of items being auctioned within each group.", "An auction in accordance with an embodiment of the present invention could also allow various improvements to the bidding rules.", "In the previous example of base contracts and peak contracts, it may be the case that even if the price of only one group goes up, a bidder may be permitted to reduce his demands in both product groups.", "For example, if the base price vector is held constant and the peak price vector goes up, a bidder may be permitted to reduce his quantity bid on both base and peak products.", "The reason for allowing this is that the base and peak product may be complements (loosely speaking, products that are useful together, such as left shoes and right shoes), so an increased price for one product could reduce a bidder's demand for both.", "Throughout this document, the terms “objects”, “items”, “units” and “goods” are used essentially interchangeably.", "The inventive system and method may be used both for tangible objects, such as real or personal property, and intangible items, such as telecommunications licenses or electric power.", "The inventive system and method may be used in auctions where the auctioneer is a seller, buyer or broker, the bidders are buyers, sellers or brokers, and for auction-like activities which cannot be interpreted as selling or buying.", "The inventive system and method may be used for items including, but not restricted to, the following: public-sector bonds, bills, notes, stocks, and other securities or derivatives; private-sector bonds, bills, notes, stocks, and other securities or derivatives; communication licenses and spectrum rights; clearing, relocation or other rights concerning encumbrances of spectrum licenses; electric power and other commodity items; rights for terminal, entry, exit or transmission capacities or other rights in gas pipeline systems; airport landing rights; emission allowances and pollution permits; and other goods, services, objects, items or other property, tangible or intangible.", "It may also be used for option contracts on any of the above.", "It may be used in initial public offerings, secondary offerings, and in secondary or resale markets.", "The present invention is useful for “reverse auctions” conducted by or for buyers to acquire various kinds of items or resources, “standard auctions” conducted by sellers in which items are offered for sale, and “exchanges” in which both buyers and sellers place bids.", "Although terms such as “items or quantities demanded” (by a bidder) and “demand curve” (of a bidder) are used to describe the present invention, the terms “items or quantities offered” (by a bidder) and “supply curve” (of a bidder) are equally applicable.", "In some cases, this is made explicit by the use of both terms, or by the use of the terms “items or quantities transacted” (by a bidder) and “transaction curve” (of a bidder).", "The term “items or quantities transacted” includes both “items or quantities demanded” and “items or quantities offered”.", "The term “bid” includes both offers to sell and offers to buy.", "The term “transaction curve” includes both “demand curve” and “supply curve”.", "Moreover, any references to “items or quantities being offered” includes both “items or quantities being sold” by the auctioneer, in the case this is a standard auction for selling items, as well as “items or quantities being bought or procured” by the auctioneer, in the case this is a reverse auction for buying items or procuring items.", "Moreover, while standard auctions to sell typically involve ascending prices and reverse auctions to buy typically involve descending prices, the present invention may utilize prices that ascend and/or descend.", "While an auction method following this description could be conducted manually, computerized implementation of the auction method on an auction system allows the auction to be conducted with all bidding information taken into account, while carefully controlling the degree to which the information itself is disclosed to the participants.", "In one embodiment, all bidding information is displayed to the bidders.", "In another embodiment, no bidding information is displayed to the bidders; only the results of the auction are displayed.", "A number of intermediate embodiments are also possible, in which some but not all bidding information is displayed to the bidders.", "For example, in one preferred embodiment, the auctioneer discloses only the aggregate quantity bid for each type of item in each round, as opposed to disclosing each individual bid.", "Computerized implementation of the auction also allows the auction to be conducted swiftly and reliably, even if bidders are not located on-site.", "The auctioneer can receive large numbers of bids, determine the auction outcome, and transmit the results to bidders with minimal labor and in a very short span of time.", "In particular, sophisticated dynamic auction methods become feasible with a computer implementation; it becomes practical to operate iterative auction procedures in which there may be ten or more auction rounds, yet bidders can participate from different parts of the globe and the entire process can conclude in a single day.", "The present invention comprises a computer that receives bids in a static or dynamic bidding process and assigns the items to bidders, and a method for receiving bids in a static or dynamic bidding process and assigning the items to bidders.", "In one embodiment, the invention comprises a Bidding Information Processor (BIP), a plurality of Bidding Terminals (BT's) communicatively coupled to the BIP, and an Auctioneer's Terminal (AT) communicatively coupled to the BIP.", "Bidders at the BT's enter bids in multiple rounds, and may observe displayed auction information.", "The BIP, BT's and AT communicate and process information in order to conduct an auction.", "The network used, if any, can be any system capable of providing the necessary communication to/from a Bidding Information Processor (BIP), a Bidding Terminal (BT), and an Auctioneer's Terminal (AT).", "The network may be a local or wide area network such as, for example, Ethernet, token ring, the Internet, the World Wide Web, the information superhighway, an intranet or a virtual private network, or alternatively a telephone system, either private or public, a facsimile system, an electronic mail system, or a wireless communications system, or combinations of the foregoing.", "The following patents and published applications are related to the present invention: Ausubel, Lawrence M., U.S. Pat.", "No.", "5,905,975, May 1999.Ausubel, Lawrence M., U.S. Pat.", "No.", "6,021,398, Feb. 2000.Ausubel, Lawrence M., U.S. Pat.", "No.", "6,026,383, Feb. 2000.Ausubel, Lawrence M., Application No.", "00304195.1 at the European Patent Office, May 2000.The following other references may be related to the present invention: Arrow, Kenneth, H. 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1962, pp.", "15-29.Vickrey, William (1976), “Auctions, Markets, and Optimal Allocation,” Bidding and Auctioning for Procurement and Allocation, New York: New York University Press 1976, pp.", "13-20.Weber, Robert J.", "(1983), “Multiple-Object Auctions,” Auctions, Bidding, and Contracting: Uses and Theory, New York: New York University Press, 1983, pp.", "165-191.Weber, Robert J.", "(1997), “Making More from Less: Strategic Demand Reduction in the FCC Spectrum Auctions,” Journal of Economics and Management Strategy, 6:529-548.Williams, Steven (1999), “A Characterization of Efficient, Bayesian Incentive Compatible Mechanisms,” Economic Theory 14: 155-180.Wilson, Robert (1979), “Auctions of Shares,” Quarterly Journal of Economics, vol.", "94, 1979, pp.675-689.BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a graphical depiction of the architecture of an exemplary computer system in accordance with an embodiment of the invention; FIG.", "2 is a graphical depiction of another exemplary computer system in accordance with an embodiment of the invention; FIG.", "3 is a detail of one element of the computer system of FIG.", "2; FIG.", "4 is a flow diagram of an exemplary auction process in accordance with one embodiment of the invention; FIGS.", "5a and 5b are flow diagrams illustrating, in greater detail, elements of the flow diagram of FIG.", "4; FIGS.", "6a and 6b are flow diagrams illustrating, in greater detail, elements of the flow diagram of FIG.", "4; FIG.", "7 is a flow diagram illustrating, in greater detail, an element of the flow diagram of FIG.", "4; and FIGS.", "8a, 8b and 8c are flow diagrams illustrating, in greater detail, elements of the flow diagram of FIG.", "4.DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Overall Structure of Auction System Earlier auction methods and systems are described in U.S. Pat.", "Nos.", "5,905,975, 6,021,398 and 6,026,383.The following description will detail the flow of the novel features of the preferred embodiments of the present method and system for an auction of multiple types of items.", "Before describing the auction process in detail, reference is made to FIG.", "1 to describe the architecture of an exemplary computer system in accordance with an embodiment of the present invention.", "In the graphical depiction of FIG.", "1, the computer system consists of multiple bidder and auctioneer computers or terminals 20a-n and 30 communicating with the server (or auction computer) 10 over a network 40.The computers or terminals 20a-n are employed by bidders, the computer or terminal 30 is employed by the auctioneer, and the server 10 is the auction computer.", "The server 10 consists of a CPU 11, memory 12, a data storage device 13, a communications interface 14, a clock 15, an operating system 16, and an auction program 17.In one embodiment, the system architecture is as a client-server system: the auction computer is a server; and the bidder and auctioneer computers are clients.", "FIG.", "2 is another graphical depiction of an exemplary computer system in accordance with an embodiment of the present invention.", "The auction system of FIG.", "2 includes an auction computer 60 (sometimes also referred to as a Bidding Information Processor or BIP), a plurality of user systems 70a, 70b and so on (sometimes also referred to as Bidder Terminal or BT), each user system 70a-n representing an individual bidder, and a user system 80 (sometimes also referred to as an Auctioneer Terminal or AT).", "The systems 60, 70a-n, and 80 communicate over a network 90.The network represents any system capable of providing the necessary communication to/from BIP, BT, and AT.", "The network may be a local or wide area network such as, for example, Ethernet, token ring, the Internet, the World Wide Web, the information superhighway, an intranet or a virtual private network, or alternatively a telephone system, either private or public, a facsimile system, an electronic mail system, or a wireless communications system.", "Each of the systems 60, 70a-n, and 80 may include a typical user interface 65, 75a-n, 85 for input/output which may include a conventional keyboard, display, and other input/output devices.", "Within each of the systems, the user interface (65, 75a-n, 85) is coupled to a network interface (64, 74a-n, 84), which in turn communicates via the network 90.Both the user interface and network interface connect, at each system, to a CPU (62, 72a-n, 82).", "Each system includes a memory (66, 76a-n, 86).", "The BIP 60 also includes a clock 61 and a data storage device 63, which will ordinarily contain a database.", "(However, in some embodiments the database might instead be stored in memory 66.)", "The memory 66 of the BIP 60 can further be broken down into a program 67, data 68 and an operating system 69.The memory (76a-n, 86) of the BT's 70a-n and the AT 80 may include a web browser (for example, Internet Explorer or Netscape) (79, 89) or other general-purpose software, but not necessarily any computer program specific to the auction process.", "In each system the CPU (62, 72a-n, 82) represents a source of intelligence when executing instructions from the memory (66, 76a-n, 86) so that appropriate input/output operations via the user interface and the network interface take place as is conventional in the art.", "The particular steps used in implementing the inventive auction system and method are described in more detail below.", "In one embodiment, each of the user systems is a personal computer or workstation.", "FIG.", "3 is a more detailed illustration of an exemplary BIP 60 showing details of the database.", "As discussed for FIG.", "2, the database is ordinarily stored on a data storage device 63, although in some embodiments it might instead be stored in memory 66.As depicted in FIG.", "3, the database includes provision for creating, storing, and retrieving records representing Items in the Auction 63-1, Status of the Items in the Auction 63-2, Auction Timetable 63-3, Current Price(s) 63-4, List of Bidder ID's 63-5, List of Passwords 63-6, Bidding History 63-7, and Constraints on Bids 63-8.The particular set of data required for any particular auction and the format of that datum or data (such as scalar, vector, list, etc.)", "is more particularly specified by the detailed description of that auction.", "Definitions Concerning “Types” of Items and “Groups” of Types of Items Many of the most useful embodiments of the present invention apply in situations where an entity wishes to sell or buy multiple “types” of items or commodities.", "Furthermore, related types of items may be usefully organized as “groups” of items.", "In order to describe the preferred embodiments, it will be helpful for us to define some terminology.", "DEFINITION 1: A type of item comprises a (nonempty) subset of the set of all items being auctioned, such that any two items within the same type are identical items or close substitutes.", "Meanwhile, types are defined so that any two items of different types exhibit significant differences in time, location or any other product characteristics.", "Typically, there are multiple units of each type of item.", "In what follows, we will assume that m (m≧1) types of items are being auctioned, and that there are n (n≧1) bidders participating in the auction.", "Whenever we state that there is a “plurality of types of items,” we are restricting attention to the case where m≧2.The items in the auction may also be said to be “homogeneous” in the case where m=1 and to be “heterogeneous” in the case where m≧2.Whenever we state that there is a “plurality of bidders,” we are restricting attention to the case where n≧2.DEFINITION 2: A group G of types comprises a (nonempty) subset of {1, .", ".", ".", ", m}, the set of all types of items.", "In other words, a group is one or more types of items that are usefully treated together.", "Typically, the reason that two types of items are included in the same group is that they are related, for example, they may be contracts for provision of the same commodity, covering different but overlapping time periods.", "In what follows, we will assume that the items in the auction are organized so that there are h (h≧1) groups of types of items.", "There is no requirement that each group contains the same number of types of items, but this often happens to be the case.", "Whenever we need notation indicating the number of types of items contained in the exemplary group G, our notation will be that there are g (g≧1) types of items in group G. In some of the preferred embodiments of the present invention, the prices for the various types of items within the same group are established so as to maintain a relationship according to a schedule.", "In others of the preferred embodiments of the present invention, a bidder submits a bid that includes the price for one type of item within a group, and a fixed relation among these prices implies the prices of the other types of item within the same group.", "In others of the preferred embodiments of the present invention, a bidder submits a bid that includes a one-dimensional price parameter, and this price parameter implies prices for all of the types of items within the group based on a fixed relation among the prices of the types of items.", "Some examples of “types” and “groups,” in situations where there may be significant commercial possibilities for embodiments of the present invention, include the following: Treasury bills or other securities: A government or central bank may wish to auction 3-month, 6-month and 12-month Treasury securities (for example, with the same starting date) together.", "Thus, there is one group of types of items (h=1), and it contains three types of items (g=3).", "In total, there are three types of items (m=3).", "Electricity contracts: An electric generating company may wish to simultaneously auction some forward contracts or options contracts for base-load and peak-load electricity generation, with durations of 2 months, 3 months, 6 months, 12 months, 24 months and 36 months, respectively.", "Thus, there are two groups of types of items (h=2), each containing six types of items (g=6).", "In total there are 2×6=12 types of items (m=12).", "Two unrelated, heterogeneous consumer commodities (e.g., apples and oranges).", "There are two groups of types of items (h=2), each containing just one type of item (g=1), for a total of two types of items (m=2).", "An auction in accordance with an embodiment of the present invention proceeds as follows.", "First, the auctioneer (i.e., the auctioneer terminal) determines a starting price vector, (P1, .", ".", ".", ",Pm), and transmits it to the bidding information processor, which in turn transmits it to bidders (i.e., bidder terminals).", "Second, a bidder responds with a bid vector indicating the quantity of each respective type of item that the bidder wishes to transact at the current price vector.", "Let the bidders be superscripted by i, where (i=1, .", ".", ".", ", n).", "The bid vector for bidder i is denoted by (Q1i, .", ".", ".", ",Qmi).", "The following definitions are helpful in describing the process associated with a first embodiment of the present invention.", "Other Definitions.", "The available quantities may, in principle, be specified for each group of types of items or for each type of items.", "In the text that follows, we will usually specify the available quantity for the group.", "The available quantity for group G will be denoted {overscore (Q)}G and this refers, in the case of an auction to sell, to the overall quantity of items in group G to be offered for sale in the auction or, in the case of an auction to buy (i.e., a procurement auction or a “reverse auction”), to the overall quantity of items in group G to be bought in the auction.", "The vector of available quantities for all groups will be denoted ({overscore (Q)}1, .", ".", ".", ", {overscore (Q)}h) Optionally, the available quantities may be allowed to depend on the prices, or otherwise be contingent on the progress of the auction.", "The current prices comprise a vector, (P1, .", ".", ".", ",Pm), whose components represent the prices for the m respective types of items.", "The current bid of bidder i comprises a vector, (Q1i, .", ".", ".", ",Qmi), whose components represent the quantities that bidder i is willing to buy (in the case of an auction to sell) or to sell (in the case of an auction to buy) at the current prices for the m respective types of items.", "The current bids comprise the collection of vectors, {Q1i, .", ".", ".", ",Qmi}i=1n consisting of the current bid of bidder i for every bidder (i=1.. .", ", n) in the auction.", "The bidding history comprises the current prices and the current bids associated with the present time and all earlier times in the current auction.", "A clock auction is a dynamic auction procedure whereby: the auctioneer announces the current prices to bidders; the bidders respond with current bids; the auctioneer determines whether the auction should continue based on the bidding history; the auctioneer updates the current prices based on the bidding history and the process repeats, if it is determined that the auction should continue; and the auctioneer allocates the items among the bidders and assesses payments among the bidders based on the bidding history, if it is determined that the auction should not continue.", "Observe that a “clock auction” differs from a standard ascending-bid electronic auction in the following important sense.", "In standard ascending-bid electronic auctions—such as in the Federal Communications Commission auctions for radio communications spectrum or in eBay auctions—the bidders name prices (and, perhaps also, quantities) that they propose to pay for the items being auctioned, in an iterative process.", "In a standard clock auction, the auctioneer sets the pace for price increases, and bidders respond only with quantities in an iterative process.", "(however, in the discussion of Intra-Round Bids, below, it will be seen that there still may be a role for bidders naming prices—to a limited extent—in a clock auction.)", "FIG.", "4 is a flow diagram of a clock auction in accordance with one embodiment of the present invention.", "The process starts with step 102, in which memory locations of a computer are initialized.", "In one preferred embodiment, the appropriate memory locations of the bidding information processor (auction computer) are initialized with information such as the types of items in the auction, the available quantity of each type of item in the auction, the data table or algorithm reflecting the “yield curve” schedule, an initial price parameter, an auction timetable, a list of bidder ID's, and a list of passwords.", "In step 104, a computer establishes the initial prices (P1, .", ".", ".", ",Pm).", "One preferred embodiment of the process of step 104 is shown in greater detail in FIG.", "5b.", "The process proceeds to step 106, in which a computer outputs auction information, including the current price vector (P1, .", ".", ".", ",Pm).", "In one preferred embodiment, the bidding information processor outputs the auction information through its network interface and transmits it via the network.", "The bidder terminals then receive the auction information through their network interfaces and display the information to bidders through their user interfaces.", "In step 108, a computer receives bids (Q1i, .", ".", ".", ",Qmi) from bidders.", "In one preferred embodiment, a bidder inputs his bids through the user interface of the bidder terminal, which then outputs the auction information through its network interface and transmits it via the network.", "The bidding information processor then receives the bids through its network interface for use in the next step.", "One preferred embodiment of the process of step 108, utilizing Intra-Round Bids, is shown in greater detail in FIG.", "8a.", "In step 110, a computer applies constraints, if any, to the received bids, and enters only those bids that satisfy said constraints.", "This process is illustrated in greater detail in FIGS.", "6a and 6b.", "In one preferred embodiment, the constraints are applied at the bidding information processor, although they may also easily be applied at the bidder terminals.", "In step 112, a computer processes the received bids and determines whether the auction should continue.", "Exemplary processes of step 112 are illustrated in greater detail in FIGS.", "7 and 8b.", "In some preferred embodiments, this determination occurs at the bidding information processor.", "If the auction should continue, the process goes to step 114, in which a computer establishes an updated price vector (P1, .", ".", ".", ",Pm).", "One preferred embodiment of the process of step 114 is shown in greater detail in FIG.", "5a.", "Then, at step 116, a computer updates other auction information, if any.", "In one preferred embodiment, the bidding information processor automatically generates a suggested revised price vector, outputs the suggested revised price vector through its network interface, and transmits it via the network.", "The auctioneer terminal then receives the suggested revised price vector through its network interface and displays it to the auctioneer through its user interface.", "The auctioneer either approves or modifies the revised price vector through the user interface of the auctioneer terminal, which then outputs the revised price vector through its network interface and transmits it via the network.", "The bidding information processor then receives the revised price vector through its network interface for use in subsequent steps.", "The process then loops to step 106.If the auction should not continue, the process goes to step 118, in which a computer outputs a final message, including the allocation of items among bidders and the payments of the bidders.", "In one preferred embodiment, the bidding information processor takes the allocation of items among bidders to be their final bids and takes the payment of each bidder i to be the dot product of the final price vector and bidder i's final quantity vector: (P1, .", ".", ".", ",Pm·(Q1i, .", ".", ".", ", Qmi).", "The bidding information processor outputs the allocation and payment outcome through its network interface and transmits it via the network.", "The bidder terminals and auctioneer terminal then receive the allocation and payment outcome through their network interfaces and display the information to bidders and the auctioneer through their user interfaces.", "One preferred embodiment of the process of step 118, utilizing Intra-Round Bids, is shown in greater detail in FIG.", "8c.", "The process then ends.", "Embodiments of the Invention Concerned with Establishing or Implying Prices FIG.", "5a is a flow diagram illustrating an exemplary process by which updated prices are established.", "Thus, FIG.", "5a illustrates, in greater detail, step 114 of FIG.", "4.The process begins with step 114-1, in which a computer selects a group G of types of items not yet considered.", "In step 114-2, a computer calculates the excess demand ZG for group G, which is given by: Z G = - Q _ G + ∑ i = 1 n ⁢ ∑ k ∈ G ⁢ Q k i .", "Thus, ZG represents the amount by which the bids for all types of items in group G, by all bidders, exceeds the available quantity {overscore (Q)}G of all items in group G. In step 114-3, a computer selects a type of item included in group G, whose “old” price is denoted by {overscore (P)}1G.", "In step 114-4, a computer sets a “new” price P1G, where P1G equals the {overscore (P)}1G plus an increment Δ.", "The increment Δ is given by Δ=c ZG, where c is a positive constant and ZG was computed in step 114-2.", "(In other embodiments, other rules for incrementing P1 are used.)", "In step 114-5, a computer looks up the new P1G in a data table reflecting the “yield curve” schedule.", "The following Table 1 is an example of such a data table for a group G containing three types of items (corresponding to the base group of the earlier example containing base and peak groups of electricity contracts): TABLE 1 P1G P2G P3G 0.00 2.00 3.00 5.00 7.00 8.00 10.00 12.00 13.00 15.00 17.00 18.00 20.00 22.00 23.00 The process continues with step 114-6 in which a computer determines whether the new P1G is an entry in the data table.", "If it is, the process continues with step 114-7 in which a computer reads across the row for the new P1G in the data table and thereby determines the new prices (P2G, .", ".", ".", ", PgG), where g denotes the number of types of items within group G. For example, with the data of Table 1, if P1G equaled 15.00, then the computer would determine “Yes” at step 114-6, and the computer would determine P2G=17.00 and P3G=18.00 at step 114-7.The process then proceeds to step 114-10.If the new P1G is not an entry in the data table, the process continues with step 114-8, in which a computer retrieves P1low, the largest entry in the data table less than P1G, and a computer retrieves P1high, the smallest entry in the data table greater than P1G.", "The process then continues with step 114-9, in which a computer determines the new prices (P2G, .", ".", ".", ", PgG) by linear interpolation, reading across the rows in the data table corresponding to P1low and P1high, and applying the new P1G.", "For example, with the data of Table 1, if P1G equaled 10.50, then the computer would determine “No” at step 114-6.The computer would then retrieve P1low=10.00 and P1high=15.00 at step 114-8, and the computer would determine P2G=12.50 and P3G=13.50 at step 114-9.The process then proceeds to step 114-10, in which a computer determines whether all groups G have been considered.", "If not, the process returns to step 114-1.However, if all groups G have been considered, then the entire step 114 has been completed, and the process continues with step 116 of FIG.", "4.Three observations should be made at this point.", "First, rather than determining prices by looking up and possibly interpolating of numbers that are contained in a data table, the relationship or relation among the prices may instead be an algorithm or a formula.", "For example, instead of using the data of Table 1, another preferred embodiment would be obtained by equivalently applying the formulae P2G=P1G+2 and P3G=P1G+3.Second, there is no particular reason that the relationship or relation among the prices need be linear, as it happens to be in Table 1 and in some applications.", "The relationship or relation (data table, or algorithm or formula) could be a nonlinear relationship between (P2G, .", ".", ".", ", PgG) and P1G.", "Third, in some embodiments, the relationship or relation (whether schedule or data table, or algorithm or formula) among prices is predetermined and fixed, and in other embodiments, the relationship or relation can be based on data which may be revised during the course of the auction.", "In typical applications, the price relationship or relation may be determined from market data external to the auction, for example, from the “yield curve” in the government securities market, expressing the differential among prices or interest rates for securities of varying durations.", "If the market data external to the auction were to markedly change during the course of the auction, then the auctioneer might wish to revise the price relationship or relation accordingly.", "FIG.", "5b is a flow diagram illustrating an exemplary process by which initial prices are established.", "Thus, FIG.", "5b illustrates, in greater detail, step 104 of FIG.", "4.The process begins with step 104-1, in which a computer selects a group G of types of items not yet considered.", "In step 104-2, a computer selects a type of item included in group G, whose price is denoted by P1G.", "In step 104-3, a computer looks up the initial value for the selected type of item in group G among the memory locations that were initialized in step 102, and uses this value as the initial P1G.", "In step 104-4, a computer looks up the initial P1G in a data table reflecting the “yield curve” schedule.", "The previous Table 1 serves as an example of such a data table, as in the description of FIG.", "5a.", "The process continues with step 104-5 in which a computer determines whether the initial P1G is an entry in the data table.", "If it is, the process continues with step 104-6 in which a computer reads across the row for the initial P1G in the data table and thereby determines the initial prices (P2G, .", ".", ".", ", PgG), where g denotes the number of types of items within group G. The process then proceeds to step 104-9.If the initial P1G is not an entry in the data table, the process continues with step 104-7, in which a computer retrieves P1low, the largest entry in the data table less than P1G, and a computer retrieves P1high, the smallest entry in the data table greater than P1G.", "The process then continues with step 104-8, in which the computer determines the initial prices (P2G, .", ".", ".", ", PgG) by linear interpolation, reading across the rows in the data table corresponding to P1low and P1high, and applying the initial P1G.", "The process then proceeds to step 104-9, in which a computer determines whether all groups G have been considered.", "If not, the process returns to step 104-1.However, if all groups G have been considered, then the entire step 104 has been completed, and the process continues with step 106 of FIG.", "4.The first two observations made after FIG.", "5a also apply to the process of FIG.", "5b.", "Elements of the Invention Concerned with Applying Constraints to Bids FIGS.", "6a and 6b are flow diagrams of two exemplary subprocesses of step 110.The process of FIG.", "6a begins with step 110a-1, in which a bidder i who has not yet been considered is selected.", "In step 110a-2, a bid (Qki,t)kεG by bidder i which has not yet been considered is selected.", "The bid (Qki,t)kεG is a bid for group G of item types at time t in the auction.", "In step I10a-3, it is checked whether each quantity Qki,t in the selected bid is a nonnegative integer.", "If each component of the bid is a nonnegative integer, the process goes to step 110a-4.In step 110a-4, it is checked whether the selected bid is consistent with bidder i's initial eligibility, that is, whether: ∑ k ∈ G ⁢ Q k i , t ≤ Q _ G i , where bidder i's initial eligibility, {overscore (Q)}Gi, for group G may, for example, be determined by the level of financial guarantee posted by bidder i.", "If the selected bid is consistent with bidder i's initial eligibility, the process goes to step 110a-5, where bidder i's most recent previously-processed bid for group G, denoted (Qki-t-1)kεG, is recalled.", "In step 110a-6, it is checked whether the selected bid is consistent with the auction's activity rule, that is, whether the constraint: ∑ k ∈ G ⁢ Q k i , t ≤ ∑ k ∈ G ⁢ Q k i , t - 1 , is satisfied.", "If it is, the process continues to step 110a-7, where the selected bid (Qki,t)kεG is entered as a valid bid by bidder i on group G. Optionally, bidder i is sent a message confirming to him that the bid is valid.", "The process then goes to step 110a-8, where it is determined whether all bids by bidder i have been considered.", "If not, the process loops back to step 110a-2.If all bids by bidder i have been considered, the process continues to step 110a-9, where it is determined whether all bidders have been considered.", "If not, the process loops back to step 110a-1.If all bidders have been considered, the process goes to step 112 of FIG.", "4.If the selected bid fails any of the checks at steps 110a-3, 110a-4 or 110a-6, the process instead goes to step 110a-10, where a message is outputted to bidder i that the selected bid is invalid.", "The selected bid then is not entered as a valid bid.", "The process then goes to step 110a-8, where it is determined whether all bids by bidder i have been considered.", "If not, the process loops back to step 110a-2.If all bids by bidder i have been considered, the process continues to step 110a-9, where it is determined whether all bidders have been considered.", "If not, the process loops back to step 110a-1.If all bidders have been considered, the process goes to step 112 of FIG.", "4.The process of FIG.", "6b begins with step 110b-1, in which a bidder i who has not yet been considered is selected.", "In step 110b-2, a bid (Qki,t)kεG by bidder i which has not yet been considered is selected.", "The bid (Qki,t)kεG is a bid for group G of item types at time t in the auction.", "In step 110b-3, it is checked whether each quantity Qki,t in the selected bid satisfies the constraint: ∑ k ∈ G ⁢ C k i , t ⁢ Q k i , t ≤ C _ G i , t , where Cki,t and {overscore (C)}Gi,t are arbitrary constants.", "If the constraint of step 110b-3 is satisfied, the process goes to step 110b-4.In step 110b-4, it is checked whether each quantity Qki,t in the selected bid satisfies the constraint: ∑ k ∈ G ⁢ C k ′ ⁢ ⁢ i , t ⁢ Q k i , t ≥ C ^ G i , t , where Ck′i,t and ĈGi,t are arbitrary constants.", "If the constraint of step 110b-4 is satisfied, the process goes to step 110b-5, where it is checked whether the selected bid was submitted at a time no earlier than the starting time of the current round.", "If it was, the process goes to step 110b-6, where it is checked whether the selected bid was submitted at a time no later than the ending time of the current round.", "If it was, the process continues to step 110b-7, where the selected bid (Qki,t)kεG is entered as a valid bid by bidder i on group G. Optionally, bidder i is sent a message confirming to him that the bid is valid.", "The process then goes to step 110b-8, where it is determined whether all bids by bidder i have been considered.", "If not, the process loops back to step 110b-2.If all bids by bidder i have been considered, the process continues to step 110b-9, where it is determined whether all bidders have been considered.", "If not, the process loops back to step 110b-1.If all bidders have been considered, the process goes to step 112 of FIG.", "4.If the selected bid fails any of the checks at steps 110b-3, 110b-4, 110b-5 or 110b-6, the process instead goes to step 110b-10, where a message is outputted to bidder i that the selected bid is invalid.", "The selected bid then is not entered as a valid bid.", "The process then goes to step 110b-8, where it is determined whether all bids by bidder i have been considered.", "If not, the process loops back to step 110b-2.If all bids by bidder i have been considered, the process continues to step 110b-9, where it is determined whether all bidders have been considered.", "If not, the process loops back to step 110b-1.If all bidders have been considered, the process goes to step 112 of FIG.", "4.Embodiments of the Invention Concerned with Whether the Auction Should Continue FIG.", "7 is a flow diagram of a subprocess of step 112 of FIG.", "4.It illustrates an exemplary process by which a computer may determine whether the auction should continue.", "Related to this will also be FIG.", "8b, below, which illustrates an exemplary process by which a computer determines whether the auction should continue, in a system where bidders are permitted to submit Intra-Round Bids.", "FIG.", "7 begins with step 112a-1, in which a group G of types of items not yet considered is selected.", "In step 112a-2, a computer determines whether the excess demand for group G of types of items is within {overscore (C)}G of the available quantity, that is, whether:  Q _ G - ∑ i = 1 n ⁢ · ∑ k ∈ G ⁢ Q k l  ≤ C _ G .", "The constant, {overscore (C)}G, has the interpretation that this is the tolerance to which the auctioneer is allowing oversell or undersell to occur.", "If the auctioneer needs to sell exactly the available quantity of the group G of item types, then {overscore (C)}G=0.If this inequality is not satisfied, then group G of item types has not yet cleared, and so the auction should continue.", "The process thus jumps immediately to step 114 of FIG.", "4.If the inequality of step 112a-2 is satisfied, the process then goes to step 112a-3, where it is determined whether all groups G of types of items have been considered.", "If not, the process loops back to step 112a-1.However, if all groups G of types of items have already been considered, then it has been found that all groups G of types of items have cleared within a tolerance of {overscore (C)}G, and so the auction should not continue.", "The process proceeds to step 118 of FIG.", "4, where the final message is generated.", "Embodiments of the Invention Concerned with Intra-Round Bids In many of the leading dynamic electronic auctions in the prior art, bidders submit bids in a sequence of discrete rounds.", "For example, in the Federal Communications Commission auctions for radio communications spectrum or in the recent UMTS auctions held by European nations, the following would be a typical bidding schedule for an auction: Round 1: 9:00-9:45 Round 2: 10:00-10:45 Round 3: 11:00-11:45 Round 4: 12:00-12:45 Round 5: 13:00-13:45 Round 6: 14:00-14:45 Round 7: 15:00-15:45 Round 8: 16:00-16:45 This bidding schedule would have the following interpretation.", "During the specified time period of each round, a bidder would be required to submit a new bid or new collection of bids (unless this bidder was already the standing high bidder on an item after the bidding of the previous round).", "If a bidder who was required to submit a new bid failed to submit a new bid, then (except for provisions in the rules concerning automatic waivers) the bidder would be eliminated from the auction.", "By contrast, some other electronic auctions in the prior art—for example, online auctions at eBay—allow bidding to occur continuously.", "Rather than adhering to any rigid round schedule, bidders may submit bids at any times that they like up to a specified closing time.", "Related to this, there is no sense that a bidder is required to bid a certain amount by any particular time in order to retain eligibility to bid at a later time in the auction.", "Many or most electronic auctions for high-valued items utilize a discrete round structure, rather than allowing bidding to occur continuously.", "There appear to be several reasons for this.", "First, a discrete round structure has desirable information properties.", "The auction can be easily structured so that the results of Round t are disseminated to bidders before the bids of Round t+1 need to be submitted.", "Second, a discrete round structure is especially conducive to enforcing “activity rules,” in which a bidder is required to be active (i.e., either be the standing high bidder or place a new high bid) on a given number of items in an earlier round of the auction in order to continue to bid on a given number of items in a later round of the auction.", "This forces bidders to effectively disclose to their opponents (through their bidding) the values that they attach to the items, helping to mitigate the well-known “Winner's Curse” present in auctions.", "Third, a discrete round structure requires a bidder to repeatedly affirm, in successive rounds, his willingness to pay a given price for an item in the auction—which may be especially desirable when items such as communications licenses may sell for millions or billions of dollars or euros.", "At the same time, the desirable properties of a discrete round structure may come at some considerable cost.", "It will typically be reasonable to hold only something like 8 to 12 rounds of bidding in a given day.", "As a result, the auctioneer must accept at least one of several problems: (1) The auction may be required to last a very long time: in some North American and European spectrum auctions, the bidding extended more than 20 business days.", "Such a lengthy auction may be rather onerous for bidders and for the seller.", "In particular, it may discourage bidder participation, causing the seller to forgo substantial revenues.", "(2) The bid increment between successive rounds may be required to be rather substantial: in some North American and European spectrum auctions, the bid increment between successive rounds never was allowed to drop below five percent of the previous bid.", "It can be argued that a seller suffers an expected revenue loss which is directly proportional to the minimum bid increment, so this may cost a seller millions of dollars or euros.", "(3) The starting price may be required to be very near to the expected closing price.", "This may discourage bidder participation, as well as potentially eliminating the possibility of bidders getting caught up in the excitement of the auction and bidding very high prices (which is one of the advantages of conducting a dynamic auction).", "This also runs the risk that the auction will fail: that is, quantities bid at the starting price being less than the available quantity at the auction.", "Moreover, in a clock auction, problem (2) above, a large bid increment, may lead to a heightened risk of “undersell”.", "Consider an auction with an available quantity of 100 units of an item, and suppose a bid increment of five percent.", "It is quite plausible that, at a price of $1,000,000 per unit, the aggregate quantity bid by all bidders would equal 110 units, but at the next price of $1,050,000 per unit, the aggregate quantity bid by all bidders would decline to only 60 units.", "The auctioneer then faces the unattractive alternatives of: selling only 60 units out of the available quantity of 100 units at a price of $1,050,000 each; rationing bidders so that only 100 units, out of the 110 demanded, are sold at $1,000,000; or restarting the auction at $1,000,000.Observe however that the “undersell” problem would in all likelihood have been substantially avoided, had a much smaller bid increment been possible.", "One embodiment of the present invention is a system and method for “Intra-Round Bids.” A discrete round structure—with all of its many advantages—is preserved for a clock auction.", "However, in each round of the clock auction, a “starting price” and “ending price” is established for each type of item.", "Bidders are permitted to submit bids at prices between the starting price and the ending price.", "In a preferred embodiment, a bidder submits a price parameter for group G representing a percentage of the distance from the starting price vector for group G and the ending price vector for group G. Bidders have every incentive to utilize Intra-Round Bids, and to the extent that bidders utilize them, the seller should be expected to attain higher auction revenues and to reduce the probability of undersell.", "Thus, a system and method for Intra-Round Bids improves upon the prior art for auction systems and methods, and has immediate practical application for dynamic auctions of radio communications spectrum, securities and other financial products, electric power, etc.", "While the previous and following description of Intra-Round Bids is framed largely in terms of regular auctions to sell (where bidders are buyers), the invention is equally applicable for reverse or procurement auctions to buy (where bidders are sellers).", "For the sake of brevity, this specification will not run through the process a second time with the roles of selling and buying reversed, but it should be clear to anybody skilled in the art that the technology can be equally used in both situations.", "Here is an example illustrating the usefulness and exact meaning of Intra-Round Bids.", "Suppose that, in a clock auction with an available quantity of 100 units, the ending price per unit associated with Round 4 is $1,000,000, and the ending price per unit associated with Round 5 is $1,050,000.In an auction with discrete bidding rounds, Bidder 1 might submit a bid quantity of 55 units for Round 4 and a bid quantity of 30 units for Round 5.If there also exists a Bidder 2 who submits the same bid quantities, then we would have exactly the “undersell” problem described above: an aggregate quantity bid by all bidders of 110 units in Round 4 but only 60 units in Round 5 (with available quantity of 100 units).", "With an auction system and method with Intra-Round Bidding, the ending price for Round 4 may be taken to be the starting price for Round 5, i.e., the starting price for Round 5 is $1,000,000.Here is an example of the bids that Bidder 1 might submit for Auction Round 5: 53 units at $1,010,000 per unit; 51 units at $1,020,000 per unit; 49 units at $1,030,000 per unit; 45 units at $1,035,000 per unit; 40 units at $1,040,000 per unit; and 30 units at $1,045,000 per unit.", "These bids have the following exact meaning: the parameters corresponding to price indicate the price at which Bidder 1 wishes to change his quantity demanded as compared to his “previous” (that is, next lower price) bid.", "Thus, in this example: Bidder 1 is willing to purchase 55 units (his previous bid from Round 4) at prices of $1,000,001-$1,009,999; Bidder 1 is willing to purchase 53 units at prices of $1,010,000-$1,019,999; Bidder 1 is willing to purchase 51 units at prices of $1,020,000-$1,029,999; Bidder 1 is willing to purchase 49 units at prices of $1,030,000-$1,034,999; Bidder 1 is willing to purchase 45 units at prices of $1,035,000-$1,039,999; Bidder 1 is willing to purchase 40 units at prices of $1,040,000-$1,044,999; and Bidder 1 is willing to purchase 30 units at prices of $1,045,000-$1,050,000.If there also exists a Bidder 2 who submits the same bid quantities, then the auctioneer would be able to declare the auction over at a price between $1,030,000 and $1,034,999, with 98 out of the 100 available units sold.", "The auction revenues are improved, and the undersell problem is greatly reduced.", "FIG.", "8a is a flow diagram of a subprocess of step 108 of FIG.", "4.It illustrates an exemplary process by which a particular bidder i may submit Intra-Round Bids.", "FIG.", "8a begins with step 108-1, in which bidder i selects a group, G, of item types on which he wishes to place a bid.", "In step 108-2, bidder i selects a price parameter for group G representing a percentage of the distance from the starting price vector for group G and the ending price vector for group G in the current round.", "For example, in a group containing three types of items, if the starting price vector is (4.00, 4.50, 4.75), if the ending price vector is (8.00, 8.50, 8.75), and if a bidder enters a price parameter of 25%, this signifies that the bidder is indicating an implied price vector of (5.00, 5.50, 5.75).", "In step 108-3, bidder i selects quantities of the item types of group G that he would like to take effect as bids at the price vector implied by the selected price parameter.", "In step 108-4, bidder i expresses whether he wishes to enter more bids.", "If so, the process loops back to step 108-1.If not, the process continues to step 108-5.In step 108-5, a computer determines whether bidder i has submitted at least one bid for each group G of item types.", "If not, the process loops back to step 108-1, and optionally a computer prompts bidder i to submit bids on the groups G of item types on which bidder i has not submitted at least one valid bid in the current round.", "If so, the process goes to step 110 of FIG.", "4.FIG.", "8b is a flow diagram of a subprocess of step 112 of FIG.", "4.It illustrates an exemplary process by which a computer determines whether the auction should continue, in a system where bidders are permitted to submit Intra-Round Bids.", "FIG.", "8b begins with step 112b-1, in which a group G of item types not yet considered is selected.", "In step 112b-2, a computer sorts all bids entered for group G in the current round.", "The sorting is done: first, by bidder ID; second, by price parameter in the entered bid (in descending order); and third, by time stamp of submission (in descending order).", "In step 112b-3, a computer selects, for each bidder i, the bid, QG,i, for group G with the highest price parameter (and then the latest time stamp).", "In step 112b-4, a computer determines whether the aggregate quantity bid for group G is no greater than the available quantity, that is, whether: ∑ i = 1 n ⁢ ∑ k ∈ G ⁢ Q k G , i ≤ Q _ G .", "If this inequality is not satisfied, then group G of item types has not yet cleared, and so the auction should continue.", "The process thus jumps immediately to step 114 of FIG.", "4.If the inequality of step 112b-4 is satisfied, the process then goes to step 112b-5, where it is determined whether all groups G of item types have been considered.", "If not, the process loops back to step 112b-1.However, if all groups G of item types have already been considered, then it has been found that all groups G of item types have cleared, and so the auction should not continue.", "The process proceeds to step 118 of FIG.", "4, where the final message is generated.", "FIG.", "8c is a flow diagram of a subprocess of step 118 of FIG.", "4.It illustrates an exemplary process by which a computer determines final allocations and payments, in a system where bidders are permitted to submit Intra-Round Bids.", "FIG.", "8c begins with step 118-1, in which for all bids entered in the current (i.e., final) round, a computer sorts the price parameters from smallest to largest, and denotes them λ1<π2<.", ".", ".", "<πR.", "In step 118-2, a computer initializes the price parameter subscript to r=1, so that in the first iteration of the remaining steps, the computer considers the smallest value, π1.In step 118-3, a group G of item types not yet considered is selected.", "In step 118-4, a computer sorts all bids entered for group G in the current round.", "The sorting is done: first, by bidder ID; second, by price parameter in the entered bid (in descending order); and third, by time stamp of submission (in descending order).", "In step 118-5, a computer selects, for each bidder i, the bid, QG,i, for group G with the highest price parameter that is less than or equal to πr (and then with the latest time stamp).", "In step 118-6, a computer determines whether the aggregate quantity bid for group G is no greater than the available quantity, that is, whether: ∑ i = 1 n ⁢ ∑ k ∈ G ⁢ Q k G , i ≤ Q _ G .", "If this inequality is not satisfied, then group G of item types has not yet cleared at price parameter πr, and so r needs to be incremented.", "The process thus goes to step 118-8, where the price parameter subscript r is advanced by 1, so that in the next iteration of these steps, the computer considers the next price parameter, πr+1.The process then loops back to step 118-3, using the new higher value of πr+1 and starting over for groups G of item types.", "If the inequality of step 118-6 is satisfied, the process continues to step 118-7, where it is determined whether all groups G of item types have been considered.", "If not, the process loops back to step 118-3.However, if all groups G of item types have already been considered, then it has been found that all groups G of item types have cleared at price parameter ;r,.", "Thus, the price parameter A, implies market-clearing prices for the auction.", "The process proceeds to calculate the price vector implied by price parameter ;r, to note the quantities bid by all bidders at this price vector, and to incorporate these computations into a final message that is outputted from a machine.", "Auction-Like Optimization Problems and Machine-Generated Bids In the course of this application, a method and apparatus for implementing auctions of multiple items has been described.", "The methods and apparatus described allow users to participate in various auctions with a level of attention which varies from continuous, down to the input of information on a single occasion.", "It should also be apparent that the required level of attention by the “auctioneer” may vary from continuous to essentially zero—aside from initiating the auction.", "Thus, for all intents and purposes, once the basic auction description is selected and the users input desired information, the auction implemented by the invention can be essentially automatic, i.e.", "devoid of human interaction.", "Because in the past auctions have generally been considered to be processes engaged in by persons, the feature of an automatic auction may be, by itself, considered relatively new.", "There are, however, many other automatic systems which interact in a way that is entirely analogous to an auction and to which the present invention could be applied.", "Hence, the present invention can be applied to improve the efficiency of computers which are used to operate the automatic systems, by economizing on the collection of informational inputs needed for the system and to speed the computational of optimal resource assignments.", "At the same time, many optimization problems encountered in the field of operations research have similar mathematical structures to the problem of determining the winners of an auction.", "Hence, the present invention can be applied to improve the efficiency of computer systems which are used to solve the similar optimization problems, by enabling the computations to be implemented on a system with parallel processing or generally by speeding the computation of solutions.", "For example, the air conditioning plant in an office building allocates cool air among individual offices in the building via a dynamic auction.", "Periodically, the central computer of the air conditioning system serves the role of the “auction computer” in a clock auction, while computers interfaced with the thermostats in each suite of offices serve the role of “bidder terminals.” Each thermostat is programmed to send back bids consisting of a desired quantity of cooled air based on: the current temperature reading of the thermostat, the desired temperature in the office, and the use (if any) to which the office is currently being put.", "Based on the parameters to which it has been programmed, the central computer of the air conditioning system then provides the results of the auction in its allocation of cooled air among the bidding offices.", "In another context, a communications, transportation or energy transmission network faces the technical problem of how to allocate its scarce network resources.", "The optimization problem in allocating its network resources (e.g., bandwidth, switches, etc.)", "has a very similar structure to the auction problem.", "Moreover, clock auctions are again well suited to the problem, since a network provider needing multiple types of network resources is able to iteratively bid on a vector of network resources, always knowing the price of each.", "Hence, the present invention can be usefully applied to improving the solution to this technical problem.", "In another context, computational resources on a distributed computer system can be allocated via a clock auction.", "Whenever a new job requiring a given quantity of CPU time enters the system, an auction is conducted.", "Each member of the distributed computer system indicates the quantity of CPU time which it can make available at a given priority level or a given price.", "In this case, the “auctioneer computer” selects and allocates the resources to be applied to the new job in accordance with some programmed timetable and hence in this fashion provides the results of the auction.", "The several examples described herein are exemplary of the invention, whose scope is not limited thereby but rather is indicated in the attached claims." ] ]	Patent_10467868
[ [ "Packet data recording method and system", "The present invention provides for a data recording system including data recording means having a plurality of network interface cards in which a plurality of network interface cards can be employed within a single recording mean and each card can be provided with a plurality of network connecting ports then, for example, for each RTP packet stream being recorded, the system can advantageously note the sequence number for the most recently accepted packet and any packet received with a lower sequence number can be readily discarded." ], [ "1.A data recording system including data recording means having a plurality of network interface cards.", "2.A system as claimed in claim 1 and including a plurality of network interface cards within a single recording means.", "3.A system as claimed in claim 2, wherein each Network interface card has multiple network connection ports.", "4.A system as claimed in claim 1, 2 or 3, wherein each individual recording means is arranged for mutual communication so as to advise each other of the presence of particular packet streams.", "5.A system as claimed in any one of claims 1 to 4, and arranged such that each recorder is advised of the addresses to be monitored; when a recorder identifies a packet with the required addresses it begins recording it and advises all other recorders to stand down; and other recorders are arranged to note these addresses as being recorded elsewhere.", "6.A system as claimed in claim 5 and further arranged such that the notification sent to other recorders include within it the sequence number of an RTP packet that was first received and the IP address of the receiving recorder; that in the event that more than one recorder receives packets prior to hearing notification from another recorder that it has commenced recording, the recorder which received the earliest packet can maintain responsibility for recording and the other recorder shall cease recording; that should more than one recorder receive the same packet and send notification of this, then the recorder with highest IP address can maintain its recording and the other(s) will stand down; and that when recording begins, a recorder will not create a record in the database of recordings until a pre-defined time has elapsed in case it receives, during this period, a notification from another recorder that it too has started recording.", "Should it receive such notification and, according to the algorithms defined in (d) to (f) above, it is to stand down, it will abandon its recording without having altered the database of recordings.", "7.A system as claimed in claim 6 and further arranged such that should a packet be lost an indication of the sequence number(s) of the lost packets can be maintained as part of the recording control structure for that IP address; and that when packets are received that are determined to be earlier than the most recently accepted packet for that address, their sequence number can subsequently be compared against the list of recently missed packets and, if found, can be stored at the appropriate offset within the recording buffer overwriting the padding that was inserted when the later packet was received and loss of packet(s) identified.", "8.A method of recording a data employing a plurality of network interface cards and including the steps carried out by the system of any one or more of claims 2 to 7.9.A data packet recording system arranged for determining an IP address of an application and including means for comparing at least a sample of received or transmitted packets with at least a set of pre-programmed signature packets.", "10.A system as claimed in claim 9 and arranged for comparing all packets received from or sent to each destination against a set of pre-programmed signature packets.", "11.A system as claimed in claim 9 and arranged such that the sustained absence of such packets is employed as an indicator of an error condition with the device that was previously active on that address.", "12.A method of recording a data packet for determining an IP address of an application and including the step of comparing at least a sample of received or transmitted packets with at least a set of pre-programmed signature packets.", "13.A data recording system arranged such that the data packets are arranged to be transmitted via a proxy server and further arranged such that the IP address of at least one party to the conversation is altered.", "14.A system as claimed in claim 13, and arranged to determine the presence and IP address of the proxy servers, to allow for recorders passing their knowledge of current mappings to other recorders allowing them to refine their filtering algorithms; and to pass examples of specific packets identified as having been mapped by the proxy server to other recorders.", "15.A data packet recording system including packet filtering means arranged for filtering on the basis of an IP address.", "16.A data packet recording system as claimed in claim 15, and arranged for filtering on the basis of an IP port number of both source and/or destination address(es).", "17.A system as claimed in claim 15 or 16, and arranged to analyse the sequence number and timestamp to determine the next packet in the expected sequence based on whether one or more packets have been missed such that performance counters can be incremented appropriately; inserting additional data in the recorded stream to pad-out the space that should have been taken up by the missing packet(s) and whether the payload of the RTP packet can be appended to a buffer and the remainder of the packet discarded.", "18.A system as claimed in claim 15 or 16 and arranged such that a signature data pattern is appended to a data buffer collecting packets to/from the specific destination; a timestamp indicating the time at which the packet was received to the buffer; a length indicator specifying the number of bytes in the recorded packet is appended to the buffer; and that the contents of the packet are appended to the buffer.", "19.A data recording system for IP telephones controlled by an IP-PBX application and wherein the controlling application is arranged to request that each phone advise it of the port number for which it can receive incoming audio data.", "20.A system as claimed in claim 19, and arranged for the identification and storage of only the payloads of successive RTP messages.", "21.A system as claimed in any one of the preceding claims, and arranged to compare the sequence number of the received packet with that of the previously received packet and to identify if a difference exists.", "22.A data recording system as claimed in any one of the preceding claims and arranged such that recordings stored independently for each direction of transmission." ], [ "The present invention relates to a packet data recording method and related system.", "The integration of computers into office communication systems has enabled many functions previously performed by separate devices to be combined into a single management system.", "For example, computer-based voice logging systems enable a computer to: receive voice communication through a hardware connection to the regular telephony network; to record either a conversation, in which at least two parties converse, or a message from at least one party to one or more parties; and to replay such recorded conversations or messages upon request.", "It is therefore appreciated that commercial entities perform substantial amounts of business via telephone or Internet contact with their customers.", "The analysis of such contact can help businesses improve the quality and efficiency of their services, and assist with customer retention and, ultimately, profitability.", "Attempts have been made previously to achieve such analysis in a satisfactory manner and to a satisfactory degree.", "For example, many businesses have, for some time, recorded some of their staff/customer interactions.", "Traditionally this was done to satisfy regulatory requirements or to help resolve disputes.", "More recently, the emphasis has moved towards the reviewing of these interactions from a quality perspective.", "This is intended to identify good and bad aspects of particular interaction calls with a view to improving the level of customer service.", "Recently, for example, recording the activity on a PC screen has been undertaken to improve the completeness of the review procedure with the reviewer able to see how accurately staff are entering information received via the telephone.", "Also, it has been known to employ Call Detail Recording (CDR) systems to prevent perceived abuse of telephone systems and to apportion costs to the department or individual making the calls.", "Originally such records were printed out directly from the Private Automatic Branch Exchange (PABX) onto a line printer.", "Later, systems were designed to store this information in a database allowing more sophisticated reporting and searching fro calls on the basis of one or more of the stored call details.", "More recently, Computer Telephony Integration (CTI) interfaces have been provided that give this information in real-time, during the call.", "Further, several systems currently exist that use call recording in combination with CDR or CTI and a database application in order to perform routine monitoring of calls with the intention of identifying weaknesses in individual Customer Service Representatives (CSR).", "Typically a small percentage of the CSR calls are reviewed and “scored” against a set of predetermined criteria to give an indication of the quality of that particular member of staff.", "Within a call-centre environment, it should also be noted that rather than simply using standard PC office automation applications when dealing with customers, staff in most call centres use increasingly sophisticated applications that help them to handle the calls more efficiently and effectively.", "Help desk applications and telemarketing call scripting applications are examples of such applications.", "U.S. Pat.", "No.", "6,122,665 discloses a method and a system for: the management of communication sessions for computer network-based telephone communication, and in particular for the identification of packets containing audio and/or video data; the storage of these packets; and for the reconstruction of selected communication sessions for audio and/or video display as needed.", "In particular, this document teaches the provision of a system and a method arranged to: record communication sessions performed over a computer network; to provide such a system and method for analysing data transmitted over the computer network in order to detect audio and video data transmitted over the computer network in order to detect audio and video data for recording; to provide such as system and method for displaying recorded video and audio data upon request; and to provide such a system and method for analysing, recording and displaying communication session conducted with a LAN-based telephone system.", "As will be appreciated, U.S. Pat.", "No.", "6,122,665 serves to illustrate a general move towards Voice over IP (VoIP) Communications as bringing together voice and data environments.", "However, there remains a need to record interactions over data networks irrespective of whether these are voice communication sessions, video or data interaction between people and systems, systems and systems or people and people.", "While it is noted that systems produced by, e.g.", "Hewlett Packard, have long been able to monitor data networks and store details and contents of the packets they observe, the primary function of such devices has normally been for network performance analysis and diagnostics.", "Disadvantageously they do not provide for the management of large volumes of recordings, and related archival and indexing.", "Thus, such systems, and related methods, do exhibit limitations and disadvantages.", "As a further illustration U.S. Pat.", "No.", "6,122,665 merely serves to address the recording of a single network segment.", "However, with the prevalence of Ethernet switches and routers, it is rare for a system of any size to be implemented as a single network segment.", "Whenever multiple segments are introduced, the packets are routed between these segments as required in order to reach their destination.", "There is therefore no single point at which all packets can be observed.", "It would therefore prove advantageous to provide a system which can be extended to tap into the network at however many points are needed to achieve the required coverage.", "Unfortunately, merely extending a simple system designed for a single tap-point will result in highly redundant recordings as many of the packets to be recorded will arise at more than one tap point.", "Further, known systems require knowledge of network addresses to operate properly.", "To determine what, and indeed how to record, some knowledge of the identity of specific IP addresses is often required.", "For example, knowing which IP address hosts an IP-PBX will allow better filtering of information and the analysis of call control packets involving that node.", "Unfortunately, many IP addresses are dynamically assigned, e.g.", "via DHCP.", "Also network configurations are regularly changed and updated as networks expand and evolve.", "Neither do known systems address the use of proxy addresses.", "In many IP networks, packets are readdressed en route by Proxy Servers.", "This makes it difficult for the controlling software to identify which packets should be recorded as a single IP address is often only appropriate for packets transmitted over the leg of the journey from which they emanate, or which they finally reach having regard to the network node having that IP address.", "The Known systems also do not support the recording of packet data streams along with voice/video.", "The system disclosed in U.S. Pat.", "No.", "6,122,665 filters packets out that are not part of a voice or video stream.", "This prohibits its use in cases where the recording of a combination of data streams is required.", "With regard to packet filtering, known systems are far from optimal.", "It is important that a recording system is able to process all the packets it receives and eliminate those that are not required as efficiently as possible.", "Often only a small proportion of received traffic is to be recorded.", "The described system performs its filtering initially by determining the type of traffic contained within the packet, for example voice or video versus other.", "In a large voice/video system with many concurrent sessions occurring, there will be many thousands of packets per second being received at the network tap point.", "In many cases only a small proportion of these will be required.", "Known systems will not filter out any of these at the first level and will pass all packets on to the following stage.", "Such manner of operation proves to be sub-optimal.", "Disadvantageously, the integration of such a system with the systems controlling call placement is not generally achievable.", "Also, the determination of which IP ports to record on the basis of H323 signalling information also received via the network tap point is often superfluous.", "Rather, better information regarding the contacts and the requirement to record them can be obtained by interfacing to real-time event outputs, i.e.", "CTI ports, of the systems that are controlling the placement of calls.", "These ACDs or PABXs will often include additional information regarding the calls that cannot be deduced by observing the session control messages.", "For example, the linkage of one call to the next as calls are transferred between people is not always available from the session control protocol.", "Also, tolerance issues such as the loss of critical packets is not addressed in known systems.", "However, in situations where session control information can be gleaned from analysis of session control packets, it can be disadvantageously adversely affected by the loss of a single packet.", "This can readily lead to a call not being recorded.", "In many systems, there can prove to be multiple ways to deduce such information, thereby making the system tolerant to the loss of certain packets.", "As with filtering, known systems prove to be sub-optimal with regard to the storage of received packet data.", "The described system does not disclose in detail how the packets are stored and it appears that these are simply kept as raw, independent packets such that a significant potential for storage overhead has been found to arise.", "Importantly, the known systems do not integrate well with traditional (non VoIP) storage and replay mechanisms.", "Again, the described system appears to store the recorded packets as packets and replays these by retransmitting them to a device similar to that which would have received them originally.", "In many systems, however, only part of the voice system is packet-based and certainly the replay mechanisms are often not packet based.", "Hence there is a need to store, retrieve and replay conversations in a way that is both efficient and compatible with existing circuit-switched voice systems.", "Still further, such known systems do not address the specific requirements of testing and monitoring, nor do they attempt performance measure.", "In any packet system, there is the chance of the loss of a particular packet and the system described makes no reference to a determination of how well it is performing and whether any packets not recorded were lost by the network or at the recording device's network interface.", "It is now appreciated that with simple extensions, such recording systems can be used to record data steams in a form suitable for use in simulated loading tests.", "The capability for advanced recording options such as stereo and multi-party (conference call) recording are also disadvantageously absent from known systems.", "In particular, the system described does not address the added value that can be obtained by recording conversations so that they may be replayed in “stereo” with multiple tracks allowing the replayer to separate the audio travelling in each direction.", "Nor does it describe the extension of this technique into the recording of conference calls where the separation and identification of individual speakers is of great benefit in aiding the listener to follow the flow of conversation.", "The present invention seeks to provide for a data recording system and method and exhibiting an advantage over such known systems.", "According to one aspect of the present invention, there is provided a data recording system including data recording means having a plurality of network interface cards.", "Of course, alternatively, where more than one segment must be tapped in order to receive all required packets, this can be done either by providing multiple systems, each tapping a single segment but this can result in the same recordings being made by multiple recorders.", "A preferred embodiment is to use multiple network interface cards (NICs) within a single recorder.", "Advantageously, each NIC may have multiple network connection ports, such as typically are used to provide fault tolerant connection to multiple Ethernet switches.", "In this case, the filtering mechanism is enhanced to allow rapid elimination of duplicate packets.", "For each RTP packet stream being recorded, the system can advantageously note the sequence number of the most recently accepted packet.", "Any packet received with a lower sequence number (account being taken of sequence numbers wrapping as they overflow their assigned binary range) will immediately be discarded.", "In this way, the system advantageously need not actively keep track of network topology and will operate regardless of how many copies of each packet it receives.", "In larger or physically separated systems which require multiple recorders, it can prove impractical to bring all packets into a single recorder.", "In such cases, the individual recorders can be arranged to communicate with each other so as to advise each other of the presence of particular packet streams.", "In a prepared example, it is desired to record packets travelling between a specific pair of addresses: a) each recorder is advised of the addresses to be monitored; b) when a recorder identifies a packet with the required addresses it begins recording it and advises all other recorders to stand down; and c) other recorders are arranged to note these addresses as being recorded elsewhere.", "Further advantageous refinements to such a scheme can seek to ensure that only one recorder records each call or session in that: d) the notification sent to other recorders call include within it the sequence number (or timestamp) of the RTP packet that was first received and the IP address (or other identifier) of the receiving recorder; e) in the event that more than one recorder receives packets prior to hearing notification from another recorder that it has commenced recording, the recorder which received the earliest packet can maintain responsibility for recording and the other recorder shall cease recording; f) should more than one recorder receive the same packet and send notification of this, then the recorder with highest IP address (or other algorithm allowing unique determination of priority) can maintain its recording and the other(s) will stand down; g) when recording begins, a recorder will not create a record in the database of recordings until a pre-defined time has elapsed in case it receives, during this period, a notification from another recorder that it too has started recording.", "Should it receive such notification and, according to the algorithms defined in (d) to (f) above, it is to stand down, it will abandon its recording without having altered the database of recordings.", "The following further refinements can be employed to ensure that packet loss is minimized: h) should a packet be lost (as can be determined from a gap in the sequence number of received packets) an indication of the sequence number(s) of the lost packets can be maintained as part of the recording control structure for that IP address; and i) when packets are received that are determined to be earlier than the most recently accepted packet for that address, their sequence number can subsequently be compared against the list of recently missed packets and, if found, can be stored at the appropriate offset within the recording buffer overwriting the padding that was inserted when the later packet was received and loss of packet(s) identified.", "It should be appreciated that the invention also provides for a data recording method employing a plurality of network interface cards and as described above.", "According to another aspect of the present invention, there is provided a data packet recording system arranged for determining an IP address of an application and including means for comparing at least a sample of received or transmitted packets with at least a set of pre-programmed signature packets.", "The system can then advantageously determine the IP addresses of significant applications by comparing all packets received from or sent to each destination against a set of pre-programmed signature packets.", "These packets are preferably chosen to be examples of the traffic expected to involve the node being sought.", "For example, in many IP-PBX systems, each IP-phone regularly heartbeats by sending a simple packet to the IP-PBX control application every minute or so.", "By monitoring for these packets it is possible to determine the location of the IP-PBXes in the system being monitored.", "Preferably, the sustained absence of such packets can also be used as an indicator of an error condition with the device that was previously active on that address and can trigger an alarm condition and/or fallback mode of operation.", "It should be appreciated that the invention also provides for a data packet recording method including a comparison step as described above.", "According to yet another aspect of the present invention, a data recording system is provided wherein data packets are arranged to be transmitted via a Proxy server and further arranged such that the IP address of at least one party to the conversation is altered.", "This advantageously allows for security and re-use of address space although difficulties can be experienced in determining which packets should be recorded.", "The invention can preferably address this by being arranged to: a) determine the presence and IP address of such Proxy servers either by explicit configuration information or, preferably, by analysing the pattern of packets entering and leaving the IP node.", "This can be achieved for example by comparing the contents of packets transmitted by each node with the contents of those received by that node.", "Should it find packets that are identical bar the IP address, it can both deduce that this node is performing a Proxy function and determine the current mapping of IP addresses that it is performing; b) allow for recorders passing their knowledge of current mappings to other recorders allowing them to refine their filtering algorithms; and c) pass examples of specific packets identified as having been mapped by the Proxy server to other recorders.", "This advantageously allows recorders to differentiate between overlapping address ranges.", "For example, two different Proxy servers may both be mapping addresses in a given range.", "By passing occasional packets to each other, along with their pre-mapping addresses, recorders can determine whether the stream of data they are observing is indeed that for this address or a completely different stream mapped by a different Proxy server.", "As before, the invention also provides for a corresponding method including IP address alteration.", "According to still another aspect of the present invention, there is provided a data packet recording system including packet filtering means arranged to perform on the basis of an IP address and preferably, an IP port number of both source and/or destination address(es).", "Having determined that the packet is one that is to be recorded, the system can preferably then store the packet in the most appropriate way.", "For example, for RTP streaming data: a) The system is arranged to analyse the sequence number and timestamp to determine whether this is the next packet in the expected sequence; b) if one or more packets have been missed then performance counters can be incremented appropriately; c) additional data can preferably be inserted in the recorded stream to pad out the space that should have been taken up by the missing packet(s).", "This advantageously avoids gradual drift between the recording and real-time which would otherwise build up and result in the call being shorter when replayed than it had been originally; and d) the payload of the RTP packet can be appended to a buffer and the remainder of the packet discarded; and e) optionally, information can be flagged from the packet header so as to be retained where this is not the default header, i.e.", "it cannot be fully deduced from knowledge of the previous header.", "As an example for other packet oriented data: a) optionally, a signature data pattern (e.g.", "well known particular 4 byte value) can be appended to the data buffer collecting packets to/from the specific destination.", "This advantageously ensures that any application reading the recording subsequently can re-synch with these inter-packet boundaries should the recording become corrupted or the start of the recording cannot be accessed; b) a timestamp indicating the time at which the packet was received is appended to said buffer.", "This will typically be precise to at least millisecond and can often achieve micro-nanosecond precision; c) a length indicator specifying the number of bytes in the recorded packet is appended to said buffer; and d) finally, the contents of the packet can be appended to said buffer Again, the invention also provides for a related method.", "In an alternative data filtering arrangement and method filtering according solely to address data, or at least before looking at packet content type can be provided.", "In some cases a given IP port might only be transmitting one type of data and so filtering on its address alone can advantageously prove sufficient in whether or not to record the packet.", "A preferred arrangement can involve looking up the address of the IP destination of each packet, or equally source data, as the primary filter.", "A “map” of known addresses is maintained and each entry serves to identify whether data going to (or from) that address should be recorded.", "Advantageously, by utilizing the IP address as the primary key into this map, extremely fast look-up can be achieved.", "Further preferred refinements to this scheme include: a) the pre-filtering of packets on IP address so as to eliminate packets sent to or from the recorder itself.", "This allows the same network interface card to be used for normal IP communications (e.g.", "with clients searching and replaying previously recorded calls) whilst it is also being used a promiscuous mode tap.", "This simple filtering out on a single (32-bit) integer IP address avoids the need to look up the address map repeatedly for all packets that are directed at other sockets on this node.", "With the introduction of IP Version 6, this will become a 128-bit integer comparison; b) in preference to hashing or sorted table indexing, the map of required addresses can be implemented as a memory array.", "By using the fact that most IP addresses required will lie within a narrow range of possible values it is faster to compare the high order bytes of the IP address with the sub net or nets on which all the target nodes are located.", "The remainder of the address can then be used as an index into a (typically sparsely populated) memory map in which each address to be recorded is represented by a binary 1 and the remainder as 0.For a Class B address as described here, only 64K bits of information are required to achieve this though the use of 65,536×32 bit words allows for each entry in the map to be either null (0x00000000) or a pointer to the structure describing the known address and its recording details e.g.", "buffer storage location.", "It should be noted that a similar scheme can be used in IP Version 6 albeit with a much smaller portion of the overall numbering range being covered by such a table; c) in RTP based systems, many of the data streams being transmitted are constant bit rate.", "In these cases, many sessions will be transmitting packets at a regular rate resulting in a strong correlation between the order in which packets are received.", "This is especially true in cases where all transmitters are of the same type e.g.", "an IP-PBX with many identical IP-phones, all configured with the same packet interval.", "This can be used to advantage in filtering the incoming packets.", "If a record is maintained of the recently received packet addresses, the following optimized search algorithm can be deployed I. note the IP destination address of the first RTP packet received at the head of a list of such addresses.", "Look up the known destination for this address and include a pointer to it at the head of a list of such pointers, II.", "as subsequent packets are received, compare their address against the packet at the head of the list, III.", "if they are different, append their addresses to the list; look up their known destination descriptor and append it to the list of such pointers, IV.", "if it matches the address at the head of the list, use the pointer to the known destination at the head of such list.", "Note that the next packet should be compared to the following entry in the list, V. subsequent packets should be compared to the next entry in the list.", "If they match, repeat step IV, if not, insert them into the list at this point.", "d) The algorithm of c above can preferably be further refined to accommodate the cessation of transmission from any address as happens when a call terminates.", "By comparing the new packets address against the following item in the list, we can determine that the previous address has probably ceased transmitting and it can be removed from the list.", "e) To allow for the case where two streams cease in one cycle of the list, it is necessary to look two addresses ahead if the new packet s address is not found at the head or subsequent entries in the list.", "This is a common case as both directions of the call will typically terminate at or about the same time.", "However, given a typical interval between transmission of 50 ms it is rare that more than one call terminates in any given cycle of the list.", "As will be appreciated, traditional telephone recording systems are often controlled by or determine call details by interacting with the telephony switch via a Computer Telephony Integration (CTI) interface.", "Such an interface typically advises the recording system of call setup and teardown as well as associated details such as dialled digits, calling line identifier (CLI) etc.", "As such systems move to packet transmission of voice, many are doing so incrementally and hence supporting a mix of packet and traditional calls.", "Where such an interface exists, it is beneficial to retain the existing investment in its design and also to allow support of VoIP and traditional calls with a single interface rather than having to support a further interface.", "The present system and related method advantageously supports the use of existing interfaces, provided that where such interface previously provided call identification in the form of telecoms circuit and timeslot information allowing the matching of recorded data stream with call details, it is enhanced to provide equivalent information in the form of IP address and, preferably IP port number.", "Ideally the addresses of both parties to the conversation (be they IP phone and/or gateway) are provided.", "However, in some cases, the telephony system is only aware of the IP address of one or other party involved in the call.", "It is a preferred feature of the present invention that the instruction to record a specified address (source or destination) can optionally be extended with the instruction to automatically start recording of the counterpart with which that address starts to exchange data.", "In the case of an IP destination being recorded, this is achieved by noting the source of the packet that is recorded and creating a corresponding known destination record for that address.", "In this way, subsequent packets sent to the newly identified address will also be recorded.", "Obviously the converse applies should packet source be specified originally.", "Further, where call control packets are analysed to determine which addresses are to be recorded, there is a danger that the failure to receive such a packet could result in the subsequent failure to record the entire conversation e.g.", "if the packet lost contained the extension number of the IP phone and hence it was not recognized that a call to a recorded extension had started.", "Fortunately, many such protocols are redundant and the required information can be deduced from several of the packets.", "According to a further aspect of the present invention, the system can specifically address the case where IP telephones are controlled by an IP-PBX application.", "In such cases it is typically possible to deduce the IP port address to be used for transmission in multiple ways.", "In one case (Cisco CallManager) the controlling application requests that each phone advise it of the port number it can receive incoming audio data.", "It then advises the other phone in the call of that number.", "Hence the recorder can determine the port number to record should it receive either of these interactions.", "Should it receive both, the redundant information can easily be recognized as such and ignored.", "Yet further, when recording RTP streams, the header of these packets can be almost entirely derived from that of the previous packet and hence contains little of value.", "By advantageously arranging for the identification and storage of only the payloads of successive RTP messages, the storage requirements for recording a session are dramatically reduced.", "This is particularly so in the case of compressed audio transmission where the audio payload may be as little as twenty bytes in a packet of over one hundred bytes in total.", "An embodiment of the present invention also addresses the issue of packet loss by being arranged to compare the sequence number of the received packet with that of the previously received packet.", "Should these differ by more than one, packet loss can be deduced.", "In addition to noting the loss in system performance counters, the missing packets can be “padded” out in the buffer being used to receive the recording.", "Aspects of the present invention can also address the use of silence suppression which results in breaks in the transmission stream.", "In such cases, the timestamps of successive packets indicate the extent of the suppressed silence whilst the successive sequence numbers let us differentiate between deliberate silence suppression and the accidental loss of packets as described above.", "When packets are suppressed in this way, the present invention provides for either: the insertion of the appropriate amount of “padding” data which may be pure silence or “noise” derived according to a compression scheme; or the insertion of explicit indication of the silent period into the recorded stream.", "Such as scheme is described by Microsoft in the definition of the “.WAV” file format.", "This provides more efficient storage of such silence periods but unfortunately, most “.WAV” file players do not support this mechanism.", "The present invention can advantageously further optimize storage by supporting a range of compression formats and acting on the received data according to user defined rules.", "These can include: compressing data received in a specified format (e.g.", "G.711 mu-law) to a specified second format (e.g.", "G.726 16 kbps); leaving data received in a compressed format in that format (to avoid the inherent quality reduction associated with decompression and further compression; optionally mixing the two halves of a communication session into a single, mono, recording (preferably) prior to compression; or optionally saving to non-volatile storage (e.g.", "disk) a summary representation of the audio levels on each of the two channels prior to compression and/or mixing.", "This Energy Envelope representation allows a compact (20 samples per second, 2 byte values) representation that permits the graphical display of the call showing which party was speaking at what level even though the two halves of the call are subsequently mixed and only available as a mono rather than stereo signal.", "Within aspects of the present invention, by storing the packet payload in a standard .WAV file, the recorded audio can be replayed alongside traditional recordings using any existing replay mechanism.", "Also, given the ability of the system to record packet data with timestamps for each packet received, the system may be run in a special mode in which all packets recorded are processed and stored as described in above for the aforesaid other packet oriented data.", "By storing all packets, including RTP streams, in this way, the recordings made can subsequently be used advantageously as known test cases for exercising and testing the invention.", "By replaying recordings made in this way over the network, recorders under test will receive exactly the same packet stream as the recorder that made the original recording did.", "Hence known test cases can be run automatically and the output of the recorders compared with that of previous test runs.", "It is a further feature of the present invention that said recording files can be arranged to be processed offline, so as to multiply the traffic and hence simulate larger systems than were actually recorded.", "By replicating the packets in the recording file and, for example, incrementing the IP port numbers for each replication, large volumes of traffic can be simulated from a single recording.", "In another aspect of the invention, it is important that such recorders do not lose data packets as this will reflect in loss of audio and/or loss of control information and hence possibly missed calls.", "The present invention therefore can be arranged to monitor its performance in the following way: 1.the gaps in the received packet stream can be determined as described above; 2.by analysing the RTCP packets received alongside the RTP packet streams it is possible to see the loss statistics being experienced by the actual participants in the call; 3.by comparing the above two figures an estimate of the packet losses occurring somewhere other than en route between the end points can be determined.", "Some of this will be due to the recorder and can be used as an indication of the quality of the recording; In yet another aspect and as discussed above, recordings may advantageously be stored independently for each direction of transmission or combined into a single, mixed recording.", "The former is highly advantageous should analysis of who was talking be required e.g.", "to identify angry callers from high interruption levels or in performing speech recognition on the files.", "The latter however is both less accurate and less useful when performed on a mixed recording since a single speaker model cannot be used and it is not clear who said each word.", "On conference calls it is particularly beneficial to retain a recording of each party involved in the call since with many speakers, it can be difficult to differentiate between speakers with similar voices, and there is often background noise from one or more parties that makes it difficult to hear the principal speaker It is a preferred feature of the present invention that the recordings of a multiparty interaction can be kept as independent streams or mixed together to optimize storage space.", "As mentioned above, it is possible to retain a summary record of the level of audio from each speaker even though the actual audio signal has been mixed.", "It is a further preferred feature of the present invention that when such conferences are handled by a conference bridge, which mixes the signals, storage of the conversations can be limited to the minimum set of data streams needed to reconstruct the conference audio.", "This advantageous aspect is achieved as follows for a conference between parties A, B and C using conference bridge X A, B and C each transmit audio to X X transmits audio from (B+C) to A X transmits audio from (A+C) to B X transmits audio from (A+B) to C The recorder may simply be instructed not to record packet streams coming from the conference bridge X and hence only records the pure audio from each of the contributing parties.", "The required mixed signals can be reconstructed at replay time if required from these three streams.", "Of course, it should be appreciated that the invention can comprise any one or more of the aspects and embodiments described above in combination or alone.", "The invention is described further hereinafter, by way of example only, with reference to the accompanying drawings in which: FIG.", "1 is a block diagram of a recording sub-system embodying the present invention; FIG.", "2 is a schematic block diagram showing the operation of an IP address filter embodying an aspect of the present invention; FIG.", "3 is a schematic block diagram showing the operation of a protocol filter embodying an aspect of the present invention; FIG.", "4 is an illustration of packet data buffering as arising in an embodiment of the present invention; FIG.", "5 is an illustration of RTP/PTCP Packet Buffering as arising in an embodiment of the present invention; and FIGS.", "6A-6C illustrate the structure of IP packets, RTP packets and RTCP packets as arising within the present invention.", "Turning now to FIG.", "1, there is illustrated a recording sub-system embodying the present invention and in which a recorder is connected to a network from which calls are to be recorded by connecting into allocated ports on one or network routers, switches or hubs such as the 10/100 Mbps Ethernet hubs [1], [2], [3].", "In the case of intelligent switches, which only transmit data packets to those ports that need to receive the data, a “SPAN” setting is normally applied to the port used for the recorder.", "This setting is normally used for network diagnostics and forces the switch to copy packets destined for one or more of its other ports to this port as well.", "Each router/switch/hub is connected to an appropriate form of network interface card (NIC), for example 10/100 Mbps Ethernet, Token Ring, Gigabit Ethernet [4], [5].", "The NIC [5] is an example of a multiport NIC which allows connection to more than one network access point.", "This allows efficient recording of larger networks where calls are spread across several network concentrator devices, without the need for a separate recorder for each of these.", "Within the operating system on the Recorder, such as Windows 2000 there will be installed one or more drivers that allow applications to communicate with the NIC(s) installed.", "A common NDIS driver (6) is illustrated.", "This typically allows other applications running on the same computer to share access to the NICs without having to be aware of each other.", "Hence this diagram only shows the recording sub-system.", "Note the archival, search and retrieval functions typical of a bulk voice recorder can still be hosted on the same PC providing of course that there are sufficient memory, processor and disk resources for these to co-exist.", "The system described communicates with the NDIS driver [6] and establishes a data channel which it places in promiscuous mode.", "This overrides the normal behaviour in which only data packets destined for this application will be delivered and instead requests that all data packets received via the network access point are passed through to an IP Address Filter mechanism [7].", "The IP Address Filter mechanism [7] is described in more detail below with reference to FIG.", "2.In summary, it compares the IP address and port number of each received IP packet against a list of known destinations.", "This could be equally well be applied to known sources rather than destinations.", "A rapid decision is made as to whether this packet is to be recorded or not.", "If the answer is yes, it will pass the packet on to a Protocol Filter [8].", "This second stage filter [8] can therefore now use both knowledge of the destination to which the packet was sent and information from the IP packet itself to determine whether the packet contains RTP or RTCP data or other packet data.", "The filter [8] passes the packet on to either a Packet Data Buffering module [9] or a RTP Packet Buffering module [10].", "Each of these two buffering modules [9], [10] applies schemes appropriate to the data type being recorded before passing on data, in relatively large, contiguous blocks, to the Record Thread Manager [14a] which, running at slightly lower priority than the previous components will write this data to the appropriate files on the hard disk.", "This results in efficient large data writes to the permanent storage such as a hard disk [11].", "The end result is typically a pair of files one containing the actual content of the data stream, for example a .WAV audio file for RTP transmitted telephony, and the other containing details of the recording such as a .XML file containing start time, duration, IP address etc.", "Alternatively the details of the recordings may be inserted directly into a database.", "In buffering the RTP data streams, the RTP Packet Buffering module analyses the sequence numbers and timestamps of the received packets.", "It compares the packet loss rates it is experiencing against those reported by the participants in the streaming interaction and can hence deduce performance levels which it logs for example to a file [14].", "It could equally well pass this information into a network management tool such as via Simple Network Management Protocol (SNMP).", "The set of known destinations that are to be recorded can be fixed in a variety of ways.", "For example, it could be achieved by direct, local configuration of the IP Address Filter [7], through blanket rules such as recording all streams; or it can be instructed explicitly by a component such as Unify [12] a CTI middleware platform which interfaces with a wide range of telephony and other customer contact service systems [13].", "By observing activity on the external system and applying business rules, the addresses to be recorded can be readily identified.", "With reference to FIG.", "2, once a packet is received and determined to be an IP packet it is examined by the IP Address Filter to determine whether or not it is to be recorded.", "It should be appreciated that, according to the network traffic patterns experienced and the proportion to be recorded, the order and/or presence of each of these filtering stages may be changed to deliver optimum performance typically measured by the packet rate that can be processed within a given proportion of the CPU time available.", "The IP packet [15] is normally first compared [17] with the IP address of the computer on which the recorder is hosted.", "This allows very rapid elimination of all packets that are intended for other processes on this computer and ensures that the hosting of, for example, a search and replay application on the same computer does not adversely impact the performance of the recording sub-system.", "The packets which were addressed to other IP addresses are then passed to a circular buffer lookup algorithm [21].", "Recently received IP destination addresses including IP port number, are noted in a circular buffer [18].", "When a packet is received, its IP address is compared with that of the entry at a Test Point [19] in the circular buffer [18].", "If a match is identified, the Test Point [19] is advanced and the entry in the circular buffer is used as a pointer to the Known Destination Object [16] for this IP address.", "An entry in the Known Destination object's [16] data structure indicates whether or not the packet is to be recorded [25].", "If it is, then it is passed through to the Protocol Filter (next section).", "If the packet's IP address does not match the next item in the Circular Buffer [18], it is compared against one or more subsequent entries and if a match is found, the test point [19] is advanced to this point.", "The entries that were tested and found not to match will be removed from the buffer as it can be deduced that they are no longer in the correct position or the stream to those addresses has terminated.", "If the packet's IP address does not match any of those within the lookahead window described above, it is passed to the following stage of the filter [22] in which the IP address is used to determine if there is an existing Known Destination record [16] for this IP address.", "The Known Destination list is typically held in memory in the form of a map with a fast indexing or hash function that allows rapid searching of many such records for the one with a specific address.", "However, the nature of IP addresses (e.g.", "10.25.34.245) is deliberately hierarchical with the earlier numbers (when represented in text string form as here) representing the larger sub-networks.", "It is therefore very common for the vast majority of traffic observed on a single network segment to involve IP addresses from that sub-net i.e.", "the first 2 or even 3 of the numbers will be common.", "In such cases it is viable to maintain one or more look-up tables of Known Destination Maps [26] which simulate content addressable memory For example, such a table, with 65,536 entries can hold pointers to all the Known Destination records [16] relating to the block of IP addresses 10.10.xxx.yyy where xxx and yyy are any number from 0 to 255.It is therefore very easy and quick to compare the top (leftmost when written as text) two bytes of an IP address against the base address of such a lookup table and, if they match, use the lower (rightmost when written as text) bytes as an index into the table.", "A 0 (null) entry in the table infers that the destination does not yet have a Known Destination record associated with it whilst a non-zero entry can contain a pointer to the relevant Known Destination object.", "If the Destination is not within the range of any such rapid lookup maps [26], the normal hash table or other indexing method will be used to determine if a Known Destination Record exists.", "If it does, it will be inserted into the Circular Buffer [18] at the insertion point normally immediately prior to the test point [19].", "If an existing Known Destination record is not found, then a new record will be created and the Record flag will be set to true or false according to the default recording rules [23].", "Having found, or created, the appropriate Known Destination Record [16], the packet will at [25] either be discarded or passed to the Protocol Filter according to the Record flag within the Known Destination Record.", "It should be noted that the Known Destination Records can also be created, deleted and their recording flag set/reset by the Unify interface [12].", "Also in order, to avoid long term build-up of redundant Known Destination Records [16], a background process can serve to periodically review the last time that a packet was observed being sent to that destination and will destroy the Known Destination Record should this exceed a pre-determined threshold.", "To enable this function, a data field within the Known Destination Record structure is updated to reflect the current system time whenever it is used to determine whether or not to record a packet.", "As illustrated in FIG.", "3, the Protocol filter stage can be embodied very simply.", "It first examines the contents of the Known Destination Record [16] that is by now associated with the packet being processed.", "This record may contain information that allows the protocol filter to determine which protocol the packet should contain.", "This is particularly advantageous in the case of RTP data streams as there is no guaranteed method for determining that a packet is indeed an RTP packet.", "Such packets are identified as UDP protocol but there is no indication within the UDP packet that the payload is indeed RTP.", "Unless an external influence such as Unify [12] has deduced that RTP is to be expected on a given destination IP address, the only alternative is to determine if the first few bytes of the UDP packet's payload represent valid entries for an RTP packet header.", "This is time consuming and prone to occasional false detection.", "Hence, if the protocol being sent to a specific port is known already, this can be used to direct the packet at either the Packet Data Buffer [30] or the RTP/RTCP Buffer [31].", "If the Known Destination Record [16] does not specify the protocol expected, the filter next examines the protocol type specified within the IP packet.", "A typical filter will simply check for TCP [28] and pass these packets to the Packet Data Buffer [30] then check for UDP packets [29] and pass these to the RTP/RTCP Packet Buffer [31].", "According to pre-configured default behaviour [32], other protocol types may be either discarded [33] or sent to one or other of the two described buffering modules or to a new buffering module specifically optimized for storage of that protocol type such as ARP, ICMP or SNMP.", "Turning now to FIG.", "4 there is illustrated the packet data buffering of the present invention.", "This can prove to be relatively straightforward with each packet simply being preceded by any or all of: A well-known synchronization value which will ensure eventual re-synchronization of a partially corrupt file; A precise time stamp; Offset of local time from Universal Co-ordinated Time (UTC); Daylight Savings Time offset; or Length of packet that follows.", "If this is the first packet being recorded for a given Known Destination [16] then the Known Destination record will not yet have had a buffer storage element [37] assigned.", "A start recording job is posted onto the queue [35] for the Record Thread Manager and a buffer [37] is assigned.", "These buffers are typically several Kbytes in length and can hence hold many individual packets.", "The packet to be stored and its associated data as listed above is appended to the buffer.", "When subsequent packets are received, these are appended to the buffer [37].", "At the point where the buffer fills, it is moved to the FIFO queue [35] to await processing by the Record Thread Manager [39].", "When the job reaches the head of the queue, it will be appended to the appropriate file on disk [34].", "Meanwhile, a new buffer [37] will be allocated to the Known Destination Record [16] and any remnants of the packet which filled the previous buffer are appended to it.", "This process repeats until the recording is terminated at which point the partially filled buffer [37], if any, is appended to the job queue [35] and a Stop Recording job is then added to the job queue.", "When the Record Thread Manager reaches a Stop Recording job, it closes the file in question.", "Additional details about the call may be included within the Start and Stop Recording messages and these, along with other derived information, such as the total data volume stored are ultimately written to a call detail file, for example in .XML format or direct to a database.", "It should be appreciated that a lower priority thread high priority still being lower than Real-time priority—is used for writing data to disk than for capturing, filtering and buffering packets into memory.", "This allows RAM buffering to absorb short term peak load conditions which would otherwise cause bottlenecks and limit the peak throughput capacity of the recorder.", "FIG.", "5 illustrates the RTP/RTCP Packet Buffering.", "This component can operate in the same way as the Packet Data Buffering component described above except for the way in which it handles the content of the data packet itself.", "Since RTP packets typically form a continuous stream of data, there is no need to store the RTP packet header information for all packets as it is only the payload of actual audio/video data that is required.", "When audio is compressed for transmission, the payload can be as little as 20 bytes (G.729A, 20 ms per packet) yet the total packet length, including Ethernet, IP, UDP and RTP headers, will typically exceed 100 bytes.", "Hence storing the whole packet is incredibly inefficient.", "In the simplest case, the payload of each RTP packet is appended to the buffer [37] and, when full, this is placed on the Record Job Queue [35].", "Recording start and stop are exactly as per the Packet Data Buffering component and these two components share a common queue ensuring that data is written to disk in a FIFO manner regardless of which type of recording i.e.", "packet or stream, is being made.", "In the case of the RTP data, the files that are written by the Record Thread Manager are typically in Microsoft .WAV format and hence readable by any player device that supports this file format and the compression standard used within the file.", "The process is complicated somewhat by the need to allow for packet loss.", "This is achieved by maintaining in memory, details of the most recently received packet sequence number and timestamp.", "It is then possible to compare the currently available packet's sequence number and timestamp with that of the previous packet.", "A gap in sequence numbers can be noted and logged to a performance log file or similar [40A].", "To avoid gradual buildup of error in the recorded file, any lost packet(s) can be compensated for by the automatic appending of the appropriate number of bytes of silence (or other pre-defined padding sound).", "Where a small number of bytes are missing (e.g.", "1 or 2 packets) it is more efficient to simply add these to the buffer [37] as if they had been received.", "However, in the case of large gaps, it is more efficient to place the current buffer [37] on the Record Job Queue [35] for processing and then place a Padding job specifying the number of bytes of silence to be inserted.", "The Record Thread Manager can then append the appropriate number of bytes to the file without using excessive memory space on the Record Job Queue as would be the case if several buffers all full of silence were queued.", "This latter technique is particularly advantageous in systems that utilize silence suppression.", "In such cases, the RTP stream will stop until the audio level reaches a threshold.", "This can save significant bandwidth in voice communications as typically only one party is speaking at a time.", "In such cases, the packet sequence numbers in the RTP packet are contiguous but the timestamps are far apart.", "Again, this can be detected and the appropriate amount of silence indicated through the placement of a Padding job on the record job queue [35].", "This then allows the Record Thread Manager to either pad the call with the appropriate amount of silence or to utilize a scheme such as the Wave List feature within the Microsoft .WAV format.", "This allows for the efficient storage of such audio as a sequence of sound, silence, sound, silence etc.", "A further refinement includes the processing of RTCP packets associated with the RTP streams being recorded.", "By examining the contents of these packets, one can determine the fraction and cumulative number of packets lost by each of the participants in the conversation.", "These details can be included with the loss rates experienced at the recorder and included in the Stop Recording job and/or entries to the log file [40A].", "This allows subsequent analysis of the recorder's performance in comparison to that experienced by the participants on the call.", "To assist with the above description, reference is now made to the general structures of the packets arising in the aspects of the present invention.", "Turning now to FIG.", "6A, a header 42 is shown as a plurality of boxes, each of which represents a portion or “field” of the header.", "The number of bytes occupied by each portion is also shown, it being understood that each layer consists of 32 bits.", "The first portion of the header, a “VERS” portion 44, is the protocol version number.", "Next, an “H.LEN” portion 46 indicates the number of 32-bit quantities in the header.", "A “SERVICE TYPE” portion 48 indicates whether the sender prefers the datagram to travel over a route with minimal delay or a route with maximal through-put.", "A “TOTAL LENGTH” portion 50 indicates the total number of octets in both the header and the data.", "In the next layer, an “IDENTIFICATION portion 52 identifies the packet itself.", "A “FLAGS” portion 54 indicates whether the datagram is a fragment or a complete datagram.", "A “FRAGMENT OFFSET” portion 56 specifies the location of this fragment in the original datagram, if the datagram is fragmented.", "In the next layer, a “TIME TO LIVE” portion 58 contains a positive integer between 1 and 255, which is progressively decremented at each route travelled.", "When the value becomes 0, the packet will no longer be passed and is returned to the sender.", "A “TYPE” portion 60 indicates the type of data being passed.", "A “HEADER CHECKSUM” portion 62 enables the integrity of the packet to be checked by comparing the actual checksum to the value recorded in portion 62.The next layer of header 42 contains the source IP address 64, after which the following layer contains the destination IP address 66.An optional IP OPTIONS portion 68 is present, after which there is padding (if necessary) and a data portion 70 of the packet containing the data begins.", "As shown in FIG.", "6B, an RTP packet header 92 features several important fields: a timestamp field 94, a synchronization source (SSRC) identifiers field 96 and a contributing source (CSRC) identifiers field 98.SSRC field 96 is used to determine the source of the RTP packets (the sender), which has a unique identifying address (the SSRC identifier).", "The CSRC identifer in CSRC field 98 is used in a conference with multiple parties, and indicates the SSRC identifier of all parties.", "Timestamp field 94 is used by an RTP software module to determine the relative time at which the data in each packet should be displayed.", "Finally FIG.", "6C shows the general structure of an RTCP packet." ] ]	Patent_10467899
[ [ "Method and system for multiple hosts anycast routing", "Conventional anycast networks provide a service which allows a sender to access the nearest of a group of receivers sharing a common anycast address.", "In contrast thereto, multicast networks establish data communications between a sender and all receivers confined by a group having the same multicast address.", "Thus, the networks providing both anycast and multicast routing only allow for accesses by a sender to one receiver being specified according to the employed anycast protocol or to a plurality of receivers forming a multicast group.", "In order to provide accesses by a sender to a specified number of nearest receivers, the present invention provides a method and a system for multiple hosts anycast routing in a network.", "An indicator specifying the number of nearest receivers to be set up for data communications with a sender is included in an anycast address or associated thereto as an extension.", "On the basis of the indicator, a corresponding number of anycast members of an anycast group identified by the anycast address are contacted/allocated for data communications with the sender." ], [ "1.A method for multiple hosts anycast routing in a network, the method comprising: communicating sender data from a sender addressed to an anycast group including anycast members, said sender data including an anycast address for addressing said anycast group and a first indicator specifying a first number of said anycast members within said anycast group for data communications with said sender, and determining a number of nearest of said anycast members within said anycast group as hosts for data communications with said sender, said number of nearest anycast members being defined by said first indicator.", "2.The method according to claim 1, comprising: communicating further sender data from said sender addressed to said anycast group including said anycast members, said further sender data further including a second indicator specifying a second number of said anycast members within said anycast group, said second number being indicative of already determined nearest of said anycast members within said anycast group, and determining said number of nearest of said anycast members within said anycast group, said number of nearest anycast members being defined by said first and said second indicator.", "3.A method for multiple hosts anycast routing in an anycast capable network, the method comprising: associating a first indicator with an anycast address identifying an anycast group having anycast members in a network, said first indicator specifying a first number of anycast members within said anycast group, communicating at least said anycast address and said first indicator in said network to said anycast group, and determining a number of nearest of said anycast members within said anycast group as hosts for data communications in said network, said number of nearest anycast members being defined by said first indicator.", "4.The method according to claim 3, comprising: associating a second indicator with said anycast address, said indicator specifying a second number of said anycast members within said anycast group, said second number being indicative of already determined nearest of said anycast members within said anycast group, communicating at least said anycast address, said first indicator and said second indicator in said network to said anycast group and determining said number of nearest of said anycast members within said anycast group, said number of nearest anycast members being defined by said first and said second indicator.", "5.The method according to claim 2, wherein said number of nearest anycast members to be determined is defined by the difference between said first number and said second number.", "6.The method according to claim 1, wherein said first and/or said second indicator is included in said anycast address.", "7.The method according to claim 1, wherein said anycast address is extended by said first and/or said second indicator.", "8.The method according to claim 1, wherein an anycast address prefix is included in said anycast address, said anycast address prefix identifying a topological region in said network confining said anycast group, said anycast address prefix including said first and/or said second indicator or being extended by said first and/or said second indicator.", "9.The method according to claim 1, wherein said anycast address and said first and/or said second indicator are communicated by communicating an Internet Protocol data packet header including said anycast address.", "10.The method according to claim 9, wherein said first and/or said second indicator is included in said Internet Protocol data packet header as a part or an extension of said anycast address, or said Internet Protocol data packet header is extended by said first and/or said second indicator by means of an options field for Internet Protocol data packet headers.", "11.The method according to claim 1, wherein said number of nearest anycast members is determined by utilizing a measure of distance of a routing protocol for said network and/or determined by utilizing a measure of distance specified by said sender.", "12.The method according to claim 1, wherein said anycast address and said first and/or said second indicator are communicated in said network between domains thereof via routers to network domains having at least one of said anycast members.", "13.The method according to claim 12, wherein an ordering of said routers is defined such that said anycast address and said first and/or said second indicator are communicated to the nearest network domains, the nearest network domains being specified according to a measure of distance of a routing protocol for said network and/or according to a measure of distance specified by said sender.", "14.The method according to claim 1, wherein said number of anycast members is contacted sequentially.", "15.", "(Cancel) 16.The method according to claim 1, wherein said first indicator is modified according to a number of already determined nearest anycast members such that said modified indicator specifies a number of said anycast members still to be determined as receivers.", "17.The method according to claim 16, wherein said first indicator is decreased by the value of said second indicator.", "18.The method according to claim 1, wherein said second indicator is decreased to a value corresponding to a number of 0 and said first indicator is decreased correspondingly to the decrease of the second indicator and said determining of said number of nearest of said anycast members within said anycast group is performed in dependence of said decreased first indicator if said second indicator has a value corresponding to a number of 0.19.The method according to claim 1, wherein said anycast address and said first and/or said second indicator are communicated to at least two network domains including at least one of said anycast members by multiplying said anycast address and said first indicator, and communicating said multiplied anycast address and said first and/or said second indicator to anycast members.", "20.The method according to claim 1, wherein said first number specified by said first indicator is decreased corresponding to a number of said anycast members in a network domain to which said anycast address and said first indicator are communicated.", "21.The method according to claim 1, wherein said communicating is performed according to the strict routing mechanism or the loose routing mechanism as defined in for the Internet Protocol.", "22.The method according to claim 1, wherein said first and said second indicator are communicated together.", "23.A system for multiple hosts anycast routing, comprising: a network a sender, and a group of receivers being identified by an anycast address, wherein data communications between said sender and a number of nearest of said receivers are established on the basis of a first indicator provided by said sender specifying said number of receivers.", "24.The system according to claim 23, wherein data communications between said sender and said number of nearest of said receivers are further established on the basis of a second indicator provided by said sender specifying a number of receivers already determined for data communications with said sender.", "25-28.", "(Cancel) 29.The method according to claim 4, wherein said number of nearest anycast members to be determined is defined by the difference between said first number and said second number.", "30.The method according to claim 3, wherein said first and/or said second indicator is included in said anycast address.", "31.The method according to claim 3, wherein said anycast address is extended by said first and/or said second indicator.", "32.The method according to claim 3, wherein an anycast address prefix is included in said anycast address, said anycast address prefix identifying a topological region in said network confining said anycast group, said anycast address prefix including said first and/or said second indicator or being extended by said first and/or said second indicator.", "33.The method according to claim 3, wherein said number of nearest anycast members is determined by utilizing a measure of distance of a routing protocol for said network and/or determined by utilizing a measure of distance specified by said sender.", "34.The method according to claim 3, wherein said first indicator is modified according to a number of already determined nearest anycast members such that said modified indicator specifies a number of said anycast members still to be determined as receivers.", "35.The method according to claim 34, wherein said first indicator is decreased by the value of said second indicator." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Technical Field The present invention relates to a method and a system for establishing data communications between a sender and receivers, the number thereof being defined by the sender, wherein the receivers are not individually addressed by the sender, but selected from a group of receivers being addressed from the sender by means of a common address.", "2.Background of the Invention An anycast network is a network providing a service which allows a sender to access the nearest of a group of receivers having a common address.", "The common address, in particular, the same anycast address for the group of receivers enables a sender to identify a plurality of receivers by providing only one address, namely an anycast address, instead of individually addressing each receiver.", "In contrast to a multicast system, wherein data communications are performed between a sender and all receivers sharing a common address, i.e.", "the same multicast address, data communications in an anycast network are performed between a sender and a particular receiver of a group of receivers sharing the same anycast address.", "In the context, data communications include any kind of data, such as alphanumerical, graphic, multimedia, audio, video, voice data, information, signals etc.", "which can be exchanged between systems, devices, network components etc.", "(e.g.", "computers, end user devices, hosts, servers, routers, modes).", "In particular, data communications in an anycast network are performed between a sender and the nearest receiver of the group of receivers sharing the same anycast address.", "In this respect, the nearest receiver in relation to a sender is defined according to a measure of a distance employed by a routing protocol or routing system used for data communications in a respective network.", "Since the routing protocol or routing system of a network which identifies the nearest receiver for accesses by a sender, the sender does not need to care about how to select the closest destination, i.e.", "the nearest receiver.", "Usually, receivers in an anycast group are so-called replicas being able to support a same network service for requesting sender.", "Examples for such replicas are mirrored web servers.", "For accessing a desired network service, among a group of receivers sharing the same anycast address, i.e.", "anycast receivers, each thereof providing the desired network service, the nearest receiver is determined and respective data communications are performed between the nearest receiver and the requesting sender.", "Thus, accessing the nearest receiver enhances the network performance perceived by the requesting sender, saves network capacities such as network's bandwidth, and provides the desired network service.", "In FIG.", "1 , the basic principle of an anycast network is illustrated.", "Both, members M 1 and M 2 are members of the same anycast group and share the same respective anycast address.", "In line with the measure of a distance employed for routing of data in the network, the distances between requesting senders S 1 and S 2 and all members of the anycast group are computed.", "Since the distance 2 calculated for the sender S 1 in relation to the anycast group members is the smallest distance, data communications will be performed between the sender S 1 and the member M 1 acting as a receiver for the sender S 1 .", "In this manner, it is possible for example to direct Internet Protocol (IP) datagrams from the sender S 1 towards the member M 1 .", "Accordingly, data communications are established between the sender S 2 and the member M 2 being a nearest receiver with respect to the sender S 2 , for example to direct IP datagrams from the sender S 2 towards the member M 2 .", "Anycast Addresses According to the Internet Protocol version 6 (IPv6), special anycast addresses have been specified in addition to unicast addresses for individually addressing single receivers and multicast addresses for addressing all receivers sharing the same multicast address.", "Anycast addresses are allocated from the address space employed for unicast addresses, using any of the formats defined for unicast addresses.", "As a result, anycast addresses cannot be syntactically distinguished from unicast addresses.", "An anycast address identifies a set of interfaces which typically belong to different nodes of a network.", "Data, e.g.", "a data packet, communicated from a sender to an anycast address is delivered to one of the interfaces identified by the respective anycast address, in particular, to the nearest interface according to the measure of a distance employed for the network routing protocol or system.", "In case, a unicast address is assigned to more than one interface, thus turning the unicast address into a respective anycast address, the nodes of the network to which the address is assigned must be explicitly configured to interpret the address as an anycast address.", "According to the IPv6, an anycast address must not be assigned to a host, but may be assigned to an IPv6 router, only.", "Usually, for any assigned anycast address, an address prefix P is utilized which identifies a topological region in which all interfaces belonging to an anycast address reside.", "In the region identified by the address prefix P, each member of the respective anycast group is advertised as separate entry in a network routing protocol or system.", "Outside the region identified by the address prefix P, the anycast address may be aggregated into the routing advertisement for the address prefix P. Anycast Host Registration In order to join an anycast group, a host instructs its first hop router, i.e.", "the closest router in relation to the host for data communications via a network, to advertise the anycast address of the anycast group on its behalf.", "This can be achieved by adding a new message type to either the Internet Group Management Protocol (IGMP) or the Neighbor Discovery Protocol.", "Then, the first hop router advertises the anycast address according to an anycast routing protocol employed for the domain including the host.", "Anycast Routing Each anycast group is confined to a particular topological region with which it shares a common address prefix.", "Within the topological region identified by the shared address prefix, each member of the anycast group is advertised as a separate entry in the employed unicast routing protocol or system.", "The principle of anycast routing within a topological region identified by an address prefix shared by an anycast group is illustrated in FIG.", "2 .", "As can be derived from the table in FIG.", "2 , there are multiple paths to the anycast address.", "In case, multiple paths exist to a destination address prefix, a routing table look up of the router will return multiple next hops.", "The selection of the next hop router to be used for data communications, e.g.", "a particular data packet, depends on the implemented routing protocol or system.", "Further, the path selected for data communications can also be effected by the type of data communications to be performed.", "For example in case of the Internet Protocol standard, the Type Of Service (TOS) field in the IP header of a data packet can be used to define a data communications path.", "Thus, a TOS designation of a data packet would help the router to chose an appropriate communication path for the given data packet.", "The selection of an appropriate path is accomplished on the basis on a determination of the distances to the destination defined by the address prefix according to a measure of distance of the respective routing protocol or system.", "In case of the Internet Protocol standard, for example, the OSPF knows the distance related to the corresponding matrix as identified in the TOS field of a data packet to the destination, i.e.", "the hop count.", "As a result, for an anycast routing it is possible to select the nearest next hop on the basis of the employed matrix.", "In this respect, it is not necessary to analyze the whole IP address of a given data packet.", "In FIG.", "2 , this in indicated by the destination address Mx wherein only the prefix “M” is analyzed.", "For an anycast routing outside the topological region identified by the shared address prefix, the anycast address may be aggregerated into the routing advertisement for the shared address prefix.", "This principle is illustrated in FIG.", "3 .", "The destination address Ax in FIG.", "3 denotes that only the shared address prefix is utilized in order to determine that data communications are to be routed to the corresponding domain.", "Since the anycast address for the anycast group shares the address prefix with network domain A, network domain B cannot aggregerate the anycast address in its address prefix.", "Therefore, network domain B should advertise the anycast address as a separate entry covering both anycast members A 4 and A 5 .", "This is indicated in FIG.", "3 by the destination address Ax being based on the address prefix for domain A and destination address Ax′ containing the complete anycast address.", "The routing for data communications within the network domains A and B is performed as described before with reference to FIG.", "2 .", "Strict and Loose Routing According to the Internet Protocol version 4 (IPv4) and the Internet Protocol version 6 (IPv6), the following options for routing data communications from a source (e.g.", "sender, requesting system) to a destination (e.g.", "receiver, accessed system) have been defined as part of the IP data packet header: Strict Routing This option defines a complete data communications path from a source to a destination by means of a sequence of IP addresses.", "Data to be communicated between the source and the destination is required to exactly follow the defined path.", "Loose Routing This option specifies a number of routers and an order thereof.", "Data to be communicated from a source to a destination is required to traverse the specified routers in the specified order, but is allowed to be communicated via other routers on its way from the source to the destination.", "Problems Existing solutions for networks being capable of anycast routing are restricted to the selection of a single nearest receiver for data communications with an accessing sender.", "In case a sender intends to access more than one receiver, existing anycast networks do no provide such a service.", "A situation for which accesses to more than one receiver are desired is, for example, a network user wanting to access a number of nearest network servers in order to negotiate for the best network service conditions.", "A further example is a network user wishing to contact a number of nearest restaurants by accessing the network servers thereof in order to check e.g.", "meals, prices, available seats, etc.", "or wishing to access a number of nearest emergency service providers in order to ensure that at least one is able to assist (note for the latter examples, sufficient information for the accessing user is only provided in case there is a geographical relationship between the restaurants and the emergency service providers and the corresponding servers).", "In conventional networks, accesses to more than one receiver can be accomplished by accessing a number of receivers by individually addressing the same on the basis of a unicast routing or by accessing a group of receivers by commonly addressing the same on the basis of a shared multicasting address.", "Such a multiple unicast routing requires that an accessing user must know each individual network address of the desired receivers and individually perform accesses thereto.", "This is a time consuming procedure which cannot lead to the desired information for a user in a case, for example, he/she is not provided the respective network addresses and/or information concerning distances to the accessed servers.", "With respect to the above given example concerning a negotiation for the best network service conditions by accessing a number of nearest servers, such a multiple unicast routing is also not appropriate since no information indicative of distances to receivers is provided and/or available.", "Accesses on the basis of a multicast routing can result in a number of contacted receivers being too high in view of the demands and desired information of a requesting sender/user.", "Further, with multicast routing it is not possible to access a desired number of receivers and, in particular, a desired number of nearest receivers, since all receivers being a member of the respective multicasting group are addressed together.", "Furthermore, a second attempt, e.g.", "by a multiple unicast routing, to access/contact receivers, the number thereof being higher compared to the number of receivers specified in a preceding, first attempt, would unnecessarily return the results from the first attempt again.", "This repetition of data communications with respect to the result of the first attempt unnecessarily utilizes network resources in case the requesting network user is not interested in being provided the first attempt results again." ], [ "<SOH> BRIEF DESCRIPTION OF THE INVENTION <EOH>The approach underlying the present invention to obtain the above object is to extend principles employed in existing anycast networks for accessing a single nearest client in order to allow for a multiple anycast routing to a specified number of nearest receivers.", "In principle, this is achieved by associating data communications from a sender directed to a network by means of an anycast routing to data which indicate the number of nearest receivers.", "In particular, such data which will be referred to as a first indicator in the following, specifies the number of nearest receivers being anycast members of an anycast group identified by an anycast address given by the sender (left) to be contacted by and/or allocated for the sender for data communications, such as requests, accesses, data transmissions, etc.", "According to the present invention, a method for multiple hosts anycast routing in a network is provided wherein sender data is communicated from a sender to an anycast group including anycast members.", "For communicating the sender data to the anycast group, the sender data includes an anycast address for addressing the anycast group.", "In order to specify a number of the anycast members for data communications with the sender, the sender data further includes a first indicator being indicative of the desired, first number of anycast members.", "On the basis of the sender data, a number of nearest anycast members is determined as hosts with respect to the sender for data communications.", "In particular, the number of nearest anycast members being determined is defined by the first indicator.", "This solution provides for data communications between a sender and a number of receivers without addressing the same separately, but addressing a plurality of possible receivers by a single address being common for all receivers and selecting a desired number therefrom by means of a data being simply amended to the single common address.", "A further benefit is that it can be employed for any network, even for networks not providing known anycast routing services.", "After having performed the above described method it may be possible that a further multiple host anycast routing should be performed wherein the number of anycast members to be determined is varied compared to the number of anycast members previously specified.", "In order to avoid that anycast members already determined in the previous multiple host anycast routing are contacted again a second indicator can be used.", "The second indicator specifies the number of anycast members previously determined in the preceding multiple host anycast routing.", "Then, the number of nearest anycast members can be determined on the basis of the first and the second indicator.", "In case the first indicator specifies a number of anycast members then the second indicator does, the number of nearest anycast members indicated by the second indicator (in the following: second number of anycast members) are skipped and not contacted, while a number of anycast member is determined for data communications with the sender up to a number obtained by a comparison of the number of anycast members specified by the first indicator (in the following: first number) and the second number.", "In particular, the number of nearest anycast members to be determined corresponds to the difference between the first number and the second number.", "In case the first number corresponds to the second number, no further anycast members have to be determined.", "The same applies for the case wherein the first number is smaller than the second number.", "In order to provide for a method according to the invention for multiple hosts anycast routing to be employed in a network providing a known anycast routing, a first indicator specifying a first number of anycast members to be contacted/allocated for data communications is associated to an anycast address.", "By means of the anycast address, an anycast group having anycast members is identified in the network.", "To establish the desired data communications, at least the anycast address and the first indicator is communicated in the network to the anycast group.", "The anycast group is identified by the anycast address, while the number of anycast members to be contacted/allocated for data communications is selected according to the first indicator.", "In particular, the first indicator defines the number of nearest anycast members and according to the first indicator, the specified number of nearest anycast members is determined as hosts for data communications in the network.", "Comparable to the embodiment of the above described method for multiple hosts anycast routing in a network, this method for multiple hosts anycast routing in an anycast capable network can employ a second indicator specifying a number of anycast members (in the following: second number) already determined for data communications in the network, e.g.", "on the basis of a previously performed multiple hosts anycast routing in a (anycast capable) network.", "For providing the first and/or the second indicator, it is possible to include the indicator(s) in the anycast address or to extend the same by the first and/or the second indicator.", "Preferably, the anycast address is included an anycast address prefix which identifies a topological region in the network, the topological region confining the anycast group.", "Here it is possible, that the anycast address prefix includes the first and/or the second indicator or is extended by the same.", "In a case of a network being operated according to an Internet Protocol standard (e.g.", "IPv4 or IPv6) the anycast address and the indicator can be communicated by a transmission of an Internet Protocol data packet header which includes at least the anycast address.", "For communicating the first and/or the second indicator, the Internet Protocol data packet header can be included the first and/or the second indicator as a part of or an extension for the anycast address.", "With respect to the options mechanisms defined for the Internet Protocols, the first and/or the second indicator can be provided by extending the Internet Protocol data packet header by an options field and, in particular, a so-called multiple anycasting options field including the first and/or the second indicator.", "It is preferred, that the property “nearest” of anycast members is determined according to a measure of distance of the respective routing protocol employed for the network.", "In particular, “nearest” may be understood as the shortest distance between a sender and anycast members.", "Additionally or as an option, “nearest” may be specified according to a distance metric requested by the sender.", "Usually, the distance metric requested by the sender, e.g.", "in the TOS field of an IP header, indicates a smaller distance metrics or nearest distance, respectively.", "In case the network comprises domains at least some of which including at least of the anycast members, the anycast address and the first and/or the second indicator can be communicated via network routers to and/or between domains having anycast members.", "Since it is assumed that a nearest network domain will also include anycast members being nearer compared to anycast members of more distant domains, it is preferred that the ordering of routers for network domain communications is set up such that the anycast address and the first and/or the second indicator are communicated to nearest network domains.", "The nearest network domains may specified according to a measure of distance of a routing protocol for the network and/or according to a (e.g.", "smallest) distance metric requested by the sender, e.g.", "in the TOS field.", "In order to provide for a fast multiple anycasting, the number of anycast members can be contacted sequentially, in particular for a small number specified by the first and/or the second indicator.", "Within a network domain, multiple anycast routing and, in particular, a communication of the anycast address and the first and/or the second indicator can be accomplished on the basis of data identifying each of the anycast members.", "Such an identifying data can be, for example, provided by means of separate entries for anycast members in a routing table according to the employed routing protocol.", "With respect to the anycast routing performed for a network domain, the first indicator can be updated by decreasing the same according to a number of anycast members comprised by a network domain being identified by means of the anycast address and receiving the first indicator.", "Further, the anycast routing can be enhanced by multiplying the anycast address and the first and/or the second indicator and communicating the multiplied anycast addresses and indicators to network domains and/or anycast members.", "Here, it is preferred to communicate the multiplied data simultaneously to the further network domain and/or anycast members.", "In order to precisely select the specified number of anycast members, while determining the number of anycast members, the indicator is modified according to a number of already determined/contacted/allocated nearest anycast members such that the modified indicator being communicated in the network specifies a number of anycast members still to be determined as receivers.", "This embodiment avoids a utilization of further data beside the indicator to provide information how many receivers are already obtained and how many are still to be selected.", "According to an Internet Protocol standard, the communication in the network can be performed by using the strict routing mechanism or the loose routing mechanism.", "Preferably, the first and the second indicator are communicated together and, advantageously in the same manner, e.g.", "both indicators being included in the anycast address or extending the same.", "Further, the present invention provides a system for multiple hosts anycast routing and an anycast routing capable network which are preferred to be operated according to the method according to the invention as set forth above.", "Moreover, the present invention provides a computer program product being adapted to carry out the method steps according to the invention and embodiments thereof as set forth above." ], [ "BACKGROUND OF THE INVENTION 1.Technical Field The present invention relates to a method and a system for establishing data communications between a sender and receivers, the number thereof being defined by the sender, wherein the receivers are not individually addressed by the sender, but selected from a group of receivers being addressed from the sender by means of a common address.", "2.Background of the Invention An anycast network is a network providing a service which allows a sender to access the nearest of a group of receivers having a common address.", "The common address, in particular, the same anycast address for the group of receivers enables a sender to identify a plurality of receivers by providing only one address, namely an anycast address, instead of individually addressing each receiver.", "In contrast to a multicast system, wherein data communications are performed between a sender and all receivers sharing a common address, i.e.", "the same multicast address, data communications in an anycast network are performed between a sender and a particular receiver of a group of receivers sharing the same anycast address.", "In the context, data communications include any kind of data, such as alphanumerical, graphic, multimedia, audio, video, voice data, information, signals etc.", "which can be exchanged between systems, devices, network components etc.", "(e.g.", "computers, end user devices, hosts, servers, routers, modes).", "In particular, data communications in an anycast network are performed between a sender and the nearest receiver of the group of receivers sharing the same anycast address.", "In this respect, the nearest receiver in relation to a sender is defined according to a measure of a distance employed by a routing protocol or routing system used for data communications in a respective network.", "Since the routing protocol or routing system of a network which identifies the nearest receiver for accesses by a sender, the sender does not need to care about how to select the closest destination, i.e.", "the nearest receiver.", "Usually, receivers in an anycast group are so-called replicas being able to support a same network service for requesting sender.", "Examples for such replicas are mirrored web servers.", "For accessing a desired network service, among a group of receivers sharing the same anycast address, i.e.", "anycast receivers, each thereof providing the desired network service, the nearest receiver is determined and respective data communications are performed between the nearest receiver and the requesting sender.", "Thus, accessing the nearest receiver enhances the network performance perceived by the requesting sender, saves network capacities such as network's bandwidth, and provides the desired network service.", "In FIG.", "1, the basic principle of an anycast network is illustrated.", "Both, members M1 and M2 are members of the same anycast group and share the same respective anycast address.", "In line with the measure of a distance employed for routing of data in the network, the distances between requesting senders S1 and S2 and all members of the anycast group are computed.", "Since the distance 2 calculated for the sender S1 in relation to the anycast group members is the smallest distance, data communications will be performed between the sender S1 and the member M1 acting as a receiver for the sender S1.In this manner, it is possible for example to direct Internet Protocol (IP) datagrams from the sender S1 towards the member M1.Accordingly, data communications are established between the sender S2 and the member M2 being a nearest receiver with respect to the sender S2, for example to direct IP datagrams from the sender S2 towards the member M2.Anycast Addresses According to the Internet Protocol version 6 (IPv6), special anycast addresses have been specified in addition to unicast addresses for individually addressing single receivers and multicast addresses for addressing all receivers sharing the same multicast address.", "Anycast addresses are allocated from the address space employed for unicast addresses, using any of the formats defined for unicast addresses.", "As a result, anycast addresses cannot be syntactically distinguished from unicast addresses.", "An anycast address identifies a set of interfaces which typically belong to different nodes of a network.", "Data, e.g.", "a data packet, communicated from a sender to an anycast address is delivered to one of the interfaces identified by the respective anycast address, in particular, to the nearest interface according to the measure of a distance employed for the network routing protocol or system.", "In case, a unicast address is assigned to more than one interface, thus turning the unicast address into a respective anycast address, the nodes of the network to which the address is assigned must be explicitly configured to interpret the address as an anycast address.", "According to the IPv6, an anycast address must not be assigned to a host, but may be assigned to an IPv6 router, only.", "Usually, for any assigned anycast address, an address prefix P is utilized which identifies a topological region in which all interfaces belonging to an anycast address reside.", "In the region identified by the address prefix P, each member of the respective anycast group is advertised as separate entry in a network routing protocol or system.", "Outside the region identified by the address prefix P, the anycast address may be aggregated into the routing advertisement for the address prefix P. Anycast Host Registration In order to join an anycast group, a host instructs its first hop router, i.e.", "the closest router in relation to the host for data communications via a network, to advertise the anycast address of the anycast group on its behalf.", "This can be achieved by adding a new message type to either the Internet Group Management Protocol (IGMP) or the Neighbor Discovery Protocol.", "Then, the first hop router advertises the anycast address according to an anycast routing protocol employed for the domain including the host.", "Anycast Routing Each anycast group is confined to a particular topological region with which it shares a common address prefix.", "Within the topological region identified by the shared address prefix, each member of the anycast group is advertised as a separate entry in the employed unicast routing protocol or system.", "The principle of anycast routing within a topological region identified by an address prefix shared by an anycast group is illustrated in FIG.", "2.As can be derived from the table in FIG.", "2, there are multiple paths to the anycast address.", "In case, multiple paths exist to a destination address prefix, a routing table look up of the router will return multiple next hops.", "The selection of the next hop router to be used for data communications, e.g.", "a particular data packet, depends on the implemented routing protocol or system.", "Further, the path selected for data communications can also be effected by the type of data communications to be performed.", "For example in case of the Internet Protocol standard, the Type Of Service (TOS) field in the IP header of a data packet can be used to define a data communications path.", "Thus, a TOS designation of a data packet would help the router to chose an appropriate communication path for the given data packet.", "The selection of an appropriate path is accomplished on the basis on a determination of the distances to the destination defined by the address prefix according to a measure of distance of the respective routing protocol or system.", "In case of the Internet Protocol standard, for example, the OSPF knows the distance related to the corresponding matrix as identified in the TOS field of a data packet to the destination, i.e.", "the hop count.", "As a result, for an anycast routing it is possible to select the nearest next hop on the basis of the employed matrix.", "In this respect, it is not necessary to analyze the whole IP address of a given data packet.", "In FIG.", "2, this in indicated by the destination address Mx wherein only the prefix “M” is analyzed.", "For an anycast routing outside the topological region identified by the shared address prefix, the anycast address may be aggregerated into the routing advertisement for the shared address prefix.", "This principle is illustrated in FIG.", "3.The destination address Ax in FIG.", "3 denotes that only the shared address prefix is utilized in order to determine that data communications are to be routed to the corresponding domain.", "Since the anycast address for the anycast group shares the address prefix with network domain A, network domain B cannot aggregerate the anycast address in its address prefix.", "Therefore, network domain B should advertise the anycast address as a separate entry covering both anycast members A4 and A5.This is indicated in FIG.", "3 by the destination address Ax being based on the address prefix for domain A and destination address Ax′ containing the complete anycast address.", "The routing for data communications within the network domains A and B is performed as described before with reference to FIG.", "2.Strict and Loose Routing According to the Internet Protocol version 4 (IPv4) and the Internet Protocol version 6 (IPv6), the following options for routing data communications from a source (e.g.", "sender, requesting system) to a destination (e.g.", "receiver, accessed system) have been defined as part of the IP data packet header: Strict Routing This option defines a complete data communications path from a source to a destination by means of a sequence of IP addresses.", "Data to be communicated between the source and the destination is required to exactly follow the defined path.", "Loose Routing This option specifies a number of routers and an order thereof.", "Data to be communicated from a source to a destination is required to traverse the specified routers in the specified order, but is allowed to be communicated via other routers on its way from the source to the destination.", "Problems Existing solutions for networks being capable of anycast routing are restricted to the selection of a single nearest receiver for data communications with an accessing sender.", "In case a sender intends to access more than one receiver, existing anycast networks do no provide such a service.", "A situation for which accesses to more than one receiver are desired is, for example, a network user wanting to access a number of nearest network servers in order to negotiate for the best network service conditions.", "A further example is a network user wishing to contact a number of nearest restaurants by accessing the network servers thereof in order to check e.g.", "meals, prices, available seats, etc.", "or wishing to access a number of nearest emergency service providers in order to ensure that at least one is able to assist (note for the latter examples, sufficient information for the accessing user is only provided in case there is a geographical relationship between the restaurants and the emergency service providers and the corresponding servers).", "In conventional networks, accesses to more than one receiver can be accomplished by accessing a number of receivers by individually addressing the same on the basis of a unicast routing or by accessing a group of receivers by commonly addressing the same on the basis of a shared multicasting address.", "Such a multiple unicast routing requires that an accessing user must know each individual network address of the desired receivers and individually perform accesses thereto.", "This is a time consuming procedure which cannot lead to the desired information for a user in a case, for example, he/she is not provided the respective network addresses and/or information concerning distances to the accessed servers.", "With respect to the above given example concerning a negotiation for the best network service conditions by accessing a number of nearest servers, such a multiple unicast routing is also not appropriate since no information indicative of distances to receivers is provided and/or available.", "Accesses on the basis of a multicast routing can result in a number of contacted receivers being too high in view of the demands and desired information of a requesting sender/user.", "Further, with multicast routing it is not possible to access a desired number of receivers and, in particular, a desired number of nearest receivers, since all receivers being a member of the respective multicasting group are addressed together.", "Furthermore, a second attempt, e.g.", "by a multiple unicast routing, to access/contact receivers, the number thereof being higher compared to the number of receivers specified in a preceding, first attempt, would unnecessarily return the results from the first attempt again.", "This repetition of data communications with respect to the result of the first attempt unnecessarily utilizes network resources in case the requesting network user is not interested in being provided the first attempt results again.", "OBJECT OF THE INVENTION The object of the present invention is to provide for a method and a system allowing for accesses of a sender to a specified number of nearest receivers and/or data communications between a sender and a specified number of nearest receivers.", "BRIEF DESCRIPTION OF THE INVENTION The approach underlying the present invention to obtain the above object is to extend principles employed in existing anycast networks for accessing a single nearest client in order to allow for a multiple anycast routing to a specified number of nearest receivers.", "In principle, this is achieved by associating data communications from a sender directed to a network by means of an anycast routing to data which indicate the number of nearest receivers.", "In particular, such data which will be referred to as a first indicator in the following, specifies the number of nearest receivers being anycast members of an anycast group identified by an anycast address given by the sender (left) to be contacted by and/or allocated for the sender for data communications, such as requests, accesses, data transmissions, etc.", "According to the present invention, a method for multiple hosts anycast routing in a network is provided wherein sender data is communicated from a sender to an anycast group including anycast members.", "For communicating the sender data to the anycast group, the sender data includes an anycast address for addressing the anycast group.", "In order to specify a number of the anycast members for data communications with the sender, the sender data further includes a first indicator being indicative of the desired, first number of anycast members.", "On the basis of the sender data, a number of nearest anycast members is determined as hosts with respect to the sender for data communications.", "In particular, the number of nearest anycast members being determined is defined by the first indicator.", "This solution provides for data communications between a sender and a number of receivers without addressing the same separately, but addressing a plurality of possible receivers by a single address being common for all receivers and selecting a desired number therefrom by means of a data being simply amended to the single common address.", "A further benefit is that it can be employed for any network, even for networks not providing known anycast routing services.", "After having performed the above described method it may be possible that a further multiple host anycast routing should be performed wherein the number of anycast members to be determined is varied compared to the number of anycast members previously specified.", "In order to avoid that anycast members already determined in the previous multiple host anycast routing are contacted again a second indicator can be used.", "The second indicator specifies the number of anycast members previously determined in the preceding multiple host anycast routing.", "Then, the number of nearest anycast members can be determined on the basis of the first and the second indicator.", "In case the first indicator specifies a number of anycast members then the second indicator does, the number of nearest anycast members indicated by the second indicator (in the following: second number of anycast members) are skipped and not contacted, while a number of anycast member is determined for data communications with the sender up to a number obtained by a comparison of the number of anycast members specified by the first indicator (in the following: first number) and the second number.", "In particular, the number of nearest anycast members to be determined corresponds to the difference between the first number and the second number.", "In case the first number corresponds to the second number, no further anycast members have to be determined.", "The same applies for the case wherein the first number is smaller than the second number.", "In order to provide for a method according to the invention for multiple hosts anycast routing to be employed in a network providing a known anycast routing, a first indicator specifying a first number of anycast members to be contacted/allocated for data communications is associated to an anycast address.", "By means of the anycast address, an anycast group having anycast members is identified in the network.", "To establish the desired data communications, at least the anycast address and the first indicator is communicated in the network to the anycast group.", "The anycast group is identified by the anycast address, while the number of anycast members to be contacted/allocated for data communications is selected according to the first indicator.", "In particular, the first indicator defines the number of nearest anycast members and according to the first indicator, the specified number of nearest anycast members is determined as hosts for data communications in the network.", "Comparable to the embodiment of the above described method for multiple hosts anycast routing in a network, this method for multiple hosts anycast routing in an anycast capable network can employ a second indicator specifying a number of anycast members (in the following: second number) already determined for data communications in the network, e.g.", "on the basis of a previously performed multiple hosts anycast routing in a (anycast capable) network.", "For providing the first and/or the second indicator, it is possible to include the indicator(s) in the anycast address or to extend the same by the first and/or the second indicator.", "Preferably, the anycast address is included an anycast address prefix which identifies a topological region in the network, the topological region confining the anycast group.", "Here it is possible, that the anycast address prefix includes the first and/or the second indicator or is extended by the same.", "In a case of a network being operated according to an Internet Protocol standard (e.g.", "IPv4 or IPv6) the anycast address and the indicator can be communicated by a transmission of an Internet Protocol data packet header which includes at least the anycast address.", "For communicating the first and/or the second indicator, the Internet Protocol data packet header can be included the first and/or the second indicator as a part of or an extension for the anycast address.", "With respect to the options mechanisms defined for the Internet Protocols, the first and/or the second indicator can be provided by extending the Internet Protocol data packet header by an options field and, in particular, a so-called multiple anycasting options field including the first and/or the second indicator.", "It is preferred, that the property “nearest” of anycast members is determined according to a measure of distance of the respective routing protocol employed for the network.", "In particular, “nearest” may be understood as the shortest distance between a sender and anycast members.", "Additionally or as an option, “nearest” may be specified according to a distance metric requested by the sender.", "Usually, the distance metric requested by the sender, e.g.", "in the TOS field of an IP header, indicates a smaller distance metrics or nearest distance, respectively.", "In case the network comprises domains at least some of which including at least of the anycast members, the anycast address and the first and/or the second indicator can be communicated via network routers to and/or between domains having anycast members.", "Since it is assumed that a nearest network domain will also include anycast members being nearer compared to anycast members of more distant domains, it is preferred that the ordering of routers for network domain communications is set up such that the anycast address and the first and/or the second indicator are communicated to nearest network domains.", "The nearest network domains may specified according to a measure of distance of a routing protocol for the network and/or according to a (e.g.", "smallest) distance metric requested by the sender, e.g.", "in the TOS field.", "In order to provide for a fast multiple anycasting, the number of anycast members can be contacted sequentially, in particular for a small number specified by the first and/or the second indicator.", "Within a network domain, multiple anycast routing and, in particular, a communication of the anycast address and the first and/or the second indicator can be accomplished on the basis of data identifying each of the anycast members.", "Such an identifying data can be, for example, provided by means of separate entries for anycast members in a routing table according to the employed routing protocol.", "With respect to the anycast routing performed for a network domain, the first indicator can be updated by decreasing the same according to a number of anycast members comprised by a network domain being identified by means of the anycast address and receiving the first indicator.", "Further, the anycast routing can be enhanced by multiplying the anycast address and the first and/or the second indicator and communicating the multiplied anycast addresses and indicators to network domains and/or anycast members.", "Here, it is preferred to communicate the multiplied data simultaneously to the further network domain and/or anycast members.", "In order to precisely select the specified number of anycast members, while determining the number of anycast members, the indicator is modified according to a number of already determined/contacted/allocated nearest anycast members such that the modified indicator being communicated in the network specifies a number of anycast members still to be determined as receivers.", "This embodiment avoids a utilization of further data beside the indicator to provide information how many receivers are already obtained and how many are still to be selected.", "According to an Internet Protocol standard, the communication in the network can be performed by using the strict routing mechanism or the loose routing mechanism.", "Preferably, the first and the second indicator are communicated together and, advantageously in the same manner, e.g.", "both indicators being included in the anycast address or extending the same.", "Further, the present invention provides a system for multiple hosts anycast routing and an anycast routing capable network which are preferred to be operated according to the method according to the invention as set forth above.", "Moreover, the present invention provides a computer program product being adapted to carry out the method steps according to the invention and embodiments thereof as set forth above.", "BRIEF DESCRIPTION OF THE FIGURES In the following description of preferred embodiments of the present invention it is referred to the accompanying figures wherein: FIG.", "1 illustrates the basic principle of anycast routing in a network according to the state of the art, FIG.", "2 illustrates an anycast routing within a topological region identified by a common address prefix according to the state of the art, FIG.", "3 illustrates an anycast routing outside a topological region identified by a common address prefix according to the state of the art, FIG.", "4 illustrates a data structure for an anycast address according to the present invention, and FIG.", "5 illustrates a multiple anycast routing according to the present invention.", "DESCRIPTION OF PREFERRED EMBODIMENTS In order to promote the understanding of the present invention preferred embodiments are described with respect to an network according to the Internet Protocol standard.", "In such a network, data communications are performed by transmitting IP data packets, short packets, each including an IP packet header, short header, between hosts, i.e.", "sending and receiving systems within the network.", "Each host is associated to a network domain defining a topological network region.", "Data communications between network domains are performed via exterior gateway routers, whereas data communications within a network domain are performed via interior gateway routers.", "Exterior gateway routers directly receiving packets from a network domain are called first exterior gateway routers and interior gateway routers directly receiving packets from an exterior gateway router are called border routers.", "Hops designate network components via which packets are communicated.", "In particular, hops include hosts and routers.", "For data communications in the network, i.e.", "routing of packets from a source to a destination, different routing protocols are used for transmission within a domain (intra-domain routing) and between domains (inter-domain routing).", "For example, an intra-domain routing can be based on interior gateway routing protocols, such as OSPF, while inter-domain routing can employ exterior gateway routing protocols such as BGP.", "According to the routing protocols, a routing table is defined which specifies a communication path for packets from a source to a destination and, in particular, hops via which packets are to be transmitted.", "As explained above, both, loose routing and strict routing are possible, whereas for a multiple hosts anycasting most probably loose routing will be applied.", "IP packet headers include information specifying a destination in the network to which packets are to be communicated from a source by means of destination addresses.", "In case of unicast routing a destination corresponds to a receiving host, short receiver, whereas in case of multicast routing, conventional anycast routing, and, specifically, multiple host anycast routing a destination comprises a group of hosts each thereof, only one thereof, and a specified number thereof acting as receiver(s) with respect to a sending host, short sender.", "In case of a multiple hosts anycast routing, short multiple anycasting, the number of hosts, i.e.", "members of an anycast group, to be contacted is specified by extending a packet and, in particular, its header by an indicator.", "The indicator providing information of the number of hosts to be contacted/allocated as originally specified by the sender and the number of hosts which still have to be contacted/allocated as receivers indicates the number of hosts that (still) have to be informed by the corresponding branch utilizing the following embodiments.", "Indicator as Part of the IP Destination Address Field in the IP Header Since anycast routing is mostly based on resolving the above described address prefix, data (e.g.", "some bits) of an IP destination address and, in particular, an address prefix is used for the indication of a sender-specified number of nearest hosts being anycast members of an anycast group indicated by the address prefix.", "In the exemplary illustration of FIG.", "4, the IP packet header includes an anycast address prefix and an indicator specifying the number of nearest anycast members (left) to be contacted/allocated.", "Initially, the indicator specifies the number of nearest anycast members as desired by a sender, while communicating the packet within the network the indicator is decreased corresponding with the number of already selected receivers (already allocated anycast members).", "For the examples given above wherein more than one host is to be contacted as receiver, it is assumed that the sender specified number of desired nearest anycast members is rather low, e.g.", "less than 10.In such applications of multiple anycasting, it might be sufficient to, for example, use 4 bits of an IP packet header for the indicator to allow for a maximum of 24(16) nearest anycast members to be contacted.", "In applications wherein a specification of a higher number of nearest anycast members should be provided, the amount of data (e.g.", "number of bits) of an IP packet header is to be set correspondingly.", "Indicator as New Options Extension to an IP Extension of an IP Header On the basis of existing IP options mechanism (e.g.", "as explained above with respect to strict and loose routing), an IP packet header is extended by a so-called “multiple anycasting” options field containing an indicator as specified above.", "In case a packet is destinated for an anycast address, the respective header including anycasting address information, anycast capable routers only analyses the extension, namely the “multiple anycasting” option field.", "Modifications of the extension can be performed, for example, to update the “multiple anycasting” option field to be in line with the number of anycast members still to be contacted/allocated in view of already contacted/allocated anycast members.", "As set forth above, data communications are differently performed for intra-domain and inter-domain communications.", "As a result, multiple anycasting will work different for intra-domain cases employing interior gateway routing protocols such as OSPF and inter-domain cases employing exterior gateway protocols such as BGP.", "In the following inter-domain multiple hosts anycasting and intra-domain multiple hosts anycasting will be described with reference to FIG.", "5 illustrating an exemplary topology and routing scenario wherein a host H in a domain C communicates an anycast packet within a network comprising domains A, B and C and exterior gateway routers R1, R2, R3 and R4.For this example it is assumed that the anycast group addressed by the host H comprises the anycast members A1, A2 and A3 in domain A and anycast members A4 and A5 in domain B.", "In particular, host H communicates an anycast packet including an indicator specifying a number of three anycast members to be contacted as receivers, i.e.", "nearest anycast members.", "Inter-Domain Multiple Hosts Anycasting Inter-domain multiple hosts anycasting is indicated by steps 1 and 3 shown in FIG.", "5.On the basis of an employed exterior gateway routing protocol, the first exterior gateway router with respect to host H in domain C, i.e.", "router R4, will check its routing tables in order to determine anycast members of the anycast group as specified by the anycast packet of host H and, in particular, all domains including the respective anycast members.", "The corresponding routers for determined domains, i.e.", "routers R1 and R3 to domains A and B, will be added to the IP packet header of the anycast packet from host H by utilizing strict or loose routing options.", "Depending on the metrics used for the employed exterior gateway routing protocol and/or the smallest distance metric requested by the sender, i.e.", "host H, (e.g.", "defined in the TOS field), the ordering of the routers will be based on distances between domains including anycast members and anycast members, respectively, and host H. According to FIG.", "5, the anycast packet of host H will be first communicated to router R3.In case the number of anycast members contacted/allocated in a domain receiving the anycast packet from router R3 is less than the number of nearest anycast members to be contacted as defined by host H, the anycast packet will be communicated to router R1.The anycast packet and, in particular, the header thereof transmitted to router R1 includes an indicator specifying the number of anycast members which are still to be contacted/allocated.", "With reference to FIG.", "5, the indicator communicated to router R1 indicates that one anycast member is to be contacted/allocated assuming that anycast members R4 and R5 of domain B are selected as nearest anycast members.", "As specified by the strict or loose routing options of the IP packet header, the specified number of anycast members may be contacted sequentially.", "Assuming the specified number being rather low, e.g.", "2 to 5 anycast members, this procedure will not effect the network service performance received by requesting host, here host H. In case a high number of anycast member should be allowed to be specified by a sender/requesting host, various known data communication methods for enhancement of data transmissions in a network can be utilized for contacting anycast members.", "Intra-Domain Multiple Hosts Anycasting Intra-domain multiple hosts anycasting will be explained with reference to steps 2 and 4 of FIG.", "5.At reception of an anycast packet in an border router for a domain, the border router will forward the anycast packet to all next hops, in particular, to the next anycast members of the respective domain.", "This can be accomplished since, as explained above, interior gateway routing protocols provide a separate entry in the routing table for each anycast member.", "For forwarding the anycast packet, the border router may multiply the anycast packet and simultaneously communicate the multiplied anycast packets to multiple destinations, i.e.", "next hops/anycast members.", "According to the number of anycast members and, in particular, of anycast members contacted/allocated in the corresponding domain, the number specified by the indicator in the header of the anycast packet is decreased.", "In case the indicator is 0 after an intra-domain multiple host anycasting, no data communications are required to contact/allocate further anycast members, e.g.", "of a different domain, since the number of nearest anycast members as desired by a requesting host is provided.", "Otherwise, the border router will forward the anycast packet including the decreased indicator to the next exterior gateway router for inter-domain multiple hosts anycasting as specified above.", "According to FIG.", "5, a border router of domain B receives the anycast packet from exterior gateway router R3 and will forward the anycast packet to the anycast members A4 and A5.For example, router R3 may serve as border router for domain B by combining exterior and interior gateway functions.", "In step 2 the border router of domain B contacts all anycast members in domain B such that they are specified for nearest anycast members for data communications with host H. As a result, still one further anycast member has to be contacted/allocated, whereby the anycast packet communicated from the border router of domain B via the exterior gateway router R3 to the exterior gateway router R1 includes a correspondingly decreased indicator, i.e.", "an indicator having the value of 1.A border router of domain A forwards the received anycast packet within domain A as specified above for contacting/allocating an anycast member.", "Comparable to router R3, router R1 combining exterior and interior gateway capabilities may be operated as border router for domain A.", "The routing within a domain may be performed in a manner comparable as the above described inter-domain routing in case hierarchies are used for the respective domain.", "Then, all levels of the same hierarchy in the domain can be contemplated as inter-domain cases, wherein levels of a lower hierarchy can be seen as intra-domain cases.", "Skipping the Number of Anycast Members Specified in a First Multiple Hosts Anycasting for Performing a Second Multiple Hosts Anycasting After having performed a first multiple hosts anycasting, it is possible that the respective host performs a second multiple hosts anycasting wherein the number of anycast members specified in the first multiple hosts anycasting should not be contacted/allocated (again).", "For that purpose it is contemplated as an ad-on to the above described multiple hosts anycast routing, but also to the existing anycast specification as known in the state of the art, that a sending/requesting host can specify the number of first anycast members (i.e.", "anycast members contacted/allocated in a first anycasting) that should be not contacted/allocated (again) in a second (multiple hosts) anycasting.", "After a previous attempt did not result in a successful anycast member search there may be no need to contact/allocate the same anycast members once more if the number of anycast members is to be extended in a subsequent attempt.", "In that case, the sending/requesting host could specify in a new optional IP header extension the number of first anycast members that should not be contacted/allocated in the subsequent attempt.", "Such an indication can be provided by a second indicator (counter) which is additionally checked, e.g.", "by routers, while performing the second (multiple hosts) anycast routing.", "In case a network domain includes anycast members, the additional indicator (counter) is first checked.", "If the second indicator indicates a number greater than 0, the second indicator is to be decreased until it reaches 0.For each decrease of the second indicator, the first multiple hosts indicator (counter), as described above, is decreased as well, but without contacting/allocating an anycast group member.", "In this way, it is ensured that the number of anycast members contaced/allocated in the first, preceding (multiple hosts) anycasting is not contacted again in the second attempt.", "As set forth above, this optional addition may already be implemented in the single hosts anycasting as known in the state of the art." ] ]	Patent_10467910
[ [ "Enhanced data storage and retrieval devices and systems and methods for utilizing the same", "The invention relates generally to store information on magnetic or optical storage media by using one or more novel approaches alone or in combination.", "These novel approaches are capable of using at least one code which may comprise more than two values (FIG.", "4d, 43).", "A first series of approaches for the storage of information applies generally to optical recording and reproducing system (FIGS.", "7-10, optical media 66), while a second series of approaches applies generally to electric or magnetic recording and reproducing systems (FIG.", "1-2, magnetic medium 1), Each series of approaches is capable of storing information data in one or more codes (FIG.", "4d, 42a, 42b, 42c, 42d) and the use of at least one higher order code which is different from the traditional binary code of “0” and “1”." ], [ "1.A method for storing data comprising: storing at least one magnetic field strength; utilizing said at least one stored magnetic field strength to cause an amount of splitting or shifting of at least one frequency in at least one material; determining the amount of shifting or splitting in said at least one material; and assigning a data value to said amount of splitting or shifting.", "2.The method of claim 1, wherein said determining comprises a means for interrogating said at least one material with electromagnetic energy to determine said amount of shifting or splitting.", "3.The method of claim 1, wherein said at least one material comprises at least one magnetic material.", "4.The method of claim 3, wherein said at least one material comprises at least one material contiguous to at least one magnetic material.", "5.The method of claim 1, wherein said method for storing data comprises a base-2 data storage system.", "6.The method of claim 1, wherein said method for storing data comprises, comprises greater than a base-2 data storage system.", "7.The method of claim 1, wherein said splitting or shifting comprises at least one Zeeman effect.", "8.A method for reading data comprising: creating at least one magnetic field strength; utilizing at least one magnetic field strength to cause an amount of splitting or shifting of at least one frequency and at least one material; determining the amount of shifting or splitting in said at least one material; and assigning a data value to said amount of splitting or shifting.", "9.A data storage apparatus comprising: means for storing at least one magnetic field strength; means for utilizing said at least one stored magnetic field strength to cause an amount of splitting or shifting of at least one frequency in said at least one material; means for determining the amount of shifting or splitting in said at least one material; and means for assigning a data value to said amount of shifting or splitting.", "10.A method for storing information optically comprising: an optical source; a means for achieving different optical intensity; a means for recording said different optical intensity; and a means for reading said different recorded intensities.", "11.A method for storing information optically comprising: an optical source; a means for achieving different optical wavelengths; a means for recording said different optical wavelengths; and a means for reading said different recorded optical wavelengths." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Data storage devices have been becoming smaller in size (as well as faster) due to, for example, the continued improvement in the ability to store more information in smaller spaces.", "For example, over the past twenty years the ability to place more data on magnetic storage media such as magnetized hard-disk drives and/or floppy disks, as well as on optical media (e.g., compact disks “CD's” and more recently, Digital Versatile Disk “DVD's”) has been increasing at a phenomenal pace.", "The impressive increase in the ability to store more data in smaller areas has been one of the main driving forces for rendering computers, and other electronic devices with reasonably sized memories, accessible to a typical home or small business consumer.", "Further, the high operating speeds of microprocessors have required stored data in computers to be rapidly accessible (e.g., both the rate at which information is delivered to a microprocessor as well as the amount of time it takes between the time that a microprocessor requests a file and when the first piece of data begins to flow from the file to the microprocessor are important).", "Magnetic memory has undergone many recent improvements and has been utilized extensively in data storage/retrieval devices because magnetic memory currently provides the least expensive alternative for the fast writing and reading capabilities for data.", "However, the various currently utilized magnetic technologies face a variety of technical challenges and appear to be headed for a stopping point.", "Specifically, these various approaches for the storage and/or retrieval of information may no longer be capable of meeting the growing demands of the industry, without experiencing significant technical enhancements.", "For applications where speed has historically been less important, various optical technologies have been growing in popularity.", "Many technical advances have been made in the recording and/or retrieval of information by optical means.", "In systems which use compact disks (“CD's”), compact disk read-only memory (“CD-ROM's”) and/or Direct Video Disks (“DVD's”), the storage and/or retrieval of information has also been of great interest.", "For example, the first CD's manufactured were CD-ROM's that are currently capable of storing about 650 MB of data.", "This equates to about 74 minutes of play-time for music that has been recorded in a typical recording mode.", "In particular, the standard current approach for utilizing CD-ROM's, includes the use of a solid state interrogating laser (or in some cases more than one laser) which has a wavelength of about 780 nanometers (0.78 μm).", "The standard CD-ROM is about 12 cm in total diameter and has a thickness of about 1.2 mm.", "The standard CD-ROM has information recorded on plateaus (“bumps”) and valleys (“pits”) that spiral from the inside of the disk toward the outside of the disk.", "The digital data stored in the tracks that comprise the spirals must be separated by enough space (e.g., tracks are typically separated radially by about 1.6 μm) so that when the data is read with an interrogating solid state laser, crosstalk from adjacent tracks does not occur (i.e., recorded information is received from only one track at a time).", "Accordingly, the placement of more information onto a typical CD has been limited by the required size (e.g., length and width) of the plateaus (“bumps”) and valleys (“pits”).", "The size of the plateaus and valleys (e.g., typically not less than 830 nanometers in length) has been a function of various factors, including the wavelength of the laser utilized to read the information from the CD.", "Various attempts have been made to increase the memory capacity of current CD's, including the stacking of a plurality of disks one on top of the other so as to provide more memory space, as well as other attempts to miniaturize the digitized information stored in the tracks.", "The most recent significant improvements in this type of optical storage/retrieval technology have been the creation of the DVD, which is known as the “Direct Video Disk” or the “Digital Versatile Disk”.", "The first commercial products of DVD's entered the marketplace in late 1997.The DVD technology is similar to the technology in CD's, except that, for example, the wavelength of lasers utilized to read information from DVD's is smaller than that used for CD's, namely, about 635 nanometers or 650 nanometers, compared to about 780 nanometers for CD's.", "The size of the plateaus (“bumps”) that make up the DVD are about 320 nanometers wide, a minimum of about 400 nanometers long and about 120 nanometers thick.", "About 740 nanometers separate one track from the next, compared to the about 1600 nanometer separation of tracks in CD's.", "A standard DVD is physically about the same size as a CD, but has a memory capacity that is about seven times greater than that of CD's.", "In particular, the memory capacity of a one-sided, one-track, DVD is now about 4.7 GB.", "However, recent improvements in DVD have allowed digital information to be placed on both sides of a single track, as well as both sides of a double track.", "In this regard, a first interrogating laser reads the digitized data on a first side of a first track (i.e., in the form of bits arranged in a spiral track that begin in the innermost portion of the DVD and spirals outward).", "Then, information can be retrieved from the opposite side of the first track in a similar manner from a second laser; however, the spiral track of information typically begins on the outermost portion of the disk and spirals inward toward the center.", "Accordingly, when dual-track, dual-sided DVD disks are used, the current amount of memory that can be achieved is about 17 GB.", "Another optical data storage technique which shows promise is known as holographic memory.", "The potential for high storage capacity and high storage retrieval rates using the holographic technique is tremendous.", "In particular, for example, recent work in a system known as Holographic Data Storage System (“HDSS”), has shown that data can be accessed in 100 microseconds or less, which is several orders of magnitude faster than the millisecond retrieval of data in typical magnetic-disk drives.", "Briefly, in this technology, an optical-interference pattern is created within a desired photosensitive material (e.g., a crystal or a polymer).", "The optical interference pattern is imprinted as a result of physical changes in such materials.", "The interference pattern is created by the interaction between an object beam and a reference beam, both of which typically originate from a single light source (e.g., a laser), but are split into two different pathways by a beam splitter.", "Specifically, the object beam is incident on a target object that contains data, and modifies the object beam, while the reference beam, taking a separate path which leaves it unmodified, interferes with the object beam at the holographic storage medium.", "When the two beams meet at the holographic storage means, the interference pattern which results from their interaction is stored on or in the holographic storage means.", "The holographic storage means is later interrogated by a reading or reference beam, which upon interacting with the holographically stored interference pattern, can then recreate the holographic image containing the data originally stored in the object beam.", "While the holographic optical technique shows great promise for the future, current industrial emphasis is being placed primarily on improving existing digital optical and magnetic storage/retrieval systems so that more information can be stored in smaller spaces, and retrieval times can be decreased without significantly changing the basic methodology and hardware for the storing and retrieving of information.", "With regard to placing more data onto magnetic recording media, effects such as the paramagnetic effect (e.g., magnetic crosstalk between magnetic domains comprising bits) are already beginning to cause problems.", "Experts have postulated that in coming years, magnetic storage technologies could reach a limit imposed by the superparamagnetic effect (“SPE”).", "The physical phenomenon known as SPE can occur in a magnetic data storage system when the magnetic spins of electrons in the domains which comprise a bit (e.g., in current technology typically either a “0” or a “1”) become unstable due to, for example, the environmental thermal energy surrounding the bits.", "Instability of bits occurs when smaller and smaller numbers of domains or atoms are used to comprise a bit and the ability for the domains or atoms to maintain their given spin directions (e.g., corresponding to, for example, a “0” or a “1”) becomes difficult.", "Non-maintenance of spin directions results in a “flipping” of spins back and forth and thus changing “0's” to “1's” and “1's” to “0's”, etc.", "Such changing or flipping of spins will result in a corruption of the data or information that the “0's” and “1's” represent.", "Accordingly, in order for smaller numbers of atoms to be used to form a bit and in order for the bit to be viable, techniques and/or materials for preventing the superparamagnetic effect need to be developed to overcome the deficiencies of the prior art.", "Another potential problem in recording information magnetically is that the write head which is used to align magnetic domains in the magnetic storage media may be limited in the ability to impart a required field strength over the entire domain area comprising a bit to cause the magnetic domains to behave as though they have been exposed to a substantially uniform field.", "In such cases, it is possible that differently oriented domains within a bit (e.g., differently oriented due to being subjected to different magnetic field strengths within a single writing head due to, for example, non-uniformity of the magnetic flux lines emanating from the write head and/or differently oriented because some domains require more energy than other domains to align due to, for example, their location within a bit) may have a higher tendency to become corrupted, as well as adding to signal/noise ratio problems.", "One attempt for miniaturizing domains involves the use of so-called “hard” magnetic materials.", "The use of hard magnetic materials is desirable when compared to “soft” magnetic materials because hard magnetic materials are more likely to maintain their magnetic spin directions when domains comprising the hard magnetic materials are closely spaced.", "In this regard, various rare-earth and transition elements have been found to be magnetically stable and are thus termed “hard”.", "Such magnetically stable materials are known to have a higher coercivity, which is typically represented by “H c ”.", "Stated in an over simplified manner, the higher the H c value, the greater the resistance of the material to outside influences, such as outside magnetic fields (e.g., the harder it is for material to lose its imprinted spin direction due to an increase in temperature and/or the influence of other magnetic fields, etc.).", "However, one drawback for utilizing “hard” magnetic materials is that hard magnetic materials are more difficult to magnetize than soft magnetic materials (e.g., in some cases, depending on the particular materials chosen, much more difficult to magnetize).", "This makes it more difficult to record data initially in hard magnetic materials.", "There are various known approaches for solving the problem of encoding data into hard magnetic materials which approaches utilize a laser in conjunction with an appropriate magnetic write head.", "These various approaches have been referred to generally as the “magneto-optical” approach.", "These magneto-optical approaches effectively reduce the H c value of the magnetic domains by causing the domains to be locally heated just prior to being subjected to the magnetic write head.", "Thus, the coercivity (H c ) of the domains is effectively reduced and when such effective reduction in H c has occurred, a somewhat standard write head, or in some cases a very different write head, can be used to alter favorably the magnetic domains.", "In other words, the use of a laser, which provides thermal energy to the domains of the hard magnetic material, causes the hard magnetic material to behave in a manner which is similar to a somewhat softer and thus easier to magnetize magnetic material.", "Each of the known approaches for utilizing the magneto-optic techniques involves the use of at least one laser and/or at least one laser focusing system.", "However, one of the common problems facing these magneto-optical approaches is that the lasers that are utilized to soften the hard magnetic material prior to subjecting the hard magnetic material to a magnetic field is that the lasers typically generate excess amounts of heat that can flow to neighboring bits or tracks (e.g., thermal energy is caused to migrate to undesirable neighboring tracks or areas resulting in a potential corruption of stored information).", "Accordingly, various laser applications and/or storage solutions have been devised to minimize the transfer of heat from the laser to neighboring tracks, and thus minimize the loss of recorded information.", "However, to date this thermal transfer of laser energy still presents problems to varying degrees in all the prior art approaches.", "One interesting technique for the reading of data in connection with one optical-magnetic system is the use of an effect known as the “Kerr effect”, and more particularly, the “magneto-optical Kerr effect”.", "Briefly, current uses of the magneto-optical Kerr effect involve incident polarized light being reflected from the surface of a magnetic domain in different ways, depending on the orientation of the magnetic domains (e.g., depending on electron spin directions).", "The changes in the reflected polarized light are made, by algorithms, to correspond to different digitized data.", "Specifically, when the incident polarized light encounters different magnetic field orientations (e.g., north or south domains comprising a bit which are ordered to represent either a “0” or a “1”) the polarization state of the reflected light is changed.", "Another approach to the digital storage of information that shows future promise is known as atomic resolution storage (“ARS”).", "This approach, similar to holographic techniques, is capable of storing tremendous amounts of information in a small amount of space.", "In this technique, generally, very small electron probes, which are used to generate electron beams, are formed into an array.", "The electron probes have tips that are roughly the size of atoms.", "The electron beams from the electron probes are made to be incident upon a storage medium so that the incident beams cause some sort of physical change in the storage medium (i.e., the medium comprising the computer memory).", "One example of such a storage medium is a material that is capable of containing at least two stable phases at ambient operating conditions so that an incident electron beam emitted from an electron probe changes the storage material from one phase to the other.", "In this technique, encoded bits of information are represented by the change in phase of the phase-change storage material.", "This technique, similar to the magneto-optical technique discussed above, also suffers from the problem of heat flowing between data spots created by the incident electron beams when the spots or bits are initially recorded.", "Accordingly, one of the challenges faced by this relatively new technology includes reducing the flow of heat between data spots which are created by the array of electron beam generators.", "The retrieval of stored information is also a challenge.", "In this regard, as bits become smaller and include lesser numbers of magnetic domains per bit and/or the domains are oriented differently, (e.g., domains are aligned perpendicular to the substrate containing at least one magnetic surface thereon instead of being parallel to the substrate surface) such bits are capable of being packed more tightly together.", "Assuming that the bits are not corrupted by their tight packing and/or smaller numbers of magnetic domains and/or non-uniform alignment within a bit, then reading of these tighter packed domains comprising the bits also becomes a challenge which the prior art is still struggling to overcome.", "Historically, large numbers of magnetic domains were present in each bit which contained digital data.", "Thus, the resulting or induced magnetic fields were relatively large and various forms of known inductive reading techniques were sufficient to ascertain whether the hundreds or thousands of aligned domains in a bit corresponded to a “0” or a “1”.", "However, as prior art miniaturization recording techniques progressed, the known inductive reading techniques were typically not sensitive enough to determine accurately the alignment of ever decreasing numbers of domains comprising a bit (e.g., signal to noise problems in the storage and/or retrieval of digital data bits were exacerbated).", "The development of magnetoresistive heads, and the subsequent development of giant magnetoresistive heads, has enabled smaller numbers of domains comprising a digital bit to be read accurately.", "In both of magnetoresistive and giant magnetoresistive heads, the electrical resistance of one or more materials comprising the read head(s) is examined.", "In particular, both magnetoresistive and giant magnetoresistive heads are made from materials that exhibit small changes in their electrical resistance as a function of magnetic fields created by oriented domains to which such heads are exposed.", "Giant magnetoresistive heads are two or three times more sensitive than magnetoresistive heads due to their novel structure which includes layering of different materials stacked on top of each other.", "One problem that continues in these technologies is the presence of magnetization vortices in the recorded domains.", "Such vortices may result from the difficulty in achieving uniform domain alignment within a bit due to, for example, the difficulty in applying a required magnetic field (e.g., uniform field) to the domains during the recording or writing process.", "While use of the magneto-optical Kerr effect and new read heads such as magnetoresistive heads and giant magnetoresistive heads have improved digital data retrieval techniques, further miniaturization is pushing the limits of these systems.", "Accordingly, additional improvements are necessary in the ability for hardware, combined with suitable programming, to detect even smaller numbers and/or smaller sizes of domains comprising digitized (or analog) data bits.", "Thus, additional innovations in the detection of stored data are required.", "In addition to the storage of information digitally, the possibility or promise of storing information in an analog manner continues to intrigue various investigators.", "While certain improvements have been made in, for example, the processing of analog information (e.g., the advent of VLSI (Very Large Scale Interrogation) chips, the techniques for the storing of analog information need much improvement.", "Particularly, while analog computing has been known for several decades, an accurate and fast-paced system for the storage and retrieval of information has continued to elude researchers.", "In this regard, digital computing has received the greatest amount of attention because of it's relative dependability for the storing and retrieving of information in a “1” or a “0” digitized format.", "Briefly, digital approaches typically include storing a sampling of the analog information that is to be recorded.", "The number of samples taken (e.g., such sampling typically occurs in predetermined units of space or time) from the analog source will correspond to the accuracy between the original analog image or sound and the stored digital data (i.e., the more digital samples that are recorded of an original analog image or sound, the more accurate the reproduction of the original analog image or sound will be).", "These techniques of predetermined quantum amounts of sampling of analog images have been adequate for most digitized applications, however, as the need for more accurate recording and retrieval of information occurs (e.g., in, for example, the fields of computing or determining various engineering and scientific relationships in the following representative areas: gravitational; electrostatic; magnetic; thermal; stress; fluid flow field analysis; wave propagation; image processing; etc.", "), the availability of computing with more accurate data becomes important.", "However, the digitization of tighter and and/or smaller sampling amounts from an analog source can result in tremendous amounts of digitized data being stored to represent a small actual amount of analog information.", "This is one area where analog computing has a distinct advantage compared to digital computing.", "In particular, an analog computer will store only a single piece of information corresponding to a single sampling area, whereas a digital computer will need to store somewhere between, for example, 8 and 16 bits of data to represent the same single piece of information stored in an analog fashion.", "The large number of bits required to store digital information corresponds to the requirement to store information in an ASCII or Unicode format.", "However, in order to escape from the current ASCII or Unicode format, analog storage requires a very large number of different data bits (e.g., data corresponding to much greater numbers than merely “0s” and “1's”) to be stored and retrieved.", "Current prior art techniques have great difficulty in storing and/or reading bits corresponding to anything other than “ 0 's” and “ 1 's”, let alone the attendant problems of storing and/or reading, for example, a near infinite number of possibilities (e.g., 1, 2, 3 .", ".", ".", "n+1).", "However, when performing complex calculations, the digital process may result in very slow processing times as well as requiring very large memories.", "It is for this, as well as other reasons, that analog computing is still very attractive.", "Thus, while advances in the processing of analog information have been made, reliable techniques for the storage and retrieval of analog information are still being sought without any good solutions currently existing.", "As stated above, with regard to digital computing, much of the recent technical emphasis in data storage and retrieval for both optical and magnetic memory enhancement has been on shrinking more information into smaller areas.", "The ability to place bits, which are the smallest elements of information used by current digital memory systems, into a binary code format, which is a combination of “0's” or “1's”, into a smaller area on, for example, magnetic and/or optical disk storage media (e.g., a disk or a tape) may soon reach a limit.", "Thus, current technologies for writing and/or reading binary data onto and/or from current storage media will be pushed to the maximum limit.", "Computer designers are already facing a performance gap whereby processors can process information faster than the information can be retrieved from computer memory.", "The majority of the currently proposed approaches for the digital storage of information, suffer from the same problem, namely, that the magnetic and/or optical information which is stored uses a binary code (i.e., a base- 2 number system) which is a combination of “0's” and “1's”.", "The predominance of the use of a “binary” code has been due to, for example, difficulty in distinguishing signals (e.g., a “0” or a “1”) from background noise.", "In this regard, many techniques have been proposed which use various hardware, as well as algorithms, which attempt to read accurately the stored information as either a “0” or a “1”.", "However, certain prior art techniques have been postulated for utilizing codes other than binary.", "In particular, U.S. Pat.", "No.", "6,154,432, entitled “Optical Storage System” discloses a holographic system which records information in very small spots and postulates that 10 or more digits may be possible to record on a single bit.", "Accordingly, this holographic technique discloses codes higher order than binary.", "Further, for example U.S. Pat.", "No.", "5,450,363, entitled “Gray Coding for a Multi Level Cell Memory System” discloses the use of a so-called “gray code” approach (also known as “gray scale” by others) for data storage.", "This approach also uses a system which is higher order than binary.", "In particular, reference is made to the storage of multiple bits of information in a single memory cell thus creating a multi-level cell.", "Still further, U.S. Pat.", "No.", "6,061,005, entitled “Encoding of Data”, discloses a method of encoding data on magnetic cards which uses symbology other than binary, such as trinary, quaternary or even higher symbology, for the recording of information.", "However, while various techniques have been postulated for systems which use codes higher than binary, none of such codes have been commercially adopted due to, for example, signal to noise ratio problems, etc.", "In particular, reading errors can occur due to lack of contrast between data values.", "Such lack of contrast can be caused by random noise (e.g., vibrating inaccurate positioning of recording/reading heads, fidelity loss during recording, etc.)", "and determinate variations.", "Accordingly, a need exists for techniques which can be commercially viable which can use codes reliably that are higher order than traditional binary codes for the storage and/or retrieval of information.", "A very particular combination of “0's” and “1's” is known as the American Standard Code for Information Exchange (i.e., the ASCII code) which assigns a unique code number between 0 and 127 to each of the 128, 7-bit, binary number characters.", "Table 1 below shows in table form the current ASCII characters arranged by Hexidecimal digits (i.e., 128 different encoding combinations of groups of seven bits).", "Reading within the Table from left to right and then down, the first 32 values are codes which correspond to various computer control functions such as line feed, carriage return, etc.", "The 33 rd value corresponds to the “space” character.", "Values 34-48 correspond to symbols and punctuation marks.", "Values 49-58 correspond to digits, and so on, until reaching value 128, which corresponds to “delete”.", "In addition, each of these values also has a corresponding hexidecimal character.", "For example, the hexidecimal digit corresponding to the “space” character is “20”, while the hexidecimal digit corresponding to the letter “n” is “6E”, and so on.", "TABLE 1 00 01 02 03 04 05 06 07 08 09 0A 0B 0C 0D 0E 0F 00 NUL SOH STX ETX EOT ENQ ACK BEL BS TAB LF VT FF CR SO SI 10 DLE DC1 DC2 DC3 DC4 NAK SYN ETB CAN EM SUB ESC FS GS RS US 20 !", "“ # $ % & ‘ ( ) * + , − .", "/ 30 0 1 2 3 4 5 6 7 8 9 : ; < = > ?", "40 @ A B C D E F G H I J K L M N O 50 P Q R S T U V W X Y Z [ \\ ] {circumflex over ( )} — 60 ’ a b c d e f g h x j k l m n o 70 p q r s t u v w x y z { \| } ˜ DEL These 128 characters are enough characters for North American English to be stored and/or retrieved, but are not enough for many other languages (Note: In ASCII, the eighth bit is used, and is thus normally set to “0”).", "To represent more than 128 characters, 8-bit (rather than 7-bit) binary numbers are utilized.", "For each digit in these eight bits there are two choices for the digit, either “0” or “1”.", "This results in a possibility of an additional 128 characters or a total of 256 different combinations of “0's” and “1's” (i.e., 2 8 ).", "Accordingly, the current data storage approaches utilize these 256 combinations of “0's” and “1's”.", "However, these 8-bit numbers still do not provide enough room for all characters that need to be used in the world (e.g., certain Asian languages have thousands of characters each).", "In addition, there is no universal agreement in the world on what all the characters should be, whether referring to the 128 characters, or the 256 characters, or any number of characters beyond these.", "However, certain approaches for dealing with all the various languages in the world, which collectively contain many thousands of characters, are beginning to utilize something referred to as the “Unicode”, which uses a 16-bit character set.", "In order to adopt something similar to the Unicode, even greater strain will be placed on current memory techniques.", "Whether using an 8-bit, 16-bit, or some other approach, all such approaches have been limited primarily to using a binary system of data storage.", "However, due to: (1) the impending size barrier for the storage of information on, for example, magnetic and/or optical media; (2) the performance gap between how fast a processor can process information versus how fast a processor can access information; and (3) the goal for a universal adoption of a common set of characters throughout the world, a different approach for the storage of information, either by digital or analog approaches, is clearly needed.", "The present invention satisfies the current and future data storage needs by using different approaches for the storage and/or retrieval of data; and/or different codes that use a code higher than the current “base- 2 code”." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the invention to provide a means for storing/retrieving digitized information optically.", "It is an object of the invention to provide a means for storing/retrieving digitized information magnetically.", "It is an object of the invention to provide a means for storing/retrieving analog information optically.", "It is an object of the invention to provide a means for storing/retrieving analog information magnetically.", "It is an object of the invention to provide a storage/retrieval means for utilizing codes other than binary.", "It is an object of the present invention to provide an increased number of characters that can be stored as a single bit of information.", "It is an object of the invention to provide the ability to store the 128 ASCII-type characters in a much smaller space than the space that is used in a standard binary code of “0's” and “1's”.", "It is an object of the invention to provide a means for storing/retrieving codes other than binary.", "It is an object of the invention to utilize at least a base- 3 optical storing/retrieving system.", "It is an object of the invention to utilize a least a base- 4 coding storing/retrieving system.", "It is an object of the invention to utilize at least a base- 6 coding storing/retrieving system.", "It is an object of the invention to utilize at least a base- 8 coding storing/retrieving system.", "It is an object of the invention to utilize at least a base- 10 coding storing/retrieving system.", "It is an object of the invention to utilize particular aspects of the Zeeman effect in connection with the storage/retrieval of digitized information.", "It is an object of the invention to utilize particular aspects of the Zeeman effect in connection with the storage/retrieval of analog information.", "It is an object of the invention to utilize different shades or colors of spots or dots in connection the optical storage of digitized information.", "It is an object of the current invention to utilize different shades or color or spots or dots in connection with the optical storage of analog information.", "It is an object of the invention to utilize either monochromatic or polychromatic light in connection with the optical storage of digital and analog information.", "It is an object of the invention to utilize an analog-like approach to the storage of data.", "The current process of storing bits, which is the smallest element of information used by current memory systems, uses a binary format of only “0's” and “1's”.", "A bit which is “off” is considered to be “false” or “not set”, while a bit that is “on” is considered to be “true” or “set”.", "Historically, because bits could only be one of two values, bits have been combined together into larger units in order to represent more characters or values.", "“Nibbles” are groups of four bits and “bytes” are groups of eight bits (i.e., two “nibbles”).", "Bytes are typically what are used to store “characters”.", "“Characters” typically include letters, numbers and/or symbols.", "For example, the ASCII code uses 128 different encoding combinations to represent: (i) The letters “A-Z” in both upper and lower case (i.e., values 65-90 and 97-122, respectively); (ii) Special characters (e.g., “<”, “.”, “%”, “$” “?”, etc) (e.g., values 33-47); (iii) The numbers 0 - 9 (i.e., values 48-57); and (iv) Certain control codes used for device control (i.e., values 0-31).", "To represent more than 128 characters, an eighth bit can be utilized.", "However, the ability to store a binary code of only “0's” and “1's” limits the ability for information to be miniaturized.", "For example, to store a single character from the ASCII code, at least seven bits of memory (typically eight bits are currently required because the last bit is usually set to zero) are needed.", "Table 2 sets forth one example of how a typical computer memory system would store the word “Patent”.", "TABLE 2 ASCII LET- CHAR- BINARY HEXADECIMEL BITS OF TER ACTER NUMBER DIGIT OR BYTE MEMORY P = 80 = 01010000 50 8 a = 97 = 01100001 61 8 t = 116 = 01110100 74 8 e = 101 = 01100101 65 8 n = 110 = 01101110 6E 8 t = 116 = 01110100 74 8 Thus, the letter “P” corresponds to the ASCII character 80 , which is represented by the binary code “01010000”.", "The first “0” in the sequence is often referred to as “the last bit” and is typically set to “0” for all ASCII characters.", "The corresponding hexidecimal digit or byte representation is “50”.", "In order for the computer memory system to store the letter “P”, eight bits must be sent to memory in the order “0-1-0-1-0-0-0-0”.", "Likewise, to store the letter “a”, eight bits must be sent to memory in the order “0-1-1-0-0-0-0-1”.", "Accordingly, 48 bits are used to store the six (6) letter word “Patent”.", "Similarly, when a microprocessor unit accesses the portion of memory containing the set of bits representing the word “Patent”, the same number of bits (i.e., 48) or six (6) bytes must be processed by the microprocessor.", "Clearly, if the word “Patent” could use a smaller amount of memory, less memory capacity would be needed in a computer and/or the rate at which data could be stored and/or retrieved from memory would be enhanced.", "In a first aspect of the invention, a coding system that is a higher order system than a binary coding system is utilized for the digitized storage of information.", "For example, any digital coding system which uses three or more different code characters (i.e., a coding system which operates in “base-3” or greater) will result in a reduction in the number of bits needed to store the same amount of digital information relative to a binary system.", "Thus, by using a higher order memory system, the possibility of storing more digitized information in a smaller memory area exists.", "Likewise, the rate of storing and/or retrieving digitized information should also increase due to memory being stored in a physically smaller area.", "This permits a data retrieval device to function more efficiently.", "The present invention permits the use of a digitized memory system which is base- 3 or greater.", "Moreover, as more code characters are used (e.g., higher order systems of more than 10 or more than 20 different characters are utilized) the digitized system begins to behave in a manner which is similar to analog systems and can thus be referred to as “analog-like”.", "In other words, as the amount of digital sampling of an analog source increases, the recorded digital signal begins to approach the storage of most features of the analog signal.", "However, current data storage/retrieval techniques utilize, for example, 8 or 16 bits to store a single piece of analog information, which limits the amount of sampling of an analog source that can be reasonably performed when creating a digital representation of the analog source.", "Accordingly, the digital data storage/retrieval techniques of the present invention can be vastly superior to current digital techniques which utilize only “0's” and “1's” and are limited to sampling much smaller areas of analog sources than would be desirable in many cases (e.g., for complex computer modeling of numerous events, etc.).", "A first embodiment of an alternative memory storage system of the present invention may function in a manner somewhat analogous to the manner in which a Compact Disk (CD) or Digital Versatile Disk (DVD) player functions.", "Current CD and DVD technologies utilize a rotating disk comprised of a number of layers of different materials.", "The layers may include, for example, polycarbonate plastic, aluminum and acrylic.", "The aluminum in the disk includes a series of plateaus and valleys sometimes referred to as “bumps” and “pits”, respectively.", "Laser light is caused to focus upon the bumps (plateaus) and pits (valleys) in the rotating disk.", "The reflection of the laser from the successive plateaus and valleys corresponds to a data stream (e.g., a binary stream) of information.", "CD's and DVD's use, for example, 16-bit strings of binary coded data.", "Specifically, laser light reflects differently from a valley compared to the reflection from a plateau and these different reflective properties of the laser, when reflected from these valleys and plateaus, correspond to the binary code data values of “0” and “1” for each bit.", "In one embodiment of the present invention, a similar concept to the CD and DVD is utilized, however, rather than utilizing valleys and plateaus (i.e., bumps and pits) in an aluminum layer embedded in a polycarbonate plastic CD or DVD, the present invention utilizes at least two data values per bit represented by different intensity, colors or color width (e.g., different wavelength or frequency).", "These data values can be optically created, stored in dots or spots, and optically examined or interrogated (e.g., by known transmission and/or reflection techniques).", "These optical dots or spots may be of a size which is approximately the same order of magnitude in size as the bumps and pits which comprise current data tracks used on CD's and DVD's, they may be larger, or they may be smaller.", "In particular, similar to current CD's and DVD's, these dots or spots can be put together to form strings or tracks of data which begin at an innermost portion of a rotatable CD/DVD disk and extend spirally out therefrom in the form of tracks.", "Accordingly, current CD's of approximately 12 centimeters in diameter would be an acceptable size.", "However, if the dots or spots of the present invention are smaller than the “bumps” of the current technology, it is possible for CD's to be smaller in size, if desired, or more data can be placed into the same amount of memory space.", "Thus, it is possible to put an amount of optical information onto a rotatable disk of approximately 12 centimeters in diameter which exceeds that amount of information which is currently stored on CD's/DVD's.", "However, it is possible for the dots or spots to be larger in size than current bumps and pits in CD's/DVD's while still storing more information relative to standard CD's/DVD's.", "This is possible due to at least two effects.", "First, tracks may be capable of being located closer together due to, for example, lesser amounts of crosstalk between bits (e.g., recording and/or reading operations can take place in a lesser amount of space).", "Second, if digital codes higher than binary are adopted, less actual bits need to be stored relative to a binary system (e.g., rather than storing 8 or 16 bits to represent a single piece of information, a lesser number of bits can be used to represent the same piece of information that is being stored).", "It should be noted that as the number of characters that can be stored in a single bit become larger, the digital storage system actually approaches behaviors similar to an analog storage system and can be referred to as “analog-like”.", "In particular, the present invention is capable of utilizing a coding system other than the binary coding system of “0's” and “1's” now used in CD/DVD systems.", "For example, a series or range of dots or spots of different color intensities (e.g., white-gray-black), and/or different colors (e.g., red-yellow-violet), and/or both as in varied intensity of polychromatic light, can be placed upon a surface (or embedded somewhere within a material), such as a suitable photoferroelectric material such as “PLZT” (i.e., lead lanthanum zirconate titanate ceramic), which is shaped in the form of, for example, a disk.", "When, for example, such a disk containing these different intensity data value dots or spots is rotated and interrogated by, for example, a known optical system (e.g., a laser system) with a suitable focusing lens and intensity detecting means (e.g., a suitable photosensitive material or photodiode), the different intensities of the dots or spots can be registered and can be coded to correspond to different values (e.g., “0”, “1”, “2”, “3”, etc.).", "Thus, rather than using a simple binary or base- 2 system where a black dot corresponds to “0” and a white dot corresponds to “1”, the present invention provides for a larger number of options for digitized data storage.", "However, as discussed in more detail herein, depending on the sensitivity of the system, this technique could also be used for actual analog computing.", "Accordingly, for example, in a simple digitized quaternary system having a quaternary code consisting of 0's, 1's, 2's and 3's, there would be 4 different choices of basic digital information storage (i.e., a “base-4” system) rather than only two choices in a binary or base- 2 digital system.", "Likewise, in a digital system which utilizes 8 different combinations of spots or dots (i.e., a “base-8” system) eight different options exist for encoding a piece of information rather than only two options in a standard binary or base- 2 digital system.", "The use of a larger number of possibilities other than “0's” and “1's” (e.g., 0, 1, 2, 3, 4, 5, 6, 7, etc.)", "render it possible to store a much greater amount of information in a much smaller physical area due to the elimination of unneeded bit space.", "Moreover, as the number of possibilities other than “0's” and “1's” increases, (e.g., systems greater than base- 10 such as base- 20 or greater systems) the closer the digital system comes to being or performing like an analog system, which has certain advantages over digital systems.", "An apparatus according to the present invention permits suitable optical data to be stored onto an appropriately constructed and shaped optical recording medium by using, for example, a laser light source.", "The optical recording media (e.g., a PLZT ceramic) can be in the form of a tape, disk, or credit card or any other acceptable shape.", "Alternatively, a photodiode or photodiode assembly can also be used.", "In all such cases, a light source (e.g., laser light source) can be used in combination with, for example, various means for controlling the direction, polarization, intensity and/or final color of the light source.", "Various examples of such systems include the following: (1) a rotatable polarizing filter (or set of filters), sometimes referred to as an analyzer, used in combination with a suitable light source which is subjected to a first polarizing filter(s) selectively modifying the polarized light from the light source.", "Specifically, once monochromatic or polychromatic light has been polarized into a first direction polarizer the polarized light is directed toward a second polarizer (sometimes referred to as an “analyzer”) which, when rotated, results in, for example, different intensities of light being emitted therefrom.", "The different intensities of light can be caused to be incident upon an appropriate recording/detecting means; (2) a light source (e.g., a laser light source), which is used in conjunction with a first polarizing filter is caused to be incident on a suitable light diffractor (e.g., a Bragg modulator) which can cause the polarized light from the light source to communicate with one or more of a series of additional polarizing filters (or analyzers) each of which analyzers are rotated, relative to the polarized source, at different angles, thus resulting an different amounts or intensities of light passing through each of the analyzers.", "Thus, by selectively transmitting the polarized light through the different analyzers (e.g., directed by, for example, the Bragg modulator) different intensities of light can be achieved.", "Such different intensities can be recorded and/or detected by a suitable means; (3) a light source (e.g., a laser light source) used in combination with an acousto-optic Bragg modulator, whereby different driving signals are input to the Bragg modulator which results in different intensities of light being emitted from the Bragg modulator.", "The multiple intensity emissions are then directed to a suitable detecting/recording means and are made to correspond to various data points in base- 2 or higher order digital data storage systems; and (4) rather than utilizing polarizing filters to polarize a monochromatic or polychromatic source of light, liquid crystal cells can also be utilized.", "In particular, light can be caused to be incident upon a liquid crystal cell.", "Depending upon whether voltage is applied to the liquid crystal cell, the output from the liquid crystal cell is oriented into one direction or another.", "Liquid crystals of this type can be made of, for example, lithium niobate.", "However, similar to the embodiments (1)-(3) discussed above, intensities can be varied due to relative polarization directions.", "The outputs of light from the liquid crystal cell can be directed, by appropriate beam displacers (e.g., a Bragg modulator), toward a suitable recording/detecting means.", "In each of the examples (1)-(3) discussed above, it is possible that a polychromatic light source could be utilized.", "In this regard, when polychromatic light is made to be incident upon a first polarizer, and thereafter upon one or more additional polarizers (e.g., analyzers), in addition to different intensities of light flowing through the analyzer(s), a spreading of these different intensities over a particular frequency or wavelength range will also occur.", "Specifically, when using polychromatic light, a spectrum of frequencies of various intensities results in the formation of a somewhat continuous curve of varying light intensities over a particular frequency or wavelength range.", "The use of a polychromatic source can therefore result in secondary indicia for determining the value assigned to stored bits of data (e.g., a “0” or a “1”) and thus enhances data storage.", "For example, not only could the highest intensity of light be recorded/detected, but also the starting and/or ending points which correspond to the lowest frequency intensity of light being emitted, and the highest frequency of light being emitted, respectively, or, alternatively, the area under each curve could also be compared.", "In other words, more than one piece of information can be checked to verify the accuracy of each data point that is stored and read.", "The presence of more than a single piece of information can result in more accurate reading of data and thus result in signal/noise improvements.", "In each of the examples (1)-(4) discussed above, the different intensities or colors of light can be made to be incident upon an appropriate receiving/recording means (e.g., an optical storage means, such as a pohtopolymerisable/photocrosslinkable material, a photodiode assembly, etc.", "), such output light being suitably focussed onto the appropriate receiving/recording means by known lens or optical systems etc., all of which are discussed in greater detail herein).", "Suitable materials for recording various intensities or colors of light include the following materials: inorganic photochromic crystals, photorefractive materials, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence.", "For example, in the simplest embodiment (i.e., that embodiment numbered “(1)” above) for understanding the basic concept for recording various intensities of light (even though this simple embodiment is not as commercially significant as the other embodiments discussed herein), each such recorded intensity corresponds to a different value for a digitized data bit.", "In this basic case, different intensities can be achieved as follows: a polarized light source, such as a laser and polarized filter or polarizer, when used in combination with at least one rotatable polarizing filter (e.g., an analyzer) can result in light of different intensities being transmitted from the at least one rotatable analyzer.", "Specifically, when the laser light source is passed through a first polarizing filter and a second polarizer (e.g., an analyzer) is sequentially rotated (e.g., from 0-90°) a predetermined amount each time relative to the first polarizing filter (i.e., either the first or second filter can be rotated), different intensity dots or spots corresponding to data values having different meanings (e.g., “0”, “1”, “2”, etc.)", "can be produced.", "The amount of rotation of the analyzer in relation to the polarizing filter can correspond to a different intensity of light being recorded on a suitable optical recording medium (e.g., a photoferroelectric material such as “PLZT”, inorganic photochromic crystals, photorefractive materals, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence).", "If a photoferroelectric material is utilized, the different intensities of light can be caused to be incident upon the photoferroelectric material by use of, for example, a suitable optic lens system or a fiberoptic cable (or a series of cables) that is capable of accurately directing the optical signal to the photoferroelectric material for the proper recording of the various optical signals.", "Thus, a large number of different intensity spots or dots corresponding to data values for a single bit can be created.", "This basic system and apparatus permits higher order coding systems (e.g., base-3 or greater) to be utilized.", "The manner in which such optical data is stored, on, for example, a rotatable disk, as well as interrogated, can be by techniques which are substantially parallel, and/or use hardware which is similar, to those techniques and/or hardware currently used in CD and DVD systems.", "For example, a series of bits of an appropriate binary or higher order digital code system can be recorded onto a rotatable disk by starting to record the different intensity dots or spots near a central potion of the disk.", "The recording continues to record data in a spiral manner to form tracks of data, such data being suitably separated, physically, to avoid corruption and crosstalk, until an outermost radial portion of the disk is encountered.", "Likewise, such a disk can be read or interrogated by, for example, a laser system which focuses light onto the dots or spots and suitably measures the intensities (e.g., reflectance or transmittance) of the spots of data.", "The different intensities can be interpreted by suitable algorithms and programs to correspond to different values in the desired coding system.", "Moreover, by utilizing a digital optical coding technique other than binary, the present invention permits a much more rapid adoption of the desired “Unicode”, which takes into account, for example, all English language characters, Chinese characters, numbers and symbols, etc., in various digitized forms.", "In another embodiment of the invention, a system which utilizes magnetic memory in combination with a suitable retrieval/reading means is utilized.", "These memory/retrieval systems can be caused to function, generally, in a manner that is somewhat similar to current magnetic memory systems (e.g., magnetic tapes, cards, disks, hard drives, etc).", "In addition, these memory systems can also use digital coding/reading systems higher than a base- 2 system, if desired.", "Still further, these memory systems can also store/retrieve information in an analog or analog-like format.", "Specifically, it is another object of the invention to utilize certain aspects of the phenomena known as the Zeeman effects to result in a means for the storage and retrieval of information onto/from suitable magnetic media.", "The term “Zeeman effects”, as used herein, denotes the effects of external magnetic fields on frequencies of atoms, molecules and components thereof, in the broadest sense.", "Thus, as used herein, the term “Zeeman effect” includes not only what may be considered to be Zeeman effects in the more narrow, classical sense but also other magnetic effects such as magnetic quadropole effects, etc.", "Digital data may be stored as either a binary code or some higher order code that is greater than the binary or “base-2” code.", "Moreover, due to the apparent sensitivities of these data storage techniques, data may be stored/retrieved in an analog or analog-like manner due to the inherent operation of the Zeeman effects.", "Specifically, without wishing to be bound by any particular theory or explanation, it appears that use of the Zeeman effects can result in a somewhat continuous set of differing data values that can be stored/retrieved.", "The present invention utilizes one aspect of the Zeeman effects in the following general manner.", "A magnetic field is applied to a suitable media that is capable of receiving and storing at least a portion of the applied magnetic field.", "One example of such a suitable magnetic material is a ferromagnetic material that is capable of exhibiting different amounts of magnetic domain alignment as a function of increasing magnetic field strength up to its natural magnetic saturation point (i.e., whereby any further increases in applied magnetic field do not result in any measurable further alignment of magnetic domains).", "Various known materials exist that meet this basic criteria, including many materials currently utilized in the magnetic storage industry (e.g., gamma Fe 2 O 3 , gamma Fe 2 O 3 modified by Co, CrO 2 , etc.).", "The various materials can be held together by a variety of organic polymers including, for example, vinyl chloride, polyvinylchloride, methylacrylate, polymethylmethyacrylate, polyurethane, epoxy, polyamides, etc.", "Suitable substrates include typical polyesters for flexible materials and aluminum for rigid materials.", "The differing amounts of stored magnetic field can result in a corresponding splitting or shifting of the electromagnetic energy frequencies or spectral frequencies (e.g., fine spectrum splitting, hyperfine spectrum splitting, vibrational spectrum splitting, etc.)", "of one or more atoms or molecules of the magnetic material itself, or similar splitting or shifting in one or more materials that are located contiguous (e.g., in the magnetic material or in a read/write head) to the material containing the stored magnetic fields.", "In the Zeeman effect, the amount of splitting or shifting of one or more frequency spectrum(s) is a function of the strength of the applied/stored magnetic fields.", "Typically, the higher the magnetic field strength that is stored/applied (e.g., stored in a first material containing magnetic domains and thereafter applied to (e.g., influencing) a second contiguous material that exhibits Zeeman splitting at the stored magnetic field strengths), the further apart the splitting or shifting will be of one or more of the electromagnetic energy frequencies of a chosen spectrum of the targeted atom(s) or molecule(s).", "In this Zeeman effect, when a relatively wide splitting of electromagnetic energy frequencies occurs due to the applied magnetic field, the amount of splitting may correspond to frequencies in the microwave region; whereas a narrow splitting will typically correspond to lower frequencies (e.g., radio).", "The amount of splitting that occurs is important because in order to determine the amount of splitting that has occurred, a suitable interrogating electromagnetic energy is caused to be incident upon, for example, the magnetic material itself or the aforementioned contiguous material.", "The contiguous material that exhibits Zeeman splitting at the stored magnetic field can be present as, for example, one or more separate sheets of material that are located adjacent to, and/or contiguous with, the material that contains the stored magnetic field.", "When multiple contiguous materials are utilized, the present invention contemplates the use of a comparative analysis of the differences in Zeeman splitting between two or more such contiguous materials.", "Alternatively, a material comprising, for example, one or more material(s) that are mixed with the magnetic material (e.g., a binder which holds together at least a portion of the material that stores the magnetic field) could also be used.", "More specifically, a particular interrogating frequency, or set or sweep of frequencies, needs to be capable of favorably interacting with one or more of the split, shifted or splitting frequencies which result in the magnetic material, or in an alternative embodiment in one or more of the contiguous material(s), due to the different magnetic fields stored therein.", "In other words, a first magnetic material is desirably chosen so that an applied magnetic field can be reasonably stored therein.", "However, it is desirable for the magnetic material to be capable of storing reliably several magnetic fields of different strength (e.g., capable of having varying numbers of magnetic domains align with an ever changing magnetic field and such domains remain frozen or pinned so that they do not change with time) and, for example, in one embodiment of the invention, the contiguous material needs to produce split, shifted and/or splitting frequencies in response to the stored magnetic fields.", "In this regard, if one or more contiguous material(s) are utilized in connection with the magnetic material(s), the contiguous material(s) needs to exhibit a splitting and/or shifting of one or more frequencies, for example, of its rotational, vibrational, etc., bands (e.g., at the atomic level) and the resultant splitting or shifting that occurs in the contiguous material needs to be detectable by an appropriate interrogating electromagnetic source (discussed in greater detail later herein).", "Alternatively, a contiguous material does not need to be utilized.", "In this case, the magnetic material per se, or at least a portion thereof, needs to exhibit desirable and detectable splitting and/or shifting of one or more frequencies at varying stored magnetic strengths.", "Still further, it should be noted that the contiguous material could actually be located in a suitable read head which, similar to magnetoresistive and giant magnetoresistive heads, is influenced by the magnetic field strengths stored in the magnetic media.", "However, rather than measuring changes in resistivity in materials comprising the read heads, the splitting or shifting of frequencies will be detected by a suitable technique, discussed elsewhere herein.", "In this embodiment of the invention, the contiguous material may be a solid, a liquid, a gas or plasma (e.g., any material that shows desirable Zeeman splitting effects).", "With specific regard to interrogating with an applied electromagnetic source to determine the amount of Zeeman splitting and/or shifting that has occurred at a spot on the material that corresponds to a stored bit of data, at least one suitable frequency can be applied as an interrogating frequency, or, alternatively a sweep or combination of frequencies can be applied as interrogating frequencies.", "In this regard, if a single suitable frequency is chosen as the interrogating frequency, then heterodyne interactions of the various split, shifted and/or splitting frequencies with the single interrogating frequency can be measured and determined.", "Alternatively, resonance interactions of the various split, shifted and/or splitting frequencies can also be measured and determined.", "Each such determined heterodyned and/or resonant frequency can be made to correspond, by suitable programming or algorithmic techniques, to various data values.", "Alternatively, a series or sweep of frequencies which may resonate with or heterodyne with various split, shifted and/or splitting frequencies achieved in the contiguous material can also be utilized.", "These particular affects will be discussed in much greater detail later herein, however, it should be understood that these data acquisition techniques could be performed using hardware which is somewhat similar to that hardware currently used for magnetoresistive and giant magnetoresistive heads.", "For example, once an interrogating frequency has been input to a material exhibiting Zeeman splitting and/or shifting, the output (e.g., a heterodyned frequency) can be used as a driving signal or input (e.g., depending on strength or frequency either amplified or unamplified) for various other devices.", "One example of such other device is an acousto-optic Bragg modulator.", "In this detection scheme, an appropriate light source is caused to be incident upon an appropriate Bragg modulator.", "Suitable materials for a Bragg modulator include, for example, lithium niobate crystals and gallium arsenide semiconductor materials.", "The incident light beam can be caused to be deflected in differing amounts due to different driving signal inputs (e.g., depending on strength or frequency either amplified or unamplified) being directed into the Bragg modulator.", "In particular, for example, the different heterodyne outputs that correspond to different Zeeman splitting amounts (and thus different data points such as a “0” or a “1”) can be input into a Bragg modulator and cause light which is incident on the Bragg modulator to be deflected in different amounts.", "Thus, the various Zeeman splitting and/or shifting frequencies that are produced can eventually result in different amounts of light deflection due to the use of a Bragg modulator.", "The amount of deflection of light can be observed by the use of, for example, the light detection devices discussed earlier herein.", "Similarly, the different heterodyne outputs could be directed into a suitable filter which, due to the different inputs, would regulate the intensity of light passing therethrough.", "Accordingly, the different intensities of light could then be detected/recorded by a suitable means discussed elsewhere herein.", "This particular embodiment of the invention which utilizes differing magnetic fields to result in differing amounts of an appropriate frequency splitting and/or shifting is very suitable to be used for the analog storage/retrieval of information.", "In particular, a substantial continuum of splitting and shifting frequencies results in most materials that are subjected to a corresponding continuum of changing magnetic field strengths.", "If a continuum of such differing split, shifted and/or splitting frequencies can be reliably detected, then analog or analog-like (e.g., higher order than binary systems) computing with this technique is facilitated.", "Additionally, many of the aforementioned embodiments of the invention can also be used in combination with various holographic and/or three dimensional memory approaches.", "To achieve all the foregoing objects and advantages, and to overcome the looming problem in the art of an impending memory barrier, the present invention discloses various means for storing and retrieving information in a digital and/or analog format.", "With regard to the digital storage of information, a code other than a binary code for the storage of information can be utilized.", "The result of using a higher order code for digital information storage (i.e., 3 or more characters) is the ability to store more information in the same amount of area or the same amount of information in a smaller area.", "Accordingly, the present invention provides significant advancement in the art of data storage and/or data retrieval which will assist in preventing the memory barrier from being realized." ], [ "TECHNICAL FIELD The present invention relates generally to the storage and/or retrieval of information on magnetic and/or optical storage media by using one or more novel approaches alone or in combination.", "These novel approaches are capable of using at least one code which may comprise more than two values (i.e., more than a “0” and a “1”).", "A first series of approaches for the storage of information applies generally to existing optical storage/retrieval systems (e.g., CD's, DVD's, etc.)", "as well as novel optical systems; while a second series of approaches applies generally to existing electric and/or magnetic storage/retrieval systems (e.g., magnetic, magneto-optic, etc.)", "as well as other novel electrical/magnetic systems.", "Each series of approaches is capable of storing information in one or more codes, wherein such approaches permit, if desired, the use of at least one higher order code which is different from the traditional binary code of “0's” and “1's” currently utilized for the storage of digital information.", "Said at least one higher order code may comprise three or more optical and/or magnetic values or bits that are used to represent, for example, ASCII or Unicode characters that are currently represented predominately by the traditional binary code.", "This higher order code may also be an analog or analog-like code.", "BACKGROUND OF THE INVENTION Data storage devices have been becoming smaller in size (as well as faster) due to, for example, the continued improvement in the ability to store more information in smaller spaces.", "For example, over the past twenty years the ability to place more data on magnetic storage media such as magnetized hard-disk drives and/or floppy disks, as well as on optical media (e.g., compact disks “CD's” and more recently, Digital Versatile Disk “DVD's”) has been increasing at a phenomenal pace.", "The impressive increase in the ability to store more data in smaller areas has been one of the main driving forces for rendering computers, and other electronic devices with reasonably sized memories, accessible to a typical home or small business consumer.", "Further, the high operating speeds of microprocessors have required stored data in computers to be rapidly accessible (e.g., both the rate at which information is delivered to a microprocessor as well as the amount of time it takes between the time that a microprocessor requests a file and when the first piece of data begins to flow from the file to the microprocessor are important).", "Magnetic memory has undergone many recent improvements and has been utilized extensively in data storage/retrieval devices because magnetic memory currently provides the least expensive alternative for the fast writing and reading capabilities for data.", "However, the various currently utilized magnetic technologies face a variety of technical challenges and appear to be headed for a stopping point.", "Specifically, these various approaches for the storage and/or retrieval of information may no longer be capable of meeting the growing demands of the industry, without experiencing significant technical enhancements.", "For applications where speed has historically been less important, various optical technologies have been growing in popularity.", "Many technical advances have been made in the recording and/or retrieval of information by optical means.", "In systems which use compact disks (“CD's”), compact disk read-only memory (“CD-ROM's”) and/or Direct Video Disks (“DVD's”), the storage and/or retrieval of information has also been of great interest.", "For example, the first CD's manufactured were CD-ROM's that are currently capable of storing about 650 MB of data.", "This equates to about 74 minutes of play-time for music that has been recorded in a typical recording mode.", "In particular, the standard current approach for utilizing CD-ROM's, includes the use of a solid state interrogating laser (or in some cases more than one laser) which has a wavelength of about 780 nanometers (0.78 μm).", "The standard CD-ROM is about 12 cm in total diameter and has a thickness of about 1.2 mm.", "The standard CD-ROM has information recorded on plateaus (“bumps”) and valleys (“pits”) that spiral from the inside of the disk toward the outside of the disk.", "The digital data stored in the tracks that comprise the spirals must be separated by enough space (e.g., tracks are typically separated radially by about 1.6 μm) so that when the data is read with an interrogating solid state laser, crosstalk from adjacent tracks does not occur (i.e., recorded information is received from only one track at a time).", "Accordingly, the placement of more information onto a typical CD has been limited by the required size (e.g., length and width) of the plateaus (“bumps”) and valleys (“pits”).", "The size of the plateaus and valleys (e.g., typically not less than 830 nanometers in length) has been a function of various factors, including the wavelength of the laser utilized to read the information from the CD.", "Various attempts have been made to increase the memory capacity of current CD's, including the stacking of a plurality of disks one on top of the other so as to provide more memory space, as well as other attempts to miniaturize the digitized information stored in the tracks.", "The most recent significant improvements in this type of optical storage/retrieval technology have been the creation of the DVD, which is known as the “Direct Video Disk” or the “Digital Versatile Disk”.", "The first commercial products of DVD's entered the marketplace in late 1997.The DVD technology is similar to the technology in CD's, except that, for example, the wavelength of lasers utilized to read information from DVD's is smaller than that used for CD's, namely, about 635 nanometers or 650 nanometers, compared to about 780 nanometers for CD's.", "The size of the plateaus (“bumps”) that make up the DVD are about 320 nanometers wide, a minimum of about 400 nanometers long and about 120 nanometers thick.", "About 740 nanometers separate one track from the next, compared to the about 1600 nanometer separation of tracks in CD's.", "A standard DVD is physically about the same size as a CD, but has a memory capacity that is about seven times greater than that of CD's.", "In particular, the memory capacity of a one-sided, one-track, DVD is now about 4.7 GB.", "However, recent improvements in DVD have allowed digital information to be placed on both sides of a single track, as well as both sides of a double track.", "In this regard, a first interrogating laser reads the digitized data on a first side of a first track (i.e., in the form of bits arranged in a spiral track that begin in the innermost portion of the DVD and spirals outward).", "Then, information can be retrieved from the opposite side of the first track in a similar manner from a second laser; however, the spiral track of information typically begins on the outermost portion of the disk and spirals inward toward the center.", "Accordingly, when dual-track, dual-sided DVD disks are used, the current amount of memory that can be achieved is about 17 GB.", "Another optical data storage technique which shows promise is known as holographic memory.", "The potential for high storage capacity and high storage retrieval rates using the holographic technique is tremendous.", "In particular, for example, recent work in a system known as Holographic Data Storage System (“HDSS”), has shown that data can be accessed in 100 microseconds or less, which is several orders of magnitude faster than the millisecond retrieval of data in typical magnetic-disk drives.", "Briefly, in this technology, an optical-interference pattern is created within a desired photosensitive material (e.g., a crystal or a polymer).", "The optical interference pattern is imprinted as a result of physical changes in such materials.", "The interference pattern is created by the interaction between an object beam and a reference beam, both of which typically originate from a single light source (e.g., a laser), but are split into two different pathways by a beam splitter.", "Specifically, the object beam is incident on a target object that contains data, and modifies the object beam, while the reference beam, taking a separate path which leaves it unmodified, interferes with the object beam at the holographic storage medium.", "When the two beams meet at the holographic storage means, the interference pattern which results from their interaction is stored on or in the holographic storage means.", "The holographic storage means is later interrogated by a reading or reference beam, which upon interacting with the holographically stored interference pattern, can then recreate the holographic image containing the data originally stored in the object beam.", "While the holographic optical technique shows great promise for the future, current industrial emphasis is being placed primarily on improving existing digital optical and magnetic storage/retrieval systems so that more information can be stored in smaller spaces, and retrieval times can be decreased without significantly changing the basic methodology and hardware for the storing and retrieving of information.", "With regard to placing more data onto magnetic recording media, effects such as the paramagnetic effect (e.g., magnetic crosstalk between magnetic domains comprising bits) are already beginning to cause problems.", "Experts have postulated that in coming years, magnetic storage technologies could reach a limit imposed by the superparamagnetic effect (“SPE”).", "The physical phenomenon known as SPE can occur in a magnetic data storage system when the magnetic spins of electrons in the domains which comprise a bit (e.g., in current technology typically either a “0” or a “1”) become unstable due to, for example, the environmental thermal energy surrounding the bits.", "Instability of bits occurs when smaller and smaller numbers of domains or atoms are used to comprise a bit and the ability for the domains or atoms to maintain their given spin directions (e.g., corresponding to, for example, a “0” or a “1”) becomes difficult.", "Non-maintenance of spin directions results in a “flipping” of spins back and forth and thus changing “0's” to “1's” and “1's” to “0's”, etc.", "Such changing or flipping of spins will result in a corruption of the data or information that the “0's” and “1's” represent.", "Accordingly, in order for smaller numbers of atoms to be used to form a bit and in order for the bit to be viable, techniques and/or materials for preventing the superparamagnetic effect need to be developed to overcome the deficiencies of the prior art.", "Another potential problem in recording information magnetically is that the write head which is used to align magnetic domains in the magnetic storage media may be limited in the ability to impart a required field strength over the entire domain area comprising a bit to cause the magnetic domains to behave as though they have been exposed to a substantially uniform field.", "In such cases, it is possible that differently oriented domains within a bit (e.g., differently oriented due to being subjected to different magnetic field strengths within a single writing head due to, for example, non-uniformity of the magnetic flux lines emanating from the write head and/or differently oriented because some domains require more energy than other domains to align due to, for example, their location within a bit) may have a higher tendency to become corrupted, as well as adding to signal/noise ratio problems.", "One attempt for miniaturizing domains involves the use of so-called “hard” magnetic materials.", "The use of hard magnetic materials is desirable when compared to “soft” magnetic materials because hard magnetic materials are more likely to maintain their magnetic spin directions when domains comprising the hard magnetic materials are closely spaced.", "In this regard, various rare-earth and transition elements have been found to be magnetically stable and are thus termed “hard”.", "Such magnetically stable materials are known to have a higher coercivity, which is typically represented by “Hc”.", "Stated in an over simplified manner, the higher the Hc value, the greater the resistance of the material to outside influences, such as outside magnetic fields (e.g., the harder it is for material to lose its imprinted spin direction due to an increase in temperature and/or the influence of other magnetic fields, etc.).", "However, one drawback for utilizing “hard” magnetic materials is that hard magnetic materials are more difficult to magnetize than soft magnetic materials (e.g., in some cases, depending on the particular materials chosen, much more difficult to magnetize).", "This makes it more difficult to record data initially in hard magnetic materials.", "There are various known approaches for solving the problem of encoding data into hard magnetic materials which approaches utilize a laser in conjunction with an appropriate magnetic write head.", "These various approaches have been referred to generally as the “magneto-optical” approach.", "These magneto-optical approaches effectively reduce the Hc value of the magnetic domains by causing the domains to be locally heated just prior to being subjected to the magnetic write head.", "Thus, the coercivity (Hc) of the domains is effectively reduced and when such effective reduction in Hc has occurred, a somewhat standard write head, or in some cases a very different write head, can be used to alter favorably the magnetic domains.", "In other words, the use of a laser, which provides thermal energy to the domains of the hard magnetic material, causes the hard magnetic material to behave in a manner which is similar to a somewhat softer and thus easier to magnetize magnetic material.", "Each of the known approaches for utilizing the magneto-optic techniques involves the use of at least one laser and/or at least one laser focusing system.", "However, one of the common problems facing these magneto-optical approaches is that the lasers that are utilized to soften the hard magnetic material prior to subjecting the hard magnetic material to a magnetic field is that the lasers typically generate excess amounts of heat that can flow to neighboring bits or tracks (e.g., thermal energy is caused to migrate to undesirable neighboring tracks or areas resulting in a potential corruption of stored information).", "Accordingly, various laser applications and/or storage solutions have been devised to minimize the transfer of heat from the laser to neighboring tracks, and thus minimize the loss of recorded information.", "However, to date this thermal transfer of laser energy still presents problems to varying degrees in all the prior art approaches.", "One interesting technique for the reading of data in connection with one optical-magnetic system is the use of an effect known as the “Kerr effect”, and more particularly, the “magneto-optical Kerr effect”.", "Briefly, current uses of the magneto-optical Kerr effect involve incident polarized light being reflected from the surface of a magnetic domain in different ways, depending on the orientation of the magnetic domains (e.g., depending on electron spin directions).", "The changes in the reflected polarized light are made, by algorithms, to correspond to different digitized data.", "Specifically, when the incident polarized light encounters different magnetic field orientations (e.g., north or south domains comprising a bit which are ordered to represent either a “0” or a “1”) the polarization state of the reflected light is changed.", "Another approach to the digital storage of information that shows future promise is known as atomic resolution storage (“ARS”).", "This approach, similar to holographic techniques, is capable of storing tremendous amounts of information in a small amount of space.", "In this technique, generally, very small electron probes, which are used to generate electron beams, are formed into an array.", "The electron probes have tips that are roughly the size of atoms.", "The electron beams from the electron probes are made to be incident upon a storage medium so that the incident beams cause some sort of physical change in the storage medium (i.e., the medium comprising the computer memory).", "One example of such a storage medium is a material that is capable of containing at least two stable phases at ambient operating conditions so that an incident electron beam emitted from an electron probe changes the storage material from one phase to the other.", "In this technique, encoded bits of information are represented by the change in phase of the phase-change storage material.", "This technique, similar to the magneto-optical technique discussed above, also suffers from the problem of heat flowing between data spots created by the incident electron beams when the spots or bits are initially recorded.", "Accordingly, one of the challenges faced by this relatively new technology includes reducing the flow of heat between data spots which are created by the array of electron beam generators.", "The retrieval of stored information is also a challenge.", "In this regard, as bits become smaller and include lesser numbers of magnetic domains per bit and/or the domains are oriented differently, (e.g., domains are aligned perpendicular to the substrate containing at least one magnetic surface thereon instead of being parallel to the substrate surface) such bits are capable of being packed more tightly together.", "Assuming that the bits are not corrupted by their tight packing and/or smaller numbers of magnetic domains and/or non-uniform alignment within a bit, then reading of these tighter packed domains comprising the bits also becomes a challenge which the prior art is still struggling to overcome.", "Historically, large numbers of magnetic domains were present in each bit which contained digital data.", "Thus, the resulting or induced magnetic fields were relatively large and various forms of known inductive reading techniques were sufficient to ascertain whether the hundreds or thousands of aligned domains in a bit corresponded to a “0” or a “1”.", "However, as prior art miniaturization recording techniques progressed, the known inductive reading techniques were typically not sensitive enough to determine accurately the alignment of ever decreasing numbers of domains comprising a bit (e.g., signal to noise problems in the storage and/or retrieval of digital data bits were exacerbated).", "The development of magnetoresistive heads, and the subsequent development of giant magnetoresistive heads, has enabled smaller numbers of domains comprising a digital bit to be read accurately.", "In both of magnetoresistive and giant magnetoresistive heads, the electrical resistance of one or more materials comprising the read head(s) is examined.", "In particular, both magnetoresistive and giant magnetoresistive heads are made from materials that exhibit small changes in their electrical resistance as a function of magnetic fields created by oriented domains to which such heads are exposed.", "Giant magnetoresistive heads are two or three times more sensitive than magnetoresistive heads due to their novel structure which includes layering of different materials stacked on top of each other.", "One problem that continues in these technologies is the presence of magnetization vortices in the recorded domains.", "Such vortices may result from the difficulty in achieving uniform domain alignment within a bit due to, for example, the difficulty in applying a required magnetic field (e.g., uniform field) to the domains during the recording or writing process.", "While use of the magneto-optical Kerr effect and new read heads such as magnetoresistive heads and giant magnetoresistive heads have improved digital data retrieval techniques, further miniaturization is pushing the limits of these systems.", "Accordingly, additional improvements are necessary in the ability for hardware, combined with suitable programming, to detect even smaller numbers and/or smaller sizes of domains comprising digitized (or analog) data bits.", "Thus, additional innovations in the detection of stored data are required.", "In addition to the storage of information digitally, the possibility or promise of storing information in an analog manner continues to intrigue various investigators.", "While certain improvements have been made in, for example, the processing of analog information (e.g., the advent of VLSI (Very Large Scale Interrogation) chips, the techniques for the storing of analog information need much improvement.", "Particularly, while analog computing has been known for several decades, an accurate and fast-paced system for the storage and retrieval of information has continued to elude researchers.", "In this regard, digital computing has received the greatest amount of attention because of it's relative dependability for the storing and retrieving of information in a “1” or a “0” digitized format.", "Briefly, digital approaches typically include storing a sampling of the analog information that is to be recorded.", "The number of samples taken (e.g., such sampling typically occurs in predetermined units of space or time) from the analog source will correspond to the accuracy between the original analog image or sound and the stored digital data (i.e., the more digital samples that are recorded of an original analog image or sound, the more accurate the reproduction of the original analog image or sound will be).", "These techniques of predetermined quantum amounts of sampling of analog images have been adequate for most digitized applications, however, as the need for more accurate recording and retrieval of information occurs (e.g., in, for example, the fields of computing or determining various engineering and scientific relationships in the following representative areas: gravitational; electrostatic; magnetic; thermal; stress; fluid flow field analysis; wave propagation; image processing; etc.", "), the availability of computing with more accurate data becomes important.", "However, the digitization of tighter and and/or smaller sampling amounts from an analog source can result in tremendous amounts of digitized data being stored to represent a small actual amount of analog information.", "This is one area where analog computing has a distinct advantage compared to digital computing.", "In particular, an analog computer will store only a single piece of information corresponding to a single sampling area, whereas a digital computer will need to store somewhere between, for example, 8 and 16 bits of data to represent the same single piece of information stored in an analog fashion.", "The large number of bits required to store digital information corresponds to the requirement to store information in an ASCII or Unicode format.", "However, in order to escape from the current ASCII or Unicode format, analog storage requires a very large number of different data bits (e.g., data corresponding to much greater numbers than merely “0s” and “1's”) to be stored and retrieved.", "Current prior art techniques have great difficulty in storing and/or reading bits corresponding to anything other than “0's” and “1's”, let alone the attendant problems of storing and/or reading, for example, a near infinite number of possibilities (e.g., 1, 2, 3 .", ".", ".", "n+1).", "However, when performing complex calculations, the digital process may result in very slow processing times as well as requiring very large memories.", "It is for this, as well as other reasons, that analog computing is still very attractive.", "Thus, while advances in the processing of analog information have been made, reliable techniques for the storage and retrieval of analog information are still being sought without any good solutions currently existing.", "As stated above, with regard to digital computing, much of the recent technical emphasis in data storage and retrieval for both optical and magnetic memory enhancement has been on shrinking more information into smaller areas.", "The ability to place bits, which are the smallest elements of information used by current digital memory systems, into a binary code format, which is a combination of “0's” or “1's”, into a smaller area on, for example, magnetic and/or optical disk storage media (e.g., a disk or a tape) may soon reach a limit.", "Thus, current technologies for writing and/or reading binary data onto and/or from current storage media will be pushed to the maximum limit.", "Computer designers are already facing a performance gap whereby processors can process information faster than the information can be retrieved from computer memory.", "The majority of the currently proposed approaches for the digital storage of information, suffer from the same problem, namely, that the magnetic and/or optical information which is stored uses a binary code (i.e., a base-2 number system) which is a combination of “0's” and “1's”.", "The predominance of the use of a “binary” code has been due to, for example, difficulty in distinguishing signals (e.g., a “0” or a “1”) from background noise.", "In this regard, many techniques have been proposed which use various hardware, as well as algorithms, which attempt to read accurately the stored information as either a “0” or a “1”.", "However, certain prior art techniques have been postulated for utilizing codes other than binary.", "In particular, U.S. Pat.", "No.", "6,154,432, entitled “Optical Storage System” discloses a holographic system which records information in very small spots and postulates that 10 or more digits may be possible to record on a single bit.", "Accordingly, this holographic technique discloses codes higher order than binary.", "Further, for example U.S. Pat.", "No.", "5,450,363, entitled “Gray Coding for a Multi Level Cell Memory System” discloses the use of a so-called “gray code” approach (also known as “gray scale” by others) for data storage.", "This approach also uses a system which is higher order than binary.", "In particular, reference is made to the storage of multiple bits of information in a single memory cell thus creating a multi-level cell.", "Still further, U.S. Pat.", "No.", "6,061,005, entitled “Encoding of Data”, discloses a method of encoding data on magnetic cards which uses symbology other than binary, such as trinary, quaternary or even higher symbology, for the recording of information.", "However, while various techniques have been postulated for systems which use codes higher than binary, none of such codes have been commercially adopted due to, for example, signal to noise ratio problems, etc.", "In particular, reading errors can occur due to lack of contrast between data values.", "Such lack of contrast can be caused by random noise (e.g., vibrating inaccurate positioning of recording/reading heads, fidelity loss during recording, etc.)", "and determinate variations.", "Accordingly, a need exists for techniques which can be commercially viable which can use codes reliably that are higher order than traditional binary codes for the storage and/or retrieval of information.", "A very particular combination of “0's” and “1's” is known as the American Standard Code for Information Exchange (i.e., the ASCII code) which assigns a unique code number between 0 and 127 to each of the 128, 7-bit, binary number characters.", "Table 1 below shows in table form the current ASCII characters arranged by Hexidecimal digits (i.e., 128 different encoding combinations of groups of seven bits).", "Reading within the Table from left to right and then down, the first 32 values are codes which correspond to various computer control functions such as line feed, carriage return, etc.", "The 33rd value corresponds to the “space” character.", "Values 34-48 correspond to symbols and punctuation marks.", "Values 49-58 correspond to digits, and so on, until reaching value 128, which corresponds to “delete”.", "In addition, each of these values also has a corresponding hexidecimal character.", "For example, the hexidecimal digit corresponding to the “space” character is “20”, while the hexidecimal digit corresponding to the letter “n” is “6E”, and so on.", "TABLE 1 00 01 02 03 04 05 06 07 08 09 0A 0B 0C 0D 0E 0F 00 NUL SOH STX ETX EOT ENQ ACK BEL BS TAB LF VT FF CR SO SI 10 DLE DC1 DC2 DC3 DC4 NAK SYN ETB CAN EM SUB ESC FS GS RS US 20 !", "“ # $ % & ‘ ( ) * + , − .", "/ 30 0 1 2 3 4 5 6 7 8 9 : ; < = > ?", "40 @ A B C D E F G H I J K L M N O 50 P Q R S T U V W X Y Z [ \\ ] {circumflex over ( )} — 60 ’ a b c d e f g h x j k l m n o 70 p q r s t u v w x y z { \| } ˜ DEL These 128 characters are enough characters for North American English to be stored and/or retrieved, but are not enough for many other languages (Note: In ASCII, the eighth bit is used, and is thus normally set to “0”).", "To represent more than 128 characters, 8-bit (rather than 7-bit) binary numbers are utilized.", "For each digit in these eight bits there are two choices for the digit, either “0” or “1”.", "This results in a possibility of an additional 128 characters or a total of 256 different combinations of “0's” and “1's” (i.e., 28).", "Accordingly, the current data storage approaches utilize these 256 combinations of “0's” and “1's”.", "However, these 8-bit numbers still do not provide enough room for all characters that need to be used in the world (e.g., certain Asian languages have thousands of characters each).", "In addition, there is no universal agreement in the world on what all the characters should be, whether referring to the 128 characters, or the 256 characters, or any number of characters beyond these.", "However, certain approaches for dealing with all the various languages in the world, which collectively contain many thousands of characters, are beginning to utilize something referred to as the “Unicode”, which uses a 16-bit character set.", "In order to adopt something similar to the Unicode, even greater strain will be placed on current memory techniques.", "Whether using an 8-bit, 16-bit, or some other approach, all such approaches have been limited primarily to using a binary system of data storage.", "However, due to: (1) the impending size barrier for the storage of information on, for example, magnetic and/or optical media; (2) the performance gap between how fast a processor can process information versus how fast a processor can access information; and (3) the goal for a universal adoption of a common set of characters throughout the world, a different approach for the storage of information, either by digital or analog approaches, is clearly needed.", "The present invention satisfies the current and future data storage needs by using different approaches for the storage and/or retrieval of data; and/or different codes that use a code higher than the current “base-2 code”.", "SUMMARY OF THE INVENTION It is an object of the invention to provide a means for storing/retrieving digitized information optically.", "It is an object of the invention to provide a means for storing/retrieving digitized information magnetically.", "It is an object of the invention to provide a means for storing/retrieving analog information optically.", "It is an object of the invention to provide a means for storing/retrieving analog information magnetically.", "It is an object of the invention to provide a storage/retrieval means for utilizing codes other than binary.", "It is an object of the present invention to provide an increased number of characters that can be stored as a single bit of information.", "It is an object of the invention to provide the ability to store the 128 ASCII-type characters in a much smaller space than the space that is used in a standard binary code of “0's” and “1's”.", "It is an object of the invention to provide a means for storing/retrieving codes other than binary.", "It is an object of the invention to utilize at least a base-3 optical storing/retrieving system.", "It is an object of the invention to utilize a least a base-4 coding storing/retrieving system.", "It is an object of the invention to utilize at least a base-6 coding storing/retrieving system.", "It is an object of the invention to utilize at least a base-8 coding storing/retrieving system.", "It is an object of the invention to utilize at least a base-10 coding storing/retrieving system.", "It is an object of the invention to utilize particular aspects of the Zeeman effect in connection with the storage/retrieval of digitized information.", "It is an object of the invention to utilize particular aspects of the Zeeman effect in connection with the storage/retrieval of analog information.", "It is an object of the invention to utilize different shades or colors of spots or dots in connection the optical storage of digitized information.", "It is an object of the current invention to utilize different shades or color or spots or dots in connection with the optical storage of analog information.", "It is an object of the invention to utilize either monochromatic or polychromatic light in connection with the optical storage of digital and analog information.", "It is an object of the invention to utilize an analog-like approach to the storage of data.", "The current process of storing bits, which is the smallest element of information used by current memory systems, uses a binary format of only “0's” and “1's”.", "A bit which is “off” is considered to be “false” or “not set”, while a bit that is “on” is considered to be “true” or “set”.", "Historically, because bits could only be one of two values, bits have been combined together into larger units in order to represent more characters or values.", "“Nibbles” are groups of four bits and “bytes” are groups of eight bits (i.e., two “nibbles”).", "Bytes are typically what are used to store “characters”.", "“Characters” typically include letters, numbers and/or symbols.", "For example, the ASCII code uses 128 different encoding combinations to represent: (i) The letters “A-Z” in both upper and lower case (i.e., values 65-90 and 97-122, respectively); (ii) Special characters (e.g., “<”, “.”, “%”, “$” “?”, etc) (e.g., values 33-47); (iii) The numbers 0 - 9 (i.e., values 48-57); and (iv) Certain control codes used for device control (i.e., values 0-31).", "To represent more than 128 characters, an eighth bit can be utilized.", "However, the ability to store a binary code of only “0's” and “1's” limits the ability for information to be miniaturized.", "For example, to store a single character from the ASCII code, at least seven bits of memory (typically eight bits are currently required because the last bit is usually set to zero) are needed.", "Table 2 sets forth one example of how a typical computer memory system would store the word “Patent”.", "TABLE 2 ASCII LET- CHAR- BINARY HEXADECIMEL BITS OF TER ACTER NUMBER DIGIT OR BYTE MEMORY P = 80 = 01010000 50 8 a = 97 = 01100001 61 8 t = 116 = 01110100 74 8 e = 101 = 01100101 65 8 n = 110 = 01101110 6E 8 t = 116 = 01110100 74 8 Thus, the letter “P” corresponds to the ASCII character 80, which is represented by the binary code “01010000”.", "The first “0” in the sequence is often referred to as “the last bit” and is typically set to “0” for all ASCII characters.", "The corresponding hexidecimal digit or byte representation is “50”.", "In order for the computer memory system to store the letter “P”, eight bits must be sent to memory in the order “0-1-0-1-0-0-0-0”.", "Likewise, to store the letter “a”, eight bits must be sent to memory in the order “0-1-1-0-0-0-0-1”.", "Accordingly, 48 bits are used to store the six (6) letter word “Patent”.", "Similarly, when a microprocessor unit accesses the portion of memory containing the set of bits representing the word “Patent”, the same number of bits (i.e., 48) or six (6) bytes must be processed by the microprocessor.", "Clearly, if the word “Patent” could use a smaller amount of memory, less memory capacity would be needed in a computer and/or the rate at which data could be stored and/or retrieved from memory would be enhanced.", "In a first aspect of the invention, a coding system that is a higher order system than a binary coding system is utilized for the digitized storage of information.", "For example, any digital coding system which uses three or more different code characters (i.e., a coding system which operates in “base-3” or greater) will result in a reduction in the number of bits needed to store the same amount of digital information relative to a binary system.", "Thus, by using a higher order memory system, the possibility of storing more digitized information in a smaller memory area exists.", "Likewise, the rate of storing and/or retrieving digitized information should also increase due to memory being stored in a physically smaller area.", "This permits a data retrieval device to function more efficiently.", "The present invention permits the use of a digitized memory system which is base-3 or greater.", "Moreover, as more code characters are used (e.g., higher order systems of more than 10 or more than 20 different characters are utilized) the digitized system begins to behave in a manner which is similar to analog systems and can thus be referred to as “analog-like”.", "In other words, as the amount of digital sampling of an analog source increases, the recorded digital signal begins to approach the storage of most features of the analog signal.", "However, current data storage/retrieval techniques utilize, for example, 8 or 16 bits to store a single piece of analog information, which limits the amount of sampling of an analog source that can be reasonably performed when creating a digital representation of the analog source.", "Accordingly, the digital data storage/retrieval techniques of the present invention can be vastly superior to current digital techniques which utilize only “0's” and “1's” and are limited to sampling much smaller areas of analog sources than would be desirable in many cases (e.g., for complex computer modeling of numerous events, etc.).", "A first embodiment of an alternative memory storage system of the present invention may function in a manner somewhat analogous to the manner in which a Compact Disk (CD) or Digital Versatile Disk (DVD) player functions.", "Current CD and DVD technologies utilize a rotating disk comprised of a number of layers of different materials.", "The layers may include, for example, polycarbonate plastic, aluminum and acrylic.", "The aluminum in the disk includes a series of plateaus and valleys sometimes referred to as “bumps” and “pits”, respectively.", "Laser light is caused to focus upon the bumps (plateaus) and pits (valleys) in the rotating disk.", "The reflection of the laser from the successive plateaus and valleys corresponds to a data stream (e.g., a binary stream) of information.", "CD's and DVD's use, for example, 16-bit strings of binary coded data.", "Specifically, laser light reflects differently from a valley compared to the reflection from a plateau and these different reflective properties of the laser, when reflected from these valleys and plateaus, correspond to the binary code data values of “0” and “1” for each bit.", "In one embodiment of the present invention, a similar concept to the CD and DVD is utilized, however, rather than utilizing valleys and plateaus (i.e., bumps and pits) in an aluminum layer embedded in a polycarbonate plastic CD or DVD, the present invention utilizes at least two data values per bit represented by different intensity, colors or color width (e.g., different wavelength or frequency).", "These data values can be optically created, stored in dots or spots, and optically examined or interrogated (e.g., by known transmission and/or reflection techniques).", "These optical dots or spots may be of a size which is approximately the same order of magnitude in size as the bumps and pits which comprise current data tracks used on CD's and DVD's, they may be larger, or they may be smaller.", "In particular, similar to current CD's and DVD's, these dots or spots can be put together to form strings or tracks of data which begin at an innermost portion of a rotatable CD/DVD disk and extend spirally out therefrom in the form of tracks.", "Accordingly, current CD's of approximately 12 centimeters in diameter would be an acceptable size.", "However, if the dots or spots of the present invention are smaller than the “bumps” of the current technology, it is possible for CD's to be smaller in size, if desired, or more data can be placed into the same amount of memory space.", "Thus, it is possible to put an amount of optical information onto a rotatable disk of approximately 12 centimeters in diameter which exceeds that amount of information which is currently stored on CD's/DVD's.", "However, it is possible for the dots or spots to be larger in size than current bumps and pits in CD's/DVD's while still storing more information relative to standard CD's/DVD's.", "This is possible due to at least two effects.", "First, tracks may be capable of being located closer together due to, for example, lesser amounts of crosstalk between bits (e.g., recording and/or reading operations can take place in a lesser amount of space).", "Second, if digital codes higher than binary are adopted, less actual bits need to be stored relative to a binary system (e.g., rather than storing 8 or 16 bits to represent a single piece of information, a lesser number of bits can be used to represent the same piece of information that is being stored).", "It should be noted that as the number of characters that can be stored in a single bit become larger, the digital storage system actually approaches behaviors similar to an analog storage system and can be referred to as “analog-like”.", "In particular, the present invention is capable of utilizing a coding system other than the binary coding system of “0's” and “1's” now used in CD/DVD systems.", "For example, a series or range of dots or spots of different color intensities (e.g., white-gray-black), and/or different colors (e.g., red-yellow-violet), and/or both as in varied intensity of polychromatic light, can be placed upon a surface (or embedded somewhere within a material), such as a suitable photoferroelectric material such as “PLZT” (i.e., lead lanthanum zirconate titanate ceramic), which is shaped in the form of, for example, a disk.", "When, for example, such a disk containing these different intensity data value dots or spots is rotated and interrogated by, for example, a known optical system (e.g., a laser system) with a suitable focusing lens and intensity detecting means (e.g., a suitable photosensitive material or photodiode), the different intensities of the dots or spots can be registered and can be coded to correspond to different values (e.g., “0”, “1”, “2”, “3”, etc.).", "Thus, rather than using a simple binary or base-2 system where a black dot corresponds to “0” and a white dot corresponds to “1”, the present invention provides for a larger number of options for digitized data storage.", "However, as discussed in more detail herein, depending on the sensitivity of the system, this technique could also be used for actual analog computing.", "Accordingly, for example, in a simple digitized quaternary system having a quaternary code consisting of 0's, 1's, 2's and 3's, there would be 4 different choices of basic digital information storage (i.e., a “base-4” system) rather than only two choices in a binary or base-2 digital system.", "Likewise, in a digital system which utilizes 8 different combinations of spots or dots (i.e., a “base-8” system) eight different options exist for encoding a piece of information rather than only two options in a standard binary or base-2 digital system.", "The use of a larger number of possibilities other than “0's” and “1's” (e.g., 0, 1, 2, 3, 4, 5, 6, 7, etc.)", "render it possible to store a much greater amount of information in a much smaller physical area due to the elimination of unneeded bit space.", "Moreover, as the number of possibilities other than “0's” and “1's” increases, (e.g., systems greater than base-10 such as base-20 or greater systems) the closer the digital system comes to being or performing like an analog system, which has certain advantages over digital systems.", "An apparatus according to the present invention permits suitable optical data to be stored onto an appropriately constructed and shaped optical recording medium by using, for example, a laser light source.", "The optical recording media (e.g., a PLZT ceramic) can be in the form of a tape, disk, or credit card or any other acceptable shape.", "Alternatively, a photodiode or photodiode assembly can also be used.", "In all such cases, a light source (e.g., laser light source) can be used in combination with, for example, various means for controlling the direction, polarization, intensity and/or final color of the light source.", "Various examples of such systems include the following: (1) a rotatable polarizing filter (or set of filters), sometimes referred to as an analyzer, used in combination with a suitable light source which is subjected to a first polarizing filter(s) selectively modifying the polarized light from the light source.", "Specifically, once monochromatic or polychromatic light has been polarized into a first direction polarizer the polarized light is directed toward a second polarizer (sometimes referred to as an “analyzer”) which, when rotated, results in, for example, different intensities of light being emitted therefrom.", "The different intensities of light can be caused to be incident upon an appropriate recording/detecting means; (2) a light source (e.g., a laser light source), which is used in conjunction with a first polarizing filter is caused to be incident on a suitable light diffractor (e.g., a Bragg modulator) which can cause the polarized light from the light source to communicate with one or more of a series of additional polarizing filters (or analyzers) each of which analyzers are rotated, relative to the polarized source, at different angles, thus resulting an different amounts or intensities of light passing through each of the analyzers.", "Thus, by selectively transmitting the polarized light through the different analyzers (e.g., directed by, for example, the Bragg modulator) different intensities of light can be achieved.", "Such different intensities can be recorded and/or detected by a suitable means; (3) a light source (e.g., a laser light source) used in combination with an acousto-optic Bragg modulator, whereby different driving signals are input to the Bragg modulator which results in different intensities of light being emitted from the Bragg modulator.", "The multiple intensity emissions are then directed to a suitable detecting/recording means and are made to correspond to various data points in base-2 or higher order digital data storage systems; and (4) rather than utilizing polarizing filters to polarize a monochromatic or polychromatic source of light, liquid crystal cells can also be utilized.", "In particular, light can be caused to be incident upon a liquid crystal cell.", "Depending upon whether voltage is applied to the liquid crystal cell, the output from the liquid crystal cell is oriented into one direction or another.", "Liquid crystals of this type can be made of, for example, lithium niobate.", "However, similar to the embodiments (1)-(3) discussed above, intensities can be varied due to relative polarization directions.", "The outputs of light from the liquid crystal cell can be directed, by appropriate beam displacers (e.g., a Bragg modulator), toward a suitable recording/detecting means.", "In each of the examples (1)-(3) discussed above, it is possible that a polychromatic light source could be utilized.", "In this regard, when polychromatic light is made to be incident upon a first polarizer, and thereafter upon one or more additional polarizers (e.g., analyzers), in addition to different intensities of light flowing through the analyzer(s), a spreading of these different intensities over a particular frequency or wavelength range will also occur.", "Specifically, when using polychromatic light, a spectrum of frequencies of various intensities results in the formation of a somewhat continuous curve of varying light intensities over a particular frequency or wavelength range.", "The use of a polychromatic source can therefore result in secondary indicia for determining the value assigned to stored bits of data (e.g., a “0” or a “1”) and thus enhances data storage.", "For example, not only could the highest intensity of light be recorded/detected, but also the starting and/or ending points which correspond to the lowest frequency intensity of light being emitted, and the highest frequency of light being emitted, respectively, or, alternatively, the area under each curve could also be compared.", "In other words, more than one piece of information can be checked to verify the accuracy of each data point that is stored and read.", "The presence of more than a single piece of information can result in more accurate reading of data and thus result in signal/noise improvements.", "In each of the examples (1)-(4) discussed above, the different intensities or colors of light can be made to be incident upon an appropriate receiving/recording means (e.g., an optical storage means, such as a pohtopolymerisable/photocrosslinkable material, a photodiode assembly, etc.", "), such output light being suitably focussed onto the appropriate receiving/recording means by known lens or optical systems etc., all of which are discussed in greater detail herein).", "Suitable materials for recording various intensities or colors of light include the following materials: inorganic photochromic crystals, photorefractive materials, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence.", "For example, in the simplest embodiment (i.e., that embodiment numbered “(1)” above) for understanding the basic concept for recording various intensities of light (even though this simple embodiment is not as commercially significant as the other embodiments discussed herein), each such recorded intensity corresponds to a different value for a digitized data bit.", "In this basic case, different intensities can be achieved as follows: a polarized light source, such as a laser and polarized filter or polarizer, when used in combination with at least one rotatable polarizing filter (e.g., an analyzer) can result in light of different intensities being transmitted from the at least one rotatable analyzer.", "Specifically, when the laser light source is passed through a first polarizing filter and a second polarizer (e.g., an analyzer) is sequentially rotated (e.g., from 0-90°) a predetermined amount each time relative to the first polarizing filter (i.e., either the first or second filter can be rotated), different intensity dots or spots corresponding to data values having different meanings (e.g., “0”, “1”, “2”, etc.)", "can be produced.", "The amount of rotation of the analyzer in relation to the polarizing filter can correspond to a different intensity of light being recorded on a suitable optical recording medium (e.g., a photoferroelectric material such as “PLZT”, inorganic photochromic crystals, photorefractive materals, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence).", "If a photoferroelectric material is utilized, the different intensities of light can be caused to be incident upon the photoferroelectric material by use of, for example, a suitable optic lens system or a fiberoptic cable (or a series of cables) that is capable of accurately directing the optical signal to the photoferroelectric material for the proper recording of the various optical signals.", "Thus, a large number of different intensity spots or dots corresponding to data values for a single bit can be created.", "This basic system and apparatus permits higher order coding systems (e.g., base-3 or greater) to be utilized.", "The manner in which such optical data is stored, on, for example, a rotatable disk, as well as interrogated, can be by techniques which are substantially parallel, and/or use hardware which is similar, to those techniques and/or hardware currently used in CD and DVD systems.", "For example, a series of bits of an appropriate binary or higher order digital code system can be recorded onto a rotatable disk by starting to record the different intensity dots or spots near a central potion of the disk.", "The recording continues to record data in a spiral manner to form tracks of data, such data being suitably separated, physically, to avoid corruption and crosstalk, until an outermost radial portion of the disk is encountered.", "Likewise, such a disk can be read or interrogated by, for example, a laser system which focuses light onto the dots or spots and suitably measures the intensities (e.g., reflectance or transmittance) of the spots of data.", "The different intensities can be interpreted by suitable algorithms and programs to correspond to different values in the desired coding system.", "Moreover, by utilizing a digital optical coding technique other than binary, the present invention permits a much more rapid adoption of the desired “Unicode”, which takes into account, for example, all English language characters, Chinese characters, numbers and symbols, etc., in various digitized forms.", "In another embodiment of the invention, a system which utilizes magnetic memory in combination with a suitable retrieval/reading means is utilized.", "These memory/retrieval systems can be caused to function, generally, in a manner that is somewhat similar to current magnetic memory systems (e.g., magnetic tapes, cards, disks, hard drives, etc).", "In addition, these memory systems can also use digital coding/reading systems higher than a base-2 system, if desired.", "Still further, these memory systems can also store/retrieve information in an analog or analog-like format.", "Specifically, it is another object of the invention to utilize certain aspects of the phenomena known as the Zeeman effects to result in a means for the storage and retrieval of information onto/from suitable magnetic media.", "The term “Zeeman effects”, as used herein, denotes the effects of external magnetic fields on frequencies of atoms, molecules and components thereof, in the broadest sense.", "Thus, as used herein, the term “Zeeman effect” includes not only what may be considered to be Zeeman effects in the more narrow, classical sense but also other magnetic effects such as magnetic quadropole effects, etc.", "Digital data may be stored as either a binary code or some higher order code that is greater than the binary or “base-2” code.", "Moreover, due to the apparent sensitivities of these data storage techniques, data may be stored/retrieved in an analog or analog-like manner due to the inherent operation of the Zeeman effects.", "Specifically, without wishing to be bound by any particular theory or explanation, it appears that use of the Zeeman effects can result in a somewhat continuous set of differing data values that can be stored/retrieved.", "The present invention utilizes one aspect of the Zeeman effects in the following general manner.", "A magnetic field is applied to a suitable media that is capable of receiving and storing at least a portion of the applied magnetic field.", "One example of such a suitable magnetic material is a ferromagnetic material that is capable of exhibiting different amounts of magnetic domain alignment as a function of increasing magnetic field strength up to its natural magnetic saturation point (i.e., whereby any further increases in applied magnetic field do not result in any measurable further alignment of magnetic domains).", "Various known materials exist that meet this basic criteria, including many materials currently utilized in the magnetic storage industry (e.g., gamma Fe2O3, gamma Fe2O3 modified by Co, CrO2, etc.).", "The various materials can be held together by a variety of organic polymers including, for example, vinyl chloride, polyvinylchloride, methylacrylate, polymethylmethyacrylate, polyurethane, epoxy, polyamides, etc.", "Suitable substrates include typical polyesters for flexible materials and aluminum for rigid materials.", "The differing amounts of stored magnetic field can result in a corresponding splitting or shifting of the electromagnetic energy frequencies or spectral frequencies (e.g., fine spectrum splitting, hyperfine spectrum splitting, vibrational spectrum splitting, etc.)", "of one or more atoms or molecules of the magnetic material itself, or similar splitting or shifting in one or more materials that are located contiguous (e.g., in the magnetic material or in a read/write head) to the material containing the stored magnetic fields.", "In the Zeeman effect, the amount of splitting or shifting of one or more frequency spectrum(s) is a function of the strength of the applied/stored magnetic fields.", "Typically, the higher the magnetic field strength that is stored/applied (e.g., stored in a first material containing magnetic domains and thereafter applied to (e.g., influencing) a second contiguous material that exhibits Zeeman splitting at the stored magnetic field strengths), the further apart the splitting or shifting will be of one or more of the electromagnetic energy frequencies of a chosen spectrum of the targeted atom(s) or molecule(s).", "In this Zeeman effect, when a relatively wide splitting of electromagnetic energy frequencies occurs due to the applied magnetic field, the amount of splitting may correspond to frequencies in the microwave region; whereas a narrow splitting will typically correspond to lower frequencies (e.g., radio).", "The amount of splitting that occurs is important because in order to determine the amount of splitting that has occurred, a suitable interrogating electromagnetic energy is caused to be incident upon, for example, the magnetic material itself or the aforementioned contiguous material.", "The contiguous material that exhibits Zeeman splitting at the stored magnetic field can be present as, for example, one or more separate sheets of material that are located adjacent to, and/or contiguous with, the material that contains the stored magnetic field.", "When multiple contiguous materials are utilized, the present invention contemplates the use of a comparative analysis of the differences in Zeeman splitting between two or more such contiguous materials.", "Alternatively, a material comprising, for example, one or more material(s) that are mixed with the magnetic material (e.g., a binder which holds together at least a portion of the material that stores the magnetic field) could also be used.", "More specifically, a particular interrogating frequency, or set or sweep of frequencies, needs to be capable of favorably interacting with one or more of the split, shifted or splitting frequencies which result in the magnetic material, or in an alternative embodiment in one or more of the contiguous material(s), due to the different magnetic fields stored therein.", "In other words, a first magnetic material is desirably chosen so that an applied magnetic field can be reasonably stored therein.", "However, it is desirable for the magnetic material to be capable of storing reliably several magnetic fields of different strength (e.g., capable of having varying numbers of magnetic domains align with an ever changing magnetic field and such domains remain frozen or pinned so that they do not change with time) and, for example, in one embodiment of the invention, the contiguous material needs to produce split, shifted and/or splitting frequencies in response to the stored magnetic fields.", "In this regard, if one or more contiguous material(s) are utilized in connection with the magnetic material(s), the contiguous material(s) needs to exhibit a splitting and/or shifting of one or more frequencies, for example, of its rotational, vibrational, etc., bands (e.g., at the atomic level) and the resultant splitting or shifting that occurs in the contiguous material needs to be detectable by an appropriate interrogating electromagnetic source (discussed in greater detail later herein).", "Alternatively, a contiguous material does not need to be utilized.", "In this case, the magnetic material per se, or at least a portion thereof, needs to exhibit desirable and detectable splitting and/or shifting of one or more frequencies at varying stored magnetic strengths.", "Still further, it should be noted that the contiguous material could actually be located in a suitable read head which, similar to magnetoresistive and giant magnetoresistive heads, is influenced by the magnetic field strengths stored in the magnetic media.", "However, rather than measuring changes in resistivity in materials comprising the read heads, the splitting or shifting of frequencies will be detected by a suitable technique, discussed elsewhere herein.", "In this embodiment of the invention, the contiguous material may be a solid, a liquid, a gas or plasma (e.g., any material that shows desirable Zeeman splitting effects).", "With specific regard to interrogating with an applied electromagnetic source to determine the amount of Zeeman splitting and/or shifting that has occurred at a spot on the material that corresponds to a stored bit of data, at least one suitable frequency can be applied as an interrogating frequency, or, alternatively a sweep or combination of frequencies can be applied as interrogating frequencies.", "In this regard, if a single suitable frequency is chosen as the interrogating frequency, then heterodyne interactions of the various split, shifted and/or splitting frequencies with the single interrogating frequency can be measured and determined.", "Alternatively, resonance interactions of the various split, shifted and/or splitting frequencies can also be measured and determined.", "Each such determined heterodyned and/or resonant frequency can be made to correspond, by suitable programming or algorithmic techniques, to various data values.", "Alternatively, a series or sweep of frequencies which may resonate with or heterodyne with various split, shifted and/or splitting frequencies achieved in the contiguous material can also be utilized.", "These particular affects will be discussed in much greater detail later herein, however, it should be understood that these data acquisition techniques could be performed using hardware which is somewhat similar to that hardware currently used for magnetoresistive and giant magnetoresistive heads.", "For example, once an interrogating frequency has been input to a material exhibiting Zeeman splitting and/or shifting, the output (e.g., a heterodyned frequency) can be used as a driving signal or input (e.g., depending on strength or frequency either amplified or unamplified) for various other devices.", "One example of such other device is an acousto-optic Bragg modulator.", "In this detection scheme, an appropriate light source is caused to be incident upon an appropriate Bragg modulator.", "Suitable materials for a Bragg modulator include, for example, lithium niobate crystals and gallium arsenide semiconductor materials.", "The incident light beam can be caused to be deflected in differing amounts due to different driving signal inputs (e.g., depending on strength or frequency either amplified or unamplified) being directed into the Bragg modulator.", "In particular, for example, the different heterodyne outputs that correspond to different Zeeman splitting amounts (and thus different data points such as a “0” or a “1”) can be input into a Bragg modulator and cause light which is incident on the Bragg modulator to be deflected in different amounts.", "Thus, the various Zeeman splitting and/or shifting frequencies that are produced can eventually result in different amounts of light deflection due to the use of a Bragg modulator.", "The amount of deflection of light can be observed by the use of, for example, the light detection devices discussed earlier herein.", "Similarly, the different heterodyne outputs could be directed into a suitable filter which, due to the different inputs, would regulate the intensity of light passing therethrough.", "Accordingly, the different intensities of light could then be detected/recorded by a suitable means discussed elsewhere herein.", "This particular embodiment of the invention which utilizes differing magnetic fields to result in differing amounts of an appropriate frequency splitting and/or shifting is very suitable to be used for the analog storage/retrieval of information.", "In particular, a substantial continuum of splitting and shifting frequencies results in most materials that are subjected to a corresponding continuum of changing magnetic field strengths.", "If a continuum of such differing split, shifted and/or splitting frequencies can be reliably detected, then analog or analog-like (e.g., higher order than binary systems) computing with this technique is facilitated.", "Additionally, many of the aforementioned embodiments of the invention can also be used in combination with various holographic and/or three dimensional memory approaches.", "To achieve all the foregoing objects and advantages, and to overcome the looming problem in the art of an impending memory barrier, the present invention discloses various means for storing and retrieving information in a digital and/or analog format.", "With regard to the digital storage of information, a code other than a binary code for the storage of information can be utilized.", "The result of using a higher order code for digital information storage (i.e., 3 or more characters) is the ability to store more information in the same amount of area or the same amount of information in a smaller area.", "Accordingly, the present invention provides significant advancement in the art of data storage and/or data retrieval which will assist in preventing the memory barrier from being realized.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a view of a typical hard-disk drive that can be used in accordance with the techniques of the present invention.", "FIG.", "2 shows a read-write head for arranging magnetic domains in a magnetic disk.", "FIG.", "3 is a graph which shows an electromagnetic frequency as a function of magnetic field strength which results in different electromagnetic splitting frequencies which can correspond to stored bits of information on magnetic storage media.", "FIGS.", "4a-4d show a polarized light source and a polarized filter utilized to create dots other than white and black in an optical system (e.g., optical CD's).", "FIG.", "4e shows a photo detector/reader which is utilized to read the optical images stored in a disk 43.FIG.", "5 shows a representative hysteresis curve for a ferromagnetic material that is used as a magnetic memory material in the present invention.", "FIG.", "6 is a schematic of a means for recording optical information in accordance with the teachings of the present invention.", "FIG.", "7 is a schematic of a means for recording optical information in accordance with the teachings of the present invention.", "FIG.", "8 is a schematic of a means for recording optical information in accordance with the teachings of the present invention.", "FIG.", "9 is a schematic of a means for recording optical information in accordance with the teachings of the present invention.", "FIGS.", "10a and 10b show a curve of polychromatic light that has been incident upon a polarizing filter and an analyzer.", "FIG.", "11 shows a fine structure spectrum for SF6 from 0-300 being magnified.", "FIGS.", "12a and 12b show the magnification of two curves from the fine structure of SF6 showing hyperfine structure frequencies.", "Note the regular spacing of the hyperfine structure curves.", "FIG.", "12a shows magnification of the curve marked with a single asterisk () in FIG.", "11 and FIG.", "12b shows the magnification of the curve marked with a double asterisk (*) in FIG.", "11.FIG.", "13 shows an energy level diagram corresponding to the hyperfine splitting for the hyperfine structure in the n=2 to n=3 transition for hydrogen.", "FIG.", "14a shows the Zeeman effect for sodium “D” lines; and FIG.", "14b shows the energy level diagram for transitions in the Zeeman effect for sodium “D” lines.", "FIG.", "15 is a graph which shows the splitting of the ground term of the oxygen atom as a function of magnetic field.", "FIG.", "16 is a graphic which shows the dependence of the Zeeman effect on magnetic field strength for the “3P” state of silicon.", "FIG.", "17a is a pictorial which shows a normal Zeeman effect and FIG.", "17b is a pictorial which shows an anomalous Zeeman effect.", "FIG.", "18 shows anomalous Zeeman effect for zinc 3P→3S.", "FIG.", "19a shows a graphic representation of four Zeeman splitting frequencies and FIG.", "19b shows a graphic representation of four new heterodyned differences.", "FIG.", "20 is a schematic of a means for recording optical information in accordance with one example of the present invention.", "FIG.", "21 is a schematic of a means for reading recorded optical information in accordance with an example of the present invention.", "FIGS.", "22a, 22b and 22c show in graphic form plots of power spectral density (i.e., intensity) for six (6) different polarizing filter settings.", "FIG.", "23 shows a graph of upper and lower intensity limit deviations corresponding to the six (6) different filter settings of FIGS.", "22a-22c.", "FIGS.", "24a, 24b and 24c show in graphic form plots of full width half max (FWHM) data points versus six (6) different polarizing filter settings.", "FIG.", "25 shows in graphic form the deviation in upper and lower limits for the data shown in FIGS.", "24a-24c.", "FIG.", "26 shows in schematic form the apparatus used to create and determine shifting/splitting frequencies according to the present invention.", "FIG.", "27 shows an optical layout of a typical holographic memory system that can be used according to the present invention.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention utilizes new and revolutionary techniques for the storage of data digitially, or in some cases, by analog or, at least a method approaching analog, such storage of data occurring optically, magnetically and/or electrically, magneto-optically and/or combinations thereof.", "The storage/retrieval methods of the present invention are capable of utilizing traditional binary or base-2 code system approaches as well as non-traditional base-3 and higher order approaches.", "The use of base-3 and higher order approaches may permit more data to be stored in approximately the same memory area relative to conventional binary techniques and/or substantially the same amount of information is capable of being stored in a smaller area relative to conventional binary techniques.", "Certain novel magnetic storage/retrieval techniques will be discussed first, and then followed by discussions of certain novel optical techniques, according to the present invention.", "FIG.", "1 shows a typical prior art hard-disk drive in a computer.", "This prior art hard-disk drive can be used substantially as shown in accordance with certain embodiments and novel techniques of the present invention.", "The prior art hard-disk drive typically comprises one or more disks 1 (in FIG.", "1, three disks are shown) that are in communication with read-write heads 2.The read-write heads are capable of communicating with each disk in one of several predetermined manners.", "Specifically, as shown generally in FIG.", "2, the heads 2 are capable of influencing magnetic domains 30 present in the disk 1.These influenced magnetic domains are represented by the number 31, while non-influenced domains are represented by the number 30, as shown in FIG.", "2.The read-write heads 2 can first influence the magnetic domains 30 on the disks 1 by applying, for example, small currents through the heads 2 to create a magnetic field in the heads 2, which heads 2 in turn implant a desired effect into said domains (e.g., the magnetic field created by heads 2 can cause the unaligned magnetic domains 30 to become oriented domains 31, such orientation occurring in a controlled manner).", "Once the domains 31 are appropriately oriented, they can then be read, if desired, by the same read-write heads 2.The domains 31 can be oriented parallel to the plane of rotation of the disk 1, or the domains 31 can be oriented perpendicular to the plane of rotation of the disk 1.When the domains 31 are oriented substantially parallel to the rotation plane of the disk 1, various electrical/magnetic induction techniques for the reading of the domains 31 are appropriate.", "However, when the domains 31 are oriented substantially perpendicular to the rotation plane of the disk 1, standard induction reading techniques do not function as well (e.g., crosstalk between closely located bits can occur and unacceptable signal to noise ratios result).", "Accordingly, development of magnetoresistive and giant magnetoresistive heads has occurred.", "Briefly, these techniques involve the detection of very small changes in the electrical resistance of one or more materials comprising the read head 2.A primary difference between magnetoresistive heads and giant magnetoresistive heads is that giant magnetoresistive heads are two or three times more sensitive than magnetoresistive heads due to their novel structure which includes the layering of different materials on top of each other.", "Somewhat conventional data storage techniques can be used in combination with the techniques of the present invention without the requirement for significant changes in current hardware being utilized.", "For example, the magnetic storage media of the prior art can be used to store data associated with Zeeman splitting and/or shifting effects.", "Moreover, the digital information that is stored on such media can be similar to or different from prior art digital data storage techniques.", "Specifically, rather than storing only a combination of sequences of “0”s and “1”s, the present invention can use more than two stored magnetic field strengths to result in different field strengths that can be applied elsewhere and which can thus result in Zeeman splitting or shifting in the magnetic media, or Zeeman splitting or shifting occurring in a contiguous media (e.g., either with one or more materials comprising the magnetic storage media or either one or more materials, such as a solid, a liquid, a gas and/or a plasma, comprising the read head).", "In other words, the different field strengths stored in a suitable magnetic media can result in differing amounts of Zeeman splitting or shifting in an appropriately chosen material, which detected Zeeman splitting or shifting corresponds to the different digital (or analog) values within a coded system.", "For example, in a base-4 system, a “0” stored on the magnetic media could correspond to a bit with no magnetic field; a “1” could correspond to a stored field strength of “x” microTesla to the bit; a “2” could correspond to a field strength of “5x” microTesla stored to the bit; and a “3” could correspond to a field strength of “10x” microTesla stored to the bit.", "The particular field strengths chosen here are exemplary, and it should be understood that differences in field strengths are simply a matter of design choice (e.g., convenient differences in magnetic field strengths should be a function of the magnetic media utilized to store information as well as the hardware utilized to store and retrieve such information and the media in which Zeeman splitting and/or shifting is desired).", "The sensitivity of such media and/or hardware may dictate how much the difference in stored field strength needs to be between each bit so that the likelihood of misreading the information stored on any one bit, or combination of bits, due to, for example, poor signal/noise ratio or crosstalk, is minimized.", "Moreover, any Zeeman splitting and/or shifting outputs can be appropriately modified or amplified by conventional techniques.", "One example of a suitable magnetic material which can be used to store a magnetic field is a ferromagnetic material.", "In particular, a ferromagnetic material that is capable of exhibiting different amounts of magnetic domain alignment as a function of increasing applied magnetic field, up to its natural magnetic saturation point.", "Examples of suitable ferromagnetic materials that can be used in connection with the present invention include gamma-Fe2O3, gamma—Fe2O3 modified by Co, CrO2, etc.", "FIG.", "5 shows a typical hysteresis curve or hysteresis loop for a typical ferromagnetic material that can be used in the present invention.", "Initially, at a point “D” no field “H” is applied and no magnetization is present in the virgin ferromagnetic material.", "However, as a field H is applied, the magnetization of domains begins at the point “D” and increases along the curve “M” up to the point of saturization (Ms).", "It should be noted that the initial magnetization or alignment of domains along the curve “M” is up to about the point “E”, in this FIG.", "4, and is reversible.", "However, when enough field H is applied to achieve a position on the curve M which corresponds to a magnetic field which is beyond the point “E”, the domains are frozen into place (i.e., will not move substantially unless influenced by, for example, another external magnetic field of greater strength or of a reverse polarity).", "This curve is known as a “hysteresis curve”.", "A suitable ferromagnetic material for use in the present invention exhibits a substantially infinite number of possible induced magnetizations along the curve M. It is these varying induced magnetizations that will be utilized to create different amounts of Zeeman splitting or shifting (discussed later herein) in the ferromagnetic materials per se, or in one or more materials which are placed contiguous to the ferromagnetic materials.", "The magnetization induced in a ferromagnetic material can be increased up to the point Ms, whereby any higher magnetic fields will not result in any additional domain alignment.", "As the applied magnetic field H is reduced to zero, the induced magnetization in the ferromagnetic material does not reduce to zero, but rather, has a remnant magnetization or resonance noted as Br.", "It should be noted that ferromagnetic materials do not typically exhibit or hold the same magnetic field that is applied to them.", "For example, if a field of 10 micro Tesla was applied, the magnetization induced in the ferromagnetic material, if measured as a field, would be less than 10 micro Tesla, for example, 7 micro Tesla.", "This factor needs to be accounted for in the present invention, but is not a substantial issue and an artisan of ordinary skill in this field will understand how to proceed in accordance with the other teachings contained herein.", "In order to understand the influence of magnetic fields on various Zeeman splitting or shifting frequencies, the following discussions of Fine Structure Frequencies, Hyperfine Structure Frequencies and Magnetic Fields are provided.", "These discussions are taken, in part, from PCT Application No.", "PCT/US01/28,392, entitled “Spectral Chemistry”, which was filed on Sep. 11, 2001, the subject matter of which is expressly incorporated herein by reference.", "Fine Structure Frequencies The field of science concerned generally with measuring the frequencies of energy and matter, known as spectroscopy is well known.", "Specifically, there are three broad classes of atomic and molecular spectra: (1) electronic spectra, which are due to electron transitions, have frequencies which occur primarily in the ultraviolet (UV), visible, and infrared (IR) regions, and are present in both atoms and molecules; (2) vibrational spectra, which are due to, for example, bond motion between individual atoms within molecules, occur primarily in the IR regions, and are present in molecules and (3) rotational spectra which are due primarily to the rotation of molecules in space and have microwave or radiowave frequencies, and also occur in molecules.", "The three broad classes of spectra discussed above have been oversimplified.", "There are actually at least three additional sets of spectra, namely, the fine structure spectra, the hyperfine structure spectra and the superfine structure spectra.", "These additional spectra occur in atoms and molecules, and extend in frequencies, for example, from the ultraviolet down to the low radio regions.", "These additional spectra are often mentioned in prior art chemistry and spectroscopy books typically as an aside, because prior art chemists usually focus more on the traditional types of spectroscopy, namely, electronic, vibrational, and rotational.", "The fine and hyperfine spectra are quite prevalent in the areas of physics and radio astronomy.", "For example, cosmologists map the locations of interstellar clouds of hydrogen, and collect data regarding the origins of the universe by detecting signals from outerspace, for example, at 1.420 GHz, a microwave frequency which is one of the hyperfine splitting frequencies for hydrogen.", "Most of the large databases concerning the microwave and radio frequencies of molecules and atoms have been developed by astronomers and physicists, rather than by chemists.", "This apparent inattention by chemists, physicists and materials scientists of the fine, hyperfine, and superfine spectra, has apparently resulted in little attention being given, if any, to these potentially useful spectra.", "In addition to the fine splitting frequencies for atoms (i.e., heterodynes), molecules also have similar fine structure frequencies.", "The origin and derivation for molecular fine structure and splitting or shifting is different from that for atoms, however, the graphical and practical results are quite similar.", "In atoms, the fine structure frequencies are said to result from the interaction of the spinning electron with its' own magnetic field.", "Basically, this means the electron cloud of a single atomic sphere, rotating and interacting with its' own magnetic field, produces the atomic fine structure frequencies.", "The prior art refers to this phenomena as “spin-orbit coupling”.", "For molecules, the fine structure frequencies correspond to the actual rotational frequencies of the electronic or vibrational frequencies.", "So the fine structure frequencies for atoms and molecules both result from rotation.", "In the case of atoms, it is the atom spinning and rotating around itself, much the way the earth rotates around its axis.", "In the case of molecules, it is the molecule spinning and rotating through space.", "Hyperfine Structure Frequencies Hyperfine structure frequencies are similar to the fine structure frequencies.", "Fine structure frequencies can be seen by magnifying a portion of a standard frequency spectrum.", "Hyperfine frequencies can be seen by magnifying a portion of a fine structure spectrum.", "Fine structure splitting frequencies occur at lower frequencies than the electronic spectra, and are located primarily in the infrared and microwave regions of the electromagnetic spectrum.", "Hyperfine splitting frequencies occur at even lower frequencies than the fine structure spectra, primarily in the microwave and radio wave regions of the electromagnetic spectrum.", "Fine structure frequencies are generally caused by at least the electron interacting with its' own magnetic field.", "Hyperfine frequencies are generally caused by at least the electron interacting with the magnetic field of the nucleus.", "An example of hyperfine splitting can be seen in FIGS.", "11 and 12.Specifically, what appears to be a single crisp curve in FIG.", "11, turns out to be a series of several curves spaced very close together.", "These are the hyperfine frequency curves.", "Accordingly, the fine structure spectra is comprised of several more curves spaced very close together.", "These other curves spaced even closer together correspond to the hyperfine frequencies.", "FIGS.", "12a and 12b show that the spacing of the hyperfine frequency curves are very close together and at somewhat regular intervals.", "The small amount that the hyperfine curves are split apart is called the hyperfine splitting frequency.", "The hyperfine splitting frequency is also a heterodyne.", "This concept is substantially similar to the concept of the fine splitting frequency.", "The difference between two curves that are split apart is called a splitting frequency.", "As before, the difference between two curves is referred to as a heterodyne frequency.", "So, hyperfine splitting frequencies are all heterodynes of hyperfine frequencies.", "Because the hyperfine frequency curves result from a magnification of the fine structure curves, the hyperfine splitting frequencies occur at only a fraction of the fine structure splitting frequencies.", "The fine structure splitting frequencies are really just several curves, spaced very close together around the regular spectrum frequency.", "Magnification of fine structure splitting frequencies results in hyperfine splitting frequencies.", "The hyperfine splitting frequencies are really just several more curves, spaced very close together.", "The closer together the curves are, the smaller the distance or frequency separating them.", "Now the distance separating any two curves is a heterodyne frequency.", "So, the closer together any two curves are, the smaller (lower) is the heterodyne frequency between them.", "The distance between hyperfine splitting frequencies (i.e., the amount that hyperfine frequencies are split apart) is the hyperfine splitting frequency.", "It can also be called a constant or interval.", "As a general example, the electronic spectrum frequency of hydrogen is 2,466 THz.", "The 2,466 THz frequency is made up of fine structure curves spaced 10.87 GHz (0.01087 THz) apart.", "Thus, the fine splitting frequency is 10.87 GHz.", "Now the fine structure curves are made up of hyperfine curves.", "These hyperfine curves are spaced just 23.68 and 59.21 MHz apart.", "Thus, 23 and 59 MHz are both hyperfine splitting frequencies for hydrogen.", "Other hyperfine splitting frequencies for hydrogen include 2.71, 4.21, 7.02, 17.55, 52.63, 177.64, and 1,420.0 MHz.", "The hyperfine structure frequencies are spaced even closer together than the fine structure frequencies, so the hyperfine splitting frequencies are smaller and lower than the fine splitting frequencies.", "Thus, the hyperfine splitting frequencies are lower than the fine splitting frequencies.", "This means that rather than being in the infrared and microwave regions, as the fine splitting frequencies can be, the hyperfine splitting frequencies are in the microwave and radiowave regions.", "These lower frequencies are in the GHz (109 hertz), MHz (106 hertz) and KHz (103 hertz) regions of the electromagnetic spectrum.", "Several of the hyperfine splitting frequencies for hydrogen are shown in FIG.", "13.(FIG.", "13 shows hyperfine structure in the n=2 to n=3 transition of hydrogen).", "Magnetic Fields In spectral terms, magnetic fields can behave in a similar manner to electric fields.", "Specifically, the spectral frequency lines of, for example, atoms and molecules, can be split and/or shifted by a magnetic field.", "In this case, the external magnetic field from outside the atom or molecule, interacts with the electric and magnetic fields already inside the atom or molecule.", "This action of an external magnetic field on spectral lines is called the “Zeeman Effect”, in honor of its' discoverer, Dutch physicist Pieter Zeeman.", "In 1896, Zeeman discovered that the yellow flame spectroscopy “D” lines of sodium were broadened when the flame was held between strong magnetic poles.", "It was later discovered that the apparent broadening of the sodium spectral lines was actually due to their splitting and shifting.", "Zeeman's original observation has evolved into a separate branch of spectroscopy relating to the study of atoms and molecules by measuring the changes of the spectral lines in atoms and molecules caused by a magnetic field.", "This in turn has evolved into the nuclear magnetic resonance spectroscopy and magnetic resonance imaging used in medicine, as well as the laser magnetic resonance and electron spin resonance spectroscopy used in physics and chemistry.", "The Zeeman effect for the famous “D” lines of sodium is shown in FIGS.", "14a and 14b.", "FIG.", "14a shows the Zeeman effect for sodium “D” lines; whereas FIG.", "14b shows the energy level diagram for the transitions in the Zeeman effect for the sodium “D” lines.", "The “D” lines are traditionally said to result from transition between the 3p2p and 3s2S electron orbitals.", "As is shown, each of the single spectral frequencies is split into two or more slightly different frequencies, which center around the original unsplit frequency.", "In this Zeeman effect, the amount that the spectral frequencies are split apart depends on the strength of the applied magnetic field.", "FIG.", "15 shows Zeeman splitting effects for the oxygen atom as a function of magnetic field.", "When there is no magnetic field, there are two single frequencies at zero and 4.8.When the magnetic field is at low strength (e.g., 0.2 Tesla) there is just slight splitting and shifting of the original two frequencies.", "However, as the magnetic field is increased, the frequencies are split and shifted farther and farther apart.", "The degree of splitting and shifting in the Zeeman effect, depending on magnetic field strength, is shown in FIG.", "16 for the 3P state of silicon.", "This Zeeman effect, due to an externally applied magnetic field, is slightly different from the Zeeman effect from an internal magnetic field depending on whether an atom or molecule is subjected to the magnetic field.", "The Zeeman effect on atoms can be divided into three different magnetic field strengths: weak; moderate; and strong.", "If the magnetic field strength is weak, the amount that the spectral frequencies will be shifted and/or split apart will be very small.", "The shifting away from the original spectral frequency will still stimulate the shifted frequencies.", "This is because they will be so close to the original spectral frequency that they will still be well within its resonance curve.", "As for the splitting, it is so small, that it is even less than the hyperfine splitting that normally occurs.", "This means that in a weak magnetic field, there will be only very slight splitting of spectral frequencies, translating into very low splitting frequencies in the lower regions of the radio spectrum and down into the very low frequency region.", "For example, the Zeeman splitting frequency for the hydrogen atom, which is caused by the earth's magnetic field, is around 30 KHz.", "Larger atoms have even lower frequencies in the lower kilohertz and even hertz regions of the electromagnetic spectrum.", "At the other end of magnetic field strength, is the very strong magnetic field.", "In this case, the splitting apart and shifting of the spectral frequencies will be very wide.", "With this wide shifting of frequencies, the difference between the split and/or shifting frequencies will be much larger than the difference between the hyperfine splitting frequencies.", "This translates to Zeeman effect splitting and/or splitting frequencies at higher frequencies than the hyperfine splitting frequencies.", "This splitting occurs somewhere around the microwave region.", "The moderate magnetic field strength case is more complicated.", "The shifting and splitting caused by the Zeeman effect from a moderate magnetic field will be approximately equal to the hyperfine splitting and/or shifting.", "Although not widely discussed in the prior art, it is possible to apply a moderate magnetic field to an atom, to produce Zeeman splitting which is substantially equivalent to its' hyperfine splitting.", "The moderate magnetic field causes low frequency Zeeman splitting that matches and hence energizes the low frequency hyperfine splitting frequency in the atom.", "However, the low hyperfine splitting frequencies actually correspond to the heterodyned difference between two vibrational or fine structure frequencies.", "When the hyperfine splitting frequency is stimulated, the vibrational or fine structure frequencies will be stimulated, and electronic frequencies will eventually be stimulated.", "This in turn causes the atom to be, for example, stimulated.", "There is also a difference between the “normal” Zeeman effect and the “anomalous” Zeeman effect.", "With the “normal” Zeeman effect, a spectral frequency is split by a magnetic field into three frequencies, with expected even spacing between them (see FIG.", "17a which shows the “normal” Zeeman effects and FIG.", "17b which shows the “anomalous” Zeeman effects).", "One of the new split frequencies is above the original frequency, and the other new split frequency is below the original frequency.", "Both new frequencies are split the same distance away from the original frequency.", "Thus, the difference between the upper and original and the lower and original frequencies is about the same.", "This means that in terms of heterodyne differences, there are at most, two new heterodyned differences with the normal Zeeman effect.", "The first heterodyne or splitting difference is the difference between one of the new split frequencies and the original frequency.", "The other splitting difference is between the upper and lower new split frequencies.", "It is, of course, twice the frequency difference between either of the upper or lower frequencies and the original frequency.", "In many instances the Zeeman splitting produced by a magnetic field results in more than three frequencies, or in splitting that is spaced differently than expected.", "This is called the “anomalous” Zeeman effect (see FIGS.", "17 and 18; wherein FIG.", "18 shows an anomalous Zeeman effect for zinc 3p→3s.", "If there are still just three frequencies, and the Zeeman effect is anomalous because the spacing is different than expected, the situation is similar to the normal effect.", "However, there are at most, two new splitting frequencies that can be used.", "If, however, the effect is anomalous because more than three frequencies are produced, then there will be a much more richly varied situation.", "Assume an easy case where there are four Zeeman splitting frequencies (see FIGS.", "19a and FIG.", "19b).", "FIG.", "19a shows four Zeeman splitting frequencies and FIG.", "19b shows four new heterodyned differences.", "In this example of anomalous Zeeman splitting, there are a total of four frequencies, where once existed only one frequency.", "For simplicity's sake, the new Zeeman frequencies will be labeled 1, 2, 3, and 4.Frequencies 3 and 4 are also split apart by the same difference “w”.", "Thus, “w” is a heterodyned splitting frequency.", "Frequencies 2 and 3 are also split apart by a different amount “x”.", "So far there are two heterodyned splitting frequencies, as in the normal Zeeman effect.", "However, frequencies 1 and 3 are split apart by a third amount “y”, where “y” is the sum of “w” and “x”.", "And, frequencies 2 and 4 are also split apart by the same third amount “y”.", "Finally, frequencies 1 and 4 are split even farther apart by an amount “z”.", "Once again, “z” is a summation amount from adding “w+x+w”.", "Thus, the result is four heterodyned frequencies: w, x, y, and z in the anomalous Zeeman effect.", "If there were six frequencies present from the anomalous Zeeman effect, there would be even more heterodyned differences.", "Thus, the anomalous Zeeman effect results in far greater flexibility in the choice of frequencies when compared to the normal Zeeman effect.", "In the normal Zeeman effect the original frequency is split into three evenly spaced frequencies, with a total of just two heterodyned frequencies.", "In the anomalous Zeeman effect the original frequency is split into four or more unevenly spaced frequencies, with at least four or more heterodyned frequencies.", "The following discussion will focus on the Zeeman effect in molecules.", "Molecules come in three basic varieties: ferromagnetic; paramagnetic; and diamagnetic.", "Ferromagnetic molecules are what comprises typical magnetic storage media and are thus capable of being used in the present invention.", "These materials typically hold a strong magnetic field due to domain alignment induced by the application of an external magnetic material and are composed of typical magnetic elements such as iron, cobalt, and/or nickel.", "Paramagnetic molecules hold only a weak magnetic field and are thus not of primary interest in the present invention as magnetic strength storage materials.", "A strong magnetic field will produce splitting greater than the hyperfine frequencies, in the microwave and infrared regions of the EM spectrum in atoms and paramagnetic molecules.", "A moderate magnetic field will produce Zeeman splitting in atoms and paramagnetic molecules at frequencies on par with the hyperfine and rotational splitting frequencies.", "Finally, consider the direction of the magnetic field in relation to the orientation of the molecule.", "When the magnetic field is parallel to an exciting electromagnetic field, π frequencies are produced.", "When the magnetic field is perpendicular to an exciting electromagnetic field, a frequencies are found.", "It is important to remember that field orientation can result in different frequencies.", "Accordingly, in a first embodiment of the present invention, in order to determine any field strength data stored on appropriate magnetic media, the magnetic media per se (or in an alternative embodiment, a material (e.g., a solid, a liquid, a gas and/or a plasma) contiguous to the magnetic media either forming a part of the magnetic media or part of an interrogating (i.e., read head) needs to be appropriately interrogated for the presence of differing amounts of Zeeman splitting and/or shifting.", "A suitable form of interrogation of such stored information could be, for example, a spectroscopic interrogation.", "For example, in reference to FIG.", "3 the magnetic media utilized to store the different induced field strengths of “0”, “x”, 5x” and “10x” microTesla, could themselves exhibit or show particular Zeeman splitting frequencies for each of the different magnetic field strengths.", "Alternatively, the different stored field strengths could result in Zeeman splitting in a contiguous material (or multiple contiguous materials).", "The contiguous material could form part of the magnetic media, or could form part of an interrogation assembly such as read/write head.", "The Zeeman splitting and/or shifting frequencies which arise are a function of the actual material which comprises the magnetic media, as well as the field strength applied thereto.", "In this regard, for example, as shown in FIG.", "3, assuming for illustrative purposes that the exemplary magnetic media exhibited a Zeeman splitting frequency of 10 KHz for a magnetic field strength of “x” microTesla, 25 KHz for a magnetic field strength of “5x” microTesla and 80 KHz for a magnetic field strength of “10x” microTesla.", "Thus, in order to read any of the bits of information stored on the exemplary magnetic media, each bit of stored information would need to be interrogated directly by applying one or more of the frequencies mentioned above (alternatively, a material contiguous (as discussed elsewhere herein) to the magnetic media could be similarly interrogated to determine Zeeman splitting and/or shifting).", "Specifically, in a first interrogation approach, a system could be designed such that each bit of information stored in a magnetic media could be interrogated with each known potential splitting frequency that could be present on each bit; or a system could be designed such that once a known splitting frequency had been found to be located at the bit, the interrogation process could stop.", "The particular approach is a matter of preference and reliability.", "For example, one interrogation process according to the present invention proceeds as follows.", "If the magnetic media per se exhibits Zeeman splitting and/or shifting, then the first bit (i.e., the bit with no magnetic field), could be interrogated with all three frequencies (i.e., 10, 25 and 80 KHz) or a sweep of frequencies (i.e., 10 KHz to 80 KHz).", "The interrogation system would recognize that none of these frequencies were present, and thus there is no stored magnetic field strength and the bit is a “0”.", "The next bit could then be interrogated with the same frequencies and it would be discovered by the interrogation that a resonant amplitude spike returned from the bit or was absorbed by the bit at a frequency of 10 KHz.", "Further interrogation of the bit would show no other resonant amplitude spikes at the interrogation frequencies of 25 and 80 KHz.", "Thus, this bit would be “1”.", "The next bit could then be interrogated with the same frequencies and it would be discovered by the interrogation process that a resonant amplitude spike returned from the bit at a frequency of 25 KHz.", "Further interrogation of the bit would show no other resonant amplitude spikes at the frequencies of 10 KHz and 80 KHz.", "Thus, this bit would be “2”.", "The final bit could then be interrogated with the same frequencies and it would be discovered that a resonant amplitude spike returned from the bit at a frequency of 80 KHz.", "Further interrogation of the bit would show no other resonant amplitude spikes at the frequencies of 10 KHz and 25 KHz.", "Thus, the bit would be a “3”.", "Accordingly, a reliable base-4 system can be achieved.", "Alternatively, another interrogation process according to the present invention could proceed as follows: if the magnetic media has a plurality of induced field strengths recorded and each of the recorded magnetic field strengths per se exhibits different amounts of Zeeman splitting in either the magnetic media or in one or more contiguous material(s), then each of the bits providing different magnetic field strengths could be interrogated with, for example, a single interrogating electromagnetic energy of a suitable frequency.", "In this regard, if a frequency of, for example, 50 KHz was used as the interrogating frequency, then heterodynes (side bands) sum and difference frequencies, etc., could be expected to occur with those Zeeman splitting frequencies which occur in the magnetic media or in the contiguous material.", "Specifically, heterodyning can occur with each of the stored frequencies, 10, 25 and 80 KHz.", "In particular, some, but not all of the possible heterodyned frequencies would be as follows: 60 KHz (i.e., 50+10); 75 KHz (i.e., 50+25); and 130 KHz (i.e., 50+80).", "In other words, the application of a 50 KHz interrogating frequency would result in at least the aforementioned heterodyned frequencies, each of which could be caused to correspond to, for example, either a “1”, a “2”, or a “3”, with no heterodynes present indicating the value of “0”.", "It should be noted that of each of the aforementioned processes, namely applying a series of frequencies or a single frequency when interrogating for heterodynes, can be used for interrogating either the magnetic material per se, or a contiguous material, said contiguous material being present in the magnetic media per se, adjacent to the magnetic media or as part of an interrogating system, such as a read/write head.", "The following brief description of heterodyning is provided to assist in understanding certain aspects of the invention.", "However, for a more complete discussion of this important area, the reader is directed toward the aforementioned PCT Application entitled “Spectral Chemistry”, which has been expressly incorporated herein by reference.", "Electromagnetic energy consists of waves.", "The number of waves in a period of time can be counted.", "Usually, time is placed on the horizontal X-axis.", "The vertical Y-axis shows the strength or intensity of the wave.", "This is also called the amplitude.", "A weak wave will be of weak intensity and will have low amplitude.", "A strong wave will have high amplitude.", "Traditionally, the number of waves per second is counted, to obtain the frequency.", "Another name for “waves per second”, is “hertz” (abbreviated “Hz”).", "Frequency=Number of waves/time=Waves/second=Hz.", "Energy waves and frequency have some interesting properties, and may interact in some interesting ways.", "The manner in which wave energies interact, depends largely on the frequency.", "For example, when two waves of energy interact, each having the same amplitude, but one at a frequency of 400 Hz and the other at 100 Hz, the waves will add their frequencies, to produce a new frequency of 500 Hz (i.e., the “sum” frequency).", "The frequency of the waves will also subtract to produce a frequency of 300 HZ (i.e., the “difference” frequency).", "All wave energies typically add and subtract in this manner, and such adding and subtracting is referred to as heterodyning.", "Common results of heterodyning are familiar to most as harmonics in music.", "There is a mathematical, as well as musical basis, to the harmonics produced by heterodyning.", "Consider, for example, a continuous progression of heterodyned frequencies.", "As discussed above, beginning with 400 Hz and 100 Hz, the sum frequency is 500 Hz and the difference frequency is 300 Hz.", "If these frequencies are further heterodyned (added and subtracted) then new frequencies of 800 (i.e., 500+300) and 200 (i.e., 500−300) are obtained.", "The further heterodyning of 800 and 200 results in 1,000 and 600 Hz as shown in FIG.", "4.A mathematical pattern begins to emerge.", "Both the sum and the difference columns contain alternating series of numbers that double with each set of heterodynes.", "In the sum column, 400 Hz, 800 Hz, and 1,600 Hz, alternates with 500 Hz, 1000 Hz, and 2000 Hz.", "The same sort of doubling phenomenon occurs in the difference column.", "Heterodyning of frequencies is the natural process that occurs whenever waveform energies interact.", "Heterodyning results in patterns of increasing numbers that are mathematically derived.", "The number patterns are integer multiples of the original frequencies.", "These multiples are called harmonics.", "For example, 800 Hz and 1600 Hz are harmonics of 400 Hz.", "In musical terms, 800 Hz is one octave above 400 Hz, and 1600 Hz is two octaves higher.", "It is important to understand the mathematical heterodyne basis for harmonics, which occurs in all waveform energies, and thus in all of nature.", "A heterodyne is a mathematical function, governed by mathematical equations, just like a fractal.", "A heterodyne also produces self-similar patterns of numbers, like a fractal.", "Heterodynes are fractals; the conclusion is inescapable.", "Heterodynes and fractals are both mathematical functions which produce a series of self-similar patterns or numbers.", "Wave energies interact in heterodyne patterns.", "Thus, all wave energies interact as fractal patterns.", "Accordingly, since energy interacts by heterodyning, matter should also be capable of interacting by a heterodyning process.", "All matter whether in large or small forms, has what is called a natural oscillatory frequency.", "The natural oscillatory frequency (“NOF”) of an object, is the frequency at which the object prefers to vibrate, once set in motion.", "The NOF of an object is related to many factors including size, shape, dimension, and composition.", "The smaller an object is, the smaller the distance it has to cover when it oscillates back and forth.", "The smaller the distance, the faster it can oscillate, and the higher its NOF.", "For example, consider a wire composed of metal atoms.", "The wire has a natural oscillatory frequency.", "The individual metal atoms also have unique natural oscillatory frequencies.", "The NOF of the atoms and the NOF of the wire heterodyne by adding and subtracting, just the way energy heterodynes.", "NOFatom+NOFwire=Sum Frequencyatom+wire and NOFatom−NOFwire=Difference Frequencyatom-wire If the wire is stimulated with the Difference Frequencyatom-wire, the difference frequency will heterodyne (add) with the NOFwire to produce NOFatom, (natural oscillatory frequency of the atom) and the atom will absorb with the energy, thereby becoming stimulated to a higher energy level.", "Cirac and Zoeller reported this phenomenon in 1995, and they used a laser to generate the Difference Frequency.", "Difference Frequencyatom-wire+NOFwire=NOFatom Matter heterodynes with matter in a manner similar to the way in which wave energies heterodyne with other wave energies.", "This means that matter in its various states may also interact in fractal processes.", "This interaction of matter by fractal processes assists in explaining why so many creatures and systems in nature exhibit fractal processes and patterns.", "Matter, as well as energy, interacts by the mathematical equations of heterodynes, to produce harmonics and fractal patterns.", "That is why there are fractals everywhere around us.", "Thus, energy heterodynes with energy, and matter heterodynes with matter.", "However, perhaps even more important is that matter can heterodyne with energy (and visa versa).", "In the metal wire discussion above, the Difference Frequencyatom-wire in the experiment by Cirac and Zoeller was provided by a laser which used electromagnetic wave energy at a frequency equal to the Difference Frequencyatom-wire.", "The matter in the wire, via its natural oscillatory frequency, heterodyned with the electromagnetic wave energy frequency of the laser to produce the frequency of an individual atom of matter.", "This shows that energy and matter do heterodyne with each other.", "In general, when energy encounters matter, one of three possibilities occur.", "The energy either bounces off the matter (i.e., is reflected energy), passes through the matter (i.e., is transmitted energy), or interacts and/or combines with the matter (e.g., is absorbed or heterodynes with the matter).", "If the energy heterodynes with the matter, new frequencies of energy and/or matter will be produced by mathematical processes of sums and differences.", "If the frequency thus produced matches an NOF of the matter, the energy will be, at least partially, absorbed, and the matter will be stimulated to, for example, a higher energy level, (i.e., it possesses more energy).", "A crucial factor which determines which of these three possibilities will happen is the frequency of the energy compared to the frequency of the matter.", "If the frequencies do not match, the energy will either be reflected, or will pass on through as transmitted energy.", "If the frequencies of the energy and the matter match either directly (e.g., are close to each other, as discussed in greater detail later herein), or match indirectly (e.g., heterodynes), then the energy is capable of interacting and/or combining with the matter.", "Another term often used for describing the matching of frequencies is resonance.", "In this invention, use of the term resonance will typically mean that frequencies of matter and/or energy match.", "For example, if the frequency of energy and the frequency of matter match, the energy and matter are in resonance and the energy is capable of combining with the matter.", "Resonance, or frequency matching, is merely an aspect of heterodyning that permits the coherent transfer and combination of energy with matter.", "In the example above with the wire and atoms, resonance could have been created with the atom, by stimulating the atom with a laser frequency exactly matching the NOF of the atom.", "In this case, the atom would be energized with its own resonant frequency and the energy would be transferred to the atom directly.", "Alternatively, as was performed in the actual wire/laser experiment, resonance could also have been created with the atom by using the heterodyning that naturally occurs between differing frequencies.", "Thus, the resonant frequency of the atom (NOFatom) can be produced indirectly, as an additive (or subtractive) heterodyned frequency, between the resonant frequency of the wire (NOFwire) and the applied frequency of the laser.", "Either direct resonance, or indirect resonance through heterodyned frequency matching, produces resonance and thus permits the combining of matter and energy.", "When frequencies match, energy transfers.", "Heterodyning produces indirect resonance.", "Heterodyning also produces harmonics, (i.e., frequencies that are integer multiples of the resonant (NOF) frequency.", "For example, the music note “A” is approximately 440 Hz.", "If that frequency is doubled to about 880 Hz, the note “A” is heard an octave higher.", "This first octave is called the first harmonic.", "Doubling the note or frequency again, from 880 Hz to 1,760 Hz (i.e., four times the frequency of the original note) results in another “A”, two octaves above the original note.", "This is called the third harmonic.", "Every time the frequency is doubled another octave is achieved, so these are the even integer multiples of the resonant frequency.", "In between the first and third harmonic is the second harmonic, which is three times the original note.", "Musically, this is not an octave like the first and third harmonics.", "It is an octave and a fifth, equal to the second “E” above the original “A”.", "All of the odd integer multiples are fifths, rather than octaves.", "Because harmonics are simply multiples of the fundamental natural oscillatory frequency, harmonics stimulate the NOF or resonant frequency indirectly.", "Thus by playing the high “A” at 880 Hz on a piano, the string for middle “A” at 440 Hz should also begin to vibrate due to the phenomenon of harmonics.", "Matter and energy in chemical reactions respond to harmonics of resonant frequencies much the way musical instruments do.", "Thus, the resonant frequency of the atom (NOFatom) can be stimulated indirectly, using one or more of its' harmonic frequencies.", "This is because the harmonic frequency heterodynes with the resonant frequency of the atom itself (NOFatom).", "For example, in the wire/atom example above, if the laser is tuned to 800 THz and the atom resonates at 400 THz, heterodyning the two frequencies results in: 800 THz−400 THz=400 THz The 800 THz (the atom's first harmonic), heterodynes with the resonant frequency of the atom, to produce the atom's own resonant frequency.", "Thus the first harmonic indirectly resonates with the atom's NOF, and stimulates the atom's resonant frequency as a first generation heterodyne.", "Of course, the two frequencies will also heterodyne in the other direction, producing: 800 THz+400 THz=1,200 THz The 1,200 THz frequency is not the resonant frequency of the atom.", "Thus, part of the energy of the laser will heterodyne to produce the resonant frequency of the atom.", "The other part of the energy of the laser heterodynes to a different frequency, that does not itself stimulate the resonant frequency of the atom.", "That is why the stimulation of an object by a harmonic frequency of particular strength of amplitude, is typically less than the stimulation by its' own resonant (NOF) frequency at the same particular strength.", "Although it appears that half the energy of a harmonic is wasted, that is not necessarily the case.", "Referring again to the exemplary atom vibrating at 400 THz, exposing the atom to electromagnetic energy vibrating at 800 THz will result in frequencies subtracting and adding as follows: 800 THz−400 THz=400 THz and 800 THz+400 THz=1,200 THz The 1,200 THz heterodyne, for which about 50% of the energy appear to be wasted, will heterodyne with other frequencies also, such as 800 THz.", "Thus, 1,200 THz−800 THz=400 THz Also, the 1,200 THz will heterodyne with 400 THz: 1,200 THz−400 THz=800 THz, thus producing 800 THz, and the 800 THz will heterodyne with 400 THz: 800 THz−400 THz=400 THz, thus producing 400 THz frequency again.", "When other generations of heterodynes of the seemingly wasted energy are taken into consideration, the amount of energy transferred by a first harmonic frequency is much greater than the previously suggested 50% transfer of energy.", "There is not as much energy transferred by this approach when compared to direct resonance, but this energy transfer is sufficient to produce a desired effect.", "While FIG.", "3 (discussed above in greater detail) shows, in graphic form, three specifically selected magnetic field strengths (i.e., 1×, 5× and 10×) and their corresponding (resultant) Zeeman splitting frequencies, it should be obvious to one of ordinary skill in this art that a virtually limitless series of possibilities exists for storage of higher order systems (i.e., higher than base-2 or a binary system), such systems being a function of the magnetic materials comprising the magnetic media, the Zeeman splitting/shifting frequencies generated in the magnetic media, or in a contiguous material, the accuracy of whatever transmitter(s) (e.g., a magnetic inductor, which generates a magnetic field) is used to induce a magnetic field in a magnetic media and/or the accuracy of the reading system which is used to detect Zeeman splitting and/or shifting effects in the magnetic media (or in a material contiguous to the magnetic material) once such magnetic media (or contiguous material) has been interrogated.", "Thus, if large numbers of stored fields can result in correspondingly large numbers of Zeeman splitting or shifting frequencies, the use of Zeeman splitting and/or shifting effects can result in an analog or analog-like storage/retrieval of information.", "For example, again referring to FIGS.", "1 and 2, the Zeeman effect can be used to read information from a series of bits on the disks 1, but such information is retrieved differently from prior art approaches.", "In this regard, by utilizing the Zeeman splitting and/or shifting effects, for example, the present invention uses the magnetic domains 31 in a manner different (e.g., the magnetic domains may comprise more than two values and/or the magnetic domains may be interrogated, directly, or indirectly through the use of one or more contiguous material(s), in a different manner) from prior art approaches.", "Specifically, the magnetic domains 31 are present on a disk 1.Current techniques allow these magnetic domains to assume “0” or “1” mode (i.e., a base-2 or binary code) and these are aligned in one of two possible combinations at pre-selected magnetic strengths.", "The present invention permits the magnetic storage media to be utilized in several unique ways.", "Specifically, for example, rather than storing only a combination or sequence of “0”s and “1”s, the present invention stores more than two different field strengths to the magnetic disk 1, by influencing the magnetic media, for example, in a somewhat standard prior art approach.", "These different field strengths are stored by utilizing a read-write head 2 that is similar to conventional heads currently being utilized.", "The read-write head 2 is capable of imparting, and thus causing storage of different field strengths of different microTesla values upon the disk 1.Any desired combination of different field strengths can be stored upon the disk 1, and can be selected as a function of convenience and/or accuracy.", "In this regard, with regard to a base-6 system (i.e., “0, 1, 2, 3, 4, and 5”), the first bit of data stored can be no magnetic field, and can be made to correspond to “0”.", "Each of the remaining five different possible values can be selected so that they correspond to different points along the curve “M” shown in FIG.", "5.The differences between the stored field strengths are a matter of material design and system designer preferences.", "For this system, the choices of magnetic materials and/or materials which exhibit desirable Zeeman splitting or shifting may assist in dictating the difference in magnetic field strengths between different values in a coded system.", "Stated more particularly, materials engineers may desire certain types of magnetic materials to be utilized as magnetic storage media and such materials may have preferential field strengths which are unique to those materials.", "Moreover, such stored field strengths may result in desirable and detectable Zeeman splitting and/or shifting in the storage media per se, or such stored field strengths may be selected to result in desirable field splitting and/or shifting in one or more materials (e.g., a solid, a liquid, a gas and/or a plasma) contiguous to the magnetic media.", "Such preferential field strengths could then be maximized as both inputs and outputs in designing systems to be efficient, accurate and inexpensive.", "Such combinations of materials and/or systems should be apparent to those of ordinary skill of the art in view of the teachings herein.", "In this Zeeman effect, when a relatively wide splitting of electromagnetic energy frequencies occurs due to the applied magnetic field, the amount of, for example, splitting may correspond to higher frequencies (e.g., microwave); whereas a narrow splitting will typically correspond to low frequencies (e.g., radio).", "The amount of splitting that occurs is important because in order to determine the amount of splitting that has occurred, a suitable interrogating electromagnetic energy is caused to be incident upon, for example, the magnetic material itself or the aforementioned contiguous material.", "The contiguous material that exhibits Zeeman splitting at the stored magnetic field can be present as, for example, one or more separate sheets of material that are located adjacent to, and/or contiguous with, the material that contains the stored magnetic field.", "When multiple contiguous materials are utilized, the present invention contemplates the use of a comparative analysis of the differences in Zeeman splitting between two or more such contiguous materials.", "Alternatively, a material comprising, for example, one or more material(s) that are mixed with the magnetic material (e.g., a binder which holds together at least a portion of the material that stores the magnetic field) could also be used.", "More specifically, a particular interrogating frequency, or set of frequencies, needs to be capable of favorably interacting with one-or more of the splitting/shifting frequencies which result in the magnetic material, or in an alternative embodiment in one or more of the contiguous materials (e.g., a solid, a liquid, a gas and/or a plasma), due to the different magnetic fields stored in the magnetic material.", "In other words, a first material is desirably chosen so that an applied magnetic field can be reasonably stored in a magnetic material.", "However, it is desirable for the magnetic material to be capable of storing reliably several magnetic fields of different strength (e.g., capable of having varying numbers of magnetic domains align with an ever increasing magnetic field and such domains remain frozen or pinned so that they do not change with time) and, for example, in one embodiment of the invention, the contiguous material needs to be sensitive to the stored magnetic fields.", "In this regard, if one or more contiguous material(s) are utilized in connection with the magnetic material(s), the contiguous material(s) needs to exhibit a splitting of one or more of its rotational, vibrational, etc., bands (e.g., at the atomic or molecular level) and the resultant splitting that occurs in the contiguous material needs to be detectable by an appropriate interrogating electromagnetic source (discussed in greater detail elsewhere herein).", "Alternatively, a contiguous material does not need to be utilized.", "In this case, the magnetic material per se, or at least a portion thereof, needs to exhibit desirable and detectable splitting or shifting of one or more frequencies.", "Accordingly, it should be clear that the Zeeman effect techniques of the present invention could be utilized in conjunction with current hardware systems.", "In this regard, so long as an appropriate field can cause to be stored in a magnetic media and the resultant Zeeman effect which occurs in the magnetic material per se, or various material(s) contiguous to the stored magnetic field, can be appropriately interrogated.", "Thus, the present invention is a significant improvement over the prior art because current read-write heads use the ASCII code which is comprised of 0's and 1's in 8 different bits resulting in 256 different combinations of 0's and 1's.", "The present invention can harvest the advantage of codes that are higher order than binary.", "The present invention also provides significant improvements over various optical memory techniques.", "Specifically, in another embodiment of an alternative memory system of the present invention, a system somewhat analogous to current optical systems utilized in compact disk (CD) players is utilized.", "Current CD technology utilizes a rotating disk comprised of a number of layers of different materials.", "The layers include polycarbonate plastic, aluminum and acrylic.", "The aluminum in the disk includes a series of plateaus and valleys sometimes referred to as “bumps”.", "Laser light is caused to focus upon the plateaus and valleys in the rotating disk.", "The reflection of the laser off of the plateaus and valleys corresponds to a binary stream of information.", "CD's use, for example, 16-bit strings of binary coded data.", "Specifically, laser light reflects differently from a valley compared to the reflection off of a plateau and these different reflective properties of the laser when shined upon these valleys and plateaus correspond to the binary code of “0's” and “1's”.", "The present invention utilizes a similar concept to the CD disk, however, rather than utilizing valleys and plateaus (i.e., bumps) in an aluminum layer embedded in a polycarbonate plastic CD, the present invention utilizes at least two dots of different intensity color, or color width.", "These dots or spots are optically created and/or optically examined or interrogated.", "These optical dots or spots may be of a size which is approximately the same order of magnitude in size as the current data tracks used on CD's, they may be larger, or they may be smaller.", "Similar to the current CD's, these dots or spots are put together in strings which begin at an innermost portion of a rotatable CD disk and extend spirally out therefrom.", "Accordingly, current CD's of approximately 12 centimeters would be an acceptable size.", "However, if the dots or spots of the present invention are smaller than the “bumps” of the current technology, it is possible for CD's to be smaller in size while maintaining the same amount of memory space.", "Thus, it is possible to put an amount of optical information onto a rotatable disk of approximately 12 centimeters in diameter which exceeds that amount of information which is currently stored on CD's.", "A first example of the invention utilizes a quaternary system which is depicted schematically in FIG.", "4.In this regard, with reference to FIG.", "4A, a polarized light source 40 (e.g., a light source from a laser that has been subjected to a polarizing filter) is projected through a polarizing filter 41 (sometimes referred to herein as an analyzer).", "When the polarized light source 40 and polarizing filter 41 are in alignment, then a white dot 42a is caused to appear on a recording media 43.This white dot may correspond to “0” in a base-4 code.", "With regard to FIG.", "4B, the polarized light source 40 is projected upon the polarizing filter 41, however, in this Example, the filter 41 has been rotated about 30° with regard to the polarized light source 40.The disk 43 has also been rotated an appropriate amount of “x” so as to permit a separate recording of the dots 42a and 42b.", "This 30° rotation of the filter 41 results in the placement or recording of a dot 42b, that is somewhat gray upon the disk 43.FIG.", "4C shows the same polarized light source 40, however, the polarizing filter 41 is now arranged at an angle of about 60° relative to the source 40 so that a somewhat darker spot 42c is created.", "Again, the disk 43 has been rotated an amount “x” so as to permit the separate recording of the spot 42c upon the disk 43.Likewise, with regard to FIG.", "4D, the polarized light source 40 and the polarizing filter 41 are now at an angle of about 90° relative to the starting position of the polarizing filter 41 in FIG.", "4a, resulting in a completely dark spot 42d being recorded upon the disk 43.FIG.", "4e shows a traditional photo detector 50 projecting a light source onto the disk 43 such that the dots 42a, 42b, 42c, and 42d on the disk 43 can be illuminated.", "Light is then reflected back to, for example, the detector portion of the photo detector 50.Specifically, the detector 50 is capable of distinguishing the white dot 42a from the black dot 42d, as well as the shades of gray dots 42b and 42c.", "It should be apparent that the example depicted in FIG.", "4 has been greatly exaggerated for clarity purposes, and, for example, the amount “x” that the disk 43 needs to move to accommodate the sequential dots 42a-42d, is minute.", "Additionally, as a result of recording the four different dots 42a-42d upon the disk 43, a base-4 or quaternary system has been utilized with the result recorded being “0”-“1”-“2”-“3”.", "Still further, any appropriate media such as, for example, a PLZT ceramic, could be utilized to store different photo intensity data or spots.", "It should be apparent that the dots 42a-42d are the beginning of a track of data which can be recorded spirally outward from a center portion of a disk 43.Another example of the invention comprises a base-8 code utilizing 8 different shades of dots.", "While this particular example is not shown in the Figures, it is clear that 8 different shades of dots would require the turning of the polarizing filter 41 shown in FIG.", "4 at approximately 12.85° each time.", "Another example, similarly, could utilize a 16 shade code system, which would require turning the polarizing filter 41 of FIG.", "4 a total of 6° each time to result in 16 different shades of dots.", "It should be clear to an artisan of ordinary skill that the amount of data storage becomes a function of how finely the polarized filter 42 is rotated relative to the polarizing light source 41 as well as how accurately an optical detector 50 can distinguish between the different light intensities that are reflected or transmitted by the different shades of gray dots.", "It is certainly possible that an entire 256 character ASCII-type code can be written into a single bit.", "In such a case, the amount of data storage is significantly improved over the current techniques.", "Still further, it should be apparent that different colors (e.g., red-yellow-violet), corresponding to different wavelengths of light can be placed upon a surface of a disk 43.When, for example, such a disk containing these different color dots or spots is rotated and interrogated by, for example, a laser lens system, the different colors (wavelengths or frequencies) of the dots or spots can be registered and could be coded to correspond to different values (e.g., “0”, “1”, “2”, “3”, etc.).", "Thus, rather than using a simple binary or base-2 system where a black dot corresponds to “0” and white dot corresponds to “1”, the present invention provides for a larger number of options for data storage by providing methods to store data based on color, intensity or both.", "Accordingly, for example, in a simple quaternary system having a quaternary code consisting of 0's, 1's, 2's and 3's, there would be 4 different choices of basic information storage (i.e., a “base-4” system) rather than only two choices in a binary or base-2 system.", "Moreover, rather than utilizing only a monochromatic light source, it is possible that a polychromatic light source could also be utilized.", "In this regard, when polychromatic light is made to be incident upon a first polarizer, and thereafter upon one or more additional polarizers (e.g., analyzers), in addition to different intensities of light flowing through the analyzer(s), a spreading of different colors over a particular frequency or wavelength range will also occur.", "Specifically, as shown in FIGS.", "10a and 10b, when using polychromatic light, a curve 90 representing a spectrum of frequencies ranging from f1 to f2 of various intensities results in the formation of a somewhat continuous curve of varying light intensities over a particular frequency or wavelength range.", "The use of a polychromatic source can therefore result in secondary indicia for determining the value assigned to stored bits of data (e.g., a “0” or a “1”) and thus enhances data storage.", "For example, not only could the highest intensity of light I1 be recorded/detected, but also the starting and/or ending points f1 and/or f2 which correspond to the lowest frequency of light being detected, and the highest frequency of light being detected, respectively.", "Alternatively, the area “A” under each curve could also be compared as, for example, with integral area denoted as power spectral density.", "Further, the FWHM (i.e., full width half max) can be used as an indicia, representing the frequency ranger, f1 to f2 at half the maximal intensity.", "A range of full widths at varying intensities (i.e., 100% maximal, 1% minimal, 50% half maximal, 75% three-fourths maximal, etc.)", "can also be used.", "In other words, more than one piece of information can be checked to verify the accuracy of each data point that is stored and read.", "The presence of more than a single piece of information can result in more accurate reading of data and thus result in signal/noise improvements.", "FIG.", "6 shows a light source 60 which emits unpolarized light 61 into a polarizing filter 62 which creates a polarized light beam 63.The polarized light 63 is incident upon a suitable light modulator 64 which causes the light beam 63 from the polarizing filter 62 to be directed to one of a plurality of different polarizing (or analyzing) filters 81, 82, 83, 84 and 85.The modulator 64 is known in the art and can function at frequencies of about 1 GHz and is thus compatible with most computer processing systems.", "While only five analyzing filters 81-85 are shown, the number of filters that can be used in accordance with this embodiment is based on the data coding system in which it is desired to work.", "This particular system which utilizes five filters 81-85 is a base-5 system.", "When the modulator 64 directs the polarized light beam 63 from the polarizing filter 62 to the different analyzing filters 81-85, the result will be different intensities of light 65 being transmitted through each of the different analyzing filters 81-85 onto (or into) a suitable optical recording medium 66.Specifically, each of the polarizing filters 81-85 are fixedly rotated a different amount relative to the polarizing direction of the light beam 63 emanating from the polarizing filter 62.Once the light 65 has been transmitted through one of the different polarizing filters 81-85, the light can then be suitably focussed and/or redirected by appropriate optical and/or magneto-optical or acousto-optical (not shown in this FIG.", "6) means onto or into the recording medium 66.Examples of suitable optical recording mediums include: inorganic photochromic crystals, photorefractive materials, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence.", "With regard to FIG.", "7, an alternative to the embodiment of FIG.", "6 is shown.", "In this embodiment, the plurality of polarizing filters 81-85 are replaced with a polarization modulator 67.In particular, the polarization modulator 67 can be made of a suitable material such as a silica glass or an X-cut lithium niobate crystal (see, for example, the disclosure in U.S. Pat.", "No.", "6,154,432 for suitable materials).", "Voltages are applied to electrodes on the modulator 67 to produce an electric field that can orient the polarization of the propagating wave in any suitable direction.", "This technique provides a very robust and mechanically stable method of polarization control.", "The polarization of the light wave 70 (as well as the light wave 65) will be maintained through appropriate transmission of the light wave through other non-polarizing optics.", "The modulator 67 has been shown to have transmission rates of over 500 Mbits/second.", "A polarizing filter 62, oriented in a fixed direction, then provides an output 65 of varying intensities to a light modulator 64.The light modulator 64 then appropriately directs the output 65′ to a similar suitable recording medium 66.FIG.", "8 shows another embodiment of the invention wherein a light source 60 emits unpolarized light 61 into an acousto-optic Bragg modulator 68.The acousto-optic Bragg modulator 68 has inputted into it different electrical driving signals which result in different intensities of light 65″ being emitted therefrom.", "The different intensities of light can then be suitably focused and/or redirected by appropriate optical and/or magneto-optical and/or acousto-optical means into or onto an appropriate recording medium 66.FIG.", "9 shows another embodiment similar to that embodiment shown in FIG.", "7, except that the polarization modulator 67, has been replaced with at least one liquid crystal cell.", "In this regard, depending upon what voltages are applied to the liquid crystal cell 69 (the output which is directed into a polarizing filter 62) will result in a variable intensity output 65′″.", "The output 65′″ can thereafter be suitably directed to an appropriate optical storage medium 66 by any of the means for directing (not sown in FIG.", "9) the output 65′″, as discussed above herein.", "It should be also be clear to an artisan of ordinary skill that the same general technique disclosed herein can be utilized for the optical layout that is shown in FIG.", "27.In this regard, FIG.", "27 shows a holographic memory system of how a crystal can be imprinted with pages of data.", "In this regard, similar to the techniques shown in FIGS.", "4a-e, the system would work just as well for a holographic memory system.", "OPTICAL EXAMPLES The following experiments were performed to show that various light intensities can be recorded by rotating at least two polarizable filters (e.g., a polarizer and an analyzer) relative to each other and recording the different resultant intensities on ordinary photographic film (i.e., color slide film).", "The recorded intensities were thereafter interrogated with an optical spectrometer by passing a light source through the recorded intensities on the slides and into a probe connected to the optical spectrometer.", "All optical recording and interrogation steps were performed on a Newport optics table having one (1) inch grids thereon.", "In particular, mounting holes were provided at each corner of one inch squares for rigidly securing all optical equipment thereto to minimize data distortion due to vibrations.", "Different optical data values were obtained and recorded by using varying amounts of light which were caused to be incident on a Canon EOS Rebel 4 camera with zoom lens, which was rigidly mounted to the optics table.", "In particular, the camera was operated in a manual mode and the manual focus and was set to 50 mm.", "The film used in the camera was ordinary color slide film known as Fujichrome Color Slide film RH-400 speed.", "The source of light for all optic experiments was a Stocker and Yale, Model 20 Imagelite, polychromatic light source.", "The fiberoptic bundle end of the light source was also rigidly mounted to the optics table, with its end being located about 3 cm from a rigidly mounted polarizer/analyzer filter set (Orion Variable Polarizing Filter Set #5558).", "In particular, as shown in FIGS.", "20 and 21, the polychromatic light source 60 was caused to communicate with a polarizing filter set 62 and 81 through a fiber optic bundle 101 (i.e., a Stocker-Yale SF 38036P single S/O; ⅜″ bundle, 36″ long, MTL-PVC over metal hose).", "The function of the fiber optic bundle 101 was to transport polychromatic light from the Imagelite light source 60 (which was operating at a setting of “4”) to the polarizing filter set 62 and 81.The polarizing filter set 62 and 81 was located at a distance of about 3.3 cm from the lens of the Canon camera 100.Light from the light source 60 was directed through the fiber optic bundle 101 and caused to be made incident upon the polarizing filter set 62 and 81.The polarizing filter set 62 and 81 was set at six (6) different relative settings which permitted six (6) different intensities of light to be transmitted therethrough.", "In particular, the polarizing directions of each filter were set at the following relative rotations to each other: 0° relative rotation, 18° relative rotation, 36° relative rotation, 54° relative rotation, 72° relative rotation, and 90° relative rotation.", "These six (6) different relative rotation filter settings resulted in six (6) different intensities of light being transmitted therethrough.", "Specifically, the different intensities of polychromatic light were caused to be incident upon the lens of the camera 100 with care being taken so that the fiber optic bundle 101 transmitted light roughly perpendicular into and through the polarizing filter set 62 and 81 and into the lens of the camera 100.The result was a different intensity spot being recorded on each of the six (6) different slides 102 corresponding to each of the six (6) different relative rotation amounts.", "The differences in each of the slides 102 was readily observable by eye.", "Once the different images were recorded onto the photographic film comprising the slides 102, the images or spots were then interrogated by a transmission technique that utilized an optic spectrometer.", "Specifically, as shown in FIG.", "21, data values were retrieved from each of the six (6) different slides 102 by using a StellarNet EPP 2000 spectrometer 104 which utilized SpectraWiz software.", "Specifically, the same light source 60 used to create the images on the slides 102 was again connected with the same fiber optic bundle 101, still rigidly mounted on the optics table.", "Each of the data slides 102 was rigidly mounted in a holder such that the output from the fiber optics bundle 101 was reproducibly and directly incident upon that portion of the slide 102 containing the data or spot, from a distance of about 7.2 cm.", "Care was taken to assure that the fiber optic bundle 101 was mounted perpendicular to the slides 102 in the slide holder.", "A fiber optic probe 103 of the StellarNet EPP 2000 spectrometer 104 was rigidly mounted on the other side of each slide 102 about 7.0 cm away, so that transmission information could be obtained.", "Again, care was taken to assure proper alignment so that transmission data would not be skewed.", "The relevant settings on the StellarNet EPP 2000 fiber optic spectrometer were as follows: Mode Scope Detector integration time 4 ms Number of scans to average 5 Pixel resolution via smoothing 0 level Temperature compensation On Spectrometer channels 1 X axis Wavelength (nm) Y axis Counts The image light source was then set at a standard setting of “7”, in order to obtain information.", "The EPP 2000 provided information regarding the maximum integral area under the curve (power spectral density, i.e., Counts2) and full width half max (“FWHM”) of the peak (in nm) for each slide via the automated Area-PSD function.", "It is noted that each slide corresponded to the six (6) different rotatable filter settings.", "Measurements were reliably reproducible.", "Measurements of the six (6) slides, corresponding to the six (6) filter positions ranged, in power spectral densities, from 347,690 at 0° to 36,674 at 90°.", "FWHM ranged from 466 nm at 0° to 160 nm at 90°.", "Using either power spectral density alone, FWRM alone, or both in combination, six (6) different and distinct data values were obtained.", "Specifically, as shown in FIGS.", "22a, 22b and 22c, three sets of measurements were taken at different times from the same aforementioned six (6) slides.", "FIGS.", "22a-22c are a plot of Power Spectral Density (i.e., intensity) versus the six (6) different filter settings.", "The deviation in the upper and/lower limits of the data in FIGS.", "22a-22c is superimposed on FIG.", "23.It is clear from FIG.", "23 that the upper and lower intensity limit deviations corresponding to each of the six (6) different filter settings do not overlap with each other.", "Thus, this technique shows that intensity alone can provide a basis for a base-6 data storage system.", "Further, optical transmission data from the six (6) slides was analyzed by a second approach.", "Specifically, FIGS.", "24a, 24b and 24c show plots of full width half max (FWHM) data points versus the six (6) different filter settings.", "Similar to the data in FIGS.", "22a-22c, these data were obtained at three (3) different times to assure the accuracy of the measurements.", "FIG.", "25 shows the deviation in upper and lower limits for the data shown in FIGS.", "24a-24c.", "It is clear from FIG.", "25 that the upper limit deviation values for FWHM corresponding to each filter setting do not overlap.", "Thus, this technique provides another method for data recognition in, for example, a higher order system.", "MAGNETIC SPLITTING/SHIFTING EXAMPLES The following magnetic experiments were performed to show that different amounts of Zeeman splitting/shifting can be achieved in a magnetic material and/or a contiguous material.", "Specifically, varying magnetic field strengths were applied to a contiguous material comprising aluminum foil to simulate one potential configuration of magnetic storage media exhibiting desirable Zeeman splitting/shifting effects.", "The source of the magnetic fields utilized were three (3) permanent magnets of different field strengths.", "In particular, the magnets were obtained from DuraMag, Inc., and were characterized as follows: Magnet A: (Low Field Strength) NS-10010050, Neodymium-Iron-Boron Magnet, 1.000″×1.000″×0.500” thick with a field strength near the center thereof of about 5,090 G, increasing to about 8,380 G at the edge; Magnet B: (Intermediate Field Strength) ND-4514 Neodymium Magnet, 1.0″×1.0″×1.0″ thick with a field strength near the center thereof of about 8,730 G, increasing to about 10,300 G at the edge; and Magnet C: (High Field Strength) ND-4514 Neodymium Magnet, 1″×1″×1.9″ thick with a field strength near the center thereof of about 11,500 G, increasing to about 11,900 G at the edge.", "All spectroscopy measurements were performed using a Hewlett-Packard microwave spectroscopy system (30-minute warm-up).", "The following are the salient features of the spectroscopy system used in these examples.", "(1) HP Display Processor—Ref 0.0 dB, S11 mode, log MAG display, 10.0 dB/and marker pips with frequency and dB readout.", "(2) HP 8510B Network Analyzer-Ch 1.", "(3) HP 8513A Reflection/Transmission Test Set—45 MHz-26.5 GHz, Port 1.", "(4) HP 8350B Sweep Oscillator—Instrument State REM, Sweep trigger—INT, Sweep—Time.", "(5) HP 83595A RF Plug-in—0.1-26.5 GHz, Power 10.0, mW RF—On, CS Filter—On, ALC—INT.", "(6) Cables—W.", "L. Gore & Associates DAS ATE (55 AT) Test cables, 24 inches long.", "(7) Waveguides (Maury Microwave, Inc., and Narda, Inc.).", "It is noted that all waveguides produced flat baselines at 0.0 dB within the various frequency ranges noted below.", "a. Narda 616: Range 1.12 to 1.7 GHz b. Maury R229A1: Range 1.7 to 2.6 GHz c. Maury S209C2: Range 2.60 to 3.96 GHz d. Maury G229A1: Range 3.95 to 5.85 GHz e. Maury C209D2: Range 6.85 to 8.2 GHz.", "FIG.", "26 shows a schematic of the basic experimental set-up that was used to determine Zeeman shifting amounts in an aluminum foil contiguous material that was subjected to different magnetic field strengths.", "A microwave spectrometer 204 was comprised of Hewlett Packard items (1)-(5) described above.", "The microwave spectrometer 204 was connected by the cable 203 described in item (6) above.", "Different waveguides 202, described in item (7) above, were placed over each magnet 201 described as Magnets A, B and C above.", "Each of the magnets 201 (i.e., A, B and C) were sequentially placed into contact with a thin sheet of commonly available aluminum foil 200 (Tamco Distr.", "), the magnets 201 being oriented with their magnetization direction pointing parallel with the electromagnetic beam propagation in the waveguide 202.The following Table 3 shows the different amounts of Zeeman shifting that were obtained with three (3) different magnetic field strengths generated by the three (3) magnets A, B and C, applied to the contiguous aluminum foil 200.The initial frequency of 1.885 GHz was obtained from the aluminum foil 200 without the application of any magnetic field.", "The other values correspond to the shifted aluminum foil 200 frequency determined by microwave spectroscopy, as well as the power frequencies in dB.", "It is clear from this simple Example that meaningful Zeeman shifting can occur in a contiguous material to which a “permanent” magnetic field has been applied.", "Accordingly, this Example demonstrates that storage of varying magnetic field strengths can function as a memory storage system and that Zeeman shifting/splitting can function as a memory retrieval system.", "More particularly, both systems can function in higher order memory systems.", "TABLE 3 Magnet A Magnet B Magnet C Waveguide No Magnet (Low) (Intermediate) (High) 1.12-1.7 GHz 1.885 GHz, 1.855 GHz, 1.645 GHz, 1.115 −4.2 dB −4.6 dB −7.2 dB GHz, −3.4 dB 1.7-2.6 GHz - 0 - - 0 - - 0 - - 0 - 2.60-3.95 GHz - 0 - - 0 - - 0 - - 0 - 3.95-5.85 GHz - 0 - - 0 - - 0 - - 0 - While there has been illustrated and described what is at present considered to be the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made, and equivalents may be substituted for elements thereof without department from the true scope of the invention.", "In addition, many modifications may be made to adapt the teachings of the invention to a particular situation without departing from the central scope of the invention.", "Therefore, it is intended that this invention not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims." ] ]	Patent_10467912
[ [ "Apparatus and method for splicing sliver of yarn during yarn formation and processing", "An apparatus (20) and methods are provided for splicing selected portions of sliver (S) such as during yarn formation and processing.", "The apparatus (20) preferably includes a needle carrying member (35) having a plurality of needles (32) to engage silver (S) when positioned adjacent thereto and a needle engaging member (45) positioned to underlie the needle carrying member (35) and to receive the plurality of needles (32) from the first needle carrying member (35).", "The apparatus (20) also preferably includes a hand-activated needle actuation device (30, 40) connected to the needle carrying member (35) and the needle engaging member (45) to position the needle carrying member (35) in an open position to allow sliver (S) to be spliced to be readily positioned therebetween and responsive to closing at least portions of the hand (H) of a user for actuating the engaging of the needle carrying member (35) with the sliver (S) and the needle engaging member (45) so that the engaging of plurality of needles (32) of the needle carrying member (35) with the needle engaging member (45) thereby defines a closed position." ], [ "1-18.", "(canceled) 19.An apparatus for splicing selected portions of sliver, the apparatus comprising: a needle carrying member having a plurality of needles to engage sliver when positioned adjacent thereto; a needle engaging member positioned to receive the plurality of needles from the needle carrying member when the plurality of needles engage the sliver in a closed position; and a hand-activated needle actuation device connected to the needle carrying member and the needle engaging member to position the needle carrying member in an open position so that the plurality of needles is spaced-apart from the needle engaging member to allow sliver to be spliced to be readily positioned therebetween, the hand-activated needle actuation device being responsive to grippingly closing at least portions of a hand of a user to actuate the engaging of the needle carrying member with the sliver and the needle engaging member when the sliver is positioned between the needle carrying member and the needle engaging member so that the engaging of the plurality of needles of the needle carrying member with the needle engaging member thereby defines a closed position.", "20.An apparatus as defined in claim 19, wherein the hand-activated needle actuation device includes a first handle portion connected to the needle carrying member and positioned to be gripped by a hand of a user, a second handle portion connected to the needle engaging member and positioned to be gripped by a hand of a user, a pivot member connected to the first and second handle portions to allow either the first or second handle portions to pivot about the pivot member between the respective open and closed positions, and a biasing member connected to the pivot member and positioned to bias the needle carrying member in the open position.", "21.An apparatus as defined in claim 19, wherein the hand-activated needle actuation device includes a first handle portion connected to the needle carrying member and positioned to be gripped by a hand of a user, a second handle portion connected to the needle engaging member and positioned to be gripped by a hand of a user, a pivot member connected to the first and second handle portions to allow either the first or second handle portions to pivot about the pivot member between the respective open and closed positions, and a biasing member associated with the first and second handle portions and positioned to bias the needle carrying member in the closed position.", "22.An apparatus as defined in claim 19, wherein the needle engaging member includes a body member and a plurality of openings formed in the body member and each extending to a depth in the body member so that none of the plurality of needles extend outwardly from the needle engaging member when in the closed position, the number of the plurality of openings being equal to the number of the plurality of needles, each of the plurality of openings being positioned to underlie a corresponding one of the plurality of needles of the needles carrying member so that the plurality of openings matingly receive the plurality of needles as the plurality of needles pass into and out of the plurality of openings to thereby engage the needles and sliver being carried therewith.", "23.An apparatus as defined in claim 22, wherein the body member of the needle engaging member includes a substantially closed bottom, and wherein the bottom underlies each of the plurality of openings so that portions of the bottom define a floor for each of the plurality of openings.", "24.An apparatus as defined in claim 19, further comprising a lock associated with the hand activated needle actuation device to lock the hand activated needle actuation device in the closed position so that none of the plurality of needles extend outwardly from the needle engaging member when locked in the closed position.", "25.An apparatus as defined in claims 19, wherein each of the plurality of needles includes a needle body and a recessed portion formed in the needle body and positioned to assist in the engaging of and interconnecting of the sliver when each needle engages sliver during movement to the closed position.", "26.An apparatus as defined in claims 20, wherein the needle carrying member is detachably connected to at least one of the first handle portion and the second handle portion and defines a needle cartridge to readily remove from the respective first handle portion and second handle portion.", "27.An apparatus as defined in claims 26, further comprising at least one replacement auxiliary needle cartridge adapted to be readily positioned in the at least one of the first handle portion and the second handle portion.", "28.An apparatus for splicing sliver, the apparatus comprising a base and a needle receiving member associated with the base and positioned to receive a plurality of needles, the apparatus being characterized by having: a first cartridge member detachably connected to the base to be readily removed therefrom, the first cartridge member having a combination of a body portion and a plurality of needles each connected to the body portion, each of the plurality of needles having a recessed portion to engage and intertwine first portions of sliver with adjacent second portions of sliver; and a second auxiliary replacement cartridge member adapted to be detachably connected to the base and to be placed in substantially the same position as the first cartridge member, the second auxiliary replacement cartridge member also having a combination of a body portion and a plurality of needles each connected to the body portion and adapted to be positioned to be received by the needle receiving member, each of the plurality of needles having a recessed portion to engage and intertwine first portions of sliver with adjacent second portions of sliver.", "29.An apparatus as defined in claim 28, further comprising a pivot member associated with the first cartridge member and the needle receiving member to allow either the first cartridge member or needle receiving member to pivot about the pivot member between respective open and closed positions, the open position being defined by portions of the first cartridge member being spaced-apart from portions of the needle receiving member to receive the first cartridge member and the closed position being defined by the first cartridge member being received by portions of the needle receiving member when positioned closely adjacent thereto.", "30.An apparatus as defined in claim 29, further comprising a biasing member associated with the pivot member and positioned to bias either the first cartridge member or the needle receiving member in a preselected biased position.", "31.An apparatus as defined in claim 28, wherein the recessed portion of each of the plurality of needles of the first and second cartridge members includes at least one of the following: a barb, a groove, and a channel.", "32.An apparatus as defined in claims 28, a lock associated with the first cartridge member and the needle receiving member to lock the first cartridge member and the needle receiving member in a closed position.", "33.A method of splicing sliver, the method being characterized by the steps of: grippingly closing a first handle portion and a second handle portion of a sliver splicer having at least one sliver engaging member by a hand of a user so that the at least one sliver engaging member engages and splices sliver positioned adjacent thereto when in a closed position, the at least one sliver engaging member having a plurality of needles each including a recessed portion to engage and intertwine first portions of the sliver with second portions of the sliver to thereby join the first portions to the second portions of the sliver; and releasingly opening the first handle portion and the second handle portion by the hand of the user from the closed position to an open position to thereby release the spliced portion of sliver from the at least one sliver engaging member.", "34.A method as defined in claim 33, further comprising the steps of removing the spliced portion from adjacent the at least one sliver engaging member, grippingly closing the first handle portion and the second handle portion to the closed position without sliver positioned therebetween, and locking the first handle portion and the second handle portion in the closed position.", "35.A method as defined in claim 34, further comprising removing the spliced portion from adjacent the at least one sliver engaging member and biasingly closing the first handle portion and the second handle portion to the closed position without sliver positioned therebetween.", "36.A method as defined in claim 35, wherein none of the recessed portion of each of the plurality of needles is exposed when in the closed position.", "37.A method of splicing sliver comprising joining first portions of sliver with a plurality of needles having a recessed portion to engage and intertwine with adjacent second portions of sliver, the method characterized by having: the plurality of needles being connected to a body portion so that the body portion and the plurality of needles in the combination define a needle cartridge member; and replacing the needle cartridge member with an auxiliary cartridge member also having a body portion and a plurality of needles connected to the body portion.", "38.A method as defined in claims 37, wherein each of the plurality of needles includes a needle body, and wherein the recessed portion is formed in the needle body and positioned to assist in the engaging of and interconnecting of the sliver when each needle engages sliver." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>In the textile industry, yarn is formed of a plurality of slivers.", "To form the yarn, however, various steps are required to obtain the type and texture of the yarn desired.", "During sliver processing, sliver is often provided for shipment and use in cans or other containers which allow a first end of the sliver to be drawn or pulled from the can.", "A second end of the sliver is often left available for splicing to the first end of another can.", "This splicing is conventionally accomplished by a hand-type braid, twist, or other connection between the second end of the first can and the first end of the second can to join these respective ends of sliver together so that when the first can of sliver empties, the second can is ready to go for additional sliver processing.", "This hand-type braiding or twisting of the sliver has also become somewhat of a specialty of different textile personnel in sliver handling and processing.", "This manual procedure, however, can be time consuming, labor intensive, costly, inconsistent from person to person forming the braid or twist, and often is not strong or secure enough when the sliver is further handled or processed.", "Other types of automatic splicing systems have been developed over the years.", "Examples can be seen in U.S. Pat.", "No.", "5,544,389 by Onoue et al.", "titled “Sliver Piecing In Spinning Machines,” U.S. Pat.", "No.", "5,140,722 by Akiyama titled “Sliver Piecing Device Having Fiber Entangling Needles And Air Jets,” U.S. Pat.", "No.", "5,058,241 by Haigh et al.", "titled “Method And Apparatus For Combining Fibres Formed Into Slivers For Supply To Textile Machinery,” U.S. Pat.", "No.", "4,445,318 by Becker et al.", "titled “Method And Device For Making A Knot-Free Thread Connection By Splicing,” U.S. Pat.", "Nos.", "4,969,323 and 4,982,563 each by Stahlecker and each titled “Sliver Splicing Arrangement For A Spinning Machine,” U.S. Pat.", "No.", "2,608,725 by Strew titled “Sliver Piecing Device,” U.S. Pat.", "No.", "3 , 308 , 520 by Gagnon titled “Process Of Splicing Tow,” U.S. Pat.", "No.", "5,359,758 by Stahlecker et al.", "titled “Process And An Arrangement For The Piercing Of A Sliver,” Japanese Patent Document Application No.", "05105652 by Takashi titled “Sliver Joining Apparatus In Spinning Machine,” and German Patent Document No.", "90-210593/28 titled “Automatic Splicer For Roving On Ring-Spinning Frame—Has Needle Arrangement To Felt Fibres.” These automated systems, however, can be quite expensive to install, can be costly to operate, can have various complex mechanical and/or electrical problems, can take up additional floor space in manufacturing environments, can be bulky and awkward to use, and can often require extensive special training for personnel or the hiring of special personnel to oversee this automated equipment." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>With the foregoing in mind, the present invention advantageously provides an apparatus and methods for splicing slivers of yarn during formation and processing which is compact, relatively simple to use, and readily portable.", "The present invention also advantageously provides an apparatus and methods for splicing various types of textile slivers which is relatively inexpensive and does not require extensive personnel training to understand and operate.", "The present invention additionally advantageously provides an apparatus and methods for splicing various types of textile sliver which allows the user to easily handle, carry, and tote and which is of such a size as to be easily inserted into a pocket of a garment or a carrying case worn by the user.", "The present invention still also advantageously provides an apparatus and methods for splicing textile sliver which forms a substantially secure connection between ends of sliver being spliced or joined for later handling and/or processing.", "The present invention further advantageously provides an apparatus and methods for splicing textile sliver which is less complex, easy to repair or replace parts, and is readily adaptable to various splicing needs and situations.", "More particularly, the present invention provides an apparatus for splicing selected portions of sliver which preferably includes a needle carrying member having a plurality of needles to engage sliver when positioned adjacent thereto, a needle engaging member positioned to receive the plurality of needles from the first needle carrying member when the plurality of needles engage the sliver in a closed position, and a hand-activated needle actuation device connected to the needle carrying member and the needle engaging member to position the needle carrying member in an open position so that the plurality of needles is spaced-apart from the needle engaging member to allow sliver to be spliced to be readily positioned therebetween and responsive to grippingly closing at least portions of the hand of a user to actuate the engaging of the needle carrying member with the sliver and the needle engaging member when the sliver is positioned between the needle carrying member and the needle engaging member so that the engaging of plurality of needles of the needle carrying member with the needle engaging member thereby defines a closed position.", "The present invention also advantageously provides an apparatus for splicing sliver which preferably includes a first handle portion having at least one sliver engaging member, a second handle portion positioned to receive the at least one sliver engaging member in a closed position, and a pivot member associated with the first and second handle portions to allow either the first or second handle portions to pivot about the pivot member between respective open and closed positions.", "The open position preferably is defined by portions of the first handle member having the at least one sliver engaging member being spaced-apart from portions of the second handle portion positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion being received by the portions of the second handle member when positioned closely adjacent thereto.", "The apparatus preferably further includes a biasing member associated with the pivot member and positioned to bias either the first or the second handle portions in a preselected biased position.", "The present invention additionally provides an apparatus for splicing sliver which preferably includes a first handle portion having a first handle body and at least one sliver engaging member detachably connected to the first handle body to thereby define a cartridge member, a second handle portion pivotally connected to and positioned to receive the at least one sliver engaging member and pivot between open and closed positions.", "The open position is preferably defined by the at least one sliver engaging member being spaced-apart from portions of the second handle portion positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion being received by the portions of the second handle member when positioned closely adjacent thereto.", "The apparatus preferably also includes a biasing member positioned to bias either the first or the second handle portions in a preselected biased position.", "The present invention further provides methods of splicing sliver.", "A first method preferably includes grippingly closing a handle portion of a sliver splicer having at least one sliver engaging member by the hand of a user so that the at least one sliver engaging member engages and splices sliver positioned adjacent thereto and releasingly opening the handle portion by the hand of the user to thereby release the spliced portion of sliver from the at least one sliver engaging member.", "Another method of splicing sliver, according to the present invention, preferably includes closing a handle portion of a needle engaging member having a plurality of needles so that the plurality of needles engages and splices sliver positioned adjacent thereto and opening the handle portion of the needle engaging member so that the plurality of needles release the spliced portions of sliver therefrom.", "Yet another method of splicing sliver according to the present invention preferably includes joining first portions of sliver with a plurality of needles each having a recessed portion to engage and intertwine with adjacent second portions of sliver, the plurality of needles being connected to a body portion so that the body portion and the plurality of needles in combination define a needle cartridge member and replacing the needle cartridge member with an auxiliary cartridge member also having a body portion and a plurality of needles connected to the body portion.", "The apparatus and methods of the present invention provide additional manufacturing, handling, processing, and formation flexibility in the use of the splicers for sliver.", "For example, manufacturing personnel can walk around a facility with an apparatus of the present invention positioned in a pocket, holster, or harness when the splicing apparatus or splicer is preferably in a locked closed position so that the manufacturing personnel can readily remove the splicer, unlock the splicer, accomplish the splicing function, relock the splicer, and return the splicer to the pocket, holster, or harness.", "Additionally, the splicing apparatus of the present invention can be strapped to a chain or belt which can enhance carrying and portability.", "Further, when one or more needles or other sliver engaging members are damaged, according to one embodiment of the present invention, a cartridge member can readily be removed which carries the needles and replaced with an auxiliary cartridge member.", "This cartridge replacement, for example, prevents the need to replace the entire splicing apparatus and saves money and reordering time.", "Also, because the splicing apparatus is portable, compact, and relatively of simple construction and low cost, many different types of manufacturing personnel can use the splicing apparatus and can readily order additional or readily replace the entire splicing apparatus if desired without incurring extensive costs." ], [ "FIELD OF THE INVENTION The present invention relates to the textile industry and, more particularly, to the field of textile splicing and methods.", "BACKGROUND OF THE INVENTION In the textile industry, yarn is formed of a plurality of slivers.", "To form the yarn, however, various steps are required to obtain the type and texture of the yarn desired.", "During sliver processing, sliver is often provided for shipment and use in cans or other containers which allow a first end of the sliver to be drawn or pulled from the can.", "A second end of the sliver is often left available for splicing to the first end of another can.", "This splicing is conventionally accomplished by a hand-type braid, twist, or other connection between the second end of the first can and the first end of the second can to join these respective ends of sliver together so that when the first can of sliver empties, the second can is ready to go for additional sliver processing.", "This hand-type braiding or twisting of the sliver has also become somewhat of a specialty of different textile personnel in sliver handling and processing.", "This manual procedure, however, can be time consuming, labor intensive, costly, inconsistent from person to person forming the braid or twist, and often is not strong or secure enough when the sliver is further handled or processed.", "Other types of automatic splicing systems have been developed over the years.", "Examples can be seen in U.S. Pat.", "No.", "5,544,389 by Onoue et al.", "titled “Sliver Piecing In Spinning Machines,” U.S. Pat.", "No.", "5,140,722 by Akiyama titled “Sliver Piecing Device Having Fiber Entangling Needles And Air Jets,” U.S. Pat.", "No.", "5,058,241 by Haigh et al.", "titled “Method And Apparatus For Combining Fibres Formed Into Slivers For Supply To Textile Machinery,” U.S. Pat.", "No.", "4,445,318 by Becker et al.", "titled “Method And Device For Making A Knot-Free Thread Connection By Splicing,” U.S. Pat.", "Nos.", "4,969,323 and 4,982,563 each by Stahlecker and each titled “Sliver Splicing Arrangement For A Spinning Machine,” U.S. Pat.", "No.", "2,608,725 by Strew titled “Sliver Piecing Device,” U.S. Pat.", "No.", "3,308,520 by Gagnon titled “Process Of Splicing Tow,” U.S. Pat.", "No.", "5,359,758 by Stahlecker et al.", "titled “Process And An Arrangement For The Piercing Of A Sliver,” Japanese Patent Document Application No.", "05105652 by Takashi titled “Sliver Joining Apparatus In Spinning Machine,” and German Patent Document No.", "90-210593/28 titled “Automatic Splicer For Roving On Ring-Spinning Frame—Has Needle Arrangement To Felt Fibres.” These automated systems, however, can be quite expensive to install, can be costly to operate, can have various complex mechanical and/or electrical problems, can take up additional floor space in manufacturing environments, can be bulky and awkward to use, and can often require extensive special training for personnel or the hiring of special personnel to oversee this automated equipment.", "SUMMARY OF THE INVENTION With the foregoing in mind, the present invention advantageously provides an apparatus and methods for splicing slivers of yarn during formation and processing which is compact, relatively simple to use, and readily portable.", "The present invention also advantageously provides an apparatus and methods for splicing various types of textile slivers which is relatively inexpensive and does not require extensive personnel training to understand and operate.", "The present invention additionally advantageously provides an apparatus and methods for splicing various types of textile sliver which allows the user to easily handle, carry, and tote and which is of such a size as to be easily inserted into a pocket of a garment or a carrying case worn by the user.", "The present invention still also advantageously provides an apparatus and methods for splicing textile sliver which forms a substantially secure connection between ends of sliver being spliced or joined for later handling and/or processing.", "The present invention further advantageously provides an apparatus and methods for splicing textile sliver which is less complex, easy to repair or replace parts, and is readily adaptable to various splicing needs and situations.", "More particularly, the present invention provides an apparatus for splicing selected portions of sliver which preferably includes a needle carrying member having a plurality of needles to engage sliver when positioned adjacent thereto, a needle engaging member positioned to receive the plurality of needles from the first needle carrying member when the plurality of needles engage the sliver in a closed position, and a hand-activated needle actuation device connected to the needle carrying member and the needle engaging member to position the needle carrying member in an open position so that the plurality of needles is spaced-apart from the needle engaging member to allow sliver to be spliced to be readily positioned therebetween and responsive to grippingly closing at least portions of the hand of a user to actuate the engaging of the needle carrying member with the sliver and the needle engaging member when the sliver is positioned between the needle carrying member and the needle engaging member so that the engaging of plurality of needles of the needle carrying member with the needle engaging member thereby defines a closed position.", "The present invention also advantageously provides an apparatus for splicing sliver which preferably includes a first handle portion having at least one sliver engaging member, a second handle portion positioned to receive the at least one sliver engaging member in a closed position, and a pivot member associated with the first and second handle portions to allow either the first or second handle portions to pivot about the pivot member between respective open and closed positions.", "The open position preferably is defined by portions of the first handle member having the at least one sliver engaging member being spaced-apart from portions of the second handle portion positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion being received by the portions of the second handle member when positioned closely adjacent thereto.", "The apparatus preferably further includes a biasing member associated with the pivot member and positioned to bias either the first or the second handle portions in a preselected biased position.", "The present invention additionally provides an apparatus for splicing sliver which preferably includes a first handle portion having a first handle body and at least one sliver engaging member detachably connected to the first handle body to thereby define a cartridge member, a second handle portion pivotally connected to and positioned to receive the at least one sliver engaging member and pivot between open and closed positions.", "The open position is preferably defined by the at least one sliver engaging member being spaced-apart from portions of the second handle portion positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion being received by the portions of the second handle member when positioned closely adjacent thereto.", "The apparatus preferably also includes a biasing member positioned to bias either the first or the second handle portions in a preselected biased position.", "The present invention further provides methods of splicing sliver.", "A first method preferably includes grippingly closing a handle portion of a sliver splicer having at least one sliver engaging member by the hand of a user so that the at least one sliver engaging member engages and splices sliver positioned adjacent thereto and releasingly opening the handle portion by the hand of the user to thereby release the spliced portion of sliver from the at least one sliver engaging member.", "Another method of splicing sliver, according to the present invention, preferably includes closing a handle portion of a needle engaging member having a plurality of needles so that the plurality of needles engages and splices sliver positioned adjacent thereto and opening the handle portion of the needle engaging member so that the plurality of needles release the spliced portions of sliver therefrom.", "Yet another method of splicing sliver according to the present invention preferably includes joining first portions of sliver with a plurality of needles each having a recessed portion to engage and intertwine with adjacent second portions of sliver, the plurality of needles being connected to a body portion so that the body portion and the plurality of needles in combination define a needle cartridge member and replacing the needle cartridge member with an auxiliary cartridge member also having a body portion and a plurality of needles connected to the body portion.", "The apparatus and methods of the present invention provide additional manufacturing, handling, processing, and formation flexibility in the use of the splicers for sliver.", "For example, manufacturing personnel can walk around a facility with an apparatus of the present invention positioned in a pocket, holster, or harness when the splicing apparatus or splicer is preferably in a locked closed position so that the manufacturing personnel can readily remove the splicer, unlock the splicer, accomplish the splicing function, relock the splicer, and return the splicer to the pocket, holster, or harness.", "Additionally, the splicing apparatus of the present invention can be strapped to a chain or belt which can enhance carrying and portability.", "Further, when one or more needles or other sliver engaging members are damaged, according to one embodiment of the present invention, a cartridge member can readily be removed which carries the needles and replaced with an auxiliary cartridge member.", "This cartridge replacement, for example, prevents the need to replace the entire splicing apparatus and saves money and reordering time.", "Also, because the splicing apparatus is portable, compact, and relatively of simple construction and low cost, many different types of manufacturing personnel can use the splicing apparatus and can readily order additional or readily replace the entire splicing apparatus if desired without incurring extensive costs.", "BRIEF DESCRIPTION OF THE DRAWINGS Some of the features, advantages, and benefits of the present invention having been stated, others will become apparent as the description proceeds when taken in conjunction with the accompanying drawings in which: FIG.", "1 is a perspective view of an apparatus for splicing sliver according to a first embodiment of the present invention; FIG.", "2 is a side elevational view of an apparatus for splicing sliver being actuated by the hand of a user according to a first embodiment of the present invention; FIG.", "3 is an enlarged fragmentary elevational view of a type of needle of an apparatus for splicing sliver according to the present invention; FIG.", "4 is a sectional view of a needle of an apparatus for splicing sliver taken along line 4-4 of FIG.", "3 according to the present invention; FIG.", "5 is an enlarged fragmentary elevational view of a needle of an apparatus for splicing sliver according to the present invention; FIG.", "6 is a sectional view of a needle of an apparatus for splicing sliver taken along line 6-6 of FIG.", "5 according to the present invention; FIGS.", "7A-7D are schematic perspective views of the operation of an apparatus for splicing sliver according to a first embodiment of the present invention; FIG.", "8A is an enlarged fragmentary perspective view of an apparatus for splicing sliver prior to engagement with sliver in an open position according to the present invention; FIG.", "8B is an enlarged fragmentary perspective view of an apparatus for splicing sliver after engagement with the sliver in a closed position according to the present invention; FIG.", "8C is an enlarged fragmentary perspective view of an apparatus for splicing sliver after engagement with the sliver in an open position according to the present invention; FIG.", "9 is an enlarged fragmentary view of sliver after being spliced with an apparatus for splicing sliver according to the present invention; FIG.", "10 is a perspective view of an apparatus for splicing sliver according to a second embodiment of the present invention; FIG.", "11 is an exploded fragmentary perspective, view of a needle cartridge for positioning in an apparatus for splicing sliver according to a second embodiment of the present invention; FIG.", "12 is a side elevational view of an apparatus for splicing sliver having a needle cartridge illustrated by broken lines for clarity according to a second embodiment of the present invention; FIG.", "13 is an exploded side elevational view of an apparatus for splicing sliver having a needle cartridge member and a plurality of replacement auxiliary needle cartridge members according to a second embodiment of the present invention; FIG.", "14 is a fragmentary perspective view a portion of an apparatus for splicing sliver illustrating a connection between first and second handle portions along a pivot member and the position of a biasing member to bias the apparatus in an open position according to the present invention; and FIG.", "15 is an apparatus for splicing sliver according to a third embodiment of the present invention.", "DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention will now be described more fully hereinafter with reference to the accompanying drawings which illustrate preferred embodiments of the invention.", "This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.", "Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.", "Like numbers refer to like elements throughout, the prime notation, if used, indicates similar elements in alternative embodiments.", "FIGS.", "1-9 illustrate a first embodiment of an apparatus 20 for splicing sliver S. The apparatus 20 preferably includes a first handle portion 30 having at least one sliver engaging member.", "The at least one sliver engaging member is preferably one or more needles 32, and more preferably a plurality of needles 32, connected to the first handle portion 30.A second handle portion 40 preferably is positioned to receive the at least one sliver engaging member, e.g., the plurality of needles 32, in a closed position such as through or within one or more openings or bores 42, 42′ as shown (see also FIG.", "10).. As perhaps be shown in FIGS.", "1-2 and 14, a pivot member 25 is associated with, and preferably connected to or mounted as illustrated, to the first and second handle portions 30, 40 to allow either the first or second handle portions 30, 40 to pivot about the pivot member 25 between respective open and closed positions.", "The open position preferably is defined by distal portions 31 of the first handle portion 30 having the at least one sliver engaging member being spaced-apart from distal portions 31 of the second handle portion 40 positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion 30 being received by the distal portions of the second handle portion 40 when positioned closely adjacent thereto.", "The first and second handle portions 30, 40 can advantageously defined a base which is advantageously hand operated, but other base structures as understood by those skilled in the art which may or may not be hand operated can be used as well according to the present invention.", "The apparatus 20 preferably includes a biasing member 28 associated with the pivot member 25 and positioned to bias either the first or the second handle portions 30, 40 in a preselected biased position.", "In the embodiment of the splicing apparatus 20 as illustrated, the first handle portion 30 is biased by the biasing member 28 to the open position.", "As shown in FIG.", "14, the biasing member 28 is preferably a spring as understood by those skilled in the art and preferably is connected to the pivot member 25 and positioned to bias the first handle portion 30 in the open position.", "Other types of biasing members, including various types of springs, can be used as well.", "Although the illustrated embodiment of biasing the first handle portion 30 in the open position is particularly advantageous due to the movement desired in the splicing process and the ease of use by a user's hand, for example, it will also be understood by those skilled in the art that the present invention would include biasing the splicing apparatus 2011 in the closed position (see also FIG.", "15).", "Further, the apparatus 20 preferably also includes a lock 50 associated with the first and second handle portions 30, 40 to lock the first and second handle portions 30, 40 in a closed position.", "In the embodiment shown, the lock 50 is primarily connected to the first handle portion 30 and slidably engages or locks, e.g., a latch, with the second handle portion 40, e.g., into a slot or channel by connecting to the needle receiving portion or body 45 of the second handle portion 40 (see FIG.", "2).", "As perhaps best illustrated in FIG.", "2, the first and second handle portions 30, 40 define a hand-activated needle actuation device which advantageously allows a ready grip by a user's hand H to apply pressure from the user's hand H to actuate the movement of the plurality of needles 32.As described above, and as illustrated in FIGS.", "7A-7D and 8A-8C, the at least one sliver engaging member preferably includes a plurality of needles 32, and the hand-activated needle actuation device is responsive to grippingly closing at least portions of the hand H of a user to actuate the engaging of the plurality of needles 32 with the sliver S to be spliced and the distal portions 45, e.g., through the openings or bores 42, 42′ of the second handle portion 40 when the sliver S is positioned between the plurality of needles 32 and the distal portions 45 of the second handle portion 40 so that the engaging of plurality of needles 32 with the distal portions 45 of the second handle portion 40 thereby further defines the closed position (see also FIG.", "10).", "Also, as perhaps best shown in FIGS.", "3-6 and 8A-8C, each of the plurality of needles 32 preferably includes a needle body 33, 33′ and a recessed portion 34, 34′ formed in the needle body 33, 33′ and positioned to assist in the engaging of and interconnecting of the sliver S when each needle 32, 32′ engages sliver S during movement to the closed position.", "The recessed portion 34, 34′ preferably includes at least one of the following: a barb, a groove, and a channel.", "The recessed portion 34, 34′ preferably has an upward slope with respect to the downward movement of the needle 32 so that the recessed portion 34, 34′ readily catches, engages, or otherwise contacts the sliver S during the downward motion of the needles and responsively releases the sliver S during upward motion.", "This process allows the intertwining or interconnecting of the sliver S to join the portions of sliver S desired to be spliced together.", "As shown in FIGS.", "10-13 and 15, according to a second embodiment of a splicing apparatus 20′ of the present invention, the at least one sliver engaging member preferably is detachably connected to the first handle portion 30′ and defines a cartridge member 35, 35′ positioned in distal portions 31′ of the first handle portion 30′ to readily remove from the first handle portion 30′.", "At least one replacement auxiliary cartridge member 38, and preferably a plurality of replacement auxiliary cartridge members 38, can have the same construction as the cartridge member 35, 35′ and can be adapted to be readily positioned in the first handle portion 30′.", "Accordingly, a kit can also be provided which has a portable splicing apparatus 20′ positioned in a container, e.g., box, bag, package, with one or more auxiliary cartridge members 38, so that when a cartridge member 35, 35′ being used is damaged, dulled, or otherwise desired to be replaced, another cartridge member 38 can be readily inserted into an opening 39 in the first handle portion 30′ of the splicing apparatus 20′ after removal of the damaged cartridge member 35, 35′ so that splicing operations proceed with substantially reduced interruptions.", "The distal portion 45′ of the second handle portion 40′ preferably has additional springs or other types of biasing members 36 which allow the needles 32′ to retract and extend from openings 37 in the distal portion 31′ as shown.", "Further still, as shown in FIG.", "15, scissor-type handles 30″, 40″, substantially closed loops, and various other types of handle or finger grips can also be used.", "In this embodiment of a splicing apparatus 20″, the pivot member 25″ is also moved forward toward a more medial portion of the handle members 30″, 40″ and the biasing member 28″ is another type of spring, as understood by those skilled in the art, which biases the scissor-type handle embodiment to a closed position so that the distal portions 31″, 45″ of the handles are positioned closely adjacent each other and the plurality of needles 32″ can engage receiving portions of the handle 40″.", "As illustrated in FIGS.", "1-15, the present invention further provides methods of splicing sliver S. A first method preferably includes grippingly closing a handle portion 30 of a sliver splicer 20 having at least one sliver engaging member 32 by the hand H of a user so that the at least one sliver engaging member 32 engages and splices sliver S positioned adjacent thereto and releasingly opening the handle portion 30 by the hand H of the user to thereby release the spliced portion of sliver S from the at least one sliver engaging member 32.Another method of splicing sliver S according to the present invention preferably includes closing a handle portion 30 of a needle engaging member having a plurality of needles 32 so that the plurality of needles 32 engages and splices sliver S positioned adjacent thereto and opening the handle portion 30 of the needle engaging member so that the plurality of needles 32 release the spliced portions of sliver S therefrom.", "Yet another method of splicing sliver S according to the present invention preferably includes joining first portions of sliver S with a plurality of needles 32 each having a recessed portion 34 to engage and intertwine with adjacent second portions of sliver S. The plurality of needles preferably is connected to a body portion so that the body portion and the plurality of needles 32 in combination define a needle cartridge member 35 and replacing the needle cartridge member 35 with an auxiliary cartridge member 38 also having a body portion and a plurality of needles 32 connected to the body portion.", "The apparatus 20, 20′, 20″ and methods of the present invention provide additional manufacturing, handling, processing, and formation flexibility in the use of the splicers for sliver.", "For example, manufacturing personnel can walk around a facility with an apparatus 20 of the present invention positioned in a pocket, holster, or harness when the splicing apparatus 20 or splicer is preferably in a locked closed position so that the manufacturing personnel can readily remove the splicer 20, unlock the splicer 20, accomplish the splicing function, relock the splicer 20, and return the splicer 20 to the pocket, holster, or harness.", "Additionally, the splicing apparatus 20 of the present invention can be strapped to a chain or belt which can enhance carrying and portability.", "Further, when one or more needles 32 or other sliver engaging members are damaged, according to one embodiment of the present invention, a cartridge member 35 can readily be removed which carries the needles 32′ and replaced with an auxiliary cartridge member 38.This cartridge replacement, for example, prevents the need to replace the entire splicing apparatus 20′ and saves money and reordering time.", "Also, because the splicing apparatus 20 is portable, compact, and relatively of simple construction and low cost, many different types of manufacturing personnel can use the splicing apparatus 20, 20′, 20″ and can readily order additional or readily replace the entire splicing apparatus 20, 20′, 20″ if desired without incurring extensive costs.", "In the drawings and specification, there have been disclosed a typical preferred embodiment of the invention, and although specific terms are employed, the terms are used in a descriptive sense only and not for purposes of limitation.", "The invention has been described in considerable detail with specific reference to these illustrated embodiments.", "It will be apparent, however, that various modifications and changes can be made within the spirit and scope of the invention as described in the foregoing specification and as defined in the appended claims." ] ]	Patent_10467924
[ [ "Production of nanoparticles from methyl vinyl ether and maleic anhydride for the administration of hydrophilic pharmaceuticals, more particularly of puric and pyrimidinic bases", "Manufacture of nanoparticles on the basis of methyl vinyl ether and maleic acid for the administration of pharmaceuticals of an hydrophilic nature, in particular analogs of puric and pyrimidinic bases.", "The nanoparticles are obtained by desolvation with an hydroalcoholic phase of a methyl vinyl ether and maleic acid copolymer solution in acetone.", "The particles obtained are next treated with cross-linking agents (diamines or proteins) for the purpose of prolonging their useful life and are, possibly, incubated with a pharmaceutical which will be transported on the surface.", "The nanoparticles can carry the pharmaceutical likewise encapsulated which would then be added during the desolvation.", "In the case of the nanoparticle-ligand conjugates, the nanoparticles previously obtained and containing inside the pharmaceutical to be transported are incubated with the ligand or molecule which will contribute the property of specifically recognising a particular receptor of the organism.", "These pharmaceutical forms have as objective to improve the transport of the pharmaceutical or biologically active molecule to its site of action and/or absorption.", "This property improves the specificity and effectiveness of said pharmaceuticals." ], [ "1.a process for the manufacture of nanoparticles and ligand-nanoparticle conjugates with site specific delivery or targeting properties, able to carry drugs or biologically active molecules on their surface or in their interior characterized by the desolvation of poly(methyl vinyl ether-co-maleic anhydride)copolymer, dissolved in an organic solvent, and subsequent cross-linkage reaction with polyfunctional chemical compounds, including polyamines and polyhydroxyls: 2.A process according to claim 1, that comprises: a) desolvation of the poly(methyl vinyl ether-co-maleic anhydride)polymer dissolved in a polar organic phase at a concentration between 0.01 and 10% w/v, which may optionally contain a drug or biologically active molecule, with a hydroalcoholic solution in an organic phase/hydroalcoholic solution ratio of 1/1 to 1/10; b) elimination of the organic solvents by conventional methods such as filtration, centrifugation or evaporation, including the use of vacuum, among others; c) stabilization of the resulting nanoparticles with cross-linking agents; d) optionally, incubation of the nanoparticles with either the drug or the biologically active molecule or, alternatively, the ligand with targeting properties; e) purification of either the obtained nanoparticles or conjugates by conventional techniques such as ultracentrifugation, centrifugation, tangential filtration, among others; f) optional freeze-drying of the obtained nanoparticles or conjugates.", "3.A process according to claim 1, characterized in that nanoparticles or conjugates are produced in a way which permits encapsulation of the drug or biologically active molecule in their interior, that comprises: i) the addition of said drug or biologically active molecule in step a) and, ii) optionally, the addition of a second drug or a different biologically active molecule or, alternatively, a ligand with targeting properties in step d).", "4.A process according to claim 1, characterized in that nanoparticles are produced with the drug or biologically active molecule on outer layer, that comprises: i) the addition of the aforesaid drug or biologically active molecule in step d) and, ii) optionally, the addition of another drug or biologically active molecule in step a).", "5.A process according to claim 1, characterized in that unloaded nanoparticles are produced consisting of carrying out step a) and step d) without adding any drug or biologically active molecule or ligand.", "6.A process according to claim 1, characterized in that step c) is carried out by using polyamine or polyhydroxyl type polyfunctional reagents, including among them proteins and polymeric macromolecules such as non-ionic surfactants or polyvinylpyrrolidone.", "7.A process according to claim 1, characterized in that step c) is carried out using the cross-linking agent 1,3-diaminopropane (DP) at a concentration ranging between 0 and 1 mg DP/mg poly(methyl vinyl ether co maleic anhydride).", "8.A process according to claim 1, characterized in that step c) is carried out using albumin, such as human serum albumin or bovine serum albumin, at a concentration ranging between 0 and 10 mg albumin/mg poly(methyl vinyl ether co maleic anhydride).", "9.A process according to claim 1, characterized in that step f) is carried out by adding mannitol or sacarose as a cryoprotector agent at a concentration ranging between 0.1 and 10% in weight.", "10.A process according to claim 1, characterized in that the ligands used in the production of the nanoparticle conjugates have the characteristics of being able to recognize specific structures, or cellular or tissular receptors located on the surface or inside specific cell types in the organism.", "11.A process according to claim 10, characterized in that the ligands used are lectins, carbohydrates, monoclonal antibodies, vitamins, amino acids, lipids or molecules of a peptidic nature.", "12.A process according to claim 11, characterized in that the ligand is the Sambucus nigra lectin which is bound at a concentration ranging between 1 and 100 mg lectin/mg nanoparticles.", "13.A process according to claim 1, characterized in that the drugs or the biologically active molecules incorporated inside the nanoparticles and conjugates possess a hydrophilic character and are soluble in organic polar solvents.", "14.A process according to claim 13, characterized in that which the drug incorporated is the anti-tumor drug 5-fluorouridine.", "15.A process according to claim 14, characterized in that 5-fluorouridine is dissolved in acetone at a concentration ranging between 0.1 and 3.33 mg/mL and then added to the poly(methyl vinyl ether co maleic anhydride)aceton solution, in order to obtain a drug/polymer ratio ranging between 0.01 and 0.4 mg/mg.", "16.A process according to claim 1, characterized in that the drug or biologically active substance is carried on the surface of the nanoparticles and in which step d) of incubation is produced in an aqueous solution.", "17.A process according to claim 16, characterized in that the drugs or biologically active molecules incorporated into the surface of the nanoparticles are of hydrophilic character or are soluble in aqueous solutions.", "18.A process according to claim 17, characterized in that the drugs or biologically active molecules are either analogs of puric or pyrimidinic bases, or they are compounds of protein nature such as peptides, proteins, glycoproteins, lipoproteins, or they are carbohydrates.", "19.A process according to claim 18, characterized in that the drug incorporated in the surface of the nanoparticles is the anti-tumor agent 5-fluorouridine.", "20.A process according to claim 19, characterized in that 5-fluorouridine is dissolved in water and, subsequently, added to the suspension of nanoparticles at a concentration ranging between 10 and 1000 μg drug/mg polymer, obtaining concentrations greater than 200 μg of drug bounded to the surface per mg nanoparticles.", "21.A process according to claim 17, characterized in that the incorporated drug is the antiviral agent ganciclovir.", "22.A process according to claim 21, characterized in that the ganciclovir is dissolved in water and then added to a suspension of nanoparticles, at a concentration ranging between 0.1 and 20 mg drug/mg polymer, obtaining entrapment efficiencies greater than 20% of the initially added ganciclovir.", "23.A process according to claim 17, characterized in that the incorporated drug is an oligonucleotide.", "24.A process according to claim 23, characterized in that the incorporated drug is an antisense oligonucleotide.", "25.A process according to claim 24, characterized in that the incorporated drug is the antisense oligonucleotide ISIS 2922.26.A process according to claim 25, characterized in that ISIS 2922 is dissolved in water and later added to the suspension of nanoparticles at a concentration ranging between 0.1 and 200 μg drug/mg polymer, producing nanoparticles whose superficially-bounded drug concentration is greater than 2 μg per mg nanoparticle.", "27.Nanoparticles or conjugates obtainable by a manufacturing process according to claim 1, characterized by having an approximate average size of less than 500 nm.", "28.Nanoparticles or conjugates according to claim 27, characterized by showing a biphasic release profile, with a first phase of immediate release of up to 60% of the loaded drug or biologically active molecule, followed by a second phase in which the drug or biologically active molecule is released slowly and in a sustained release manner.", "29.Nanoparticles or conjugates characterized in that they have the following composition: 13-99% w/w poly(methyl vinyl ether co maleic anhydride), 0.001-15% w/w cross-linking agent, optionally, 0.001-15% w/w drug or biologically active molecule, optionally, 0.01-4.5% w/w ligand with specific targeting properties, optionally, 70-82% w/w cryoprotector.", "30.Use of A method comprising using the nanoparticles and conjugates according to claim 27 for the administration of drugs or biologically active molecules to a patient.", "31.A method comprising using the nanoparticles and conjugates according to claim 27 in the administration of third generation colloidal pharmaceutical forms to a patient.", "32.A method comprising using the nanoparticles and conjugates of claim 27 for the pellicular coating of macroscopic pharmaceutical forms such as tablets, granules, granulates and pellets.", "33.A method comprising using the nanoparticles and conjugates of claim 27 loading 5-fluorouridine in the preparation of compositions that are useful in the treatment of certain diseases such as colon cancer, cancers of the gastrointestinal tract, breast cancer, cancers of the cervix and endometrium, as well as cancers of the head and neck, liver, ovary, pancreas, prostrate and skin.", "34.A method comprising using the nanoparticles and conjugates of claim 27 loading ganciclovir in the preparation of compositions which are useful for the treatment of infections induced by human cytomegalovirus.", "35.A method comprising using the nanoparticles and conjugates of claim 27, loading ganciclovir, as adjuvant, in the preparation of gene therapy compositions, which incorporate suicide genes and, in particular, the thymidine quinase gene.", "36.A method comprising using the nanoparticles and conjugates of claim 27 loading the antisense oligonucleotide ISIS 2922 in the preparation of compositions useful for the treatment of infections induced by human cytomegalovirus." ], [ "<SOH> TECHNICAL FIELD OF THE INVENTION <EOH>The present invention is within the scope of the procedures for the production of non-biological vectors, nanoparticles and nanoparticle-ligand conjugates, for the transport and administration of pharmaceuticals or active biological molecules of hydrophilic nature through the use of the methyl vinyl ether and maleic anhydride copolymer, poly(methyl vinyl ether-co-maleic anhydride), as priority element." ], [ "TECHNICAL FIELD OF THE INVENTION The present invention is within the scope of the procedures for the production of non-biological vectors, nanoparticles and nanoparticle-ligand conjugates, for the transport and administration of pharmaceuticals or active biological molecules of hydrophilic nature through the use of the methyl vinyl ether and maleic anhydride copolymer, poly(methyl vinyl ether-co-maleic anhydride), as priority element.", "STATE OF THE ART The behavior of a drug after its administration in the organism is determined by a combination of different processes: distribution and elimination when the drug is administered by intravenous route; absorption, distribution, and elimination when an extravascular route is used.", "Each one of these processes mainly depends on the physico-chemical properties of the drug substance which are determined by its chemical structure.", "After administering the drug by conventional means, it is distributed throughout the organism according to its molecular structure, which determines its physicochemical properties (molecular mass, lipophilia, pKa, crystalline structure, solubility), biochemical properties (affinity with plasmatic or tissue proteins, affinity with the membrane or intracellular receptors, sensibility to the enzymes responsible for its biotransformation, etc.)", "and pharmacological properties (ability to clear biological membranes, intensity of the action, etc.).", "Numerous studies have shown that the classical pharmaceutical forms (tablets, capsules, suppositories, injections, etc.)", "only permit the modulation of the intensity and/or speed of release of the biologically active molecule into the blood circulation (absorption), but they have no effect on the subsequent stages of in vivo behavior of the drug (distribution and elimination).", "During the distribution stage, the drug spreads to a large number or to every anatomical area of the organism.", "This implies: (i) the administration of doses higher to those which would theoretically be needed to obtain the required effect or (ii) the repeated administration of the drug.", "The therapeutic effect is attained in spite of the loss of an important fraction of the drug dose in different tissues other than those where it should act, resulting in unwanted side effects.", "This nonspecific distribution of the drug, tolerable for those with high therapeutic indexes (quotient between the dose that provokes the toxic effects and the therapeutic dose), could become the limiting factor for the clinical use of certain biologically active molecules (example: the 5-fluorouridine).", "So as to obtain a more rational and better adapted therapeutic use, one of the most promising possibilities is that which uses the concept of vectorization for the increase in the efficiency and specificity of action of the drugs.", "This concept consists in associating a drug or a biologically active molecule to an appropriate vector or carrier.", "Although there are different types of vectors, in this study only the characteristics and properties of the non-biological vectors (liposomes, nanoparticles and conjugates) will be studied.", "The use of these new pharmaceutical forms permits the association between the drug and a system whose mission is to carry the drug to its pharmacological target instead of remaining in a free state.", "In fact, the use of non-biological vectors permits the masking of the physicochemical properties of the drug and, logically, the characteristics of the carrier are what permit control over the distribution stage.", "In the best of cases, the vector-drug pair should be inactive and remain stable in the different biological media between the site of administration and the place of action.", "The most important potentialities that these vectors or non-biological carriers provide are the following (Couvreur & Puisieux.", "Adv.", "Drug Del.", "Rev., 10 (1993) 141-162): protection against the chemical, enzymatic or immunological inactivation of the drug between the administration site and the place of action; improvement in the transport of the biologically active molecule to places of difficult access and its penetration into the cell (in the case of infections located in the intracellular territories which are inaccessible by simple diffusion); increase in the specificity of action by a selective, effective, and regular concentration of drug in the cellular and/or molecular target.", "In this way, with smaller doses, the obtained therapeutic activity is, at least, identical and the side effects are milder; decrease in the toxicity for certain organs by means of modifying the tissular distribution of the biologically active molecule that is carried; in some cases, a lengthening of the time of residence of the drug in the organism (interesting for those molecules with high clearance and a low biological half-life) and, control of its release.", "All of this implies a decrease of the frequency of drug intake and indirectly, an increase in the observance of the treatment on the part of the patient.", "Within the group of non-biological vectors, colloidal systems of solid particle type with a size smaller than a micrometer stand out; they are also called nanoparticles.", "These are subdivided into matrix nanospheres and vesicular nanocapsules (Orecchioni e Irache, Formes pharmaceutiques pour application locale.", "Lavoisier Tech & Doc., Paris, 1996, 441-457).", "The nanocapsules are vesicular systems formed by an internal cavity which is surrounded by a membrane or polymeric wall.", "The nanospheres are matrix forms, formed by a tridimensional polymeric net.", "In both cases, the molecules of the biologically active substance can be dissolved, entrapped or adhered to the macromolecular structure (in the nanospheres) or encapsulated by the polymeric membrane (in the nanocapsules).", "Also, the biologically active substance may remain adsorbed on the surface of the nanoparticles.", "Depending on the used material, the nanospheres can be prepared from: reactions of monomer polymerization; macromolecules of natural origin; preformed synthetic polymers.", "The nanoparticles produced from preformed polymers are generally obtained by two different procedures: (i) application of techniques based on the emulsion of an organic solution of the polymer (Vanderhoff et al, U.S. Pat.", "No.", "4,177,177 (1979)) and (ii) by processes derived from the desolvation of the polymer (Fessi et al, FRENCH PAT, (1986) 2 608 988).", "On the contrary, nanoparticles of natural macromolecules are generally obtained by means of (i) gelification techniques (Rajaonari-vony et al, J. Pharm.Sci, 82 (1993) 912-917), (ii) emulsion (Vanderhoff et al, U.S. Pat.", "No.", "4,177,177 (1979) and (iii) controlled desolvation (Marty et al, Pharm Acta Helv., 53 (1978) 79-82).", "Generally, a supplementary stage is needed to estabilize the aforesaid systems.", "In the organism, the distribution of the nanoparticles generally depends on their physicochemical characteristics (mainly their size and their surface properties).", "By intravenous route, the submicronic vectors (for example, the nanoparticles) are captured by the endothelial reticular system (ERS), also called mononuclear phagocyte system, principally located in the liver, spleen, lungs and marrow.", "This property that nanoparticles possess converts them into very interesting pharmaceutical forms for the administration of drugs destined to the treatment of diseases located in the ERS or in the treatment of diseases in which the cells of ERS possess a function (infections, cancerous processes, etc.).", "On the other hand, administration of nanoparticles through the mucous membranes (oral, nasal, ocular, etc.", "routes) enables the development of adhesive interactions which increase the time of residence of the pharmaceutical form in contact with the site of absorption or action of the drug (Ponchel & Irache, Adv.", "Drug Del.", "Rev., 34 (1998) 191-219).", "For the design of pharmaceutical forms (based on nanoparticles) capable of vectorizing cells or tissues that are different from those which the nanoparticles normally distribute, active vectors (also called third generation vectors or nanoparticle-ligand conjugates can be used) are used.", "These systems are obtained by means of the binding of molecules capable of interacting specifically with cellular or molecular receptors to the surface of the nanoparticles.", "Among these molecules or ligands, polyethylenglycoles (“stealth” vectors; Allen et al., Cancer Res., 52 (1992) 2431-2439), certain monoclonal antibodies (Weinstein et al, Pharm.", "Therap., 24 (1984) 207-233), lectins (Irache et al, Biomaterials, 15 (1994), carbohydrates (Maruyama et al, Biomaterials, 15 (1994) 1035-1042), amines (Roser & Kissel, Eur.", "J. Pharm.", "Biopharm., 39 (1993) 8-12), vitamins (Russell-Jones & Westwood, U.S. Pat.", "No.", "07/956.003; (1992) WO 92/17167), etc.", "can be used.", "Nowadays, the preparation of nanoparticle pharmaceutical forms containing hydrophilic drugs is limited by several aspects of their formulation.", "In general, hydrophilic substances or drugs do not show any affinity for matrix lipophylic systems (for example, nanospheres).", "Therefore, production yields are very low and, logically, can not be applied to drug administration.", "Analogs of puric and pirimidinic bases such as oligonucleotides, 5-fluouridine or ganciclovir are included in this category of hydrophilic drugs.", "Poly(methyl vinyl ether-co-maleic anhydride)synthetic copolymers have a wide range of applications in the agricultural field (commercial name: Agrimer® VEMA, ISP) as adyuvants and estabilizers in veterinary solutions, in seed, granules and tablet coatings, etc.", "These copolymers are also used in chemical and pharmaceutical fields (commercial name: Gantrez® Copolymers, ISP), mainly due to their capacity to form coating films and their high bioadhesive capacity (Esposito er al., Biomaterials, 15 (1994) 177-182).", "They have also been described as thickeners, complexing agents and hydrophilic colloids.", "Likewise, their use in sustained release systems such as transdermic patches or ocular matrixes has been proposed (Finne et al., Int.", "J.", "Pharm., 78 (1992) 237-241).", "Their main advantages are their relatively low toxicity (LD50=8-9 g/Kg by oral route) and their easy production, regarding both quantity and price.", "Poly(methyl vinyl ether-co-maleic anhydride)copolymers have also been used to prepare microparticle vectorial systems by a process of complex coacervation (Mortada et al., J.", "Microencas., 4 (1987) 11-21).", "Poly(methyl vinyl ether-co-maleic anhydride)copolymer nanospheres are new matrix systems of vectors of submicronic size.", "These are able to retain the biologically active substance thanks to (i) solution or entrapping inside the macromolecular structure or matrix, (ii) covalent bonds result of the interaction of the drug and the polymer anhydride groups and (iii) processes of adsorption by means of weak bonds.", "The extremely high reactivity of this copolymer due to its cyclic anhydride groups makes it perfect to retain hydrophilic substances in its structure.", "They are also very interesting systems for the fixation of ligands to the nanoparticle surface, without having to use highly toxic organic reactants (glutaraldehyde and carbodiimides) .", "A variety of current drugs (for example, 5-fluouridine, ganciclovir) and biotechnological products (for example, antisense oligonucleotides) can be mentioned as hydrophilic active ingredients.", "All of them are analogs of puric and pirimidinic bases.", "Other kind of molecules such as proteins, peptides, lectins and, in general, all those substances which have alcohol groups and/or primary amines (for example: lectins) can also be fixed.", "The majority of the vectorial systems that have been described in the literature and are used to administrate hydrophilic substances require laborious manufacturing processes.", "Thus, for example, quite complex bonds with toxic reactions (Rimoli et al., J.", "Controlled Rel., 58 (1999) 61-68), multiple emulsions (Jeffery et al., Pharm.", "Res., 10 (1993) 362-367) or working with very high drug concentrations in conditions not applicable to the industrial field have been proposed.", "The conditions of applicability that allow to ascertain of the industrial interest of poly(methyl vinyl ether-co-maleic anhydride)nanoparticle vectors, could be the following: 1.The obtaining of homogeneous populations with submicronic particle size.", "This favors the distribution, improves the transport of active substances to its place of action and increases the penetration in the target cells.", "2.The use in the chemical-pharmaceutical field of abundant preformed copolymers with a low expense, for the administration of drugs.", "3.The obtaining of particles in which part of the drug will be entrapped and other part will be bound to the matrix by a covalent bond.", "This favors the bi-phasic release of the drug.", "The entrapped fraction enables a very quick initial release, while the bound fraction is released in a controlled way over a long period of time.", "This period will depend on particle degradability and on the breakage of the bounds between the polymer and the biologically active molecule.", "4.The obtaining of particles in which the drug is bound by a covalent bond to the surface.", "Like in the previous case, the drug will be released in a controlled way and depending on the velocity of the breakage of the bonds between the groups located on the nanoparticle surface and the biologically active molecule.", "5.The use of the aforementioned nanoparticles, as a base to produce third generation vectors or nanoparticle-ligand conjugates capable of carrying the active principle to certain cells or tissues by a specific recognition system.", "6.Working conditions at room temperature that avoid the possible degradation of thermolabile active substances of hydrophilic nature, and of chemical and/or biotechnological origin.", "7.The obtaining of particles capable of protection against premature inactivation of the encapsulated active principles.", "DESCRIPTION OF THE INVENTION Poly(methyl vinyl ether-co-maleic anhydride)nanoparticles are obtained by a desolvation method consisting in the addition of a polar solvent (miscible with the copolymer solution) to an organic solution of the copolymer.", "Subsequently, a second non-solvent liquid is added, in this case an hydroalcoholic solution.", "In order to do this, the copolymer is dissolved in a predetermined volume of acetone and then an ethanol solution is added.", "Then a similar volume of water is added to this solution.", "The particles are instantly formed in the medium, which takes on the appearance of a milky suspension.", "The organic solvents (ethanol and acetone) are then eliminated by evaporation under reduced pressure.", "The particles remain in a stable aqueous suspension.", "The poly(methyl vinyl ether-co-maleic anhydride)copolymer is insoluble in water, but after some time it hydrolyzes to an acid form which is soluble in water.", "Although this hydration process starts on contacting with water, it lasts approximately 10 hours.", "This is the reason why a supplementary stage is required.", "This stage consists in the stabilization of the particles by the cross-linkage of the particles with the purpose of increasing their stability in aqueous media.", "This also allows the modulation and control of the release of the encapsulated drug.", "As cross-linking agents, polyfunctional chemical compounds, including a number of polyamines and polyhydroxyl molecules, can be used.", "The main cross-linking agents used are human serum albumin (HSA) and 1,3-diaminopropane (DP).", "Other macromolecules, such as bovine serum albumin (BSA), povidone and gelatin, have also shown to be good stabilizing agents.", "The next manufacturing step consists in purifying the resultant nanoparticles in order to eliminate all the remains of the process, mainly polymer and cross-linker residues.", "To do this, different techniques such as ultracentrifugation or tangential filtration may be used.", "Finally, the purified nanoparticles may be lyophilized or freeze-dried for their long term storage and preservation.", "Depending on the aqueous solubility of the drug, two different procedures may be used to associate a drug to the nanoparticles.", "The first one consists in binding the drug (hydrophilic or with a low aqueous solubility) to the polymer in an organic solution.", "In order to do this, poly(methyl vinyl ether-co-maleic anhydride) and the drug are dissolved in a given volume of acetone.", "This solution is incubated and stirred at a predetermined temperature and for a fixed time.", "Then a given volume of ethanol is added, followed by the addition of a similar volume of water.", "The particles are immediately formed in the medium, and this suspension takes on a milky appearance.", "The organic solvents (ethanol and acetone) are removed by means of evaporation under reduced pressure and the particles remain in a stable aqueous suspension.", "The particles are cross-linked with one of the aforementioned products (mainly HSA and DP) and purified by ultracentrifugation or tangential filtration.", "Finally, the particles can be lyophilized.", "The second one is a process for the loading of molecules with a high polarity, in which the drug is bound to the surface of freshly prepared nanoparticles.", "In this case, the poly(methyl vinyl ether-co-maleic anhydride)copolymer is dissolved in a given volume of acetone.", "Then, a given amount of ethanol is added followed by the addition of a similar amount of water.", "The particles are immediately formed in the medium, and this suspension takes on a milky appearance.", "The organic solvents (ethanol and acetone) are removed by means of evaporation under reduced pressure, and the particles remain in a stable, aqueous medium.", "The freshly prepared nanoparticles are then incubated at a fixed temperature for a predetermined time with an aqueous solution of the drug and the solution is incubated at a predetermined temperature and for a fixed time.", "Afterwards, particles are stabilized by cross-linkage using one of the aforementioned agents (mainly HSA and DP) and purified by ultracentrifugation or tangential filtration.", "Finally, nanoparticles can be lyophilized.", "Finally, to prepare ligand-nanoparticle conjugates, the manufacturing process is quite simple and is based on the incubation between the nanoparticles and the ligand to be bound to the surface of the carriers.", "Then, a supplementary purifying step is needed to eliminate the unbound ligand remains.", "As in the previous cases, the obtained conjugates can be lyophilized.", "The following examples illustrate the present invention, but in no way do they pretend to limit the possible uses of the invention.", "In these examples, three different pharmaceuticals, either derivatives from puric or pyrimidinic bases, have been used.", "These drugs are the anti-cancer agent 5-fluorouridine, the antiviral ganciclovir and the antisense oligonucleotide ISIS 2922.5-fluorouracile (2,4-dioxo-5-fluropyrimidine) and, its derivative, 5-fluorouridine (2,4-dioxo-5-fluoropyrimidine riboside) are analogs of the pyrimidines, and their molecules contain a fluoride atom.", "These drugs are usually used in the treatment of colon cancer, either alone or in combination with other drugs.", "They are also used in the treatment of breast, cervix, endometrium, gastrointestinal, head and neck, liver, ovary, pancreas, prostrate and skin tumors.", "The 5-fluorouridine has an antitumoral activity which is 100-times more potent than 5-fluorouracile and other derivatives of this drug (Jampel et al.", "Arch.", "Ophtal., 108 (1990) 430-435).", "However, its clinical use is limited due to the severe side effects (leukopenia, thrombocytopenia and gastrointestinal toxicity) when administered in a traditionally pharmaceutical form (Brusa et al., II Farmaco, 52 (1997) 71-81).", "Administration of this drug in the form of nanoparticles would allow an increase of the therapeutic index of this drug.", "Ganciclovir, (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine, is a synthetic nucleoside analogue.", "This molecule exhibits antiviral activity against some herpes viruses (Martin et al., J. Med.", "Chem., 26 (1983) 759-761).", "Furthermore, it is the most frequently used antiviral drug in the treatment of infections caused by human cytomegalovirus (CMV) in immunocompromised patients, mainly in those with the acquired immunodeficiency syndrome (AIDS), congenital immunodeficiency or in individuals who have undergone organ transplantation.", "The principal clinical manifestation is retinitis, which if left untreated, results in irreversible blindness (Markham & Faulde, Drugs, 48 (1994) 455-460).", "The use of nanoparticles would permit the obtaining of pharmaceutical dosage forms showing sustained release properties and controlled drug release.", "Besides, and given its small size, they would minimally affect vision when administered by intravitreous route.", "In addition, during the last few years, ganciclovir has been used as an adjuvant for gene therapy involving suicide genes.", "This new therapeutical technique consists of administrating retrovirus, adenovirus or herpes simplex incorporating the thymidine quinase (tk) gene, followed by the adminisration of ganciclovir to the patient.", "The viral vectors permit the incorporation of tk into the dividing cells and, after expression, the ganciclovir is metabolized by the expressed enzyme in its phosphate derivative.", "This derivative is then transformed into ganciclovir-diphosphate and finally in ganciclovir-triphosphate, which is toxic for the cell and induces its apoptosis.", "This is being successfully used for the treatment of pancreas tumors (Carrio et al., Gene Ther., 6 (1999) 547-553), prostate adenocarcinomas (Herman et al., Hum.", "Gene Ther., 10 (1999) 1239-1249) and hepatocarcinomas (Qian et al., Hum.", "Gene.", "Ther., 10 (1997) 349-358).", "Antisense oligonucleotides are new therapeutic agents which are able to regulate the genetic expression of living organisms.", "This technology consists in the use of synthetic fragments of DNA or RNA, called oligonucleotides, which allow the stoppage of the production of illnesses related to the synthesized proteins.", "Antisense compounds block the transmission of genetic information between the nucleus and the place where the protein is produced inside the cell.", "There is a variety of applications that include the treatment of certain types of cancer, inflammatory pathologies and viral illnesses.", "ISIS 2922 is an antisense oligonucleotide with a sequence complementary to messenger RNA in the human citomegalovirus region IE1 (Anderson et al., Antimicrob.", "Agents Chemother., 40 (1996), 2004-2011).", "This oligonucleotide blocks the expression of a protein essential for viral replication, thereby inhibiting the replication of the virus (Leeds et al., Drug Metabol.", "Dispos., 25 (1997) 921-926) with an in vitro efficiency of about 30-times greater than traditional therapies.", "However, physico-chemical oligonucleotide characteristics (high mass, charged, hydrophilic molecules) do not allow its free access into the cell where they have to act.", "The use of nanoparticles permits an increase of the penetration capacity of those substances and a protection against exo- and endonucleases.", "The size and zeta potential of the nanoparticles were determined in a Zetamaster apparatus (Malvern Instruments/Optilas, Spain).", "The drugs, 5-fluoroudine (provided by Sigma; Alcobendas, Spain) and ganciclovir (Cymevene®, Roche; Madrid, Spain), were analyzed by High Performance Liquid Chromatography (HPLC), using a Hewlett Packard series 1050 (Germany).", "The antisense oligonucleotide, ISIS 2922, was obtained from Pharmacia Biotech (Cambridge, United Kingdom) and analyzed by zone capillary electrophoresis.", "The Sambucus nigra lectin was obtained from Vector Laboratories (USA) and determined by gel permeability chromatography.", "The samples of nanoparticles were lyophilized using a Virtis Genesis 12EL apparatus (USA).", "EXAMPLE 1 Preparation of Nanoparticles from poly(methyl vinyl ether-co-maleic anhydride)copolumer The process described below is valid for the preparation of colloidal pharmaceutical forms, nanoparticle type, which can be used for the administration of drugs or biologically active molecules.", "In addition, these nanoparticles can also be used for the film coating of macroscopic pharmaceutical forms, such as tablets, granules, granulates, and minigranules.", "1.1.Cross-Linkage with 1,3-diaminopropane (DP) 100 mg of poly(methyl vinyl ether-co-maleic anhydride)copolymer are dissolved in 5 mL organic solvent (acetone).", "Then, under stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added.", "The resulting mixture is stirred for homogenization during 5 minutes and the suspension of nanoparticles is evaporated under reduced pressure for organic solvents elimination.", "The final volume is adjusted with water (or an aqueous solution) to 10 mL.", "The next step is to cross-link the resulting nanoparticles with 1,3-diaminopropane (DP).", "In order to do this, a DP solution is prepared in water (1% v/v), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.", "The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.", "The supernatants are eliminated and the residues are resuspended in water or in an aqueous solution of mannitol (5% w/v).", "Finally, the resulting suspension of nanoparticles is lyophilized maintaining all its properties.", "The resulting formulation of the nanoparticles suspension before liophilization is: Poly(methyl vinyl ether-co-maleic anhydride) 1.0% w/v Mannitol 5.0% w/v 1,3-diaminopropane c.s.", "Purified water c.s.p.", "10 mL The mean size of the obtained nanoparticles is lower than 300 nm and the carriers display a negative superficial charge.", "Table 1 shows the influence of the amount of DP (used to cross-link the nanoparticles) on the size and zeta potential of the resulting nanoparticles.", "From these results, it is clear that an increase in the amount of cross-linking agent used to harden the nanoparticles increases the particle size and decreases negative zeta potential of these colloidal carriers.", "This last fact proves the reaction between the anhydride groups from the surface of the particles and the cross-linking agent.", "TABLE 1 Effect of the quantity of the cross-linking agent (DP) on the size and zeta potential of the resulting nanoparticles.", "DP Quantity (mL) Size (nm) Zeta potential (mV) 0 206.7 ± 1.13 −51.7 ± 2.45 2 239.8 ± 5.16 −37.8 ± 1.13 3 270.9 ± 4.13 −27.7 ± 2.26 5 270.1 ± 10.53 −23.7 ± 0.85 10 275.0 ± 11.33 −23.7 ± 0.78 The yield of the nanoparticles manufacturing process was calculated as the quotient between the weight of the freeze-dried samples and the initial amount of polymer used to prepare the nanoparticles, either with mannitol or without cryoprotector.", "From a previously known amount of the polymer (100 mg) the amount that had been transformed in nanoparticles at the end of the process was determined by gravirnetry.", "In any case, the yield of the manufacturing process (expressed in % with respect to the initial mass of polymer) was high; although higher efficiencies were obtained when the manufacturing process was carried out at room temperature rather than at 40° C. (see FIG.", "1).", "1.2.Cross-Linkage with Human Serum Albumin (HSA) 100 mg of poly(methyl vinyl ether-co-maleic anhydride)copolymer are dissolved in 5 mL of an organic solvent (acetone).", "Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added.", "The resulting mixture is stirred for 5 minutes, and the suspension of nanoparticles is evaporated under reduced pressure for organic solvent elimination.", "The final volume is adjusted with water (or an aqueous solution) to 10 mL.", "The next step is to harden the resulting nanoparticles with human serum albumin (HSA).", "In order to do this, a HSA solution is prepared in water (30 mg/mL), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 120 minutes.", "The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.", "The supernatants are eliminated and the residues resuspended in water or in an aqueous solution of mannitol (5% w/v).", "Finally, the resulting suspension of nanoparticles is freeze-dried maintaining all its properties.", "The resulting formulation of the nanoparticles solution before liophilization is: Poly(methyl vinyl ether-co-maleic anhydride) 1.0% w/v Mannitol 5.0% w/v Human serum albumin c.s.", "Purified water c.s.p.", "10 mL The mean size of the obtained nanoparticles is lower than 300 nm and the carriers display a negative superficial charge.", "Table 2 shows the influence of the amount of HSA (used to cross-link the nanoparticles) on the size and zeta potential of the resulting nanoparticles.", "It was observed that an increase in the amount of cross-linking agent used to harden the nanoparticles slightly increases the size of the resulting nanoparticles and decreases their negative zeta potential.", "These results prove that the anhydride groups on the surface of the particles react with the cross-linking agent.", "TABLE 2 Effect of the quantity of the cross-linking agent HSA on the size and zeta potential of the resulting nanoparticles.", "Amount HSA Zeta (mg/mg polymer) Size (nm) potential (mV) 0 206.7 ± 1.13 −51.7 ± 2.45 10 239.8 ± 5.16 −42.7 ± 1.13 15 240.9 ± 15.05 −42.7 ± 1.00 The yield of the nanoparticles manufacturing process was calculated as the quotient between the weight of the freeze-dried samples and the initial amount of polymer used to prepare the nanoparticles, either with mannitol or without cryoprotector.", "The amount of poly(methyl vinyl ether-co-maleic anhydride)copolymer that had been transformed in nanoparticles at the end of the process was determined by gravimetry from a previously known amount of the polymer (100 mg).", "In any case, the yield of the manufacturing process (expressed in % with respect to the initial mass of polymer) was high; although higher efficiencies were obtained when the manufacturing process was carried out at room temperature rather than at 40° C. (see FIG.", "2).", "EXAMPLE 2 Manufacturing Process of Nanoparticle Conjugates Based on the Binding of Lectin to the Surface of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles The process described below permits the preparation of third generation pharmaceutical colloidal forms for the site specific delivery or targeting of drugs.", "These forms are formed by the binding of a ligand to the surface of copolymer nanoparticles.", "The ligand offers the possibility of driving the pharmaceutical dosage form to a specific site in the organism.", "In fact, the ligand should specifically recognize certain receptors located on the surface or inside a certain type of cells.", "These forms are known as conjugates.", "In the next example, the model ligand used was the lectin of Sambucus nigra agglutinin (SNA).", "First of all, 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).", "Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added.", "The resulting mixture is stirred for 5 minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvent elimination.", "The final volume is adjusted with water (or an aqueous solution) to 10 mL.", "The next step is to harden the resulting nanoparticles with 1,3-diaminopropane (DP).", "In order to do this, a solution of DP is prepared in water (1% v/v), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.", "The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.", "The supematants are eliminated and the residues resuspended in water.", "Then the purified nanoparticles are incubated with the lectin.", "In order to do this, a variable amount of SNA is added to the nanoparticle suspension and this mixture is magnetically stirred at room temperature for 1 hour.", "The conjugates are centrifuged twice at 10,000 rpm for 12 minutes, in order to remove the unbound lectin (5% w/v).", "Finally, the resulting suspension of conjugates is freeze-dried after the addition of sacarose or mannitol as cryoprotectors.", "FIG.", "3 shows the influence of the initial amount of cross-linking agent on the lectin bound to the poly(methyl vinyl ether-co-maleic anhydride)nanoparticles.", "The lectin was determined by HPLC (High performance Liquid Chromatography).", "It was evident that as the amount of cross-linking agent increases, the amount of lectin being loaded by the nanoparticles decreases.", "Likewise, the amount of lectin bound to the nanoparticles is higher when the initial amount of lectin increases.", "The quantity of active lectin bound to the nanoparticles was determined by means of an erythrocyte agglutination test.", "It was observed that by increasing the initial quantity of lectin, a greater amount of lectin bound to the nanoparticles remained active.", "When analyzing the influence of the amount of cross-linking agent, it was observed that there are certain ideal conditions that allow the optimal binding of lectins to the nanoparticle surfaces, around 30 μg/mg nanoparticle (FIG.", "4).", "Similar results were obtained when using human serum albumin (HSA) or bovine serum albumin (BSA) as cross-linking agents.", "EXAMPLE 3 Manufacturing Process for the Preparation of 5-fluorouridine-loading poly(methyl vinyl ether-co-maleic anhydride)nanoparticles The process described below permits the preparation of colloidal pharmaceutical forms (i.e., nanoparticles and ligand-nanoparticle conjugates) carrying 5-fluorouridine or any other active molecule with a chemical structure similar to or derived from said drug.", "3.1.Cross-Linkage with 1,3-diaminopropane (DP) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 2 mL organic solvent (acetone).", "A predetermined amount of 5-fluorouridine is dissolved in 3 mL acetone, until a theoretical maximum of 10 mg/mL is reached.", "The two aforementioned solutions are mixed and then incubated at room temperature for 2 hours.", "Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution.", "The resulting mixture is stirred for 5 more minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvents elimination.", "The final volume is adjusted with water (or an aqueous solution) to 10 mL.", "The next step is to harden the resulting nanoparticles with 1,3-diaminopropane (DP).", "In order to do this, a solution of DP is prepared in water (1% v/v), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.", "The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.", "Optionally, the resulting nanoparticles (loaded with 5-fluorouridine) are incubated with the Sambucus nigra lectin in order to obtain the nanoparticle-lectin conjugates, as described in example 2.The supernatants are analyzed by HPLC.", "The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.", "The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).", "Finally, the resulting suspension of nanoparticles is freeze-dried.", "FIG.", "5 shows the influence of the incubation time between 5-fluorouridine and the poly(methyl vinyl ether-co-maleic anhydride) on the drug loading.", "In the example of the SNA-nanoparticle conjugates containing 5-fluorouridine (experimental conditions: 200 μg initial lectin), the amount of encapsulated fluorouridine does not change and the amount of lectin bound to the surface of nanoparticles was calculated to be around 40 μg/mg polymer.", "3.2.Cross-Linkage with Human Serum Albumin (HSA) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 2 mL organic solvent (acetone).", "A predetermined amount of 5-fluorouridine is dissolved in 3 mL acetone, until a theoretical maximum of 10 mg/mL is reached.", "The two aforementioned solutions are mixed and then incubated at room temperature for 2 hours.", "Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution.", "The resulting mixture is stirred for 5 more minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvent elimination.", "The final volume is adjusted with water (or an aqueous solution) to 10 mL.", "The next step is to harden the resulting nanoparticles with human serum albumin (HSA).", "In order to do this, a solution of HSA is prepared in water (30 mg/mL), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 120 minutes.", "The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.", "The supernatants are analyzed by HPLC.", "The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.", "The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).", "Finally, the resulting suspension of nanoparticles is freeze-dried maintaining all its properties.", "FIG.", "6 shows the influence of the incubation time between 5-fluorouridine and the copolymer on the drug loading of nanoparticles when the carriers where cross-linked with HSA.", "EXAMPLE 4 Manufacturing Process for the Preparation of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles Coated with 5-fluorouridine The process described below permits the preparation of colloidal pharmaceuticai forms (i.e., nanoparticles and ligand-nanoparticle conjugates) containing 5-fluorouridine or any other active molecule with a chemical structure similar to or derived from said drug.", "4.1.Cross-Linkage with 1,3-diaminopropane (DP) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).", "A predetermined amount of 5-fluorouridine is dissolved in 2 mL water.", "Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution of the copolymer.", "The resulting mixture is stirred for 5 more minutes, and the suspension of nanoparticles is evaporated under reduced pressure for organic solvents elimination.", "The final volume is adjusted with water (or an aqueous solution) to 10 mL, and the suspension is then incubated with the 5-fluorouridine aqueous solution.", "The next step is to harden the resulting nanoparticles with 1,3-diaminopropane (DP).", "In order to do this, a solution of DP in water (1% DP v/v water) is prepared and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.", "The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.", "The supernatants are analyzed by HPLC.", "The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.", "The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).", "Finally, the resulting suspension of nanoparticles is freeze-dried maintaining all its properties.", "FIG.", "7 shows the influence of the initial amount of 5-fluorouridine (FU) that is adsorbed or interacts with the anhydride groups on the nanoparticle (NP) surface.", "It is clear that when the initial amount of the drug increases, the amount of FU carried by the nanoparticles also increases.", "Likewise, the higher the initial amount of FU loaded in the nanoparticles, the higher the amount that is released quickly.", "In the case of the administration of these colloidal pharmaceutical forms, the drug fraction that is released immediately would act as burst dose.", "4.2.Cross-Linkage with Human Serum Albumin (HSA) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).", "A predetermined amount of 5-fluorouridine is dissolved in 2 mL water.", "Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution of the copolymer.", "The resulting mixture is stirred during 5 minutes more and the suspension of nanoparticles is concentrated under reduced pressure for organic solvents elimination.", "The final volume is adjusted with water (or an aqueous solution) to 10 mL and the suspension is, then, incubated with the 5-fluorouridine aqueous solution.", "The next step is to harden the resulting nanoparticles with human serum albumin (HSA).", "In order to do this, a solution of HSA is prepared in water (30 mg/mL), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 120 minutes.", "The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.", "The supernatants are analyzed by HPLC.", "The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.", "The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).", "Finally, the resulting suspension of nanoparticles is freeze-dried maintaining all its properties.", "FIG.", "8 shows the influence of the initial concentration of 5-fluorouridine that is adsorbed and interacts with the reactive groups on the nanoparticle surface.", "It is clear that when the initial amount of drug increases, the amount of FU carried by nanoparticles cross-linked with HSA also increases.", "As expected, the immediately released fraction increased by increasing the amount of drug incubated with the freshly prepared nanoparticles.", "This burst effect would be acting as an initial dose.", "4.3.In Vitro Release Studies For these studies, the nanoparticles were prepared following the conditions described in 4.1.The in vitro release assay was carried out in a temperature-controlled bath, with constant stirring and a constant temperature of 37±1° C. FIG.", "9 shows the results obtained for two different batches of nanoparticles prepared from 1 and 2 mg FU per mL suspension.", "It can be observed that a fraction of the drug (approximately 30-50% of the drug) is adsorbed, in a labile way, onto the surface of the nanoparticles and is immediately released.", "In addition, there is another fraction, which is strongly bound to the nanoparticles.", "This second fraction was not released during the time in which the release study was performed.", "However, it can be expected that in vivo conditions, the bond between the drug and the copolymer would break given its lability.", "This would permit the here described nanoparticles to act as a biphasic releasing drug.", "The great potential of this system is based on this property, together with their capacity to target certain organs and inflamed regions of the organism.", "EXAMPLE 5 Manufacturing Process for the Preparation poly(methyl vinyl ether-co-maleic anhydride)nanoparticles Coated with Ganciclovir The process described below permits the preparation of colloidal pharmaceutical forms (i.e., nanoparticles and ligand-nanoparticle conjugates) containing ganciclovir or any other active molecule with a chemical structure similar to or derived from said drug.", "5.1.Cross-Linkage with Human Serum Albumin (HSA) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).", "A predetermined amount of ganciclovir is dissolved in water.", "Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution of the copolymer.", "The resulting mixture is stirred during 5 more minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvents elimination.", "The final volume is adjusted with water (or an aqueous solution) to 10 mL, and the suspension is then incubated with the ganciclovir aqueous solution.", "The next step is to harden the resulting nanoparticles with human serum albumin (HSA).", "In order to do this, a solution of HSA is prepared in water (30 mg HSA/mL water) and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 120 minutes.", "The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.", "The supernatants are analyzed by HPLC.", "The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.", "The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).", "Finally, the resulting suspension of nanoparticles is freeze-dried maintaining its properties.", "This manufacturing process gives similar results when nanoparticles are cross-linked with 1,3-diaminopropane (DP) in the conditions indicated in the previous examples.", "FIG.", "10 shows the influence of the bulk concentration of ganciclovir incubated with the nanoparticles in the percentage of drug bound to them.", "It can be observed that an initial ganciclovir amount greater, than 4-5 mg, does not have much effect on the percentage of entrapped drug.", "5.2.In Vitro Release Studies These in vitro release studies were carried out with nanoparticulate batches prepared as described in 5.1.In order to do this, 100 μL of different formulation (with similar drug entrapped efficiency but increasing amounts of drug loaded) were dispersed in 1 mL phosphate buffer solution containing tripsin.", "The in vitro release assay was carried out in a temperature-controlled bath, with constant stirring and a constant temperature of 37±10° C. taking samples at different predetermined times.", "A biphasic release profile was observed for all of the formulations tested.", "This profile was characterized by a first phase of rapid (almost immediate) release (also know as a “burst effect”), followed by a second slow and continuous release.", "During the first release stage (in the first eight hours), the percentages of drug released ranged between 40 and 50% of the loaded drug.", "The second part of the curve was adjusted to a zero-order kinetic (see FIG.", "11) that continued for more than 7 days.", "FIG.", "11 shows the ganciclovir release kinetic from the poly(methyl vinyl ether-co-maleic anhydride)nanoparticles, for a formulation containing 90 μg ganciclovir bound to the nanoparticles per niL suspension.", "EXAMPLE 6 Manufacturing Process for the Preparation poly(methyl vinyl ether-co-maleic anhydride)nanoparticles Coated with the antisense oligonucleotide ISIS 2922 The process described below permits the preparation of colloidal pharmaceutical forms (i.e., nanoparticles and ligand-nanoparticle conjugates) containing antisense oligonucleotides or any other active molecule of DNA or RNA.", "The example described below was carried out with the oligonucleotide ISIS 2922.ISIS 2922 is an oligonucleotide of 21 bases which possesses antiviral activity against cytomegalovirus.", "6.1.Cross-Linkage with 1,3-diaminopropane (DP) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).", "A predetermined amount of ISIS 2922 is dissolved in water.", "Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution of the copolymer.", "The resulting mixture is stirred during 5 more minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvents elimination.", "The final volume is adjusted with water (or an aqueous solution) to 10 mL, and the suspension is then incubated with the ISIS 2922 aqueous solution for 20 minutes.", "The next step is to harden the resulting nanoparticles with 1,3-diaminopropane (DP).", "For this purpose, a solution of DP is prepared in water (1% DP v/v water), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.", "The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.", "The supernatants are analyzed by capilar electrophoresis.", "The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.", "The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).", "Finally, the resulting suspension of nanoparticles is freeze-dried maintaining its properties.", "FIG.", "12 shows the influence of the initial amount of ISIS 2922 incubated with the nanoparticles on the amount of oligonucleotide loaded.", "This procedure gives similar results when cross-linkage is carried out with (DP), in the conditions described in the previous examples.", "DESCRIPTION OF THE FIGURES FIG.", "1.Influence of the incubation time (abscissas) and temperature on the yield of the manufacturing process (ordinates) of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with DP.", "FIG.", "2.Influence of the incubation time (abscissas) and temperature on the yield of the manufacturing process (ordinates) of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with HSA.", "FIG.", "3.Influence of the initial amount of the cross-linking agent (1,3-diaminopropane in μLsol/mg polymer; abscissas) on the amount of lectin bound (μg/mg polymer; ordinates) to the poly(methyl vinyl ether-co-maleic anhydride)nanoparticles.", "FIG.", "4.Influence of the cross-linking agent (1,3-diaminopropane in μL sol 1% v/v/mg polymer; abscissas) on the amount of active Sambucus nigra lectin (μg/mg polymer; ordinates) bound to the poly(methyl vinyl ether-co-maleic anhydride)nanoparticles.", "FIG.", "5.Influence of the incubation time (abscissas) on the anti-cancer 5-fluorouridine loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with DP (drug loading in μg FU/mg nanoparticles; ordinates).", "Experimental conditions: initially 10 mg drug.", "FIG.", "6.Influence of the incubation time (abscissas) on the 5-fluorouridine loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with HSA (Drug loading in μg FU/mg nanoparticles; ordinates).", "Experimental conditions: initially 10 mg drug.", "FIG.", "7.Influence of the initial amount of 5-fluorouridine (FU) (μ/mg polymer; abscissas) on the drug loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with 1,3-diaminopropane (μg FU/mg NP; ordinates).", "The squares represent the total drug loading and the circles represent the drug immediately released by dilution.", "Experimental conditions: Initially 10 mg drug.", "FIG.", "8.Influence of the initial amount of 5-fluorouridine (μg FU/mg polymer; abscissas) on the drug loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with HSA (μg FU/mg NP; ordinates).", "The squares represent the total drug loading and the circles represent the drug immediately released by dilution.", "Experimental conditions: Initially 10 mg of drug.", "FIG.", "9.Study of the release (abscissas) of two formulations cross-linked with DP and prepared from two different concentrations of 5-fluouridine (FU 5 ordintaes).", "FIG.", "10.Influence of the initial amount of ganciclovir (Bulk GCV in mg; abscissas) on the drug encapsulation efficiency (in %, ordinates) in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with HSA.", "FIG.", "11.In vitro release kinetic (hours in abscissas) in an aqueous solution of tripsin, for a formulation of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles with ganciclovir (GCV-μg ordinates).", "FIG.", "12.Influence of the initial amount of ISIS 2922 (μg in abscissas) on its loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles (μg ISIS/mg NP; ordinates).", "Initial amount of nanoparticles (NP)=100 mg." ] ]	Patent_10467983
[ [ "Method for the administration of a subscriber card for mobile telephony equipment of the type with auxiliary reader and embedded system for the implementation of the method", "The invention concerns a method for the administration of a subscriber SIM card 3 inserted in the main smartcard reader of a mobile telephone of the type including a second smartcard reader, using an additional administrator smartcard temporarily inserted in this second reader.", "The administrator smartcard includes at least one specific loader (Ch) applet and one or more files (fl-fn) corresponding to the applets (A1-An) which can be loaded in the SIM card under the control of the loader (Ch).", "The administrator card complies with the Sim Toolkit standard.", "The method can also be used to carry out the deletion, modification and various operations on data or applets (A1-An) present in the SIM card: display, determination of the memory space, etc.", "The invention also concerns an embedded system, more particularly a smartcard, for the implementation of the method." ], [ "1.Administration method for an embedded system for a device of type including a first embedded system reader, the main reader, designed to take the said embedded system and a second embedded system reader, the auxiliary reader, the said embedded system including means for the computer processing and storage of data, wherein it consists of loading in the said storage means of the said embedded system at least one item of digital data contained in an additional embedded system, the administration system, inserted in the said auxiliary embedded system reader, under the control of a specific software module contained in the said additional embedded system.", "2.Method according to claim 1, wherein it consists of performing the addition, deletion, modification and management/display administration operations under the control of the said specific software module.", "3.Method according to claim 1, wherein it includes at least the following steps: temporary insertion of the said administration embedded system in the said auxiliary embedded system reader; conversion of the said digital data item(s) in the first format into a series of digital data in a second format under the control of the said specific software module; and the selective transfer of the said digital data item(s) in the said second format to the said embedded system and its loading in the said storage means of this embedded system, via the said auxiliary and main embedded system readers, under the control of the said specific software module.", "4.Method according to claim 1, wherein the same administration embedded system becomes “master” on powering up although inserted in the said auxiliary embedded system reader.", "5.Method according to claim 1, wherein the said device transmits, on being put into service, a command for automatic activation of the said specific software module and in that, the said device including display means, it initialises the display of an interactive menu on the said display means, enabling a user to select one of the said items of digital data for loading in the said storage means of the said embedded system, guiding the user through the loading operations.", "6.Method according to claim 1, wherein the said specific software module and the said digital data item(s) to be loaded in the said storage means of the said embedded system are applets encoded in JAVA (registered trademark) language.", "7.Method according to claim 1, wherein the said administration of the said embedded system includes the scanning, under the control of the said specific software module, of its storage means, in order to determine predetermined attributes associated with the said digital data item(s) loaded in these storage means and the display of these attributes on the display means of the said device.", "8.Method according to claim 1, wherein it includes an additional step, before the loading, which consists of determining whether the remaining memory space is sufficient to perform the said loading, and, in case of negative result, execution of an additional step which consists of the selective and optional deletion, after the choice by a user of the said device and under the control of the said specific software module, of at least one of the said digital data items, in order to release sufficient memory space to carry out the said loading.", "9.Method according to claim 1, wherein the said device is a mobile telephony device, the said embedded system is a subscriber embedded system, the said administration embedded system complies with the so-called SIM Toolkit standard, in that it includes an operating system in compliance with this standard enabling the transmission of at least so-called proactive commands in compliance with standard ETSI 11.14, in that the communication protocol used for the loading complies with standard ISO 7816, including the so-called APDU commands, and in that the said proactive commands can be used to activate and send APDU commands, in order to set up a communication session between the said administration and subscriber embedded systems, and obtain the said selective transfer of at least one digital data item and their loading in the storage means of the said subscriber embedded system.", "10.Embedded system including means for the computer processing and storage of digital data, characterised in that it includes, in its storage means, a specific software module, including at least one component consisting of a loader program (Ch) and at least one item of digital data in order to load at least one of the said digital data items in the said storage means of a second embedded system, for a device when the said system is inserted in an auxiliary reader of the said device.", "11.Embedded system according to claim 10, wherein the said specific software module stored in the said storage means of the said administration embedded system and the said digital data item(s) to be loaded in the said storage means of the said embedded system are applets encoded in JAVA (registered trademark)." ], [ "The invention concerns a method for the administration of an embedded system with electronic chip, more especially a subscriber card for mobile telephony equipment of the type with auxiliary reader.", "It is more especially, although not exclusively, intended for mobile telephony terminals complying with the GSM standard, henceforth referred to simply as “mobile telephone”.", "One of the most frequently used standards in Europe is the GSM transmission standard (Global System for Mobile communications, public radiocommunications operating in the 900 MHz band).", "It must be clearly understood however that the invention is not limited to this standard alone.", "In particular, it may be used in the standards under development such as GPRS or UTMS.", "The invention concerns an embedded system, more particularly an electronic smartcard, for the implementation of the method.", "Henceforth, for simplification, the name “smartcard” will be used, without limiting in any way whatsoever the scope of the invention.", "Some mobile telephones are known as “dual slot” since they include a first smartcard reader, which will henceforth be called the main reader, accepting a card equipped with information processing and storage means, including a functional module known by the abbreviation SIM (Subscriber Identification Module).", "This smartcard, which will henceforth be called “subscriber” card, may be replaced by a simple electronic module acting as smartcard.", "If the smartcard has the format known as ID-1, it includes a card as such made from plastic material on which the above-mentioned module with electronic chip is positioned.", "The assembly respects a certain number of well known standards, whether regarding physical (dimensions, location of the chip, etc.", "), electrical and/or electronic aspects, which require no further mention.", "Otherwise, the module alone, in the Plug-in SIM format, can be inserted in the main reader.", "Generally, the latter simply consists of a chamber fitted with a cover.", "A back wall has contact studs used to connect the module with the electronic circuits equipping the telephone terminal.", "The smartcard inserted in the main reader, or SIM card, stores a certain number of data items concerning the telephone subscription as such the name(s) of the operator(s) that the subscription(s) have been taken out with, subscription, subscriber identification data, etc.", "), as well as so-called embedded applications.", "These are, in particular, applets providing access to services present on remote servers, via the telephone network, or just executed locally.", "The interactive video games displayed on the screen of the mobile telephone are examples of this last category of applications.", "To do this, the SIM cards implement the so-called SIM Toolkit technology which complies with standard ETSI 11.14.These cards are programmable.", "Like all programmable cards, they not only receive commands from the host terminal, i.e.", "the mobile telephone, but they also transmit commands to this terminal.", "These commands are known as “proactive”.", "As an illustration, a proactive command called “DISPLAY TEXT”, with as parameter, for example, the following text: “Welcome”, will request the mobile telephone, under the control of the applet contained in the card, to display the above-mentioned text “Welcome” on its display screen.", "Amongst the proactive commands described by the above-mentioned standard ETSI 11.14, some of them are used to activate and then send so-called APDU (Application Protocol Data Unit) commands, i.e.", "complying with standard ISO 7816, to a second smartcard inserted in a second reader of the mobile equipment, henceforth called the auxiliary reader.", "To understand the proactive commands sent by the SIM card, the GSM terminals must of course also comply with the standard ETSI 11.14.In the known state of the art, the second smartcard generally consists of a credit card, for example a standard bank card.", "The auxiliary reader generally consists of a traditional smartcard reader which can read a smartcard of this type.", "In order to do this, it has a slot into which the smartcard is inserted whilst the transaction is being carried out.", "For example, the transaction could consist of debiting the credit card or the bank card by a certain amount to reload the SIM card with telephone communication units.", "The second smartcard operates in “slave” mode, since it is under the control of the first smartcard.", "In the context of the invention, the term “embedded applications” must be taken in its broadest sense.", "It normally concerns applets or similar programs, but also includes all types of digital data.", "For example, the entries of a telephone directory stored in the SIM card or any other data file.", "Henceforth, the term “digital data” will designate either “applications” (or “programs”) or “data or simple data files” stored in the memory means of the SIM card.", "We can easily see that for various reasons, it may be useful, or even necessary, to add, delete and/or modify applications or data stored in the SIM card.", "It is known that applets need to be added in a SIM card.", "Generally, a computer (e.g.", "a traditional microcomputer) is used to load applets.", "The applet to be loaded is, for example, stored on a hard disk of the computer.", "A resident program in the computer, known as the “Loader” receives in input a file containing the applet and converts it into a set of commands that it sends to the SIM card.", "These commands can be sent in two main ways: locally, from a smartcard reader connected to the computer.", "remotely, for example by using the technology known as Over The Air (O.T.A.)", "in GSM terminology.", "These methods present several disadvantages.", "Firstly, the SIM card is inserted in the smartcard reader of the computer used for loading.", "It can be seen immediately that this methods presents the disadvantage of requiring specific hardware.", "At least, the subscriber, holder of the SIM card, must physically go to a place where this type of hardware is available.", "As regards the second method, it is worth pointing out that digital data is generally transmitted via the Short Message Service (SMS) technology (GSM-Data Service) in compliance with the two standards ETSI 03.40 and ETSI 03.48.Generally, the maximum length of useful in these messages is 160 septets or 140 octets, depending on the applications.", "Although with current technology, smartcards can only store programs requiring relatively a limited amount of memory, as is the case with the applets, they often come in the form of files occupying about 10 kB.", "Clearly, it would take about 80 messages to send a file of this length.", "It must also be pointed out that with transmissions implementing OTA technology, the security and/or reliability rates are not very high.", "In particular, especially when a set of SIM cards must be updated with data, even identical, the process has to be repeated for each mobile telephone, since the links between a remote server and a mobile telephone are “point to point”.", "Consequently, the above-mentioned method is, in practice, extremely complicated and possibly even impossible to implement.", "Since the mobile telephones concerned by the invention are dual slot type, as pointed out earlier, one possible method could have been to use the auxiliary reader to load applets in the SIM card inserted in the main reader.", "An obvious solution would in fact be to implement a loader program in the subscriber's SIM card.", "The code of the applet to be loaded could then be stored in another smartcard which could be inserted in the auxiliary reader.", "The loader program in the SIM card could then transmit one or more proactive command(s) in order to read the code of the applet to be loaded.", "Once the program has read the applet code, it can then load it using a suitable means.", "Initially, this method seems to offer advantages: 1) The loader program of the SIM card is compatible with the card itself since stored in it; 2) The auxiliary smartcard containing the applet code does not need to comply with the Sim Toolkit standard.", "However, this method presents significant disadvantages: 1) Since the loader program takes up a non-negligible amount of space in the SIM card, there is less space for the applets which have to be stored there.", "Even though the capacity of the memories used on smartcards has significantly increased over the last few years, it remains relatively limited for this type of application.", "2) This solution cannot be considered using the present technology, since: it is impossible to send loading commands.", "The commands to be sent by the application to the operating system in the SIM card are in fact specified in the standard ETSI 03.19.Loading commands cannot be sent under this standard.", "in addition, the applet cannot request the mobile telephone to send commands to the card it hosts.", "The current standards would therefore have be modified in order to consider this type of operation.", "it is known from the european patent application EP 0 869 691 a mobile phone in which the SIM card communicates with an external component.", "The SIM card behaves as a gateway between the external component and the GSM network and/or the mobile phone so that the external component controls completely the mobile phone.", "The modified functionalities are transferred through applets via the SIM card to the external component.", "it is also known from the international patent application WO 00/154530 a system allowing to program a set of selectable optional features into the memory of a cellular telephone independtly of the main program.", "The systems is constituted of a programmer and a port.", "The programmer located outside the housing of the telephone sends the set of selectable optional features to the port for storage in the memory.", "The invention aims to overcome the disadvantages of the methods and devices of the known state of the art, some of which have just been described.", "The purpose of the invention, for a mobile device of type including two card readers, is to define a method for the administration of digital data, applications and/or simple data, stored on a first smartcard, called subscriber or SIM smartcard, inserted in a first smartcard reader, called the main reader, via a second smartcard, called administration card, inserted in a second smartcard reader, called auxiliary smartcard reader.", "The method according to the invention can be used not only to add one or more applets in the SIM card, i.e.", "to load applets, but also provides true management or administration of digital data, applications and/or simple data, stored in it.", "Apart from loading digital data in the SIM card, the method according to the invention can be used to delete digital data, display it on the screen of the mobile device and modify it.", "This method can also be used to obtain information concerning various parameters and/or attributes about the said digital data and/or the SIM card as such, for example by displaying the names or statuses of the applets already stored in the SIM card, the memory space available in the SIM card, etc.", "The method according to the invention does not involve any modification of the hardware implemented (ordinary telephony equipment can be used) and remains fully compatible with current standards whether regarding the transmissions the smartcards (SIM Toolkit technology) or the communications between the smartcards and the circuits of the mobile device, which invoke APDU standards.", "To do this, according to a first characteristic, an “administrator” smartcard will be used, which implements a specific software module.", "It is this specific program module which is used to administer the SIM card.", "If the loading function is available, the software module includes at least a loader program and one or more applet codes which can be loaded selectively into the SIM card.", "Preferably, the loader program consists of an applet.", "Preferably, this applet and the other applets are programmed in JAVA (registered trademark).", "If the subscriber SIM card only stores simple digital data (i.e.", "no applications), then it does not necessarily need to comply with the SIM Toolkit technology.", "The invention also concerns a mobile telephony device to implement the method.", "Apart from the numerous possibilities offered by the method and its high flexibility, it must also be pointed out that the administrator card is not a “proprietary” type card.", "It does not in fact have to be provided by a telephone network operator.", "In all cases, it can be obtained, for example purchased, from various sources: supermarkets, post offices, etc., or sent through the post.", "Once the user has simply inserted it in the auxiliary reader; the entire loading process, or more generally the administration process, is carried out automatically (as will be fully detailed below) and/or according to the instructions displayed on the mobile telephone screen.", "Once again preferably, the instructions for using the administrator card can be printed on it or displayed on screen when it is inserted in the auxiliary reader, after switching on the mobile telephone.", "Still preferably, the administrator card is in ID-1 format, which means that it can be inserted instead of a credit card or a standard bank card.", "The main purpose of the invention is therefore to define an administration method for an embedded subscriber system for a mobile telephone device of type including a first embedded system reader, the main reader, designed to take the said subscriber embedded system and a second embedded system reader, the auxiliary reader, the said subscriber embedded system including computer processing and data storage means, the said data possibly including software applications, characterised in that it includes at least the following steps: initial loading, in the digital data storage means of an additional system, the administration system, of a specific software module, including at least one component consisting of a loader program and at least one digital data file in a first format; temporary insertion of the said administration embedded system in the said auxiliary embedded system reader; conversion of the said digital data in the first format into a series of digital data in a second format under the control of the said specific software module; and the selective transfer of at least one series of digital data in the said second format to the said subscriber embedded system and its loading in the said storage means of this embedded system, via the said auxiliary and main embedded system readers, under the control of the said specific software module; and in that the said transfer is carried out according to a given communication protocol.", "The invention also concerns an embedded system for the implementation of the method.", "The invention will now be described in more detail, referring to the attached drawings, amongst which: FIG.", "1 is a diagrammatic representation of a configuration example for mobile telephone including two smartcard readers with a subscriber SIM card and a smartcard according to the invention; FIG.", "2 is a block diagram explaining the main steps and phases of the method according to the invention and the interactions between the components implemented; FIG.", "3 is a block diagram schematising the main administration operations possible through the use of the method according to the invention; FIG.", "4 is a block diagram illustrating a practical example of an applet in the subscriber SIM card, according to the method of the invention; and FIG.", "5 is a block diagram illustrating a practical example of deleting an applet in the subscriber SIM card, according to the method of the invention.", "Henceforth, without limiting in any way whatsoever the scope of the invention, we will consider the preferred application of the invention, unless otherwise specified, i.e.", "the context of a dual slot mobile telephone complying with the GSM standard.", "FIG.", "1 is a diagrammatic representation of a mobile telephone 1 and the two smartcards implemented, 2 and 3.We have assumed that the SIM subscriber card 3 is a module in Plug-in SIM format.", "As such, this SIM card corresponds to the known state of the art.", "It includes a support 30 in the above-mentioned Plug-in SIM format, on which is positioned an electronic chip 31 including input-output contact studs, with unique reference 310.The assembly is defined by various standards and/or norms, whether regarding the physical (dimensions, location of the chip, etc.", "), electrical and/or electronic aspects.", "The electronic chip includes in particular data processing means in a stored program (microprocessor or microcontroller) and non volatile (ROM, EPROM) and volatile (RAM and various registers) storage means.", "These various computer resources, also well known, are not shown on FIG.", "1.Apart from programs and digital data directly associated with the telephone transmissions, carried out for example in compliance with the above-mentioned GSM standard, the storage means of the module 3 can store various items of digital data, and especially applets, for example n applets, A1 to An.", "As such, this characteristic also corresponds to the known state of the art.", "If module 3 stores applets, it must comply with the Sim Toolkit technology so that it can receive commands and transmit proactive commands.", "In the example shown on FIG.", "1, the mobile telephone 1 includes a main body 10 and an auxiliary body 11, which can be folded back by rotation around an axis 110 at the bottom of the main body 10.The main smartcard reader 12 consists of an internal chamber 12 housing the SIM module 3 (represented on FIG.", "1 enlarged and outside its housing).", "The main body 10 is equipped in particular with a display screen 100, generally liquid crystal type.", "The auxiliary smartcard reader 13 is located in the auxiliary folding body 11 It communicates with the exterior via a slot 130 in which a smartcard in ID-1 format, and therefore compatible with standard smartcards, bank or credit cards, can be inserted.", "The mobile telephone according to the invention therefore remains fully compatible with the norms and standards of the known state of the art.", "It requires no modifications.", "In a preferred mode of realisation of the invention, the smartcard used as administration card also complies with the above-mentioned standard ID-1.The auxiliary reader 13 can therefore accept either standard smartcards or smartcards 2 complying with the characteristics of the invention and which are detailed below.", "Initially, we assume that the main function of the smartcard 2 is to load new applets in the SIM module 3.Preferably, the smartcard 2 complies with the Sim Toolkit technology.", "It can send proactive type commands to the host mobile telephone 1 and read and/or write commands to the files in its Sim Toolkit operating system OSST.", "According to a first characteristic of the invention, apart from the above-mentioned Sim Toolkit operating system OSST, the electronic chip 20 of the smartcard 2 contains at least one software module known as the loader Ch and a certain number of files, f1 to fn, capable of being transferred into the SIM module 3, in order to load applications there in addition to any applications already resident (not shown).", "Preferably, these applications consist of applets, advantageously coded in JAVA (registered trademark) language.", "In this case, the files, f1 to fn, are advantageously coded according to the CAP file format defined by the Java Card forum (registered trademark).", "The loader program Ch itself is also a Sim Toolkit applet.", "According to a characteristic of the method of the invention, the subscriber's SIM module or card 3 becomes the receiving card.", "The administrator card 2, according to another characteristic of the method of the invention becomes the main card during the loading, although it is inserted in the auxiliary smartcard reader 13.In a preferred mode of realisation, due to the fact that the administrator card uses Sim Toolkit technology, it can, unlike the known state of the art, become priority when the mobile telephone 1 is switched on, i.e.", "it can become “master”.", "The card inserted in the auxiliary reader 13, i.e.", "the administrator card 2, then takes priority over the SIM module 3: it is the applets of this administrator card 2 which are proposed to the user and not those resident in the SIM module 3 (receiving card).", "However, it must be made quite clear that this arrangement is not mandatory.", "The user could, for example, be requested to press a key or a key combination 101 on the keyboard of the mobile telephone 1 so that the process starts “manually” after this mobile telephone 1 is switched on.", "For example, the instructions for use may be supplied independently from the administrator card 2 or printed or engraved on one side of it.", "In the preferred mode of realisation, since the administrator card 2 takes priority, an initial interactive menu can be displayed on the screen 100 showing in particular the loader applet Ch installed on this card.", "Once the user has selected this application, it can read the files present, f1 to fn, so that it can then execute APDU commands on the receiving card, i.e.", "the SIM module 3, and thereby load the applet(s) corresponding to the files present on the administrator card.", "These operations are carried out in co-operation with the Sim Toolkit operating system OSST.", "More precisely, the card inserted in the auxiliary reader 13, i.e.", "the administrator card 2, can read the files present on its operating system via one or more traditional APDU commands, such as “READ BINARY”.", "Reading the system files, f1 to fn, enables the administrator card 2 to collect all the information required concerning the applet to be installed on the subscriber's SIM module or card 3.In addition, the administrator card 2 can open a session in order to access the subscriber's SIM module 3 via the proactive command “POWER ON CARD”.", "When this has been done, the administrator card 2 sends APDU loading commands defined by the standards ETSI 03.48 and ISO/IEC 7816-4 in order to install the applet read previously.", "The APDU commands are therefore sent to the SIM module via the proactive command “PERFORM CARD APDU”.", "Once the applet has been installed, the administrator card 2 of the auxiliary reader 13 closes the session with the command “POWER OFF CARD”.", "The user can now withdraw the administrator card 2 from the auxiliary reader 13 in order to reconnect on the SIM module 3.On la FIG.", "1, the applets referenced A1 to An, loaded in addition in the SIM module 3 correspond to files f1 to fn of the administrator card 2.All these commands or sessions comply with the usual standards and norms concerning the communication protocol between a card reader and a smartcard.", "As pointed out above, APDU commands in particular are used.", "The invention therefore involves no modifications.", "It should also be pointed out that, once the administrator card 2 according to the invention has been withdrawn, a traditional smartcard, for example a bank card or a credit card, can be inserted in the auxiliary reader 13.In this case, operation corresponds once again to the known state of the art: the smartcard in the auxiliary reader 13 no longer takes priority.", "It remains under the control of the SIM module or card 3.FIG.", "2 is a block diagram explaining in more detail the main interactions between the various components implemented.", "The main steps and phases of the method are also shown.", "We also assumed that that the administrator card can be used not only to load one or more applets in the SIM module 3, but also for other operations: deletion, etc., which will be detailed below, especially in reference to FIG.", "3.The software module specific to the invention includes several components, under the single reference 40.Preferably, as indicated, it is activated automatically when the mobile telephone 1 is switched on (FIG.", "1: 1).", "To do this, a command called “TERMINAL PROFILE” is used, generated by the mobile telephone 1 (FIG.", "1) precisely when switching on.", "The files stored in the administrator card 2 under the single reference 41, are generally associated not only with applets likely to be loaded in the SIM module 3, but also with digital data.", "For example, this data could consist of entries in the telephone directory.", "The administration card 2 may also be intended to load data associated with a new telephony operator in the SIM module 3.A priori, since the standards used for the communications between smartcard and reader implement APDU commands, the data and/or applications 41 cannot be loaded directly into the SIM module 3.A prior operation 42 must be carried out, which will be called formatting, under the specific program command 40.Note that are of course, as with any program during execution, interactions between the operating system OSST and this specific program 40.Once the formatting has been carried out, still under the control of the specific program 40, communication sessions, which may be bidirectional, are set up between the administrator card 2 and the module 3, via the auxiliary reader 13 and main reader 12, using a series of APDU commands (standardised communication protocol).", "Consequently, one or more additional applets may be loaded in the SIM module 3.However, the method according to the invention is not limited to loading applets.", "As shown by the block diagram on FIG.", "3, the card 2 can perform the following main administration operations 200: “addition” 201 (as shown above), as well as “deletion” 202 and “modification” 203.These operations can be carried out on applications (applets) 410 or various items of digital data 411.Lastly, the administrator card 2 can perform administration operations as such, which may be called “management” operations, especially various displays (for example multiple choice interactive menus) on the screen 100 (FIG.", "1), search and display of the memory space occupied by the applets present in the SIM module 3, search and display of the name and logical status of these applets, etc.", "These operations are all performed under the control of the specific program 40 and implement standard APDU commands and sessions.", "As an illustration, we will now describe two examples of practical implementation, referring to FIGS.", "4 and 5.The first concerns more specifically the selective addition of an applet, which in this case is used for local execution of the well known game called “MINEFIELD”.", "The second concerns more specifically the selective deletion of an applet present in the SIM module 3, after detecting insufficient memory space, in order to add a new applet (an address manager in this case).", "EXAMPLE 1 Adding an Applet (FIG.", "4) We assume that a user has already loaded an applet concerning the well known game “HANGMAN” on the SIM module 3 (FIG.", "1).", "He buys an administrator card according to the invention in order to load an extra game, in this game the game “MINEFIELD”.", "When the user inserts the loader card, i.e.", "the administrator card 2 (FIG.", "1) in the auxiliary reader 13 (FIG.", "1) with the “Loader” applet Ch (FIG.", "1) and the file corresponding to the “MINEFIELD” applet, according to the preferred mode, an initial interactive menu is automatically displayed on the screen 100 (FIG.", "1), without the user having to press any key 101 (FIG.", "1) on the mobile telephone 1 (FIG.", "1).", "This initial menu indicates to the user the name of the applet that can be loaded and requests whether or not to continue the loading: step 50.The text displayed is as follows: “Do you want to install Minefield on your SIM card?", "OK CANCEL”.", "If the user refuses (right hand branch: “CANCEL validated by user”), a new menu prompts the user to withdraw the loader card, i.e.", "the administrator card 2 (FIG.", "1): step 55.The text displayed is as follows: “You can withdraw your loader card.", "Goodbye”.", "The user withdraws the administrator card 2 from the auxiliary reader 13 (FIG.", "1).", "Following this operation, the mobile telephone 1 (FIG.", "1) switches back over to the SIM module 3 (FIG.", "1) and requests the user's PIN code (Personal Identifier Number): step 56.After entering a correct PIN code, the user can use the SIM module or card 3 again and select an applet from those already resident, for example the “HANGMAN” applet: step 57.If the user accepts to load the new applet (left hand branch, “OK” validated by user), the administrator card 2 (FIG.", "1) loads the “MINEFIELD” applet on the SIM module 33 (FIG.", "1), and informs the user that the operation has been carried out successfully: steps 51 and 52.The following messages are displayed: step 51: “Please wait.", "Loading .", ".", ".", "”, and step 52: “Minefield has been loaded correctly.", "You can withdraw the loader card”.", "When the user has withdrawn the administrator card 2 (FIG.", "1), he is prompted to enter his PIN code: step 53.After entering a correct PIN code, the user can use the SIM module or card 3 (FIG.", "1) again and select an applet from those already resident.", "After selection of the applet menu the two applets present on the SIM module are displayed: “HANGMAN”, previously resident, and “MINEFIELD” which has just been loaded: step 54.He can use this new applet, i.e.", "execute the game “MINEFIELD” on his mobile telephone 1 (FIG.", "1).", "EXAMPLE 2 Deleting an Applet (FIG.", "5) We now assume that the user has two applets installed on the SIM module 3 (FIG.", "1), for example the above-mentioned “HANGMAN” and “MINEFIELD”, and he wants to install an additional applet, for example an “address manager”.", "As before, the user inserts an administrator card 2 (FIG.", "1) which has a specific program 40 (FIG.", "2), especially including a component to load the “address manager” applet (one of the files 41: FIG.", "2).", "At step 60, a message is displayed: “Do you want to install the address manager on your SIM card?", "OK CANCEL”.", "If the user accepts the option presented (left hand branch, “OK” validated), the specific program 40 (FIG.", "2) scans the memory of the SIM module 3 (FIG.", "2): step 61.If there is sufficient memory space left on the SIM module 3 (FIG.", "1) for this third applet, the “address manager” applet can be loaded on the SIM module 3 (FIG.", "1).", "The execution of this operation is similar to that which has been described with respect to FIG.", "4.Consequently, the corresponding steps will not be described again.", "Otherwise, if there is not sufficient memory space, a message is displayed to the user indicating that at least one of the two applets already present must be deleted.", "In the example described, the message is as follows: “Insufficient memory.", "You must first delete an applet OK CANCEL”.", "If the user accepts this possibility (“OK” validated), he is prompted to select one of the applets, in response to the message displayed: “Select the applet to be deleted: HANGMAN or MINEFIELD-OK CANCEL”: step 62.If the user accepts one of the options presented, for example if he selects “HANGMAN”, the specific program 40 (FIG.", "2) starts a session in order to delete the “HANGMAN” applet.", "After deleting one of the applets loaded, the user is prompted to continue the procedure, just as in the previous example (FIG.", "4): loop back to step 60.The following message is displayed: “The HANGMAN applet has been deleted from memory.", "Do you want to continue?", "OK CANCEL”: step 63.In steps 60, 61, 62 and 63, the user has the possibility of refusing the choice proposed (“CANCEL” validated).", "The process then continues at step 64.The user is prompted to withdraw the administrator card 2 (FIG.", "1) from the auxiliary reader 13 (FIG.", "1).", "The following message is displayed: “You can withdraw your loader card.", "Goodbye”.", "The user must then enter his PIN code in order to use the SIM module 3 (FIG.", "1): step 65.After reading the above, it is easy to see that the invention does in fact reach the objectives set.", "With the method according to the invention, it is possible in particular to use a standard mobile telephone since no modifications are required to the equipment.", "It complies fully with current norms and standards.", "It is therefore fully compatible with the known state of the art.", "However, it offers numerous advantages.", "Not only can it be used to add (load) but also to delete and modify applications and/or digital data, as well as to perform various management operations: display, scanning the SIM memory, etc.", "It therefore provides true management of the subscriber's SIM card or module.", "No other equipment is required, as with certain methods of the known art, since the auxiliary reader of the mobile telephone is used as receiver of the administrator card.", "Neither does it rely on downloads from remote servers, with the disadvantages inherent to this type of method, which were pointed out in the preamble of this description.", "Lastly, the administrator card can be issued by any entity, it is not only a proprietary card issued by a mobile telephone operator.", "The applications and/or data stored are a priori independent from those specific to the telephony operators.", "However, the method enables an administrator card issued by a particular operator to be used in order to update its own data or to add a subscription to this operator in the SIM card, or on the contrary to delete it.", "The administrator card may be available from various types of point of sale or procurement, or even sent through the post.", "The operations possible, especially the loading of additional applets, require no special knowledge.", "In a preferred mode of realisation, certain operations are fully automatic, in particular the activation of the loader program or, more generally, of the specific program.", "The following steps are interactive, the user being guided by menu.", "However, the invention is of course not limited to only those examples of realisation explicitly described, especially in relation to FIGS.", "1 to.", "In particular the text of the messages displayed was only indicated to describe the method of the invention more clearly.", "These messages are related to the particular applications (applets) loaded on the administrator card.", "A priori, any messages would be possible without leaving the scope of the invention.", "Similarly, the method according to the invention does not interfere in any way with the technology used for the transmissions: GSM, GPRS or UTMS, for example, since the operations are all carried out locally through the implementation of two readers: the main reader containing the SIM module or card and the auxiliary reader intended to receive a traditional smartcard, for example a bank card.", "Moreover, the invention is not exclusively intended for mobile telephony.", "The invention concerns too for example an administration method for a data processing device as a computer, an organizer, an encoder reader or others." ] ]	Patent_10468033
[ [ "Estimating signal strength measurements in a telecommunications system", "The number of measurements being performed in a telecommunications system can be reduced by performing a predetermined measurement in Cell A and in co-located Cell B, step 1, for example a received signal code power (RSCP) measurement.", "The difference between the two signals is determined, and stored for future reference, step 3.", "Once the difference value has been established, the mobile station no longer needs to perform measurements on both Cell A and Cell B.", "Instead, if a subsequent measurement is made on Cell A, step 5, the network can estimate the measurement for Cell B based on the difference signal previously determined, step 7." ], [ "1.A method of estimating a signal measurement in a telecommunications system, the method comprising the steps of: performing a first measurement in first and second co-located cells of the telecommunications system; determining a difference value between the measurements; and using the difference value to estimate a second measurement for the second cell, based on a second measurement being made in the first cell.", "2.A method as claimed in claim 1, wherein the second measurement is the same type of measurement as the first measurement.", "3.A method as claimed in claim 1, wherein the second measurement is a different type of measurement to the first measurement, but having a predetermined relationship to the first measurement.", "4.A method as claimed in claim 3, wherein the second measurement is a CPICH Ec/No measurement.", "5.A method as claimed in any one of claims 1 to 4, wherein the first measurement is a received signal code power (RSCP) measurement.", "6.A method as claimed in any one of the preceding claims, wherein the measurements are performed at a mobile station in the telecommunications system and the difference value stored at a network side of the telecommunications system.", "7.A method as claimed in any one of the preceding claims, wherein the method of estimating a measurement is used to evaluate which cells should belong to an active set on a frequency other than the frequency being used.", "8.A method of handover in a cellular telecommunications system, the method of handover using a method of estimating a signal strength measurement as defined in any one of claims 1 to 7.9.A telecommunications system in which signal strength measurements are performed in two or more co-located cells, the system comprising: means for measuring a first measurement in first and second co-located cells of the telecommunications system; means for determining a difference value between the measurements; and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.", "10.A system as claimed in claim 9, wherein the second measurement is the same type of measurement as the first measurement.", "11.A system as claimed in claim 9, wherein the second measurement is a different type of measurement to the first measurement, but having a predetermined relationship to the first measurement.", "12.A system as claimed in claim 11, wherein the second measurement is a CPICH Ec/No measurement.", "13.A system as claimed in any one of claims 9 to 12, wherein the first measurement is a received signal code power (RSCP) measurement.", "14.A system as claimed in any one of claims 9 to 13, wherein the measurements are performed at a mobile station in the telecommunications system and the difference value stored at a network side of the telecommunications system.", "15.A system as claimed in any one of claims 9 to 14, having means for evaluating which cells should belong to an active set on a frequency other than the frequency being used.", "16.A method of estimating a signal measurement substantially as hereinbefore described, with reference to, and as shown in FIG.", "1 or 2 of the accompanying drawings.", "17.A telecommunications system substantially as hereinbefore described, with reference to, and as shown in FIG.", "1 or 2 of the accompanying drawings.", "18 A communications node for a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the communications node comprising: means for receiving a first measurement from first and second co-located cells of the telecommunications system; means for determining a difference value between the measurements; and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.", "19.A communications node as claimed in claim 18, wherein the second measurement is the same type of measurement as the first measurement.", "20.A communications node as claimed in claim 18, wherein the second measurement is a different type of measurement to the first measurement, but having a predetermined relationship to the first measurement.", "21.A communications node as claimed in claim 20, wherein the second measurement is a CPICH Ec/No measurement.", "22.A communications node as claimed in any one of claims 18 to 21, wherein the first measurement is a received signal code power (RSCP) measurement.", "23.A communications node substantially as hereinbefore described, with reference to, and as shown in FIG.", "1 or 2 of the accompanying drawings.", "24.A method of estimating a signal measurement in a communications node of a telecommunications system, the method comprising the steps of: receiving a first measurement from first and second co-located cells of the telecommunications system; determining a difference value between the measurements; and using the difference value to estimate a second measurement for the second cell, based on a second measurement being made in the first cell.", "25.A method as claimed in claim 24, wherein the second measurement is the same type of measurement as the first measurement.", "26.A method as claimed in claim 24, wherein the second measurement is a different type of measurement to the first measurement, but having a predetermined relationship to the first measurement.", "27.A method as claimed in claim 26, wherein the second measurement is a CPICH Ec/No measurement.", "28.A method as claimed in any one of claims 24 to 27, wherein the first measurement is a received signal code power (RSCP) measurement.", "29.A method of estimating a signal measurement in a communications node of a telecommunications system, the method being substantially as hereinbefore described, with reference to, and as shown in FIG.", "1 or 2 of the accompanying drawings.", "30.A mobile communications terminal for use in a telecommunications system as described in any one of claims 9 to 15." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>In a telecommunications system, for example a wideband CDMA system (WCDMA), there is always a desire to communicate using the least amount of power.", "Therefore, all transmissions should ideally be performed at the lowest possible power level, while still maintaining an acceptable quality level.", "However, there are certain factors which act against this general desire to reduce power levels.", "Certain communication techniques have an inherent need for the power levels to be increased, both in the mobile station and the network.", "For example, data compression can be used to transmit a given amount of data in less time, resulting in gaps being created to allow inter-frequency or inter-RAT (Radio Access Technology) measurements to be carried out.", "In addition to the peak power being increased during the data compression, the average power also needs to be increased to compensate for the channel estimates that are not being updated during the time gaps, resulting in the receiver not being optimally tuned after the time gaps.", "While increased inter-frequency and inter-RAT measurements typically have the disadvantage of increasing the average power consumption when using real time services with requirements of low delays, for example speech, they also have a degrading effect in that they reduce the available channelization-codes.", "During data compression a code for a lower spreading factor needs to be used, which typically blocks several codes used for higher spreading factors in the downlink.", "The gaps in transmission mentioned above can also occur in other ways.", "For example, reducing the number of bits to be transmitted by reducing the number of extra bits which are used for error correction during some frames (i.e.", "code puncturing), or scheduling data transmission from a higher layer.", "It is known to reduce the power requirement (and in turn improve data throughput) by reducing the number of measurements carried out in the telecommunications system.", "One known example of how this may be achieved is based on the use of neighbouring cell lists, and thresholds for starting measurements.", "This type of solution suffers from the disadvantage that each neighbouring cell is treated equally as if they were adjacent neighbours using different antennas covering different, but to some extent overlapping, geographical areas.", "The reduction in these schemes is based on limiting the number of neighbouring cells included in the list, and on handover statistics and propagation predictions when cell planning is performed.", "The aim of the present invention is to provide a method of estimating measurements in a telecommunications system, thereby reducing the number of measurements being performed.", "This has the advantage of reducing power consumption and increasing the capacity of the system." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>According to a first aspect of the present invention, there is provided a method of estimating a signal measurement in a telecommunications system, the method comprising the steps of: performing a first measurement in first and second co-located cells of the telecommunications system; determining a difference value between the measurements; and using the difference value to estimate a second measurement for the second cell, based on a second measurement being made in the first cell.", "According to another aspect of the present invention, there is provided a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the system comprising: means for measuring a first measurement in first and second co-located cells of the telecommunications system; means for determining a difference value between the measurements; and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.", "According to another aspect of the present invention, there is provided a communications node for a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the communications node comprising means for receiving a first measurement from first and second co-located cells of the telecommunications system, means for determining a difference value between the measurements, and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value." ], [ "FIELD OF THE INVENTION The present invention relates to performing measurements in a telecommunications system, and in particular, to estimating measurements in a telecommunications system, thereby reducing the number of measurements being performed.", "BACKGROUND OF THE INVENTION In a telecommunications system, for example a wideband CDMA system (WCDMA), there is always a desire to communicate using the least amount of power.", "Therefore, all transmissions should ideally be performed at the lowest possible power level, while still maintaining an acceptable quality level.", "However, there are certain factors which act against this general desire to reduce power levels.", "Certain communication techniques have an inherent need for the power levels to be increased, both in the mobile station and the network.", "For example, data compression can be used to transmit a given amount of data in less time, resulting in gaps being created to allow inter-frequency or inter-RAT (Radio Access Technology) measurements to be carried out.", "In addition to the peak power being increased during the data compression, the average power also needs to be increased to compensate for the channel estimates that are not being updated during the time gaps, resulting in the receiver not being optimally tuned after the time gaps.", "While increased inter-frequency and inter-RAT measurements typically have the disadvantage of increasing the average power consumption when using real time services with requirements of low delays, for example speech, they also have a degrading effect in that they reduce the available channelization-codes.", "During data compression a code for a lower spreading factor needs to be used, which typically blocks several codes used for higher spreading factors in the downlink.", "The gaps in transmission mentioned above can also occur in other ways.", "For example, reducing the number of bits to be transmitted by reducing the number of extra bits which are used for error correction during some frames (i.e.", "code puncturing), or scheduling data transmission from a higher layer.", "It is known to reduce the power requirement (and in turn improve data throughput) by reducing the number of measurements carried out in the telecommunications system.", "One known example of how this may be achieved is based on the use of neighbouring cell lists, and thresholds for starting measurements.", "This type of solution suffers from the disadvantage that each neighbouring cell is treated equally as if they were adjacent neighbours using different antennas covering different, but to some extent overlapping, geographical areas.", "The reduction in these schemes is based on limiting the number of neighbouring cells included in the list, and on handover statistics and propagation predictions when cell planning is performed.", "The aim of the present invention is to provide a method of estimating measurements in a telecommunications system, thereby reducing the number of measurements being performed.", "This has the advantage of reducing power consumption and increasing the capacity of the system.", "SUMMARY OF THE INVENTION According to a first aspect of the present invention, there is provided a method of estimating a signal measurement in a telecommunications system, the method comprising the steps of: performing a first measurement in first and second co-located cells of the telecommunications system; determining a difference value between the measurements; and using the difference value to estimate a second measurement for the second cell, based on a second measurement being made in the first cell.", "According to another aspect of the present invention, there is provided a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the system comprising: means for measuring a first measurement in first and second co-located cells of the telecommunications system; means for determining a difference value between the measurements; and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.", "According to another aspect of the present invention, there is provided a communications node for a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the communications node comprising means for receiving a first measurement from first and second co-located cells of the telecommunications system, means for determining a difference value between the measurements, and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.", "BRIEF DESCRIPTION OF THE DRAWINGS For a better understanding of the present invention, and to show more clearly how it may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:— FIG.", "1 shows the steps involved in a method of estimating a measurement according to a first aspect of the present invention; and, FIG.", "2 shows the steps involved in a method of estimating a measurement according to a second aspect of the present invention.", "DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE PRESENT INVENTION In a WCDMA based radio access network, several cells may be co-located using different frequencies.", "The network will have information that a particular cell, say Cell A, is co-located with one or more other cells, say Cell B.", "A mobile station positioned in a given location is able to make signal measurements on common pilot channels (CPICHs) transmitted from a serving cell and any co-located cells.", "FIG.", "1 shows the steps involved in estimating a measurement according to a first aspect of the present invention.", "In step 1, the network instructs a mobile station to perform a predetermined measurement in Cell A and in co-located Cell B, for example a received signal code power (RSCP) measurement, hereinafter referred to as RSCPA and RSCPB, respectively.", "These measurements are reported by the mobile station to the network.", "The network determines the difference between the two signals (RSCPA−B) which is stored for future reference, step 3.Once the difference value has been established, the mobile station no longer needs to perform measurements on both Cell A and Cell B.", "The network can therefore suspend inter-RAT or inter-frequency RSCP measurements.", "Instead, if a subsequent measurement is made on Cell A (RSCPA+t), step 5, the network can estimate the measurement for Cell B based on the difference signal previously determined.", "For example, if the difference signal was originally determined by subtracting RSCPB from RSCPA, then the estimated value for Cell B is determined by subtracting the difference value RSCPA−B from the new measurement for Cell A, RSCPA+t, step 7.Thus, RSCPB+t=(RSCPA+t−RSCPA−B) Since Cell A is co-located with Cell B, the path loss difference will remain constant for all locations of the mobile station, which means that the RSCP difference RSCPA−B also remains constant.", "This difference value can therefore provide an accurate estimate of a measurement for one cell, using the measurement of a co-located cell.", "Measurements in Cell B can be restarted at any time, for example if the estimated measurement approaches a critical value.", "According to another aspect of the invention, the difference value described above can be used to provide further reductions in measurements.", "In addition to RSCP measurements, a mobile station may perform any one, or any combination, of the following measurements, depending upon the processing utilised in the mobile station: 1.UTPA carrier RSSI (this stands for received signal strength indicator, and relates to signal strength plus interference on a particular frequency from any intra frequency cell) 2.CPICH RSCP (this stands for received signal code power, and relates to signal strength from a particular cell's CPICH on a particular frequency) 3.CPICH Ec/No (this is basically the signal-to-noise ratio used for representing the “cell quality” for handover evaluation).", "Each of the measurements described above imposes a different demand on the telecommunications system in terms of the processing power required, and/or the time needed to perform a given measurement.", "In other words, certain measurements are more “expensive” than others.", "For example, the CPICH Ec/No measurement requires typically more processing power that the CPICH RSCP measurement, which is turn requires more processing power than the UTRA carrier RSSI measurement.", "The measurements described above have a predetermined relationship whereby: CPICH Ec/No=(CPICH RSCP)/(UTRA carrier RSSI) Using this relationship, the estimation method according to the first aspect of the invention can be used to reduce the number of “expensive” measurements being made, for example by reducing the number of CPICH Ec/No measurements being performed in the telecommunications system.", "Referring to FIG.", "2, a difference signal is first determined, as mentioned above, by calculating the difference between the RSCPA and RSCPB measurements, (RSCPA−B), step 101.The RSSIA measurement and (Ec/No)A measurement for Cell A are also performed, step 103.Depending upon the particular implementation in a given mobile station, the (Ec/No)A measurement can either be calculated directly, or calculated from the RSCPA and RSSIA values.", "Next, when it is determined that an Ec/No measurement is subsequently required for Cell B, step 105, instead of measuring this value directly, the measurement can be estimated as follows.", "First, the RSSI value for cell B is measured, RSSIB, as shown in step 107.The Ec/No measurement can then be estimated from the RSSIB measurement, using a RSCPA measurement for cell A and the previously stored difference value RSCPA−B, as follows: (Ec/No)B=(RSCPA−RSCPA−B)/(RSSIB) Thus, the Ec/No measurement for cell B can be estimated without having to perform a relatively costly CPICH RSCP measurement for cell B.", "Instead, only the less costly UTRA RSSI measurement needs to be made for cell B.", "Also, since the RSCP and Ec/No measurements for cell A are being made in the same cell that the mobile station is currently using, these measurements are also less costly than the corresponding measurements for cell B (i.e.", "being the cell which is not currently being used by the mobile station).", "The Ec/No measurement for cell A and the estimated Ec/No measurement for cell B may be used to distribute traffic between cells, for example, to trigger handover between cell A and cell B.", "As noted above, since the RSSI measurement for cell B is less demanding than the Ec/No measurement, power consumption is thereby reduced.", "For example, assume that one 7 slot wide measurement gap is used every third frame (each frame consisting of 15 slots) for inter-frequency compressed mode measurements on CPICH Ec/No, and that the average DL power needs to be increased by 3 dB to maintain the same connection quality.", "The width of the measurement gap can be decreased to one slot used every third frame, and, if the power increase required to maintain the same quality can be assumed to be decreased by {fraction (1/7)} because of that, the following difference in power can be calculated.", "This corresponds to a 0.6 dB increase (i.e.", "1.143 times increase) instead of 3 dB increase (i.e.", "2 times increase) when making CPICH Ec/No measurements.", "Thus, if it is assumed that all the mobiles in a cell are using compressed mode at all times then, if the nominal amount of mobiles is planned to be limited to 20 per cell using 1 watt each when not using compressed mode, the estimated amount of mobiles when using compressed mode and the same DL power margins would be: 20/2=10 mobiles when the mobiles are performing inter-frequency CPICH Ec/No measurements all of the time, and 20/1.143=17.5 mobiles when using estimation according to the second aspect of the invention, whereby RSSI measurements are carried out instead.", "According to the first and second aspects of the invention, rather than having a mobile station perform measurements on each of the co-located cells, the mobile station determines a relationship between the cells, assumes that this relationship remains constant, and then performs measurements on just one cell—the other cell's measurements being estimated from the known relationship with the first cell.", "Also, if different measurements within a given cell have different power requirements, the high power measurements can be estimated by measuring the lower power measurements, and calculating the higher power measurements accordingly.", "As can be seen from the above, estimating measurements in this way has the effect of reducing the number of measurements being performed.", "This has the effect of reducing power consumption and increasing the capacity of the telecommunications system, thereby enabling a greater number of mobile stations to be supported.", "Although the preferred embodiment refers to the difference value being calculated with respect to the respective RSCP measurements from each cell, the invention is equally applicable to other signals being used in this manner, for example determining a difference signal using the BCCH frequency RXLEV in a GSM system, or measured despread signal strength on a IS95 system.", "Also, although the invention has been described as performing measurements between two co-located cells, the invention may be used with just one cell, or with any number of co-located cells.", "Furthermore, the invention can be used in the same way for evaluating cells on a frequency other than the frequency being used, to determine whether they should belong to the active set.", "For example, by using knowledge about the active set on the currently used frequency and also the knowledge that the cells on used and unused frequencies are co-located, the measurement requirements on the unused frequency will only be UTRA Carrier RSSI and information on RSCPB+t, allowing the CPICH Ec/No value to be used for evaluating the cells on the unused frequency, to determine whether they should belong to the active set." ] ]	Patent_10468046
[ [ "Device with flexible roller shutter", "This invention concerns a shutter system comprising, firstly, drive systems allowing the movement of the shutter (1) along a path between a closed and an open position, said shutter (1) being intended to close an opening (3) or other aperture and, secondly, detection systems (16) allowing determination of deflection of the shutter (1) from its path, for example due to the presence of an obstruction (13) below the shutter (1), during its movement into its closed or open position, the detection systems (16) combining with the drive systems in such a way as to permit stopping or opening of the shutter (1), each of the side edges of the shutter (1) running in a guide track (9).", "This system is characterised by the fact that the detection systems (16) are fitted in such a way as to be able to combine them with the guide track (9) in order to detect whether the shutter (1) is deflected from its path in the vicinity of the guide track (9)." ], [ "1.Shutter system consisting of, firstly, drive systems permitting movement of the shutter (1) along a path between a closed and an open position, intended to close an opening (3) or other aperture and, secondly, detection systems (16) allow determination of whether the shutter (1) is being deflected from its path, for example, by the presence of an obstruction (13) below the shutter (1) during its movement into its closed position or open position, said detection systems (16) being linked to the drive systems in such a way as to stop or open the shutter (1), each of the side edges of the shutter (1) running in a guide track (9), characterised by the fact that the detection systems (16) are fitted in a way permitting their combination with the guide track (9) in order to detect whether the shutter (1) is being deflected from its path in the vicinity of the guide track (9).", "2.System in accordance with claim 1, characterised by the fact that the detection systems include at least one switch (16) fitted in such a way as to permit the shutter (1) undergoing deflection from its path to operate the switch (16) and interrupt movement of the shutter (1) towards its closed or open position.", "3.System in accordance with claims 1 or 2, characterised by the fact that the part of the path alongside the opening (3) or other aperture is demarcated by a guide track (9) extending downwards from the drive systems (2).", "4.System in accordance with claim 3, characterised by the fact that the aforementioned detection systems (16) are fitted in such a way as to permit deflection of the shutter (1) from its path at a point located outside the guide track (9).", "5.System in accordance with claim 3, characterised by the fact that the detection systems (16) are mounted on the guide track (9) in the vicinity of that part of the path located outside the guide track (9).", "6.System in accordance with claim 5, characterised by the fact that the guide track (9) is elastically mounted on a bracket (20) in a flexible way and/or one which will allow it to pivot about an axis approximately parallel to the longitudinal axis of the guide track (9) so that if a force is applied to the plane of the shutter (1) when it is not fully open, the detection systems (16) will move with the guide track (9) to avoid being operated by the movement of the shutter (1) due to said force.", "7.System in accordance with claim 3, characterised by the fact that the guide tracks (9) have a recess (23) which permits the shutter (1) to be deflected across said recess (23) when normal opening or closure of the shutter (1) is disturbed, e.g.", "due to the presence of an obstruction (13) below the shutter (1), the aforementioned detection systems (16) being fitted in a way to allow such deflection to be detected.", "8.System in accordance with claim 7, characterised by the fact that the guide tracks (9) essentially take the form of U-shaped channel, the aforementioned recess (23) being provided in one of the walls (25) of the U.", "9.System in accordance with claim 3, characterised by the fact that the guide tracks (9) are flexibly mounted on a bracket (20) so that if the shutter (1) is deflected from its path when running in the guide tracks (9), the latter are deflected from the bracket (20), the detection systems (16) being fitted in such a way as to permit detection of such movement.", "10.System in accordance with claim 9, characterised by the fact that the guide tracks (9) linked to a spring (8) apply a force to the guide tracks (9) towards the bracket (20) in order to flatten the shutter (1).", "11.System in accordance with claim 1 or claim 10, characterised in that the aforementioned drive systems include a drum (2) which can be rotated about its centre line (12), the aforementioned detection systems (16) being mounted to the side of the drum (2) and at a certain distance from it, so that if the shutter (1) undergoes specific deflection from its path at the level of the drum (2), the detection systems (16) combine with the drive systems by stopping movement of the shutter (1) into its closed position.", "12.System in accordance with claim 11, characterised by the fact that the detection systems include at least one switch (16) mounted to the side of the drum (2) at a certain distance from it and fitted in such a way as to permit the shutter (1) undergoing deflection from its path at the level of the drum (2) to operate the switch (16) and interrupt the movement of the shutter (1) towards its closed position.", "13.System in accordance with claim 1, characterised by the fact that a flexible stiffened area (17) is provided on the shutter (1), extending in the direction of movement of the shutter (1).", "14.System in accordance with claim 1, characterised by the fact that a flexible stiffened area (17) extends into the vicinity of both the side edges (7) of the shutter (1).", "15.System in accordance with one of either claims 13 or 14, characterised by the fact that the stiffened area combines with the detection systems in order to detect deflection of the shutter (1) from its path.", "16.Flexible shutter for use in a closure system and intended for the closure of an opening (3) or other aperture in which the shutter (1) is unwound from a drum (2) along a path during closure of the opening (3) or other aperture by moving in guide tracks (9) which are arranged along the opening (3) or other aperture, characterised by the fact that a flexible stiffened area (17) extends into the vicinity of at least one side edge (7) of the shutter (1) in order to be able to bend if the shutter (1) is deflected from its path and to be able to combine with detection systems (16) detecting the curvature (26) of the stiffened area (17)." ], [ "This invention concerns a flexible shutter system combined with drive systems in such a way as to permit the shutter to move along a specific path between a closed and an open position.", "The said shutter is intended to close an opening or other aperture.", "One of the essential objectives of this invention is the provision of a system of the aforementioned type which provides maximum safety when the shutter is moving towards its closed position and its path is obstructed, or if movement of the shutter is prevented for another reason during closure or opening.", "For this purpose, inventive detection systems are provided which allow deflection of the shutter from its path to be determined, e.g.", "due to the presence of an obstruction below the shutter when it moves towards its closed or open position.", "The detection systems combine with the drive systems to stop or open the shutter.", "In an advantageous embodiment of the inventive system, the aforementioned drive systems include a drum which may be rotated about its centre line, the aforementioned detection systems being fitted at the side of the drum at a certain distance from it so that if the shutter undergoes specific deflection from its path at the level of the drum, the detection systems will act upon the drive systems, stopping movement of the shutter towards its closed position.", "In a particularly advantageous embodiment of the inventive system, a stiffened area is provided on the shutter, extending in the direction of movement of the latter.", "Other details and particular features of the invention emerge from the description provided below, in non-exhaustive examples of several embodiments with references to the drawings appended hereto.", "FIG.", "1 is a diagrammatic vertical section through a first embodiment of the inventive system with the shutter in its closed position.", "FIG.", "2 is a view analogous to that in FIG.", "1, with the shutter in its open position.", "FIG.", "3 is a view analogous to that in FIGS.", "1 and 2 while the shutter is moving into its closed position and an obstruction is present in its path.", "FIG.", "4 is a partial lateral section through a shutter of which the side edges are held in guide tracks, on a larger scale.", "FIG.", "5 is a view, analogous to that in FIG.", "4, of a second embodiment of the invention in which a switch is mounted on the guide track.", "FIG.", "6 is a front elevation of part of a shutter in accordance with the second embodiment of the invention, as shown in FIG.", "5.FIG.", "7 is a view analogous to that in FIG.", "5, of the second embodiment of the invention in which a transverse force is exercised on the plane of the shutter.", "FIG.", "8 is a diagrammatic vertical section through the second embodiment of the inventive system while the shutter is moving into its closed position and an obstruction is present in its path.", "FIG.", "9 is a diagrammatic vertical section through a third embodiment of the inventive system with the shutter in its closed position.", "FIG.", "10 is a horizontal section through the guide track along the line X-X in FIG.", "9.FIG.", "11 is a horizontal section through the guide track along the line XI-XI in FIG.", "9.FIG.", "12 is a view analogous to that in FIG.", "9 while the shutter is moving into its closed position and an obstruction is present in its path.", "FIG.", "13 is a diagrammatic horizontal section through part of a guide track in a fourth embodiment of the inventive system.", "FIG.", "14 is a horizontal section through the guide track along the line XIV-XIV in FIG.", "13.FIG.", "15 is a view analogous to that in FIG.", "13 when the shutter is deflected from its normal path.", "FIG.", "16 is a horizontal section through the guide track along line XVI-XVI in FIG.", "15.The same reference numbers refer to the same or analogous components in the various drawings.", "In general, this invention concerns a shutter system 1 combined with drive systems such as a drum 2 of which the centre line is connected to the shaft of a motor (not shown).", "The shutter 1, which can move between a closed position and an open position, is intended to close an opening 3 in a wall 4 or any aperture.", "In FIG.", "1, the system is shown in its closed position with the lower edge 5 of the shutter 1 against the floor 6.For the purposes of this invention, the word “shutter” should be understood as any component at least partially pliant, flexible, rigid or semi-rigid, such as a tarpaulin, a strip of plastic material, an assembly of articulated lamellae, a grille, etc.", "More specifically, it involves a shutter which may be folded or wound around a spindle at right angles to its direction of movement between its open and closed positions.", "However, it should be noted that pronounced preference is given to pliant shutters formed by a tarpaulin.", "The drawings thus concern a shutter made up of a tarpaulin 1 of which the side edges 7 have, for example, a bead or a series of blocks 8, as shown in FIG.", "6.The shutter 1 is guided by its side edges 7 in guide tracks 9 extending approximately vertically on either side of the opening 3.When the shutter 1 opens, as shown in FIG.", "2, its side edges 7 are moved along the guide tracks 9 in the direction indicated by the arrow 10 as a consequence of the shutter 1 being wound on the drum 2, which rotates around its centre line 12 in the direction indicated by the arrow 11.The guide tracks 9 are fixed to the wall 4 by systems not shown.", "The shutter 1 is moved along a specific path during normal opening and closure.", "In a standard procedure, during normal operation of the shutter system, within the meaning of this description, this path corresponds to that followed by the shutter 1 during its movement between its closed position, as shown in FIG.", "1, and its open position, as shown in FIG.", "2.Should any interference with normal operation of the shutter take place during movement of the latter in the direction indicated by the arrow 14 into its closed position, e.g.", "by an obstruction 13 located below the shutter 1 which consequently prevents movement of said shutter 1, part of the shutter 1 will be deflected from its normal path, by forming a bulge or gatherings located outside its path.", "This bulge makes contact with detection systems, such as a switch 16, which acts upon the drive systems of the shutter 1 in such a way as to interrupt its descent.", "In the embodiment shown in FIGS.", "1 to 8, the guide tracks 9 extend along part of the path of the shutter 1 to a certain distance from the drive systems so that the shutter 1 is not guided over this distance.", "A gap 22 is thus formed between the upper end of the guide tracks 9 and the drive systems in which the shutter 1 is not guided laterally and into which the shutter 1 may be deflected if its movement is retarded, e.g.", "by the presence of an obstruction below the shutter 1.As shown in FIG.", "3, if an obstruction 13, such as a fork-lift truck, is fouled by the lower edge 5 of the shutter 1, the latter will form a bulge 15 above the guide tracks 9.This is caused by the fact that the drive systems will not stop immediately when the lower edge 5 of the shutter 1 encounters the obstruction 13.In particular, the drum 2 will continue to unroll the shutter 1 of which the movement of the lower edge 5 is prevented by the presence of the obstruction 13 in the path of the shutter 1.Inventive detection systems are provided which allow determination of whether the shutter 1 is being deflected from its normal path.", "It is advantageous for these detection systems to be fitted so that deflection of the shutter 1 from its path can be detected outside the guide track 9.As shown in FIGS.", "1 to 3, the detection systems include a switch 16 mounted to the side of the drum 2 at a certain distance from the latter and fitted against the wall 4 so that if the shutter 1 undergoes specific deflection from its path in the gap 22 at the level of the drum 2 due to the presence of an obstruction 13 below the shutter 1, the latter will encounter and operate the switch 16 by forming a bulge 15.The switch 16 combines with the drive systems to cause movement of the shutter 1 into its closed position to be interrupted.", "In order to remedy the interference caused by the obstruction 13 immediately, shutter 1 is preferably returned to its open position automatically by the drive systems .", "In some cases, use of a highly pliant shutter 1 to obtain a very light shutter system which can open and close rapidly is indispensable.", "In this case, instead of forming a bulge 15, there is a risk that the shutters will gather if the lower edge 5 fouls an obstruction 13 when moving into its closed position.", "In order to ensure that the switch 16 will be operated by the shutter 1 when the latter is deflected from its path, it is advantageous to provide a flexible stiffened area 17 on the shutter 1, as shown in diagrammatic form in FIG.", "4.Said stiffened area 17 preferably extends into the vicinity of each of the side edges 7 of the shutter 1.The stiffened area 17 may consist of a continuous plastic or metallic strip affixed to the shutter 1.In a specific embodiment of the invention, the stiffened area 17 is formed by a series of rigid elements which are mounted alongside each other on one surface of the shutter 1.Said rigid elements are mounted on the surface of the shutter 1 facing the switch 16.Such a series of rigid elements ensures that the shutter will not gather and render it flexible in only one direction, so that a bulge 15 of a specific shape will be formed which will encounter the switch 16 if there is an obstruction below the shutter 1.A different embodiment of the inventive shutter system is shown in FIGS.", "5 to 8.In this embodiment, the detection systems include a switch 16 which is mounted on the guide track 9.The switch 16 is mounted in the vicinity of the path of the shutter I which is located outside the guide track 9, i.e.", "in the aforementioned gap 22 located between the drum 2 and the free upper end of the guide tracks 9, more specifically in the vicinity of said free end.", "In this way, the switch 16 can detect whether the shutter 1 is being deflected from the section of the path located above the guide track 9.The fact that the detection systems are mounted on the guide track 9 prevents the shutter 1 from encountering the switch 16 if a force is exercised on shutter 1 in a direction approximately transverse to the plane of the latter, e.g.", "by wind.", "In FIG.", "7, the arrows 19 represent the forces exercised on the shutter 1 by wind.", "As illustrated, the shutter 1 is bent in the direction indicated by the arrow 19 by these forces and the guide track 9 is pivoted about its longitudinal axis from the position shown in FIG.", "5.Due to the fact that the switch 16 is mounted on the guide track 9, it combines with the guide track 9, moving with it.", "However, the distance between the shutter 1 and the switch 16 remains approximately constant.", "In this embodiment of the inventive shutter system, the guide track 9 is mounted on a bracket 20 which is fixed to the wall 4 in a flexible way and/or one which will allow it to pivot about an axis approximately parallel to the longitudinal axis of said guide track 9.Such a method of mounting guide tracks 9 has already been described in international patent application no.", "PCT/BE92/00017.The switch 16 has an arm 21 which extends above the guide track 9, as clearly illustrated in FIGS.", "6 and 8.In this way, if a bulge 15 forms above the guide track 9 due to the presence of an obstruction 13 below the shutter 1, it will encounter the arm 21 which is linked to the switch 16.Consequently, movement of the shutter 1 will be stopped and it will preferably be immediately wound on to the drum 2.A third embodiment of the inventive shutter system is shown in FIGS.", "9 to 12.In this embodiment, the guide tracks 9 form an essentially U-shaped channel, as shown in FIG.", "10.The shutter 1 is thus guided in the guide tracks 9 by a block 24 which is located in the channel and which is provided on the side edges of the shutter 1.In order to permit detection of whether an obstruction is located below the shutter 1 during closure of the latter, each guide track 9 contains a recess 23 over part of its length.", "Said recess 23 is, in particular, provided in one of the walls 25 of the guide track 9, as shown in FIGS.", "9 and 11.If closure of the shutter 1 is retarded by the presence of an obstruction, as shown in FIG.", "12, shutter 1 may be deflected from its normal path by forming a bulge 26 extending across the recess 23.A switch 16 is fitted opposite the recess 23 in order to detect such deflection of the shutter 1.In this way, the shutter 1, which has been deflected from its path, encounters the switch 16 which combines with the drive systems, thus causing the shutter 1 to stop or open.", "In a variation of this inventive embodiment of the system, the guide track 9 has a cover at the height of the recess 23, such as a plate, which will become detached or move easily when the lower edge 5 of the shutter 1 encounters an obstruction and a bulge 26 is formed across said recess 23.The cover then encounters the switch 16 and sends a signal to the drive systems.", "FIGS.", "13 to 16 show a fourth embodiment of the inventive system, in which the guide tracks 9 are elastically mounted on a bracket 20 which is affixed to the wall.", "In this way, the guide tracks 9 can move relative to the bracket 20 if the shutter 1 gathers or if a force is applied to the plane of the shutter.", "The shutter 1 gathers, for example, if it is moving along the guide tracks 9 into its closed position and the lower edge 5 of the shutter 1 fouls an obstruction.", "Due to the gathering of the shutter 1, its side edges 7 move towards each other and the shutter 1 is deflected from its normal path, moving the guide tracks 9 relative to the bracket 20 in the direction indicated by the arrow 27 in FIG.", "16.Such movement of the guide tracks 9 relative to the path is detected by the detection systems, particularly by a switch 16 mounted on the bracket 20 and thus linked to the guide tracks 9.The guide tracks 9 are linked to a spring 28 forcing the former towards the bracket 20, to ensure that the shutter 1 is flat.", "It is self-evident that the invention is not restricted to the various embodiments described above and that other variations may be envisaged without departing from the scope of this invention, particularly in respect of the position of the switch.", "It is possible to install several switches, for example on the two side edges of the shutter 1.In other respects, the detection systems may include any means which allow the presence of a bulge to be detected, such as, for example, optical detection systems, particularly an electronic eye.", "In addition, the stiffened area may be affixed to the shutter continuously, or intermittently by discrete fixings.", "For example, the stiffened area may consist of a reinforced side edge of the shutter 1." ] ]	Patent_10468129
[ [ "Plasma surface graft process for reducing thrombogenicity", "In accordance with the present invention, there is provided a novel process for modifying the surface properties of a material that is suitable for contact with animal tissue so as to enhance its hemocompatibility and make it less thrombogenic when in use.", "This process comprises: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on said surface; activating a molecule with an activator to produce a reactive molecular species capable of forming convalent bonds with the reactive groups created on the surface of the material to form convalent bonds.", "The invention further encompasses the materials produced by this process as well as devices, such as vascular prosthesis, that are comprised of these process-modified materials." ], [ "1.A process for modifying the surface properties of a material suitable for contact with a living tissue comprising: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on said surface; Activating a molecule with an activator to produce a reactive molecular species capable of forming strong bonds with the reactive groups created on the surface of the material; and Contacting the reactive molecular species with the reactive groups created on the surface of the material to form strong bonds.", "2.A process as defined in claim 1, wherein said strong bonds are covalent bonds.", "3.A process as defined in claim 1 or 2, wherein the resulting surface of the material is compatible with living tissue.", "4.A process as defined in claim 3, wherein said tissue is blood tissue.", "5.A process as defined in claim 1, wherein: said plasma is ammonia Radio Frequency (RF) plasma and said reactive groups are amine groups; said molecule is selected from the group consisting of choline, heparin, and other molecules known to those skilled in the art for their hemocompatibility, said activator is phosphoryl chloride (POCl3) and said reactive molecular species is the oxyphosphorodichlorinated derivative of said molecule; and said strong bonds are phosphoamide-type covalent bonds.", "6.A process as defined in claim 5, wherein said plasma treatment conditions consist of the application of a RF power of between about 5 watts to about 500 watts for a time of about 10 seconds to about 30 minutes at a pressure of about 50 mtorr to about 5 torr.", "7.A process as defined in claim 6, wherein said plasma treatment conditions consist of the application of a RF power of about 20 watts for a time of about 250 seconds at a pressure of about 300 mtorr.", "8.A process as defined in claim 6, wherein said plasma treatment conditions consist of the application of a RF power of about 15 watts for a time of about 100 seconds at a pressure of about 250 mtorr.", "9.A process as defined in claim 7 or 8, wherein the third step is performed within about 2 hours of the first step.", "10.A process as defined in any one of claims 1 to 9, wherein said material suitable for contact with a living tissue is material implantable in an animal's body.", "11.A process as defined in claim 10, wherein said material implantable in an animal's body is selected from the group consisting of: Microporous expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyethylenes, polyesters (such as polyethylene terephtalate—PET), polypropylenes, polyurethanes, polycarbonates, silicones, PVDF and polymer-coated materials, such as metals and ceramics for example.", "12.A process as defined in claim 11, wherein said material implantable in an animal's body is ePTFE.", "13.A process as defined in claim 12, wherein said ePTFE is that of the internal surface of a vascular prosthesis.", "14.A process as defined in claim 13, wherein said vascular prosthesis has an inner diameter of about 1 to 30 mm.", "15.A material implantable in an animal's body produced by the process of any one of claims 1 to 14.16.A vascular prosthesis produced by the process of claim 13 or 14.17.A device comprising a material as defined in claim 15.18.A device as defined in claim 17, wherein said device is to be used in contact with an animal's tissue.", "19.A device as defined in claim 18, wherein said tissue is blood tissue.", "20.A device as defined in claim 18 or 19, wherein said device is selected from the group consisting of: An implant, a prosthesis, an artificial organ, a stent, a cardiac valve, an apparatus contacting blood during an extra-corporal blood circulation, an apparatus contacting blood during a dialysis treatment or any other material with surfaces coming in contact with blood.", "21.Use of a device as defined in any one of claims 17 to 20 for preventing thrombosis.", "22.A material suitable for contact with a living tissue characterized in having a surface that is resistant to neutrophil adhesion, platelet adhesion and activation but that enhances the development of fibroblasts and endothelial cells.", "23.A material as defined in claim 22 which is plasma-treated ePTFE with grafted PRC.", "24.A device comprising a material as defined in claim 22 or 23.25.A device as defined in claim 24, wherein said device is selected from the group consisting of: An implant, a prosthesis, an artificial organ, a stent, a cardiac valve, an apparatus contacting blood during an extra-corporal blood circulation, an apparatus contacting blood during a dialysis treatment or any other material with surfaces coming in contact with blood.", "26.Use of a device as defined in claim 24 or 25 for preventing thrombosis." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Approximately 350 000 synthetic vascular prostheses are implanted each year as arterial bypasses in Western countries and Japan.", "Made of Dacron™ (polyethyleneterephthalate) or microporous Teflon™ (expanded polytetrafluoroethylene, or ePTFE), they perform quite well in large-diameter vessels under high flow conditions but have a low patency rate when used as small-diameter bypasses (i.e., coronaries) or as medium-diameter bypasses in low-flow conditions and high-resistance locations (i.e., leg arteries).", "(The patency rate relates to a biomaterial's ability to remain pervious to blood flow when replacing an artery).", "A study on 398 ePTFE vascular prostheses implanted as medium-diameter bypasses and retrieved following complications revealed that 65% of them had to be explanted due to thrombosis.", "(1 ) Endothelial cell lining is the natural blood-compatible surface that covers the inside surface of blood vessels and heart chambers, and it is recognized to be the best possible hemocompatible surface.", "The principal techniques that have been proposed in the past to promote endothelial cell adhesion and spreading onto the luminal surfaces of vascular prostheses are: (1) endothelial cell seeding; (2) graft pre-treatment with endothelial cell mitogens; (3) increasing structural porosity; and (4) coating the surface with a protein matrix.", "While several attempts have been made, the complete endothelial cell coverage of the blood-exposed inside prosthetic surfaces has never been observed in humans.", "It is therefore evident that increasing the patency rate of arterial prostheses is a high priority in current cardiovascular research.", "It has been shown in many instances that surface modification through plasma treatment is one of the most promising techniques that may be used to improve specific hemocompatibility and general biocompatibility of polymeric materials.", "(2,3) One advantage of the plasma treatment is that it allows formation of a covalent bond between the modifier and the surface to be treated, unlike other approaches where a coating is deposited inside the prosthesis without formation of a covalent bond (4,5) , where the bond is ionic (6) or where the bioactive substance is incorporated in the biomaterial.", "(7) Following to the covalent bonding of the modifier to the surface of the material, made possible by plasma treatment, a molecule chosen for its biocompatibility properties can be covalently attached to the modifier either directly or through a spacer molecule.", "Clinically, the increased stability of a surface treatment involving plasma treatment could result in improved limb salvage rates and a possible future use in small-diameter coronary bypass operations.", "This, in turn, could minimize the incidence of re-operation for patients and lower social healthcare costs related to surgical interventions of this type.", "Plasma treatment of biomaterials is not the only process that can yield covalent bonding of molecules possessing desirable hemocompatibility and/or biocompatibility.", "However, the processes that are described in the relevant prior art frequently involve several steps.", "(8-14) To date, most of the experiments performed to improve the performance of biomaterial surfaces have been based on flat specimens of material, particularly polymeric sheets.", "Unfortunately, few experiments involving the use of plasma to activate the surface have been carried out directly on commercial prostheses with a non-planar geometry.", "Experiments done at the University of Washington, (2,15-20) which consisted mainly in treating commercial Dacron™ arterial prostheses with a plasma treatment in a tetrafluoroethylene (TFE) gas environment so as to coat the internal surfaces of the prostheses with potentially more biocompatible CF 3 groups, were a precursor to the development of the new commercial Radio Frequency Glow Discharge (RFGD)-treated vascular grafts.", "(17) Yet, despite the existence of many surface treatment systems for biomaterial surfaces, few have resulted in the treatment, as opposed to the coating, of the internal surface of tubular devices.", "(21-23) Over the last 30 years, a number of studies have been designed to test the ability of plasma to either treat or coat the surfaces of biomedical devices with a view to enhancing their biocompatibility or to modulate the interactions of polymers and tissue when prostheses are implanted in situ.", "(16,24-30) Plasma polymerizable gas has been used to coat various substrates with a thin polymeric layer, (15,17-19,24,31,32) while non-polymerizable gas has been applied to treat substrate surfaces.", "(33-38) Despite the fact that many approaches have been developed to coat or treat the surface of biomaterials, the introduction of amino groups through RFGD treatment is particularly interesting for two reasons.", "First, amino groups are known to facilitate cellular spreading on a biomaterial surface.", "(39-41) Second, amino groups react readily with other chemical functional groups, allowing for the attachment of specific molecules through interactions sufficiently strong to prevent the leaching of these molecules by the blood stream.", "(42) For example, amino groups can be used to ionically immobilize heparin, a well-known polysaccharide anticoagulant.", "(22) While many efforts have been made in the last 20 years in the field of cardiovascular prosthetic devices to develop surfaces with improved human blood compatibility, an acceptable long-term blood-compatible synthetic material has yet to be achieved.", "The complexity of the interactions between blood and synthetic materials constitutes a major interfacial problem in this respect." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Surface treatments allow a modulation of the surface properties of a biomaterial to enhance its interfacial reaction with a biological environment.", "Low pressure plasma surface treatments are particularly advantageous in the design and development of new biocompatible materials, since they permit surface modifications without altering the bulk of the materials' properties.", "(43) In the context of the present invention, they are particularly useful for modulating different tissue/biomaterial interface properties, but their ultimate utility may reside in their significant improvement of the hemocompatibility of vascular prostheses.", "Thus, in accordance with the present invention, there is provided a novel process for modifying the surface properties of a material that is to be placed in contact with living tissues.", "This process comprises: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on the surface of the material; Activating a molecule with an activator to produce a reactive molecular species capable of forming strong bonds with the reactive groups created on the surface of the material; and Contacting the reactive molecular species with the reactive groups created on the surface of the material to form strong bonds.", "Preferably, the strong bonds formed are covalent bonds.", "In a preferred embodiment, the molecule selected for activation is compatible with living tissue, including human tissue.", "In a specific embodiment the novel process of the present invention further comprises the following features: the plasma is ammonia Radio Frequency (RF) plasma and the reactive groups are amine groups; the molecule selected for activation is chosen from the group consisting of choline, heparin, and other molecules known to those skilled in the art for their hemocompatibility, the activator is phosphoryl chloride (POCl 3 ) and the reactive molecular species is the oxyphosphorodichlorinated derivative of the molecule; and the strong bonds are phosphoamide-type covalent bonds.", "In a preferred embodiment of the specific embodiment above, the RF power is between about 5 watts to about 500 watts and is applied for a time of about 10 seconds to about 30 minutes at a pressure of about 50 mtorr to about 5 torr.", "Those skilled in the art will recognize that care has to be taken in order to avoid combinations of time and power that would deliver excessive amounts of energy to the treated surface.", "Such conditions lead to a microscopically brittle surface.", "Preferably, the RF power is about 20 watts and is applied for a time of about 250 seconds at a pressure of about 300 mtorr.", "In yet another preferred embodiment, the RF power is about 15 watts and is applied for a time of about 100 seconds at a pressure of about 250 mtorr.", "Ideally, the third step of the process is performed within about 2 hours of the first step.", "The novel process of the present invention is particularly suitable for an implantable material selected from the following group: expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), polyethylenes, polyesters, polypropylenes and polyurethanes.", "In a preferred embodiment, the implantable material is ePTFE, and the treated surface is the internal surface of a vascular prosthesis.", "In a more preferred embodiment, the vascular prosthesis has an inner diameter of about 1 to 30 mm.", "The present invention further comprises the materials produced by the novel process, as well as devices comprising these materials, including any of the following: an implant, a prosthesis, an artificial organ, a stent, a cardiac valve, an apparatus contacting blood during an extra-corporal blood circulation, an apparatus contacting blood during a dialysis treatment or any other material with surfaces coming in contact with blood.", "Additionally, the present invention is meant to cover the use of any such device, particularly to prevent thrombosis in an animal, including a human being.", "By controlling the RF power, the ammonia pressure, and the treatment duration, the atomic substitution percentage may be modulated and up to 15% of the surface atoms may be substituted with nitrogen, as probed by X-ray photoelectron spectroscopy (XPS).", "On the surface, different chemical species are present after the treatment, such as amine and imine groups.", "Storage in air for up to 80 days shows a defluorination of the surface and a slow decrease of the surface nitrogen concentration during this storage.", "Ammonia RF Plasma treatment can be successfully used to uniformly treat the internal surface of an ePTFE arterial prosthesis despite the fact that care has to be taken to prevent a significant surface reorganization upon exposure to the atmosphere.", "(44,45) The ammonia RF plasma-treated internal surfaces of vascular prostheses may be reacted with a substance whose molecular structure can render the prostheses more hemocompatible and non-thrombogenic when in use.", "The molecule is preferably activated phosphorylcholine (PRC) or any other molecule with similar properties, wherein the two hydroxyl groups on the phosphate moiety have been substituted with chlorine to form the dichloro derivative of the molecule.", "This dichloro derivative readily reacts with the amino groups resulting from the ammonia RF plasma treatment of the inside surfaces of the vascular prostheses, allowing for the effective anchoring of the molecule.", "It is an object of the present invention to provide a novel process for producing a material suitable for contact with a living tissue.", "This material is characterized in having a surface that is resistant to neutrophil adhesion and thrombosis but that enhances the development of fibroblasts and endothelial cells.", "The process may be used, for example, to treat the inside surfaces of devices, such as vascular prostheses, so as to enhance their hemocompatibility and make them less thrombogenic when in use.", "A further object of the invention is therefore to provide devices treated with the novel process of the present invention.", "Other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings." ], [ "FIELD OF THE INVENTION The present invention relates to a novel process for modifying the surface properties of a material that is suitable for contact with living tissue comprising: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on said surface; activating a molecule to produce a reactive molecular species capable of forming strong bonds with the reactive groups created on the surface of the material; and contacting the reactive molecular species with the reactive groups created on the surface of the material to form strong bonds.", "The invention further encompasses the materials produced by this process as well as devices that are comprised of these process-modified materials, such as vascular prostheses.", "BACKGROUND OF THE INVENTION Approximately 350 000 synthetic vascular prostheses are implanted each year as arterial bypasses in Western countries and Japan.", "Made of Dacron™ (polyethyleneterephthalate) or microporous Teflon™ (expanded polytetrafluoroethylene, or ePTFE), they perform quite well in large-diameter vessels under high flow conditions but have a low patency rate when used as small-diameter bypasses (i.e., coronaries) or as medium-diameter bypasses in low-flow conditions and high-resistance locations (i.e., leg arteries).", "(The patency rate relates to a biomaterial's ability to remain pervious to blood flow when replacing an artery).", "A study on 398 ePTFE vascular prostheses implanted as medium-diameter bypasses and retrieved following complications revealed that 65% of them had to be explanted due to thrombosis.", "(1) Endothelial cell lining is the natural blood-compatible surface that covers the inside surface of blood vessels and heart chambers, and it is recognized to be the best possible hemocompatible surface.", "The principal techniques that have been proposed in the past to promote endothelial cell adhesion and spreading onto the luminal surfaces of vascular prostheses are: (1) endothelial cell seeding; (2) graft pre-treatment with endothelial cell mitogens; (3) increasing structural porosity; and (4) coating the surface with a protein matrix.", "While several attempts have been made, the complete endothelial cell coverage of the blood-exposed inside prosthetic surfaces has never been observed in humans.", "It is therefore evident that increasing the patency rate of arterial prostheses is a high priority in current cardiovascular research.", "It has been shown in many instances that surface modification through plasma treatment is one of the most promising techniques that may be used to improve specific hemocompatibility and general biocompatibility of polymeric materials.", "(2,3) One advantage of the plasma treatment is that it allows formation of a covalent bond between the modifier and the surface to be treated, unlike other approaches where a coating is deposited inside the prosthesis without formation of a covalent bond(4,5), where the bond is ionic(6) or where the bioactive substance is incorporated in the biomaterial.", "(7) Following to the covalent bonding of the modifier to the surface of the material, made possible by plasma treatment, a molecule chosen for its biocompatibility properties can be covalently attached to the modifier either directly or through a spacer molecule.", "Clinically, the increased stability of a surface treatment involving plasma treatment could result in improved limb salvage rates and a possible future use in small-diameter coronary bypass operations.", "This, in turn, could minimize the incidence of re-operation for patients and lower social healthcare costs related to surgical interventions of this type.", "Plasma treatment of biomaterials is not the only process that can yield covalent bonding of molecules possessing desirable hemocompatibility and/or biocompatibility.", "However, the processes that are described in the relevant prior art frequently involve several steps.", "(8-14) To date, most of the experiments performed to improve the performance of biomaterial surfaces have been based on flat specimens of material, particularly polymeric sheets.", "Unfortunately, few experiments involving the use of plasma to activate the surface have been carried out directly on commercial prostheses with a non-planar geometry.", "Experiments done at the University of Washington,(2,15-20) which consisted mainly in treating commercial Dacron™ arterial prostheses with a plasma treatment in a tetrafluoroethylene (TFE) gas environment so as to coat the internal surfaces of the prostheses with potentially more biocompatible CF3 groups, were a precursor to the development of the new commercial Radio Frequency Glow Discharge (RFGD)-treated vascular grafts.", "(17) Yet, despite the existence of many surface treatment systems for biomaterial surfaces, few have resulted in the treatment, as opposed to the coating, of the internal surface of tubular devices.", "(21-23) Over the last 30 years, a number of studies have been designed to test the ability of plasma to either treat or coat the surfaces of biomedical devices with a view to enhancing their biocompatibility or to modulate the interactions of polymers and tissue when prostheses are implanted in situ.", "(16,24-30) Plasma polymerizable gas has been used to coat various substrates with a thin polymeric layer,(15,17-19,24,31,32) while non-polymerizable gas has been applied to treat substrate surfaces.", "(33-38) Despite the fact that many approaches have been developed to coat or treat the surface of biomaterials, the introduction of amino groups through RFGD treatment is particularly interesting for two reasons.", "First, amino groups are known to facilitate cellular spreading on a biomaterial surface.", "(39-41) Second, amino groups react readily with other chemical functional groups, allowing for the attachment of specific molecules through interactions sufficiently strong to prevent the leaching of these molecules by the blood stream.", "(42) For example, amino groups can be used to ionically immobilize heparin, a well-known polysaccharide anticoagulant.", "(22) While many efforts have been made in the last 20 years in the field of cardiovascular prosthetic devices to develop surfaces with improved human blood compatibility, an acceptable long-term blood-compatible synthetic material has yet to be achieved.", "The complexity of the interactions between blood and synthetic materials constitutes a major interfacial problem in this respect.", "SUMMARY OF THE INVENTION Surface treatments allow a modulation of the surface properties of a biomaterial to enhance its interfacial reaction with a biological environment.", "Low pressure plasma surface treatments are particularly advantageous in the design and development of new biocompatible materials, since they permit surface modifications without altering the bulk of the materials' properties.", "(43) In the context of the present invention, they are particularly useful for modulating different tissue/biomaterial interface properties, but their ultimate utility may reside in their significant improvement of the hemocompatibility of vascular prostheses.", "Thus, in accordance with the present invention, there is provided a novel process for modifying the surface properties of a material that is to be placed in contact with living tissues.", "This process comprises: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on the surface of the material; Activating a molecule with an activator to produce a reactive molecular species capable of forming strong bonds with the reactive groups created on the surface of the material; and Contacting the reactive molecular species with the reactive groups created on the surface of the material to form strong bonds.", "Preferably, the strong bonds formed are covalent bonds.", "In a preferred embodiment, the molecule selected for activation is compatible with living tissue, including human tissue.", "In a specific embodiment the novel process of the present invention further comprises the following features: the plasma is ammonia Radio Frequency (RF) plasma and the reactive groups are amine groups; the molecule selected for activation is chosen from the group consisting of choline, heparin, and other molecules known to those skilled in the art for their hemocompatibility, the activator is phosphoryl chloride (POCl3) and the reactive molecular species is the oxyphosphorodichlorinated derivative of the molecule; and the strong bonds are phosphoamide-type covalent bonds.", "In a preferred embodiment of the specific embodiment above, the RF power is between about 5 watts to about 500 watts and is applied for a time of about 10 seconds to about 30 minutes at a pressure of about 50 mtorr to about 5 torr.", "Those skilled in the art will recognize that care has to be taken in order to avoid combinations of time and power that would deliver excessive amounts of energy to the treated surface.", "Such conditions lead to a microscopically brittle surface.", "Preferably, the RF power is about 20 watts and is applied for a time of about 250 seconds at a pressure of about 300 mtorr.", "In yet another preferred embodiment, the RF power is about 15 watts and is applied for a time of about 100 seconds at a pressure of about 250 mtorr.", "Ideally, the third step of the process is performed within about 2 hours of the first step.", "The novel process of the present invention is particularly suitable for an implantable material selected from the following group: expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), polyethylenes, polyesters, polypropylenes and polyurethanes.", "In a preferred embodiment, the implantable material is ePTFE, and the treated surface is the internal surface of a vascular prosthesis.", "In a more preferred embodiment, the vascular prosthesis has an inner diameter of about 1 to 30 mm.", "The present invention further comprises the materials produced by the novel process, as well as devices comprising these materials, including any of the following: an implant, a prosthesis, an artificial organ, a stent, a cardiac valve, an apparatus contacting blood during an extra-corporal blood circulation, an apparatus contacting blood during a dialysis treatment or any other material with surfaces coming in contact with blood.", "Additionally, the present invention is meant to cover the use of any such device, particularly to prevent thrombosis in an animal, including a human being.", "By controlling the RF power, the ammonia pressure, and the treatment duration, the atomic substitution percentage may be modulated and up to 15% of the surface atoms may be substituted with nitrogen, as probed by X-ray photoelectron spectroscopy (XPS).", "On the surface, different chemical species are present after the treatment, such as amine and imine groups.", "Storage in air for up to 80 days shows a defluorination of the surface and a slow decrease of the surface nitrogen concentration during this storage.", "Ammonia RF Plasma treatment can be successfully used to uniformly treat the internal surface of an ePTFE arterial prosthesis despite the fact that care has to be taken to prevent a significant surface reorganization upon exposure to the atmosphere.", "(44,45) The ammonia RF plasma-treated internal surfaces of vascular prostheses may be reacted with a substance whose molecular structure can render the prostheses more hemocompatible and non-thrombogenic when in use.", "The molecule is preferably activated phosphorylcholine (PRC) or any other molecule with similar properties, wherein the two hydroxyl groups on the phosphate moiety have been substituted with chlorine to form the dichloro derivative of the molecule.", "This dichloro derivative readily reacts with the amino groups resulting from the ammonia RF plasma treatment of the inside surfaces of the vascular prostheses, allowing for the effective anchoring of the molecule.", "It is an object of the present invention to provide a novel process for producing a material suitable for contact with a living tissue.", "This material is characterized in having a surface that is resistant to neutrophil adhesion and thrombosis but that enhances the development of fibroblasts and endothelial cells.", "The process may be used, for example, to treat the inside surfaces of devices, such as vascular prostheses, so as to enhance their hemocompatibility and make them less thrombogenic when in use.", "A further object of the invention is therefore to provide devices treated with the novel process of the present invention.", "Other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1 is a schematic representation of the Radio Frequency Glow Discharge treatment system used in the process of the invention.", "FIG.", "2 shows the XPS spectrum of the inside surface of an untreated ePTFE vascular prosthesis and the XPS spectrum of the inside surface of an ammonia plasma treated ePTFE vascular prosthesis.", "FIG.", "3 shows the effect on the surface composition of treatments performed at 300 mtorr of ammonia by varying the power (20, 40 and 50 W) and treatment times (10, 120 and 275 seconds).", "FIG.", "4 shows the relationship between the pressure within the chamber and the modification of the ePTFE prosthetic surface.", "FIG.", "5 shows the percentage of nitrogen and the N/C and F/C ratios measured on the surface as a function of ammonia plasma treatment time.", "FIG.", "6 shows the derivatization reaction of a plasma treated PTFE film with chlorobenzaldehyde.", "FIG.", "7 shows the relationship between the atomic surface concentration of phosphorous grafted onto the surface (% P) and the intensity of the N+ peak at 402 eV.", "FIG.", "8 shows the cellular growth determined by the fluorescence of DNA strands using specific markers with untreated and treated prostheses as well as with “control” cell cultures.", "FIG.", "9 reproduces SEM micrographs showing the degree of accumulation of blood platelets within (a) a prosthesis treated in accordance with the process of the invention and (b) an untreated prosthesis.", "FIG.", "10 shows the quantification of the adherent blood platelets on untreated and treated prostheses.", "FIG.", "11 shows the quantification of the adherent neutrophils on untreated and treated prostheses.", "FIG.", "12 shows the influence of a treated and untreated prostheses on the clotting time.", "FIG.", "13 shows the influence of a treated and untreated prostheses on the thrombogenic index.", "FIG.", "14 displays microscopic slides of the growth of fibroblasts within a prosthesis treated in accordance with the process of the invention ((a) and (c)) and an untreated prosthesis ((b) and (d)).", "FIG.", "15 SEM images at 20× and 2000× magnification for ANBIOPA™-treated prostheses and commercially-available untreated prostheses after a one-month implantation in a canine model: a) ANBIOPA™-treated, 20×; b) untreated, 20×; c) ANBIOPA™-treated, 2000×; and d) untreated, 2000×.", "DESCRIPTION OF A PREFERRED EMBODIMENT For the present purposes, we give the following definitions of the terms “untreated” and “treated”.", "“Untreated” means a commercially available vascular prosthesis, to which the inventors did not apply any surface modification treatment.", "“Treated” means a commercially available vascular prosthesis to which the inventors applied the surface modification procedure hereafter described.", "The term “animal” as used herein is meant to signify human beings, primates, domestic animals (such as horses, cows, pigs, goats, sheep, cats, dogs, guinea pigs, mice, etc.)", "and other mammals.", "Generally, this term is used to indicate living creatures having highly-developed vascular systems.", "I. Pre-Treatment of the Inside Surface of Vascular Prostheses with Ammonia Radio Frequency (RF) Plasma Experimental Method (The Experimental Method is described with reference to FIG.", "1.)", "Materials Microporous ePTFE vascular prostheses 6 (6 and 10 mm ID) with a fibril average length of 25 microns were used, and the method is described in relation to a single prosthesis.", "A microporous ePTFE arterial prosthesis (length 3 to 10 cm) was first washed with high purity methanol for 10 minutes and vacuum-dried.", "This procedure was performed to remove any contaminants which may have been present on the surface of the “as-received” prosthesis.", "Following this washing procedure, two small holes were punctured at one end of the prosthesis, facing each other, and a polypropylene monofilament (5-0—Ethicon) 3 was inserted through both holes and a knot tied loosely above the prosthesis.", "The other end of the monofilament was rolled around a pulley 5, itself located on the horizontal shaft of an electric motor 36 to allow a vertical displacement of the sample prosthesis inside the Pyrex™ tube 2 of the RFGD treatment system.", "Gravity caused the vascular prosthesis to move down inside the Pyrex™ tube.", "For vascular grafts longer than 3 cm, it was preferable, in addition to the aforementioned knot and its attachment via the monofilament to the pulley, to attach a polypropylene monofilament 3 at the bottom of the vascular prosthesis and to attach the monofilament to the pulley by first letting the monofilament run down the tube and then up to the pulley.", "With this set-up, the motor can pull on the prosthesis when it is going down the Pyrex™ tube, and therefore friction of the prosthesis on the inside wall of the tube ceases to be a problem when moving the prosthesis down the tube.", "Prior to closing the system, a container and its cap were placed inside a bell jar 1 to collect and store the prosthesis in an argon atmosphere following the RFGD treatment, thereby minimizing any contact of the active sites on the surface of the prosthesis with air.", "Plasma Equipment As shown in FIG.", "1, the RFGD treatment system used can be generally represented by the numeral 10.This system is basically comprised of a Pyrex™ glass tube (400 mm long, 12.5 mm i.d.", "for 10 mm i.d.", "prostheses and 8 mm i.d.", "for 6 mm i.d.", "prostheses) 2 with an internal diameter 4 slightly greater than the external diameter of the prosthesis 6, a configuration which allows for RFGD generation on the internal side of the prosthesis only.", "Indeed, the path length between the external side of the prosthesis 6 and the Pyrex™ tube 4 is not long enough to allow the electron-induced cascade ionization which is necessary for plasma formation.", "(11) Therefore, the treatment of prostheses of various diameter requires the selection of a Pyrex™ tube of the appropriate diameter.", "As mentioned above, the criteria of selection is that no plasma is ignited on the outside surface of the prosthesis during its treatment.", "A space of about 1 mm all around the outside diameter of the prosthesis and the inside wall of the Pyrex™ glass tube 2 has proven to be appropriate.", "The Pyrex™ glass tube 2 has an inlet for gas injection 8 located 5 cm below its upper end.", "Three capacitively coupled copper electrodes 12a, 12b and 12c, spaced from 3 to 5 cm from each other (depending on the length of the prosthesis to treat), are attached to the middle of the tube 2.The vacuum within the chamber was created by a turbo-molecular pump 14 with a pumping speed of 60 liters/s (Model TCP015, Balzers Pfeiffer) which, in turn, is connected to a mechanical pump 16 (Model 1402, Welch Scientific Company).", "This set-up allows a pressure of close to 4×10−5 torr to be reached.", "During treatment, pressure is kept constant within the chamber by means of a capacitance pressure gauge/electromechanical valve controller 18 (type 250-1A, MKS Instruments) connected to an electromechanical valve 20 (type 248A-00200SV, MKS Instruments) and a capacitance manometer 22 (Baratron type 626A01TAE, MKS Instruments).", "Finally, gaseous molecules are excited through a radio-frequency field made by an RF generator 24 (YAESU HF transceiver FT-840, YAESU) that transmits the power, through a power meter 26, to an automatic matching network (Smartuner SG-230, SGC) 28 which then couples the RF power with an antenna made of three copper electrodes 12a-c. Electrodes 12a and 12c, located at both ends of the antenna, are connected to a ground (not shown) while the RF power is brought through the central electrode 12b.", "The treatment can be operated in a RF power range from 10 to 100 W at 13.56 MHz.", "Plasma Treatment Initially, all valves on the system are closed.", "After the introduction of the prosthesis into the bell jar 1, valve 30 is opened and the mechanical pump 16 is activated to reach a vacuum of approximately 10−3 torr.", "As soon as this pressure is attained inside the system, valve 30 is closed, valve 32 is opened and after a few seconds the turbo-molecular pump 14 is activated.", "After about one minute, valve 34 is opened and pumping inside the bell jar continued until it reaches a vacuum greater than 5×10−5 torr.", "The chamber is then isolated from the turbo pump 14 by closing valve 34 and high purity ammonia is introduced into the bell jar 1 via electromechanical valve 20 and, when the pressure is above 0.1 torr, the bell jar 1 is pumped by the mechanical pump 16 alone, through valve 30.The electromechanical valve controller 18 automatically regulates the amount of gas entering the chamber and the pressure, read by the capacitance gauge 22, settles within 1 minute to the value selected on the capacitance gauge/electromechanical valve controller.", "Typically, this configuration allows plasma generation within a pressure range of 10−3 to 1 torr.", "With the above parameters established, the plasma is ignited by turning the radio-frequency generator 24 on.", "Sufficient time is allowed for the matching network 28 to reach a standing wave ratio (SWR) signal of between 1 and 1.15, as read on the power meter 26 in order to provide optimal matching of the power input to the discharge.", "RFGD treatments are then performed using a static treatment: the prosthesis 6 is initially located just under the inlet 8 while the plasma parameters are adjusted, it is then moved from this position in the Pyrex™ glass tube 2 into the middle of the lighted area (between the three electrodes 12a, 12b and 12c) using the electric motor 36, and is maintained within the plasma for predetermined periods of time.", "Prostheses longer than 10 cm could be treated by slowly moving the prostheses across the lighted area using electric motor 36, resulting in similar grafting efficiencies.", "(45) At the end of the treatment, the Radio-Frequency generator 24 is turned off and the gas flow stopped by closing the electromechanical valve 20 via its controller 18.The mechanical pump 16 is kept in operation to remove residual gaseous ammonia from the bell-jar 1 until a pressure of approximately 50 mtorr is reached.", "It is then flushed with argon for 2 minutes by carefully opening valve 38.This is done in order to allow the excited species resulting from the plasma treatment to reach their ground state.", "After isolating the mechanical pump 16 from the bell-jar by closing valve 30, argon flow is continued inside the chamber to bring the bell jar back to atmospheric pressure.", "The prosthesis 6 is then lowered to the bottom of the Pyrex™ tube 2, the bell jar 1 is quickly opened and the prosthesis 6 is disconnected from the polypropylene microfilament 3 and put inside a capped container.", "It is then placed in a glove box purged with nitrogen where a small sample is taken for XPS studies in order to assess the success of the ammonia treatment.", "The prosthesis is then prepared for the anchoring process as described below.", "II.", "Anchoring of Molecules Mimicry of the non-thrombogenic surface of the erythrocyte has been advocated as the starting point for the development of non-thrombogenic biomaterials.", "Since phosphorylcholine (PRC) is a major component of the outer surface of the erythrocyte, materials containing it would be expected to be non-thrombogenic.", "(46-50) Moreover, it is known that PRC groups attached to polymer surfaces improve hemocompatibility.", "(51-57) In order to enhance the hemocompatibility and non-thrombogenicity of the interior surfaces of vascular prostheses, prostheses which had been pre-treated with plasma (as described above) were made to react with activated analogues of molecules such as phosphorylcholine (PRC).", "Since the ultimate goal is the implantation of such prostheses into the human body, the molecules must be solidly attached to the inside walls of the prostheses.", "Consequently, it is necessary to form strong bonds (preferably, covalent bonds) between the molecules and the amine groups created by the plasma treatment rather than to rely on the formation of ionic bonds or adsorption phenomenon to secure the molecules onto the inside surfaces of the prostheses.", "Additionally, it is desirable that any chemical treatment of this type be simple and inexpensive.", "The general structure of the molecules is represented below: Direct reaction between PRC and the amino groups present on the surface of the plasma-treated prostheses results in ionic bonds and/or surface adsorption neither of which, as previously stated, is sufficiently strong or stable for the applications contemplated.", "It is therefore necessary to enhance the reactivity of these molecules by synthesizing reactive analogues so that they may partake in the creation of phosphoamide-type covalent bonds with the amino groups on the surface of the ammonia plasma pre-treated prostheses.", "In this way, the molecules may be anchored effectively to the interior surface of the prostheses.", "In order to achieve the desired covalent reaction, the dichlorinated derivative RPO(Cl)2 of the target molecule RPO(OH)2 must first be synthesized.", "The synthesis of the dichlorinated derivatives of the molecules suitable for reaction with the amino groups on the inside surface of the pre-treated vascular prostheses may be achieved in at least two ways, as revealed diagrammatically below with PRC.", "In the first pathway, PRC, which is available commercially in the form of a calcium salt, is reacted with an acidic resin(58) to eliminate the calcium.", "The product is then made to react with phosphorus trichloride or pentachloride (or any halogenation agent such as phosgene, oxalyl chloride, thionyl chloride) to obtain the dichlorinated derivative.", "An alternative synthesis is shown in the second pathway.", "Here, the dichlorinated derivative of PRC is synthesized directly from phosphoryl chloride and choline in a single step.", "In a preferred embodiment, the preferred pathway is the second one described above which has the advantage of requiring a lesser number of steps.", "This experimental protocol takes into account such factors as reproducibility and toxicity, as well as feasibility on an industrial scale.", "Experimental Method The dichloro derivative of PRC is synthesized by the addition of phosphoryl chloride (POCl3) in chloroform solution to a chloroform solution containing choline with glass beads (3 mm diameter) at 0° C. (ice bath) in a closed glass flask, under a dry nitrogen stream to exclude the presence of water and displace the HCl resulting from the reaction.", "The mixture is then stirred at 0° C. for 1 hr, and at room temperature for 12 hours to allow the reaction to proceed, as indicated by dissolution of the choline crystals and the formation of a oily phase.", "A slight excess of choline is used to ensure that all the phosphoryl chloride is consumed during the activation reaction.", "Immediately following the synthesis of the dichloro derivative of PRC, the 3 cm prostheses are immersed at room temperature in the abovementioned glass flask and agitation is continued for an additional 2 hours.", "In the case of longer vascular prostheses (>3 cm) a simple system was designed with a 1-liter commercial glass reactor with an outlet at the bottom.", "In this case, following the 12 hours of mechanical agitation, the chloroform layer at the bottom was removed via the bottom outlet of the flask and was replaced by fresh chloroform in order to remove any remaining POCl3.A PTFE diaphragm pump is then used to circulate the activated PRC solution from the bottom of the reactor, through a section of PTFE tubing, then through a vertical glass tube in which the prosthesis is held.", "Finally, a section of PTFE tubing brings back the solution in the glass reactor for recirculation.", "When the grafting of the activated PRC has occurred, the prostheses are washed many times in water to eliminate all traces of chlorine that may remain on the phosphoamide moiety of the anchored molecule (see Step 2 of the complete reaction, below) as well as all traces of reactants and solvent.", "The prostheses are then dried under vacuum overnight before being analyzed by XPS and static SIMS to determine the level of grafting.", "Overall Reaction for ePTFE Vascular Prostheses Inner Surface Treatment The process of the present invention may be extended to bind other molecules that are highly compatible with biological tissue, such as blood tissue.", "In fact, preliminary results(63) indicate that the internal surface of commercial ePTFE arterial prostheses that were previously treated with the ammonia plasma treatment described herein can be homogeneously coated, using a grafting method different from the one presented here, with polyethylene-glycol (PEG), which is another highly biocompatible molecule.", "Characterization of Ammonia RFGD Plasma Treated Surfaces and PRC-Grafted Surfaces X-Ray Photoelectron Spectroscopy (XPS) Analyses X-ray photoelectron spectroscopy (XPS) was used to characterize the modified surfaces.", "XPS spectra were recorded using a PHI 5600-ci spectrometer (Physical Electronics).", "A monochromatic aluminum X-ray source (1486.6 eV) was used to record the survey and high resolution spectra of the prosthetic surface.", "For both type of analyses, the power source was kept at 100 W to minimize X-ray induced modifications of the specimen and the detection was performed at 45° with respect to the surface.", "The pressure within the XPS analytical chamber was kept at about 5×10−9 torr.", "Ammonia RFGD Plasma Treated ePTFE Surfaces After RFGD ammonia plasma treatments, specimens were longitudinally opened in a chamber under nitrogen positive pressure and their internal surfaces were analyzed by XPS within 30 minutes of the RFGD treatment.", "Surface atomic concentration of oxygen was always lower than 5%.", "In order to collect data of the surface modifications along the longitudinally-treated length of a specimen, the XPS analysis area was moved along the length of the prosthesis.", "To validate that the RFGD system resulted in the treatment of the internal surface only, XPS analyses of the external surface of a prosthetic specimen treated at 20 W, for 250 seconds with an ammonia pressure of 300 mtorr were conducted.", "The data (not shown) confirmed that the external surface was not modified by the glow discharge when the internal diameter of the Pyrex™ tube was chosen to be only slightly larger (1 mm) than the external diameter of the prosthesis.", "Under the pressure and power conditions used, the Debye length did not allow the formation of a stable plasma between the external surface of the prosthesis and the internal surface of the tube, because the ionization processes were insufficient to compensate for the recombination processes.", "(60) All results are discussed by considering the percentage of atomic concentration of nitrogen on the surface, and the N/C, F/C and O/C surface concentration ratios.", "It was observed that because of experimental and instrumental uncertainties, atomic concentration values above 5% are reliable to ±1% (absolute) or better.", "As gaseous ammonia was used to create the plasma, it is clear that the percentage of nitrogen would be a valid indicator of the extent of the RFGD treatment.", "Moreover, the N/C ratio probes the extent of nitrogen atom insertion onto the ePTFE surface, while the F/C ratio monitors the level of defluorination and is indicative of the amount of fluorine atoms substituted by other atoms and/or multiple bond formations within or between the chains.", "Relationship Between the Nature of the Nitrogen-Containing Species Grafted onto the Surface of the Vascular Prostheses and Treatment Parameters With a view to understanding the relationship between the nature of the nitrogen-containing species grafted onto the surface of the RFGD treated ePTFE samples, different tests were performed to investigate the effects of variable parameters such as gas pressure, RF power and treatment time.", "Prostheses measuring 3 cm in length were treated under conditions where two of the variables were kept constant while the third was varied.", "As may be seen in FIG.", "2, the RF plasma treatment allowed a surface atomic nitrogen concentration of up to 15% to be reached, with an average N/C ratio of up to 0.3, at 20 W for 250 seconds and with an ammonia pressure of 300 mtorr.", "This represents a significant improvement over previously-reported results following treatment aimed at incorporating nitrogen on ePTFE surfaces, where published data indicates a percentage of nitrogen between 1 and 3%(26,42,61) or a N/C ratio between 0.06 and 0.18(28-43,59).", "A possible explanation for the present results may reside in the cylindrical configuration of the reactor chosen maximizes the efficiency of the treatment(59).", "The three surface characterization parameters (F/C, N/C and % N) indicate that the surface modifications induced on any given prosthesis are fairly homogenous: for example, (13±1)% atomic nitrogen concentration on a 7 cm-segment of a 6 mm ID prosthesis (250 s, 300 mtorr, 15 W).", "The degree of homogeneity required for subsequent grafting of the hemocompatible molecule and obtaining of an improved performance of the vascular prostheses can only be properly determined by hemocompatibility tests.", "However, the observed relative uniformity of surface modifications following ammonia plasma treatment provides a good starting point for the subsequent grafting.", "FIG.", "3 shows the effect on the surface composition of treatments performed by keeping the pressure constant (300 mtorr) while varying the power (20, 40 and 50 W) and treatment times (10, 120 and 275 seconds).", "It appears that the nitrogen concentration reached its highest value for a RF power of 40 W. The N/C ratio measured for the experiments performed for 275 seconds was significantly higher than that measured following treatment for 120 seconds.", "On the other hand, the F/C ratio was identical for these two treatment times for all RF powers tested.", "Thus, this data suggests that, despite leading to a higher nitrogen surface concentration, longer treatment times did not promote increased fluorine substitution.", "This result could mean that different treatment times lead to the formation of different chemical species on the surface.", "As may be seen in FIG.", "4, increasing the ammonia pressure from 100 to 600 mtorr (RF power kept at 20 W and treatment time at 10 s) lead to only a slight increase of the nitrogen content: from lower to higher pressures, this content ranged from 5 to 7%, respectively and the N/C ratio which ranged from 0.14 to 0.16.However, it is clear that the defluorination of the surface is highly pressure-dependent; the F/C ratio decreased from 1.45, for a 100 mtorr pressure, to 1.1 at 600 mtorr.", "Therefore, it is evident that despite a similar nitrogen insertion, the chemical moieties formed on the surface depend upon the pressure within the chamber.", "Further experiments were carried out to determine the time required to reach an approximately constant surface concentration of nitrogen.", "The nitrogen concentration reached a plateau after a treatment time of 160 seconds, as shown by the percentage of nitrogen and the N/C ratio measured on the surface (FIG.", "5).", "Defluorination of the surface, despite showing a more complex behavior, appears to follow a similar pattern.", "The above results have shown that the pressure, the duration and the RF power of the treatment all have an effect on the chemical composition as seen from the surface characterization parameters given by the survey spectra.", "In particular, these results raise the possibility of presence of different nitrogen species as a function of the above three treatment parameters.", "As several nitrogen containing species may be formed upon the ammonia RFGD plasma treatment, we have performed surface derivatization in order to quantify the percentage of the nitrogen present as NH2 species.", "The results of the surface derivatization on model surfaces (not shown) demonstrated that chlorobenzaldehyde specifically reacts with the amine moieties as depicted in FIG.", "6.Therefore, the chlorine XPS signal of the derivatized surface allows the determination of the surface amine concentration as described by d'Agostino et al.(62).", "Using the surface derivatization technique, it was possible to show that up to 45% of all the nitrogen species grafted onto the surface during the plasma treatment are amine moieties (as shown in Table 1 by % of N0 present as NH2).", "In addition, the data presented in the Table 1 also indicate that the surface amine concentration may be controlled by changing the plasma treatment duration.", "For example, the surface concentration of NH2 on the surface goes from 3.6% for a 50 second treatment duration to 6.0% when the surface is treated for 250 seconds.", "However, too long exposures to the plasma environment should be avoided to minimise the occurrence of polytetrafluoroethylene chain scission or formation of double bonds.", "(64) TABLE 1 Quantification of amine groups on plasma-treated PTFE film Plasma treatment % of N0 Surface concentration Duration F/C N/C % N0 present as NH2 of NH2 250 s 0.499 0.269 14.3 42 6.0 100 s 0.626 0.229 11.9 42 5.0 50 s 0.865 0.249 11.6 31 3.6 One skilled in the art will appreciate that the surface amine groups can be created on the surface by methods different from the one presented here without departing from the spirit of the present invention.", "For example, amine groups can be created on the surface not only by using an ammonia plasma, but also by using plasmas of N2H4 (hydrazine),(65,66) aliphatic amines,(67) coating-forming gases such as allylamine,(68-71) to name a few non-restrictive possibilities.", "U.S. Pat.", "No.", "6,159,531,(72) for example, briefly discusses the relative merits of these gases in view of their use as precursors to surface amine groups.", "Various methods of activating the surface can also be used, other than the continuous capacitively coupled RF plasma used here.", "Without departing from the spirit of the present invention, these include, but are not limited to, pulsed plasma,(68,70,73) inductively coupled RF plasma,(74-76) corona discharge,(77,78) microwave plasma(79,80) and UV irradiation.", "(81,82) As an example, one can read U.S. Pat.", "No.", "5,922,161,(83) which teaches the use of such treatments for the partial surface oxidation of polymers.", "Furthermore, without departing from the spirit of the present invention, one skilled in the art will appreciate that surface reactive groups other than amine can successfully react with the oxyphosphorodichlorinated derivative form of phosphorylcholine or another so-activated molecule.", "Hydroxyl and thiol groups(84-87) are two non-restrictive examples of such surface groups.", "Conversely, different pathways, in addition to the one proposed here, can be followed to obtain an activated form of phosphorylcholine, as one skilled in the art knows.", "These additional activation methods include, but are not limited to, using reagents such as PCl5, PCl3, PBr5, PBr3, PI3, SOCl2, SOBr2, COCl2, COBr2, (COCl)2, (COBr)2 to directly activate phosphorylcholine through the formation of at least a reactive P-halogen bond.", "(87,88) Storage in Atmosphere of the Ammonia Plasma Treated Prostheses Surface Atomic Concentrations as a Function of the Storage Time in Air The surface atomic concentration of nitrogen on the surface of a treated prosthesis tends to slowly decrease (from 14% initially to 12% after 80 days of storage in air or a decrease of N/C from 0.3 to 0.2) and oxygen uptake is also observed (O/C increased from 0.05 to 0.3 after 20 days)(45).", "Therefore, it is preferable to perform the grafting of the molecule shortly after the plasma treatment, preferably within two hours(45).", "PRC Grafted ePTFE Surfaces The atomic surface compositions were also determined by XPS spectroscopy.", "First, the survey spectra clearly showed that there is phosphorus and chlorine atom integration in approximately similar concentrations, as expected from the molecular structure of PRC (chlorine is present as the counterion of the N(CH3)3+ moiety of PRC).", "The extent of grafting of the PRC was therefore estimated from the percentage of phosphorus detected by XPS.", "Moreover, FIG.", "7 shows that the N 1 s High Resolution XPS spectra clearly indicates that there is a good correlation between the trimethylammonium peak at 402 eV (a peak which is not present on an ammonia plasma-treated prosthesis) and the percentage of phosphorus grafted onto the prosthesis.", "Indeed, there is a linear relation between the extent of PRC grafting (characterized by % P), and the trimethylammonium peak, +N(CH3)3 (FIG.", "7).", "The C 1 s peak (not shown) for each of the three methyl groups from the trimethylammonium moiety, +N(CH3)3, followed the same trend and provided further indication of the successful grafting of PRC onto the surface.", "The PRC grafting on PTFE films was also confirmed by static SIMS (static Secondary Ion Mass Spectroscopy).", "This method identifies molecules adsorbed on a surface by recording the mass spectra of atomic or molecular particles which are emitted when a surface is bombarded by energetic primary particles.", "The secondary ions emitted from the surface result from the fragmentation of the initial surface molecules and are only detected under their ionized state (positive and negative state).", "The peaks are given in Dalton (molecular weight—gmol−1).", "Thus, from an analysis of these spectra it is possible to determine the initial structure of the molecules grafted onto the surface.", "The spectra of a PRC-grafted PTFE film (not shown) gave the following fragments and confirmed the PRC grafting.", "122.1 78.8 PO3− 58.1 +N—(CH3)3 62.8 PO2− 43 +N—(CH3)2 O—CH2—CH+ 29 +N—CH3 15 CH3+ III.", "Sterilization of Inner-Surface Modified Vascular Prostheses For the present method of surface modification to be useful, it is required that sterilization not remove the grafted molecules.", "A very widely used method for sterilization is autoclaving.", "At least two possible methods of autoclaving may be contemplated: 1) sterilization under vapor pressure: 120° C., 15 PSI, 20 min; and 2) dry sterilization: short duration and high temperature (180° C., 1 h) or for a longer duration at a lower temperature (150° C., 2 h).", "According to scientific literature, PRC derivatives are stable when subjected to the first sterilization procedure.", "In addition, ePTFE prostheses are more frequently sterilized in this manner.", "This method is recognized and used by hospitals.", "Sample PRC-grafted films and prostheses were placed in a sterilization bag and sealed.", "A sterilization marker, a piece of adhesive tape, was placed on the bag; this indicator darkens when sterilization is successful.", "Following the sterilization procedure, the sample prostheses were analyzed by XPS to determine whether their compositions and the distribution of the phosphorus atoms had been altered.", "The results are summarized in Table 2 below.", "TABLE 2 Effect of Sterilization on PRC-Grafted Prostheses Sterilization Before After After 6 months % P 5 ± 1 4 ± 1 3.4 ± 0.6* This data was obtained from a 8 cm-segment of a 6 mm ID prosthesis (yielding 60 data points) whereas the other values were estimated from a shorter segment (1 cm, yielding 6 data points), hence the difference in the standard deviations.", "Despite the slight decrease in the percentage of phosphorus, which is taken as a measure of the amount of PRC on the surface because it is otherwise absent, PRC grafting was found to be stable when subjected to sterilization under vapor pressure and the distribution of the grafting on the surface remained homogeneous.", "The surface concentrations were stable after 6 months and the distribution remained homogeneous (the same was observed for intermediate times of 1 and 2 months for different prostheses).", "Consequently, the sterilization procedure most practiced by hospitals does not appear to adversely modify the quantity and distribution of the grafted molecules on the PRC-treated vascular prostheses.", "IV.", "In Vitro Testing of Inner-Surface Modified Vascular Prostheses A. Non-Toxicity of Ammonia RFGD Plasma Treated Prostheses To verify the non-toxicity of ammonia plasma treated prostheses, we used the diffusion test which determines whether cellular growth is inhibited by the biomaterial by using a coloring agent specific to DNA (Hoechst).", "The elution test, also known as “extract dilution”, was also used; it determines whether there is inhibition of cellular growth by looking for an extract of biomaterial.", "a) Diffusion Test Human endothelial cells derived from an umbilical cord were cultured in a 24-well plate (1×104 cells/well), taking care to pre-treat each well with gelatin before use.", "The cells were allowed to grow in Medium 199 containing 10% FBS, heparin (90 g/ml), L-Glutamine (2 mM), penicillin G (100 I.U./ml), streptomycin sulfate (100 μg/ml), amphotericin B (250 ng/ml) and ECGS (20 μg/ml).", "Circular prosthetic samples measuring 7 mm in diameter from both treated and untreated prostheses were placed in the 24 wells.", "The cultures were incubated for 3 days with 5% CO2 in a humid atmosphere.", "Subsequently, the cultures were washed, lysed, treated with Hoechst #33358 and finally transferred to another plate designed for fluorescent studies.", "Readings were taken with a Bio-Tek FL600 fluorometer, and the quantity of DNA calculated from a calibration curve.", "At the same time, cells were cultured in wells without prosthetic samples (control).", "b) Elution Test Two samples, one from a pre-treated prosthesis and the other from an untreated prosthesis, were incubated in M199 for 7 days.", "Twenty-four hours before the end, endothelial cells were cultured in a 24-well plate.", "At the seventh day, the cells were cultured in the M199 medium used with the prostheses.", "(It is important to note here that, in contrast to what is done in the Diffusion Test, only the medium is used; the sample prosthetic devices are set aside following the incubation period.)", "The endothelial cells are incubated for 3 days under the same conditions as those for the Diffusion Test and analyzed in the same way (fluorometry).", "Table 3 shows the results for the Diffusion and Elution Tests on untreated (virgin) prostheses, on ammonia plasma treated prostheses and on control samples.", "(All experiments were done in duplicate, series A and B.)", "These results demonstrate that there are no significant variations in the level of cellular proliferation.", "It may therefore be concluded that ammonia plasma treated ePTFE vascular prostheses are no more toxic than untreated ePTFE vascular prostheses.", "TABLE 3 Results of in vitro Cellular Cultures Analyzed by Fluorometry Ammonia Plasma Untreated Prostheses treated Prostheses Control Test (104) (104) (104) Diffusion Series A 54 ± 1 52 ± 2 Series B 43.7 ± 0.5 43.1 ± 0.5 40 ± 1 Elution Series A 32.9 ± 0.4 33 ± 1 Series B 28 ± 1 32 ± 1 31 ± 2 Cytotoxicity of the PRC-Grafted Vascular Prostheses As discussed above, toxicity tests performed on prostheses following plasma treatment but prior to the anchoring of the molecules revealed that the plasma-treated prostheses were non-toxic, or at least no more toxic than virgin prostheses.", "The next logical step was to determine whether prostheses which had been completely treated by the process of the present invention were also non-toxic to cells, and particularly to endothelial cells which naturally line the internal surface of blood vessels.", "Endothelial cells are currently used to verify the toxicity of certain products as well as to check the efficiency of sterilization.", "In cytotoxicity testing, the prostheses are not in direct contact with endothelial cells and the incubation period is 3 days at 37° C. Cellular growth is determined by the fluorescence of DNA strands using specific markers.", "Testing was repeated several times in order to obtain the best statistical average for the cell culture results.", "To gain a better appreciation of the effect of PRC grafting on the cells, tests were also performed on untreated prostheses as well as with “control” cell cultures.", "The histogram in FIG.", "8 clearly shows that treated prostheses have no more effect on cell growth than do untreated prostheses and neither of them have a significant effect on the growth of the cells.", "Hence, the prostheses treated with the process of the invention are non-cytotoxic and in fact seem to slightly favor endothelial cell growth.", "These findings are promising given that endothelial cells must line the internal surfaces of the prostheses.", "B) Blood Platelets Adhesion Adhesion and Aggregation of Blood Platelets The adhesion and aggregation of blood platelets on prosthetic devices has a direct effect on hemocompatibility, since the platelets are involved in the coagulation cascade.", "Indeed, the adherence of platelets on the internal surfaces of prostheses may prevent the adherence of endothelial cells (the natural lining of blood vessels), and may result in thrombosis if the accumulation is too important.", "In vitro studies were performed several times in the following manner.", "Following the centrifugation of human blood, the platelet-rich plasma was extracted and placed on prostheses for one hour at 37° C. The prostheses were then treated with different solutions before analyses by SEM.", "FIG.", "9 shows SEM photographs of treated and untreated prosthetic devices.", "A comparison of the two photographs reveals that the untreated prosthesis has a significantly greater accumulation and activation of platelets than the prosthesis that had been treated with the process of the invention.", "Hence, the treated prosthesis appears to prevent much more effectively the accumulation of blood platelets.", "Quantification of Blood Platelets After centrifugation of human blood, the platelet-rich plasma (5*104 platelets/μl count by a hemocytometer) was extracted, radiolabelled with 51Cr and placed on prostheses for two hours at 37° C. The prostheses were then washed three times with sterile PBS before analyses and the amount of platelet uptake was evaluated by a gamma scintillation counter adjusted to 270-370 keV.", "This test was performed 3 times with human blood from six donors (FIG.", "10).", "In all cases, no significant difference in the adherent platelets was noticed for untreated and treated prostheses.", "C) Neutrophils Neutrophils are characteristic of the inflammatory response of an individual to a foreign material.", "After centrifugation of human blood, the leukocyte-rich suspension was extracted, radiolabelled with 111In and placed on prostheses for two hours at 37° C. The prostheses were then analyzed and the adherent neutrophils were evaluated by a gamma scintillation counter adjusted to 260-480 keV.", "This test was performed three times with blood from six different donors (FIG.", "11).", "For every individual, there were fewer adherent neutrophils on the treated prosthesis than on the untreated prosthesis.", "Thus, it appears that the inflammatory response was reduced by the PRC grafted on the prosthesis.", "D) Clotting Time Platelet adhesion to biomaterials is often used as an indication of blood compatibility, but a more clinically relevant issue is whether the adherent platelets are able to promote clot formation.", "Indeed, clotting events require the presence of other blood elements such as fibrinogen to catalyze the clotting cascade.", "Longer clotting times are taken as indicative of a better thrombogenic resistance of a prosthesis in contact with blood.", "Normal human blood was collected into a citrate anticoagulant tube and a 1-ml sample was deposited onto the surface of the prosthesis.", "The clotting time was determined visually: the blood was deemed to have clotted when there was no longer a movement of the blood in response to a tilting motion of the sample prosthesis.", "This test was performed three times with blood from six human donors, on untreated and treated (PRC-grafted) prostheses (FIG.", "12).", "As observed in FIG.", "12, clotting times were significantly longer on PRC-grafted prostheses.", "Thus, it appears that the treated prosthesis is less thrombogenic when compared to the untreated prosthesis.", "E) Thromboelastography Another important measure of the thrombogenic potential of a material is the evolution of the elastic properties of whole blood clots that form on the surface of the material.", "For a given material in contact with blood, the thrombogenicity index takes into account the coagulation time as well as the elasticity of the blood clot.", "Surfaces with good blood compatibility should exhibit low thrombogenecity indexes, as measured by a thromboelastograph.", "This technique uses a steel piston placed in a cuvette having a 1-mm clearance between the piston and the inner surface of the cuvette.", "Blood is placed inside the cuvette.", "As coagulation proceeds, the elasticity of the forming blood clot varies and these changes are detected by the torsion wire to which the piston is suspended.", "This is converted to an electric signal which is sent to a chart recorder.", "The resulting trace is called the thromboelastogram (TEG), which records the elasticity of the formed blood clot.", "This test was performed using the blood of six human donors, on samples of untreated and treated prostheses (FIG.", "13).", "Comparison of the index of thrombogenic potential for treated and untreated prostheses showed a lower thrombogenic potential for the PRC-grafted prostheses.", "Values ranged from 17 to 45% lower, except for one individual where the value was 30% higher.", "Thus, the treated prostheses improved the non-thrombogenic potential of the initial material.", "F) Fibroblast Cultures The development of an endothelium, which is the natural hemocompatible surface, on the surface of prostheses would render them more hemocompatible.", "Fibroblasts, which are cells capable of differentiation, are commonly used for in vitro hemocompatibility tests.", "By testing the adsorption of fibroblasts, one can measure the ability of a material to act as a viable substrate for the adsorption and spreading of cells.", "The adsorption and spreading of cells represent conditions that are necessary for the development of the endothelium.", "Fibroblasts were placed in direct contact with the prostheses in order to observe the ability of cells to develop on different materials.", "The cells were allowed to grow for 7 days at 37° C. The fibroblasts were then observed through a microscope using two coloring agents: the Hoëscht reagent marked the nucleus (allowing the cells to be counted) and rhodamine dyed the actin filaments in the cytoplasm (allowing the shape of the cells to be determined).", "As shown in FIG.", "14, the development of fibroblasts on the surface of the prostheses treated by the process of the present invention was greatly enhanced in comparison to the untreated prosthesis: the number of nuclei is greater, and the shape of the fibroblasts is more elongated, which shows that the cells adhere to and develop on the surface.", "G) Endothelial Cell Cultures Endothelial cells are the cells that naturally line the internal surface of blood vessels; they therefore constitute the ideal hemocompatible surface.", "Because endothelial cells are known to be more fragile and sensitive than fibroblasts, this test is more severe.", "Endothelial cells were cultured following the same protocol as that used for the fibroblasts.", "The incubation period was 3 days at 37° C. The development of endothelial cells was greatly enhanced on the prosthesis treated by the process of this invention than on the untreated one.", "FIG.", "14 shows the optical microscope image obtained after using the Hoëscht reagent to mark the nucleus of the endothelial cells.", "The number of cell nucleus is clearly more important on the treated prosthesis, as is the spreading of the cells, as observed by SEM (FIG.", "14).", "V. In Vivo Testing of Inner-Surface Modified Vascular Prostheses The in vivo response of the ANBIOPA™-treated prostheses was tested in a canine model.", "The implantation period was one month and six dogs received a prosthesis: three received an untreated Gore-Tex™ ePTFE prosthesis while the three others received an ANBIOPA™-treated prosthesis.", "Surgery (The following procedure, while described in relation to a single dog, was used on all six dogs that participated in the trial.)", "After a 24-hour fast, a conditioned mongrel dog having a weight of between 20 and 25 kg was pre-anaesthetized by intra-veinous administration of atropin sulfate (0.05 mg/kg) and acepromazin maleate (0.1 mg/kg).", "The dog's abdomen was shaved and disinfected by an application of Hibitane and Proviodine.", "The dog was then placed on a surgical table in decubitus position, anaesthetized by intra-veinous administration of Penthothal (10 mg/kg) and then ventilated with a mix of air and isofuran to maintain anaesthesia.", "After abdominal incision, the sub-renal aorta was localized and cleared of surrounding structures before the collateral arteries were tied.", "Prior to clamping, heparin (0.5 mg/kg) was injected as an anticoagulant.", "The aorta was clamped upstream and downstream of a 5 cm segment in an aorto-aortic position and this segment was replaced with a 6 mm diameter vascular prosthesis (untreated commercially available prothesis or ANBIOPA™-treated prosthesis) of the same length by termino-terminal anastomosis using a Surgilene™ monofilament.", "The dog was stitched and butorphanol tartrate (0.2 mg/kg) was subcutaneously injected as an analgesic for three days after surgery.", "Following surgery, the dog was fed with a standard diet until explantation of the prosthesis one (1) month later.", "Image Analysis SEM observation on the explanted prostheses shows clear differences between the untreated commercially available prostheses and the ANBIOPA™-treated prostheses (FIG.", "15).", "At low magnification, the surface of an ANBIOPA™-treated prosthesis remains uniform with a smooth surface and is almost deposit free (FIG.", "15a).", "Further observations at higher magnification reveal a coating consisting of several layers of red blood cells (FIG.", "15c).", "The lack of activated platelets and fibrin clot on an ANBIOPA™-treated prosthesis surface suggests an anti-thrombogenic property.", "On the other hand, an untreated commercially available prosthesis shows important deposits on the surface at low magnification (FIG.", "15b) with deposits mainly composed of a network of activated platelets and red blood cells trapped in a fibrin clot (FIG.", "15d), thus providing evidence of thrombus formation.", "Although the present invention has been described by way of preferred embodiments thereof, it can be modified without departing from the spirit and nature of the subject invention, as defined in the appended claims.", "LIST OF REFERENCES 1.R.", "Guidoin, N. 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[ [ "Central Fastening Element for an Axially Symmetric Gas Spring", "In a central fastening element for an axially symmetric, vehicle gas spring, which includes a bellows that has central bores or cutouts in the region of its end faces, the fastening element being fixed to the vehicle body, protruding from the surroundings of the attachment point in a direction normal to it, and being encompassed by the bores or cutouts.", "The fastening element includes a shaped stud or a shaped cap, the maximum outer diameter of the stud or the cap being at least less than one fifth of the maximum outer diameter of the gas-spring bellows.", "A device may be provided which allows a gas spring to be installed in a simple manner and allows the spring bellows to rotate with respect to the suspension and/or the vehicle body during the initial installation and/or the initial operation." ], [ "1-9.", "(canceled) 10.A central fastening element for an axially symmetric, vehicle gas spring including a bellows having one of central bores and cutouts in a region of an end face, the fastening element configured to be fixed to a vehicle body and to protrude from surroundings of an attachment point in a normal direction and to be encompassed by the one of the bores and cutouts, comprising: at least one of a stud and a cap, one of the at least one of the stud and the cap shaped, a maximum outer diameter of the one of the stud and the cap at least less than one fifth of a maximum outer diameter of the bellows, the one of the stud and the cap including at least one necked-down portion having a diameter less than the maximum outer diameter of the one of the stud and the cap, the end face elastic in a zone of contact with the one of the stud and the cap.", "11.The fastening element according to claim 10, wherein a base of the bellows axially and radially surrounds the one of the stud and the cap without a sealing joint.", "12.The fastening element according to claim 10, further comprising an integrated supply line.", "13.The fastening element according to claim 10, wherein the cap includes an internal thread, the stud having an external thread, the cap substantially completely surrounding the stud, the cap arranged to one of directly and indirectly contact the vehicle body.", "14.The fastening element according to claim 10, wherein the stud is arranged to be screwed into a tapped hole of the vehicle body, the stud having a surface oriented in a direction normal to an axis of the stud, the surface arranged to one of directly and indirectly rest against the vehicle body.", "15.The fastening element according to claim 10, wherein the stud is arranged to be screwed into a nut fastened to the vehicle body.", "16.The fastening element according to claim 10, wherein a base of the bellows includes at least two superposed layers, at least one of the layers made of metal, at least one of the layers made of metal.", "17.The fastening element according to claim 10, wherein a base of the bellows is arranged to rest against at least two surface sections of the one of the stud and the cap oriented in axially opposite directions.", "18.The fastening element according to claim 10, wherein a base of the bellows is arranged to rest against a surface section of the one of the stud and the cap oriented in a direction of the vehicle body and against the vehicle body in a region of attachment of the one of the stud and the cap.", "19.The fastening element according to claim 10, wherein the cap includes an internal thread, the stud having an external thread, the cap substantially completely surrounding the stud, the cap one of directly and indirectly contacting the vehicle body.", "20.The fastening element according to claim 10, wherein the stud is screwed into a tapped hole of the vehicle body, the stud having a surface oriented in a direction normal to an axis of the stud, the surface one of directly and indirectly resting against the vehicle body.", "21.The fastening element according to claim 10, wherein the stud is screwed into a nut fastened to the vehicle body.", "22.The fastening element according to claim 10, wherein a base of the bellows rests against at least two surface sections of the one of the stud and the cap oriented in axially opposite directions.", "23.The fastening element according to claim 10, wherein a base of the bellows rests against a surface section of the one of the stud and the cap oriented in a direction of the vehicle body and against the vehicle body in a region of attachment of the one of the stud and the cap.", "24.The fastening element according to claim 10, wherein the fastening element is fixed to a vehicle body.", "25.The fastening element according to claim 10, wherein the fastening element protrudes from the surroundings of the attachment point in the normal direction.", "26.The fastening element according to claim 10, wherein the fastening element is encompassed by the one of the bores and cutouts.", "27.A device, comprising: an axially symmetric, vehicle gas spring including a bellows having one of central bores and cutouts in a region of an end face; and a fastening element configured to be fixed to a vehicle body and to protrude from surroundings of an attachment point in a normal direction, the fastening element encompassed by the one of the bores and cutouts, the fastening element including at least one of a stud and a cap, one of the stud and the cap shaped, a maximum outer diameter of the one of the stud and the cap at least less than one fifth of a maximum outer diameter of the bellows, the one of the stud and the cap including at least one necked-down portion having a diameter less than the maximum outer diameter of the one of the stud and the cap, the end face elastic in a zone of contact with the one of the stud and the cap." ], [ "<SOH> BACKGROUND INFORMATION <EOH>Such a device is described in European Published Patent Application No.", "0 123 171.The gas spring includes, inter alia, a vertical U-bellows and two axially symmetric bodies terminating it on the upper and lower sides.", "On the upper side, the gas spring is fixed to the vehicle body, e.g., to the vehicle frame, with the aid of a bolt.", "This bolt is seated in a bore of the frame and is screwed to the axially symmetric upper body, a so-called plug.", "The U-bellows itself is fastened to the axially symmetric, upper member with the aid of a tension band.", "In this case, the diameter of the plug is as large as the inner diameter of the U-bellows.", "This type of fastening requires that the bolthead on the upper side of the frame be accessible during installation.", "This makes it difficult to automate the installation of the gas spring.", "The gas spring tends to twist during installation, and while compressing and rebounding.", "Therefore, the tightening torque of the bolt must be selected to be high enough to prevent the connection between the gas spring and the vehicle body from loosening or releasing in response to vibrations and shock.", "It is an object of the present invention to provide a fastening element, which renders simple installation possible and allows the bellows to rotate with respect to the suspension and/or the vehicle frame during the initial installation and/or the initial operation." ], [ "<SOH> SUMMARY <EOH>This object may be achieved by providing a fastening element as described herein.", "To this end, the fastening element may include a shaped stud or a shaped cap, the maximum outer diameter of the stud or the cap being at least less than one fifth of the maximum outer diameter of the gas-spring bellows.", "The cap or the stud has at least one necked-down portion or waist, whose outer diameter is less than the above-mentioned, maximum diameter of the cap or stud.", "The end face is elastic in the zone in which it comes into contact with the stud or the cap.", "The central fastening element may be attached to the vehicle body prior to the installation of the gas spring and may protrude from the vehicle body in a direction normal to it.", "During installation, the gas spring is attached to, for example, the strut of the suspension and, e.g., pressed, together with it, against the shaped stud or the shaped cap.", "In the following, the term, stud, also includes the shaped cap.", "The elastic zone of the end face of the gas spring comes into contact with the stud in response to being slid up, and then engages with it in the manner of a snap fastener.", "The stud has a necked-down portion, which is encompassed by the bore or the cutout in a form-locked or force-locked manner.", "This type of fastening may allow the gas spring to be installed in an automated manner.", "In this case, and during initial operation, the gas spring may rotate on the stud, which means that the twisting of the U-bellows and the increased wear caused by it are prevented.", "The keyed connection between the stud and spring prevents vibrations and shock from detaching the fastening element.", "The base of the gas-spring bellows surrounds the stud axially and radially.", "Consequently, the gas spring is fixed in position in the axial and radial directions after installation.", "For example, it may not detach when the vehicle is jacked up, or in response to a pressure drop.", "The gas spring may be attached at its upper and lower ends in the same manner.", "Supply lines may be run through the fastening element into the interior of the gas spring.", "At least some regions of the base of the gas-spring bellows may be made of an elastic material, e.g., rubber.", "This may allow the base to act as a damping layer.", "The base may also be made up of multiple layers, e.g., a rubber layer and a metallic layer.", "In this case, the gas-spring bellows is attached to the metallic layer.", "The metallic layer is simultaneously used for increasing the strength of the gas-spring base.", "Several rubber and metallic layers may also be combined.", "To attach the gas spring, e.g., this rubber layer is compressed between two surface sections of the stud oriented in opposite, axial directions, or between a surface section oriented in the direction of the vehicle body, and the vehicle body.", "If the base of the gas spring is made out of multiple layers, the inner layer may be, for example, a metallic layer.", "The gas-spring bellows is then attached to this.", "This layer may be constructed in such a manner, that it supports the rubber layer and holds the position of the gas spring on the stud.", "Further details of the fastening element according to the present invention are set forth below in the subsequent description of several schematically represented, example embodiments." ], [ "FIELD OF THE INVENTION The present invention relates to a central fastening element for an axially symmetric, vehicle gas spring, which includes a bellows that has central bores or cutouts in the region of its end faces, the fastening element being fixed to the vehicle body, protruding from the surroundings of the attachment point in a direction normal to it, and being encompassed by the bores or cutouts.", "BACKGROUND INFORMATION Such a device is described in European Published Patent Application No.", "0 123 171.The gas spring includes, inter alia, a vertical U-bellows and two axially symmetric bodies terminating it on the upper and lower sides.", "On the upper side, the gas spring is fixed to the vehicle body, e.g., to the vehicle frame, with the aid of a bolt.", "This bolt is seated in a bore of the frame and is screwed to the axially symmetric upper body, a so-called plug.", "The U-bellows itself is fastened to the axially symmetric, upper member with the aid of a tension band.", "In this case, the diameter of the plug is as large as the inner diameter of the U-bellows.", "This type of fastening requires that the bolthead on the upper side of the frame be accessible during installation.", "This makes it difficult to automate the installation of the gas spring.", "The gas spring tends to twist during installation, and while compressing and rebounding.", "Therefore, the tightening torque of the bolt must be selected to be high enough to prevent the connection between the gas spring and the vehicle body from loosening or releasing in response to vibrations and shock.", "It is an object of the present invention to provide a fastening element, which renders simple installation possible and allows the bellows to rotate with respect to the suspension and/or the vehicle frame during the initial installation and/or the initial operation.", "SUMMARY This object may be achieved by providing a fastening element as described herein.", "To this end, the fastening element may include a shaped stud or a shaped cap, the maximum outer diameter of the stud or the cap being at least less than one fifth of the maximum outer diameter of the gas-spring bellows.", "The cap or the stud has at least one necked-down portion or waist, whose outer diameter is less than the above-mentioned, maximum diameter of the cap or stud.", "The end face is elastic in the zone in which it comes into contact with the stud or the cap.", "The central fastening element may be attached to the vehicle body prior to the installation of the gas spring and may protrude from the vehicle body in a direction normal to it.", "During installation, the gas spring is attached to, for example, the strut of the suspension and, e.g., pressed, together with it, against the shaped stud or the shaped cap.", "In the following, the term, stud, also includes the shaped cap.", "The elastic zone of the end face of the gas spring comes into contact with the stud in response to being slid up, and then engages with it in the manner of a snap fastener.", "The stud has a necked-down portion, which is encompassed by the bore or the cutout in a form-locked or force-locked manner.", "This type of fastening may allow the gas spring to be installed in an automated manner.", "In this case, and during initial operation, the gas spring may rotate on the stud, which means that the twisting of the U-bellows and the increased wear caused by it are prevented.", "The keyed connection between the stud and spring prevents vibrations and shock from detaching the fastening element.", "The base of the gas-spring bellows surrounds the stud axially and radially.", "Consequently, the gas spring is fixed in position in the axial and radial directions after installation.", "For example, it may not detach when the vehicle is jacked up, or in response to a pressure drop.", "The gas spring may be attached at its upper and lower ends in the same manner.", "Supply lines may be run through the fastening element into the interior of the gas spring.", "At least some regions of the base of the gas-spring bellows may be made of an elastic material, e.g., rubber.", "This may allow the base to act as a damping layer.", "The base may also be made up of multiple layers, e.g., a rubber layer and a metallic layer.", "In this case, the gas-spring bellows is attached to the metallic layer.", "The metallic layer is simultaneously used for increasing the strength of the gas-spring base.", "Several rubber and metallic layers may also be combined.", "To attach the gas spring, e.g., this rubber layer is compressed between two surface sections of the stud oriented in opposite, axial directions, or between a surface section oriented in the direction of the vehicle body, and the vehicle body.", "If the base of the gas spring is made out of multiple layers, the inner layer may be, for example, a metallic layer.", "The gas-spring bellows is then attached to this.", "This layer may be constructed in such a manner, that it supports the rubber layer and holds the position of the gas spring on the stud.", "Further details of the fastening element according to the present invention are set forth below in the subsequent description of several schematically represented, example embodiments.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 schematically illustrates the attachment of a gas spring to a vehicle body.", "FIG.", "2 schematically illustrates an alternative manner of attaching a gas spring to a vehicle body.", "FIGS.", "3 to 7 schematically illustrate alternative example embodiments of the fastening elements for the arrangements illustrated in FIGS.", "1 and 2.DETAILED DESCRIPTION FIG.", "1 illustrates the attachment of a gas spring 70 to a vehicle body 5, or to a strut.", "In this case, gas spring 70, e.g.", "an air spring, is made up of, inter alia, a U-bellows 72, which is attached to a damper plate 50.In this connection, damper plate 50 forms the base of gas-spring bellows 72.A fastening element 10 is mounted to vehicle body 5, e.g., to the vehicle frame, a body member, or a reinforced part of the body paneling.", "On vehicle body 5, a bolt 15 is situated in a concave and, optionally, axial asymmetric depression 6.For example, this bolt 15 may be welded on.", "Bolt 15 has a thread 16.A mushroom-shaped cap 19 is screwed onto this thread 16 via an internal thread 21.Cap 19 is axially asymmetric and has a cylindrical outer contour 23 in the upper region.", "In the middle region, the contour changes, e.g., discontinuously, into a circular arc 24.This transition is referred to in the following as 35 necked-down portion 25, undercut, or waist.", "In this case, the center point of circular arc 24 is, e.g., on the radius of cylindrical outer contour 23 or below it.", "Curved section 24 is referred to below as a toroid.", "Cap 19 changes continuously or discontinuously between toroid 24 and a conical section 28 and tapers to approximately three quarters of its upper diameter.", "Instead of being semicircular, the contour of the partial cross-section of toroid 24 may also be triangular.", "In the case of this contour, toroid 24 has a frustoconical lateral surface, whose imaginary apex points to the center of the U-bellows interior.", "For example, the bottom surface of this frustoconical apex discontinuously joins up with necked-down portion 25 to form a barb-shaped undercut.", "The frustoconical apex may also be an imaginary enveloping surface for a series of resilient barbs positioned about cap 19.After installation, these barbs sink into optionally sharp-edged groove 54 of damper plate 50, while elastically recovering.", "For example, a hexagonal recess 26 is centrally situated in cap 19, cf.", "FIG.", "3.Damper plate 50 is positioned around cap 19.The upper side of damper plate 50 is designated as outer side 59 and the lower side is designated as inner side 58.Cylindrical damper plate 50 is made of rubber and has a central bore 52, which is smaller that the maximum outer diameter of cap 19.A recess in the form of a circular groove 54 is situated approximately halfway up bore 52.This has a contour opposite to that of toroid 24 and therefore may ensure that damper plate 50 grips cap 19 from behind.", "In this connection, the region of damper plate 50 between groove 54 and outer side 59 may be compressed.", "Bore 52 changes into a chamfer 57 at the upper end face of damper plate 50.Damper plate 50 is also chamferred at the lower end face.", "The thickness of damper plate 50 in the middle region approximately corresponds to the length of cap 19, but the thickness in the outer region is reduced to approximately two thirds of the overall height.", "In this connection, inner side 58 is approximately planar.", "A metallic disk 62 and a thin rubber layer 63 are situated on the outer side 59 of damper plate 50.The two parts may be cemented on or vulcanized on.", "A machined disk 64, whose central region is formed in the shape of a frustoconical shell in the direction of the center, is situated on inner side 58.The top edge of the frustoconical shell may be oriented, e.g., along groove 54 and may thus reinforce the rubber layer between bore 52 and machined disk 64.This improves, for example, the rear gripping action.", "For example, this machined disk 64 may be vulcanized into damper plate 50.At least two bores 65 are situated in damper plate 50.These are cylindrical in the region of upper metallic disk 62, thin rubber layer 63 and damper plate 50.The diameter of bores.", "65 in machined disk 64 is adapted to cover bolts 68.Gas-spring bellows 72 is attached to damper plate 50 by these cover bolts 68.Damper plate 50 rests against vehicle body 5 in the region of vehicle-frame depression 6, and in the region of rubber sheet 63.FIG.", "2 illustrates an alternative manner of attaching gas spring 70 to vehicle body 5.As in the exemplary embodiment illustrated in FIG.", "1, a bolt 15 having external thread 16 is situated in a depression 6 of vehicle body 5.A cap 19 is screwed onto a bolt 15.In this case, this cap 19 has two circumferential toroids 24 at the transition from conical part 28 to cylindrical outer contour 23, the outer diameter being greater in the region of the cap 19 of the two circumferential toroids than in the region of cylindrical outer contour 23.The spacing of the two toroids 24 is, for example, approximately as large as half the difference of the diameter of a toroid 24 and cylindrical outer contour 23.Conical part 28 of cap 19 is tapered in the downward direction.", "Damper plate 50 of gas spring 70 is a cylindrical, axially symmetric rubber sheet, which has a central bore 52.Bore 52 has two circumferential grooves 54.These have a contour opposite to that of toroids 24 and therefore may ensure that damper plate 50 elastically grips cap 19 from behind.", "In this connection, the region of damper plate 50 between grooves 54 is optionally compressed.", "In the region of the cap, the thickness of damper plate 50 corresponds to approximately two-thirds of the length of cap 19.In this case, outer side 59 of damper plate 50 is planar.", "Situated in damper plate 50 is a, e.g., metallic, machined disk 80, which may be vulcanized in.", "It has a central bore 82.Near the bore 82, the thickness of this machined disk 80 is approximately twice as much as in the remainder of machined disk 80.The diameter of bore 82 is approximately one third of the overall diameter of machined disk 80.A recess 83 is situated approximately in the center of this cylindrical bore 82.Damper plate 50 engages with the former and thus allows axial, form-locked engagement.", "Bore 82 of machined disk 80 surrounds damper plate 50 to provide stiffness in the region of grooves 54.Machined disk 80 has at least two countersunk bores 86 in the outer region.", "Cover bolts 68 are situated in these countersunk bores 86, cf.", "FIG.", "1.In the exemplary embodiment illustrated in FIG.", "2, metallic disks 62, 64 and/or rubber disks 63 illustrated in FIG.", "1 may also be vulcanized into damper plate 50.In the two exemplary embodiments, gas-spring bellows 72 pre-mounted to damper plate 50 is installed in the designated position.", "In this connection, central bore 52 of damper plate 50 is aligned with and centered on cap 19.Chamfer 57 of bore 52 is then seated on conical part 28 of cap 19.For installation purposes, damper plate 50 is pushed, optionally together with spring bellows 72, against cap 19, and pushed over toroid(s) 24, as damper plate 50 elastically expands.", "Cap in this case, grooves 54 rest against toroid(s) 24.During the automatic assembly of the axle, this gas spring 70 may be snapped into place in the nonpressurized state.", "Additional fastening measures, such as bonding or the application of a torque, may not be necessary for installation.", "In addition, a special tool may not be required for the installation or the detachment of gas spring 70 in the exemplary embodiments.", "During installation, gas spring 70 centers itself on cap 19, using chamfer 57 of damper plate 50.No torsional stress is generated in spring bellows 72 in response to it being filled with gas, since spring 70 may align itself about fastening element 10.Due to its rear engagement, gas spring 70 may not detach from its mounting in response to a drop in pressure.", "If gas spring 70 is equipped, on its end faces, with one of the mountings described in the exemplary embodiments, upper or lower damper plate 50 of gas spring 70 may also be arranged to have a blind hole in place of a bore 52.In this case, the sealing of the interior of gas spring 70 may be eliminated in the region of attachment.", "Damper plate 50 may also be part of spring bellows 72.In this case, the need for pre-mounting spring bellows 72 to damper plate 50 may be eliminated.", "In this exemplary embodiment, the need for the upper and/or lower gaskets in the region of the cover bolts may be eliminated.", "The rubber-elastic seating of damper plate 50 on vehicle body 5 allows it to acoustically decouple vehicle body 5 from the suspension.", "FIGS.", "3 to 7 illustrate exemplary embodiments of the form and the attachment of bolt 15, i.e., of the shaped stud, and cap 19 to vehicle frame 5.In FIG.", "3, bolt 15 is welded, for example, to vehicle frame 5.Bolt 15 may also be arranged as a sleeve.", "Cap 19 has a cylindrical inner bore 22.As seen from above, the final third of inner bore 22 is arranged to be a tapped hole 21.In FIG.", "4, stud 15 is screwed into a thread in vehicle frame 5.To secure the connection, stud 15 is braced against vehicle frame 5 at collar 17.FIG.", "5 illustrates a bolt 95, which is inserted from above, through a bore 8 of vehicle frame 5.Bolt 95 may have a special head shape.", "Head 96 of bolt 95 is welded to vehicle frame 5 from the top.", "Cap 19 is screwed onto bolt 15 from below.", "In this case, this cap 19 has the same exemplary embodiment as in FIG.", "3.Cap 19 is fastened to bolt 95 by bracing it.", "In FIG.", "6, a nut 93 is welded to vehicle frame 5.Stud 15 is screwed into the nut.", "In this connection, stud 15 may be arranged to have or not have a collar.", "When stud 15 is constructed without a collar, stud 15 is secured by bracing stud 15 against the root of the thread.", "When stud 15 has a collar, stud 15 is secured by bracing the shaft collar against nut 93.FIG.", "7 corresponds to FIG.", "6, the difference being that a weld nut 92 is attached to the upper side of vehicle frame 5.Stud 15 is secured, for example, by tack-welding it to weld nut 92, or by cold-working the ends of the thread, e.g., using a special tool.", "Other attachment variations are possible.", "These may combine, for example, the elements described above." ] ]	Patent_10468176
[ [ "Methods and compositions for inhibiting hiv-coreceptor interactions", "Novel methods and compositions are provided for inhibiting interactions between human immunodeficiency viruses (HIVs) and viral coreceptors, including CXCR4 and/or CCR5 coreceptors.", "The anti-coreceptor binding agent includes a novel peptide portion of the gp120 envelope protein of HIV-1, as well as peptide analogs and mimetics of this peptide, that specifically binds to, or modulates activity of, the coreceptors(s).", "The anti-coreceptor binding agent is useful as a prophylactic or therapeutic treatment to prevent or inhibit HIV binding to a susceptible cell and thereby reduces infection and/or moderates or treats related diseases.", "In alternative embodiments, the peptides, analogs and mimetics are effective to inhibit direct co-receptor binding by HIV virus, coreceptor binding by HIV gp120 proteins or peptides, HIV fusion with target host cells, HIV virion entry into host cells, HIV replication, and HIV transmission between cells and hosts.", "In more detailed embodiments, the anti-coreceptor binding agents of the invention are multi-tropic by exhibiting activity against HIV interactions with multiple, CXCR4 and CCR5, coreceptors." ], [ "1.A composition comprising an effective amount of an anti-coreceptor binding agent to inhibit binding of a CXCR4 and/or CCR5 coreceptor of a subject by an HIV virus or viral protein, wherein the anti-coreceptor binding agent is a gp120 peptide, peptide analog or mimetic that specifically binds the CXCR4 and/or CCR5 coreceptor.", "2.The composition of claim 1, wherein the gp120 peptide, peptide analog or mimetic is between about 12 and about 24 amino acid residues in length and comprises a conserved CXXXXXXW amino acid sequence motif, wherein X is any naturally occurring or synthetic amino acid or amino acid analog.", "3.The composition of claim 1, wherein the peptide, peptide analog or mimetic is modified by addition, admixture, or conjugation of additional amino acids, peptides, proteins, chemical reagents or moieties which do not substantially alter the anti-coreceptor binding activity of the peptide.", "4.The composition of claim 1, wherein the anti-coreceptor binding agent is a peptide comprising an allelic variant among native HIV gp120 peptide sequences.", "5.The composition of claim 1, wherein the anti-coreceptor binding agent is formulated for delivery to subject selected from an isolated or bound coreceptor, a membrane or cell preparation comprising the coreceptor, a cell population, tissue or organ expressing the coreceptor, or a mammalian patient.", "6.The composition of claim 5, wherein the anti-coreceptor binding agent is combined with a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant for administration in a prophylactic or therapeutic effective dose to a mammalian patient to prevent or inhibit HIV infection or a related disease condition or symptom in the patient.", "7.The composition of claim 1, wherein the anti-coreceptor binding inhibits one or more biological activities mediated by or associated with HIV-coreceptor interactions selected from (a) direct co-receptor binding by HIV virus, (b) coreceptor binding by a HIV gp120 protein or a peptide fragment or derivative thereof, (c) HIV fusion with target host cells, (d) HIV virion entry into host cells, (e) HIV replication, and/or (f) HIV cell-cell or host-host transmission.", "8.The composition of claim 1, wherein the anti-coreceptor binding agent comprises an effective formulation of an HIV-1 peptide, peptide analog or mimetic for in vivo administration to inhibit one or more biological activities selected from (a) direct co-receptor binding by HIV-1 virus, (b) coreceptor binding by a HIV-1 gp120 protein or a peptide fragment or derivative thereof, (c) HIV-1 fusion with target host cells, (d) HIV-1 virion entry into host cells, (e) HIV-1 replication, and/or (f) HIV-1 cell-cell or host-host transmission.", "9.The composition of claim 1, wherein the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic formulated for administration to a mammalian patient in a prophylactically or therapeutically effective dose to prevent or inhibit HIV-1 infection or an HIV-1-related disease condition or symptom.", "10.The composition of claim 1, wherein the anti-coreceptor binding agent is a peptide that includes a conserved “CXXXXXXW” amino acid sequence motif, wherein X is any amino acid, and wherein the peptide is from about 12-17 amino acids in length and is selected from peptide 15K, comprising an amino acid sequence IRKAHCNISRAKWND (SEQ ID NO:8), or a corresponding or overlapping native peptide sequence or peptide analog that shares substantial sequence identity to the reference peptide sequence of 15K.", "11.The composition of claim 10, wherein the peptide includes one or more residues occurring naturally or by substitution at a relative, aligned position corresponding to a designated position for peptide 15K, selected from: Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q, N, I, or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, V, Y, D, A; Position 9—S, N, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A, R, K, T, S, G, E, D, N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; and/or Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. 12.The composition of claim 1, wherein the anti-coreceptor binding agent exhibits multi-tropic activity characterized by effective inhibition of HIV viral, or gp120 protein or peptide binding to multiple, CXCR4 and CCR5, coreceptors.", "13.The composition of claim 12, wherein the multi-tropic anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic effective to inhibit one or more biological activities of both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV-1 viruses selected from (a) direct co-receptor binding by viruses, (b) coreceptor binding by viral gp120 proteins or peptide fragments or derivatives thereof, (c) viral fusion with target host cells, (d) virion entry into host cells, (e) viral replication, and/or (f) viral cell-cell or host-host transmission.", "14.A method for inhibiting human immunodeficiency virus (HIV) interaction with a CXCR4 and/or CCR5 coreceptor comprising exposing a subject to an effective amount of an anti-coreceptor binding agent to inhibit binding of the CXCR4 and/or CCR5 coreceptor by an HIV virus or viral protein, wherein the anti-coreceptor binding agent is a gp120 peptide, peptide analog or mimetic that specifically binds the CXCR4 and/or CCR5 coreceptor.", "15.The method of claim 14, wherein the gp120 peptide, peptide analog or mimetic is between about 12 and about 24 amino acid residues in length and comprises a conserved CXXXXXXW amino acid sequence motif, wherein X is any naturally occurring or synthetic amino acid or amino acid analog.", "16.The method of claim 14, wherein the peptide, peptide analog or mimetic is modified by addition, admixture, or conjugation of additional amino acids, peptides, proteins, chemical reagents or moieties which do not substantially alter the anti-coreceptor binding activity of the peptide.", "17.The method of claim 14, wherein the anti-coreceptor binding agent is a peptide comprising an allelic variant among native HIV gp120 peptide sequences.", "18.The method of claim 14, wherein the subject is an isolated or bound CXCR4 and/or CCRS coreceptor, a membrane or cell preparation comprising the coreceptor, a cell population, tissue or organ expressing the coreceptor, or a mammalian patient.", "19.The method of claim 18, wherein the subject comprises a cell population, tissue or organ selected for in vivo or ex vivo treatment or diagnostic processing.", "20.The method of claim 18, wherein the subject is a mammalian patient susceptible to HIV infection and the anti-coreceptor binding agent is administered in a prophylactic or therapeutic effective dose to prevent or inhibit HIV infection or a related disease condition or symptom.", "21.The method of claim 14, wherein the anti-coreceptor binding agent is administered to the subject in an amount effective to inhibit one or more biological activities mediated by or associated with HIV-coreceptor interactions selected from (a) direct co-receptor binding by HIV virus, (b) coreceptor binding by a HIV gp120 protein or a peptide fragment or derivative thereof, (c) HIV fusion with target host cells, (d) HIV virion entry into host cells, (e) HIV replication, and/or (f) HIV cell-cell or host-host transmission.", "22.The method of claim 14, wherein the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to the subject in an amount effective to inhibit one or more biological activities selected from (a) direct co-receptor binding by HIV-1 virus, (b) coreceptor binding by a HIV-1 gp120 protein or a peptide fragment or derivative thereof, (c) HIV-1 fusion with target host cells, (d) HIV-1 virion entry into host cells, (e) HIV-1 replication, and/or (f) HIV-1 cell-cell or host-host transmission.", "23.The method of claim 14, wherein the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to a mammalian patient in a prophylactically or therapeutically effective dose to prevent or inhibit HIV-1 infection or an HIV-1-related disease condition or symptom.", "24.The method of claim 14, wherein the anti-coreceptor binding agent is a peptide that includes a conserved “CXXXXXXW” amino acid sequence motif, wherein X is any amino acid, and wherein the peptide is from about 12 to about 17 amino acids in length and is selected from peptide 15K, comprising an amino acid sequence IRKAHCNISRAKWND (SEQ ID NO:8), or a corresponding or overlapping native peptide sequence or peptide analog that shares substantial sequence identity to the peptide sequence of 15K.", "25.The method of claim 24, wherein the peptide includes one or more residues occurring naturally or by substitution at a relative, aligned position corresponding to a designated position for peptide 15K, selected from: Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q, N, I or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, Y, D, A; Position 9—S, N, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A,R,K,T, S, G, E, D,N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; and/or Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. 26.The method of claim 14, wherein the anti-coreceptor binding agent exhibits multi-tropic activity characterized by effective inhibition of HIV viral, or gp120 protein or peptide binding to multiple, CXCR4 and CCR5, coreceptors.", "27.The method of claim 26, wherein the multi-tropic anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to the subject in an amount effective to inhibit one or more biological activities of both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV-1 viruses selected from (a) direct co-receptor binding by viruses, (b) coreceptor binding by viral gp120 proteins or peptide fragments or derivatives thereof, (c) viral fusion with target host cells, (d) virion entry into host cells, (e) viral replication, and/or (f) viral cell-cell or host-host transmission." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The envelope glycoprotein of the human immunodeficiency virus type I (HIV-1) mediates in the fusion of viral and host cell membranes necessary for virion entry (Freed et al., J. Biol.", "Chem.", "270:23883-23886, 1995).", "The envelope glycoprotein of HIV-1 is produced by the enzymatic cleavage from a gp160 precursor protein to produce the external gp120 protein and the transmembrane gp41 protein (Capon et al., Annu.", "Rev.", "Immunol.", "9:649-678, 1991).", "Several studies have identified specific portions or domains of the gp120 protein that may elicit humoral and/or cell-mediated immune responses to HIV in susceptible host subjects, and may therefore be useful to formulate anti-HIV reagents and methods for prevention and treatment of HIV infection and related diseases.", "These general HIV peptide studies describe a large, diverse assemblage of gp120 peptides that are proposed as candidates for therapeutic use, primarily in vaccine formulations to prevent and treat HIV infection and related disease.", "For example, U.S. Pat.", "No.", "5,691,135 describes various peptides that are selected for the ability to inhibit HIV infection by stimulating VH3 and VH4 antibody responses.", "The peptides are proposed for administration to a patient as an antigen in sufficient quantity to induce antibodies that exhibit superantigen binding to gp120.The disclosure considers 31 peptides obtained from the AIDS Research and Reference Program, NIH, which peptides correspond to sequences from gp120 and gp41 from different strains of HIV.", "Additional gp120 peptides are described in U.S. Pat.", "No.", "5,939,074.In particular, this references describes peptides that are useful as “multideterminant peptide antigens” for eliciting both cell-mediated and humoral immune responses against HIV.", "Additional studies have identified particular regions of the gp120 that are proposed to determine specific interactions between gp120 and CD4, which is the primary receptor on target cells mediating cellular entry by HIV-1 (Arthos et al., Cell 57:469481, 1989; Clayton et al., Nature 339:548-551, 1989; Kwong et al., Nature 393:648-659, 1998; and Landau et al., Nature 334:159-162, 1988).", "In addition to CD4 binding, cellular entry by HIV-1 is thought to require additional interactions between the virus and one or more “coreceptors” on the surfaces of target cells.", "Briefly, the HIV-1 particles are proposed to bind initially to the CD4 receptor, and then subsequently to a chemokine receptor present on the target cells, which is used by the virus as a “coreceptor” for mediating cellular entry.", "Different strains of HIV-1 preferentially utilize different chemokine receptors that are variably expressed among HIV-1 target cells.", "In particular, some strains of HIV-1, referred to as “lymphotropic” strains, bind CXCR4 chemokine receptors predominantly expressed on lymphocyte target cells, while other HIV-1 strains, termed “monocyte tropic” strains, bind CCR5 receptors predominantly expressed on cells of monocyte lineage.", "Thus, the major viral coreceptors are CXCR4 (Berson et al., J. Virol.", "70:6288-6295, 1996), which has a native function as a chemokine receptor for stromal derived factor-1 (SDF-1), and CCR5 (Alkhatib et al., Science 272:1955-1958, 1996; Deng et al., Nature 381:661-666, 1996; and Dragic et al., Nature 381:667-673, 1996), which functions naturally as a receptor for several chemokines, including macrophage inflammatory protein-1β (MIP-1β).", "It is proposed that a conformational change occurs in gp120 following HIV binding of CD4, and that this conformation change exposes one or more binding sites on the gp120 molecule that mediate additional interactions between HIV and chemokine receptor, which are referred to in this context as “viral coreceptors” (Kwong et al., Nature 393:648-659, 1998, incorporated herein by reference).", "Recent studies suggest a complicated, multi-step binding and activation model, wherein the association of gp120 with CD4 yields a CD4-gp120 complex, which subsequently associates with the viral coreceptor resulting in a structural rearrangement (e.g., conformational change) of gp120 that facilitates interaction of the gp41 envelope protein subunit with the host cell membrane, leading to viral entry (Helseth et al., J. Virol.", "64:6314-6318, 1990; and Weissenhom et al., Nature 387:426-430, 1997).", "Within this emergent field of investigation, numerous studies have focused on structure-function mapping of chemokine receptors, with the goals of determining binding determinants and mechanisms of action of the receptors.", "Yet additional research has focused on chemokine mapping studies.", "From these studies chemokine-derived peptides have been identified that reportedly comprise binding determinants of the chemokines capable of blocking native chemokine receptor-ligand interactions.", "Further studies, focusing alternatively on HIV gp120 mapping, report production and testing of gp120 peptides capable of blocking HIV-coreceptor binding and HIV infectivity.", "With regard to chemokine receptor studies, numerous articles report that chemokine receptors play a direct role as coreceptors for HIV cell entry.", "Briefly, as noted above, it is reported that specific interactions between the HIV envelope glycoprotein gp120 and one or more chemokine receptors mediate viral entry into target cells.", "More specifically, monocyte-tropic or “m-tropic” HIV strains bind to a distinct chemokine receptor, CCR5, for cell entry, while T cell lymphotropic or “T-tropic” virus use mainly CXCR4 receptors for cell entry.", "More detailed, structure-function analyses have been directed toward identifying specific HIV binding determinants of chemokine receptors mediating their activity as coreceptors for HIV entry.", "In conjunction with these reports, certain references focus on a concept of blocking chemokine receptor activity (e.g., receptor binding with chemokines, or with gp120) using competitive inhibitors.", "Proposed inhibitors of chemokine receptor binding interactions include peptide inhibitors that mimic structures of chemokines, or of gp120, binding determinants or related structural domains.", "These studies follow more basic research which shows that intact chemokines, the normal ligands of chemokine receptors, can compete with cognate chemokines, or with HIV, for binding to the target receptors.", "Referring specifically to chemokine mapping studies focusing on chemokine structure-function, various publications attempt to identify and characterize receptor binding determinants of chemokines.", "As noted above, certain of these references also describe chemokine-based peptides reportedly capable of blocking chemokine-receptor binding and other activities mediated by receptor-ligand (chemokine or HIV) interactions.", "One such study is presented in a publication by Reckless et al., ( Biochem.", "J.", "340:803-811, 1999).", "This study identifies a number of chemokine-derived peptides, including a peptide designated “peptide 3”, based on a human chemokine, monocyte chemotactic protein-1 (MCP-1).", "Reckless and colleagues report that the peptide 3 inhibits cell migration induced by a wide range of chemokines.", "Moreover, peptide 3 reportedly binds to THP-1 cells and inhibits THP-1 migration, reportedly by acting as a chemokine receptor antagonist.", "On this basis, the authors propose that peptide 3 and its derivatives, including peptides ranging from 6-15 residues in length, may be useful as chemokine inhibitors.", "A number of related reports focus on a distinct portion of chemokines as prospective receptor binding determinants.", "In particular, the reports focus on the N-terminal region of chemokines comprising a structural element called the “N-loop”.", "This distinct element follows the first two cysteine residues of a model chemokine, and is proposed to play an important role in chemokine-receptor interactions.", "Other parts of chemokine molecules have also been proposed to contribute significantly as structural determinants of interactions between chemokines and their cognate receptors.", "For example, Crump et al., ( EMBO J.", "16:6996-7007, 1997), teach that the N-terminal eight residues of the chemokine SDF-1 form an important receptor binding site.", "At this site, two residues (Lys-1 and Pro-2) were proposed to be directly involved in receptor activation.", "Disruption of these residues reportedly abolished activation.", "It has further been reported that SDF-1 includes a second receptor binding motif at residues 12-17 of the chemokine loop region, termed the “RFFESH motif”.", "Thus, it has been widely considered that the N-terminal region and so called N-loop following the first two cysteine residues of chemokines play the most important role in mediating the interactions between chemokines and their cognate receptors (Clark-Lewis et al., J. Biol.", "Chem.", "266:23128-23134, 1991; Crump et al., EMBO J.", "16:6996-7007, 1997; and Pakianathan et al., Biochemistry 36:9642-9648, 1997).", "In one of these studies, Clark-Lewis et al.", "reported that a C-terminally truncated form of IL-8 missing the α-helix and β-turn within this region manifested greatly reduced chemotactic activity.", "Additional confirmation of the primary significance of this segment of chemokines for receptor binding is provided by a recent observation that the biological activity of human MIP-1β is strongly reduced by substitutions of Arg-45, and Arg-47, with Serine (Czaplewski et al., J. Biol.", "Chem.", "274:16077-16084, 1999, incorporated herein by reference).", "Finally, a peptide corresponding to the MCP-1 sequence just preceding the C-terminal α-helix was reported to inhibit chemotaxis of THP-1 cells indicating the importance of this region for chemokine function (Reckless et al., Biochem.", "J.", "340:803-1121, 1999).", "Additional structure-function data for the CC chemokine RANTES have been reported by Pakianathan et al., ( Biochemistry 36:9642-9648, 1997).", "Notably, a number of the foregoing publications point to a complex, multi-determinant model of interactions between chemokines and their native receptors.", "In this context, Pakianathan et al.", "believe the data indicates that RANTES interacts with each of its receptors in a distinct and specific manner and supports a two-site model of interaction between chemokines and their receptors.", "Protein mapping studies of HIV gp120 envelope have reported identification of structural determinants of gp120 responsible for mediating HIV-coreceptor interactions.", "Among these studies, Rizzuto et al., Science 280:1949-1953, 1998, have described a conserved gp120 structure that is reportedly important for binding to CCR5, and have postulated generally that this structural determinant should facilitate development of pharmacologic or immunologic inhibitors of virus-receptor interactions.", "Rizzuto et al., suggested “that the CCR5-binding site is likely composed of conserved gp120 elements near or within the bridging sheet and V3 loop residues.” In another report addressing gp120 structure-function analysis relating to coreceptor binding, Verrier et al., ( AIDS Res.", "Hum.", "Retroviruses 15:731-743, 1999), studied the effect of linear V3 peptides (21-30 amino acids in length) on infectivity of different strains of HIV-1.These studies also pointed to the V3 loop as an important determinant of coreceptor choice, whereby single amino acid substitutions in V3 were reported to dramatically alter coreceptor usage.", "In conjunction with this disclosure, Verrier et al., reported that artificial, linear peptides of V3 could compete with intact gp120 for binding to CCR5 and CXCR4 and block HIV entry into cells.", "More specifically, the authors reported that the most efficient peptides for blocking fusion were derived from the middle of V3, and therefore did not include sequences at the C-terminal or N-terminal ends of V3 that form the base of the V3 loop.", "Also significantly, Verrier et al., (supra) pointed to a “pattern of restriction” between multiple gp120 binding determinants, whereby peptides from different HIV strains (m-tropic versus lymphotropic) discriminate in their fusion-blocking activity in a pattern that “follows the coreceptor usage of the parental envelopes from which the peptides were derived.” This indicates that the candidate peptides described by Verrier et al., (supra) would not exhibit multi-specific blocking potential against both m-tropic and lymphotropic HIV-coreceptor interactions.", "In a related study, Sakaida et al., ( J. Virol.", "72:9763-9670, 1998) reported that synthetic cyclized (but not linear) V3 peptides of CXCR4 and dual-tropic strains of HIV-1 (but not a CCR5 strain) can prevent binding of anti-CXCR4 antibodies, potentially by binding to the coreceptor and acting as a competitive inhibitor.", "These same peptides reportedly inhibited calcium mobilization by the chemokine SDF-1 in a T cell line.", "The reference proposes that V3 loop peptides can directly bind to the relevant chemokine receptor and determine coreceptor usage, and postulates that such peptides can serve as HIV-1 reagents.", "Like Verrier et al., (supra) Sakaida and coworkers conclude that the coreceptor binding activities of V3 peptides are strain-dependent.", "More detailed reports pertaining to the role of V3 in mediating HIV-coreceptor interactions focus on specific sites or residues within the V3 domain as reportedly critical or important residues for coreceptor binding.", "For example, Tugarinov et al., ( Structure Fold Des.", "8:385-395, 2000), report that a conserved, central loop sequence, GPG, within the V3 loop plays a primary role in maintaining the conformation of the loop to mediate coreceptor binding.", "Kato et al.", "( J. Virol.", "73:5520-5526, 1999) suggest that three specific residues, confined to the central, loop portion of V3 and located distant from the C-terminal segment, are particularly important for coreceptor usage.", "Xiao et al., ( Virology 240:83-92, 1998), identify a consensus motif S/GXXXGPGXXXXXXXE/D, covering the central portion of the V3 loop and excluding the C-terminal portion, as a critical motif for coreceptor binding.", "Wang et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 95:5740-5745, 1998), studied whether certain V3 residues conserved among HIV-1, HIV-2 and SIV determine the utilization of CCR5 as a coreceptor.", "They concluded that Arg-298 (at the beginning of V3 loop) has an importance for CCR5 utilization.", "The authors suggested that this residue may represent a highly conserved structural element and a useful target for developing anti-viral therapies.", "In another study by Wang et al., ( Proc.", "Natl.", "Acad.", "Sci.", "USA 96: 4558-4562, 1999), additional V3 residues reported to be critical for CCR5 utilization are identified by alanine scanning mutagenesis.", "One of the identified residues, A 328 , is located at the C-terminus of V3, and its substitution reportedly results in a 1,000-fold reduction of CCR5 binding activity.", "However, this residue is only one of two “highly conserved” residues and six “critical” residues identified for CCR5 utilization.", "Most of these residues are distinctly located at the C-terminal or central loop portion of V3, including R 298 , K 305 , I 307 , R 313 , and F 315 .", "Wang and colleagues propose that “these highly invariable residues” as well as others identified in a “bridging sheet” portion of the molecule may represent “targets for antiviral designs aimed at blocking the coreceptor entry step of HIV-1 replication.” Based on the foregoing articles, there is a broadly-confirmed disclosure of V3 as a critical domain for mediating HIV-coreceptor interactions.", "A large number of important or critical residues are indicated, with a substantial range of discrepancy between reports implicating different residues as being important, or critical, for coreceptor binding functions.", "These references that focus on V3 uniformly teach away from excluding important “critical” or “highly conserved” V3 residues.", "At the same time, these references focusing on the V3 domain uniformly fail to implicate other sequence elements that may be present in the gp120 molecule outside the V3 loop portion thereof.", "Coupled with these teachings, a host of related publications point to yet additional components of gp120 that may be essential for HIV-coreceptor interactions.", "Because these components appear to be complex and potentially interact to achieve coreceptor binding, their disclosure adds further complexity to understanding gp120 structure-function relationships for mediating coreceptor binding and cell entry.", "In particular, Tugarinov et al., ( Structure Fold Des.", "8:385-395, 2000), teach the importance of the GPG motif of V3, but also teach that “[h]igh affinity binding of gp120 to the chemokine receptors requires participation of other domains in gp120 such as the CD4i epitope.” Rizzuto et al., ( Science 280:1949-1953, 1998), suggest that the “CCR5-binding site is likely composed of conserved gp120 elements near or within the bridging sheet and V3 loop residues.” They further proposed that CD4 binding may distort the V1/V2 stem, repositioning the stem allowing the formation of the β-sheet important for CCR5 binding.", "They also noted that substitution of Asp for Thr 123 , located in the V1/V2 stem and which contact CD4, significantly decreased CCR5 binding.", "This report parallels others which point to a favored model of a “conformational binding site” in gp120 for mediating HIV-coreceptor interactions.", "According to this model, effective binding of coreceptors by gp120 involves initial binding of the gp120 to a CD4 receptor, which brings about a “CD4 induced” conformational change in gp120 involving distant residues—that in turn leads to formation of a conformational binding determinant on gp120 to mediate HIV-coreceptor interactions.", "In this context, Wu et al., ( Nature 384:179-183, 1996) suggested that HIV-1 attachment to CD4 creates a high-affinity binding site for CCR-5, leading to membrane fusion and virus entry.", "Consistent with this model, recent studies have identified yet additional sites of gp120 which are reportedly important, or essential, for mediating HIV-coreceptor interactions.", "These sites are generally distinct from V3, and also from the V3-flanking regions.", "For example, Cho et al., ( J. Virol.", "72:2509-2515, 1998), report that V1/V2 and V3 confer on HIV-1 the ability to use CXCR4 coreceptors, but that the V4 and V5 regions are also “required in conjunction with regions V1 and V3 of HIV-1DH 12 gp120 for efficient utilization of CXCR4.” Este et al., ( Mol.", "Pharmacol.", "52:98-104, 1997), point to structural determinants in each of the V2, V3 and C3 regions of gp120 for determining cell tropism and coreceptor utilization.", "Hoffman et al., ( Mol.", "Membr.", "Biol.", "16:57-65, 1999), concluded the V3 loop was implicated in regulating viral tropism, but, that other regions of Env, such as the V1- and V2-loops, modulated the effects of the V3-loop.", "They also acknowledged that some important exceptions to this model suggested that understanding of virus tropism and Env-chemokine receptor interactions was incomplete.", "Collectively, these studies point to numerous components of gp120 that may be important or essential for HIV-coreceptor interactions, and which may in fact involve multiple interactions whereby different parts of gp120 are required to work together to achieve efficient HIV-coreceptor interactions.", "This complexity and the proposed requirement for multiple, distant binding determinants on gp120 to form a conformational binding determinant for coreceptor usage, may be generally considered to teach away from the utility of small peptides for effectively blocking HIV-coreceptor binding interactions, particularly to block multiple HIV strains infecting different target cell types.", "Moreover, while the foregoing, separate bodies of literature individually discuss models of conserved binding elements of chemokines, or of gp120, as determinants for mediating coreceptor interactions, these reports do not teach or suggest identification of conserved binding determinants shared between gp120 and chemokines.", "On the contrary, complex functional interactions and distinct binding mechanisms and determinants previously proposed for gp120 and chemokines may be considered to lead away from investigations aimed at identifying common binding determinants or mechanisms between gp120 and chemokines.", "This conclusion is well supported in the literature reviewed herein above.", "Based on these and other teachings, there does not appear to be a clear suggestion in the literature to use a common blocking agent for coreceptor interactions by chemokines and HIV aimed at, or patterned after, a shared binding determinant between these two distinct molecules.", "The literature does not point toward any such common structural domain or binding determinant between HIV gp120 and chemokines, and the distinct structure-function reports for how HIV and chemokines are thought to interact with the subject receptors leads away from this path of inquiry.", "In view of the foregoing teachings and unsolved questions in the literature, there remains an urgent need in the art for additional tools and methods to combat HIV infection and related disease.", "Related to this fundamental goal, there remains a clear need for additional therapeutic agents and methods targeting HIV viral entry into host cells, preferably that will include compositions and methods to block gp120 binding to different coreceptors to inhibit viral entry and infection and ameliorate HIV-related disease.", "Surprisingly, the instant invention fulfills these objects and satisfies additional objects and advantages which will become apparent from the following description." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The instant invention provides novel methods and compositions for inhibiting interactions between human immunodeficiency viruses (HIVs) and chemokine receptors, termed “viral coreceptors” in this context.", "The methods of the invention generally comprise exposing a CXCR4 or CCR5 coreceptor of a subject to an effective amount of an anti-coreceptor binding agent of the invention to inhibit binding of the coreceptor by an HIV virus or viral protein.", "Typically, the anti-coreceptor binding agent is a gp120 peptide, peptide analog or mimetic that specifically binds the coreceptor.", "Within certain methods of the invention, the subject is an isolated or bound coreceptor, a membrane or cell preparation comprising the coreceptor, a cell population, tissue or organ expressing the coreceptor, or a mammalian patient.", "In more detailed embodiments, the subject comprises a cell population, tissue or organ selected for in vivo or ex vivo treatment or diagnostic processing.", "Alternatively, the subject may be a mammalian patient susceptible to HIV infection and the anti-coreceptor binding agent is administered in a prophylactic or therapeutically effective dose to prevent or inhibit HIV binding to a susceptible cell and thereby preventing or inhibiting infection or a related disease condition or symptom.", "In typical aspects of the invention, the anti-coreceptor binding agent is administered to the subject in an amount effective to inhibit one or more biological activities mediated by or associated with HIV-coreceptor interactions selected from (a) direct co-receptor, e.g., CXCR4 and/or CCR5, binding by HIV virus, (b) coreceptor binding by a HIV gp120 protein or a peptide fragment or derivative thereof, (c) HIV fusion with target host cells, (d) HIV virion entry into host cells, (e) HIV replication, and/or (f) HIV cell-cell or host-host transmission.", "In more specific embodiments, the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic and is administered to the subject in an amount effective to inhibit one or more biological activities selected from (a) direct co-receptor, i.e., CXCR4 and/or CCR5, binding by HIV-1 virus, (b) coreceptor binding by a HIV-1 gp120 protein or a peptide fragment or derivative thereof, (c) HIV-1 fusion with target host cells, (d) HIV-1 virion entry into host cells, (e) HIV-1 replication, and/or (f) HIV-1 cell-cell or host-host transmission.", "Often, the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to a mammalian patient in a prophylactically or therapeutically effective dose to prevent or inhibit HIV-1 binding infection, to a susceptible host, HIV-1 or an HIV-1-related disease condition or symptom.", "In more detailed methods and compositions of the invention, the gp120 peptide, peptide analog or mimetic is between about 12 and about 24 amino acid residues in length and comprises a conserved CXXXXXXW amino acid sequence motif identified within the amino acid sequence of gp120 proteins of HIV isolates and also among diverse chemokines, wherein X is any naturally occurring or synthetic amino acid or amino acid analog.", "The peptide, peptide analog or mimetic can be modified in a wide variety of ways and forms, e.g., by addition, admixture, or conjugation of additional amino acids, peptides, proteins, chemical reagents or moieties which do not substantially alter the anti-coreceptor binding activity of the peptide.", "Often, the anti-coreceptor binding agent of the invention is a peptide comprising an allelic variant that is found among native HIV gp120 peptide sequences.", "Within more detailed embodiments, the anti-coreceptor binding agent is a peptide of between about 12-17 amino acids in length that includes a conserved “CXXXXXXW” amino acid sequence motif, which is selected from an exemplary “reference” peptide designated 15K comprising an amino acid sequence IRKAHCNISRAKWND (SEQ ID NO:8), or is alternatively represented by a corresponding or overlapping native peptide sequence or peptide analog that shares substantial sequence identity to the reference amino acid sequence of 15K.", "In various specific embodiments, the peptide includes one or more residues occurring naturally or by substitution at a relative, aligned position corresponding to a designated position for peptide 15K, selected from: Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q. N, I, or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, V, Y, D, A; Position 9—S, N, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A, R, K, T, S, G, E, D, N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; and/or Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. In yet additional embodiments of the invention, the anti-coreceptor binding agent exhibits multi-tropic activity characterized by effective inhibition of HIV viral, or gp120 protein or peptide binding to multiple, CXCR4 and CCR5, coreceptors.", "Often, the multi-tropic anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to the subject in an amount effective to inhibit one or more biological activities of both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV-1 viruses selected from (a) direct co-receptor binding by viruses, (b) coreceptor binding by viral gp120 proteins or peptide fragments or derivatives thereof, (c) viral fusion with target host cells, (d) virion entry into host cells, (e) viral replication, and/or (f) viral cell-cell or host-host transmission.", "Within the methods and compositions of the invention, the anti-coreceptor binding agent may be formulated in various combinations with a pharmaceutically acceptable carrier, diluent, excipient, adjuvant or other active or inactive agents, in an amount or dosage form sufficient to prevent, reduce or even alleviate HIV infection or related disease conditions or symptoms.", "In yet additional aspects of the invention, the anti-coreceptor binding agent of the invention is administered according to the foregoing methods in a combinatorial formulation or coordinate treatment with one or more additional anti-HIV, antibacterial, antiviral or other therapeutically active agent(s).", "Within related methods and compositions, the anti-coreceptor binding agent is admixed or co-administered, simultaneously or sequentially with one or more additional anti-HIV, antibacterial, antiviral or other therapeutically active agent(s) to prevent, reduce or even alleviate HIV infection or related conditions in a mammalian patient.", "The instant invention also includes kits, packages and multicontainer units containing the anti-coreceptor binding agent, optionally with other active or inactive ingredients, and/or means for administering the same for use in the diagnosis, management and/or prevention and treatment of HIV and related conditions.", "Typically, these kits include a diagnostic or pharmaceutical preparation of the anti-coreceptor binding agent, typically formulated with a biologically suitable carrier and optionally contained in a bulk dispensing container or unit or multi-unit dosage form.", "Optional dispensing means can be provided, for example an intranasal spray applicator.", "Packaging materials optionally include a label or instruction which indicates a desired use of the kit as described herein below.", "Additional aspects of the invention include polynucleotide molecules and vector constructs encoding anti-coreceptor binding peptides and peptide analogs.", "Also provided are peptide vaccines and other immunogenic compositions that elicit an immune response involving production of antibodies targeting one or more epitopes of gp120 recognized by antibodies that specifically bind an anti-coreceptor binding peptide of the invention.", "In addition, the invention provides, antibodies, including monoclonal antibodies, and immunotherapeutic methods and compositions comprising such antibodies that specifically recognize anti-coreceptor binding peptides of the invention, for use as diagnostic and therapeutic reagents.", "Also provided within the invention are a variety of additional diagnostic and therapeutic tools and reagents as set forth in detail in the following description." ], [ "RELATED APPLICATION This application claims priority to provisional application Ser.", "No.", "60/269,534 filed on Feb. 15, 2001.BACKGROUND OF THE INVENTION The envelope glycoprotein of the human immunodeficiency virus type I (HIV-1) mediates in the fusion of viral and host cell membranes necessary for virion entry (Freed et al., J. Biol.", "Chem.", "270:23883-23886, 1995).", "The envelope glycoprotein of HIV-1 is produced by the enzymatic cleavage from a gp160 precursor protein to produce the external gp120 protein and the transmembrane gp41 protein (Capon et al., Annu.", "Rev.", "Immunol.", "9:649-678, 1991).", "Several studies have identified specific portions or domains of the gp120 protein that may elicit humoral and/or cell-mediated immune responses to HIV in susceptible host subjects, and may therefore be useful to formulate anti-HIV reagents and methods for prevention and treatment of HIV infection and related diseases.", "These general HIV peptide studies describe a large, diverse assemblage of gp120 peptides that are proposed as candidates for therapeutic use, primarily in vaccine formulations to prevent and treat HIV infection and related disease.", "For example, U.S. Pat.", "No.", "5,691,135 describes various peptides that are selected for the ability to inhibit HIV infection by stimulating VH3 and VH4 antibody responses.", "The peptides are proposed for administration to a patient as an antigen in sufficient quantity to induce antibodies that exhibit superantigen binding to gp120.The disclosure considers 31 peptides obtained from the AIDS Research and Reference Program, NIH, which peptides correspond to sequences from gp120 and gp41 from different strains of HIV.", "Additional gp120 peptides are described in U.S. Pat.", "No.", "5,939,074.In particular, this references describes peptides that are useful as “multideterminant peptide antigens” for eliciting both cell-mediated and humoral immune responses against HIV.", "Additional studies have identified particular regions of the gp120 that are proposed to determine specific interactions between gp120 and CD4, which is the primary receptor on target cells mediating cellular entry by HIV-1 (Arthos et al., Cell 57:469481, 1989; Clayton et al., Nature 339:548-551, 1989; Kwong et al., Nature 393:648-659, 1998; and Landau et al., Nature 334:159-162, 1988).", "In addition to CD4 binding, cellular entry by HIV-1 is thought to require additional interactions between the virus and one or more “coreceptors” on the surfaces of target cells.", "Briefly, the HIV-1 particles are proposed to bind initially to the CD4 receptor, and then subsequently to a chemokine receptor present on the target cells, which is used by the virus as a “coreceptor” for mediating cellular entry.", "Different strains of HIV-1 preferentially utilize different chemokine receptors that are variably expressed among HIV-1 target cells.", "In particular, some strains of HIV-1, referred to as “lymphotropic” strains, bind CXCR4 chemokine receptors predominantly expressed on lymphocyte target cells, while other HIV-1 strains, termed “monocyte tropic” strains, bind CCR5 receptors predominantly expressed on cells of monocyte lineage.", "Thus, the major viral coreceptors are CXCR4 (Berson et al., J. Virol.", "70:6288-6295, 1996), which has a native function as a chemokine receptor for stromal derived factor-1 (SDF-1), and CCR5 (Alkhatib et al., Science 272:1955-1958, 1996; Deng et al., Nature 381:661-666, 1996; and Dragic et al., Nature 381:667-673, 1996), which functions naturally as a receptor for several chemokines, including macrophage inflammatory protein-1β (MIP-1β).", "It is proposed that a conformational change occurs in gp120 following HIV binding of CD4, and that this conformation change exposes one or more binding sites on the gp120 molecule that mediate additional interactions between HIV and chemokine receptor, which are referred to in this context as “viral coreceptors” (Kwong et al., Nature 393:648-659, 1998, incorporated herein by reference).", "Recent studies suggest a complicated, multi-step binding and activation model, wherein the association of gp120 with CD4 yields a CD4-gp120 complex, which subsequently associates with the viral coreceptor resulting in a structural rearrangement (e.g., conformational change) of gp120 that facilitates interaction of the gp41 envelope protein subunit with the host cell membrane, leading to viral entry (Helseth et al., J. Virol.", "64:6314-6318, 1990; and Weissenhom et al., Nature 387:426-430, 1997).", "Within this emergent field of investigation, numerous studies have focused on structure-function mapping of chemokine receptors, with the goals of determining binding determinants and mechanisms of action of the receptors.", "Yet additional research has focused on chemokine mapping studies.", "From these studies chemokine-derived peptides have been identified that reportedly comprise binding determinants of the chemokines capable of blocking native chemokine receptor-ligand interactions.", "Further studies, focusing alternatively on HIV gp120 mapping, report production and testing of gp120 peptides capable of blocking HIV-coreceptor binding and HIV infectivity.", "With regard to chemokine receptor studies, numerous articles report that chemokine receptors play a direct role as coreceptors for HIV cell entry.", "Briefly, as noted above, it is reported that specific interactions between the HIV envelope glycoprotein gp120 and one or more chemokine receptors mediate viral entry into target cells.", "More specifically, monocyte-tropic or “m-tropic” HIV strains bind to a distinct chemokine receptor, CCR5, for cell entry, while T cell lymphotropic or “T-tropic” virus use mainly CXCR4 receptors for cell entry.", "More detailed, structure-function analyses have been directed toward identifying specific HIV binding determinants of chemokine receptors mediating their activity as coreceptors for HIV entry.", "In conjunction with these reports, certain references focus on a concept of blocking chemokine receptor activity (e.g., receptor binding with chemokines, or with gp120) using competitive inhibitors.", "Proposed inhibitors of chemokine receptor binding interactions include peptide inhibitors that mimic structures of chemokines, or of gp120, binding determinants or related structural domains.", "These studies follow more basic research which shows that intact chemokines, the normal ligands of chemokine receptors, can compete with cognate chemokines, or with HIV, for binding to the target receptors.", "Referring specifically to chemokine mapping studies focusing on chemokine structure-function, various publications attempt to identify and characterize receptor binding determinants of chemokines.", "As noted above, certain of these references also describe chemokine-based peptides reportedly capable of blocking chemokine-receptor binding and other activities mediated by receptor-ligand (chemokine or HIV) interactions.", "One such study is presented in a publication by Reckless et al., (Biochem.", "J.", "340:803-811, 1999).", "This study identifies a number of chemokine-derived peptides, including a peptide designated “peptide 3”, based on a human chemokine, monocyte chemotactic protein-1 (MCP-1).", "Reckless and colleagues report that the peptide 3 inhibits cell migration induced by a wide range of chemokines.", "Moreover, peptide 3 reportedly binds to THP-1 cells and inhibits THP-1 migration, reportedly by acting as a chemokine receptor antagonist.", "On this basis, the authors propose that peptide 3 and its derivatives, including peptides ranging from 6-15 residues in length, may be useful as chemokine inhibitors.", "A number of related reports focus on a distinct portion of chemokines as prospective receptor binding determinants.", "In particular, the reports focus on the N-terminal region of chemokines comprising a structural element called the “N-loop”.", "This distinct element follows the first two cysteine residues of a model chemokine, and is proposed to play an important role in chemokine-receptor interactions.", "Other parts of chemokine molecules have also been proposed to contribute significantly as structural determinants of interactions between chemokines and their cognate receptors.", "For example, Crump et al., (EMBO J.", "16:6996-7007, 1997), teach that the N-terminal eight residues of the chemokine SDF-1 form an important receptor binding site.", "At this site, two residues (Lys-1 and Pro-2) were proposed to be directly involved in receptor activation.", "Disruption of these residues reportedly abolished activation.", "It has further been reported that SDF-1 includes a second receptor binding motif at residues 12-17 of the chemokine loop region, termed the “RFFESH motif”.", "Thus, it has been widely considered that the N-terminal region and so called N-loop following the first two cysteine residues of chemokines play the most important role in mediating the interactions between chemokines and their cognate receptors (Clark-Lewis et al., J. Biol.", "Chem.", "266:23128-23134, 1991; Crump et al., EMBO J.", "16:6996-7007, 1997; and Pakianathan et al., Biochemistry 36:9642-9648, 1997).", "In one of these studies, Clark-Lewis et al.", "reported that a C-terminally truncated form of IL-8 missing the α-helix and β-turn within this region manifested greatly reduced chemotactic activity.", "Additional confirmation of the primary significance of this segment of chemokines for receptor binding is provided by a recent observation that the biological activity of human MIP-1β is strongly reduced by substitutions of Arg-45, and Arg-47, with Serine (Czaplewski et al., J. Biol.", "Chem.", "274:16077-16084, 1999, incorporated herein by reference).", "Finally, a peptide corresponding to the MCP-1 sequence just preceding the C-terminal α-helix was reported to inhibit chemotaxis of THP-1 cells indicating the importance of this region for chemokine function (Reckless et al., Biochem.", "J.", "340:803-1121, 1999).", "Additional structure-function data for the CC chemokine RANTES have been reported by Pakianathan et al., (Biochemistry 36:9642-9648, 1997).", "Notably, a number of the foregoing publications point to a complex, multi-determinant model of interactions between chemokines and their native receptors.", "In this context, Pakianathan et al.", "believe the data indicates that RANTES interacts with each of its receptors in a distinct and specific manner and supports a two-site model of interaction between chemokines and their receptors.", "Protein mapping studies of HIV gp120 envelope have reported identification of structural determinants of gp120 responsible for mediating HIV-coreceptor interactions.", "Among these studies, Rizzuto et al., Science 280:1949-1953, 1998, have described a conserved gp120 structure that is reportedly important for binding to CCR5, and have postulated generally that this structural determinant should facilitate development of pharmacologic or immunologic inhibitors of virus-receptor interactions.", "Rizzuto et al., suggested “that the CCR5-binding site is likely composed of conserved gp120 elements near or within the bridging sheet and V3 loop residues.” In another report addressing gp120 structure-function analysis relating to coreceptor binding, Verrier et al., (AIDS Res.", "Hum.", "Retroviruses 15:731-743, 1999), studied the effect of linear V3 peptides (21-30 amino acids in length) on infectivity of different strains of HIV-1.These studies also pointed to the V3 loop as an important determinant of coreceptor choice, whereby single amino acid substitutions in V3 were reported to dramatically alter coreceptor usage.", "In conjunction with this disclosure, Verrier et al., reported that artificial, linear peptides of V3 could compete with intact gp120 for binding to CCR5 and CXCR4 and block HIV entry into cells.", "More specifically, the authors reported that the most efficient peptides for blocking fusion were derived from the middle of V3, and therefore did not include sequences at the C-terminal or N-terminal ends of V3 that form the base of the V3 loop.", "Also significantly, Verrier et al., (supra) pointed to a “pattern of restriction” between multiple gp120 binding determinants, whereby peptides from different HIV strains (m-tropic versus lymphotropic) discriminate in their fusion-blocking activity in a pattern that “follows the coreceptor usage of the parental envelopes from which the peptides were derived.” This indicates that the candidate peptides described by Verrier et al., (supra) would not exhibit multi-specific blocking potential against both m-tropic and lymphotropic HIV-coreceptor interactions.", "In a related study, Sakaida et al., (J. Virol.", "72:9763-9670, 1998) reported that synthetic cyclized (but not linear) V3 peptides of CXCR4 and dual-tropic strains of HIV-1 (but not a CCR5 strain) can prevent binding of anti-CXCR4 antibodies, potentially by binding to the coreceptor and acting as a competitive inhibitor.", "These same peptides reportedly inhibited calcium mobilization by the chemokine SDF-1 in a T cell line.", "The reference proposes that V3 loop peptides can directly bind to the relevant chemokine receptor and determine coreceptor usage, and postulates that such peptides can serve as HIV-1 reagents.", "Like Verrier et al., (supra) Sakaida and coworkers conclude that the coreceptor binding activities of V3 peptides are strain-dependent.", "More detailed reports pertaining to the role of V3 in mediating HIV-coreceptor interactions focus on specific sites or residues within the V3 domain as reportedly critical or important residues for coreceptor binding.", "For example, Tugarinov et al., (Structure Fold Des.", "8:385-395, 2000), report that a conserved, central loop sequence, GPG, within the V3 loop plays a primary role in maintaining the conformation of the loop to mediate coreceptor binding.", "Kato et al.", "(J. Virol.", "73:5520-5526, 1999) suggest that three specific residues, confined to the central, loop portion of V3 and located distant from the C-terminal segment, are particularly important for coreceptor usage.", "Xiao et al., (Virology 240:83-92, 1998), identify a consensus motif S/GXXXGPGXXXXXXXE/D, covering the central portion of the V3 loop and excluding the C-terminal portion, as a critical motif for coreceptor binding.", "Wang et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 95:5740-5745, 1998), studied whether certain V3 residues conserved among HIV-1, HIV-2 and SIV determine the utilization of CCR5 as a coreceptor.", "They concluded that Arg-298 (at the beginning of V3 loop) has an importance for CCR5 utilization.", "The authors suggested that this residue may represent a highly conserved structural element and a useful target for developing anti-viral therapies.", "In another study by Wang et al., (Proc.", "Natl.", "Acad.", "Sci.", "USA 96: 4558-4562, 1999), additional V3 residues reported to be critical for CCR5 utilization are identified by alanine scanning mutagenesis.", "One of the identified residues, A328, is located at the C-terminus of V3, and its substitution reportedly results in a 1,000-fold reduction of CCR5 binding activity.", "However, this residue is only one of two “highly conserved” residues and six “critical” residues identified for CCR5 utilization.", "Most of these residues are distinctly located at the C-terminal or central loop portion of V3, including R298, K305, I307, R313, and F315.Wang and colleagues propose that “these highly invariable residues” as well as others identified in a “bridging sheet” portion of the molecule may represent “targets for antiviral designs aimed at blocking the coreceptor entry step of HIV-1 replication.” Based on the foregoing articles, there is a broadly-confirmed disclosure of V3 as a critical domain for mediating HIV-coreceptor interactions.", "A large number of important or critical residues are indicated, with a substantial range of discrepancy between reports implicating different residues as being important, or critical, for coreceptor binding functions.", "These references that focus on V3 uniformly teach away from excluding important “critical” or “highly conserved” V3 residues.", "At the same time, these references focusing on the V3 domain uniformly fail to implicate other sequence elements that may be present in the gp120 molecule outside the V3 loop portion thereof.", "Coupled with these teachings, a host of related publications point to yet additional components of gp120 that may be essential for HIV-coreceptor interactions.", "Because these components appear to be complex and potentially interact to achieve coreceptor binding, their disclosure adds further complexity to understanding gp120 structure-function relationships for mediating coreceptor binding and cell entry.", "In particular, Tugarinov et al., (Structure Fold Des.", "8:385-395, 2000), teach the importance of the GPG motif of V3, but also teach that “[h]igh affinity binding of gp120 to the chemokine receptors requires participation of other domains in gp120 such as the CD4i epitope.” Rizzuto et al., (Science 280:1949-1953, 1998), suggest that the “CCR5-binding site is likely composed of conserved gp120 elements near or within the bridging sheet and V3 loop residues.” They further proposed that CD4 binding may distort the V1/V2 stem, repositioning the stem allowing the formation of the β-sheet important for CCR5 binding.", "They also noted that substitution of Asp for Thr123, located in the V1/V2 stem and which contact CD4, significantly decreased CCR5 binding.", "This report parallels others which point to a favored model of a “conformational binding site” in gp120 for mediating HIV-coreceptor interactions.", "According to this model, effective binding of coreceptors by gp120 involves initial binding of the gp120 to a CD4 receptor, which brings about a “CD4 induced” conformational change in gp120 involving distant residues—that in turn leads to formation of a conformational binding determinant on gp120 to mediate HIV-coreceptor interactions.", "In this context, Wu et al., (Nature 384:179-183, 1996) suggested that HIV-1 attachment to CD4 creates a high-affinity binding site for CCR-5, leading to membrane fusion and virus entry.", "Consistent with this model, recent studies have identified yet additional sites of gp120 which are reportedly important, or essential, for mediating HIV-coreceptor interactions.", "These sites are generally distinct from V3, and also from the V3-flanking regions.", "For example, Cho et al., (J. Virol.", "72:2509-2515, 1998), report that V1/V2 and V3 confer on HIV-1 the ability to use CXCR4 coreceptors, but that the V4 and V5 regions are also “required in conjunction with regions V1 and V3 of HIV-1DH12 gp120 for efficient utilization of CXCR4.” Este et al., (Mol.", "Pharmacol.", "52:98-104, 1997), point to structural determinants in each of the V2, V3 and C3 regions of gp120 for determining cell tropism and coreceptor utilization.", "Hoffman et al., (Mol.", "Membr.", "Biol.", "16:57-65, 1999), concluded the V3 loop was implicated in regulating viral tropism, but, that other regions of Env, such as the V1- and V2-loops, modulated the effects of the V3-loop.", "They also acknowledged that some important exceptions to this model suggested that understanding of virus tropism and Env-chemokine receptor interactions was incomplete.", "Collectively, these studies point to numerous components of gp120 that may be important or essential for HIV-coreceptor interactions, and which may in fact involve multiple interactions whereby different parts of gp120 are required to work together to achieve efficient HIV-coreceptor interactions.", "This complexity and the proposed requirement for multiple, distant binding determinants on gp120 to form a conformational binding determinant for coreceptor usage, may be generally considered to teach away from the utility of small peptides for effectively blocking HIV-coreceptor binding interactions, particularly to block multiple HIV strains infecting different target cell types.", "Moreover, while the foregoing, separate bodies of literature individually discuss models of conserved binding elements of chemokines, or of gp120, as determinants for mediating coreceptor interactions, these reports do not teach or suggest identification of conserved binding determinants shared between gp120 and chemokines.", "On the contrary, complex functional interactions and distinct binding mechanisms and determinants previously proposed for gp120 and chemokines may be considered to lead away from investigations aimed at identifying common binding determinants or mechanisms between gp120 and chemokines.", "This conclusion is well supported in the literature reviewed herein above.", "Based on these and other teachings, there does not appear to be a clear suggestion in the literature to use a common blocking agent for coreceptor interactions by chemokines and HIV aimed at, or patterned after, a shared binding determinant between these two distinct molecules.", "The literature does not point toward any such common structural domain or binding determinant between HIV gp120 and chemokines, and the distinct structure-function reports for how HIV and chemokines are thought to interact with the subject receptors leads away from this path of inquiry.", "In view of the foregoing teachings and unsolved questions in the literature, there remains an urgent need in the art for additional tools and methods to combat HIV infection and related disease.", "Related to this fundamental goal, there remains a clear need for additional therapeutic agents and methods targeting HIV viral entry into host cells, preferably that will include compositions and methods to block gp120 binding to different coreceptors to inhibit viral entry and infection and ameliorate HIV-related disease.", "Surprisingly, the instant invention fulfills these objects and satisfies additional objects and advantages which will become apparent from the following description.", "SUMMARY OF THE INVENTION The instant invention provides novel methods and compositions for inhibiting interactions between human immunodeficiency viruses (HIVs) and chemokine receptors, termed “viral coreceptors” in this context.", "The methods of the invention generally comprise exposing a CXCR4 or CCR5 coreceptor of a subject to an effective amount of an anti-coreceptor binding agent of the invention to inhibit binding of the coreceptor by an HIV virus or viral protein.", "Typically, the anti-coreceptor binding agent is a gp120 peptide, peptide analog or mimetic that specifically binds the coreceptor.", "Within certain methods of the invention, the subject is an isolated or bound coreceptor, a membrane or cell preparation comprising the coreceptor, a cell population, tissue or organ expressing the coreceptor, or a mammalian patient.", "In more detailed embodiments, the subject comprises a cell population, tissue or organ selected for in vivo or ex vivo treatment or diagnostic processing.", "Alternatively, the subject may be a mammalian patient susceptible to HIV infection and the anti-coreceptor binding agent is administered in a prophylactic or therapeutically effective dose to prevent or inhibit HIV binding to a susceptible cell and thereby preventing or inhibiting infection or a related disease condition or symptom.", "In typical aspects of the invention, the anti-coreceptor binding agent is administered to the subject in an amount effective to inhibit one or more biological activities mediated by or associated with HIV-coreceptor interactions selected from (a) direct co-receptor, e.g., CXCR4 and/or CCR5, binding by HIV virus, (b) coreceptor binding by a HIV gp120 protein or a peptide fragment or derivative thereof, (c) HIV fusion with target host cells, (d) HIV virion entry into host cells, (e) HIV replication, and/or (f) HIV cell-cell or host-host transmission.", "In more specific embodiments, the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic and is administered to the subject in an amount effective to inhibit one or more biological activities selected from (a) direct co-receptor, i.e., CXCR4 and/or CCR5, binding by HIV-1 virus, (b) coreceptor binding by a HIV-1 gp120 protein or a peptide fragment or derivative thereof, (c) HIV-1 fusion with target host cells, (d) HIV-1 virion entry into host cells, (e) HIV-1 replication, and/or (f) HIV-1 cell-cell or host-host transmission.", "Often, the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to a mammalian patient in a prophylactically or therapeutically effective dose to prevent or inhibit HIV-1 binding infection, to a susceptible host, HIV-1 or an HIV-1-related disease condition or symptom.", "In more detailed methods and compositions of the invention, the gp120 peptide, peptide analog or mimetic is between about 12 and about 24 amino acid residues in length and comprises a conserved CXXXXXXW amino acid sequence motif identified within the amino acid sequence of gp120 proteins of HIV isolates and also among diverse chemokines, wherein X is any naturally occurring or synthetic amino acid or amino acid analog.", "The peptide, peptide analog or mimetic can be modified in a wide variety of ways and forms, e.g., by addition, admixture, or conjugation of additional amino acids, peptides, proteins, chemical reagents or moieties which do not substantially alter the anti-coreceptor binding activity of the peptide.", "Often, the anti-coreceptor binding agent of the invention is a peptide comprising an allelic variant that is found among native HIV gp120 peptide sequences.", "Within more detailed embodiments, the anti-coreceptor binding agent is a peptide of between about 12-17 amino acids in length that includes a conserved “CXXXXXXW” amino acid sequence motif, which is selected from an exemplary “reference” peptide designated 15K comprising an amino acid sequence IRKAHCNISRAKWND (SEQ ID NO:8), or is alternatively represented by a corresponding or overlapping native peptide sequence or peptide analog that shares substantial sequence identity to the reference amino acid sequence of 15K.", "In various specific embodiments, the peptide includes one or more residues occurring naturally or by substitution at a relative, aligned position corresponding to a designated position for peptide 15K, selected from: Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q. N, I, or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, V, Y, D, A; Position 9—S, N, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A, R, K, T, S, G, E, D, N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; and/or Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. In yet additional embodiments of the invention, the anti-coreceptor binding agent exhibits multi-tropic activity characterized by effective inhibition of HIV viral, or gp120 protein or peptide binding to multiple, CXCR4 and CCR5, coreceptors.", "Often, the multi-tropic anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to the subject in an amount effective to inhibit one or more biological activities of both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV-1 viruses selected from (a) direct co-receptor binding by viruses, (b) coreceptor binding by viral gp120 proteins or peptide fragments or derivatives thereof, (c) viral fusion with target host cells, (d) virion entry into host cells, (e) viral replication, and/or (f) viral cell-cell or host-host transmission.", "Within the methods and compositions of the invention, the anti-coreceptor binding agent may be formulated in various combinations with a pharmaceutically acceptable carrier, diluent, excipient, adjuvant or other active or inactive agents, in an amount or dosage form sufficient to prevent, reduce or even alleviate HIV infection or related disease conditions or symptoms.", "In yet additional aspects of the invention, the anti-coreceptor binding agent of the invention is administered according to the foregoing methods in a combinatorial formulation or coordinate treatment with one or more additional anti-HIV, antibacterial, antiviral or other therapeutically active agent(s).", "Within related methods and compositions, the anti-coreceptor binding agent is admixed or co-administered, simultaneously or sequentially with one or more additional anti-HIV, antibacterial, antiviral or other therapeutically active agent(s) to prevent, reduce or even alleviate HIV infection or related conditions in a mammalian patient.", "The instant invention also includes kits, packages and multicontainer units containing the anti-coreceptor binding agent, optionally with other active or inactive ingredients, and/or means for administering the same for use in the diagnosis, management and/or prevention and treatment of HIV and related conditions.", "Typically, these kits include a diagnostic or pharmaceutical preparation of the anti-coreceptor binding agent, typically formulated with a biologically suitable carrier and optionally contained in a bulk dispensing container or unit or multi-unit dosage form.", "Optional dispensing means can be provided, for example an intranasal spray applicator.", "Packaging materials optionally include a label or instruction which indicates a desired use of the kit as described herein below.", "Additional aspects of the invention include polynucleotide molecules and vector constructs encoding anti-coreceptor binding peptides and peptide analogs.", "Also provided are peptide vaccines and other immunogenic compositions that elicit an immune response involving production of antibodies targeting one or more epitopes of gp120 recognized by antibodies that specifically bind an anti-coreceptor binding peptide of the invention.", "In addition, the invention provides, antibodies, including monoclonal antibodies, and immunotherapeutic methods and compositions comprising such antibodies that specifically recognize anti-coreceptor binding peptides of the invention, for use as diagnostic and therapeutic reagents.", "Also provided within the invention are a variety of additional diagnostic and therapeutic tools and reagents as set forth in detail in the following description.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 demonstrates inhibition of specific chemokine binding to CXCR4 and CCRS expressing cells by exemplary HIV gp120-derived anti-coreceptor binding peptides 15K and 15D.", "FIG.", "2 documents inhibition of the chemotactic response of HEK-CCR5 cells to the chemokine RANTES by the exemplary anti-coreceptor binding peptides 15K and 15D.", "Migration of HEK-CCR5 cells to RANTES alone, or the combination of RANTES and designated concentrations of either 15K or 15D peptides, were determined in micro-chemotaxis chambers.", "The 15K and 15D peptides did not induce detectable chemotaxis when tested at any concentration.", "The results are representative of three independent experiments.", "p<0.05.FIG.", "3 demonstrates inhibition of monocyte-tropic HIV-1 infection of susceptible target cells by the exemplary anti-coreceptor binding peptides 15K and l5D.", "Monocyte-derived macrophages were treated with the designated concentrations of the indicated peptide for a period of 1 hr prior to addition of HIV-1JRFL.", "After 2 hr, cells were washed, and viral replication was determined after 72 hr by p24 analysis.", "Results are representative of 4 independent experiments.", "FIG.", "4 shows inhibition of T cell-tropic HIV-1 infection by the exemplary anti-coreceptor binding peptides 15K, l5D and scrambled peptide 15KS.", "Peripheral blood mononuclear cells were treated with designated concentrations of peptide for a period of 1 hr prior to addition of HIV-1IIIB.", "After 2 hr, cells were washed, and viral replication was determined after 48 hr by analysis of p24 levels.", "Results are representative of 4 independent experiments.", "FIGS.", "5A through 5C demonstrate the effect of peptides 15K, 15D and scrambled peptide 15KS on the binding of anti-CXCR4 antibody 12G5 to CEMx174 cells.", "FIG.", "5A.", "Dose dependent effect of peptide 15K in comparison with SDF-1α.", "FIG.", "5B.", "The comparison of the effects of 15K and 15D.", "FIG.", "5C.", "The comparison of the effects of 15K and 15KS.", "Cells were preincubated at 22° C. with peptides at designated concentrations of SDF-1α for 60 min.", "Then cells were incubated with FITC-labeled anti-human CXCR4 monoclonal antibodies 12G5 for 40 min at 22° C. and washed with FACS buffer prior to flow cytometry.", "FIGS.", "6A through 6D depict the effect of peptide pre-treatment on the mobilization of intracellular Ca2+ in THP-1 cells in response to SDF-1α.", "Cells loaded with fura-2 were preincubated with peptide 15K (FIG.", "6C), 15D (FIG.", "6B) and 15CW (FIG.", "6D) at a final concentration of 500 μM for 3 min prior to stimulation with SDF-1α (100 ng/ml).", "FIGS.", "7A through 7C depicts the inhibition of chemokine receptor ligand binding by peptide pre-treatment.", "FIG.", "7A.", "Effect of pre-treatment with peptides 15K and 15D on the binding of MIP-1β to SupT1/CCR5 cells; FIG.", "7B.", "Effect of pre-treatment with peptides 15K and 1SD on the binding of SDF-1α to CEMx174 cells; and FIG.", "7C.", "Effect of pre-treatment with 15K and control peptide 15GIG on the binding of SDF-1α to CEmx174 cells.", "Binding studies were performed as described in Materials and Methods.", "DETAILED DESCRIPTION The present invention provides novel methods and compositions for clinical and diagnostic use in the evaluation, treatment and prevention of HIV infection and related disease conditions by reducing the ability of HIV to bind and fuse to cells.", "Typically, the compositions and procedures of the invention are directed toward prophylaxis or treatment of HIV-1 infection and related clinical conditions.", "The methods of the invention involve administering an effective amount of an anti-coreceptor binding agent to a subject to inhibit HIV-coreceptor interaction.", "Typically, the anti-coreceptor binding agent is administered in a prophylactic or therapeutically effective dose to a mammalian patient susceptible to HIV infection.", "Alternatively, the subject may comprise a susceptible cell population, tissue or organ, selected for in vivo or ex vivo treatment or diagnostic processing involving exposure of the subject to an anti-coreceptor binding agent (for example, bone marrow or other tissue or organ materials treated ex vivo before re-implantation or transplant).", "The methods and compositions of the invention involve delivery or formulation of an anti-coreceptor binding agent in an amount that is effective to inhibit HIV-coreceptor interactions, and/or to inhibit one or more selected biological activities or conditions associated with, or mediated by, HIV-coreceptor interactions.", "Selected biological activities include direct co-receptor binding by HIV viruses, coreceptor binding by selected viral proteins or peptides (including gp120 proteins and peptide fragments or derivatives of gp120), as well as binding by antibodies that recognize epitopes on gp120 or on a selected chemokine or HIV coreceptor.", "Additional biological activities in this context include HIV infection and related activities and conditions, for example HIV fusion with target host cells, HIV virion entry into host cells, HIV propagation and related HIV infective events, as well as specific HIV-related disease conditions including the full range of clinical conditions and disease states associated with AIDS and AIDS Related Complex (ARC) (for example, Kaposi's sarcoma and opportunistic viral (e.g., herpes) and bacterial (e.g., pneumonia infections).", "As used herein, the term “anti-coreceptor binding agent” is meant to include anti-coreceptor binding HIV peptides, as well as peptide analogs and peptide mimetics which exhibit comparable, or substantially the same, anti-coreceptor binding activity as a selected anti-coreceptor binding HIV peptide as described herein.", "The term anti-coreceptor binding peptide includes all of the reference peptides described herein, as well as other natural or artificially selected mutant or allelic forms and derivatives of these reference peptides having the desired anti-coreceptor binding activity.", "The term peptide analog refers to such artificially modified peptide analogs as chemically cleaved peptide fragments, chemically modified peptide derivatives, site directed mutant peptide variants having one or more amino acid insertions, substitutions or deletions, and the like.", "The anti-coreceptor binding peptides, analogs and mimetics of the invention function to specifically inhibit HIV-1 gp120 binding interactions with chemokine receptors (HIV-1 coreceptors).", "In addition, the anti-coreceptor binding agents are effective to inhibit or block HIV-1 cell fusion and virion entry, thereby impairing viral replication and transmission.", "Correlated with these activities, the anti-coreceptor binding agents and methods of the invention provide for safe and effective treatment of HIV-1 infection and related diseases.", "Ancillary uses are also readily implemented using these compositions and methods for diagnosing and evaluating HIV infection and related activities and disease mechanisms.", "The description herein illustrates production and characterization of peptides modeled after a novel structural motif identified in the HIV-1 gp120 envelope protein.", "This novel structural motif shares similarities with a corresponding structural motif identified as a conservative feature among diverse chemokines.", "This conservative motif is represented in one aspect by the reference sequences identified for HIV strains HXB2, IIIB, and JRFL, which represent operable anti-coreceptor binding peptides within the invention provided as native fragments of the corresponding gp120 proteins of these strains.", "As shown in Table 1, below, these sequences aligned with MIP-1β and SDF-1α embrace a novel, HIV-1 coreceptor binding motif.", "TABLE AMINO ACID ALIGNMENT OF PARTIAL SEQUENCES OF GP120 FROM REPRESENTATIVE HIV STRAINS WITH PARTIAL SEQUENCES OF MIP-1β AND SDF-1α HIV strain 326 340 HXB2 M R Q A H C N I S R A K W N N SEQ ID NO:1 326 340 III B M R Q A H C N I S R A K W N A SEQ ID NO:2 322 336 JR FL I R Q A H C N I S R A K W N D SEQ ID NO:3 Consensus I R Q A H C N V S R S E W N K SEQ ID NO:4 HIV A Consensus I R Q A H C N I S R A Q W N N SEQ ID NO:5 HIV B chemokine 47 60 MIP-1β S K Q - V C A D P S E S W V Q SEQ ID NO:6 48 61 SDF-1α N R Q - V C I D P K L K W I Q SEQ ID NO:7 *The residues numbered according to (Korber, B. and Los Alamos National Laboratory - Theoretical Biology and Biophysics Group T-10, Human retroviruses and AIDS.", "1998: a compilation and analysis of nucleic acid and amino acid sequences.", "Published by Theoretical Biology and Biophysics Group T-10 Los Alamos National Laboratory, Los Alamos, N.M., 1998, incorporated herein by reference).", "The residues that are shared between identified regions of HIV-1 gp120 and chemokines are shown in bold.", "This alignment illustrates the finding herein that the amino acid sequences of nearly all chemokines feature a Trp residue located at the beginning of a C-terminal α-helix and separated by six intervening residues from a conserved Cys residue.", "Comparing this conserved motif with the gp120 molecule of HIV-1, it is further demonstrated that all HIV isolates have a similar structural motif adjacent the V3 loop.", "As described in further detail below, computer modeling based on the sequence of HIV-1 strain JRFL which closely relates to a Consensus HIV-1 sequence, demonstrates that a fragment of gp120 containing a conserved “CXXXXXXW motif” can be “template forced” onto the homologous portion of the chemokine MIP-1β.", "Thus, the anti-coreceptor binding peptides of the invention typically feature the above noted CXXXXXXW motif, with additional amino acids, peptides, proteins, chemical reagents or moieties combined or conjugated therewith.", "These peptides include native HIV-1 peptides, such as those identified above in Table 1 and other known HIV-1 peptides that correspond to, or include, a partial or complete, homologous sequence to these exemplary peptides.", "Other peptides comprising the novel structural motif that includes the above noted CXXXXXXW anti-coreceptor binding determinant can comprise allelic variants among native peptide sequences, or synthetic or mutant peptide analogs of selected native HIV-1 gp120 peptides.", "Two exemplary peptides within the invention, designated 15K and 15D, include the C-terminal part of the V3 loop of gp120 and the N-terminal part of the C3 segment of the protein (Table 2).", "These peptides differ from each other by the presence of a D or K (Asp or Lys) residue at the twelfth position, which difference reflects allelic sequence variation among natural HIV-1 isolates.", "In addition, both of these exemplary synthetic peptides differ from corresponding, native HIV-1 sequences by a site-directed mutation comprising a substitution of K for Q (Lys for Gln) at the third residue position in the peptides, which reflects allelic sequence variation among natural HIV-1 isolates and also results in reduced susceptibility of the peptides to hydrolysis.", "TABLE 2 SEQUENCES OF EXEMPLARY SYNTHETIC PEPTIDES Design- tion Sequence 15K I R K A H C N I S R A K W N D (SEQ ID NO:8) 15D I R K A H C N I S R A D W N D (SEQ ID NO:9) These exemplary synthetic peptides of the invention inhibit or block binding or “docking” interactions between the HIV-1 envelope protein gp120 and chemokine receptors (e.g., CXCR4 and CCR5) that function as “coreceptors” for HIV entry on the surface of target cells (macrophages and T lymphocytes).", "The natural ligands of the coreceptors are chemokines (e.g., SDF-1α and MIP-1β), and it is further demonstrated herein that the conserved structural elements identified for the HIV gp120 protein motif that are shared with chemokines mediate critical HIV-coreceptor interactions.", "Detailed functional analyses of the peptides show that both are effective in competing with chemokines for binding to CCR5- and CXCR4-expressing cells.", "The peptides also inhibit monocyte chemotaxis stimulated by the chemokine RANTES (Regulated upon Activation Normal T-cell Expressed and Secreted).", "In addition, the anti-coreceptor binding peptides of the invention are shown herein to be potent inhibitors of HIV replication.", "In certain aspects, the peptides effect this inhibition in a multi-tropic or multi-specific manner to prevent or treat infection by both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV strains by blocking HIV interactions with distinct (CXCR4 and CCR5) coreceptors.", "This ability to use small peptides to achieve multi-tropic (i.e., multi-receptor) blockade is a surprising and important advantage satisfied by the invention.", "The anti-coreceptor binding peptides of the present invention include naturally occurring peptide variants, e.g., naturally occurring allelic variants and mutant proteins, as well as synthetic, e.g., chemically or recombinantly engineered, peptide fragments and analogs.", "As used herein, anti-coreceptor binding peptide “analogs” is meant to include a modified gp120 peptide incorporating one or more amino acid substitutions, insertions, rearrangements or deletions as compared to a native amino acid sequence of an HIV-1 gp120 anti-coreceptor binding peptide domain, fragment or motif, as described herein.", "Anti-coreceptor binding peptide analogs thus modified exhibit substantial anti-coreceptor binding activity comparable to that of a corresponding native peptide, which is activity that at least 50%, typically at least 75% or greater, compared to activity of the corresponding native peptide (e.g., as determined by an in vitro coreceptor binding assay or HIV-1 infection assay).", "As used herein, the term “biologically active anti-coreceptor binding analogs and mimetics” refers to analogs or mimetics of native peptides which encompass the entire length, sequence or chemical structure of the corresponding native peptide but which nevertheless maintains substantial anti-coreceptor binding activity as described above in an appropriate assay system.", "For purposes of the present invention, the term anti-coreceptor binding peptide “analog” thus includes derivatives or synthetic variants of a native HIV-1 gp120 anti-coreceptor binding peptide, such as amino and/or carboxyl terminal deletions and fusions, as well as intrasequence insertions, substitutions or deletions of single or multiple amino acids.", "Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein.", "Random insertion is also possible with suitable screening of the resulting product.", "Deletional variants are characterized by removal of one or more amino acids from the sequence.", "Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.", "Where a native HIV-1 gp120 anti-coreceptor binding peptide is modified by amino acid substitution, amino acids are generally replaced by other amino acids having similar, conservatively related chemical properties such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like.", "Residue positions which are not identical to the native peptide sequence are thus replaced by amino acids having similar chemical properties, such as charge or polarity, which changes are not likely to substantially effect the properties of the peptide analog.", "These and other minor alterations substantially maintain the immunoidentity (e.g., recognition by one or more monoclonal antibodies that recognize a native HIV-1 gp120 anti-coreceptor binding peptide) and other biological activities of the native peptide.", "In this context, the term “conservative amino acid substitution” refers to the general interchangeability of amino acid residues having similar side chains.", "For example, a group of amino acids having aliphatic side chains is alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.", "Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another.", "Likewise, the present invention contemplates the substitution of a polar (hydrophilic) residue such as between arginine and lysine, between glutamine and asparagine, and between threonine and serine.", "Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another or the substitution of an acidic residue such as aspartic acid or glutamic acid for another is also contemplated.", "Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.", "Anti-coreceptor binding peptide analogs also include modified forms of a native HIV-1 gp120 anti-coreceptor binding peptide incorporating stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, or unnatural amino acids such as a, α-disubstituted amino acids, N-alkyl amino acids, lactic acid.", "These and other unconventional amino acids may also be substituted or inserted within native HIV-1 gp120 anti-coreceptor binding peptides of the present invention.", "Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and other similar amino acids and amino acids (e.g., 4-hydroxyproline).", "For purposes of the present invention, analogs of native HIV-1 gp120 anti-coreceptor binding peptides also include single or multiple substitutions, deletions and/or additions of carbohydrate, lipid and/or proteinaceous moieties that occur naturally or artificially as structural components of gp120 peptides or are bound or otherwise associated with the peptide analog.", "To facilitate production and use of anti-coreceptor binding peptide analogs within the invention, reference can be made to molecular phylogenetic studies that characterize conserved and divergent protein structural and functional elements between different HIV-1 strains, and between HIV-1 and other HIV taxa and more distantly related retroviruses.", "In this regard, available studies provide detailed assessments of gp120 protein structure-function relationships on a fine molecular level.", "These studies include detailed sequence comparisons identifying conserved and divergent structural elements among a large number of HIV-1 gp120 allelic variants, for example.", "Each of these conserved and divergent structural elements facilitate practice of the invention by pointing to useful targets that for modifying native HIV-1 gp120 anti-coreceptor binding peptides to confer desired structural and/or functional changes.", "In this context, existing sequence alignments may be analyzed, or conventional sequence alignment methods may be employed to yield sequence comparisons for analysis, to identify corresponding protein regions and amino acid positions between native HIV-1 gp120 anti-coreceptor binding peptides and related or homologous peptides bearing a structural element of interest for incorporation within an anti-coreceptor binding peptide analog.", "Typically, one or more amino acid residues marking a structural element of interest in a different reference peptide sequence is incorporated within the anti-coreceptor binding peptide analog.", "For example, a cDNA encoding a native HIV-1 gp120 anti-coreceptor binding peptide may be recombinantly modified at one or more corresponding amino acid position(s) (i.e., corresponding positions that match or span a similar aligned sequence element according to accepted alignment methods to residues marking the structural element of interest in a heterologous reference peptide sequence) to encode an amino acid deletion, substitution, or insertion that alters corresponding residue(s) in the native HIV-1 gp120 anti-coreceptor binding peptide to generate an operable peptide analog within the invention having an analogous structural and/or functional element as the reference peptide or protein.", "Within this rational design method for constructing anti-coreceptor binding peptide analogs, the native or wild-type identity of residue(s) at amino acid positions corresponding to a structural element of interest in a heterologous reference peptide or protein may be altered to the same, or a conservatively related, residue identity as the corresponding amino acid residue(s) in the reference peptide or protein.", "However, it is often possible to alter native amino acid residues non-conservatively with respect to the corresponding reference protein residue(s).", "In particular, many non-conservative amino acid substitutions, particularly at divergent sites suggested to be more amenable to modification, may yield a moderate impairment or neutral effect, or even enhance a selected biological activity, compared to the function of a native HIV-1 gp120 anti-coreceptor binding peptide.", "Extensive protein structure-function data are available in the literature pertaining to HIV that will serve and guide the artisan to prepare functional anti-coreceptor binding peptide analogs for use within the invention.", "Structure-function relationships between HIV-1 strains and between HIV-1 and more distantly related retroviruses may be elucidated using conventional molecular phylogenetic analysis coupled with functional assays for determining anti-coreceptor binding activities described herein.", "Sequence alignment and comparisons to forecast useful peptide analogs and mimetics will be further refined by analysis of crystalline structure (see, e.g., Löebermann et al., J. Mol.", "Biol.", "177:531-556, 1984; Huber et al., Biochemistry 28:8951-8966, 1989; Stein et al., Nature 347:99-102, 1990; Wei et al., Structural Biology 1:251-255, 1994, each incorporated herein by reference) coupled with computer modeling methods known in the art.", "These analyses allow detailed structure-function mapping to identify desired structural elements and modifications for incorporation into anti-coreceptor binding peptide analogs and mimetics that will exhibit substantial anti-coreceptor binding activity for use within the methods of the invention.", "Native HIV-1 gp120 anti-coreceptor binding peptides and peptide analogs within the invention are typically between about 6-35 amino acid residues in length, more typically between about 10 and 21 or 22 amino acid residues in length.", "Within certain embodiments, the native peptides and analogs are between about 10-17 residues in length.", "In more specific embodiments, peptides are 10, 12, 13, 15, 17, 21, or 22 amino acid residues in length.", "Anti-coreceptor binding peptide analogs of the invention typically show substantial sequence identity to a corresponding native HIV-1 gp120 anti-coreceptor binding peptide sequence.", "Within the foregoing length ranges, both the native HIV-1 gp120 anti-coreceptor binding peptide and the peptide analogs typically comprise the conserved “CXXXXXXW” motif, described above, which may be extended to include additional residues from the native HIV-1 gp120 anti-coreceptor binding peptide sequence or non-native residues, fusion protein members, chemical moieties and the like.", "As applied to anti-coreceptor binding peptide analogs and fragments, these analogs and fragments typically exhibit substantial amino acid sequence identity to a corresponding native HIV-1 gp120 anti-coreceptor binding peptide sequence.", "The term “substantial sequence identity” as used herein means that the two subject amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap penalties, share at least 80 percent sequence identity, often at least 90-95 percent or greater sequence identity.", "“Percentage amino acid identity” refers to a comparison of the amino acid sequences of two peptides which, when optimally aligned, have approximately the designated percentage of the same amino acids.", "Sequence comparisons are generally made to a reference sequence over a comparison window of at least 10 residue positions, frequently over a window of at least 15-20 amino acids, wherein the percentage of sequence identity is calculated by comparing a reference sequence to a second sequence, the latter of which may represent, for example, a peptide analog sequence that includes one or more deletions, substitutions or additions which total 20 percent, typically less than 5-10% of the reference sequence over the window of comparison.", "The reference sequence may be a subset of a larger sequence, for example, as a segment of the HIV-1 gp120 protein.", "Optimal alignment of sequences for aligning a comparison window may be conducted according to the local homology algorithm of Smith and Waterman (Adv.", "Appl.", "Math.", "2:482, 1981), by the homology alignment algorithm of Needleman and Wunsch (J. Mol.", "Biol.", "48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc.", "Natl.", "Acad.", "Sci.", "USA 85:2444, 1988), or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).", "By aligning a peptide analog optimally with a corresponding native HIV-1 gp120 anti-coreceptor binding peptide, and by using appropriate assays, e.g., coreceptor binding assays, to determine a selected biological activity, one can readily identify operable peptide analogs for use within the methods and compositions of the invention.", "Anti-coreceptor binding peptide analogs are typically specifically immunoreactive with antibodies raised to the corresponding native HIV-1 gp120 anti-coreceptor binding peptide.", "Likewise, nucleic acids encoding functional anti-coreceptor binding peptide analogs will typically selectively hybridize to nucleic acid sequences encoding a corresponding native HIV-1 gp120 anti-coreceptor binding peptide under accepted, moderate or high stringency hybridization conditions (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference).", "The phrase “selectively hybridizing to” refers to a selective interaction between a nucleic acid probe that hybridizes, duplexes or binds preferentially to a particular target DNA or RNA sequence, for example when the target sequence is present in a heterogenous preparation such as total cellular DNA or RNA.", "Generally, nucleic acid sequences encoding functional anti-coreceptor binding peptide analogs and fragments will hybridize to nucleic acid sequences encoding native HIV-1 gp120 anti-coreceptor binding peptides under stringent conditions selected to be about 5° C. lower than the thermal melting point (Tm) for the subject sequence at a defined ionic strength and pH.", "The Tm is the temperature (under defined ionic strength and pH) at which 50% of the complementary or target sequence hybridizes to a perfectly matched probe.", "For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, 1989 or Current Protocols in Molecular Biology F. Ausubel et al, ed., Greene Publishing and Wiley-Interscience, New York, 1987, each of which is incorporated herein by reference.", "Typically, stringent or selective conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60° C. Less stringent selective hybridization conditions can also be chosen.", "As other factors may significantly affect the stringency of hybridization, including, among others, base composition and size of the complementary strands, the presence of organic solvents and the extent of base mismatching, the combination of parameters is more important than the specific measure of any one.", "Anti-coreceptor binding peptide analogs of the invention typically show substantial sequence identity to a corresponding native HIV1 gp120 anti-coreceptor binding peptide sequence.", "Within the foregoing length ranges, both the native HIV-1 gp120 anti-coreceptor binding peptide and the peptide analogs typically comprise the conserved “CXXXXXXW” motif, described above, which may be extended to include additional residues from the native HIV-1 gp120 anti-coreceptor binding peptide sequence, non-native residues, fusion protein members, chemical moieties, and the like.", "As applied to anti-coreceptor binding peptide analogs and fragments, these analogs and fragments typically exhibit substantial amino acid sequence identity to a corresponding native HIV-1 gp120 anti-coreceptor binding peptide sequence.", "Various peptide analogs are contemplated within the scope of the invention, which typically satisfy the foregoing length criteria of 10 to and include the conserved “CXXXXXXW” motif, but which incorporate selected sequence modifications at one or more positions to yield a desired structural or activity modification.", "To illustrate the breadth of different peptides and peptide analogs that are useful within the invention, the following table (Table 3) provides a map of anti-coreceptor binding peptide sequence variants based on a detailed analysis of different strains of HIV-1, along with structure-function analysis directed to both gp120 and chemokine structure-function, further guided by general rules of peptide structure-function.", "TABLE 3 MENU FOR CONSTRUCTING OPERABLE HIV gp120 PEPTIDES AND PEPTIDE ANALOGS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Peptide 15K I R Q A H C N I S R A K W N D SEQ ID NO:8 Peptide 15D D SEQ ID NO:9 M G K T Y D L N K R R Q N K E R P F H V D G K E D K S K L V Q T Y T S T D E E T S E S N K D K A S Q K T L T P I E A Y E G N R Q A I A V S I D E T A R V V I P I D S S S A S Q T N G T A P T V W Q A G I N H G N H V M M D A V L Y P I I H H V In accordance with the foregoing table, peptides which satisfy the above-described length criteria and include the conserved “CXXXXW” motif can also be selected from corresponding or overlapping native peptides among any known HIV isolate having a variant sequence as compared to a corresponding “reference” peptide.", "As illustrated in Table 3, one reference peptide that may be used in this context is the 15K peptide, which provides an exemplary reference sequence for comparison with other peptide sequences within the invention.", "Against this reference sequence, the table provides a range of amino acid residues that can be effectively substituted at the indicated residue positions (numbered in reference to the 15K peptide) which, for the substitutions designated by regular font single amino acid code, follow natural variations in the HIV gp120 available in the various HIV sequence data banks (e.g., http://hiv-web.lanl.gov/., incorporated herein by reference).", "Each of these residues can be incorporated, in a native HIV gp120-derived peptide, or in a single- or multiple-substituted peptide analog of the invention, to yield effective anti-coreceptor binding agents.", "Thus, peptides and peptide analogs are provided which satisfy the above-described length criteria and include the conserved “CXXXXXXW” motif, and which include any one, or any combination, of the following alternative residues occurring naturally or by substitution at the indicated positions (as designated for peptide 15K in Table 3 and determined for the subject peptide by conventional comparison and alignment against, e.g., the 15K reference sequence, as described herein): Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q, N, I, or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, V, Y, D, A; Position 9—S, N+EE, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A, R, K, T, S, G, E, D, N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. Certain of the foregoing residue/position alternatives, for example, the residue/position alternatives designated by underscoring in the preceding paragraph, may be selected in a greater number of peptides and analogs of the invention compared to other alternatives.", "This preference may be guided based on the greater degree of conservation of the indicated residue at corresponding positions among different HIV-1 isolates, or based on a convergent or homologous occurrence of the indicated residue at corresponding positions in gp120 of a selected HIV isolate and in a selected chemokine.", "Additional selections within the foregoing “menu” will be guided to favor conservative structural relationships between original and substitute residues in the preparation of anti-coreceptor binding peptide analogs, as described herein.", "In accordance with these selection principals, the above-noted, alternative residues may be included in peptides and peptide analogs of the invention, singly or in any combination of 2, 3, 4 or more and up to 13 residues (e.g., as exemplified by the various combinations shown in the separate lines of Table 3), selected or substituted at the indicated positions.", "Within additional aspects of the invention, peptide mimetics are provided which comprise a peptide or non-peptide molecule that mimics the tertiary binding structure and activity of the anti-coreceptor binding peptides described herein.", "These peptide mimetics include recombinantly or chemically modified peptides, as well as non-peptide anti-coreceptor binding agents such as small molecule drug mimetics, as further described below.", "In one aspect, peptides of the invention are modified to produce peptide mimetics by replacement of one or more naturally occurring side chains of the 20 genetically encoded amino acids (or D amino acids) with other side chains, for instance with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclics.", "For example, proline analogs can be made in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members.", "Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic.", "Heterocyclic groups can contain one or more nitrogen, oxygen, and/or sulphur heteroatoms.", "Examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl.", "These heterocyclic groups can be substituted or unsubstituted.", "Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.", "The peptide compounds of the invention, including peptidomimetics, can also be covalently bound to one or more of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkenes, in the manner set forth in U.S. Pat.", "No.", "4,640,835; U.S. Pat.", "No.", "4,496,689; U.S. Pat.", "No.", "4,301,144; U.S. Pat.", "No.", "4,670,417; U.S. Pat.", "No.", "4,791,192; or U.S. Pat.", "No.", "4,179,337, all which-are incorporated by reference in their entirety herein.", "Other peptide analogs and mimetics within the invention include glycosylation variants, and covalent or aggregate conjugates with other chemical moieties.", "Covalent derivatives can be prepared by linkage of functionalities to groups which are found in amino acid side chains or at the N- or C-termini, by means which are well known in the art.", "These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g., lysine or arginine.", "Acyl groups are selected from the group of alkyl-moieties including C3 to C18 normal alkyl, thereby forming alkanoyl aroyl species.", "Covalent attachment to carrier proteins, e.g., immunogenic moieties may also be employed.", "In certain embodiments, glycosylation alterations of anti-coreceptor binding agents are included, which can be made, e.g., by modifying the glycosylation patterns of a peptide during its synthesis and processing, or in further processing steps.", "Particularly preferred means for accomplishing this are by exposing the peptide to glycosylating enzymes derived from cells which normally provide such processing, e.g., mammalian glycosylation enzymes.", "Deglycosylation enzymes are also contemplated.", "Also embraced are versions of the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine, or other moieties, including ribosyl groups or cross-linking reagents.", "Peptidomimetics may also have amino acid residues which have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate groups.", "In some embodiments, the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity ligands.", "A major group of peptidomimetics within the invention are covalent conjugates of the anti-coreceptor binding peptides, or fragments thereof, with other proteins or peptides.", "These derivatives can be synthesized in recombinant culture such as N- or C-terminal fusions or by the use of agents known in the art for their usefulness in cross-linking proteins through reactive side groups.", "Preferred peptide and protein derivatization sites for targeting by cross-linking agents are at free amino groups, carbohydrate moieties, and cysteine residues.", "Fusion polypeptides between anti-coreceptor binding peptides and other homologous or heterologous peptides and proteins are also provided.", "Many growth factors and cytokines are homodimeric entities, and a repeat construct of anti-coreceptor binding peptide linked to form “cluster peptides” will yield various advantages, including lessened susceptibility to proteolytic degradation.", "Various alternative multimeric constructs comprising peptides of the invention are also provided.", "In one embodiment, various polypeptide fusions are provided as described in U.S. Pat.", "Nos.", "6,018,026 and 5,843,725, by linking one or more anti-coreceptor binding peptides of the invention with a heterologous, multimerizing polypeptide or protein, for example, immunoglobulin heavy chain constant region, or an immunoglobulin light chain constant region.", "The biologically active, multimerized polypeptide fusion thus constructed can be a hetero- or homo-multimer, e.g., a heterodimer or homodimer, which may each comprise one or more distinct anti-coreceptor binding peptide(s) of the invention.", "Other heterologous polypeptides may be combined with the anti-coreceptor binding agents to yield fusions comprising, e.g., a hybrid protein exhibiting heterologous (e.g., CD4) receptor binding specificity.", "Likewise, heterologous fusions may be constructed exhibit a combination of properties or activities of the derivative proteins.", "Other typical examples are fusions of a reporter polypeptide, e.g., CAT or luciferase, with a peptide of the invention, to facilitate localization of the fused protein (see, e.g., Dull et al., U.S. Pat.", "No.", "4,859,609, incorporated herein by reference).", "Other gene/protein fusion partners useful in this context include bacterial beta-galactosidase, trpE, Protein A, beta-lactamase, alpha amylase, alcohol dehydrogenase, and yeast alpha mating factor (see, e.g., Godowski et al., Science 241:812-816, 1988, incorporated herein by reference).", "The present invention also contemplates the use of anti-coreceptor binding agents modified by covalent or aggregative association with chemical moieties.", "These derivatives generally fall into the three classes: (1) salts, (2) side chain and terminal residue covalent modifications, and (3) adsorption complexes, for example with cell membranes.", "Such covalent or aggregative derivatives are useful for various purposes, for example as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of ligands or other binding ligands.", "For example, an anti-coreceptor binding agent can be immobilized by covalent bonding to a solid support such as cyanogen bromide-activated Sepharose, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of antibodies that specifically bind the anti-coreceptor binding agent.", "The anti-coreceptor binding agent can also be labeled with a detectable group, for example radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.", "Those of skill in the art recognize that a variety of techniques are available for constructing peptide mimetics with the same or similar desired biological activity as the corresponding peptide compound but with more favorable activity than the peptide with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis (see, e.g., Morgan and Gainor, Ann.", "Rep. Med.", "Chem.", "24:243-252, 1989, incorporated herein by reference).", "The following describes methods for preparing peptide mimetics modified at the N-terminal amino group, the C-terminal carboxyl group, and/or changing ore or more of the amido linkages in the peptide to a non-amido linkage.", "It being understood that two or more such modifications can be coupled in one peptide mimetic structure (e.g., modification at the C-terminal carboxyl group and inclusion of a —CH2-carbamate linkage between two amino acids in the peptide.", "For N-terminal modifications, the peptides typically are synthesized as the free acid but, as noted above, can be readily prepared as the amide or ester.", "One can also modify the amino and/or carboxy terminus of the peptide compounds of the invention to produce other compounds of the invention.", "Amino terminus modifications include methylating (i.e., —NHCH3 or —NH(CH3)2), acetylating, adding a carbobenzoyl group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO—, where R is selected from the group consisting of naphthyl, acridinyl, steroidyl, and similar groups.", "Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints.", "Amino terminus modifications are as recited above and include alkylating, acetylating, adding a carbobenzoyl group, forming a succinimide group, and the like.", "Specifically, the N-terminal amino group can then be reacted as follows: (a) to form an amide group of the formula RC(O)NH— where R is as defined above by reaction with an acid halide (e.g., RC(O)Cl) or acid anhydride.", "Typically, the reaction can be conducted by contacting about equimolar or excess amounts (e.g., about 5 equivalents) of an acid halide to the peptide in an inert diluent (e.g., dichloromethane) preferably containing an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to scavenge the acid generated during reaction.", "Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes).", "Alkylation of the terminal amino to provide for a lower alkyl N-substitution followed by reaction with an acid halide as described above will provide for N-alkyl amide group of the formula RC(O)NR—; (b) to form a succinimide group by reaction with succinic anhydride.", "As before, an approximately equimolar amount or an excess of succinic anhydride (e.g., about 5 equivalents) can be employed and the amino group is converted to the succinimide by methods well known in the art including the use of an excess (e.g., ten equivalents) of a tertiary amine such as diisopropylethylamine in a suitable inert solvent (e.g., dichloromethane) (see, for example, U.S. Pat.", "No.", "4,612,132, incorporated herein by reference).", "It is understood that the succinic group can be substituted with, for example, C2-C6 alkyl or —SR substituents which are prepared in a conventional manner to provide for substituted succinimide at the N-terminus of the peptide.", "Such alkyl substituents are prepared by reaction of a lower olefin (C2-C6) with maleic anhydride in the manner described by (U.S. Pat.", "No.", "4,612,132) and —SR substituents are prepared by reaction of RSH with maleic anhydride where R is as defined above; (c) to form a benzyloxycarbonyl-NH— or a substituted benzyloxycarbonyl-NH-group by reaction with approximately an equivalent amount or an excess of CBZ-Cl (i.e., benzyloxycarbonyl chloride) or a substituted CBZ-Cl in a suitable inert diluent (e.g., dichloromethane) preferably containing a tertiary amine to scavenge the acid generated during the reaction; (d) to form a sulfonamide group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R—S(O)2Cl in a suitable inert diluent (dichloromethane) to convert the terminal amine into a sulfonamide where R is as defined above.", "Preferably, the inert diluent contains excess tertiary amine (e.g., ten equivalents) such as diisopropylethylamine, to scavenge the acid generated during reaction.", "Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes); (e) to form a carbamate group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R—OC(O)Cl or R—OC(O)OC6H4-p-NO2 in a suitable inert diluent (e.g., dichloromethane) to convert the terminal amine into a carbamate where R is as defined above.", "Preferably, the inert diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to scavenge any acid generated during reaction.", "Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes); and (f) to form a urea group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R—N═C═O in a suitable inert diluent (e.g., dichloromethane) to convert the terminal amine into a urea (i.e., RNHC(O)NH—) group where R is as defined above.", "Preferably, the inert diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine.", "Reaction conditions are otherwise conventional (e.g., room temperature for about 30 minutes).", "In preparing peptide mimetics wherein the C-terminal carboxyl group is replaced by an ester (i.e., —C(O)OR where R is as defined above), resins as used to prepare peptide acids are employed, and the side chain protected peptide is cleaved with base and the appropriate alcohol, e.g., methanol.", "Side chain protecting groups are then removed in the usual fashion by treatment with hydrogen fluoride to obtain the desired ester.", "In preparing peptide mimetics wherein the C-terminal carboxyl group is replaced by the amide —C(O)NR3R4, where R3 and R4 independently are hydrogen or a lower alkyl, a benzhydrylamine resin is used as the solid support for peptide synthesis.", "Upon completion of the synthesis, hydrogen fluoride treatment to release the peptide from the support results directly in the free peptide amide (i.e., the C-terminus is —C(O)NH2).", "Alternatively, use of the chloromethylated resin during peptide synthesis coupled with reaction with ammonia to cleave the side chain protected peptide from the support yields the free peptide amide and reaction with an alkylamine or a dialkylamine yields a side chain protected alkylamide or dialkylamide (i.e., the C-terminus is —C(O)NRR1 where R and R1 independently are hydrogen or a lower alkyl).", "Side chain protection is then removed in the usual fashion by treatment with hydrogen fluoride to give the free amides, alkylamides, or dialkylamides.", "In another alternative embodiment, the C-terminal carboxyl group or a C-terminal ester can be induced to cyclize by internal displacement of the —OH or the ester (—OR) of the carboxyl group or ester respectively with the N-terminal amino group to form a cyclic peptide.", "For example, after synthesis and cleavage to give the peptide acid, the free acid is converted to an activated ester by an appropriate carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene chloride (CH2Cl2), dimethyl formamide (DMF) mixtures.", "The cyclic peptide is then formed by internal displacement of the activated ester with the N-terminal amine.", "Internal cyclization as opposed to polymerization can be enhanced by use of very dilute solutions.", "Such methods are well known in the art.", "One can also cyclize the anti-coreceptor binding peptides of the invention, or incorporate a desamino or descarboxy residue at the termini of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the peptide.", "C-terminal functional groups of the compounds of the present invention include amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.", "Other methods for making peptide derivatives and mimetics of the are described in Hruby et al., (Biochem J.", "268:249-262, 1990, incorporated herein by reference).", "According to these methods, the anti-coreceptor binding peptide compounds of the invention also serve as structural models for non-peptide mimetic compounds with similar biological activity.", "Those of skill in the art recognize that a variety of techniques are available for constructing compounds with the same or similar desired biological activity as the lead peptide compound but with more favorable activity than the lead with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis (see, e.g., Morgan and Gainor, Ann.", "Rep. Med.", "Chem.", "24:243-252, 1989, incorporated herein by reference).", "These techniques include replacing the peptide backbone with a backbone composed of phosphonates, amidates, carbamates, sulfonamides, secondary amines, and/or N-methylamino acids.", "Peptide mimetics wherein one or more of the peptidyl linkages —C(O)NH— have been replaced by such linkages as a —CH2-carbamate linkage, a phosphonate linkage, a —CH2-sulfonamide linkage, a urea linkage, a secondary amine (—CH2NH—) linkage, and an alkylated peptidyl linkage —C(O)NR6— where R6 is lower alkyl are prepared during conventional peptide synthesis by merely substituting a suitably protected amino acid analogue for the amino acid reagent at the appropriate point during synthesis.", "Suitable reagents include, for example, amino acid analogues wherein the carboxyl group of the amino acid has been replaced with a moiety suitable for forming one of the above linkages.", "For example, if one desires to replace a —C(O)NR— linkage in the peptide with a —CH2-carbamate linkage (—CH2OC(O)NR—), then the carboxyl (—COOH) group of a suitably protected amino acid is first reduced to the —CH2OH group which is then converted by conventional methods to a —OC(O)Cl functionality or a para-nitrocarbonate —OC(O)O—C6H4-p-NO2 functionality.", "Reaction of either of such functional groups with the free amine or an alkylated amine on the N-terminus of the partially fabricated peptide found on the solid support leads to the formation of a —CH2OC(O)NR— linkage.", "For a more detailed description of the formation of such —CH2-carbamate linkages, see, e.g., Cho et al., (Science 261:1303-1305, 1993, incorporated herein by reference).", "Replacement of an amido linkage in an anti-coreceptor binding peptide with a —CH2-sulfonamide linkage can be achieved by reducing the carboxyl (—COOH) group of a suitably protected amino acid to the —CH2OH group, and the hydroxyl group is then converted to a suitable leaving group such as a tosyl group by conventional methods.", "Reaction of the tosylated derivative with, for example, thioacetic acid followed by hydrolysis and oxidative chlorination will provide for the —CH2—S(O)2Cl functional group which replaces the carboxyl group of the otherwise suitably protected amino acid.", "Use of this suitably protected amino acid analogue in peptide synthesis provides for inclusion of an —CH2S(O)2NR— linkage which replaces the amido linkage in the peptide thereby providing a peptide mimetic.", "For a more complete description on the conversion of the carboxyl group of the amino acid to a —CH2S(O)2Cl group, see, e.g., Weinstein and Boris (Chemistry & Biochemistry of Amino Acids.", "Peptides and Proteins, Vol.", "7, pp.", "267-357, Marcel Dekker, Inc., New York, 1983, incorporated herein by reference).", "Replacement of an amido linkage in an anti-coreceptor binding peptide with a urea linkage can be achieved in the manner known to the skilled artisan.", "Secondary amine linkages wherein a —CH2NH— linkage replaces the amido linkage in the peptide can be prepared by employing, for example, a suitably protected dipeptide analogue wherein the carbonyl bond of the amido linkage has been reduced to a CH2 group by conventional methods.", "For example, in the case of diglycine, reduction of the amide to the amine will yield after deprotection H2NCH2CH2NHCH2 COOH which is then used in N-protected form in the next coupling reaction.", "The preparation of such analogues by reduction of the carbonyl group of the amido linkage in the dipeptide is well known in the art.", "The anti-coreceptor binding agents of the present invention may exist in a monomeric form with no disulfide bond formed with the thiol groups of the cysteine residue(s).", "Alternatively, an intermolecular disulfide bond between the thiol groups of cysteines on two or more peptides can be produced to yield a multimeric (e.g., dimeric, tetrameric or higher oligomeric) compound.", "Certain of these peptides can be cyclized or dimerized via displacement of the leaving group by the sulfur of a cysteine or homocysteine residue (see, e.g., Barker et al., J. Med.", "Chem.", "35:2040-2048, 1992; and/or et al., J. Org.", "Chem.", "56:3146-3149, 1991, each incorporated herein by reference).", "Thus, one or more native cysteine residues may be substituted with a homocysteine.", "Intramolecular or intermolecular disulfide derivatives of anti-coreceptor binding agents provide analogs in which one of the sulfurs has been replaced by a CH2 group or other isostere for sulfur.", "These analogs can be made via an intramolecular or intermolecular displacement, using methods known in the art as shown below.", "One of skill in the art will readily appreciate that this displacement can also occur using other homologs of the a-amino-g-butyric acid derivative shown above and homocysteine.", "All of the naturally occurring, recombinant, and synthetic peptides and peptide analogs and mimetics of the invention can be used for screening (e.g., in kits and/or screening assay methods) to identify additional compounds, including other peptides and peptide mimetics, that will function as anti-coreceptor binding agents within the methods and compositions of the invention.", "Several methods of automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period (see, e.g., Fodor et al., Science 251:767-773, 1991, and U.S. Pat.", "Nos.", "5,677,195; 5,885,837; 5,902,723; 6,027,880; 6,040,193; and 6,124,102, each incorporated herein by reference).", "For example, large combinatorial libraries of the compounds can be constructed by the encoded synthetic libraries (ESL) method described in, e.g., WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503, and WO 95/30642 (each incorporated by reference).", "Peptide libraries can also be generated by phage display methods (see, e.g., WO 91/18980, incorporated herein by reference).", "Many other publications describing chemical diversity libraries and screening methods are also considered reflective of the state of the art pertaining to these aspects of the invention and are generally incorporated herein.", "In one general screening strategy within the invention, new agonists and antagonists against HIV-coreceptor binding can be readily identified using the anti-coreceptor binding peptides of the invention incorporated within highly automated assay methods, e.g., using a purified chemokine receptor.", "Of particular importance are antagonist compounds that have multi-tropic or multi-specific binding affinity or activity, i.e., which inhibit or block HIV interactions with distinct (e.g., both CXCR4 and CCR5) coreceptors and thereby inhibit or prevent infection by both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV strains.", "One method of screening for new anti-coreceptor binding agents (e.g., small molecule drug peptide mimetics) utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing an anti-coreceptor binding peptide.", "Such cells, either in viable or fixed form, can be used for standard ligand/receptor binding assays (see, e.g., Parce et al., Science 246:243-247, 1989; and Owicki et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 87:4007-4011, 1990, each incorporated herein by reference).", "Competitive assays are particularly useful, where the cells are contacted and incubated with a labeled receptor or antibody having known binding affinity to the peptide ligand, and a test compound or sample whose binding affinity is being measured.", "The bound and free labeled binding components are then separated to assess the degree of ligand binding.", "The amount of test compound bound is inversely proportional to the amount of labeled receptor binding to the known source.", "Any one of numerous techniques can be used to separate bound from free ligand to assess the degree of ligand binding.", "This separation step can involve a conventional procedure such as adhesion to filters followed by washing, adhesion to plastic followed by washing, or centrifugation of the cell membranes.", "Viable cells can also be used to screen for the effects of drugs on coreceptor-mediated functions and biological activities, e.g., HIV viral fusion, cell entry, replication, and the like.", "Some detection methods allow for elimination of a separation step, e.g., a proximity sensitive detection system.", "Another technique for drug screening within the invention involves an approach which provides high throughput screening for compounds having suitable binding affinity to a target molecule, e.g., a chemokine receptor, and is described in detail in European Patent Application 84/03564, published on Sep. 13, 1984.First, large numbers of different test compounds, e.g., small peptides, are synthesized on a solid substrate, e.g., plastic pins or some other appropriate surface, (see, e.g., Fodor et al., Science 251:767-773, 1991, and U.S. Pat.", "Nos.", "5,677,195; 5,885,837; 5,902,723; 6,027,880; 6,040,193; and 6,124,102, each incorporated herein by reference).", "Then all of the pins are reacted with a solubilized anti-coreceptor binding agent of the invention, and washed.", "The next step involves detecting bound anti-coreceptor binding agent.", "Rational drug design may also be based upon structural studies of the molecular shapes of the anti-coreceptor binding agents.", "Various methods are available and well known in the art for characterizing, mapping, translating, and reproducing structural features of anti-coreceptor binding agents to guide the production and selection of new anti-coreceptor binding mimetics, including for example x-ray crystallography and 2 dimensional NMR techniques.", "These and other methods, for example, will allow reasoned prediction of which amino acid residues present in a selected anti-coreceptor binding peptide forms molecular contact regions necessary for peptide-coreceptor binding and specificity (see, e.g., Blundell and Johnson, Protein Crystallography, Academic Press, N.Y., 1976, incorporated herein by reference).", "Operable anti-coreceptor binding analogs and mimetics within the invention retain partial, complete or enhanced activity compared to native anti-coreceptor binding peptides, for example, partial or complete activity for inhibiting HIV-coreceptor binding, HIV viral fusion, cell entry, and/or replication, or HIV-related disease occurrence or progression.", "In this regard, operable anti-coreceptor binding analogs and mimetics for use within the invention will retain at least 50%, often 75%, and up to 95-100% or greater levels of one or more selected activities as compared to the same activity observed for a selected native HIV-1 gp120 anti-coreceptor binding peptide.", "These biological properties of altered peptides or non-peptide mimetics can be determined according to any suitable assay disclosed or incorporated herein, for example, by determining the ability of an anti-coreceptor binding analog or mimetic to inhibit HIV viral fusion to coreceptor positive target cells.", "In accordance with the description herein, the compounds of the invention are useful in vitro as unique tools for analyzing the nature and function of gp120 interactions with chemokine receptors, and will also serve as leads in various programs for designing additional peptide and non-peptide (e.g., small molecule drug) inhibitors of HIV-1.In addition, the anti-coreceptor binding peptides and peptide analogs of the invention are useful as immunogens, or components of immunogens, for eliciting an immune response in mammalian subjects, for example, to provide a protective immune response to prevent or treat HIV infection.", "In this aspect, the peptides of the invention can be administered alone or in a formulation comprising the peptide and a pharmaceutically acceptable carrier or adjuvant, with or without additional active or inactive ingredients such immune modulatory agents (e.g., cytokines).", "In certain embodiments, the peptides are administered as immunogens in the form of a conjugate (e.g., a multimeric peptide, or a peptide/carrier or peptide/hapten conjugate).", "In one embodiment, a peptide is conjugated with a multimerizing polypeptide as described above.", "Alternatively, a multimeric construct of immunogenic peptides, for example, comprising repeat peptide subunits, or containing two or more different peptides, can be employed, which contain one or multiple immunogenic epitope(s) that elicit a specific, humoral and/or cell-mediated (e.g.", "CTL) immune response directed against the immunizing peptide(s).", "Typically, the immune response will be marked by production of antibodies that bind the immunizing peptide(s) or peptide conjugate(s) with high affinity or avidity, but do not similarly recognize unrelated peptides.", "In other embodiments, the antibodies recognize the immunizing peptide(s) or peptide conjugate(s) but fail to bind with high affinity or avidity to chemokines, or to peptides derived from chemokines.", "In yet additional embodiments, antibodies generated against the anti-coreceptor binding peptide(s) or conjugate(s) bind to gp120 only when the gp120 protein is in a “bound” or “activated” state.", "For example, when gp120 is bound to a CD4 receptor this may result in a conformational change or other activation event that exposes a gp120 coreceptor binding domain.", "Antibodies that specifically target gp120 during this vulnerable activation period, (e.g., by recognizing one or more “masked” or “hidden” epitopes of gp120 that are not exposed for immune targeting when gp120 is in a “free” conformation or activity state, are particularly useful within the invention.", "Thus, the invention also provides diagnostic and therapeutic antibodies, including monoclonal antibodies, and related compositions and methods for use in the management and treatment of HIV infection and related disease.", "The antibodies specifically recognize anti-coreceptor binding peptides of the invention and are therefore useful for blocking HIV-coreceptor interactions when administered in vivo.", "For example, monoclonal antibodies can be generated that specifically bind the anti-coreceptor binding peptides of the invention, which antibodies can be purified and administered to a patient to block or inhibit HIV-coreceptor interactions, including binding or “docking,” and HIV viral fusion, entry, replication and HIV-related disease occurrence or progression.", "These activities are typically mediated, at least in part, by in vivo binding of the antibodies to gp120 (and therefore to intact HIV virus), at least in the protein's activated state upon binding to CD4.These immunotherapeutic reagents often include humanized antibodies, and can be combined for therapeutic use with additional active or inert ingredients as disclosed herein, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, and optionally with adjunctive or combinatorially active agents such as antiretroviral drugs.", "The production of non-human monoclonal antibodies, e.g., murine or rat, can be accomplished by, for example, immunizing the animal with a preparation comprising purified peptides of the invention, or purified gp120.The immunogen, often comprising a peptide/hapten complex or other conjugate as described herein, can be obtained from a natural source, by peptides synthesis or preferably by recombinant expression.", "Antibody-producing cells obtained from the immunized animals are immortalized and screened for the production of an antibody which binds to gp120 or a specific anti-coreceptor binding peptide (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, Cold Springs Harbor Press, Cold Spring Harbor, N.Y., 1988, incorporated by reference for all purposes).", "Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques (see, e.g., Queen et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 86:10029-10033, 1989 and WO 90/07861, each incorporated by reference).", "Human antibodies can be obtained using phage-display methods (see, e.g., WO 91/17271; WO 92/01047, each incorporated herein by reference).", "In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces.", "Antibodies are usually displayed as Fv or Fab fragments.", "Phage displaying antibodies with a desired specificity are selected by affinity enrichment to human cytochrome P450 or a fragment thereof.", "Human antibodies are selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody.", "The invention further provides fragments of the intact antibodies described above.", "Typically, these fragments compete with the intact antibody from which they were derived for specific binding to anti-coreceptor binding peptides and/or gp120.Antibody fragments include separate heavy chains, light chains Fab, Fab′ F(ab′)2, Fv, and single chain antibodies.", "Fragments can be produced by enzymic or chemical separation of intact immunoglobulins.", "For example, a F(ab′)2 fragment can be obtained from an IgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 using standard methods such as those described in Harlow and Lane, supra.", "Fab fragments may be obtained from F(ab′)2 fragments by limited reduction, or from whole antibody by digestion with papain in the presence of reducing agents.", "Fragments can also be produced by recombinant DNA techniques.", "Segments of nucleic acids encoding selected fragments are produced by digestion of full-length coding sequences with restriction enzymes, or by de novo synthesis.", "Often fragments are expressed in the form of phage-coat fusion proteins.", "This manner of expression is advantageous for affinity-sharpening of antibodies.", "To produce antibodies of the invention recombinantly, nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions, are inserted into expression vectors.", "The light and heavy chains can be cloned in the same or different expression vectors.", "The DNA segments encoding antibody chains are operably linked to control sequences in the expression vector(s) that ensure the expression of antibody chains.", "Such control sequences include a signal sequence, a promoter, an enhancer, and a transcription termination sequence.", "Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosome.", "E. coli is one prokaryotic host particularly useful for expressing antibodies of the present invention.", "Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.", "In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication) and regulatory sequences such as a lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.", "Other microbes, such as yeast, may also be used for expression.", "Saccharomyces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.", "Mammalian tissue cell culture can also be used to express and produce the antibodies of the present invention (see, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., 1987, incorporated herein by reference).", "Eukaryotic cells are preferred, because a number of suitable host cell lines capable of secreting intact antibodies have been developed.", "Preferred suitable host cells for expressing nucleic acids encoding the immunoglobulins of the invention include: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293) (Graham et al., J. Gen. Virol.", "36:59, 1977, incorporated herein by reference); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin, Proc.", "Natl.", "Acad.", "Sci.", "USA 77:4216, 1980, incorporated herein by reference); mouse sertoli cells (TM4, Mather, Biol.", "Reprod.", "23:243-251, 1980, incorporated herein by reference); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); and, TRI cells (Mather et al., Annals N.Y. Acad.", "Sci.", "383:44-46, 1982, incorporated herein by reference); and baculovirus cells.", "The vectors containing the polynucleotide sequences of interest (e.g., the heavy and light chain encoding sequences and expression control sequences) can be transferred into the host cell.", "Calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation can be used for other cellular hosts (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference).", "When heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins.", "After introduction of recombinant DNA, cell lines expressing immunoglobulin products are cell selected.", "Cell lines capable of stable expression are preferred (i.e., undiminished levels of expression after fifty passages of the cell line).", "Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, e.g., Scopes, Protein Purification, Springer-Verlag, N.Y., 1982, incorporated herein by reference).", "Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred.", "The anti-coreceptor binding agents of the invention can also generally be used in drug screening compositions and procedures, as noted above, to identify additional compounds having binding affinity to chemokine receptors and/or act as agonists or antagonists to gp120-coreceptor or chemokine-chemokine receptor interactions and related biological activities.", "Various screening methods and formats are available and well known in the art.", "Subsequent biological assays can then be utilized to determine if the screened compound has intrinsic coreceptor binding, agonist or antagonist, or other desired activity useful within the invention.", "Thus, in one example, the anti-coreceptor binding agents of the invention are useful as competitive binding agents in assays to screen for new HIV coreceptor agonists and antagonists.", "In such assays, the compounds of the invention can be used without modification or can be modified in a variety of ways; for example, by labeling, such as covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal.", "Direct labeling moieties include, for example, radiolabels, enzymes such as peroxidase and alkaline phosphatase (see, e.g., U.S. Pat.", "No.", "3,645,090; and U.S. Pat.", "No.", "3,940,475, each incorporated herein by reference), and fluorescent labels.", "Moieties for indirect labeling include biotin and avidin, a binding pair that can be coupled to one constituent and the other to a label.", "The compounds may also include spacers or linkers in cases where the compounds are to be attached to a solid support.", "The anti-coreceptor binding agents of the invention can also be employed, based on their ability to bind HIV coreceptors (chemokine receptors), as reagents for detecting and/or quantifying HIV coreceptors on living cells, fixed cells, in biological fluids, in tissue homogenates, in purified, natural biological materials, and the like.", "For example, by labeling such peptides, one can identify and/or quantify cells having HIV coreceptors on their surfaces.", "In addition, based on their ability to bind HIV coreceptors, the peptides of the present invention can be used in in situ staining, FACS (fluorescence-activated cell sorting), Western blotting, ELISA, and the like.", "Further, the peptides of the present invention can be used in receptor purification, or in purifying cells expressing HIV coreceptors.", "The compounds of the present invention can also be utilized as commercial reagents for various medical research and diagnostic uses.", "Such uses include but are not limited to: (1) use as a calibration standard for quantitating the activities of candidate HIV-coreceptor binding agonists and antagonists in a variety of functional assays; (2) use in structural analysis of HIV coreceptors through co-crystallization; and (3) use to investigate the mechanism of HIV coreceptor binding and activation.", "The present invention also provides reagents, formulations, kits, and methods which provide significant prophylactic and therapeutic values.", "In one embodiment of the invention, methods and compositions employ an anti-coreceptor binding agent for preventing and/or inhibiting HIV-1 binding to a host cell thereby ameliorating HIV, i.e., HIV-1 infection or a selected disease or condition associated therewith.", "Additional methods and compositions are provided to treat, prevent or delay the occurrence or AIDS or ARC.", "In yet additional embodiments, the methods and compositions of the invention can be used to treat other diseases and conditions which benefit from the compositions and methodologies disclosed herein, for example, a specific HIV-related disease or condition, such as Kaposi's sarcoma or an opportunistic viral (e.g., herpes) or bacterial (e.g., pneumonia) infection or condition.", "In accordance with the various treatment methods of the invention, the anti-coreceptor binding agent is delivered to a patient or other subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought.", "In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of an anti-coreceptor binding agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent and/or inhibit HIV binding to a host cell thereby preventing and/or inhibiting HIV infection and ameliorating a selected disease or condition.", "The term “subject” as used herein means any mammalian patient to which the compositions of the invention may be administered.", "Typical subjects intended for treatment with the compositions and methods of the present invention include humans, as well as non-human primates and other animals.", "Alternate subjects for administration of anti-coreceptor binding agents of the invention (either for a diagnostic, analytic, disease management or therapeutic purpose) include cells, cell explants, tissues and organs, particularly those originating from mammalian subjects at risk of developing or presently suffering from HIV infection.", "To identify subject patients for prophylaxis or treatment according to the methods of the invention, accepted screening methods are employed to determine risk factors associated with HIV infection, or to determine the status of an existing HIV infection or related condition in a subject.", "These screening methods include, for example, conventional work-ups to determine sexual and drug-use related risk factors, as well as diagnostic methods such as various ELISA immunoassay methods, which are available and well known in the art to detect and/or characterize HIV infection and related disease.", "These and other routine methods allow the clinician to select patients in need of therapy using the anti-coreceptor binding agents of the invention.", "In accordance with these methods and principles, anti-coreceptor binding agent therapy can be implemented as an independent prophylaxis or treatment program or as a follow-up, adjunct or coordinate treatment regimen to other treatments, for example other anti-HIV treatments such as drug therapy (AZT, DDI, protease inhibitors and other anti-retroviral drugs), surgery, vaccination, immunotherapy, hormone treatment, cell, tissue, or organ transplants, and the like.", "Within the compositions and methods of the invention, the anti-coreceptor binding agent is typically formulated with a pharmaceutically acceptable carrier and administered in an amount sufficient to inhibit virus binding and initiation or progression of HIV infection or a related disease or condition in the subject.", "According to the methods of the invention, the anti-coreceptor binding agent can be administered to subjects by a variety of administration modes, including by intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, intraperitoneal, parenteral, oral, rectal, intranasal, intrapulmonary, transdermal or topically to the eyes, ears, skin or mucosal surfaces.", "Alternatively, the anti-coreceptor binding agent may be administered ex vivo by direct exposure to cells, tissues or organs originating from a mammalian subject, for example, as a component of an ex vivo tissue or organ treatment formulation that contains the anti-coreceptor binding agent in a biologically suitable, liquid or solid carrier.", "For prophylactic and treatment purposes, the anti-coreceptor binding agent can be administered to the subject in a single bolus delivery, via continuous delivery (e.g., continuous intravenous or transdermal delivery) over an extended time period, or in a repeated administration protocol (e.g., on an hourly, daily or weekly basis).", "The various dosages and delivery protocols contemplated for administration of anti-coreceptor binding agents are therapeutically effective to inhibit the occurrence or alleviate one or more symptoms of HIV infection.", "An “anti-HIV therapeutically effective amount” of the anti-coreceptor binding agent thus refers to an amount that is effective, at dosages and for periods of time necessary, to achieve detectable inhibition of HIV binding and/or infection (initiation or progression) or a related condition (e.g., in HIV-exposed versus unexposed, or treated versus untreated, test and control subjects).", "In certain embodiments, a therapeutically effective amount of the anti-coreceptor binding agent, depending on the selected mode, frequency and duration of administration, will be effective to reduce or prevent HIV binding and infection of cells of the patient.", "Alternatively or in addition to these effects, a therapeutically effective dosage of the anti-coreceptor binding agent, which can include repeated doses within an prolonged prophylaxis or treatment regimen, will alleviate one or more symptoms or detectable conditions associated with HIV infection.", "Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by determining effective dosages and administration protocols that significantly reduce the occurrence or severity of HIV infection or related disease symptoms or conditions in the subject, which may be any of a range of accepted, e.g., murine or non-human primate, animal model subjects known in the art.", "Alternatively, effective dosages can be determined using in vitro models (e.g., immunologic and histopathologic assays).", "Using such models, only ordinary calculations and adjustments are typically required to determine an appropriate concentration and dose to administer an effective amount of anti-coreceptor binding agent (e.g., intranasally effective, transdermally effective, intravenously effective, or intramuscularly effective) to a subject to elicit a desired response.", "In alternative embodiments, an “effective amount” or “effective dose” of the anti-coreceptor binding agent may simply inhibit one or more selected biological activity(ies) correlated with HIV-coreceptor binding, as set forth above.", "The actual dosage of anti-coreceptor binding agent will of course vary according to factors such as the risk or state of infection or disease, the subject's age, and weight, as well as the established potency of the anti-coreceptor binding agent for eliciting the desired activity or biological response in the subject.", "Dosage regimens can be adjusted to provide an optimum prophylactic therapeutic response.", "A therapeutically effective amount is also one in which any toxic or detrimental side effects of the anti-coreceptor binding agent is outweighed by therapeutically beneficial effects.", "A non-limiting range for a therapeutically effective amount of the anti-coreceptor binding agent 0.01 μg/kg-10 mg/kg, more typically between about 0.05 and 5 mg/kg, and in certain embodiments between about 0.2 and 2 mg/kg.", "Dosages within this range can be achieved by single or multiple administrations, including, e.g., multiple administrations per day, daily or weekly administrations.", "Per administration, it is desirable to administer at least one microgram of anti-coreceptor binding agent, more typically between about 10 μg and 5.0 mg, and in certain embodiments between about 100 μg and 1.0 or 2.0 mg to an average human subject.", "It is to be further noted that for each particular subject, specific dosage regimens should be evaluated and adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the anti-coreceptor binding agent compositions.", "Dosage of the anti-coreceptor binding agent can be varied by the attending clinician to maintain a desired concentration at the target site.", "For example, if an intravenous mode of delivery is selected local concentration of the anti-coreceptor binding agent in the bloodstream at a selected target tissue (e.g., circulating blood) can be between about 1-50 nanomoles of anti-coreceptor binding agent per liter, sometimes between about 1.0 nanomelia per liter and 10, 15 or 25 nanomoles per liter, depending on the subject's status and projected or measured response.", "Higher or lower concentrations can be selected based on the mode of delivery, e.g., trans-epidermal delivery versus delivery to a mucosal surface.", "Dosage should also be adjusted based on the release rate of the administered formulation, e.g., nasal spray versus powder, sustained release oral versus injected particle or transdermal delivery formulations, and the like.", "To achieve the same serum concentration level, for example, slow-release particles with a release rate of 5 nanomolar (under standard conditions) would be administered at about twice the dosage of particles with a release rate of 10 nanomolar.", "Anti-coreceptor binding agents comprising HIV-1 gp120 peptides and peptide analogs can be readily constructed using peptide synthetic techniques, such as solid phase peptide synthesis (Merrifleld synthesis), and the like, or by recombinant DNA techniques, that are well known in the art.", "Techniques for making substitution mutations at predetermined sites in DNA include for example M13 mutagenesis.", "Manipulation of DNA sequences to produce substitutional, insertional, or deletional variants are conveniently described elsewhere such as Sambrook et al., (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989).", "In accordance with these and related teachings, defined mutations can be introduced into a native gp120 peptide to generate analogs of interest by a variety of conventional techniques, e.g., site-directed mutagenesis of a cDNA copy of a portion of the gp120 gene encoding a selected peptide fragment, domain or motif.", "This can be achieved through and intermediate of single-stranded form, such as using the MUTA-gen® kit of Bio-Rad Laboratories (Richmond, Calif.), or a method using the double-stranded plasmid directly as a template such as the Chameleon® mutagenesis kit of Strategene (La Jolla, Calif.), or by the polymerase chain reaction employing either an oligonucleotide primer or a template which contains the mutation(s) of interest.", "A mutated subfragment can then be assembled into a complete anti-coreceptor binding peptide analog-encoding cDNA.", "A variety of other mutagenesis techniques are known and can be routinely adapted for use in producing the mutations of interest in a anti-coreceptor binding peptide analog-encoding cDNA and corresponding peptide analog of the invention.", "In accordance with the present invention, anti-coreceptor binding agents are isolated and purified before administration to a subject so that contaminants are removed.", "In one method for obtaining purified anti-coreceptor binding peptides, a polynucleotide molecule, for example, a deoxyribonucleic acid (DNA) molecule, that defines a coding sequence for a selected anti-coreceptor binding peptide or peptide analog (e.g., a biologically active mutant or fragment of a peptide as disclosed herein bearing a deletion or substitution of 1, 2, 3 or more residues) is operably incorporated in a recombinant polynucleotide expression vector that direct expression of the peptide or analog in a suitable host cell.", "Exemplary methods for cloning and purifying anti-coreceptor binding peptides and analogs employing these novel polynucleotides and vectors are widely known in the art.", "Briefly, a polynucleotide of the invention encoding an anti-coreceptor binding peptide or peptide analog is amplified by well known methods, such as the polymerase chain reaction (PCR).", "In this way the polynucleotide encoding the anti-coreceptor binding peptide, or a recombinantly modified version or portion thereof, is obtained for expression and purification according to conventional methods.", "A DNA vector molecule that incorporates a DNA sequence encoding the subject peptide or analog can be operatively assembled, e.g., by linkage using appropriate restriction fragments from various plasmids which are described elsewhere.", "Also contemplated by the present invention are ribonucleic acid (RNA) equivalents of the above described polynucleotides comprising a coding sequence for the subject anti-coreceptor binding peptide operatively linked in a polynucleotide expression construct for recombinant expression of the peptide or peptide analog.", "Once a polynucleotide molecule encoding an anti-coreceptor binding peptide or analog is isolated and cloned, the peptide or analog can be expressed in a variety of recombinantly engineered cells.", "Numerous expression systems are available for expressing a DNA encoding anti-coreceptor binding peptide.", "The expression of natural or synthetic nucleic acids encoding an anti-coreceptor binding peptide is typically achieved by operably linking the DNA to a promoter (which is either constitutive or inducible) within an expression vector.", "By expression vector is meant a polynucleotide molecule, linear or circular, that comprises a segment encoding the anti-coreceptor binding peptide of interest, operably linked to additional segments that provide for its transcription.", "Such additional segments include promoter and terminator sequences.", "An expression vector also may include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like.", "Expression vectors generally are derived from plasmid or viral DNA, and can contain elements of both.", "The term “operably linked” indicates that the segments are arranged so that they function in concert for their intended purposes, for example, transcription initiates in the promoter and proceeds through the coding segment to the terminator (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference).", "Expression vectors can be constructed which contain a promoter to direct transcription, a ribosome binding site, and a transcriptional terminator.", "Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway as described by Yanofsky, (J. Bacteriol.", "158:1018-1024, 1984, incorporated herein by reference) and the leftward promoter of phage lambda (Pk) as described by Herskowitz and Hagen, (Ann.", "Rev.", "Genet.", "14:399-445, 1980, incorporated herein by reference).", "The inclusion of selection markers in DNA vectors transformed in E. coli is also useful.", "Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.", "Vectors used for expressing foreign genes in bacterial hosts generally will contain a selectable marker, such as a gene for antibiotic resistance, and a promoter which functions in the host cell.", "Plasmids useful for transforming bacteria include pBR322 (Bolivar et al., Gene 2:95-113, 1977, incorporated herein by reference), the pUC plasmids (Messing, Meth.", "Enzymol.", "101:20-77, 1983; Vieira and Messing, Gene 19:259-268.1982, each incorporated herein by reference), pCQV2, and derivatives thereof Plasmids may contain both viral and bacterial elements.", "A variety of prokaryotic expression systems can be used to express anti-coreceptor binding peptides and peptide analogs.", "Examples include E. coli, Bacillus, Streptomyces, and the like.", "Detection of the expressed peptide is achieved by methods such as radioimmunoassay, Western blotting techniques or immunoprecipitation.", "For expression in eukaryotes, host cells for use in practicing the invention include mammalian, avian, plant, insect, and fungal cells.", "Fungal cells, including species of yeast (e.g., Saccharomyces spp., Schizosaccharomyces spp.)", "or filamentous fungi (e.g., Aspergillus spp., Neurospora spp.)", "can be used as host cells within the present invention.", "Strains of the yeast Saccharomyces cerevisiae can be used.", "As explained briefly below, anti-coreceptor binding peptides and analogs can be expressed in these eukaryotic systems.", "Suitable yeast vectors for use in the present invention include YRp7 (Struhl et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 76:1035-1039, 1978, incorporated herein by reference), YEp13 (Broach et al., Gene 8:121-133, 1979, incorporated herein by reference), POT vectors (U.S. Pat.", "No.", "4,931,373, incorporated herein by reference), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978, incorporated herein by reference) and derivatives thereof.", "Such vectors generally include a selectable marker, which can be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected.", "Often, the selectable marker will be one that complements host cell auxotrophy, provides antibiotic resistance and/or enables a cell to utilize specific carbon sources, for example, LEU2 (Broach et al., Gene 8:121-133, 1979), URA3 (Botstein et al., Gene 8:17, 1979, incorporated herein by reference), HIS3 (Struhl et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 76:1035-1039, 1978) or POT1 (U.S. Pat.", "No.", "4,931,373).", "Another suitable selectable marker available for use within the invention is the CAT gene, which confers chloramphenicol resistance on yeast cells.", "Examples of promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol.", "Chem.", "255:12073-12080, 1980; Alber and Kawasaki, J. Mol.", "Appl.", "Genet.", "1:419434, 1982; U.S. Pat.", "No.", "4,599,311) or alcohol dehydrogenase genes (Young et al., Genetic Engineering of Microorganisms for Chemicals, Hollaender et al., eds., p. 355, Plenum, N.Y., 1982; Ammerer, Meth.", "Enzymol.", "101:192-201, 1983).", "The TPI1 promoter (U.S. Pat.", "No.", "4,599,311) and the ADH2-4c promoter (Russell et al., Nature 304:652-654, 1983; and EP 284,044) also can be used.", "The expression units can also include a transcriptional terminator.", "An example of such a transcriptional terminator is the TPI1 terminator (Alber and Kawasaki, J. Mol.", "Appl.", "Genet.", "1:419-434, 1982).", "In addition to yeast, anti-coreceptor binding peptides and peptide analogs of the present invention can be expressed in filamentous fungi, for example, strains of the fungi Aspergillus (U.S. Pat.", "No.", "4,935,349, which is incorporated herein by reference).", "Examples of useful promoters include those derived from Aspergillus nidulans glycolytic genes, such as the ADH3 promoter and the tpiA promoter.", "An example of a suitable terminator is the ADH3 terminator (McKnight et al., EMBO J.", "4: 2093-2099, 1985, incorporated herein by reference).", "The expression units utilizing such components are cloned into vectors that are capable of insertion into the chromosomal DNA of Aspergillus.", "Techniques for transforming fungi are well known in the literature, and have been described, for instance, by Beggs (Nature 275:104-108, 1978), Hinnen et al., (Proc.", "Natl.", "Acad.", "Sci.", "USA 75:1929-1933, 1978), Yelton et al., (Proc.", "Natl.", "Acad.", "Sci.", "USA 81:1740-1747, 1984), and Russell (Nature 301:167-169, 1983), each incorporated herein by reference.", "The genotype of the host cell generally contains a genetic defect that is complemented by the selectable marker present on the expression vector.", "Choice of a particular host and selectable marker is well within the level of ordinary skill in the art.", "In addition to fungal cells, cultured mammalian cells can be used as host cells within the present invention.", "Examples of cultured mammalian cells for use in the present invention include the COS-1 (ATCC CRL 1650), BHK, and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol.", "6:59-72, 1977, incorporated herein by reference) cell lines.", "An example of a BHK cell line is the BHK 570 cell line (deposited with the American Type Culture Collection under accession number CRL 10314).", "In addition, a number of other mammalian cell lines can be used within the present invention, including rat Hep I (ATCC CRL 600), rat Hep II (ATCC CRL 1548), TCMK (ATCC CCL 139), human lung (ATCC CCL 75.1), human hepatoma (ATCC HTB-52), Hep G2 (ATCC HB 8065), mouse liver (ATCC CCL 29.1), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (Urlaub and Chasin, Proc.", "Natl.", "Acad.", "Sci USA 77:4216-4220, 1980, incorporated herein by reference).", "Mammalian expression vectors for use in carrying out the present invention include a promoter capable of directing the transcription of a cloned cDNA.", "Either viral promoters or cellular promoters can be used.", "Viral promoters include the immediate early cytomegalovirus (CMV) promoter (Boshart et al., Cell 41:521-530, 1985, incorporated herein by reference) and the SV40 promoter (Subramani et al., Mol.", "Cell.", "Biol.", "1:854-864, 1981, incorporated herein by reference).", "Cellular promoters include the mouse metallothionein-1 promoter (U.S. Pat.", "No.", "4,579,821, incorporated herein by reference), a mouse V1 promoter (Bergman et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 81:7041-7045, 1983; Grant et al., Nuc.", "Acids Res.", "15:5496, 1987, each incorporated herein by reference), a mouse V1 promoter (Loh et al., Cell 33:85-93, 1983, incorporated herein by reference), and the major late promoter from Adenovirus 2 (Kaufman and Sharp, Mol.", "Cell.", "Biol.", "2:1304-13199, 1982, incorporated herein by reference).", "Cloned DNA sequences can be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973; each incorporated by reference herein in their entirety).", "Other techniques for introducing cloned DNA sequences into mammalian cells can also be used, such as electroporation (Neumann et al., EMBO J.", "1:841-845, 1982, incorporated herein by reference) or cationic lipid-mediated transfection (Hawley-Nelson et al., Focus 15:73-79, 1993, incorporated herein by reference) using, e.g., a 3:1 liposome formulation of 2,3-dioleyloxy-N-[2 (sperminecarboxyamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate and dioleoly-phosphatidylethanolamine in water Lipofectamine reagent, GIBCO-BRL).", "To identify cells that have integrated the cloned DNA, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest.", "Examples of selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate.", "The selectable marker can be an amplifiable selectable marker, for example the DHFR gene.", "Additional selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., which is incorporated herein by reference).", "The choice of selectable markers is well within the level of ordinary skill in the art.", "Selectable markers can be introduced into the cell on a separate plasmid at the same time as the polynucleotide encoding the anti-coreceptor binding peptide, or they may be introduced on the same plasmid.", "If on the same plasmid, the selectable marker and the peptide-encoding polynucleotide can be under the control of different promoters or the same promoter.", "Constructs of this latter type are known in the art (for example, U.S. Pat.", "No.", "4,713,339).", "It also can be advantageous to add additional DNA, known as “carrier DNA” to the mixture which is introduced into the cells.", "Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, to begin expressing the polynucleotide sequence(s) of interest.", "Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion.", "For cells that have been transfected with an amplifiable selectable marker the drug concentration is increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels.", "Host cells containing polynucleotide constructs of the present invention are then cultured to produce anti-coreceptor binding peptide.", "The cells are cultured according to standard methods in a culture medium containing nutrients required for growth of the host cells.", "A variety of suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors.", "The growth medium generally selects for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct.", "Recombinantly produced anti-coreceptor binding peptides and peptide analogs as described above can be purified by techniques well known to those of ordinary skill in the art.", "For example, recombinantly produced peptides can be directly expressed or expressed as fusion proteins.", "The proteins can then be purified by a combination of cell lysis (e.g., sonication) and affinity chromatography.", "For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired peptide.", "The phrase “substantially purified” when referring to anti-coreceptor binding peptides or peptide analogs (including peptide fusions with other peptides and/or proteins) of the present invention, means a composition which is essentially free of other cellular components with which the peptides or analogs are associated in a non-purified, e.g., native state or environment.", "Purified peptide is generally in a homogeneous state although it can be in either in a dry state or in an aqueous solution.", "Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.", "Generally, substantially purified anti-coreceptor binding peptide comprises more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the peptide with a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient.", "More typically, the peptide is purified to represent greater than 90% of all proteins present in a purified preparation.", "In specific embodiments, the peptide is purified to greater than 95% purity or may be essentially homogeneous wherein other macromolecular species are not detectable by conventional techniques.", "The peptides and analogs of the present invention can be purified to substantial purity by standard techniques well known in the art.", "Useful purification methods include selective precipitation with such substances as ammonium sulfate; column chromatography; affinity methods, including immunopurification methods; and others.", "See, for instance, R. Scopes, Protein Purification: Principles and Practice, Springer-Verlag: N.Y., 1982, incorporated herein by reference.", "In general, anti-coreceptor binding peptides can be extracted from tissues or cell cultures that express the peptides and then immunoprecipitated, whereafter the peptides can be further purified by standard protein chemistry/chromatographic methods.", "For therapeutic administration, anti-coreceptor binding peptides, analogs and mimetics of the invention are typically formulated with a pharmaceutically acceptable carrier.", "As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents, and other excipients or additives that are physiologically compatible.", "In specific embodiments, the carrier is suitable for intranasal, intravenous, intramuscular, subcutaneous, parenteral, oral, transmucosal or transdermal administration.", "Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound.", "In preparing pharmaceutical compositions of the present invention, it may be desirable to modify the anti-coreceptor binding agent, or combine or conjugate the peptide or mimetic compound with other agents, to alter pharmacokinetics and biodistribution of the anti-coreceptor binding agent.", "A number of methods for altering pharmacokinetics and biodistribution are known to persons of ordinary skill in the art.", "Examples of such methods include protection of peptides, proteins or complexes thereof in vesicles composed of other proteins, lipids, carbohydrates, or synthetic polymers.", "For example, anti-coreceptor binding agents can be incorporated into liposomes in order to enhance pharmacokinetics and biodistribution characteristics.", "A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., (Ann.", "Rev.", "Biophys.", "Bioeng.", "2:467, 1980; U.S. Pat.", "Nos.", "4,235,871; 4,501,728 and 4,837,028, each incorporated herein by reference).", "For use with liposome delivery, the anti-coreceptor binding agent is typically entrapped within the liposome, or lipid vesicle, or is bound to the outside of the vesicle.", "Several strategies have been devised to increase the effectiveness of liposome-mediated delivery by targeting liposomes to specific tissues and specific cell types.", "Liposome formulations, including those containing a cationic lipid, have been shown to be safe and well tolerated in human patients (Treat et al., J. Natl.", "Cancer Instit.", "82:1706-1710, 1990, incorporated herein by reference).", "The compositions of the invention may alternatively contain as pharmaceutically acceptable carriers, substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.", "For solid compositions, conventional nontoxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.", "For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95%, more typically 25% to 75% of active ingredient.", "Therapeutic compositions for administering the anti-coreceptor binding agent can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients.", "The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.", "Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants.", "In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.", "Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.", "In certain embodiments of the invention, the anti-coreceptor binding agent is administered in a time release formulation, for example in a composition which includes a slow release polymer, or by depot injection.", "The active peptide, analog or mimetic can be prepared with carriers that will protect against rapid release, for example a controlled release vehicle such as implants, transdermal patches, or microencapsulated delivery system.", "Prolonged delivery of the anti-coreceptor binding agent, or a biologically active analog or mimetic thereof, in various compositions of the invention can be brought about by including in the composition agents that delay absorption, for example, aluminum monosterate hydrogels and gelatin.", "When controlled release formulations of anti-coreceptor binding agents is desired, controlled release binders suitable for use in accordance with the invention include any biocompatible controlled-release material which is inert to the active ingredient and which is capable of incorporating the anti-coreceptor binding agent.", "Numerous such materials are known in the art.", "Useful controlled-release binders are materials which are metabolized slowly under physiological conditions following their subcutaneous or intramuscular injection in mammals (i.e., in the presence of bodily fluids which exist there).", "Appropriate binders include but are not limited to biocompatible polymers and copolymers previously used in the art in sustained release formulations.", "Such biocompatible compounds are non-toxic and inert to surrounding tissues, e.g., following subcutaneous or intramuscular injection, and do not trigger significant adverse effects such as immune response, inflammation, or the like.", "They are metabolized into metabolic products which are also biocompatible and easily eliminated from the body.", "For example, a polymeric matrix derived from copolymeric and homopolymeric polyesters having hydrolysable ester linkages may be used.", "A number of these are known in the art to be biodegradable and to lead to degradation products having no or low toxicity.", "Exemplary polymers include polyglycolic acids (PGA) and polylactic acids (PLA), poly(DL-lactic acid-co-glycolic acid)(DL PLGA), poly(D-lactic acid-coglycolic acid) (D PLGA) and poly(L-lactic acid-co-glycolic acid)(L PLGA).", "Other useful biodegradable or bioerodable polymers include but are not limited to such polymers as poly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid), poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate), hydrogels such as poly(hydroxyethyl methacrylate), polyamides, poly(amino acids) (i.e., L-leucine, glutamic acid, L-aspartic acid and the like), poly(ester urea), poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides and copolymers thereof.", "Many methods for preparing such formulations are generally known to those skilled in the art (see, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, incorporated herein by reference).", "Useful formulations include controlled-release compositions such as are known in the art for the administration of leuprolide (trade name: Lupron.RTM.", "), e.g., microcapsules (U.S. Pat.", "Nos.", "4,652,441 and 4,917,893, each incorporated herein by reference), injectable formulations (U.S. Pat.", "No.", "4,849,228, incorporated herein by reference), lactic acid-glycolic acid copolymers useful in making microcapsules or injectable formulations (U.S. Pat.", "Nos.", "4,677,191 and 4,728,721, each incorporated herein by reference), and sustained-release compositions for water-soluble peptides (U.S. Pat.", "No.", "4,675,189, incorporated herein by reference).", "A long-term sustained release implant also may be used.", "These can be readily constructed to deliver therapeutic levels of the anti-coreceptor binding agent for at least 10 to 20 days, often at least 30 days, up to 60 days or longer.", "Long-term sustained release implants are well known to those of ordinary skill in the art and can incorporate some of the absorption delaying components described above.", "Such implants can be particularly useful by placing the implant near or directly within the target tissue or cell population, thereby affecting localized, high-doses of the anti-coreceptor binding agent at one or more sites of interest.", "In alternate embodiments, anti-coreceptor binding agent may be delivered to a mucosal surface, e.g., orally, intranasally, or rectally, for prophylaxis or treatment of HIV infection and related disease.", "For mucosal administration, the peptide, analog or mimetic is typically combined with an inert diluent or an assimilable edible carrier.", "The anti-coreceptor binding agent may be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into a subject's diet.", "For oral therapeutic administration, the anti-coreceptor binding agent may be incorporated with excipients and used in the form of ingestable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.", "Of course, taste-improving substances can be added in the case of oral administration forms.", "The percentage (e.g., by weight or by volume) of the anti-coreceptor binding agent in these compositions and preparations may, of course, be varied.", "As noted above, the amount of anti-coreceptor binding agent in such therapeutically useful compositions is generally such that a therapeutically effective dosage will be delivered.", "For oral or rectal administration, the anti-coreceptor binding agent can be worked into tablets or other solid forms by being mixed with solid, pulverulent carrier substances, such as sodium citrate, calcium carbonate or dicalcium phosphate, and binders such as polyvinyl pyrrolidone, gelatin or cellulose derivatives, possibly by adding also lubricants such as magnesium stearate, sodium lauryl sulfate, “Carbowax” or polyethylene glycol.", "Solid delivery vehicles may contain a protein or peptide in a mixture with fillers, such as lactose, saccharose, mannitol, starches, such as potato starch or amylopectin, cellulose derivatives or highly dispersed silicic acids.", "In soft-gelatin capsules, the active substance is dissolved or suspended in suitable liquids, such as vegetable oils or liquid polyethylene glycols.", "As further forms, one can use plug capsules, e.g., of hard gelatin, as well as dosed soft-gelatin capsules comprising a softener or plasticizer, e.g., glycerin.", "Alternatively, liquid dosage forms for delivering anti-coreceptor binding agents to mucosal surfaces include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.", "Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.", "Oral dosage forms optionally contain flavorants and coloring agents.", "Parenteral and intravenous forms would also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.", "The therapeutic compositions of the invention typically must be sterile and stable under all conditions of manufacture, storage and use.", "Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.", "Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.", "In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.", "The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.", "In certain embodiments of the invention, the anti-coreceptor binding agent is administered by topical delivery to a mucosal surface of the patient, for example, via intranasal or intrapulmonary delivery in the form of an aerosol spray or powder.", "According to one aspect of the invention, the anti-coreceptor binding agent is delivered in an intranasally or intrapulmonarily effective amount, typically in a selected volume of administered spray or powder, to achieve a desired therapeutic result.", "In related aspects of the invention, novel pharmaceutical compositions are provided for intranasal or intrapulmonary delivery that incorporate the anti-coreceptor binding agent in a powder or aqueous formulation.", "Intranasal or intrapulmonary administration allows self-administration of treatment by patients, provided that sufficient safeguards are in place to control and monitor dosing and side effects.", "Nasal or intrapulmonary administration also overcomes certain drawbacks of other administration forms, such as injections, that are painful and expose the patient to possible infections and may present drug bioavailability problems.", "Systems for aerosol dispensing of therapeutic liquids as a spray are well known.", "In one embodiment, metered doses of aerosolized anti-coreceptor binding agent are delivered by means of a specially constructed mechanical pump valve (See, for example, U.S. Pat.", "No.", "4,511,069, incorporated herein by reference).", "This hand-held delivery device is uniquely nonvented so that sterility of the solution in the aerosol container is maintained indefinitely.", "Certain nasal and intrapulmonary spray solutions within the invention comprise an anti-coreceptor binding agent in a liquid carrier that optionally includes a nonionic surfactant for enhancing absorption of the drug and one or more buffers or other additives to minimize nasal or pulmonary irritation.", "In some embodiments, the nasal or pulmonary spray solution further comprises a propellant.", "Thus, in certain embodiments the pharmaceutical compositions comprising an anti-coreceptor binding agent administrable in fine particulate form (e.g., between about 0.5-5.0 μm, more typically between about 1.0-2.5 μm diameter particles) comprising a surfactant and propellant as pharmaceutically acceptable carriers (see, e.g., U.S. Pat.", "No.", "5,902,789, incorporated herein by reference).", "The surfactant must be nontoxic and soluble in the propellant.", "Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.", "Mixed esters, such as mixed or natural glycerides, can be employed.", "Additional carrier can be included as desired, for example lecithin for intranasal delivery.", "The pH of the intranasal or intrapulmonary spray solution is typically between pH 6.8 and 7.2.Alternative means of mucosal administration for the anti-coreceptor binding agents of the invention may involve the use of powder carriers, for example ion exchange resins or adsorbent resin powders (see, e.g., U.S. Pat.", "No.", "5,942,242, incorporated herein by reference).", "In more detailed aspects of the invention, the anti-coreceptor binding agent is stabilized to extend its effective half-life following delivery to the subject, particularly for extending metabolic persistence in an active state within the physiological environment (e.g., in the bloodstream, at a mucosal surface, or within a connective tissue compartment or fluid-filled body cavity).", "For this purpose, the anti-coreceptor binding agent can be modified by chemical means, e.g., chemical conjugation, N-terminal capping, PEGylation, or recombinant means, e.g., site-directed mutagenesis or construction of fusion proteins, or formulated with various stabilizing agents or carriers.", "Thus stabilized, the anti-coreceptor binding agent administered as above retains anti-coreceptor activity for an extended period (e.g., 2 to 3, up to 5 to 10 fold greater stability) under physiological conditions compared to its non-stabilized form.", "Numerous reports in the literature describe the potential advantages of pegylated proteins, which include their increased resistance to proteolytic degradation, increased plasma half-life, increased solubility and decreased antigenicity and immunogenicity (Nucci et al., Advanced Drug Deliver Reviews 6:133-155, 1991; Lu et al., Int.", "J. Peptide Protein Res.", "43:127-138, 1994, each incorporated herein by reference).", "A number of proteins, including L-asparaginase, strepto kinase, insulin, and interleukin-2 have been conjugated to a poly(ethyleneglycol) (PEG) and evaluated for their altered biochemical properties as therapeutics (see, e.g., Ho et al., Drug Metabolism and Disposition 14:349-352, 1986; Abuchowski et al., Prep.", "Biochem.", "9:205-211, 1979; and Rajagopaian et al., J. Clin.", "Invest.", "75:413-419, 1985, each incorporated herein by reference).", "Although the in vitro biological activities of pegylated proteins may be decreased, this loss in activity is usually offset by the increased in vivo half-life in the bloodstream (Nucci, et al., Advanced Drug Deliver Reviews 6:133-155, 1991, incorporated herein by reference).", "Several procedures have been reported for the attachment of PEG to proteins and peptides and their subsequent purification (Abuchowski et al., J. Biol.", "Chem.", "252:3582-3586,1977; and Beauchamp et al., Anal.", "Biochem, 131:25-33, 1983, each incorporated herein by reference).", "Lu et al., (Int.", "J. Peptide Protein Res.", "43:127-138, 1994) describe various technical considerations and compare PEGylation procedures for proteins versus peptides (see also, Katre et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 84:1487-1491, 1987; Becker et al., Makromol.", "Chem.", "Rapid Commun.", "3:217-223, 1982; Mutter et al., Makromol.", "Chem.", "Rapid Commun.", "13:151-157, 1992; Merrifield, R. B., J.", "Am.", "Chem.", "Soc.", "85:2149-2154, 1993; Lu et al., Peptide Res.", "6:142-146, 1993; Lee et al., Bioconiugate Chem.", "10:973-981, 1999; Nucci et al., Adv.", "Drug Deliv.", "Rev.", "6:133-151, 1991; Francis et al., J.", "Drug Targeting 3:321-340, 1996; Zalipsky, Bioconjugate Chem.", "6:150-165, 1995; Clark et al., J. Biol.", "Chem.", "271:21969-21977, 1996; Pettit et al., J. Biol.", "Chem.", "272:2312-2318, 1997; Delgado et al., Br.", "J.", "Cancer 73:175-182, 1996; Benhar et al., Bioconjugate Chem.", "5:321-326, 1994; Benhar et al., J. Biol.", "Chem.", "269:13398-13404, 1994; Wang et al., Cancer Res.", "53:4588-4594, 1993; Kinstler et al., Pharm.", "Res.", "13:996-1002, 1996, Filpula et al., Exp.", "Opin.", "Ther.", "Patents 2:231-245, 1999; Pelegrin et al., Hum.", "Gene Ther.", "9:2165-2175, 1998, each incorporated herein by reference).", "Following these and other teachings in the art, the conjugation of anti-coreceptor binding peptides and analogs with poly(ethyleneglycol) polymers, is readily undertaken with the expected result of prolonging circulating life and/or reducing immunogenicity while maintaining an acceptable level of activity of the PEGylated anti-coreceptor binding agent.", "Amine-reactive PEG polymers for use within the invention include SC-PEG with molecular masses of 2000, 5000, 10000, 12000, and 20000; U-PEG-10000; NHS-PEG-3400-biotin; T-PEG-5000; T-PEG-12000; and TPC-PEG-5000.Chemical conjugation chemistries for these polymers have been published (see, e.g., Zalipsky, Bioconjugate Chem.", "6:150-165, 1995; Greenwald et al., Bioconjugate Chem.", "7:638-641, 1996; Martinez et al., Macromol.", "Chem.", "Phys.", "198:2489-2498, 1997; Hermanson, Bioconjugate Techniques, pp.", "605-618, 1996; Whitlow et al., Protein Eng.", "6:989-995, 1993; Habeeb, Anal.", "Biochem.", "14:328-336, 1966; Zalipsky et al., Poly(ethyleneglycol) Chemistry and Biological Applications, pp.", "318-341, 1997; Harlow et al., Antibodies: A Laboratory Manual pp.", "553-612, Cold Spring harbor Laboratory, Plainview, N.Y., 1988; Milenic et al., Cancer Res.", "51:6363-6371, 1991; Friguet et al., J. Immunol.", "Methods 77:305-319, 1985, each incorporated herein by reference).", "While phosphate buffers are commonly employed in these protocols, the choice of borate buffers may beneficially influence the PEGylation reaction rates and resulting products.", "PEGylation of anti-coreceptor binding peptides and analogs may be achieved by modification of carboxyl sites (e.g., aspartic acid or glutamic acid groups in addition to the carboxyl terminus).", "The utility of PEG-hydrazide in selective modification of carbodimide-activated protein carboxyl groups under acidic conditions has been described (Zalipsky, Bioconjugate Chem.", "6:150-165, 1995; Zalipsky et al., Poly(ethyleneglycol) Chemistry and Biological Applications, pp.318-341, American Chemical Society, Washington, D.C., 1997, each incorporated herein by reference).", "Alternatively, bifunctional PEG modification of anti-coreceptor binding peptides can be employed.", "In some procedures, charged amino acid residues, including lysine, aspartic acid, and glutamic acid, have a marked tendency to be solvent accessible on protein surfaces.", "Conjugation to carboxylic acid groups of proteins is a less frequently explored approach for production of protein bioconjugates.", "However, the hydrazide/EDC chemistry described by Zalipsky et al., (Zalipsky, Bioconjugate Chem.", "6:150-165, 1995; Zalipsky et al., Poly(ethyleneglycol) Chemistry and Biological Applications, pp.", "318-341, American Chemical Society, Washington, D.C., 1997, each incorporated herein by reference) offers a practical method of linking PEG polymers to protein carboxylic sites.", "For example, this alternate conjugation chemistry has been shown to be superior to amine linkages for PEGylation of brain-derived neurotrophic factor (BDNF) while retaining biological activity (Wu et al., Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 96:254-259, 1999, incorporated herein by reference).", "Maeda and colleagues have also found carboxyl-targeted PEGylation to be the preferred approach for bilirubin oxidase conjugations (Maeda et al., Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications, J. M. Harris, ed., pp.", "153-169, Plenum Press, New York, 1992, incorporated herein by reference).", "In addition to PEGylation, anti-coreceptor binding agents of the invention can be modified to enhance circulating half-life by shielding the peptide or analog via conjugation to other known protecting or stabilizing compounds, or by the creation of fusion proteins with the peptide or analog linked to one or more carrier proteins, such as one or more immunoglobulin chains (see, e.g., U.S. Pat.", "Nos.", "5,750,375; 5,843,725; 5,567,584 and 6,018,026, each incorporated herein by reference).", "These modifications will decrease the degradation, sequestration or clearance of the anti-coreceptor binding agent and result in a longer half-life in a physiological environment (e.g., in the circulatory system, or at a mucosal surface).", "The anti-coreceptor binding agents modified by PEGylation and other stabilizing methods are therefore useful with enhanced efficacy within the methods of the invention.", "In particular, the anti-coreceptor binding agents thus modified maintain activity for greater periods at a target site of delivery compared to the unmodified peptide or analog.", "Even when the anti-coreceptor binding agents are thus modified, they retain substantial biological activity for inhibiting HIV-coreceptor interactions and HIV infection and related disease.", "In yet additional aspects of the invention, an anti-coreceptor binding agent is administered according to the foregoing methods in a coordinate therapy protocol with one or more additional anti-HIV agents or treatment steps.", "Thus, in various embodiments an anti-coreceptor binding agent is administered coordinately with one or more adjunct or combinatorially (e.g., additively or synergistically) effective treatment agents selected from anti-retroviral drugs (e.g., nucleoside reverse transcriptase inhibitors such as AZT (zidovudine), Videx® (ddI or didanosine), or Hivid® (ddC or zalcitabine); antiviral drugs (e.g., acyclovir); antibiotics (e.g., penicillins, cephalosporins, macrolides and lincosamides such as erythromycin, gentamycin, ceftriaxone, cefixime, azithromycin, spectinomycin, ofloxacin, ciprofloxacin, cefoxitin, clindamycin, metronidazole, amoxicillin, tetracycline, doxycycline); immunomodulatory agents (e.g., interferons (IFNα, IFNβ, IFNγ), interleukins (such as IL-1 or IL-2); G-CSF, GM-CSF, levamisole) particularly interferon); or vaccine agents.", "Other immune modulatory agents that are useful for coordinate administration with anti-coreceptor binding agents include corticosteroids, cytotoxic drugs, T-cell specific inhibitors, antisera, replacement immune globulins, and monoclonal antibodies.", "At times, specific immunosuppressive drugs can be used in conjunction with anti-coreceptor binding agent therapy, for example, azathioprine, cyclophosphamide, or cyclosprine.", "Some cytotoxic drugs may also be used for coordinate administration with anti-coreceptor binding agents, for example azathioprine, azathioprine sodium, chlorambucil, cyclophosphamide, methotrexate, methotrexate sodium, and/or clyclosprorine.", "Additional combinatorial or adjunctive therapeutic agents for use within coordinate formulations and treatment methods of the invention may comprise a glucocorticoid or non-steroidal anti-inflammatory drug (NSAID).", "Glucocorticoids useful within this aspect of the invention include short-acting glucocorticoids (e.g., cortisone and hydrocortisone), intermediate-acting glucocorticoids (e.g., prednisone, prednisolone, meprednisone, methylprednisolone and triamcinolone), and long-acting glucocorticoids (betamethasone, dexamethasone and paramethasone).", "NSAID's useful within the invention include, aspirin, salicylates, naproxen, indomethacin, piroxicam, oxaprozin, phenylbutazone, ibuprofen, flurbiprofen, fenoprofen, and ketoprofen, and the like.", "Additional therapeutic agents for use in conjunction with anti-coreceptor binding agent therapy may include the anti-malarial drug hydroxychloroquine, or sulfasalazine.", "Also useful in coordinate therapy protocols with anti-coreceptor binding agents are antihistamines, including amino alkyl ethers (e.g., diphenhydramine, clemasine), ethylenediamines (e.g., pyrilamine, tripelennamine), alkylamines (e.g., brompheniramine, chlorpheniramine, dexchlorpheniramine, triprolidine), and phenothiazines (e.g., methdilazine, promethazine, trimeprazine).", "Within alternate methods and compositions of the invention, the anti-coreceptor binding agent is administered as above coordinately, admixed or separately, simultaneously or sequentially, with of one or more of the foregoing “combinatorially effective or adjunct treatment agents”, in respective amounts sufficient (independently sufficient or combinatorially sufficient) to prevent or alleviate HIV infection or a disease condition or symptom associated therewith.", "The instant invention also includes kits, packages and multicontainer units containing the above described pharmaceutical compositions, active ingredients, and/or means for administering the same for use in the prevention and treatment of HIV infection and related disease conditions.", "Briefly, these kits include a container or formulation which contains an anti-coreceptor binding agent, typically formulated in a pharmaceutical preparation with a biologically suitable carrier.", "The anti-coreceptor binding agent is optionally contained in a bulk dispensing container or unit or multi-unit dosage form.", "Optional dispensing means may be provided, for example, an intranasal or intrapulmonary spray applicator.", "Packaging materials optionally include a label or instruction which indicates that the pharmaceutical agent packaged therewith can be used for treating HIV and related disease conditions.", "In more detailed embodiments of the invention, kits include reagents and/or devices for detecting the presence and/or status of HIV infection or related disease in a subject.", "For example, an immunological or molecular probe that binds or reacts with an HIV-specific marker detectable in blood or other biological samples to be obtained from the subject.", "Thus, the kits may contain ELISA probes for detecting HIV antigens, as well as additional, optional kit materials for collecting and/or processing samples for ELISA and other diagnostic assays.", "The kits may also contain suitable buffers, preservatives such as protease inhibitors, direct or sandwich-type labels for labeling probes, and/or developing reagents for detecting a signal from the label.", "Thus, a broad selection of therapeutic and diagnostic kits are provided within the invention based on the description herein, including kits that contain specific instructions for carrying out the prophylactic and treatment protocols and associated assays of the invention.", "The invention is further illustrated by the following specific examples which are not intended in any way to limit the scope of the invention.", "EXAMPLES Materials and Methods The following examples describe computer modeling, synthesis and testing of co-receptor agents that inhibit the binding of HIV-1 to CXCR4 and CCR5.Synthetic Peptides Exemplary peptides 15D, 15K and 15KS, were synthesized using known methods by Macromolecular Resources (Fort Collins, Colo.), peptide 15CW was synthesized by the Basic Research Laboratory National Cancer Institute, (Frederick, MD).", "The peptides were purified by reverse-phase HPLC and the homogeneity of the peptide preparations was confirmed by mass-spectrometry.", "Computer Modeling Computer-generated structural models were derived using non-hydrogen atom superimposition of homologous residues during an optimization protocol of constrained consistent valence force field (CVFF) (Dauber-Osguthorpe et al., Proteins 4:31-47, 1988; Hagler et al., J.", "Am.", "Chem.", "Soc.", "96:5319-27, 1974; and Hagler et al., Science 227:1309-15, 1985, each incorporated herein by reference), molecular dynamics sampling and conjugate gradients minimization of sampled structures.", "Cells and Culture Conditions HEK-293 cells expressing human CCR5 (HEK-CCR5) and CXCR4 (HEK-CXCR4) (Howard et al., J. Biol.", "Chem.", "274:16228-16234, 1999, incorporated herein by reference) were cultured in Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, Md.)", "containing 10% fetal bovine serum (HyClone, Logan, Utah), 2 mM glutamine, 100 units/ml penicillin and streptomycin (Quality Biologicals, Gaithersburg, Md.)", "and 400 mg/ml Geneticin (Life Technologies, Inc., Rockville, Md.)", "at 37° C. in a humidified 5% CO2 atmosphere.", "CEMx174 and THP-1 cell lines were obtained from ATCC (Rockville, Md.).", "Sup-T1 cells expressing CCR5 was the gift of James Hoxie.", "Cells were cultured in RPMI-1640 medium (BioWhittaker, Walkersville, Md.)", "containing 10% fetal bovine serum (FBS; HyClone, Logan, Utah), 2 mM glutamine, 100 units/ml penicillin and streptomycin (Quality Biologicals, Gaithersburg, Md.)", "at 37° C. in a humidified 5% CO2 atrnosphere.", "Binding Assays Binding assays were performed with HEK-CCR5 or HEK CXCR4 in triplicate by adding unlabeled competitor (anti-coreceptor binding peptide or control chemokine) and radiolabeled chemokine, 0.2 ng/ml (125I-MIP-1β or SDF-1α, specific activity 2000 Ci/mmol, NEN Life Science Products) to a cell suspension (4×105 cells in 200 μl) in RPMI 1640 supplemented with 1% bovine serum albumin and 25 mM HEPES pH 8.0 (binding medium).", "Cells were then incubated at 22° C. for 40 min with continuous rotation.", "After incubation cells were transferred to the tubes containing 800 μl of 10% sucrose in PBS and harvested by centrifugation.", "The supernatant was aspirated and the cell-associated radioactivity was measured using a 1272 Wallac gamma counter.", "Binding assays were performed with the 174xCEM and Sup-T1 cells expressing CCR5 in duplicates by adding unlabeled competitor (peptide or control chemokine) and radiolabeled chemokine, 0.2 ng/ml (125I-MIP-1β or 125I-SDF-1α, specific activity 2000 Ci/mmol, NEN life Science Products) to 300 μl cell suspension (4×106 cells/ml) in RPMI 1640 supplemented with 1% bovine serum albumin, 0.1% sodium azide and 25 mM HEPES pH 8.0 (binding medium).", "Cells were then incubated at 22° C. for 30 min with continuous rotation.", "After incubation cells were transferred to the tubes containing 800 μl of 10% sucrose in PBS and harvested by centrifugation, the supematant was aspirated and the cell-associated radioactivity was measured using a 1272 Wallac gamma counter.", "The data were analyzed by nonlinear regression using the computer program GraphPad Prizm 3.0.Chemotaxis Assays Chemotaxis assays for HEK-293 cells transfected with CCRS was performed as previously described (Howard et al., J. Biol.", "Chem.", "274:16228-16234, 1999, incorporated herein by reference).", "Briefly, the chemokine RANTES, diluted in binding medium to 100 ng/ml, was placed in the lower wells of a microchemotaxis chamber.", "Polycarbonate membrane pretreated with rat tail collagen type 1 was placed over the chemokine solution.", "The cells (1×106/ml) were suspended in binding medium along with the exemplary peptides 15D or 15K at designated concentrations and placed in the upper wells of the chamber.", "After incubation for 3 hr the membrane was removed, stained using a Diff-Quik kit (Trends Scientific, Kalamazoo, Mich.) and counted.", "The results are expressed as the “chemotaxis index”, which represents the ratio of the number of cells in high powered field in test versus control samples.", "Flow Cytometric Analysis CEMx174 cells were pelleted and resuspended at 106 cells/ml in PBS containing 1% BSA and 0.1% sodium azide (FACS buffer).", "Cells were then preincubated with designated concentrations of peptides or SDF-1-α (1 μg/ml) at 22° C. for 60 min.", "FITC-labeled anti-human CXCR4 antibody (clone 12G5, BD PharMingen, San Diego, Calif.) was added to the cells per the manufactures instructions and further incubated at 22° C. for 40 minutes.", "Cells were extensively washed with FACS buffer and analyzed using a FACS Calibur flowcytometer (Becton Dickinson).", "HEK/CCR5 cells were suspended in Dulbecco's PBS containing 1% FCS and 0.05% NaN3 (104 cells in 100 μl) and incubated with or without 5 μg of MIP-1β (PeproTech, Rocky Hill, N.J.), 0.1 mM peptide 15D or 15K for 45 min at 22° C. Cells were then treated with FITC-conjugated anti-human CCR5 antibody (2D7; BD PharMingen, San Diego, Calif.), and incubated for 30 min at 22° C. Cells were washed twice, and analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.).", "CA2+ Mobilization Assay THP-1 cells (12×106/ml in RPMI 1640 containing 10% FSB) were loaded in the presence of 5 μM fura-2 AM (Molecular Probes, Eugene, Oreg.)", "at 22° C. for 30 min in the dark.", "Subsequently, cells were washed three times and resuspended (106 cells/ml) in the buffer containing 138 mM sodium chloride, 6 mM potassium chloride, 1 mM calcium chloride, 1 mM magnesium chloride, 10 mM HEPES, pH 7.4,5 mM glucose, and 0.1% bovine serum albumin.", "1.93 ml of loaded cells was transferred into a quartz cuvette.", "50 μl of a peptide stock solution (2×10−2 M) was added to the cells, and after 3 min of incubation 20 μl of human SDF-1α (10 μg/ml, PeproTech) was added to a stirred cuvette.", "The measurements were performed using luminescence spectrophotometer LS50 B (Perkin-Elmer).", "Ca2+ mobilization in the cells in response to SDF-1α was measured by analysis of the ratio of fluorescence emitted at 510 nm after sequential excitation at 340 and 380 nm.", "Assays of HIV Infectivity PBMCs were obtained from whole blood of normal donors and isolated by Ficoll-Paque Plus (Pharmacia Biotech, Piscataway, N.J.) density gradient centrifugation, and plated at a cell density of 2×106 cells/ml.", "Monocyte-derived macrophages (MDM) were generated from adherent human peripheral blood mononuclear cells by culture for 7 days with M-CSF (100 ng/ml).", "Cultures were maintained in RPMI-1640 medium (Life Technologies, Rockville, Md.)", "supplemented with 10% heat-inactivated endotoxin-free FCS (Hyclone, Logan, Utah), 10 μg/ml gentamicin, and 1 mM glutamine.", "Cells were treated with designated concentrations of the exemplary peptides 15K or 15D, and after 1 hr cells were infected with HIV-1IIIB (T cell tropic) or HIV-1JRFL (monocyte tropic) at an multiplicity of infection (MOI) of 0.1.After 2 hr, cells were washed, and cultured for additional 48-72 hr, followed by analysis of HIV replication as determined by quantification of accumulated p24 in the supernatant.", "The production of p24 was determined by conventional sandwich ELISA, using ELISA plates pre-coated with capture anti-p24 antibodies provided by the AIDS Vaccine Program (SAIC Frederick, NCI-FCRDC, Frederick, Md.).", "The captured p24 antigen was detected using rabbit anti-HIV-1 anti-p24 antibody, and a secondary goat anti-rabbit IgG (peroxidase-labeled) antibody.", "The captured p24 protein was detected using a 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide detection system (KPL Laboratories, Gaithersburg, Md.).", "The reaction was read spectrophotometrically at 450 nm.", "No cytotoxicity was detectable following treatment of PBMC with the peptides even at a concentration of 100 μg/ml.", "Example I Structural Analysis of gp120 and Chemokines As noted above, a number of recent studies point to a role of chemokine receptors as coreceptors for HIV cell entry.", "Additional studies suggest that protein fragments or peptides from chemokines or HIV corresponding to structural determinants involved in chemokine receptor (coreceptor) binding, may be useful to block HIV-coreceptor binding and therefore serve as anti-HIV reagents.", "Considering that both chemokines and the HIV envelope protein gp120 are thought to directly interact with chemokine receptors, it is conceivable that this direct interaction may involve a part of the gp120 polypeptide chain that is structurally similar to (e.g., by homologous or convergent evolutionary relationship) receptor binding determinants of chemokines.", "However, conventional amino acid sequence comparison between gp120 and chemokines undertaken by the present inventors did not reveal any significant amino acid identity or similarity relationship that would point to a convergent, or conservative, chemokine receptor binding domain.", "Despite this apparent lack of structural homology, the present investigation focused on a putative structural element found to be conserved within the amino acid sequences of nearly all chemokines, and also shared in a corresponding structural motif postulated for the HIV-1 gp120 protein.", "In particular, all chemokines studied were found to possess a Trp residue located at the beginning of a C-terminal alpha-helix.", "This conserved Trp residue is separated by six residues from a 4th Cys residue, to comprise what is characterized herein as a conserved chemokine structural motif.", "By comparison, in the amino acid sequences of gp120 proteins of all HIV-1 isolates there is a very similar motif in a spanning part of the conserved region 3 (C3) adjacent to the V3 loop.", "Surprisingly, this motif was also found to include one or more residues within a C-terminal portion of the variable loop 3 (V3) of gp120 that are conserved between different HIV-1 strains and exhibit some homology with chemokines (Table 1).", "Example II Computer Modeling of gp120 and Chemokines Computer modeling further demonstrates that novel peptide fragments of gp120 can be template forced onto a “homologous loop” of the known structure of an exemplary chemokine, MIP-1β without violation of the general rules of protein structure.", "To assess the possibility that a selected region of gp120 may potentially assume a structure similar to that of chemokines, a model of the corresponding structure of gp120 was built using the structure of MIP-1β (Lodi et al., Science 263:1762-1767, 1994, incorporated herein by reference) as the template.", "Based on this analysis, it was determined that a model of the three-dimensional structure of the selected gp120 segment could be generated without violation of protein stereochemistry.", "In an effort to examine the functional activity of this region, peptides corresponding to the selected gp120 sequence were synthesized for further studies.", "Example III Peptide Design and Synthesis Two exemplary peptides were synthesized based on a reference peptide comprising a C-terminal portion of the V3 loop and an N-terminal of the C3 domain of gp120 (see Table 4, above).", "The sequence of HIV-1JRFL, which is very close to the consensus B of HIV-1 sequences (Korber and Los Alamos National Laboratory-Theoretical Biology and Biophysics Group T-10, Human Retroviruses and AIDS, 1998, A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, Los Alamos National Laboratory, Los Alamos, NW, 1998), was used as a template for the design of anti-HIV peptides.", "Synthesized peptides corresponding to a conserved structural motif of the HlV1JRFL gp120 protein, bearing the above-noted structural similarity to a corresponding, conserved motif identified in chemokines, are shown in Table 4.Because gp120 of different HIV-1 strains have some sequences variabilities even in conserved regions, peptides were designed with amino acid sequences similar to the widest range of naturally existing variants.", "In many HIV strains there is a lysine (k) residue instead of glutamine (Q) in position 3 (Table 4).", "Moreover, it was reasonable to change glutamine for lysine in the synthetic peptide to avoid hydrolysis and conversion to glutamic acid near the conserved positively changed arginine.", "The lysine residue preceding the tryptophan (W) in the peptide designated 15K was changed to aspartic acid (D) in the peptides designated 15D because some HIV-1 isolates have aspartic acid in this position and the change provided an opportunity to explore the significance of this substitution.", "For control experiments, peptides with “scrambled” sequences were also synthesized (peptide 15CW with the same amino acid composition as 15D, and 15KS with the same amino acid composition as 15K).", "In some control experiments a 15 amino acid peptide, 15GIG, with an amino acid sequence unrelated to gp120 was also used (See Table 4).", "TABLE 4 SEQUENCE OF SYNTHESIZED PEPTIDES SEQ Amino Acid Sequence ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 NO: HIVJRFL I R Q A H C N I S R A K W N D SEQ ID NO:3 15 K I R K A H C N I S R A K W N D SEQ ID NO:8 15 D I R K A H C N I S R A D W N D SEQ ID NO:9 15CW I R K A H C W I D R A D N N S SEQ ID NO:10 15KS K I N S W R A D N I H C K A R SEQ ID NO:11 15GIG G I G D P V T C L K S G A I A SEQ ID NO:12 As further detailed below, these exemplary peptides exhibited surprising anti-coreceptor binding activities, including competition with chemokines for binding to CCR5- and CXCR4-expressing cells, and inhibition of chemotaxis in chemokine-responsive cells.", "Correlated with these activities, the peptides mediate potent inhibition of HIV replication in macrophages and T lymphocytes, evincing their efficacy for prophylaxis and treatment of HIV infection and related disease conditions within the compositions and methods of the invention.", "This and related description herein further provides abundant structural information to enable the design and construction of a large assemblage of effective anti-coreceptor binding peptides, peptide analogs and mimetics, to serve as potent inhibitors of HIV-coreceptor interactions.", "Example IV Anti-Coreceptor Binding Activity of gp120 Peptides as Demonstrated by Inhibition of Chemokine Binding to CCR5 and CXCR4 Expressing Cells Considering that the 15K and 15D peptides were designed in part based on their structural similarity to corresponding chemokine fragments, the binding activity of these peptides for CCR5 and/or CXCR4 receptors, and corresponding anti-coreceptor binding activity against chemokines, was assessed.", "Experiments were carried out to determine the ability of these exemplary peptides to competitively inhibit binding of radiolabeled MIP-1β or SDF-1α to cells expressing CCR5 or CXCR4, respectively.", "The results, shown in FIG.", "1, demonstrate that both peptides significantly inhibit chemokine binding, although high concentrations (e.g., 100 μM) of peptide are required to mediate this effect.", "Example V Anti-Coreceptor Binding Activity of gp120 Peptides as Demonstrated by Inhibition of CCR5-Mediated Chemotaxis The inhibition of binding of MIP-1β and SDF-1α to their respective receptors suggests that the exemplary peptides 15K and 15D might also inhibit chemotaxis of cells expressing cognate chemokine receptors for appropriate ligands.", "To achieve this goal, the capacity of these peptides to inhibit the chemotactic response of HEK-CCR5 cells to a cognate ligand of CCR5 receptors, the chemokine RANTES, was assayed.", "The results demonstrated that RANTES-directed chemotactic responses were completely blocked by the addition of either 15K or 15D (FIG.", "2).", "Interestingly, one of the peptides, 15D, consistently exhibited greater inhibitory activity in these experiments.", "These results show that the 15K and 15D peptides interfere with chemokine receptor function.", "Example VI Anti-Coreceptor Binding Activity of gp120 Peptides Mediates Inhibition of HIV Infection in Monocyte-Tropic Cells The results described in the preceding Examples indicate that the 15K and 15D peptides effectively block the ability of CCR5 to serve as a receptor to mediate chemotactic responses.", "Additional experiments were thus carried out to determine the capacity of these peptides to inhibit coreceptor activity mediating cellular entry and infection by HIV-1JRFL (a monocyte-tropic HIV strain), using peripheral blood monocyte-derived macrophages.", "The results, presented in FIG.", "3, show that the addition of either 15K or 15D significantly reduced monocyte lineage target cell infection by HIV-1.Significant inhibition was exhibited at concentrations as low as 10 ng/ml.", "Interestingly, the CCR5-selective chemokine ligand MIP-1β and the 15K and 15D peptides exhibited comparable inhibitory activities (FIG.", "3).", "Example VII Anti-Coreceptor Binding Activity of gp120 Peptides Mediates Inhibition of HIV Infection in Lymphocyte-Tropic Cells In view of the foregoing binding inhibition studies, and considering the homology between the 15K and 15D peptides and the CXCR4 ligand SDF-1α, it was further undertaken to determine the capacity of the synthetic peptides to inhibit CXCR4 co-receptor function.", "In particular, experiments were carried out to assess the ability of 15K or 15D to alter the infection of PBMCs with the T cell-tropic HIV-1β strain.", "The results, presented in FIG.", "4, demonstrate that both peptides significantly reduced HIV-1 infection, in a dose-dependent manner.", "Significant inhibition of HIV-1 infection was detected with concentrations of peptides as low as 10 ng/ml.", "The scrambled peptide 15KS with the same amino acid composition as 15K but a randomized sequence manifested significantly less inhibition of virus infectively (FIG.", "4) demonstrating the importance of the sequence of amino acids for inhibiting activity.", "Example VIII Activity of gp120 Peptides in Relation to Binding of Anti-Coreceptor Antibodies To determine if peptides 15D and 15K interact directly with chemokine receptors the effect of these and control peptides on the binding of anti-chemokine receptor antibodies to CXCR4 and CCR5 was studied.", "The monoclonal antibody 12G5 recognizes a conformational extracellular epitope on CXCR4.This antibody blocks the infectivity of some X4 strains of HIV-1 and HIV-2 (Endres et al., Cell 87:745-756, 1996; Hoxie et al., J. Reprod.", "Immunol.", "41:197-211(1998); McKnight et al., J. Virol.", "74:1692-1696(1997), 19, 26).", "It also inhibits the binding of SDF-1α, a natural CXCR4 ligand Schols et al., J. Exp.", "Med.", "186:1383-1388, 1997 (Haste et al., Mol.", "Pharmacol.", "60:164-173(2001), 33).", "Further, the binding of 12G5 to CXCR4 receptor-expressing cells is prevented by anti-HIV compounds.", "The results show that peptide 15K inhibited the binding of the 12G5 antibody to CXCR4 in a dose-dependent manner (FIG.", "5A), and at a concentration of 50 μg/ml approached the efficacy of SDF-1α at 5 mg/ml.", "Peptide 15D appeared to be less effective in blocking of 12G5 antibody binding to CXCR4 (FIG.", "5B), which is not surprising since the binding site of CXCR4 includes several negatively charged residues Duranz et al., J. Virol.", "73:2752-2761, 1999.To determine the importance of the particular amino acid sequence of the inhibitory activity of the peptides.", "The effects of peptide 15K and the corresponding scrambled peptide 15KS on binding of 12G5 to CXCR4 was compared.", "The scrambled peptide 15KS manifest significantly less inhibitory activity in comparison with 15K suggesting the importance of the specific peptide sequence (FIG.", "5C).", "Apparently there is a direct correlation between the ability of 15K to inhibit the anti-CXCR4 antibody binding and its ability to inhibit HIV-1 infectivity since the scrambled peptide 15KS with the same amino acid comparison has reduced ability to manifest both these activities.", "The effect of the exemplary peptides 15K and 15D on the binding of 2D7 antibodies to CCR5 was assayed to further evaluate the mechanism of anti-coreceptor activity of the peptides.", "The 2D7 monoclonal antibody binds to a conformational epitope on CCR5, and inhibits Ca2+ flux induced by RANTES.", "This antibody has also been shown to inhibit HIV infection in vitro (Wu et al., J. Exp.", "Med.", "186:1373-81, 1997, incorporated herein by reference).", "It does not, however, inhibit binding of gp120-SCD4 complexes to CCR5-expressing L1.2 murine cells (Olson et al., J. Virol.", "73:4145-4155, 1999, incorporated herein by reference).", "The effects of peptides 15D and 15K on 2D7 antibody binding to HEK293/CCR5 cells were analyzed by flow cytometry.", "No significant effect of either of the two peptides on anti-CCR5 antibody binding was observed.", "These results indicate that the peptides bind to an epitope on the CCR5 receptor that is spatially and sterically distinct from the antibody binding site.", "Considering that both the peptides and the 2D7 antibodies inhibit HIV infection in vitro, these results may be interpreted as underscoring the unexpected nature and advantages of the present invention.", "Example IX Effects of Peptides on Induction of Intracellular Ca2+ Concentration in CXCR4 Expressing THP-1 Cells The increase of intracellular Ca2+ concentration mediated by a chemokine receptor in response to a cognate chemokine is a reliable assay for measurement of chemokine agonist activity.", "The human monocytic cell line THP-1 expresses a high level of CXCR4 and responds well to SDF-1α stimulation (FIG.", "6A), so these cells were used to determine whether peptides 15D and 15K could block the activation of CXCR4.Preincubation of cells with peptide 15D (at a concentration 500 μM) for 2 min completely inhibited cellular response to SDF-1α (FIG.", "6B).", "Peptide 15K also inhibited Ca2+ mobilization although less efficiently than 15D (FIG.", "6C).", "There is a probable direct correlation between the greater ability of 15D to inhibit Ca2+ mobilization in response to SDF-1α and anti-HIV-1 activity because in some experiments higher anti-HIV-1 activity of 15D was observed in comparison with 15K (data not shown).", "The scrambled peptide 15CW, with amino acid composition identical to 15D (Table 4), was less effective at inhibiting the Ca2+ mobilization response (FIG.", "6D) suggesting the importance of the specific peptide sequence.", "The peptide treatment alone did not induce Ca2+ mobilization.", "Example X Effect of gp120 Derived Peptides 15K and 15D on Binding of Chemokines to CCRS and CXCR4 Expressing Cells Since the 15K and 15D peptides were designed based on their structural homology with a chemokine fragment, whether these peptides might exhibit binding activity for CCR5 or CXCR4 was determined.", "Experiments were carried out to assess the ability of the peptides to competitively inhibit binding of radiolabeled MIP-1β or SDF-1α to cells expressing CCR4 or CXCR4, respectively.", "To determine the effect of peptides exclusively on chemokine binding and to exclude the contribution of chemokine internalization, cells were incubated at room temperature in the presence of 0.1% sodium azide.", "Under such conditions all cell-bound ligand could be removed by rinsing the cells with 0.1 M Gly-HCl pH 2.5, excluding the contribution of internalization to the chemokine binding.", "The binding experiments demonstrated that the 15D and 15K peptides competitively inhibited chemokine binding to CCR5 (FIG.", "7A) and CXCR4 (FIG.", "7B).", "The binding experiments did not reveal any difference in the inhibitory activity of peptides 15K and 15D in comparison with their scrambled analogs (data not shown).", "The control 15-mer peptide 15GIG with a different amino acid composition manifested significantly lower competing activity (FIG.", "7C).", "It is possible that the particular scrambling of the peptide sequence that was performed did not change the sequences of the scrambled peptides sufficiently to significantly reduce their capacity to bind to the receptors because scrambling of these peptides may have created similar amino acid triplets in other positions of the peptides (like RAK and KAR in 15K and 15KS, Table 4).", "It is possible that because of this peptide 15KS also manifested a low level of inhibitory activity (although significantly less than original peptide) on anti-CXCR4 antibody binding to the receptor (FIG.", "5C).", "Additional structure-functional studies are necessary to determine the minimum sequence of the peptides sufficient for inhibition of virus.", "To briefly summarize the foregoing description, the interaction of HIV envelope glycoprotein gp120 with a chemokine receptor is known to be a prerequisite for viral attachment and entry into target cells (Alkhatib et al., Science 272:1955-1958, 1996; Berson et al., J. Virol.", "70:6288-6295, 1996; Deng et al., Nature 381:661-666, 1996; and Dragic et al., Nature 381:667-673, 1996, each incorporated herein by reference).", "Monocyte-tropic viruses (R5 strains) utilize a distinct chemokine receptor, CCR5, for cell entry, while T cell tropic viruses (X4 strains) utilize CXCR4 receptors.", "Chemokine receptor binding site(s) on gp120 are thought to be formed, or exposed, after binding of gp120 to CD4 (Wu et al., Nature 384:179-183, 1996, incorporated herein by reference).", "However, gp120 binding to the chemokine receptor in certain HIV strains does not require interaction with CD4 (Hoffman et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 96:6359-6364, 1999; and Hoxie et al., J. Reprod.", "Immunol.", "41:197-211, 1998, each incorporated herein by reference).", "The V3 loop of gp120 has been identified as the major determinant of cellular tropism and coreceptor specificity (Cocchi et al., Nat.", "Med.", "2:1244-1247, 1996; and Hwang, et al., Science 253:71-74, 1991, each incorporated herein by reference).", "Nevertheless, the precise region or residues within this 35-37 amino acid V3 loop responsible for mediating these phenotypic effects has not yet been established (Hung et al., J. Virol.", "73:8216-8226, 1999, incorporated herein by reference).", "Synthetic cyclized peptides corresponding to the V3 loop of gp120 of X4 and dual strains of HIV-1 (but not an R5 strain) at micromolar concentrations could prevent binding of anti-CXCR4 antibodies.", "Some of these peptides at micromolar concentrations inhibited the infectivity of HIV-IIIB (Sakaida et al., J. Virol.", "72:9763-9770, 1998, incorporated herein by reference).", "Synthetic polymer preparations including a putative V3 consensus sequence (GPGRAF, SEQ ID NO:13) of HIV-1 were reported to inhibit HIV-1 infection by an unknown mechanism.", "However, it is unlikely that this inhibition was due to competition with gp120 binding to the chemokine receptor, or to CD4 (Moulard et al., J. Pept.", "Res.", "53:647-655, 1999, incorporated herein by reference).", "Although the influence of V3 on Hw coreceptor utilization is well established, other more conservative regions of gp120 are also proposed to be involved in coreceptor usage (Rizzuto et al., Science 280:1949-1953, 1998, incorporated herein by reference).", "Within the present invention, a conservative chemokine receptor binding motif of gp120 was identified which shares certain similarity in amino acid sequence that appears conserved among relevant chemokines.", "More specifically, the present inventors observed that in the amino acid sequences of most chemokines there is a conserved Trp residue separated by six amino acid residues from a fourth Cys residue.", "In comparison, the gp120 of all HIV isolates analyzed exhibited a comparable structural motif in the C3 region following the V3 loop.", "Moreover, several residues of the C-terminal part of V3 loop also manifest some homology with a canonical chemokine sequence (Table 1).", "Computer modeling as provided herein further demonstrated that the corresponding fragment of gp120 could be template forced onto a “homologous” loop of the known three dimensional structure of an exemplary chemokine MIP-1β (for specific MIP-1β structural detail, see, e.g., Lodi et al., Science 263:1762-7, 1994, incorporated herein by reference) without violation of protein stereochemistry.", "Prompted in part by these discoveries, two candidate peptides were synthesized which exemplify the proposed gp120 structural motif (Table 2).", "Both candidate peptides were demonstrated to compete with chemokines for binding to CCR5- and CXCR4-expressing cells.", "In addition, the exemplary peptides each inhibited CCR5-directed chemotaxis.", "Correlated with these anti-coreceptor activities, the peptides mediated potent inhibit replication of both monocyte-tropic HIV-1JRFL and T cell tropic HIV-1IIIB.", "The X-ray structure of an HIV-1 gp120 core (not including V-loops), complexed with a two-domain fragment of CD4, and an antigen-binding fragment of neutralizing antibody, has been reported (Kwong et al., Nature 393:648-659, 1998).", "Although the structure does not include variable loops of gp120 it did include the C3 portion of the fragment of gp120, which the present invention considered to be structurally similar to the chemokines and potentially involved in interaction with chemokine receptors.", "In fact, the three-dimensional structure of this fragment (HIVHXB2, residues 331-340, Table 1) appeared to be similar to the corresponding MIP-1β fragment.", "Both motifs are characterized by an α-helix preceded by a turn.", "Both contain a Trp residue with the indole ring buried in the interior region of the turn and with the α-carbon present on an exterior turn of the helix.", "The surface residues on the helices are characterized by long aliphatic side chains, terminated with polar groups, including Lys, Glu, Gln, and Asn.", "Importantly, this fragment is located within the region of gp120 molecule, which was implicated in CCR5 binding (Rizzuto et al., Science 280:1949-1953, 1998).", "Since the peptides 15D and 15K include only 5 residues from the C-terminal part of the V3 loop, which is more conserved than other parts of V3 loop, and 9 residues from the N-terminal part of the conservative C3 region, it was reasonable to expect that anti-HIV activity of these peptides may not be dependent on virus tropism.", "The effects of the peptides on HIV-1 infectivity using R5 and X4 viruses confirmed this prediction (FIGS.", "3 and 4).", "The data above demonstrating that peptides 15K and 15D compete with anti-CXCR4 antibody 12G5 suggests a direct interaction of the peptide with this receptor.", "Although the 15D peptide had reduced ability to block binding of the 12G5 antibody, the infectivity data indicates that the low affinity interaction of 15D was still sufficient to interfere with viral infection.", "Moreover, the inhibition of the SDF-1α induced intracellular Ca2+ influx by peptides 15D and 15K also supports the conclusion that both antiviral peptides interact with chemokine receptors.", "The affinity of interaction of the peptides 15D and 15K with chemokine receptors is apparently low, as evidenced by the high concentration of the peptides required to inhibit anti-CXCR4 antibody binding, mobilization of intracellular Ca2+ in response to SDF-1α, and chemokine binding.", "However, even this low affinity interaction is sufficient for interference with HIV-1 infection based on the low concentration of peptides required for blocking of the viral infection of macrophages and T lymphocytes.", "Indeed, it is not necessary for an efficient inhibitor of virus interaction with the coreceptor to also be a strong competitor of chemokines binding to the same receptor, because the envelope protein of HIV-1 only mimicks the highly specific binding of a chemokine with its receptor and the affinity of this interaction can be quite low (Hoffman et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 99:11215-11220, 2000).", "The use of low affinity anti-viral drugs interfering with HIV-1 coreceptor interaction may allow targeting multiple cellular receptors while maintaining the ability to inhibit interactions of a viral glycoprotein, which is subject to frequent mutation.", "This may provide the basis for the capacity of the present peptides to inhibit infection by viruses using different coreceptors.", "Moreover, the rather weak competition of peptides 15D and 15K with chemokines for receptor binding, together with potent inhibition of HIV-1 infectivity, can be therapeutically preferable to high affinity inhibitors of CXCR4 and CCR5, because 15K and 15D peptides would not compromise functions of potentially critical chemokines such as SDF-1α or the CCR5 ligands.", "The present invention has identified a region of HIV-1 gp120, which is structurally similar to chemokines, and appears to be directly involved in the interaction with certain chemokine receptors.", "The above findings that these peptides inhibit HIV-1 infection of human monocyte-derived macrophages and T-lymphocytes at low nanomolar concentrations suggest that these peptides, their analogs, and peptide menetics, can be used to dissect gp120 interactions with different chemokine receptors and could serve as not only leads for the design of new peptide inhibitors of HIV-1 not restricted by viral tropism, but are useful themselves as therapeutic agents to prevent binding of HIV-1 to a susceptible cell thereby reducing infection and viral replication in the treatment and prevention of HIV infection and related disease.", "Moreover, it may also be possible that antibodies raised against this sequence of HIV-1 gp120 can also have anti-HIV protective and therapeutic activity by reducing or preventing HIV binding to a susceptible cell.", "Although the foregoing invention has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to the artisan that certain changes and modifications are comprehended by the disclosure and may be practiced without undue experimentation within the scope of the appended claims, which are presented by way of illustration not limitation.", "All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes." ] ]	Patent_10468182

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